Tissue Engineering Strategies for Cardiac Regeneration

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2 Tissue Engineering Strategies for Cardiac Regeneration A dissertation submitted to the Division of Research and Advanced Studies of the University of Cincinnati in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY (Ph.D.) in the Biomedical Engineering Program in the School of Energy, Environmental, Biological & Medical Engineering of the College of Engineering and Applied Science October 2011 by Jennifer R. Hurley B.S., Illinois Institute of Technology, Chicago, IL, 2002 Dissertation Committee: Daria A. Narmoneva, PhD, Committee Chair, Associate Professor, Biomedical Engineering, School of Energy, Environmental, Biological & Medical Engineering, University of Cincinnati. Ronald Millard, PhD, Professor, Pharmacology & Cell Biophysics, University of Cincinnati College of Medicine, Co-Principal Investigator of NSF IGERT, Bio-Applications of Membrane Science and Technology. Jason Shearn, PhD, Assistant Professor, Biomedical Engineering, School of Energy, Environmental, Biological & Medical Engineering, University of Cincinnati.

3 ABSTRACT Cardiovascular disease (CVD) is a significant health burden and is the leading cause of death in the United States. Cardiac pathologies which lead to cardiovascular disease, including myocardial infarction and diabetes, often result in damaged, ischemic and/or fibrotic tissue and would benefit from a therapeutic approach to promote cardiac regeneration. The use of a tissue engineering approach, where the principles of biology and engineering are combined to develop functional tissue substitutes, is a very promising and active field of study. However, cardiac tissue engineering approaches to date have been limited due to 1) insufficient vascularization of engineered tissues (essential for the supply of oxygen, nutrients, and immune cells and waste removal) and 2) impaired regulation of extracellular matrix remodeling and turnover (leading to damaging structural, geometric and functional changes in the heart). Therefore, promoting both vascularization and reparative matrix remodeling is one of the key requirements for successful cardiac regeneration via cardiac tissue engineering approaches. Our long-term goal is to develop a new cardiac tissue engineering approach to treat cardiovascular diseases, including myocardial infarction and diabetic cardiomyopathy, by applying recent advances in nanobiotechnology to modify the microenvironment of heart muscle and promote cardiac regeneration. The studies in this dissertation contribute to this goal by investigating the interactions between cardiac cells, including endothelial cells and fibroblasts, as well as the use of RAD16-II peptide nanofibers and mechanical strain to promote angiogenesis and reparative matrix remodeling by these cells in vitro. The central hypothesis of this research is that RAD16-II peptide nanofibers can be used as a microenvironment for a cardiac tissue engineering approach which promotes cardiac regeneration via revascularization and reparative matrix remodeling by cardiac fibroblasts. i

4 The results from this dissertation research help identify the role of fibroblasts in temporal regulation of the angiogenic process and in reparative matrix remodeling. Additionally, the effects of diabetes on the matrix remodeling response by fibroblasts and potential compensatory tissue engineering strategies (i.e. mechanical stretch, peptide nanofiber microenvironment) were investigated as well. The culture system of RAD16-II nanofibers was utilized as both a controlled microenvironment for study and as a system which supports matrix metalloproteinasemediated extracellular matrix remodeling by cardiac fibroblasts in vitro. The findings of this research will contribute towards developing an optimal microenvironment to enhance cardiac regeneration after injury and for cardiac tissue engineering applications. ii

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6 ACKNOWLEDGMENTS The author would like to acknowledge the National Science Foundation (NSF-IGERT ) and the American Heart Association (GRA 09PRE ) for pre-doctoral fellowship support. Additional student support was provided by the University Research Council s Graduate Student Research Fellowship, National Institutes of Health (NIH 1R21DK A1) and the University of Cincinnati Department of Biomedical Engineering. The research performed in this dissertation was funded by the American Heart Association (AHA BGIA B), the National Institutes of Health (NIH 1R21DK A1), and the University of Cincinnati Department of Biomedical Engineering. The author has many people to thank for their support, guidance, and friendship during the past five years. Dr. Daria Narmoneva for welcoming me into her lab with open arms and providing unending support, direction and guidance for my intellectual endeavors. As well as being my most important mentor, thank you for being a supportive friend throughout it all. Dr. Jason Shearn, Dr. Ronald Millard, and Dr. William Ball for their tenure on my qualifying and/or dissertation committees. Your feedback, guidance and support have helped shape me into the scientist/engineer I am today. Abdul Sheikh, Hongkwan Cho, Varun Krishnamurthy, Toloo Taghian, and Swathi Balaji for being valued colleagues and more importantly friends. You were always there to bounce ideas off of, talk through problems, help out with experiments or just be a friend. All of the undergraduate students that I have mentored over the years and that have been involved in this research Justin Wong and Thea Zimnicki (NSF REU Summer students), iv

7 Cameron Ingram and Meredith Beckenhaupt (Academic Year REU students), Andrew Mutchler (UC BME Co-op student), Kristof Nolan and Brenton Huxel (UC BME Independent Study students). Thank you for your time, contributions, dedication and interest in the research. To everyone else in the Biomedical Engineering and IGERT programs staff, faculty, graduate students and undergraduate students. While there are too many individuals to name, you ve all played an important role in getting me this point today and I thank you. Finally, to my wonderful husband Kevin. You re fantastic and you ve put up with me through thick and through thin. I couldn t have asked for a more supportive partner in all this. v

8 TABLE OF CONTENTS Abstract i Acknowledgements iv Table of Contents vi List of Figures and Tables vii Chapter 1 Introduction and Specific Aims 1 Chapter 2 Background 11 Chapter 3 Complex temporal regulation of capillary morphogenesis by fibroblasts 32 Chapter 4 Effects of mechanical strain and diabetic phenotype on fibroblast 63 matrix remodeling response Chapter 5 Self-assembling peptide nanofibers for MMP-mediated matrix 85 remodeling in diabetic cardiomyopathy Chapter 6 Discussion and Conclusions 112 Chapter 7 Future Directions 122 Bibliography 131 Appendix Publications, peer-reviewed abstracts and presentations 146 vi

9 LIST OF TABLES AND FIGURES CHAPTER 2 Figure 1. Components of cardiac tissue engineering strategies. 16 Table 1. Major challenges for development of successful cardiac tissue 17 engineering strategies. Figure 2. Structural organization of the myocardial extracellular matrix. 19 Figure 3. Cardiac fibroblast functions associated with matrix remodeling and 22 cardiac regeneration. CHAPTER 3 Figure 4. Capillary morphogenesis in endothelial and endothelial-fibroblast 42 cultures on peptide nanofibers. Figure 5. Long term cell viability and apoptosis levels in peptide nanofiber 44 cultures. Figure 6. Temporal profiles of Vascular Endothelial Growth Factor and 47 Angiopoietin-1 expression in the medium and matrix-bound. Figure 7. Protein expression of matrix metalloproteinase -2 and Figure 8. Collagen I deposition. 51 Figure 9. Stiffness of cell-nanofiber constructs measured using rheometry. 54 Figure 10. Proposed schematic of angiogenic regulation by fibroblasts in 56 endothelial-fibroblast co-cultures in vitro. CHAPTER 4 Table 2. Effects of magnitude and duration of strain on matrix remodeling 66 responses of fibroblasts. vii

10 Figure 11. Evidence of cardiac fibrosis in the diabetic heart 6 weeks 71 post STZ injection. Figure 12. Phalloidin staining of static and strained samples at 24 hrs. 72 Figure 13. Protein expression of matrix metalloproteinase Figure 14. Protein expression of collagen I. 75 Figure 15. Protein expression of vascular endothelial growth factor. 76 Figure 16. Cell proliferation. 78 Figure 17. Cell apoptosis. 79 CHAPTER 5 Figure 18. MMP-2 release kinetics from peptide nanofibers. 94 Figure 19. Staining for fibroblast phenotype. 95 Figure 20. Cell apoptosis levels in cell-scaffold cultures. 97 Figure 21. Cell proliferation levels in cell-scaffold cultures. 98 Figure 22. Native protein expression of matrix metalloproteinase Figure 23. Collagen I deposition. 102 Figure 24. Collagen IV deposition. 103 Figure 25. Stiffness of cell-scaffold constructs measured using rheometry. 105 Figure 26. Proposed schematic of matrix remodeling response by cardiac 106 fibroblasts (both wild type and diabetic) in the peptide nanofiber scaffold. CHAPTER 7 Figure 27. Myocardial injection of peptide nanofibers and exogenous MMP in a wild type rat animal model. viii

11 CHAPTER 1 Introduction and Specific Aims Cardiovascular disease (CVD) is a significant health burden and is the leading cause of death in the United States, accounting for 33.6% of all deaths in 2007 (1). Two distinct cardiac pathologies which lead to cardiovascular disease are coronary heart disease (CHD) and diabetic cardiomyopathy (DCM). CHD and DCM often result in damaged, ischemic and/or fibrotic tissue resulting from a loss of vascularization and aberrant matrix remodeling either after an acute attack or with the disease progression and both would benefit from a therapeutic approach to promote cardiac regeneration via revascularization and reparative matrix remodeling. The use of a tissue engineering approach, where the principles of biology and engineering are combined to develop functional tissue substitutes (2), is very promising and various strategies have been intensively investigated (3-10). However, cardiac tissue engineering approaches to date have been limited due to 1) insufficient vascularization of engineered tissues (essential for the supply of oxygen, nutrients, and immune cells and waste removal) and 2) impaired regulation of extracellular matrix remodeling and turnover (leading to damaging structural, geometric and functional changes in the heart (11, 12)). Therefore, promoting both vascularization and reparative matrix remodeling is one of the key requirements for successful cardiac regeneration via cardiac tissue engineering approaches. Our long-term goal is to develop a new cardiac tissue engineering approach to treat cardiovascular diseases, including myocardial infarction and diabetic cardiomyopathy, by applying recent advances in nanobiotechnology to modify the microenvironment of heart muscle and promote cardiac regeneration. The studies reviewed in this dissertation contribute to this 1

12 goal by investigating the interactions between cardiac cells and also the use of RAD16-II peptide nanofibers and mechanical strain to promote angiogenesis and reparative matrix remodeling in vitro. The central hypothesis of this research is that RAD16-II peptide nanofibers can be used as a microenvironment for a cardiac tissue engineering approach which promotes cardiac regeneration via revascularization and reparative matrix remodeling by cardiac fibroblasts. This dissertation research was investigated via four Aims which are outlined in this chapter. AIM 1 Coronary heart disease (CHD) leads to a narrowing of the blood vessels that supply blood and oxygen to the heart. In a myocardial infarction (MI), the flow of blood and oxygen through these vessels is actually blocked for enough time that part of the heart muscle becomes ischemic and is damaged or dies. The heart is limited in its ability to repair and regenerate itself, resulting in the formation of non-functional scar tissue and a dearth of living cells. Therefore, there is great interest in using cardiac tissue engineering strategies to enhance cardiac function and regeneration after myocardial injury. In order to develop novel strategies to promote vascularization and remodeling in damaged myocardial tissue, it is first necessary to understand the interactions between the major cardiac cell types, in particular the interactions between endothelial cells and cardiac fibroblasts. Previous findings suggest a possible dual role chemical and mechanical for fibroblasts during the process of vasculature assembly and remodeling in the environment of healing tissue (12, 13). However, our knowledge of these processes remains incomplete and may benefit from the use of a three-dimensional culture system which mimics the native cell environment in pore size and gross structure while uncoupling scaffold-triggered signaling (not present in peptide 2

13 nanofibers) from cell-cell interactions. Previous studies have shown that synthetic RAD16-II peptide nanofibers provide a pro-angiogenic microenvironment, enhancing capillary-like network formation in vitro without the addition of external growth factors and allowing for long term study of cell-cell interactions (14, 15), and thus represent an appropriate in vitro system to study endothelial-fibroblast interactions during angiogenesis for cardiac tissue engineering applications. The objective and hypothesis for Aim 1 are as follows. Aim 1: Elucidate the mechanisms for fibroblast-mediated temporal regulation of angiogenesis and matrix remodeling using the culture system of RAD16-II nanofibers. Hypothesis 1: Fibroblasts temporally regulate capillary morphogenesis chemically via growth factor expression and mechanically via cell-mediated scaffold disruption, extracellular matrix deposition and remodeling. The results from this study (16) suggest that at the early stages of the tissue repair process, fibroblasts play a major role in capillary morphogenesis both by paracrine growth factor signaling as well as mechanical disruption of the extracellular matrix (ECM) to lead the way for the formation of endothelial cell networks. At the later stages, the role of fibroblasts as regulators of the mechanical microenvironment becomes more prominent. Endothelial-fibroblast interactions appeared to help maintain the balance in ECM homeostasis, enhancing both matrix metalloproteinase-2 and collagen I production and resulting in improved integrity and a more stable microenvironment by day 6. The results of Aim 1 provide important insight into the temporal manner in which fibroblasts regulate capillary morphogenesis and matrix remodeling in vitro in the controlled microenvironment of the RAD16-II peptide nanofiber scaffold. The published study (16) is provided in its entirety in Chapter 3 of this manuscript. 3

14 AIM 2 The context in which Aim 1 was investigated was damaged or scarred myocardial tissue, particularly after myocardial infarction. Excessively fibrotic and stiff repair tissue often results from the healing process after infarction, and this condition naturally led to a further research question regarding how matrix compliance and stiffness may contribute to our findings. Recent studies indicate that more compliant matrices, including scaffolds made from RAD16-II nanofibers, better promote in vitro network formation and cell migration, as compared to more rigid ones (17, 18). Therefore, two concentrations of peptide nanofibers were investigated: 1% (10 mg/ml), which is more stiff to better represent the fibrotic microenvironment of the healing heart, and more compliant nanofibers (0.6%, or 6 mg/ml). While the peptide concentrations investigated resulted in stiffness values significantly lower than that of cardiac muscle ( kpa (19)), they do represent a promising synthetic biomaterial with considerable structural integrity for a hydrogel scaffold. Aim 2 was performed concurrently with the previous aim and the objective and hypothesis were as follows. Aim 2: Determine the effects of matrix stiffness on cell-cell interactions and mechanical regulation of the microenvironment. Hypothesis 2: A more compliant nanofiber matrix will promote in vitro network formation and cell migration. In this study (16), the results showed that the compliant nanofibers allowed for coordinated migration of both endothelial cells and fibroblasts as well as scaffold disruption. In contrast, in the stiff nanofibers, only the highly migratory fibroblasts appeared able to move through the scaffold, resulting in lower stiffness in the fibroblast constructs. These results are in agreement with previously reported enhanced capillary morphogenesis in more compliant 4

15 RAD16-II scaffolds (17), and confirm that in this culture system matrix stiffness does affect cell behavior, with fibroblasts playing a more significant role in mechanical regulation in denser nanofibers. However, the results suggest that more compliant nanofibers better support migration of both endothelial cells and fibroblasts, allowing for a better balance between cell migration and ECM remodeling and may ultimately be better suited for improved angiogenesis and long term stability. The results of Aim 2 suggest that the angiogenic response by endothelial cells and fibroblasts may be controlled through simple changes to the matrix stiffness and has important implications for the development of cardiac tissue engineering strategies. The published study (16) is provided in its entirety in Chapter 3 of this manuscript. AIM 3 While our overarching clinical interest remained cardiovascular disease, the research focus for Aims 3 and 4 shifted towards the serious health concern of diabetes and in particular the cardiac condition of diabetic cardiomyopathy (DCM). DCM is a diabetes-associated cardiovascular condition defined as ventricular dysfunction in the absence of other etiological factors (20-23). Pathological alterations to the myocardium resulting from DCM include circulatory defects, impaired heart muscle contraction, and progressive fibrosis. In DCM, similar to CHD and MI, there exists a need for cardiac regeneration due to excessive fibrosis of the cardiac tissue. In particular, the promotion of reparative matrix remodeling to reduce cardiac fibrosis appears to be a promising therapeutic strategy. 5

16 While many factors regulate extracellular matrix turnover, impaired matrix metalloproteinase (MMP) activity and increased collagen accumulation in diabetic human patients and animal models represent a novel therapeutic target for DCM. Recent studies have shown that cardiac dysregulation of MMP-2 expression contributes to the increased collagen deposition, progressive fibrosis, increased ventricular stiffness, and cardiac dysfunction seen in diabetic cardiomyopathy (24-27). Detrimental changes to the cardiac fibroblast phenotype resulting from diabetic conditions include decreased MMP expression and activity (24-26, 28, 29) and increased collagen synthesis (29-31). Therefore, overcoming the inhibitory effects of diabetic conditions on matrix remodeling by cardiac fibroblasts represents a novel target for therapeutic treatment of DCM. One possible strategy to overcome the diabetes-associated deficiencies in matrix remodeling by cardiac fibroblasts may be via stimulation by environmental stimuli which are experienced in the native environment, such as mechanical strain. Mechanical stimulation plays an important role in tissue development and repair and cells adapt and respond to changes in their mechanical environment via morphological and phenotypic alterations (32). In vitro studies have shown that the application of mechanical strain on fibroblasts in culture leads to a variety of responses related to ECM remodeling (33, 34), including increased gene expression and matrix deposition of extracellular matrix proteins (35-41), increased expression of growth factors (including insulin-like growth factor (IGF-1) (42, 43), vascular endothelial growth factor (VEGF) (44), and transforming growth factor (TGF-β) (36)), and increased MMP-2 activation (45). While such studies provide important information regarding the effect of mechanical strain on the matrix remodeling response of fibroblasts, there remains much more to learn, in particular 6

17 with regards to the effect of diabetic phenotype in conjunction with mechanical strain. Therefore, the objective and hypotheses of Aim 3 are as follows. Aim 3: Determine the effect of diabetes and mechanical strain on the reparative matrix remodeling response of cardiac fibroblasts in vitro. Hypothesis 3: Diabetic phenotype will result in a diminished reparative matrix remodeling response by cardiac fibroblasts in vitro, with decreased MMP expression and increased extracellular matrix deposition. Hypothesis 4: Stimulation of cardiac fibroblasts with mechanical strain will result in enhanced matrix remodeling response and attenuate diabetes-induced phenotypic cell alterations. In this study, a commercially available Flexcell system was used to apply uniaxial stretch to rat cardiac fibroblasts. The fibroblast matrix remodeling response due to strain was measured via MMP-2 (ECM remodeling), collagen I (ECM deposition), and VEGF expression (cell migration stimulus) as well as fibroblast morphology and orientation and cell proliferation and apoptosis. Interestingly, the results from this study did not agree with the hypotheses. Neither model of in vitro diabetic phenotype investigated resulted in a clear and complete reduction in matrix remodeling response, with cells cultured in high glucose exhibiting only increased ECM deposition and cells harvested from diabetic animals exhibiting only decreased MMP expression. However, the results do demonstrate that cardiac fibroblasts harvested from diabetic animals maintain aspects of in vivo matrix remodeling deficiencies, including decreased proliferation and MMP-2 expression, for extended in vitro culture periods. Additionally, the results from this study also demonstrated that application of cyclic strain resulted in limited improvements in matrix remodeling response, both by wild type fibroblasts and those of diabetic phenotype. 7

18 Therefore, while mechanical stretch may prove to be an effective tissue engineering strategy to compensate for the dysregulation of matrix remodeling by diabetic cardiac fibroblasts, the results of Aim 3 do not support this observation in our particular experimental setup. Increased magnitude and duration of strain may prove to be a more efficacious approach, resulting in an improved matrix remodeling response by cardiac fibroblasts. The study is provided in its entirety in Chapter 4 of this manuscript. AIM 4 While Aim 3 investigated mechanical strain as one possible strategy to overcome diabetes-associated deficiencies in matrix remodeling in fibroblasts, there remain a number of alternative approaches. Overcoming the inhibitory effects of diabetic conditions on matrix remodeling by cardiac fibroblasts through stimulation of native MMP-2 expression or delivery of exogenous MMP-2 represents a novel target for therapeutic treatment of DCM (27). The results obtained in Aim 1 (16) demonstrate that RAD16-II nanofibers promote fibroblast expression of native MMP-2 without detriment to angiogenesis and overall matrix stability, suggesting their potential to attenuate MMP-2 deficiency and thus improve matrix remodeling in diabetic conditions. Additionally, RAD16-II nanofibers can potentially be used as a protein delivery vehicle (46) for exogenous MMP-2 to increase local cardiac MMP-2 concentration. Therefore RAD16-II peptide nanofibers appear to be a promising cardiac tissue engineering approach to improve reparative matrix remodeling and cardiac regeneration in DCM. The objective and hypotheses of Aim 4 are as follows. Aim 4: Quantify the effect of the peptide nanofiber microenvironment on MMP2-mediated extracellular matrix remodeling by normal and diabetic cardiac fibroblasts in vitro. 8

19 Hypothesis 5: The peptide nanofiber microenvironment will attenuate matrix remodelingrelated deficiencies observed in diabetic cardiac fibroblasts. Hypothesis 6: The peptide nanofiber microenvironment will promote native MMP-2 expression and improve the matrix remodeling response by cardiac fibroblasts in vitro. Hypothesis 7: Delivery of exogenous MMP-2 using the peptide nanofibers will promote in vitro matrix remodeling by cardiac fibroblasts and that the effect will be additive to that of nanofibers alone. The results of this study demonstrate that nanofibers support temporal in vitro matrix remodeling by cardiac fibroblasts both wild type and diabetic. Importantly, no significant differences were observed between wild type and diabetic fibroblast remodeling response, indicating that the nanofiber environment may attenuate diabetes-induced phenotypic cell alterations (28). For both wild type and diabetic fibroblasts, increased native MMP-2 expression and ECM deposition was observed at day 1. By day 6, MMP-2 expression was still prominent with changes in stiffness values consistent with changes in ECM levels due to deposition and remodeling. However, by day 14 a shift towards more active matrix remodeling was observed, with decreased ECM levels and decreased stiffness. However, while the RAD16-II nanofibers are clearly an effective protein delivery vehicle, no clear improvement in fibroblast matrix remodeling response was seen as a result of exogenous MMP-2 incorporation into nanofibers. Therefore, the results from this study suggest that using the nanofibers alone to stimulate native MMP-2 expression by diabetic cardiac fibroblasts may ultimately be the more suitable strategy to improve reparative matrix remodeling. The results of Aim 4 provides insight into how the RAD16-II peptide nanofibers may be used in cardiac tissue engineering approaches for cardiac regeneration, particularly for treatment 9

20 of diabetes and diabetic cardiomyopathy. The study is provided in its entirety in Chapter 5 of this manuscript. The format of the dissertation will be as follows. Chapter 2 will provide background information closely related to the research. Chapters 3, 4 and 5 will discuss the findings from Aims 1-4. The chapters will be presented in publication format, as all studies are either published, submitted for publication or in preparation. Chapter 6 and 7 will provide discussion and concluding remarks as well as suggestions for future studies. 10

21 CHAPTER 2 Background Tissue engineering aims to combine the principles of biology and engineering to develop functional tissue substitutes (2). Myocardial infarction and other cardiac pathologies often result in ischemic tissue and would benefit from a tissue engineering approach to promote cardiac regeneration. One significant limitation in cardiac tissue engineering approaches to date has been insufficient vascularization of engineered tissues, which is essential for the supply of oxygen, nutrients, and immune cells as well as the removal of cellular by-products and waste. Studies indicate that vascularization due to blood vessel in-growth is usually insufficient (47-51) and scaffold prevascularization (5, 52-54) results in limited cell survival. Furthermore, the regulation of extracellular matrix remodeling and turnover is also impaired in ischemia as well as other cardiac pathologies, leading to damaging structural, geometric and functional changes in the heart (11, 12). Therefore, developing tissue engineering strategies which promote both vascularization and reparative matrix remodeling is one of the key requirements for successful myocardial regeneration and are the focus of this dissertation research. The findings of this work will contribute towards creating the optimal microenvironment to enhance cardiac regeneration and for cardiac tissue engineering applications. 1. Clinical Problem Heart disease is a significant health burden and is the leading cause of death in the United States. In 2007, 813,804 people died of heart disease, representing 33.6% of all deaths in the United States (1). It is estimated that in 2010, heart disease cost the United States $316.4 billion 11

22 (1). While the US heart disease mortality rate has decreased from approximately 500 deaths per 100,000 Americans in 1979 to 250 deaths per 100,000 Americans in 2007 (55), cardiovascular disease remains an important clinical focus. Cardiac pathologies which lead to heart disease, including myocardial infarction and diabetes, often result in damaged and ischemic tissue and would benefit from a cardiac tissue engineering approach to promote cardiac regeneration. 1a. Coronary Heart Disease and Myocardial Infarction Coronary heart disease (CHD) is a narrowing of the small blood vessels that supply blood and oxygen to the heart. In 2007, CHD was the cause of 406,351 deaths in the United States, or 1 in 6 of all total deaths (56). Myocardial infarctions, known as heart attacks, occur when the blood flow to the heart is actually blocked for a long enough time that part of the heart muscle is damaged or dies. Every year in the United States, about 785,000 people have a first heart attack. Additionally, another 470,000 people who have already had one or more heart attacks have another attack (56). In the US, approximately every 25 seconds, someone will have a coronary attack, and approximately every minute, someone will die as a result of one (56). In the last 30 years, the mortality rate due to CHD in the US has decreased from approximately 330 deaths per 100,000 Americans in 1979 to 120 deaths per 100,000 Americans in 2007 (55), partially due to well-established surgical and pharmacological treatment options which are available. However, there still exists an obvious need for new strategies and technologies as seen by the high morbidity and mortality rates. After myocardial injury, the normal healing response occurs in stages including acute inflammation, cell migration and infiltration, granulation tissue formation, and ultimately scar formation (57). There is significant evidence to suggest that reduced angiogenesis and aberrant matrix remodeling occur during this healing response, leading to fibrotic scar tissue which lacks the contractile, mechanical and 12

23 electrical properties of healthy myocardium. This loss of function leads to further injury and often death and therefore represents a promising therapeutic target. 1b. Diabetes and Diabetic Cardiomyopathy Diabetes mellitus is a significant problem in the United States, estimated to afflict 25.8 million individuals or 8.3% of the population (58). In 1980, 5.6 million Americans had been diagnosed with diabetes. That number significantly increased to 19.7 million individuals diagnosed in 2009 (55). Diabetes was the sixth leading cause of death in the United States in 2006 (59), responsible for 72,449 or 3% of all reported deaths. Annual medical costs were estimated at $174 billion in 2007 (58). Untreated diabetes can lead to many serious medical problems, including heart disease and stroke. Diabetic cardiomyopathy (DCM) is a diabetes-associated cardiovascular condition defined as ventricular dysfunction in the absence of other etiological factors, such as hypertension or coronary heart disease (20-23). Pathological alterations to the myocardium resulting from DCM include circulatory defects, impaired heart muscle contraction, and progressive fibrosis. Patients with diabetes and DCM experience a high incidence of congestive heart failure (60, 61), often leading to death. The prevention and treatment of DCM is a clinically relevant and active research focus, with studies suggesting that glycemic control is beneficial early in myocardial dysfunction (22, 61), however late diagnoses of diabetes and/or DCM may limit this preventative measure. Neurohormonal antagonism has demonstrated preserved diastolic function in the diabetic heart in animal models (22, 62), however, it has not yet been translated to clinical studies. While strategies such as these appear promising, there exists a need for novel treatment approaches which focus on alternative molecular mechanisms for the disease. 13

24 Functionally, DCM leads to excess collagen deposition, myocardial fibrosis, and cardiac hypertrophy (11, 20, 22). Matrix remodeling and extracellular matrix (ECM) turnover are essential in many physiological processes yet regulation is impaired in DCM, leading to damaging structural, geometric and functional changes in the heart (11, 12). ECM turnover can be regulated by many factors, including angiotensin II (62-64), aldosterone (21), TGF-β1 (26), nitric oxide (65), advanced glycation end products (11, 66), kinins (67, 68), and matrix metalloproteinases (12). These factors represent any number of therapeutic targets for the development of treatment strategies for DCM. 1c. Research Goal - Promoting Cardiac Regeneration The cardiac pathologies of myocardial infarction and diabetic cardiomyopathy as discussed above often result in damaged, fibrotic, and/or ischemic tissue resulting from a loss of vascularization and aberrant matrix remodeling. The heart has limited regenerative ability (4, 69) and such damage to the myocardium would benefit from therapeutic approaches which promote cardiac regeneration via revascularization and reparative matrix remodeling. 2. Cardiac Tissue Engineering Cardiovascular disease is a serious health concern and current treatment strategies include pharmacological options, for example β-blockers and anti-platelet agents, and surgical options, including angioplasty, artery bypass and transplantation. However, due to limited effectiveness, availability and finances, there clearly exists a clear need for novel therapeutic approaches. Tissue engineering combines the principles of biology and engineering to develop functional tissue substitutes (2), with important breakthroughs occurring in the development of skin, cartilage, bone and bladder substitutes, among others (70, 71). Damaged heart tissue 14

25 which results from multiple cardiac pathologies would greatly benefit from a cardiac tissue engineering approach to promote tissue repair via implantation, regeneration and/or mobilization of native cells. Cardiac tissue engineering is an extraordinarily active research focus (3-10). However, while cardiac tissue engineering strategies have shown promise, they are generally not yet clinically viable due to the numerous challenges faced in successfully engineering a tissue for myocardial repair (8, 9, 71). Cardiac tissue engineering strategies or approaches vary widely, but all follow a similar approach or paradigm. There are four major approaches which can be used to promote cardiac regeneration. They include 1) stimulation of endogenous repair mechanisms, 2) cell transplantation, 3) tissue engineered graft implantation, and 4) nanobiotechnology-based approaches (3, 8). In these major approaches to cardiac tissue engineering, cells, scaffolds, growth factors/cytokines and/or environmental stimuli can be used together or independently to promote myocardial regeneration (3), as seen in Figure 1. These various components of cardiac tissue engineering will be covered in more detail later in this chapter as they relate to the research performed for this dissertation. 15

26 Figure 1. Components of cardiac tissue engineering strategies. Cells, scaffolds, proteins and environmental stimuli can be used independently or together to engineer myocardial tissue in vitro for transplantation or in vivo in the native environment. Adapted from (3). There have been a number of key advances in the cardiac tissue engineering field, including in vitro development and transplantation of three-dimensional cardiac tissue into animal models (53, 72-74) and notably the first human clinical trial (the MAGNUM trial) (75). However, there still exist a number of major difficulties and challenges in cardiac tissue engineering approaches, as outlined in Table 1. This includes challenges related to the choice of cells, scaffold, and delivery and functionality in vivo. It is clear that much remains to be 16

27 investigated in order to develop the most effective and clinically relevant cardiac tissue engineering approaches, including determination of functional standards for engineered cardiac tissue. Major challenges for development of successful cardiac tissue engineering strategies Cells Source Ease of harvest Expansion Differentiation ability/control Survival Integration Immunogenecity Retention Cardiomyogenic potential Scaffold Cell-friendly Toxicity Immunogenecity Biodegradable Bioactive Flexible Mechanically stable Integration Biomimetic Diffusion Fabrication - ease and cost Delivery of bioactive molecules Support electro-mechanical coupling Animal model Delivery methods Surgical implantation Injectability Time of delivery post injury Vascularization and integration Construct/patch size Oxygen delivery Nutrient delivery Infection/Rejection Long-term outcomes Table 1. Major challenges for development of successful cardiac tissue engineering strategies. Adapted and compiled from (3, 7, 9, 10, 76). 17

28 3. Cells for Cardiac Tissue Engineering Strategies One of the most crucial challenges faced in cardiac tissue engineering is selecting the appropriate population of cells. For clinical success, regenerated tissue should ultimately consist of phenotypically stable cardiac cell populations, including cardiomyocytes, fibroblasts and vascular cells (9). Therefore, the optimal cell source should be easy to harvest, proliferative, autologous or non-immunogenic, and be able to differentiate into mature and functional cardiac cells. Extensive research has been performed to explore the regenerative ability of a wide variety of potential cell sources, including fetal cardiomyocytes, skeletal myoblasts, mesenchymal stem cells, smooth muscle cells, endothelial progenitor cells, bone marrow-derived stem cells, umbilical cord cells, fibroblasts, human embryonic stem cells, and cloned cells (3). To date, no perfect cell source for cardiac regeneration has been identified as all seemingly have both advantages and disadvantages associated with their use. For the research performed in this dissertation, the primary cell type investigated was the fibroblast. While fibroblasts are autologous, easily harvested, and highly proliferative, they do lack the ability to differentiate into the other cardiac cell populations and thus are not the most appropriate sole cell source for cardiac tissue engineering strategies, including tissue graft development and implantation. However, as an integral component of the myocardium, fibroblasts have an important role cardiac regeneration via vascularization and matrix remodeling, which was investigated throughout this research. 3a. Cardiac Fibroblasts and Cardiac Regeneration Fibroblasts represent two-thirds of the total cardiac cell number and are intricately organized into a three-dimensional network in the heart where they are connected to each other 18

29 as well as cardiomyocytes and endothelial capillary networks through the connective tissue, as seen in Figure 2 (77-79). Figure 2. Structural organization of the myocardial extracellular matrix. Identifying the primary cardiac cell types large bodied cardiac myocytes, cardiac fibroblasts in the interstitial tissue, and capillaries composed of endothelial cells. a) Schematic adapted from (79). b) Normal rabbit myocardium, stained with lectin (brown) to identify capillaries (endothelial cells). Courtesy of R. Lee Lab, Harvard University. Cardiac fibroblasts are important components of the myocardium and are primarily responsible for both physiological and pathological extracellular matrix homoeostasis (78, 80-83). Fibroblasts maintain the mechanical extracellular microenvironment via controlled proliferation, extracellular matrix deposition and metalloproteinase-mediated ECM turnover or remodeling (12, 79, 80, 84). Additionally, fibroblasts provide chemical signaling via expression of growth factors (13, 16, 85, 86) and play a role in cytokine signaling (87). Due to the crucial 19

30 role that cardiac fibroblasts play in cardiac regeneration, they are the primary cell type investigated in the following dissertation studies. 3a. Cardiac Fibroblasts in Revascularization Angiogenesis, the formation of new capillaries from existing ones, is critical for cardiac regeneration of ischemic or damaged tissue. Revascularization is necessary for the supply of oxygen, nutrients, and immune cells as well as the removal of cellular by-products and waste in regenerating tissue. While capillaries themselves are composed of endothelial cells, they are surrounded and supported by stromal cells, particularly cardiac fibroblasts (85). Therefore, in order to develop successful cardiac tissue engineering strategies which promote revascularization, it is first critical to understand the interactions between the major cardiac cell types and how these cell-cell interactions affect cardiac healing. While previous studies show the importance of cardiomyocyte-endothelial interactions (14, 88), limited information is available on endothelial-fibroblast interactions. Studies have demonstrated increased endothelial sprouting and migration and decreased endothelial apoptosis in the presence of fibroblasts (42, 89, 90) as well as increased expression of important angiogenic factors (13, 85, 86). Additionally, fibroblasts regulate angiogenesis through alterations to the mechanical environment via myocardial remodeling (85). Capitalizing on fibroblast regulation of angiogenesis while limiting detrimental myocardial remodeling as discussed further in the next section, will likely prove clinically relevant for cardiac tissue engineering strategies. 3b. Cardiac Fibroblasts in Reparative Matrix Remodeling Fibroblasts are responsible for ongoing physiological extracellular matrix homoeostasis (78, 80, 81). This occurs via highly regulated matrix deposition (80% of newly synthesized ECM is collagen I (34), a major component of the heart and substrate of MMP-2) and 20

31 metalloproteinase-mediated ECM remodeling (12, 83, 84, 91). In response to myocardial injury or infarction, cardiac fibroblasts are activated, often differentiating to myofibroblast phenotype, and participate in the wound healing reponse (79, 92, 93) via migration to the wound site, cell proliferation, secretion of various bioactive molecules, and increased ECM deposition and subsequent remodeling, as seen in Figure 3. While reparative fibroblast-mediated remodeling may initially improve cardiac performance, over time pathological or aberrant remodeling and excessive ECM deposition and the persistance of myofibroblasts in scar tissue results in increased mechanical stiffness, lack of vasculature, and contributes to cardiac dysfuction and failure (57, 79, 94-96). Therefore, it is important to make a distinction between reparative matrix remodeling, where structural scar replaces areas of cell and tissue death and can allow for revascularization and regeneration, and pathological or maladaptive remodeling, where a net accumulation of ECM results in significant fibrosis and loss of cardiac function (79, 83, 97). 21

32 Figure 3. Cardiac fibroblast functions associated with matrix remodeling and cardiac regeneration. Cardiac fibroblast response to environmental stimuli or altered cardiac microenvironment due to injury includes cell proliferation, migration, differentiation to myofibroblast phenotype, expression of cytokines and growth factors and increased matrix sythensis and degredation. Adapted from (83). 3c. Diabetes Alters the Fibroblast Phenotype and Matrix Remodeling Response Diabetic cardiomyopathy often leads to increased collagen accumulation and fibrosis in both diabetic human patients and animal models (20, 21). This is likely due to chronic hyperglycemic conditions leading to a dysregulation of physiological cardiac matrix remodeling via changes to the fibroblast phenotype. Studies on fibroblasts from db/db (genetically diabetic type II diabetes) murine models demonstrated impairments in vital cellular processes, including reduced growth factor expression, reduced cellular migration and decreased matrix 22

33 metalloproteinase (MMP) activation as compared to normal fibroblasts in vitro (28, 81). Studies have shown that cardiac dysregulation of MMP-2 expression and activity contributes to the increased collagen deposition, progressive fibrosis, increased ventricular stiffness, and cardiac dysfunction seen in diabetic cardiomyopathy (24-27). Importantly, studies indicate that hyperglycemic conditions increase collagen synthesis and crosslinking (29-31, 98) and decrease MMP-2 expression and expression in fibroblasts (24-27), resulting in the increased collagen accumulation, progressive fibrosis, increased ventricular stiffness, and cardiac dysfunction seen in diabetic cardiomyopathy. Therefore, overcoming the effects of hyperglycemic conditions on matrix remodeling by cardiac fibroblasts represents a novel therapeutic approach for treatment of DCM as well as other cardiac conditions in the context of diabetes. 4. Scaffolds for Myocardial Tissue Engineering Cell-matrix interactions are essential in both normal and healing tissue. Therefore, the biomaterial scaffold is a key component of most cardiac tissue engineering strategies. The scaffold should be cell-friendly and provide structural support as well as biological and chemical guidance to cells during tissue development or regeneration (99). Additionally, potential scaffolds should be non-toxic, non-immunogenic, biodegradable, and easy to deliver (46). There are a vast number of natural and synthetic materials which have been investigated for potential use in cardiac tissue engineering strategies (8). Native materials investigated include collagen, fibrin, alginate, Matrigel, and gelatin, among others. Native biomaterials are advantageous for a number of reasons, including availability of receptor-binding ligands and natural degradation by proteases. However, disadvantages associated with native scaffolds include purification, immunogenicity and infection. Alternatively, synthetic scaffolds include a variety of polymers 23

34 and peptide nanofibers and are generally biocompatible and highly customizable with respect to mechanics, chemistry and degradation. However, without additional modifications, many synthetic scaffolds are not bioactive and limited in functional cell-scaffold interactions. In an effort to overcome perceived scaffold limitations, various combinations of natural and synthetic scaffolds have also been investigated. Clearly, the sheer magnitude of potential biomaterial scaffolds for use in cardiac tissue engineering applications makes it very difficult to clearly ascertain the most effective alternatives. This research investigated the use of the synthetic biomaterial class of peptide nanofiber scaffolds. Peptide nanofibers belong to a novel class of biomaterials which are made via the spontaneous assembly of self-complementary oligopeptides consisting of 8 to 16 alternating ionic hydrophilic and hydrophobic amino acids (100). These oligopeptides form β-sheets in water which spontaneously assemble into stable, three-dimensional hydrogels after exposure to physiological ph. Once formed, the β-sheets are stable across a wide range of temperatures and ph (100). The scaffold structure consists of interwoven nanofibers with nanofiber diameter of ~10 nm and pore size of ~5 to 200 nm (100), which mimics the porosity and gross structure of the native extracellular matrix. Peptide nanofibers are commercially available and produced under sterile conditions. Additionally, there is extreme flexibility in design and modification of amino acid sequence, allowing for tailoring and functionalization to any number of applications. Upon formation into a hydrogel, nanofibers can be easily administered in vivo via injection with little to no immune response ( ). Peptide nanofiber scaffolds have been shown to support multiple cell types, including fibroblasts, endothelial cells, cardiomyocytes, neurons and neural progenitor cells, liver progenitor cells, chondrocytes, and osteoblasts (14, 16, 100, ). Additionally, nanofibers have been used for the local cardiac delivery of both proteins (102,

35 113) and cells (48, 101, 114, 115). For our research, RAD16-II (RARADADA) 2 peptide nanofibers were investigated as a potential biomaterial scaffold to promote cardiac regeneration in the injured heart. RAD16-II nanofibers have been studied primarily for use in cardiac tissue engineering applications due to their pro-angiogenic nature (14, 101), although they likely also have potential in tissue systems beyond the heart. 4a. Peptide Nanofibers for the Study of Cell-Cell Interactions Revascularization is an important key to regenerating injured cardiac tissue. In order to develop strategies which promote revascularization in vivo, it is important to study the role of the cardiac cells and the cell-cell interactions in the angiogenic process. Experimental systems used for in vitro studies of angiogenesis have produced important results but are not without limitations. Native systems such as collagen, fibrin and Matrigel are subject to excessive or uncontrolled scaffold contraction, proteolytic degradation, and often require the addition of external growth factors to promote in vitro angiogenesis. Therefore, a three-dimensional culture system, which mimics the native cell environment while uncoupling scaffold-triggered signaling from cell-cell interactions, may provide important insights into the role of cell-cell interactions during the angiogenic process. Important properties of the RAD16-II nanofiber system include the absence of significant cell-induced scaffold contraction and resistance to proteolysis due to a lack of MMP degradation sites (100). RAD16-II nanofibers have been shown to provide an angiogenic microenvironment which enhances capillary-like network formation in vitro without the addition of external growth factors and allows for long term study of cell-cell interactions (14, 15). Furthermore, these biocompatible nanofibers can also serve as an angiogenic microenvironment for cardiac regeneration in vivo (46, 116), and therefore represent an 25

36 appropriate in vitro system to study cell-cell interactions during capillary morphogenesis for cardiac tissue engineering applications. 4b. Peptide Nanofibers for the Induction of Endogenous Repair Mechanisms Peptide nanofibers may also prove to be an important tool for stimulation of endogenous repair mechanisms in injured cardiac tissue. This potential approach for cardiac regeneration challenges the belief that the myocardium lacks regenerative capabilities, instead focusing on the potential of cardiac progenitor cells (117, 118) as well as resident cardiac cells, including cardiac fibroblasts and myofibroblasts (79, 83, 92, 119), and their role in tissue repair. However, the exact mechanisms of this endogenous repair process remain unclear. Interestingly, recent studies by our group and others have suggested that RAD16-II peptide nanofibers may actually help to promote endogenous repair. Injection of RAD16-II nanofibers into the myocardium of mice resulted in the recruitment of progenitor cells expressing endothelial markers (101). Additionally in wound healing in a genetically diabetic (db/db) murine model, delivery of RAD16-II nanofibers enhanced infiltration of endogenous endothelial precursor cells and endothelial cells to the injured tissue (103). Furthermore, in vitro studies demonstrated increased production of angiogenic growth factors (vascular endothelial growth factor and angiopoietin-1) and proteases (matrix metalloproteinase-2) by endothelial cells and fibroblasts cultured in nanofibers, as compared to collagen controls (120). This suggests that peptide nanofibers may induce native expression of important regenerative factors by resident cells upon in vivo delivery. Therefore, RAD16-II peptide nanofibers may have the potential to induce endogenous repair in the injured myocardium via either infiltration by cardiac precursor cells and/or resident cardiac cells as well as enhanced expression of important regenerative factors by those infiltrating cells. 26

37 5. Protein Delivery to Stimulate Cardiac Regeneration Localized delivery of small molecules and proteins to injury sites are a popular therapeutic option. However, without a supporting scaffold factors are quickly washed away from the site of delivery and into the bloodstream (121). Therefore, site-specific delivery and retention of regenerative factors from biomaterials or engineered tissue may provide an effective means of stimulating local regenerative responses such as angiogenesis and matrix remodeling as well as enhancing cell infiltration and survival. Bioactive molecules can be incorporated into biomaterial scaffolds a number of ways, including patterning, tethering, and non-covalent binding, allowing for spatial and temporal delivery control (46). Any number of bioactive molecules have been investigated for their regenerative capacity, including insulin like growth factor (IGF-1) (111, 114), platelet derived growth factor (PDGF-BB) (102), stromal cell derived factor (SDF-1) (112), vascular endothelial growth factor (VEGF) (122), bone morphogenic protein (BMP-2) (123), transforming growth factor (TGF-β1) (113), and p38 MAPK inhibitor (124), among many others. These factors have been delivered via a multitude of biomaterial scaffold options, including but not limited to liposomes, naturally derived matrices, synthetic polymers, and peptide nanofiber scaffolds. The requirements for an effective system for protein delivery are the same as those for an effective biomaterial scaffold non-toxic, nonimmunogenic, non-infections and biodegradable (46). The biomaterial delivery system also needs to have appropriate mechanical properties, strong protein binding interactions, resistance against proteases, and an easy delivery method. For the studies performed in this dissertation, peptide nanofibers were chosen for study as a potential protein delivery system of matrix metalloproteinase-2 to promote cardiac regeneration by fibroblasts in vitro and in the diabetic heart. 27

38 5a. Delivery of Matrix Metalloproteinases to Promote Cardiac Regeneration Matrix metalloproteinases (MMPs) are a family of zinc-dependant proteases which participate in the degradation and remodeling of the ECM and the activation of other proteins (12, 125). There are more than 25 members of the MMP family, divided based on substrate specificity into five major groups: collagenases, gelatinases, stromelysins, membrane-type, and other. Normal reparative healing processes require active MMPs for remodeling. Precise MMP control is essential, as both loss of activity and enhanced expression can result in diseases including cancer, arthritis and cardiovascular disease (12, 125). MMPs are regulated in vivo at the levels of transcription, activation, and inhibition (125, 126). In normal tissue, regulation via the various pathways keeps precise control over MMP activity. MMPs are particularly important in cardiac regeneration strategies, as they play an important role in both angiogenesis and matrix remodeling, via proteolytic basement membrane (BM) and extracellular matrix degradation and remodeling and release of angiogenic factors (127). This balance is disrupted in pathological remodeling and often leads to detrimental healing. Importantly, recent studies in diabetic human patients ( ) and animal models (24-27, 62, 131, 132) suggest that MMP expression and activity is impaired in diabetes, thus highlighting this particular mechanism as a novel therapeutic target. In particular, studies have shown that dysregulation of cardiac MMP-2 expression contributes to the increased collagen deposition, progressive fibrosis, increased ventricular stiffness, and cardiac dysfunction seen in diabetic cardiomyopathy in rodent animal models (24-27). Therefore, overcoming the inhibitory effects of diabetic conditions on matrix remodeling by cardiac fibroblasts through delivery of exogenous MMP-2 may represent a novel protein delivery strategy for therapeutic treatment of diabetes-related cardiac fibrosis. 28

39 5b. Peptide Nanofibers for Myocardial Protein Delivery An RAD16-II scaffold-based approach for protein delivery allows for spatiotemporal controlled delivery, and can be achieved via either diffusion from or tethering to a scaffold, depending on protein size and binding affinity (46). RAD16-II and similar nanofibers have been extensively studied in vitro for controlled protein delivery ( ). Recent in vitro studies have examined the release of functional proteins and cytokines, including lysozyme, bovine serum albumin (BSA), fibroblast growth factor (βfgf), VEGF, and brain-derived neurotrophic factor (BDNF), from similar nanofiber scaffolds and have shown slow and sustained release profiles over 2 to 3 weeks ( ). These studies suggest that diffusion through nanofibers is dependent primarily on protein size (133, 135), although lipophilicity and charge density does have an effect dependent on peptide amino acid sequence ( ). Additionally, increased peptide nanofiber density results in decreased protein diffusion (134, 135), suggesting a straightforward strategy for controlling protein release kinetics. Multiple in vivo studies have shown that RAD16-II peptide nanofibers serve as a unique protein delivery vehicle and can be easily administered via single injection for cardiac tissue engineering applications. Studies have shown that nanofibers remain present 28 days after myocardial injection in mice without significant immune response, while allowing for cellular infiltration and matrix deposition (101). Importantly, RAD16-II and similar peptide scaffolds have been successfully used for the local cardiac delivery of many different proteins, including IGF-1 (111, 114), PDGF-BB (48, 102), and SDF-1 (112), resulting local retention of measurable levels of exogenously delivered protein (~25% of initial levels) at 14 days post-injection (102) and modest improvements in cardiac healing. Therefore, the RAD16-II nanofiber 29

40 microenvironment may represent a novel tissue engineering scaffold which can lead to increased local protein concentration via delivery of exogenous protein. 6. Environmental Stimuli to Stimulate Cardiac Regeneration In developing strategies to promote cardiac regeneration, it is important to consider not only the need of functional myocardial tissue to support all resident cardiac cells and extracellular matrix components, but also to propagate electrical impulses and replicate contractile function. Studies have shown that engineered constructs exposed to electric field stimulation or mechanical stretch exhibited electromechanical properties approaching those in the native heart (53, 137, 138). Therefore environmental stimuli which occur in the healthy myocardium, such as mechanical stretch and electrical stimulation may prove to be another approach to stimulate cardiac regeneration. 6a. Mechanical Stretch for Promotion of Cardiac Regeneration In particular, mechanical stretch may be a potential strategy to compensate for the dysregulation of matrix remodeling by diabetic cardiac fibroblasts. Mechanical stimulation plays an important role in tissue development and repair and cells adapt and respond to changes in their mechanical environment via morphological and phenotypic alterations (32). In vitro studies have shown that the application of mechanical strain on fibroblasts in culture leads to a variety of responses related to extracellular matrix remodeling (Figure 3) (33, 34). Interestingly, fibroblasts exposed to cyclic strain experience decreased proliferation in two-dimensional culture (38, 139) and increased proliferation in a three-dimensional setting (39, 40). When exposed to cyclic strain, rat cardiac fibroblast collagen I gene expression and deposition was significantly upregulated (35-40, 140), as well as increased extracellular matrix protein expression (41, 141). 30

41 Importantly for matrix remodeling in DCM, mechanical strain stimulated fibroblast expression of MMP-2 (140, 142) and membrane-type matrix metalloproteinase (MT-MMP, an activator of MMP-2) (45). Growth factor expression by fibroblasts is affected by cyclic stretch as well, with increases observed in IGF-1 (42, 43), VEGF (44), and TGF-β (36). These studies provide important information regarding how fibroblasts respond to mechanical strain with regards to matrix remodeling, including cell proliferation, expression of proteases and growth factors and increased matrix sythensis and degredation. However, there remains much more to learn, in particular with regards to the effect of diabetic phenotype in conjunction with cyclic strain. Additionally, while the matrix remodeling response may be reparative at first with application of mechanical strain, prolonged exposure beyond the initial repair stage may lead to pathological remodeling and a loss of function. Nonetheless, stimulation with environmental stimulus such as mechanical stretch may prove to be an effective strategy to promote cardiac regeneration, potentially by promoting the matrix remodeling response of fibroblasts either in vitro or within a tissue engineered construct. 31

42 CHAPTER 3 Complex Temporal Regulation of Capillary Morphogenesis by Fibroblasts Jennifer R. Hurley, Swathi Balaji, Daria A. Narmoneva. Department of Biomedical Engineering, University of Cincinnati, Cincinnati, Ohio. Published in American Journal of Physiology - Cell Physiology, vol. 299, C444-C453, Permission not required for reprinting in dissertation. ABSTRACT Interactions between endothelial and stromal cells are important for vascularization of regenerating tissue. Fibroblasts are responsible for expression of angiogenic growth factors and matrix metalloproteinases, as well as collagen deposition and fibrotic myocardial remodeling. Recently, self-assembling peptide nanofibers were described as a promising environment for cardiac regeneration due to its synthetic nature and control over physicochemical properties. In this study, peptide nanofibers were used as a model system to quantify the dual role of fibroblasts in mediating angiogenesis chemically via expression of angiogenic factors and mechanically via cell-mediated scaffold disruption, extracellular matrix deposition and remodeling. Human microvascular endothelial cells (EC), fibroblasts (FB), or co-cultures were cultured in three-dimensional nanofibers for up to 6 days. The peptide nanofiber microenvironment supported cell migration, capillary network formation and cell survival in the absence of detectable scaffold contraction and proteolytic degradation. FBs enhanced early capillary network formation by assisting EC migration and increasing VEGF and Ang1 32

43 expression in a temporal manner. EC-FB interactions attenuated fibroblast MMP-2 expression while increasing collagen I deposition, resulting in greater construct stiffness and a more stable microenvironment in co-cultures. While FBs are critical for the initial steps of angiogenesis in the absence of external angiogenic stimulation, coordinated efforts by ECs and FBs are required for a balance between cell-mediated scaffold disruption, ECM deposition and remodeling at later time points. The findings of this study also emphasize the importance of developing a microenvironment which supports cell-cell interactions and cell migration, thus contributing towards an optimal environment for successful cardiac regeneration strategies. INTRODUCTION Tissue engineering aims to combine the principles of biology and engineering to develop functional tissue substitutes (2). Thus far, one major limitation has been insufficient vascularization of engineered tissues, which is essential for the supply of oxygen, nutrients, and immune cells as well as the removal of cellular by-products and waste. This is particularly important in cardiac tissue engineering approaches, where recent studies have demonstrated the feasibility of engineering functional cardiac muscle in vitro, with promising results after graft implantation in animal models (49, 50, 54, 143, 144). These studies indicate that revascularization of an engineered graft is critical for successful graft implantation and regeneration of ischemic cardiac tissue. However, vascularization due to blood vessel in-growth is usually insufficient (49-51) and scaffold pre-vascularization (5, 54) results in limited cell survival. Therefore, developing engineering strategies which promote vascularization is one of the key requirements for successful graft implantation and myocardial regeneration. 33

44 In order to develop novel strategies to promote vascularization in engineered cardiac grafts, it is necessary to understand the interactions between the major cardiac cell types. Normal mammalian myocardium has a complex cellular organization, where three major cell types cardiomyocytes, endothelial cells and cardiac fibroblasts are arranged in an intricate spatial network and communicate constantly. The importance of cardiomyocyte-endothelial interactions in angiogenesis has been previously documented (101, 145). However, less is known about interactions between endothelial and stromal cells, cardiac fibroblasts in particular. To study such cell-cell interactions, in vitro co-culture assays have been commonly used to represent angiogenesis in vivo (146), with many recent studies applying various threedimensional systems (including collagen I, fibrin, Matrigel, and scaffold-free approaches) (42, 64, 89, 90, ). Such in vitro studies have demonstrated that the formation of capillary-like structures is enhanced by the presence of fibroblasts, with increased endothelial sprouting and migration and decreased endothelial apoptosis (42, 89, 90). Additionally, studies indicate that during capillary assembly, fibroblasts provide chemical signaling via expression of angiogenic factors (13, 85), including vascular endothelial growth factor (VEGF) (89, 150, 151), angiopoietin-1 (89, 152), stromal cell derived factor-1 (153), hepatocyte growth factor (154, 155), interleukin-8 (156, 157), transforming growth factor -β (158, 159), and matrix metalloproteinases (MMPs) (12, 84). Studies also suggest that fibroblasts can regulate angiogenesis by changing the mechanical extracellular microenvironment via matrix deposition and metalloproteinase-mediated extracellular matrix (ECM) remodeling (12, 84). However, in addition to pro-angiogenic effects of ECM remodeling shown in vitro ( ), fibroblasts are also the major contributors to excessive fibrotic myocardial remodeling in vivo, which can lead to detrimental collagen matrix deposition and contraction, increased cardiac stiffness, lack of 34

45 vasculature and ultimately heart failure (57, 95, 96). Overall, these findings suggest a possible dual role chemical and mechanical for fibroblasts during the process of vasculature assembly and remodeling in the environment of healing tissue, such as in the regenerating heart. However, our knowledge of these processes remains incomplete. In particular, the effects of fibroblastendothelial interactions on the mechanical microenvironment and how these interactions can affect angiogenesis are not well understood. This is partially due to the limitations of the experimental systems used for in vitro studies of angiogenesis. Native systems such as collagen, fibrin and Matrigel are subject to excessive or uncontrolled scaffold contraction, proteolytic degradation, and often require the addition of external growth factors to promote in vitro angiogenesis, all of which can be hurdles for in vitro studies of fibroblast-mediated angiogenic signaling. Therefore, a three-dimensional culture system, which mimics the native cell environment while uncoupling scaffold-triggered signaling from cell-cell interactions, can provide important insights into the role of endothelial-fibroblast interactions during the angiogenic process. It has previously been shown that the scaffold made from synthetic RAD16-II peptide nanofibers (NFs) provides an angiogenic microenvironment which enhances capillary-like network formation in vitro without the addition of external growth factors and allows for long term study of cell-cell interactions (14, 15). Importantly, these biocompatible nanofibers can also serve as an angiogenic microenvironment and drug delivery tool for cardiac regeneration in vivo (46, 116), and therefore represent an appropriate in vitro system to study cell-cell interactions during angiogenesis for cardiac tissue engineering applications. Specific properties of this system, including the absence of significant cell-induced scaffold contraction and resistance to proteolysis due to lack of MMP degradation sites (100), may allow for the uncoupling of 35

46 chemical signaling due to cell-scaffold interactions from cell-cell signaling, thus enabling quantitation of changes in cell behavior and protein expression not masked by scaffold-induced signaling associated with scaffold degradation. Therefore, the goal of this study was to elucidate the mechanisms of temporal regulation of the angiogenic process by fibroblasts using a comprehensive approach and the culture system of RAD16-II nanofibers. We tested the hypothesis that fibroblasts regulate capillary morphogenesis chemically via growth factor expression and mechanically via cell-mediated scaffold disruption, ECM deposition and remodeling in a temporal manner. Our results suggest that at the early stages of tissue repair process, fibroblasts may play a major role in capillary morphogenesis via both paracrine growth factor signaling and mechanical disruption of ECM to lead the way for the formation of endothelial cell networks. At the later stages, the role of fibroblasts as regulators of the mechanical microenvironment becomes more prominent. Interestingly, endothelial-fibroblast interactions appeared to help maintain the balance in ECM homeostasis, enhancing both MMP-2 and collagen I production and resulting in improved integrity and a stable microenvironment by day 6, as demonstrated by the rheometry analyses. MATERIALS AND METHODS Cell culture. Human microvascular endothelial cells (ECs; Cascade Biologics, Portland, OR) and human dermal fibroblasts (FBs; Cascade Biologics, Portland, OR) were cultured in Medium 199 (HyClone, Logan, UT) containing 10% fetal bovine serum (FBS; Atlanta Biologicals, Lawrenceville, GA), 1% Antibiotic-Antimycotic (Atlanta Biologicals, Lawrenceville, GA), 10 µg/ml heparin (Sigma-Aldrich, St. Louis, MO), and 0.2 ng/ml growth supplement (Sigma- 36

47 Aldrich, St. Louis, MO). Cell cultures were maintained at 37ºC in 100% humidified air containing 5% CO 2. Cells of passage 4-12 were used in all experiments. Experimental groups. Experimental groups included EC (endothelial cells only), FB (fibroblasts only), and EC+FB (endothelial cells and fibroblasts in a 1:1 ratio). Sample preparation. For all experiments, a three-dimensional nanofiber scaffold made from RAD16-II peptide nanofibers (RARADADARARADADA; SynBioSci Corporation, Livermore, CA) served as a controlled microenvironment. In network formation and cell proliferation experiments, cells were surface seeded on 10 mg/ml peptide nanofibers in culture plate inserts (13 mm diameter, 0.4 µm pore size; Millipore, Billerica, MA) at a cell seeding density of 1.0x10 5 cells/cm 2. For ELISA experiments, cells were embedded in 10 mg/ml peptide nanofibers in culture plate inserts at a density of 5.0x10 6 cells/ml, and cultured up to 6 days with daily media changes. For mechanical testing of cell-scaffold constructs using rheometry, cells were embedded in 6 mg/ml or 10 mg/ml peptide nanofibers at 2.5x10 6 cells/ml and cultured for 1, 3, and 6 days with medium changes every 24 hrs. For analyses of cell apoptosis, samples were fixed and embedded in paraffin for staining at 3 and 6 days. Capillary morphogenesis. Prior to seeding, endothelial cells and fibroblasts were labeled with CellTracker Dyes (Invitrogen Corporation, Carlsbad, CA). Cells were seeded on the nanofibers and cultured for 24 hrs to allow capillary-like network formation. After fixation with 2% paraformaldehyde, images were taken (n=5 per sample) using an inverted fluorescent microscope (Olympus IX81; Olympus America Inc., Center Valley, PA). Correlation analysis with MATLAB (The MathWorks Inc., Natick, MA) was used to characterize capillary-like endothelial network size as previously described (14). For 3-dimensional network characterization, a built-in integrated 3D imaging system was used (Plus Imaging System, 37

48 Olympus) which included motorized Z-drive with 10 nanometer step size and imaging/analysis software (ImagePro, Media Cybernetics, Inc.). Lumens were visualized using Z-stack images of cells stained with endothelial cell marker lectin (fluorescein ulex europaeus agglutinin I; Vector Labs, Burlingame, CA) with a spacing of 0.8 µm between frames. Cell proliferation. CellTiter 96 Aqueous non-radioactive cell proliferation assay (Promega Corporation, Madison, WI) was used to assess cell viability and proliferation at 1, 3, and 6 days in culture. Cells were embedded in the nanofibers at a density of 5.0x10 6 cells/ml and cultured with daily medium changes. At each time point, samples were incubated in medium containing MTS/PMS solution for 3 hours per manufacturer instructions. Media samples from culture inserts were placed in a 96-well plate and absorbance was measured at 490 nm using an ELISA plate reader. All data were normalized to day 1 endothelial cell values for analyses. After testing, MTS/PMS medium was aspirated and fresh medium was added to the samples. Cell apoptosis. Cell-nanofiber constructs were fixed at days 3 and 6, and embedded in paraffin (Fisher Scientific, Pittsburgh, PA). Staining was performed on 5 µm sections. Staining with lectin (Vector Labs, Burlingame, CA) was performed to identify endothelial cells. Anti- ACTIVE Caspase-3 (Promega Corporation, Madison, WI) and DAPI (Invitrogen Corporation, Carlsbad, CA) staining was performed to assess cell death, and apoptotic cells were counted and compared to total cell numbers. Sample protein content determination. Cell-nanofiber constructs were cultured in no growth factor medium (cell culture medium without additional growth factor supplementation) and collected at day 6 and stored in TriReagent (Molecular Research Center, Cincinnati, OH) at - 80ºC until testing. Protein isolation was performed per the manufacturer s protocol. Total 38

49 protein content in the samples was determined using Coomassie Plus Assay Kit (Thermo Fisher Scientific, Rockford, IL). Protein content using Enzyme-Linked Immunosorbent Assay (ELISA). For all ELISA experiments, cell culture medium (M199 with 10% FBS, 1% Antibiotic-Antimycotic, and 10 µg/ml heparin) without growth supplement was used with daily medium changes. Medium and matrix samples were collected at days 1, 3 and 6 and stored at -80ºC until testing. ELISA kits (R&D Systems, Minneapolis, MN) were used per manufacturer s protocol to determine protein concentrations in medium samples (Human VEGF, Angiopoietin-1, Total MMP-9 and MMP- 2/TIMP-2) and matrix samples (Human VEGF and Angiopoietin-1). Collagen I expression was quantified using an ELISA for human collagen I in both medium and matrix samples, as described in (160) (human collagen I, antibodies and substrates from Southern Biotechnology, Birmingham, AL). Protein expression in matrix samples was normalized using total protein content. For all ELISAs, additional controls of the cell culture medium alone (containing 10% serum) were included to confirm that growth factors in the serum would not affect protein expression and detection, with no differences observed between medium samples and the 0 pg/ml standard. Mechanical testing of cell-scaffold constructs using rheometry. Elastic moduli (G ) of peptide nanofibers (6 or 10 mg/ml) with living cells or nanofibers alone (controls) at 1, 3, and 6 days were measured with a parallel-plate rheometer (Bohlin Instruments Inc., East Brunswick, NJ). Using molds, circular constructs of 8 mm diameter and approximately 500 µm height were formed on glass slides. The glass slides were covered with cell culture medium and cultured with in an incubator with daily medium changes. For testing, glass slides were transferred and secured to the bottom plate of the rheometer. The top parallel plate was lowered to a gap height 39

50 which ensured complete contact with the sample and a constant strain amplitude (γ=0.01) frequency sweep (f= Hz) was performed. The elastic modulus (G ) served as an indicator of overall cell-seeded construct stiffness, and moduli values measured at 0.1 Hz are reported.in the text. Statistical analysis. The results are reported as average ± standard deviation. Statistical comparisons between experimental groups were performed using either two-way ANOVA or Student s t-test as appropriate. Additionally, multi-factor ANOVA with post-hoc tests with Bonferroni corrections (SPSS, SPSS Inc., Chicago, IL) were used to test the effects of cell type, peptide concentration and time in culture on the material properties of cell-nanofiber constructs. Results were considered statistically significant at p<0.05. All experiments were performed in triplicates and repeated at least 2 times (minimum n=6). RESULTS Endothelial-fibroblast cell migration regulates capillary morphogenesis. Immediately after seeding, both endothelial cells and fibroblasts attached and spread uniformly across the peptide nanofibers (Figure 4a). By 24 hrs, coordinated migration of ECs and FBs was seen, with FBs surrounding the nascent endothelial networks and a distinct lack of cells outside of these networks (Figure 4b). The appearance of the endothelial networks (Figure 4b, green) in EC+FB co-cultures was similar to the capillary morphogenesis in this material by endothelial cells alone, as reported previously by Sieminski et al 2007 (17) and our group (14). Consistent with previous results (14, 145), Z-stack images of endothelial structures clearly show formation of hollow lumens (Figure 4c). Interestingly, noticeable scaffold disruption likely 40

51 resulting from extensive cell migration was observed in FB-containing constructs, with a lesser effect in EC-only samples. Correlation analysis of capillary networks (14) demonstrated that networks formed faster and were significantly larger (Figure 4d) when ECs were cultured in the presence of fibroblasts at all time points (p<0.001). As early as 3.5 hrs, EC+FB co-cultures were significantly larger than EC cultures (20.4 ± 4.7 µm vs ± 0.4 µm, respectively, p<0.001). This trend continued and by 24 hrs, EC+FB networks remained significantly larger (42.6 ± 10.4 µm), as compared to EC-only networks (24.7 ± 4.5 µm, p<0.001). 41

52 Figure 4. Capillary morphogenesis in endothelial and endothelial-fibroblast cultures on peptide nanofibers. a) Uniform distribution of endothelial cells (lectin - green) and fibroblasts (CellTracker - orange) immediately (1 hr) after cell seeding. Scale bar = 50 µm. b) At 24 hrs after cell seeding, coordinated cell migration and formation of multi-cell networks is seen in co-cultures, with fibroblasts (CellTracker orange) surrounding the nascent endothelial cell (lectin green) networks. Scale bar = 50 µm. c) 3-D reconstruction 42

53 of Z-stack images of single endothelial structure from sample in panel b was used to visualize lumen formation. Hollow endothelial lumens were observed in (xy) plane (along the structure, bottom left) and through (xz) plane (through the structure, bottom right). Scale bar = 10 µm. d) Characteristic size (microns) of capillary-like networks is significantly larger in endothelial-fibroblast co-cultures (EC+FB) as compared to endothelial cell cultures (EC) at 3.5, 6, 9, and 24 hrs. * indicates p<0.001 between EC and EC+FB at all time points. Peptide nanofibers provide a stable microenvironment which supports long term cell survival. In contrast to significant cell-seeded construct contraction reported previously for collagen-containing gels, the peptide nanofibers experienced limited scaffold contraction in vitro with less than <15% contraction at day 7. This is in agreement with the values for RAD16-II peptide contraction reported previously (17), as compared to ~70% for collagen I gel (17). To determine if this stable microenvironment supports long-term cell survival, an MTS-based cell viability assay was performed on cells embedded in nanofibers and cultured up to 6 days. Results demonstrated that the viable cell number (Figure 5a) remained at 80% or greater of the 225,000 total cells initially embedded within the peptide nanofibers, in culture medium containing 10% FBS with no additional growth factors. The percentage of viable cells was not significantly different between the experimental groups at days 3 and 6 (92 ± 3 % and 93 ± 5 % of EC+FB co-cultures vs. 82 ± 6 % and 80 ± 5 % of endothelial cells vs. 87 ± 11 % and 97 ± 2 % of fibroblasts at day 3 and 6, respectively). Staining for Caspase-3 showed no significant difference in cell apoptosis levels (Figure 5b) in cells embedded in the peptide nanofibers (21 ± 13 % and 24 ± 3% of EC+FB co-cultures vs. 22 ± 7 % and 20 ± 8 % of endothelial cells vs. 19 ± 43

54 4 % and 18 ± 2 % of fibroblasts at days 3 and 6, respectively). Overall, these results indicate the balance between cell proliferation and apoptosis and an overall stability in the peptide nanofiber microenvironment. Figure 5. Long term cell viability and apoptosis levels in peptide nanofiber cultures. a) Cell viability was measured using MTS-based viability assay at days 3 and 6. The number of viable cells in culture remained at 80% or greater of the 225,000 total cells initially seeded for all experimental groups up at days 3 and 6. b) Cell apoptosis was measured by staining for Caspase-3 positive cells. The percentage of apoptotic cells was 20-30% for all experimental groups at days 3 and 6. 44

55 Fibroblasts mediate capillary morphogenesis via modulating temporal profiles of VEGF and Angiopoietin-1 expression. Vascular endothelial growth factor (VEGF) is a major angiogenic stimulus and activation signal for endothelial migration and sprouting (13). Cellular VEGF expression in the sample medium was measured using ELISA (Figure 6a). The results showed that VEGF levels in the medium were largely determined by fibroblasts. At day 1, VEGF expression was significantly higher in EC+FB and FB cultures (362 ± 75 pg/ml and 995 ± 153 pg/ml, respectively) as compared to EC cultures (109 ± 38 pg/ml, p<0.05). At day 3, VEGF expression in EC+FB and FB cultures (226 ± 58 pg/ml and 470 ± 100 pg/ml, respectively) was significantly reduced from day 1 (p<0.05) but still remained higher than expression by ECs (119 ± 42 pg/ml, p<0.05). Beyond day 3, protein expression of soluble VEGF remained low in all experimental groups. Matrix-bound VEGF expression was also measured using ELISA (Figure 6b). In EC cultures, matrix-bound VEGF levels at day 1 (0.142 ± pg VEGF/µg total protein) and day 3 (0.109 ± pg VEGF/µg total protein) were significantly higher than at day 6 (0.055 ± pg VEGF/µg total protein, p<0.05). Similarly, protein levels in FB cultures at day 1 (0.103 ± pg VEGF/µg total protein) and day 3 (0.109 ± pg VEGF/µg total protein) were significantly greater than levels at day 6 (0.057 ± pg VEGF/µg total protein, p<0.05). In EC+FB co-cultures, a significant decrease in matrix-bound VEGF was observed from day 1 (0.111 ± pg VEGF/µg total) to both day 3 (0.067 ± pg VEGF/µg total protein, p<0.05) and day 6 (0.055 ± pg VEGF/µg total protein, p<0.05). No significant difference in matrix-bound VEGF was observed between EC+FB, EC and FB cultures at any time point. Angiopoietin-1 (Ang1) is an angiogenic factor which is implicated in stabilization of nascent capillaries (13). Cellular Ang1 expression in the sample medium was measured using 45

56 ELISA (Figure 6c). Similar to the results for VEGF expression, the presence of fibroblasts resulted in higher protein levels of Ang1, as compared to endothelial-only constructs. However, the temporal profiles of Ang1 expression were different from those for VEGF, consistent with different functions for VEGF and Ang1 during angiogenesis as an angiogenic activation signal and capillary stabilization signal, respectively. At day 1, Ang1 expression in FB cultures (828 ± 241 pg/ml) was significantly higher than expression in EC+FB cultures (375 ± 178 pg/ml) and EC cultures (322 ± 201 pg/ml, p<0.05). Ang1 expression increased from day 1 to day 3 and expression in FB and EC+FB cultures (2965 ± 674 pg/ml and 1393 ± 312 pg/ml, respectively) remained significantly higher than expression in EC cultures (625 ± 211 pg/ml, p<0.05). At later time points, Ang1 expression was reduced in FB cultures and increased in EC+FB cultures, though both remained higher than EC cultures. ELISA was also used to measure matrix-bound Ang1 levels (Figure 6d). In EC cultures, no difference was observed in matrix-bound Ang1 levels from day 1 (14.3 ± 2.0 pg Ang1/µg total protein) to day 3 (14.2 ± 5.3 pg Ang1/µg total protein), however a significant decrease was observed from day 1 to day 6 (9.4 ± 1.8 pg Ang1/µg total protein, p<0.05). Similarly, no difference was observed in protein levels in FB cultures from day 1 (11.3 ± 0.4 pg Ang1/µg total protein) to day 3 (12.0 ± 0.7 pg Ang1/µg total protein), while levels at day 6 (8.7 ± 0.3 pg Ang1/µg total protein, p<0.05) were significantly decreased from day 1. The same trend was seen in EC+FB co-cultures, where no difference was seen in matrix-bound Ang1 levels from day 1 (10.6 ± 1.8 pg Ang1/µg total protein) to day 3 (13.0 ± 6.1 pg Ang1/µg total protein). However, matrix-bound Ang1 was significantly decreased from day 1 to day 6 (8.0 ± 0.6 pg Ang1/µg total protein). Additionally, at day 1 EC cultures had significantly greater matrix-bound Ang1 levels than both FB and EC+FB cultures (p<0.05), 46

57 however, no significant differences were observed between EC+FB, EC and FB cultures at later time points. Figure 6. Temporal profiles of Vascular Endothelial Growth Factor and Angiopoietin-1 expression in the medium and matrix-bound. ELISA was performed on samples collected from cell-nanofiber constructs at days 1, 3 and 6 to determine concentration of Vascular Endothelial Growth Factor (VEGF, panels a and b) and Angiopoietin-1 (Ang1, panels c and d). a) VEGF expression in the medium was significantly higher in FB cultures and EC+FB cultures as compared to EC controls. Temporal decreases in VEGF expression in FB and EC+FB cultures were also observed. b) No significant difference in matrix-bound VEGF was observed between EC+FB, EC and FB cultures at any time point. However, a 47

58 temporal decrease in matrix-bound VEGF expression was observed in all experimental groups. c) Ang1 protein expression in the medium was significantly higher in FB cultures (all time points) and EC+FB cultures (days 3 and 6) as compared to EC controls. A temporal increase was also observed in Ang1 expression in EC+FB, but not EC- or FB-only cultures. d) Expression of matrix-bound Ang1 was significantly lower at day 1 in FB and EC+FB cultures as compared to EC controls, however no significant differences were observed between the experimental groups at later time points. A temporal decrease in matrix-bound Ang1 expression was observed in all experimental groups. * p<0.05 when compared to EC samples, + p<0.05 when compared to FB samples, # p<0.05 when compared to day 1 samples of same experimental group, ^ p<0.05 when compared to day 3 samples of same experimental group. Endothelial-fibroblast interactions play a role in maintaining the balance for ECM turnover. To characterize ECM turnover, protein expression of MMP-2/TIMP-2 and MMP-9 (proteases which participate in the degradation and remodeling of the ECM (12)), and collagen I (a major component of the ECM) was measured using ELISA (Figures 7 and 8, respectively). At early time points, no significant differences in MMP-2/TIMP-2 expression exist between the experimental groups. Expression of MMP-2/TIMP-2 at day 3 (Figure 7a) in EC+FB and FB cultures (47 ± 6 ng/ml and 62 ± 3 ng/ml, respectively) was significantly higher compared to EC samples (30 ± 0.3 ng/ml, p<0.05). Expression of MMP-2/TIMP-2 at day 6 in EC+FB and FB cultures (37 ± 3 ng/ml and 56 ± 5 ng/ml, respectively) was significantly higher compared to EC samples (1.2 ± 0.3 ng/ml, p<0.05). Interestingly, MMP-2/TIMP-2 levels in EC+FB co-cultures 48

59 were more than twice the values for EC cultures at day 6, suggesting that this difference is not simply due to half of the cells in EC+FB constructs being fibroblasts and that endothelialfibroblast interactions may play a role in regulation of extracellular MMP-2 levels. In contrast to MMP-2/TIMP-2 expression, the protein levels for MMP-9 were low, and no significant differences between cell types were observed (Figure 7b). Figure 7. Protein expression of matrix metalloproteinase -2 and -9. ELISA was performed on medium samples collected from cell-nanofiber constructs at days 3 and 6 to determine concentration of MMP-2/TIMP-2 (panel a) and MMP-9 (panel b). a) At each time point, 49

60 MMP-2/TIMP-2 protein expression was significantly higher in FB cultures and EC+FB cocultures as compared to EC cultures. There was a temporal decrease observed in MMP- 2/TIMP-2 protein expression observed in EC+FB and EC cultures. b) There were no differences observed in MMP-9 expression. * p<0.05 when compared to EC samples, + p<0.05 when compared to FB samples, ^ p<0.05 when compared to day 3 samples of same experimental group. Similar to MMP-2/TIMP-2 data, the results for collagen I expression gave evidence of the role of endothelial-fibroblast interactions in regulation of ECM deposition (Figure 8a). Early expression of collagen I in the matrix at day 3 demonstrated significantly higher levels in EC+FB co-cultures (179 ± 71 pg col I/µg total protein) as compared to EC and FB cultures (49 ± 27 pg col I/µg total protein and 77 ± 58 pg col I/µg total protein, respectively, p<0.05). By day 6, EC+FB co-cultures collagen I levels in the matrix had significantly decreased from day 3 (62 ± 12 pg col I/µg total protein, p<0.05) and were similar to FB levels (62 ± 15 pg col I/µg total protein), with both significantly higher than EC levels (34 ± 9 pg col I/µg total protein, p<0.05). These observed levels for EC+FB co-cultures suggest the non-linear effect of EC-FB interactions on collagen I deposition, because in the absence of these interactions the expected values of collagen I protein in EC+FB co-cultures would be half-way between EC and FB levels. Low collagen I levels in the medium were observed (Figure 8b), with no significant differences detected between experimental groups (EC+FB, EC, FB) at either day 3 (1.06 ± 0.70 ng/ml, 1.04 ± 0.59 ng/ml, and 1.28 ± 0.34 ng/ml, respectively) or day 6 (0.87 ± 0.56 ng/ml, 1.12 ± 0.49 ng/ml, 1.19 ± 0.89 ng/ml, respectively). 50

61 Figure 8. Collagen I deposition. ELISA was performed on cell-nanofiber matrix samples (panel a) and medium samples (panel b) at days 3 and 6 to determine protein levels of collagen I (col I). a) Protein levels of col I in the matrix are significantly higher in EC+FB co-cultures in the peptide nanofiber microenvironment at days 3 and 6 in culture as compared to EC only cultures. A temporal decrease in col I protein levels was also observed in EC+FB cultures. b) Protein levels of col I in the medium were low, with no significant differences detected between experimental groups at either day 3 or day 6. Expression levels were normalized using total protein content as measured using Bradford 51

62 assay. * p<0.05 when compared to EC samples, + p<0.05 when compared to FB samples, ^ p<0.05 when compared to day 3 samples of same experimental group. Matrix permissiveness for cell migration is a regulator of extracellular mechanical environment. A parallel-plate rheometer (Figure 9a) was used to measure the mechanical properties of cell-nanofiber constructs (10 mg/ml peptide nanofibers) at days 1, 3, and 6 (Figure 9b), with elastic modulus (G ) serving as an indicator of construct stiffness. All cell-nanofiber constructs (EC+FB, EC, FB) had higher stiffness values than nanofiber only controls (1.97 ± 0.19 kpa at day 1), indicating the contribution of cell stiffness to overall stiffness. At day 1, only the highly migratory fibroblasts appeared able to easily move through the peptide nanofibers, resulting in scaffold disruption and a significantly lower FB construct stiffness (2.16 ± 0.38 kpa) as compared to EC and EC+FB constructs (4.29 ± 0.11 kpa and 4.28 ± 0.59 kpa, respectively, p<0.05). At day 3, the stiffness values are influenced by cell migration and subsequent peptide nanofiber disruption, with lower stiffness values measured for the FB and EC+FB constructs (2.91 ± 0.65 kpa and 2.95 ± 0.70 kpa, respectively) as compared to the less migratory EC constructs (3.63 ± 0.25 kpa). At day 6, EC+FB co-culture stiffness values (6.02 ± 0.71 kpa) are significantly increased from days 1 and 3 (p<0.05) and are higher than ECs (4.14 ± 0.22 kpa) and significantly higher than more migratory FBs (2.63 ± 0.61 kpa, p<0.05). 52

63 A more compliant nanofiber environment results in long term maintenance of greater construct stiffness in EC+FB co-cultures. Recent studies indicate that more compliant matrices promote in vitro network formation and cell migration (17, 18). Therefore, the effect of the scaffold stiffness on cell mechanical microenvironment was investigated in rheometry experiments using both a compliant nanofiber scaffold (6 mg/ml peptide concentration) and the stiffer peptide nanofiber scaffold (10 mg/ml peptide concentration), which better represents the stiff fibrotic microenvironment of the healing heart. In the compliant nanofiber concentration (Figure 9c), again all cell-nanofiber constructs (EC+FB, EC, FB) had higher stiffness values than nanofiber only controls (0.62 ± 0.02 kpa at day 1). Coordinated endothelial cell and fibroblast migration and peptide nanofiber disruption in the EC+FB constructs led to lower stiffness (0.64 ± 0.13 kpa) as compared to EC and FB constructs (0.97 ± 0.11 kpa and 0.77 ± 0.19 kpa, respectively) at day 1. The stiffness values at day 3 of all cell-nanofiber constructs (EC+FB, EC, FB) are similar (0.71 ± 0.03 kpa, 0.72 ± 0.10 kpa, and 0.70 ± 0.03 kpa, respectively), indicating that the effects of cell proliferation, cell migration and ECM remodeling are roughly equivalent at this time. By day 6, EC+FB cocultures have significantly increased stiffness values (0.94 ± 0.09 kpa) as compared to days 1 and 3 (p<0.05). Also at day 6, EC+FB stiffness is higher than ECs (0.87 ± 0.02 kpa) and significantly higher than more migratory FBs (0.64 ± 0.05 kpa, p<0.05). Statistical analyses showed that cell composition, time in culture and peptide nanofiber concentration were all significant factors in the stiffness of cell-nanofiber constructs (p<0.001 for each factor, ANOVA). Further analysis revealed that significant differences existed in the stiffness values between days 1 and 6 and days 3 and 6 (p<0.001, Bonferroni post hoc t-test). In 53

64 the cell composition, significant stiffness differences existed between fibroblasts cultures and both endothelial cell cultures and EC+FB co-cultures (p<0.001, Bonferroni post hoc t-test). Figure 9. Stiffness of cell-nanofiber constructs measured using rheometry. a) Schematic of parallel plate rheometer setup, showing living cells embedded in the peptide-nanofiber construct for testing. b) Elastic moduli (G ) values for cell-nanofiber constructs (10 mg/ml peptide nanofibers). Fibroblasts play a more significant role in the denser concentration, as FB only constructs displayed lowest stiffness values at all time points. EC+FB cocultures have significantly greater stiffness values at day 6 compared to other experimental groups as well as previous time points. c) Elastic moduli (G ) values for cell-nanofiber constructs (6 mg/ml peptide nanofibers). The more compliant peptide nanofibers result in increased construct stiffness in EC+FB co-cultures at day 6 as compared to previous time points, similar to the denser concentration. G (kpa) is reported at frequency of 0.1 Hz. * p<0.05 when compared to EC samples, + p<0.05 when compared to FB samples, # p<0.05 when compared to day 1 samples of same experimental group, ^ p<0.05 when compared to day 3 samples of same experimental group. 54

65 DISCUSSION In this study, both chemical and mechanical regulation of capillary morphogenesis by fibroblasts were for the first time investigated simultaneously in a temporal manner. Recent studies have established the effect of the mechanical environment on angiogenesis in vitro by showing that extracellular matrix stiffness modulates formation of endothelial networks via altering traction forces exerted by the cells (17, 161). While there is literature regarding fibroblast roles in capillary morphogenesis either as a source of angiogenic growth factors (89) or as a substrate for endothelial culture and migration in scaffold-free in vitro cultures (42), the possible effects of fibroblasts on angiogenesis via modulation of the mechanical environment and the interplay between mechanical and chemical signaling have not been studied in the same system. Therefore, the goal of this study was to elucidate the mechanisms of fibroblast regulation of angiogenic process using a comprehensive approach and a recently described culture system. The scaffold made from RAD16-II peptide nanofibers was used to mimic the in vivo threedimensional matrix architecture and support cell functions and interactions (162). Importantly, cell viability was stable within the nanofiber culture system, indicating that the system supports cell survival and that the observed results do not directly stem from significant cell proliferation and/or death. The obtained results demonstrate that fibroblasts provide a complex timedependent regulation of in vitro capillary morphogenesis and overall cell-scaffold homeostasis, as seen in the proposed temporal schematic (Figure 10). The results show that secretion of soluble growth factors (VEGF, Ang1) by and migration of fibroblasts and corresponding scaffold disruption promote capillary morphogenesis and stabilization at early time points. At the later stages, the role of fibroblasts as regulators of mechanical microenvironment becomes more 55

66 prominent via maintaining the balance in ECM turnover via MMP-2 and collagen I production, which resulted in improved integrity and a stable microenvironment. Figure 10. Proposed schematic of angiogenic regulation by fibroblasts in endothelialfibroblast co-cultures in vitro. Arrows denote the relative levels of protein expression and stiffness in the co-cultures at days 1, 3 and 6. The results show that fibroblasts mediate angiogenic process in vitro via two major mechanisms: direct regulation of capillary morphogenesis via expression of angiogenic factors, and indirect regulation by altering mechanical microenvironment via matrix disruption, deposition and remodeling. Initially, the process is regulated by chemical signaling from fibroblasts (a sharp increase in VEGF levels, as compared to EC-only cultures) and EC-FB co-migration at day 1. Gradually, this initial response is replaced by stabilizing factors (increases in Ang1 and decreases in VEGF levels) and signaling via alterations in mechanical microenvironment, where endothelialfibroblast interactions appeared to help maintain the balance in ECM homeostasis, enhancing both MMP-2 and collagen I production. 56

67 Interestingly, the effect of fibroblasts on the protein expression of two major angiogenic factors, VEGF and Ang1, in the co-culture with endothelial cells in the nanofiber culture system appears to be precisely orchestrated to parallel stages of angiogenic response in vivo. Indeed, the angiogenic process in vivo (163) starts with VEGF-induced activation of endothelial cells and capillary sprouting. The elevated levels of VEGF gradually decrease, when sprouting is replaced by capillary stabilization, which is largely mediated by pericyte-produced Ang1 and is accompanied by increased levels of this protein. Consistent with this view, the results of this study show that in endothelial-fibroblast co-cultures, VEGF levels are highest at day 1 and then gradually decrease. Interestingly, VEGF levels in EC+FB co-cultures are slightly lower at all time points than expected based on cell numbers alone, suggesting that in co-culture cellular VEGF production may be reduced, although this difference is not pronounced. In contrast to the observed VEGF temporal pattern, Ang1 expression steadily rises from day 1 to day 6, implying reduced endothelial migration and stabilization of newly formed endothelial networks. In fact, Ang1 levels in co-culture at day 6 are much higher than what would be expected based on cell numbers alone, suggesting that in co-culture Ang1 production is enhanced. Importantly, this pattern of Ang1 expression does not occur in either EC- or FB-only cultures and has not been observed previously in other in vitro systems, where addition of angiogenic factors is usually required to induce angiogenesis, precluding quantitation of endogenous (cell-induced) Ang1 levels. A new finding of this study is the role that fibroblasts play in facilitating endothelial cell migration during active stages of capillary morphogenesis. Indeed, the significantly larger and faster forming networks seen in EC-FB co-cultures indicate that migratory cells such as fibroblasts promote endothelial cell organization and assembly into larger networks in this 57

68 system. Interestingly, the peptide nanofibers lack MMP degradation sites by design (100), so a mechanism other than proteolytic migration (161) is responsible for cell migration within the nanofiber matrix. Amoeboid migration driven both by cells squeezing through the matrix pores and through deformation of the ECM network (164) (allowing for circumnavigation rather than degradation of ECM barriers) is the likely mechanism of cell migration within the nanofiber scaffold. Thus, these results suggest a novel mechanism for fibroblasts to regulate network formation by endothelial cells via mechanical disruption of the scaffold and thus leading the way for endothelial networks to spread. Recent studies demonstrated that ECM mechanical environment affects angiogenesis via regulating contractile forces generated by the endothelial cells (161). Therefore, our finding that migratory fibroblasts promote capillary morphogenesis via mechanical ECM disruption may have an important implication for cardiac regeneration, where endothelial cells need to overcome the stiffness barrier of fibrotic scarring in order to penetrate the ischemic area. The overall effect of endothelial-fibroblast interactions on the extracellular mechanical environment was studied by employing a rheometry approach to measure the mechanical stiffness of three-dimensional cell-scaffold constructs. The elastic modulus (G ) served as an indicator of the cell-construct stiffness, reflecting the balance between scaffold disruption due to cell (mostly FB) migration and extracellular matrix remodeling due to deposition and proteolysis of new matrix components (such as collagen I). To the best of our knowledge, this is the first time that rheometry has been used to measure the material properties of constructs seeded with live cells. The rheometry results showed that trends seen in network formation and protein expression were accompanied by the corresponding changes in stiffness values of cell-seeded nanofiber constructs. The results suggest that cell migration and associated scaffold disruption 58

69 play the major role in overall stability of mechanical microenvironment, and that this effect depends on time in culture and the cell type. For highly migratory fibroblasts, stiffness values do not change significantly throughout the experimental duration and are the lowest of all three groups, with no significant differences in collagen I and MMP-2 levels between the time points. In endothelial-only constructs, there is initial endothelial cell activation, enhanced migration and associated scaffold disruption, resulting in decreased stiffness at day 3, as compared to day 1. At later times, slightly increased levels of Ang1 and capillary stabilization are associated with less migration, and with reduced collagen I concentration and MMP-2 remodeling, resulting in overall higher values of stiffness at day 6. However, in EC+FB constructs, endothelial-fibroblast interactions result in a significantly different temporal pattern for alterations in the mechanical environment. Thus, the results show that initially (from day 1 to day 3), in the stiffer scaffold, mechanical stiffness of the EC+FB constructs is significantly decreasing, even though the collagen concentration is the highest at day 3. This sharp decrease in stiffness between day 1 and day 3 does not occur in more compliant scaffold, where there is less resistance for cell migration, suggesting that migration-induced scaffold disruption is mostly responsible for the decreased mechanical stiffness at day 3. Additionally, these high collagen I levels (more than expected based on cell numbers alone) are paralleled by high MMP-2/TIMP-2 levels in EC+FB cocultures at day 3, consistent with increased MMP-2 production by endothelial cells in collagen I- rich environment (165). Interestingly, collagen I has also been shown to enhance endothelial production of MT1-MMP and subsequently MMP-2 activation (165, 166), which may explain observed decreases in both MMP-2/TIMP-2 and collagen I levels at day 6. However, increasing levels of Ang1 and stabilization of endothelial networks due to endothelial-fibroblast interactions from day 3 on are associated with significant increases in stiffness of EC+FB constructs at day 6 59

70 in both stiff and compliant scaffolds, even as compared with day 3. These results suggest that a combination of ECM deposition and remodeling overcomes the structural nanofiber disruption caused by cell migration, resulting in greater stiffness values, better structural integrity and a stabilization of the overall microenvironment at day 6, as compared with earlier time points. In this study, the effects of matrix concentration on cell-cell interactions and mechanical regulation of the microenvironment were studied by comparing stiffness values for cell-seeded constructs made using two peptide nanofiber concentrations: a stiffer nanoscaffold (10 mg/ml peptide nanofibers) which better represents the microenvironment of the healing heart, and a more compliant nanoscaffold (6 mg/ml peptide nanofibers) in order to investigate previous observations that more compliant matrices promote in vitro network formation and cell migration (17, 18). In these studies, the compliant nanofibers allowed for coordinated endothelial cell and fibroblast migration and scaffold disruption. In contrast, in the stiff nanofibers, only the highly migratory fibroblasts appeared able to easily move through the peptide, resulting in lower stiffness in the fibroblast constructs. These results are in agreement with previously reported enhanced capillary morphogenesis in more compliant RAD16-II scaffolds (102). In the compliant nanofibers, there was a steady increase in cell-seeded construct stiffness from day 1 to day 3, in contrast to sharp decrease and subsequent increase in modulus of the stiffer nanofibers. Therefore, the data confirm that in this culture system, similar to others (17, 18, 148), matrix stiffness does affect cell behavior, with fibroblasts playing a more significant role in mechanical regulation in denser nanofibers. Physiologically, this is more representative of the scar environment in the healing heart where fibroblasts often play a role in aberrant matrix remodeling. In contrast, our results suggest that more compliant nanofibers better support migration of both endothelial cells and fibroblasts, allowing for a better balance between cell 60

71 migration and ECM remodeling and may ultimately be better suited for improved angiogenesis and long term stability. To accurately represent the process of cardiac tissue revascularization and remodeling in vitro, an ideal system would include all three major cardiac cell types (cardiomyocytes, endothelial cells and cardiac fibroblasts) from the same organism. In this study, we chose to use human endothelial cells and fibroblasts in a 1:1 ratio to represent cell behavior in healing human cardiac tissue (78, 167). While human microvascular endothelial cells are readily available, human cardiac fibroblasts are not. Therefore, human dermal fibroblasts were used, which may be a limitation of the study. However, given similarities that exist between cardiac and dermal wound healing (168), we believe that this study provides important findings which reflect phenotypic fibroblast behavior and are representative of endothelial-fibroblast interactions during cardiac tissue revascularization. In summary, this study demonstrates that fibroblasts mediate the angiogenic process in vitro via two major mechanisms: direct regulation of capillary morphogenesis via expression of angiogenic factors, and indirect regulation by altering mechanical microenvironment via matrix disruption, deposition and remodeling. The results show that both of these mechanisms are time dependent, and that at each time point, endothelial cell behavior is likely to be controlled by a combination of factors, such as concentration of angiogenic growth factors, ECM composition, stiffness and remodeling, all of which are in turn influenced by the presence of fibroblasts. Thus, our findings provide insight into the complex intercellular signaling occurring between fibroblasts and endothelial cells in the context of angiogenesis and cardiac regeneration. The use of peptide nanofibers in this study effectively allowed for the uncoupling of cell-cell interactions from cell-scaffold interactions and allowed for the simultaneous investigation of both the 61

72 chemical and mechanical roles of fibroblasts in angiogenesis in an in vitro system. The results of this study, along with recent in vivo studies in mouse cardiac tissue (101, 102) and during wound healing in diabetic mice (103), suggest that the peptide nanofiber environment may be uniquely suited as a tissue engineering substrate for regeneration of vascular tissues. The findings of this study emphasize the importance of creating the microenvironment which supports cell-cell interactions and is permissive for cell migration, thus contributing towards creating the optimal environment for cardiac tissue engineering applications. 62

73 CHAPTER 4 Effects of Mechanical Strain and Diabetic Phenotype on Fibroblast Matrix Remodeling Response Jennifer R. Hurley, Abdul Q. Sheikh, Daria A. Narmoneva. School of Energy, Environmental, Biological & Medical Engineering. University of Cincinnati, Cincinnati, Ohio. In preparation for submission. ABSTRACT Overcoming the effects of hyperglycemic conditions on matrix remodeling by cardiac fibroblasts represents a novel therapeutic approach for treatment of cardiac conditions in the context of diabetes. Mechanical stretch may represent a potential strategy to compensate for the dysregulation of matrix remodeling by diabetic cardiac fibroblasts, as studies have shown that the application of mechanical strain on fibroblasts in culture leads to a variety of responses related to extracellular matrix remodeling. The purpose of this study was to investigate the effects of mechanical stretch on the matrix remodeling response of cardiac fibroblasts, including those of diabetic phenotype. A commercially available Flexcell system was used to apply uniaxial stretch to rat cardiac fibroblasts. The fibroblast matrix remodeling response due to strain was measured via MMP-2 (ECM remodeling), collagen I (ECM deposition), and VEGF (cell migration stimulus) expression as well as fibroblast morphology and orientation and cell proliferation and apoptosis. Our results demonstrate that in our experimental setup, the 63

74 application of cyclic strain resulted in limited improvements in matrix remodeling response, both by wild type fibroblasts and those of diabetic phenotype. INTRODUCTION Diabetes is one of the most common chronic illnesses in the world with 25.8 million individuals (8.3% of the population) afflicted in the United States (58). Annual medical costs associated with diabetic care were estimated at $174 billion in 2007 (58). While there are many life-threatening complications associated with diabetes, heart disease is one of the most severe with a high incidence of congestive heart failure (60, 61), often leading to death. Diabetic cardiomyopathy (DCM) is a diabetes-associated cardiovascular condition defined as ventricular dysfunction in the absence of other etiological factors, such as hypertension or coronary heart disease (20-23), and pathological alterations to the myocardium resulting from DCM include circulatory defects, compromised heart muscle contraction, and progressive fibrosis. Cardiac fibroblasts are especially important in diabetic alterations to the heart, particularly fibrosis where impaired matrix remodeling and extracellular matrix (ECM) turnover lead to damaging structural, geometric and functional changes in the heart (11, 12). Fibroblasts represent two-thirds of total cardiac cell number and are primarily responsible for both physiological and pathological extracellular matrix homoeostasis (78, 80-82). Studies suggest that fibroblasts maintain the mechanical extracellular microenvironment via matrix deposition and metalloproteinase-mediated ECM remodeling (12, 84). However, altered fibroblast phenotype associated with diabetes and chronic hyperglycemic conditions can lead to dysregulation of cardiac regeneration and matrix remodeling by fibroblasts, with increased collagen accumulation and fibrosis seen in both diabetic human patients ( ) and animal 64

75 models (24-27, 62, 131, 132). Therefore, overcoming the effects of hyperglycemic conditions on matrix remodeling by cardiac fibroblasts represents a novel therapeutic approach for treatment of not only diabetic cardiomyopathy but other cardiac conditions in the context of diabetes as well. Application of environmental stimuli which occur in the healthy myocardium, such as mechanical stretch and electrical stimulation, may prove to be potential therapeutic approaches for stimulation of cardiac regeneration. In particular, mechanical stretch may be a promising strategy to compensate for the dysregulation of matrix remodeling by diabetic cardiac fibroblasts. Mechanical stimulation plays an important role in tissue development and repair and cells adapt and respond to changes in their mechanical environment via morphological and phenotypic alterations (32). In vitro studies have shown that the application of mechanical strain on fibroblasts in culture leads to a variety of responses related to extracellular matrix remodeling (33, 34), including increased extracellular matrix expression, deposition, and density (35-38, 40, 41, 141), increased activation of MMP-2 (142), and increased growth factor expression (36, 42-44). The major findings from these previous studies as well as cell source, strain magnitude and duration are compiled in Table 2. Clearly, while a number of studies provide valuable information regarding the matrix remodeling response of wild type fibroblasts to mechanical strain, there remains much more to learn, in particular with regards to the effect of diabetic phenotype in conjunction with cyclic strain. 65

76 Cell source Strain magnitude Duration of strain Substrate/scaffold Major findings Reference canine ACL and MCL FBs 5% 3 days fibronectin, collagen I decreased cell proliferation (139) human foreskin FBs 2, 4, 8, 16% 8 days fibrin gels collagen density increased with magnitude and duration of stretch (40) human gingival FBs, periodontal ligament FBs 7, 14, 21% 24 hrs collagen I increased VEGF (44) human myofbs 4, 8, 12% 2 weeks PGA constructs induced crosslinked collagen production (169) human scleral FBs 15% 12, 24, 48 hrs collagen I increased prommp-2, reduced TIMP-2 (142) murine embryonic FBs 5% 7 days collagen I gel increased collagen and proteoglycan content, increased material strength (141) rat cardiac FBs 10% hrs collagen I increased procollagen transcription (36) rat cardiac FBs 20% 1-4 days elastin increased procollagen synthesis (35) Table 2. Effects of magnitude and duration of strain on matrix remodeling responses of fibroblasts. Compilation of previous studies which investigated the effect of mechanical strain on various matrix remodeling responses (ECM remodeling, ECM deposition, growth factor expression, cell proliferation) by fibroblasts from a variety of sources. The purpose of this study was to investigate the effects of mechanical strain on the matrix remodeling response of cardiac fibroblasts, including those of diabetic phenotype. A commercially available Flexcell system was used to apply uniaxial stretch to rat MMP-2 (ECM remodeling), collagen I (ECM deposition), and VEGF (cell migration stimulus) expression as well as fibroblast morphology and orientation and cell proliferation and apoptosis. For this study, we tested two hypotheses; first, that diabetic phenotype will result in a diminished reparative matrix remodeling response by cardiac fibroblasts in vitro, with decreased MMP expression and increased extracellular matrix deposition, and second, that stimulation of cardiac fibroblasts with mechanical strain will result in enhanced matrix remodeling response and 66

77 attenuate diabetes-induced phenotypic cell alterations. Our results demonstrate that application of cyclic strain results in limited improvements in matrix remodeling response, both by wild type fibroblasts and those of diabetic phenotype. METHODS Cell isolation. All animal procedures were performed using protocols approved by the University of Cincinnati Institutional Animal Care and Use Committee. Diabetic (db) and wild type (wt) cardiac fibroblasts were isolated from week old female Sprague Dawley rats (SAS SD Strain 400, Charles River, Wilmington, MA). Type I diabetes was induced in 8 week old rats using a single intraperitoneal injection of streptozotocin (70 mg/kg, Sigma-Aldrich, St. Louis, MO) (23-27, 67, 170). The streptozotocin rat model was chosen as it closely mimics the time-dependent disease progression of diabetic cardiomyopathy (171). Immediate onset of diabetes was confirmed with serum glucose levels >450mg/dl and diabetic animals were sacrificed 6 weeks after injection. For cell isolation, hearts were harvested and washed in cold PBS several times, finely chopped and digested first in trypsin (1 mg/ml, Sigma-Aldrich) at room temperature for 1 hour and then collagenase I (172 U/ml, Worthington Biochemical Corp., Lakewood, NJ) at 37 C for 45 minutes. Supernatant containing cells was separated from remaining tissue using a 70 μm disposable cell strainer (Becton Dickinson Labware, Bedford, MA) and centrifuged at 800 rpm for 5 minutes at 4 C. The cell pellet was resuspended and added to uncoated cell culture dishes for 2 hours to allow Medium 199 (HyClone, Logan, UT) containing 10% fetal bovine serum (FBS; Atlanta Biologicals, Lawrenceville, GA), 1% Antibiotic-Antimycotic (Atlanta Biologicals, Lawrenceville, GA), 10 µg/ml heparin (Sigma- Aldrich), and 0.2 ng/ml cell growth supplement (Sigma-Aldrich) as previously described (14, 67

78 16). Cell cultures were maintained at 37ºC in 100% humidified air containing 5% CO 2. Cells of passage 4-10 were used in all experiments. Experimental groups. Three days prior to experiments, the cell culture media was changed to reflect the appropriate experimental group. The first group was normal or wild type cells cultured in standard low glucose media (wt; cell culture media supplemented with 5.5 mm D- glucose, Sigma-Aldrich). Two groups represented diabetic phenotype: wild type cells cultured in high glucose media (wt HG; cell culture media supplemented with 22.2 mm D-glucose) and cells harvested from diabetic animals and cultured in standard low glucose media (db; cell culture media supplemented with 5.5 mm D-glucose). An additional group of wild type cells was cultured in osmotic control media (wt L-control; cell culture media supplemented with 5.5 mm D-glucose and 15 mm L-glucose, Sigma-Aldrich). D- and L- glucose are stereoisomers of glucose, with only the D-glucose isomer occurring naturally and the L-glucose isomer commonly used as a control group. Results from the osmotic control group are not reported in the text. Application of cyclic strain. Cells were seeded onto 6-well UniFlex Culture Plates pre-coated with collagen I (Flexcell International Corp., Hillsborough, NC) at a seeding density of 150,000 cells/well in the appropriate experimental group cell culture media. Additional six-well tissue culture plates were coated with collagen I (PureCol, Advanced BioMatrix, San Diego, CA) for 30 min at 37 C prior to cell seeding (150,000 cells/well) with appropriate media for static controls. In the heart, 80% of all newly synthesized collagen is collagen I (34), making it an appropriate substrate for cardiac cell studies. All culture plates were cultured overnight without stimulation to allow for cell attachment. UniFlex Culture Plates were then loaded onto the Flexcell FX-5000 Tension System outfitted with ArcTangle Loading Stations (24 mm; Flexcell International Corp.) for application of uniaxial strain and housed at 37ºC in 100% 68

79 humidified air containing 5% CO 2. Strain parameters were set at 5% elongation at 1 Hz and 60 cycles/minute for 24 hours. These strain parameters were selected to fall within the range of previous studies (Table 2) as well as physiological in vivo cardiac parameters (33). Static controls were maintained in the same incubator for the experiment duration. Cell morphology and orientation. At 24 hrs, both strained and static cell samples were fixed with 2% paraformaldehyde. Cell samples were stained with phalloidin-tetramethylrhodamine-b isothiocyanate (phalloidin-tritc, Sigma-Aldrich) as per manufacturer s protocol, followed by DAPI (Invitrogen Corporation, Carlsbad, CA). Images were taken (n=5 per sample) using an inverted fluorescent microscope (Olympus IX81; Olympus America Inc., Center Valley, PA). Protein expression using Enzyme-Linked Immunosorbent Assay (ELISA). At the end of 24 hour strain application, cell culture media was collected from the wells and stored at -80ºC until testing. Rat VEGF ELISA (R&D Systems, Minneapolis, MN) was performed on media samples as per manufacturer s protocol. Additional MMP-2 and collagen I ELISAs were performed as described in (160) to determine protein concentrations in medium samples (Rat MMP-2 - antibodies from R&D Systems, Rat Collagen I antibodies from Pierce Biotechnology, Rockford, IL). Collagen I ELISA was not performed on matrix samples, as it would not have been possible to differentiate native cell collagen I deposition from the collagen I pre-coated on both the static and strained culture dishes. For all ELISAs, additional controls of the cell culture medium alone (containing 10% serum) were included to confirm that protein content in the serum would not affect protein expression and detection, with no differences observed between control samples and the 0 pg/ml standard. Cell proliferation. Staining for proliferation marker Ki67 (Abcam, Cambridge, MA) and DAPI (Invitrogen Corporation) was performed to assess cell growth. After application of strain for 24 69

80 hrs, cells from both strained and static controls were trypsinized and replated onto gelatin coated 24-well tissue culture plates at a seeding density of 20,000 cells/well (n=6 per experimental group). Cells were fixed with 2% paraformaldehyde at 24 hrs. Samples were immunostained with rabbit anti-rat Ki67 (1:50; Abcam), followed by fluorescent goat anti-rabbit AlexaFluor secondary antibody (1:250; Invitrogen Corporation) and DAPI, to identify total and proliferating cells. The average absolute number and percentages (number of positive cells to the total number of cells) of proliferating cells were quantified in 3 non-overlapping, randomly selected fields per sample at 20X magnification. Cell apoptosis. Anti-ACTIVE Caspase-3 (Promega Corporation, Madison, WI) and DAPI staining was performed to assess cell death. After application of strain for 24 hours, cells from both strained and static controls were trypsinized and replated onto gelatin coated 24-well tissue culture plates at a seeding density of 20,000 cells/well (n=6 per experimental group). Cells were fixed with 2% paraformaldehyde at 24 hours and immunostained with Anti-ACTIVE Caspase-3 (1:250; Promega Corp.), followed by goat anti-rabbit AlexaFluor secondary antibody (1:250; Invitrogen Corporation) and DAPI, to identify total and apoptotic cells. The average absolute number and percentages (number of positive cells to the total number of cells) of apoptotic cells were quantified in 3 non-overlapping, randomly selected fields per sample at 20X magnification. Statistical analyses. The results are reported as average ± standard deviation. Each experiment was performed in triplicate and repeated three times (N=3, minimum n=9). ANOVA and posthoc tests assuming equal variances were used to determine the effects of stimulation (static or cyclic strain) and diabetic condition (wt, wt HG or db) on matrix remodeling (MMP-2 and collagen I expression) and cell responses (proliferation, apoptosis and VEGF expression). All tests were run at a significance level of α =

81 RESULTS Picosirius red staining of wild type and diabetic heart tissue sections demonstrated that significant cardiac fibrosis is evident in the diabetic heart at 6 weeks post STZ injection (Figure 11), validating both the use of the STZ type I diabetic rat model as well as the 6 week time frame for cardiac fibrosis and diabetic cardiomyopathy development. Cardiac fibroblasts harvested from hearts of wild type and STZ type I diabetic rats were used for the following experiments. Figure 11. Evidence of cardiac fibrosis in the diabetic heart 6 weeks post STZ injection. Picosirius red staining of the rat myocardium shows the presence of cardiac fibrosis (golden red) in the left ventricle of heart tissue from STZ type I diabetic animals (right panel), as compared to heart tissue from age- and strain-matched wild type controls (left panel). Scale bar represents 200 μm. Staining courtesy of Dr. Yigang Wang, University of Cincinnati College of Medicine. Static and strained samples were stained with phalloidin at 24 hrs to visualize actin filaments within the cell body (Figure 12). No qualitative differences were apparent in cell 71

82 morphology and orientation in wt, wt HG, and db samples in static conditions (Figure 12, top panels) or after application of strain (Figure 12, bottom panels). Figure 12. Phalloidin staining of static and strained samples at 24 hrs. Top panel) Static cultures of wt, wt HG, and db fibroblasts (left to right). Bottom panel) Strained cultures of wt, wt HG, and db fibroblasts (left to right). No differences were apparent in cell morphology and orientation in either static conditions or after application of strain. Scale bar represents 100 microns. Protein expression of matrix metalloproteinase-2, a protease which participates in the degradation and remodeling of the ECM (12), was measured in media samples from static and strained fibroblast cultures using ELISA. In static samples (Figure 13a), MMP-2 concentration in wt HG ( ± 92.5 pg/ml) was significantly greater than in wt and db samples ( ± 60.5 and ± 34.4 pg/ml, respectively, p<0.05). In samples subjected to cyclic strain (Figure 13b), MMP-2 levels in wt and wt HG samples ( ± 67.8 and ± pg/ml, respectively) were significantly higher than that in db samples ( ± 70.3 pg/ml, p<0.05). 72

83 No differences existed between the different experimental groups between static and strained samples. Figure 13. Protein expression of matrix metalloproteinase -2. ELISA was performed on media samples collected from static and strained fibroblast cultures (wt, wt HG, and db) to determine MMP-2 concentration (pg/ml). a) MMP-2 protein expression (pg/ml) in static fibroblast cultures. MMP-2 expression was significantly increased in wt HG samples as compared to the other experimental groups. b) MMP-2 protein expression (pg/ml) in strained fibroblast cultures. MMP-2 expression was significantly decreased in db samples as compared to the other experimental groups. * indicates p<

84 Collagen I (col I) is a major component of the extracellular matrix and substrate of MMP- 2 and col I concentration was measured in media samples from static and strained fibroblast cultures using ELISA. In static samples (Figure 14a), collagen I concentration in db samples (17.9 ± 8.1 pg/ml) was significantly lower than in wt and wt HG samples (37.5 ± 5.0 and 42.3 ± 5.4 pg/ml, respectively, p<0.05). In samples subjected to cyclic strain (Figure 14b), collagen I levels in wt HG samples (43.5 ± 5.8 pg/ml) was significantly greater than db low samples (29.6 ± 5.9 pg/ml, p<0.05). No differences were observed between wt samples (36.7 ± 8.7 pg/ml) and the other experimental groups. Collagen I levels were also significantly higher in strained db samples than in static db samples (p<0.05). 74

85 Figure 14. Protein expression of collagen I. ELISA was performed on media samples collected from static and strained fibroblast cultures (wt, wt HG, and db) to determine collagen I concentration (pg/ml). a) Collagen I protein expression (pg/ml) in static fibroblast cultures. Collagen I expression was significantly decreased in db samples as compared to the other experimental groups. b) Collagen I protein expression (pg/ml) in strained fibroblast cultures. Collagen I expression was significantly increased in wt HG samples as compared to db samples. * indicates p<0.05. Vascular endothelial growth factor (VEGF) is a major regenerative stimulus and activation signal for cell migration, including fibroblast migration (13, 172). The concentration 75

86 of VEGF was measured in media samples from static and strained fibroblast cultures using ELISA. In static samples (Figure 15a), VEGF concentration in wt HG (344.0 ± pg/ml) was significantly greater than in wt and db samples (50.7 ± 18.6 and 50.5 ± 25.8 pg/ml, respectively, p<0.05). In samples subjected to cyclic strain (Figure 15b), no difference was observed between wt, wt HG and db samples (38.3 ± 11.4, 31.0 ± 12.7 and 41.9 ± 22.9 pg/ml, respectively). VEGF levels were significantly decreased in strained wt HG samples as compared to static wt HG samples (p<0.05). Figure 15. Protein expression of vascular endothelial growth factor. ELISA was performed on media samples collected from static and strained fibroblast cultures (wt, wt HG, and db) to determine VEGF concentration (pg/ml). a) VEGF protein expression 76

87 (pg/ml) in static fibroblast cultures. VEGF expression was significantly increased in wt HG samples as compared to the other experimental groups. b) VEGF protein expression (pg/ml) in strained fibroblast cultures. No differences were observed among experimental groups. * indicates p<0.05. Fibroblast proliferation was measured in samples from static and strained fibroblast cultures by staining for Ki67, a cell proliferation marker. In static samples (Figure 16a), the percentage of proliferating cells in wt cells (22.0 ± 2.6 %) was significantly greater than in wt HG and db samples (14.8 ± 0.7 and 5.2 ± 3.5 %, respectively, p<0.05). In samples subjected to cyclic strain (Figure 16b), no difference in cell proliferation was observed between wt, wt HG and db samples (13.2 ± 1.8, 14.3 ± 8.4 and 9.3 ± 5.8 %, respectively). The percentage of proliferating cells was also significantly higher in static wt samples than in strained wt samples (p<0.05), with no differences observed in wt HG and db samples. 77

88 Figure 16. Cell proliferation. Ki67 staining was performed on cells subjected to 24 hrs of strain and static controls (wt, wt HG and db) to determine the percentage of proliferating cells. a) Proliferating cells (%) in static fibroblast cultures. Cell proliferation was significantly higher in wt samples as compared to the diabetic experimental groups. b) Proliferating cells (%) in strained fibroblast cultures. No differences were observed among experimental groups. * indicates p<0.05. Fibroblast apoptosis was measured in samples from static and strained fibroblast cultures by staining for Caspase-3, a cell apoptosis marker. In static samples (Figure 17a), no difference in cell apoptosis was observed between wt, wt HG and db samples (3.1 ± 1.1, 2.8 ± 0.8 and 4.0 ± 0.2 %, respectively). In samples subjected to cyclic strain (Figure 17b), again no difference in 78

89 cell apoptosis was observed between wt, wt HG and db samples (3.2 ± 1.8, 7.3 ± 2.5 and 3.8 ± 3.5 %, respectively). The percentage of apoptotic cells was significantly lower in static wt HG samples than in strained wt HG samples (p<0.05), with no differences observed in wt and db samples. Figure 17. Cell apoptosis. Caspase-3 staining was performed on cells subjected to 24 hrs of strain and static controls (wt, wt HG and db) to determine the percentage of apoptotic cells. a) Apoptotic cells (%) in static fibroblast cultures. No differences were observed among experimental groups. b) Apoptotic cells (%) in strained fibroblast cultures. No differences were observed among experimental groups. * indicates p<

90 DISCUSSION In this study, the effect of cyclic strain and diabetic phenotype on the matrix remodeling response by cardiac fibroblasts was investigated in a systematic manner. Characterization of the effect of diabetic phenotype on remodeling response by cardiac fibroblasts was performed using static cell cultures. Strain was applied to cell samples using a commercially available Flexcell system, applying 5% uniaxial stretch for 24 hrs. Out results indicate that the application of cyclic strain resulted in limited improvement in matrix remodeling response by cardiac fibroblasts, both wild type and diabetic, which may be attributable to the short duration of strain application and/or or the magnitude of strain. The effect of diabetic phenotype on the matrix remodeling response by cardiac fibroblasts was elucidated using static cultures of normal or wild type cardiac fibroblasts cultured in standard low glucose media (wt) and two models of diabetic phenotype wild type fibroblasts cultured in high glucose media (wt HG) and fibroblasts harvested from diabetic animals and cultured in standard low glucose media (db). While no differences were observed in cell apoptosis levels between wild type and diabetic fibroblasts, our results did demonstrate significantly decreased proliferation in both models of diabetic phenotype as compared to the wild type phenotype. This is consistent with previous observations of decreased proliferation in stromal cells from diabetic mice (173). Additionally, previous studies have demonstrated reduced growth factor expression, reduced cellular migration, decreased MMP activation, and increased collagen synthesis by cardiac fibroblast cultures in high glucose conditions (28-31). Interestingly however, in our study, we observed significantly increased collagen I levels (consistent with previous studies in high glucose conditions (29-31)) along with significantly increased MMP-2 and VEGF levels (in contrast to previous studies in cells from db/db mice 80

91 (28)) in fibroblasts cultured in high glucose conditions (wt HG) as compared to the wild type controls. Alternatively, we observed significantly decreased MMP-2 levels (consistent with previous studies in cells from db/db mice (28)) along with significantly decreased collagen I and no difference in VEGF levels (in contrast to previous studies in cells from db/db mice (28)) in diabetic fibroblasts (db) as compared to the wild type controls. These are interesting observations, in particular with regards to investigating different in vitro models of diabetic phenotype. While high glucose conditions mimic some of the responses seen in previous studies (increased collagen I levels), it also resulted in significant increases in growth factor and protease expression indicating that the high glucose conditions may actually be stimulating cells in vitro in opposition to the behavior of diabetic cells in vivo. On the other hand, the cells harvested from diabetic animals had decreased MMP-2 levels as expected, however with no change in VEGF expression. While collagen I expression was also decreased in db cells, this may be a result of the short time duration (24 hrs) of the experiment not allowing for significant ECM deposition. Therefore, our results do suggest that cardiac fibroblasts harvested from diabetic animals maintain aspects of their diabetic phenotype in culture for multiple cell passages and an extended period. However, high glucose conditions may initiate uncontrolled protein expression by fibroblasts and may not be the most appropriate in vitro diabetic model for the purposes of our study. After investigating the effect of diabetic phenotype on cardiac fibroblasts and matrix remodeling response, the effect of cyclic strain on wild type fibroblasts was explored. A number of previous studies have explored the effect of cyclic strain on fibroblasts and responses associated with strain. For example, cyclic strain led to decreased proliferation of 2-D cultured ligament fibroblasts (139) and cardiac fibroblasts (38), although increased proliferation was 81

92 observed in 3-D seeded human fibroblasts (39, 40). Studies demonstrate increased collagen I gene expression (35-38), increased collagen matrix and protein deposition (39-41), and greater collagen fibril density and proteoglycan content (141) by fibroblasts exposed to cyclic strain. Additionally, growth factor production and expression by fibroblasts is affected by cyclic stretch as well, with increases observed in insulin growth factor-1 (IGF-1) (42, 43), vascular endothelial growth factor (VEGF) (44), and transforming growth factor beta (TGF-β) (36) levels. Mechanical strain also stimulated the activation of MMP-2, decreased TIMP-2 (142) and induced production of membrane MMP (activator of MMP-2) (45) in fibroblasts. Finally, mechanical strain results in orientation and migration parallel to applied strain by fibroblasts (174, 175) in 3-D gels. While clearly these studies demonstrate significant regulation of the matrix remodeling response by fibroblasts with exposure to cyclic strain, our study did not produce similar results. In our experimental setup, wild type cardiac fibroblasts (wt) exposed to 24 hrs of cyclic strain experienced a significant decrease in cellular proliferation as compared to static wild type controls, consistent with previous studies (38, 139). However, no significant differences in MMP-2, collagen I, or VEGF expression or cellular morphology or orientation were observed in strained wild type samples as compared to static wild type controls. This lack of response may be attributable to the short duration of strain application and/or or the magnitude of strain. Finally, our study focused on the combined effect of cyclic strain and diabetic phenotype on the matrix remodeling response by cardiac fibroblasts, a research focus area in which less is known. A recent study on the combined effect of mechanical stretch and high glucose demonstrated decreased proliferation and increased apoptosis in bovine retinal pericytes (176). While we observed no difference in cellular proliferation in either model of diabetic phenotype 82

93 in strained samples as compared to static controls, we did observe a similar significant increase in apoptosis levels in high glucose cells (wt HG) subjected to mechanical strain as compared to static controls. However, no differences in MMP-2 expression were observed in cells of diabetic phenotype subjected to mechanical strain as compared to static controls, with increased MMP-2 expression still observed in cells cultured in high glucose conditions (wt HG) and decreased expression in cells from diabetic animals (db) as compared to wild type samples. Collagen I levels were increased in db fibroblasts exposed to cyclic strain as compared to static controls, but still significantly lower than that of wt HG samples. Finally, application of cyclic strain resulted in significantly decreased VEGF levels in high glucose conditions as compared to static controls, resulting in similar levels of VEGF expression in wild type cells and both experimental groups of diabetic phenotype. Again, exposure of strain did not result in any observable difference in cellular morphology or orientation in either diabetic phenotype experimental group. One possible reason for the limited improvements observed in the matrix remodeling response by cardiac fibroblasts may have been the experimental parameters selected for in vitro application of cyclic strain. In the myocardium, fibroblasts are constantly exposed to cyclic strain, with the magnitude and frequency varying with heart rate and pressure load (33). In the healthy heart, all cardiovascular cells experience strain with every heartbeart (i.e. a frequency close to 1 Hz) (33) and the strains experienced by the cardiac wall cells are approximately 10% in magnitude (177, 178). In our application of cyclic strain, a 5% magnitude strain was applied for 24 hrs. While this strain is slightly lower than the physiological strain experienced by the cells in vivo, it allows for cell adherence throughout the duration of the experiment. However, a slightly higher strain may be more physiologically representative and produce more clear improvements in the matrix remodeling response by cardiac fibroblasts. Additionally, the 83

94 duration of the experiments (24 hrs) may not have been long enough for the effects of cyclic strain to become apparent, especially in response measures such as protein expression. Additionally, previous studies have demonstrated that increased magnitude and increased duration of strain does lead to more pronounced matrix remodeling responses (40). Therefore, while mechanical stretch may prove to be an effective strategy to compensate for the dysregulation of matrix remodeling by diabetic cardiac fibroblasts, our results do not support this observation in our particular experimental setup. Increased magnitude and duration of strain may prove to be a more efficacious approach, resulting in significantly improved matrix remodeling response by cardiac fibroblasts and the development of tissue engineering strategies for cardiac regeneration in the diabetic heart. 84

95 CHAPTER 5 Self-Assembling Peptide Nanofibers for MMP-Mediated Matrix Remodeling in Diabetic Cardiomyopathy Jennifer R. Hurley, Abdul Q. Sheikh, Daria A. Narmoneva. School of Energy, Environmental, Biological & Medical Engineering. University of Cincinnati, Cincinnati, Ohio. Submitted to Cells Tissues Organs, September ABSTRACT In the diabetic heart, increased collagen accumulation, stiffness and cardiac dysfunction may be linked to the reduced expression and activity of matrix metalloproteinase-2 (MMP-2), suggesting that diabetes-associated cardiac fibrosis may be attenuated through stimulation of native MMP-2 expression or delivery of exogenous MMP-2. Peptide nanofibers were investigated as a microenvironment for endogenous MMP-2 stimulation or exogenous MMP-2 delivery to promote matrix remodeling by wild type and diabetic cardiac fibroblasts. Cells were isolated from wild type or diabetic rat hearts and embedded in nanofibers, nanofibers with exogenous MMP-2, and Matrigel controls for 1, 6 and 14 days. Responses associated with matrix remodeling were assessed, including cell survival, native MMP-2 expression, ECM deposition and construct stiffness. The results demonstrate that nanofiber scaffolds provide an effective delivery vehicle with gradual MMP-2 release, while supporting long term survival and temporal matrix remodeling by cardiac fibroblasts. Nanofiber scaffolds maintained the balance between cell proliferation and apoptosis, in contrast to increased apoptosis with time in culture in 85

96 Matrigel. Diabetic and wild type fibroblasts showed different temporal trends for MMP-2 expression, collagen I deposition and scaffold stiffness, indicating increased matrix remodeling by diabetic cells in the nanofiber microenvironment. The data suggest that stimulation of native MMP expression by the nanofibers alone may be the more suitable strategy to improve reparative matrix remodeling. Overall, the results suggest that peptide nanofibers may be uniquely suited to increase local MMP-2 concentration in the diabetic heart and may be promising for applications focused on therapeutic matrix remodeling and cardiac regeneration. INTRODUCTION Diabetes is one of the most common chronic illnesses in the world and 25.8 million individuals 8.3% of the population are afflicted in the United States alone with annual medical costs estimated at $174 billion in 2007 (58). While there are many life-threatening complications associated with diabetes, heart disease is one of the most severe with a diabetesassociated death rate 2 to 4 times higher than normal (60, 61). Diabetic cardiomyopathy (DCM) is a diabetes-associated cardiovascular condition defined as ventricular dysfunction in the absence of other etiological factors, such as hypertension or coronary heart disease (20-23), which results in pathological alterations to the myocardium including circulatory defects, impaired heart muscle contraction, and progressive fibrosis. The elusive and poorly defined nature of DCM indicates that there exists a need for novel treatment approaches which focus on alternative molecular mechanisms for the disease. Extracellular matrix (ECM) turnover and remodeling are essential in many physiological processes yet their regulation is impaired in DCM, leading to damaging structural, geometric and functional changes in the heart (11, 12, 20, 22). ECM turnover can be regulated by many factors, 86

97 including matrix metalloproteinases (MMPs) (12), angiotensin II (62, 63, 179), aldosterone (21), TGF-β1 (26), nitric oxide (65), advanced glycation end products (11, 66), and kinins (67, 68). Importantly, recent studies of diabetic human patients ( ) and in animal models (24-27, 62, 131, 132) suggest that MMP activity is impaired in diabetes, thus highlighting this particular mechanism as a novel therapeutic target. In particular, studies such as these have shown that dysregulation of cardiac MMP-2 expression contributes to the increased collagen deposition, progressive fibrosis, increased ventricular stiffness, and cardiac dysfunction seen in diabetic cardiomyopathy in rodent animal models. This MMP-2 deficiency likely stems from diabetesrelated changes to the cardiac fibroblast phenotype, such as increased collagen synthesis in cardiac fibroblasts under diabetic conditions (29-31). At the same time, studies in murine models of type II (db/db) diabetes have shown decreased MMP activation in diabetic fibroblasts, as well as impairments in vital cellular processes, including reduced growth factor expression and reduced cellular migration (28). Therefore, overcoming the inhibitory effects of diabetic conditions on matrix remodeling by cardiac fibroblasts through stimulation of native MMP-2 expression or delivery of exogenous MMP-2 represents a novel target for therapeutic treatment of DCM. Peptide nanofibers, such as RAD16-II, represent a scaffold-based tissue engineering approach which can be effectively used for local and controlled delivery of proteins to the myocardium (102, 111, 112, 114). RAD16-II nanofibers are made via the spontaneous assembly of self-complementary oligopeptides consisting of alternating hydrophilic and hydrophobic amino acids (100). The material and biochemical properties of the nanofibers can be easily tailored via sequence alterations and peptide concentration. RAD16-II and similar nanofibers have been extensively studied in vitro for controlled protein delivery (102, 113, ). 87

98 Additionally, these nanofibers have been used in vivo for temporally-controlled and localized cardiac delivery of proteins, including growth factors IGF-1 (111, 114), PDGF-BB (102), and SDF-1 (112). Importantly, nanofiber-based scaffolds represent a potentially very attractive proteolytically-stable microenvironment suitable for MMP delivery, in contrast to native ECMbased materials which are rapidly degraded by the proteases expressed by the cells or present in the extracellular environment. Our previous studies have demonstrated that RAD16-II peptide nanofibers significantly enhance native expression of MMP-2 by human dermal fibroblasts as compared with collagen I controls, while maintaining long-term cell survival and scaffold stability (16, 180). The goal of this study was to determine if self-assembling peptide nanofibers could be used to create a proteolytically stable extracellular microenvironment for long-term MMP delivery and enhancement of cardiac remodeling. This study tested the hypothesis that increased MMP-2 concentration, either native or exogenous, in the nanofiber microenvironment would promote matrix remodeling by diabetic cardiac fibroblasts in vitro. Our results suggest that a nanofiber-based approach may be a promising cardiac tissue engineering strategy to stimulate reparative matrix remodeling in the diabetic heart. MATERIALS AND METHODS MMP-2/NF delivery system. To quantify protein release in vitro from peptide nanofibers (NFs), active human MMP-2 was incorporated into the NFs. Human MMP-2 was selected in order to distinguish it from the native rat MMP-2 expressed by fibroblasts in subsequent experiments. Active human MMP-2 (EMD Chemicals, Gibbstown, NJ) was incorporated into nanofibers (RAD16-II, (RARADADA) 2, 1.0% w/w, SynBioSci, Livermore, CA) via non-covalent binding 88

99 at 100 ng/ml. This concentration was selected as it is falls well below the peptide nanofiber protein binding capacity (~1.0 ng protein/μg peptide (102)) and is similar to normal cardiac MMP-2 levels (181, 182). Nanofibers without MMP-2 and Matrigel (BD Biosciences, Bedford, MA) served as controls. 75 µl of scaffold solution was added to culture inserts and incubated in phosphate-buffered saline (PBS) at 37ºC to form three-dimensional scaffolds. Supernatant (PBS after incubation) was collected and replaced at 0.5, 1, 2, 4, 6, 12, 24, 36, 48, and 72 hrs. Supernatant samples were tested using Human MMP-2 Quantikine ELISA Kit (R&D Systems, Minneapolis, MN) to determine MMP-2 release kinetics from NFs. Cell isolation and culture. All animal procedures were performed using protocols approved by the University of Cincinnati Institutional Animal Care and Use Committee. Diabetic (db) and wild type (wt) cardiac fibroblasts were isolated from week old female Sprague Dawley rats (SAS SD Strain 400, Charles River, Wilmington, MA). Type I diabetes was induced in 8 week old rats using a single intraperitoneal injection of streptozotocin (70 mg/kg, Sigma) (23-27, 67, 170). Immediate onset of diabetes was confirmed with serum glucose levels >450mg/dl and diabetic animals were sacrificed 6 weeks after injection. For cell isolation, hearts were harvested and washed in cold saline several times, finely chopped and digested first in trypsin (1 mg/ml, Sigma-Aldrich, St. Louis, MO) and then collagenase I (172 U/ml, Worthington Biochemical Corp., Lakewood, NJ). Supernatant containing cells was separated from remaining tissue using a 70 μm disposable cell strainer (Becton Dickinson Labware, Bedford, MA) and centrifuged. The cell pellet was resuspended and added to uncoated cell culture dishes for 2 hours to allow selective adhesion of fibroblasts. Dishes were washed and attached cells were cultured in Medium 199 (HyClone, Logan, UT) containing 10% fetal bovine serum (FBS; Atlanta Biologicals, Lawrenceville, GA), 1% Antibiotic-Antimycotic (Atlanta Biologicals, 89

100 Lawrenceville, GA), 10 µg/ml heparin (Sigma-Aldrich), and 0.2 ng/ml growth supplement (Sigma-Aldrich) as previously described (14, 16). Cell cultures were maintained at 37ºC in 100% humidified air containing 5% CO 2. Cells of passage 4-10 were used in all experiments. Sample preparation. To quantify the effects of peptide nanofibers (NFs) on native MMP release and matrix remodeling by wild type (wt) and diabetic (db) cardiac fibroblasts, a total of six experimental groups were created: NF, NF+MMP, Matrigel, each with wt or db cells. Active human MMP-2 was incorporated into the NFs for exogenous protein delivery in order to distinguish it from the native rat MMP-2 expressed by the cells. Matrigel was selected as a control three-dimensional microenvironment as it does not induce MMP-2 expression and activation in vitro (183, 184). Diabetic and wild type fibroblasts (passages 4-10) were three-dimensionally embedded in nanofibers (NF, RAD16-II, (RARADADA) 2, 1.0% w/w, SynBioSci), NFs ng/ml active human MMP-2 (NF+MMP, EMD Chemicals), and Matrigel (BD Biosciences) at a density of 2.5x10 6 cells/ml (protein expression, rheometry, and cell proliferation) or surface seeded at 1.0x10 4 cells/insert (cell apoptosis). Scaffolds without cells served as controls for protein expression and rheometry experiments. Samples were cultured for 1, 6 and 14 days with daily media changes. For all ELISA experiments, cell culture medium (M199 with 10% FBS, 1% Antibiotic-Antimycotic, and 10 µg/ml heparin) without growth supplement was used with daily medium changes. Cell phenotype. Staining was performed to confirm fibroblast phenotype of the primary cells and for visualization of cells within the three-dimensional scaffolds. Wild type and diabetic fibroblasts were either cultured in gelatin-coated wells in 24-well tissue culture plates until near confluency or embedded in NF, NF+MMP and Matrigel scaffolds at a density of 2.5x

101 cells/ml and cultured with daily media changes for 1, 6 and 14 days. Samples were fixed with methanol/acetone. Samples were stained with vimentin (a fibroblast marker (93, 185, 186)) and α-smooth muscle actin (α-sma, a marker of myofibroblasts (92, 187)) primary antibodies (Sigma-Aldrich) and appropriate fluorescent secondary antibodies (Alexa Fluor, Invitrogen Corp., Carlsbad, CA) and DAPI (Invitrogen Corporation). Imaging of stained cells was performed with an inverted fluorescent microscope (Olympus IX81; Olympus America Inc., Center Valley, PA) and positive stained cells were counted (3 images/sample). Cell apotosis. LIVE/DEAD Kit (Molecular Probes, Carlsbad, CA) was used to assess cell apoptosis at 1, 6 and 14 days in culture. The kit was used per manufacturer instructions with 30 minute incubation with Calcein AM and Ethidium homodimer-1 followed by imaging of stained cells with an inverted fluorescent microscope (Olympus IX81). Total numbers of live and dead cells were counted (3 images/sample) to determine percentage of apoptotic cells. Cell proliferation. CellTiter 96 Aqueous non-radioactive cell proliferation assay (Promega Corporation, Madison, WI) was used to assess cell proliferation at 1, 6, and 14 days in culture. Cells were embedded in scaffolds at a density of 2.5x10 6 cells/ml and cultured with daily medium changes. At each time point, samples were incubated in medium containing MTS/PMS solution for 3 hours per manufacturer instructions. Media samples from culture inserts were placed in a 96-well plate and absorbance was measured at 490 nm using an ELISA plate reader. All data were normalized to day 1 values for analyses. After testing, MTS/PMS medium was aspirated and fresh medium was added to the samples. Sample total protein content determination. Cell-scaffold constructs were cultured in no growth factor medium (cell culture medium without additional growth factor supplementation) and collected at days 1, 6 and 14. Samples were stored in TriReagent (Molecular Research Center, 91

102 Cincinnati, OH) at -80ºC until testing. Protein isolation was performed per the manufacturer s protocol. Total protein content in the samples was determined using Coomassie Plus Assay Kit (Thermo Fisher Scientific, Rockford, IL). Protein expression using Enzyme-Linked Immunosorbent Assay (ELISA). Cell-scaffold constructs were cultured in no growth factor medium (cell culture medium without additional growth factor supplementation) and media and matrix samples were collected at days 1, 6 and 14 and stored at -80ºC until testing. ELISAs were performed as described in (160) to determine protein concentrations in medium samples (Rat MMP-2 - antibodies from R&D Systems) and matrix samples (Rat MMP-2, Rat Collagen I antibodies from Pierce Biotechnology, Rockford, IL, and Rat Collagen IV antibodies from Abcam, Cambridge, MA). Protein expression in matrix samples was normalized using total protein content. For all ELISAs, additional controls of the cell culture medium alone (containing 10% serum) and non-cellular scaffolds were included to confirm that protein content in both the serum and scaffold would not affect protein expression and detection, with no differences observed between the control samples and the 0 pg/ml standard. Mechanical testing of cell-scaffold constructs using rheometry. Elastic moduli (G ) of NF, NF+MMP and Matrigel with living cells or scaffolds alone (controls) at 1, 6, and 14 days were measured with a parallel-plate rheometer (Bohlin Instruments Inc., East Brunswick, NJ) as described previously (16). Briefly, circular constructs of 8 mm diameter and approximately 500 µm height were formed on glass slides using molds, covered with cell culture medium and cultured within an incubator with daily medium changes. For testing, glass slides were transferred and secured to the bottom plate of the rheometer and the top parallel plate was lowered to a gap height which ensured complete contact with the sample. A constant strain 92

103 amplitude (γ=0.01) frequency sweep (f= Hz) was applied, with the measured elastic modulus (G ) serving as an indicator of overall cell-seeded construct stiffness. Moduli values measured at 0.1 Hz are reported in the text. Statistical analyses. The results are reported as average ± standard deviation. For each experiment, the sample size was four, and all experiments were repeated twice (minimum n=8). Multi-factor ANOVA and post-hoc tests were used to determine the effects of scaffold type (NF, NF+MMP, or Matrigel), diabetic condition (db or wt), and culture time (1, 6, or 14 days) on matrix remodeling (MMP-2, collagen I and IV expression), cell proliferation and apoptosis, and mechanical stiffness. All tests were run at a significance level of α = RESULTS Peptide nanofibers provide a stable microenvironment for controlled released of MMP-2. ELISA was used to measure the release kinetics of human MMP-2 from RAD16-II nanofibers (Figure 18). Results showed a higher immediate release initially at 0.5 hr (4.6±2.0 % of total MMP-2), 1 hr (2.5±2.3 % of total MMP-2) and 2 hrs (1.1±0.5 % of total MMP-2). After this initial burst, release slowed and remained relatively constant around 1% of total MMP-2 incorporated even at the time between supernatant collections increased. By 72 hrs, the total cumulative release was approximately 16% of the total MMP-2 incorporated initially into the nanofiber scaffold. This indicates successful MMP-2 incorporation into the NFs which allows for sustained protein release. 93

104 Figure 18. MMP-2 release kinetics from peptide nanofibers. Active human MMP-2 was incorporated into NFs at a concentration of 100 ng/ml and release kinetics were measured by performing ELISA on supernatant samples at 0.5, 1, 2, 4, 6, 12, 24, 36, 48, and 72 hours. There was a higher immediate release initially at 0.5 hr, 1 and 2 hrs. After this initial burst, release slowed and remained relatively constant. By 72 hrs, the total cumulative release was approximately 16% of the total MMP-2 incorporated initially into the nanofiber scaffold. Peptide nanofibers provide a stable microenvironment, supporting long term cardiac fibroblast survival. Staining of wt and db cells both plated and embedded in NF, NF+MMP and Matrigel was performed to confirm fibroblast phenotype and morphology within the scaffolds (Figure 19). All cells were positive for vimentin, an intermediate filament associated protein expressed by 94

105 fibroblasts (93, 185, 186). Additionally, approximately 70-80% of cells were also positive for α- SMA, an intermediate filament associated protein expressed by myofibroblasts (92, 187). These observations are consistent with previous studies, where primary fibroblasts expressed a myofibroblast phenotype in culture, characterized by increased α-sma expression ( ). This activated fibroblast phenotype is critical in wound healing and results in a more active matrix remodeling response by the fibroblasts in the nanofiber scaffold, which is important for the results of this study. Figure 19. Staining for fibroblast phenotype. Cells were stained with antibodies for fibroblast marker vimentin (red) and myofibroblast marker α-smooth muscle actin (α- SMA, green) and DAPI (blue). The top panels are wild type cells and the bottom panels are diabetic cells. Cells were either plated on gelatin coated dishes (left panels) or embedded in NF, NF+MMP, and Matrigel scaffolds (higher magnification panels, from left to right). All cells were positive for fibroblast marker vimentin and approximately 70-80% 95

106 of cells were also positive for myofibroblast marker α-sma. Scale bar in left panels is 100 μm. Scale bar in higher magnification panels is 25 μm. To determine if the peptide microenvironment supports long-term cardiac fibroblast survival and assess cell proliferation, LIVE/DEAD and MTS-based cell proliferation assays were performed on cells embedded in NF, NF+MMP and Matrigel and cultured up to 14 days. Results from the LIVE/DEAD assay demonstrated that wild type and diabetic cell apoptosis levels (Figure 20) were less than 15% in NF, NF+MMP and Matrigel scaffolds. The number of apoptotic diabetic cells at day 1 was significantly higher than that in wild type cells in Matrigel scaffolds (p<0.05), with a similar trend observed between db and wt cells cultured in NF-based scaffolds. The results for later time points (days 6 and 14) demonstrate that this trend was actually reversed, with higher levels of apoptosis in the wt cells as compared to db cells (p<0.05 for NF+MMP and Matrigel scaffolds). Interestingly, this increase in apoptosis at the later time points seemed to be compensated for by increases in cell proliferation in the NF-based scaffolds, but not in Matrigel, with the total number of viable cells remaining at 95% or greater of the 125,000 total cells initially embedded within NF and NF+MMP scaffolds, with no significant differences observed between wild type and diabetic fibroblasts. Cell number (Figure 21) increased in both NF and NF+MMP scaffolds from day 1 to day 6 in both wt (p<0.05) and db cells (p<0.05 for NF+MMP scaffold). No differences in numbers of either wild type or diabetic cells were observed with the addition of exogenous MMP-2 to the NFs. In contrast, in Matrigel cultures, the number of viable cells by day 14 was significantly decreased in both wild type and diabetic fibroblasts (p<0.05), with the cell number significantly lower than in NF and NF+MMP scaffolds (p<0.05). 96

107 Figure 20. Cell apoptosis levels in cell-scaffold cultures. Cell apoptosis was measured using LIVE/DEAD staining. a) The percentage of apoptotic wild type (wt) fibroblasts was less than 15% for in NF, NF+MMP and Matrigel scaffolds at days 1, 6 and 14. While no difference was observed in apoptosis in NF scaffold, there was a significant increase in apoptosis in both NF+MMP and Matrigel from day 1 to days 6 and 14. b) The percentage of apoptotic diabetic (db) fibroblasts was less than 15% for in NF, NF+MMP and Matrigel scaffolds at days 1, 6 and 14. Initial (day 1) apoptosis levels in db fibroblasts were significantly higher and decreased at day 6 in all scaffolds. * p<0.05 when compared to NF-based samples, + p<0.05 when compared to NF samples, ^ p<0.05 when compared to day 1 samples of same experimental group, # p<0.05 when compared to wt samples of same experimental groups. 97

108 Figure 21. Cell proliferation levels in cell-scaffold cultures. Cell viability was measured using MTS-based proliferation assay at days 1, 6, and 14. a) The number of viable wild type (wt) cells in culture remained at 95% or greater of the 125,000 total cells initially embedded within NF and NF+MMP scaffolds. This was significantly higher than Matrigel cultures, where the number of viable cells by day 14 was less than 70% of initial cells numbers. b) The number of viable diabetic (db) cells in culture remained at 95% or greater of the 125,000 total cells initially embedded within NF and NF+MMP scaffolds. This was significantly higher than Matrigel cultures, where the number of viable cells by day 14 was at 82% of initial cells numbers. All data were normalized to day 1 values for analyses. * p<0.05 when compared to NF-based samples, + p<0.05 when compared to NF samples, ^ p<0.05 when compared to day 1 samples of same experimental group, # p<0.05 when compared to wt samples of same experimental groups. Overall, these results indicate a balance between cell proliferation and apoptosis in the NF and NF+MMP scaffolds, in contrast to the shift toward more apoptosis with time in culture in Matrigel. Therefore, these results suggest an overall stability of the peptide nanofiber 98

109 microenvironment both with and without added MMP-2, which supports long term cell survival in both wild type and diabetic fibroblast in vitro. Expression of native MMP-2 by cardiac fibroblasts is increased in the peptide nanofiber microenvironment. Native rat MMP-2 concentration expressed by cardiac fibroblasts was measured in both the media and matrix using a rat MMP-2 ELISA (Figure 22). In media samples (Figure 22a-b), there was no difference in MMP-2 expression levels by wt cells between different time points in NF-based scaffolds. However, MMP-2 expression by db cells in these scaffolds was lower at day 1 (p<0.05 vs. wt NF and NF+MMP) and subsequently increased in the NF group (p<0.05 vs. day 1). No differences in native MMP-2 media expression by wild type cells was observed as a result of the addition of exogenous MMP-2 to the NFs, although in diabetic cells a significant increase at day 1 was observed between NF and NF+MMP scaffolds (p<0.05). In contrast to the MMP-2 levels observed in NF-based samples, MMP-2 levels in Matrigel samples were lowest at day 1 in both db and wt cells and increased with time (p<0.05 vs. day 1). Additionally, MMP-2 expression by wt cells in Matrigel scaffolds at day 1 was significantly less than NF-based scaffolds (p<0.05). The amount of MMP-2 bound to the NF and NF+MMP matrix (Figure 22cd) showed an opposite trend from that in the media, with the highest MMP-2 concentration at day 1, which significantly decreased by day 6 in both wild type and diabetic fibroblasts (p<0.05). Additionally, matrix-bound MMP-2 levels by both wt and db fibroblasts were significantly higher in both NF and NF+MMP scaffolds than in Matrigel at each time point (p<0.05). No differences in native MMP-2 levels in the matrix by wild type or diabetic fibroblasts were observed as a result of the addition of exogenous MMP-2 to the NFs. 99

110 Figure 22. Native protein expression of matrix metalloproteinase -2. ELISA was performed on samples collected from cell-scaffold constructs at days 1, 6 and 14 to determine concentration of native rat MMP-2 in the media (panels a and b) and matrixbound (panels c and d). a) Native MMP-2 protein expression (ng MMP-2/ml) by wild type (wt) fibroblasts in NF, NF+MMP and Matrigel scaffolds as measured in media samples. MMP-2 expression was increased at day 1 in NF as compared to Matrigel, with no effect of exogenous MMP-2 (NF+MMP). b) Native MMP-2 protein expression (ng MMP-2/ml) by diabetic (db) fibroblasts in NF, NF+MMP and Matrigel scaffolds as measured in media samples. A temporal increase in MMP-2 expression was observed in NF scaffolds. c) Native MMP-2 protein expression (pg MMP-2/ug total protein) by wild type (wt) fibroblasts in NF, NF+MMP and Matrigel scaffolds as measured in matrix samples. MMP- 100

111 2 expression was increased in NF-based scaffolds as compared to Matrigel, with no effect of exogenous MMP-2 (NF+MMP). A temporal decrease was observed in MMP-2 expression in NF scaffolds. d) Native MMP-2 protein expression (pg MMP-2/ug total protein) by diabetic (db) fibroblasts in NF, NF+MMP and Matrigel scaffolds as measured in matrix samples. MMP-2 expression was again increased in NF-based scaffolds as compared to Matrigel, with no effect of exogenous MMP-2 (NF+MMP). A temporal decrease was again observed in MMP-2 expression in NF scaffolds. * p<0.05 when compared to NF-based samples, + p<0.05 when compared to NF samples, ^ p<0.05 when compared to day 1 samples of same experimental group, # p<0.05 when compared to wt samples of same experimental groups. Native extracellular matrix deposition by cardiac fibroblasts is supported by peptide nanofiber microenvironment. Collagen I (col I) and collagen IV (col IV) ELISAs on matrix samples were performed to determine the concentration of native ECM deposited by wild type and diabetic fibroblasts. Due to the fact that the Matrigel scaffold itself contains a significant amount of col I and IV, no comparison with NF and NF+MMP groups were made. There were no differences seen in collagen I deposition by wild type cells at any time point in NF and NF+MMP scaffolds (Figure 23). Additionally, no significant differences in collagen I deposition by wild type fibroblasts were observed as a result of the addition of exogenous MMP-2 to the NFs. However, in diabetic cells, there was a significant temporal decrease in collagen I levels in NF scaffolds (p<0.05), where collagen I levels were significantly lower at day 14 as compared to NF+MMP scaffolds (p<0.05). 101

112 Figure 23. Collagen I deposition. ELISA was performed on cell-scaffold matrix samples for wild type fibroblasts (panel a) and diabetic fibroblasts (panel b) at days 1, 6, and 14 to determine protein levels of collagen I (col I). a) Collagen I concentration (pg col I/ug total protein) for wild type (wt) fibroblasts in NF and NF+MMP scaffolds at days 1, 6 and 14. Steady collagen I levels were observed in NF, with no significant effect of exogenous MMP- 2 (NF+MMP). b) Collagen I concentration (pg col I/ug total protein) for diabetic (db) fibroblasts in NF and NF+MMP scaffolds at days 1, 6 and 14. A significant decrease in collagen I levels was observed in NF by day 14 consistent with ECM remodeling. The addition of exogenous MMP-2 (NF+MMP) resulted in significantly higher collagen I levels at day 14 as compared to NF. Expression levels were normalized using total protein content as measured using Bradford assay. + p<0.05 when compared to NF samples, ^ p<0.05 when compared to day 1 samples of same experimental group, # p<0.05 when compared to wt samples of same experimental groups. Interestingly, both wt and db cells demonstrated a temporal decrease in collagen IV levels (Figure 24), similar to that observed in collagen I levels in diabetic cells only. This trend was 102

113 significant in wild type cells, where deposition of col IV was significantly higher in NF-based scaffolds initially at day 1 than later time points (p<0.05). There was significantly more collagen IV deposited by wild type cells at day 1 in NF than in NF+MMP scaffolds (p<0.05). Figure 24. Collagen IV deposition. ELISA was performed on cell-scaffold matrix samples for wild type fibroblasts (panel a) and diabetic fibroblasts (panel b) at days 1, 6, and 14 to determine protein levels of collagen IV (col IV). a) Collagen IV concentration (pg col IV/ug total protein) for wild type (wt) fibroblasts in NF and NF+MMP scaffolds at days 1, 6 and 14. Collagen IV levels at day 1 were higher in NF than in NF+MMP scaffolds and decreased with time, indicating ECM remodeling. b) Collagen IV concentration (pg col IV/ug total protein) for diabetic (db) fibroblasts in NF and NF+MMP scaffolds at days 1, 6 and 14. Collagen IV levels were highest at day 1 in NF scaffolds, with no effect of exogenous MMP-2 (NF+MMP). Expression levels were normalized using total protein content as measured using Bradford assay. + p<0.05 when compared to NF samples, ^ p<0.05 when compared to day 1 samples of same experimental group, # p<0.05 when compared to wt samples of same experimental groups. 103

114 The extracellular mechanical environment is regulated by matrix remodeling by cardiac fibroblasts. A parallel-plate rheometer was used to measure the mechanical properties of cell-scaffold constructs at days 1, 6, and 14, with elastic modulus (G ) serving as an indicator of construct stiffness (Figure 25). Previous studies indicate that overall cell-scaffold stiffness is regulated by a combination of extracellular matrix deposition, remodeling and scaffold disruption due to cell migration (16). Scaffold-only controls at day 1 served as a baseline of scaffold stiffness (NF: 1.93±0.78 kpa, NF+MMP: 1.38±0.21 kpa, Matrigel: 0.09±0.03 kpa), with NF-based scaffolds significantly stiffer than Matrigel (p<0.05). For wt cells, no difference in NF scaffold stiffness was observed from day 1 to day 6. However, day 14 stiffness was significantly decreased from day 6 in NF scaffolds (p<0.05). In db cells, there was a significant increase in stiffness from day 1 to 6 (p<0.05), and again a significant decrease from day 6 to day 14 (p<0.05). In NF+MMP scaffolds, there was no difference in scaffold stiffness observed with time in either wild type or diabetic cell constructs. At all time points for both wild type and diabetic fibroblasts, both NF and NF+MMP scaffolds were significantly stiffer than Matrigel scaffolds (p<0.05), indicating the robust structural integrity of the peptide nanofibers. 104

115 Figure 25. Stiffness of cell-scaffold constructs measured using rheometry. a) Elastic moduli (G ) values in kpa for wild type (wt) fibroblasts in NF, NF+MMP and Matrigel scaffolds at days 1, 6 and 14. NF and NF+MMP scaffolds are significantly stiffer than Matrigel at all time points. Decreased stiffness was observed in NF scaffolds at day 14. No changes in stiffness were observed in NF+MMP scaffolds. b) Elastic moduli (G ) values in kpa for diabetic (db) fibroblasts in NF, NF+MMP and Matrigel scaffolds at days 1, 6 and 14. Again, NF and NF+MMP scaffolds are significantly stiffer than Matrigel at all time points. NF scaffolds exhibited a significant increase in stiffness at day 6 followed by a significant decrease at day 14. No changes in stiffness were observed in NF+MMP scaffolds. G (kpa) is reported at frequency of 0.1 Hz. * p<0.05 when compared to NFbased samples, + p<0.05 when compared to NF samples, ^ p<0.05 when compared to day 1 samples of same experimental group, $ p<0.05 when compared to day 6 samples of same experimental group, # p<0.05 when compared to wt samples of same experimental groups. DISCUSSION The results of this study demonstrate that the nanofiber microenvironment supports longterm survival and temporal in vitro matrix remodeling by cardiac fibroblasts both wild type and 105

116 diabetic (Figure 26). Early at day 1, we see increased native MMP-2 expression and ECM deposition. By day 6, MMP-2 expression is still prominent with stiffness values consistent with ECM levels. However, by day 14 we see a shift towards more active matrix remodeling, with decreased MMP-2 and ECM levels and decreased stiffness. These results are consistent with the normal wound healing response in cardiac tissue in vivo, which is by characterized initial increased MMP expression leading to net ECM degradation (79). Importantly, our data show that NF-based scaffolds support slow MMP-2 release without detrimental effects of increased MMP-2 levels (both native and exogenous) on cell viability, indicating the promise of this material for therapies which may involve MMP-mediated tissue remodeling. Interestingly, there was no apparent effect of the presence of exogenous MMP in the NF scaffold on the matrix remodeling response, with both of the NF-based scaffolds stimulating significantly improved matrix remodeling response by cardiac fibroblasts as compared to Matrigel controls. Figure 26. Proposed schematic of matrix remodeling response by cardiac fibroblasts (both wild type and diabetic) in the peptide nanofiber scaffold. Increased native MMP-2 106

117 expression and ECM deposition by cardiac fibroblasts is seen early at day 1. By day 6, MMP-2 expression is still prominent with stiffness values consistent with observed ECM levels. However, a shift towards more active matrix remodeling is seen by day 14, with decreased MMP-2 and ECM levels and decreased stiffness. RAD16-II nanofibers were investigated in this study as a biomaterial strategy to increase local MMP-2 concentration, either via stimulation of native expression or delivery of exogenous protein, in order to promote matrix remodeling by cardiac fibroblasts in vitro. While numerous biomaterials have been successfully used for various cardiac tissue engineering approaches including protein and drug delivery (116, 122, ), an RAD16-II peptide nanofiber approach was chosen based on ease of handling, proteolytic stability, and proven myocardial protein delivery capability (102, 111, 112). Importantly, RAD16-II peptide nanofibers have been shown to significantly enhance native expression of MMP-2 by human dermal fibroblasts as compared with collagen I controls (16, 180), which is particularly interesting as collagen I has been identified as a stimulus for MMP-2 activation (80, 183). The results from this study extend this prior observation to another control scaffold, with the RAD16-II nanofibers stimulating significantly higher native MMP-2 expression by cardiac fibroblasts, as compared to Matrigel controls. Furthermore, the measurement of cell-scaffold stiffness indicates that NF scaffolds retained their structural integrity and stability in the presence of high MMP-2 levels, likely due to absence of proteolytic degradation sites in the peptide sequence. Importantly, our results also demonstrate that the NF microenvironment is supportive for diabetic, as well as normal, cardiac fibroblasts, in contrast to significantly increased death for both cell types in Matrigel controls at day 14 in culture. This indicates a balance between cell proliferation and apoptosis and an overall 107

118 stability in the peptide nanofiber microenvironment which supports long term cell survival in cardiac fibroblasts in vitro, consistent with the previous studies of long-term culture of endothelial cells in this microenvironment (16). While our results demonstrate that NFs alone do increase native MMP-2 expression, we were also interested in the use of NFs as an exogenous protein delivery system. An RAD16-II scaffold-based approach for protein delivery is particularly attractive, because it allows for temporally-controlled and localized delivery, and can be achieved via either diffusion from or tethering to a scaffold, depending on protein size and binding affinity (46). Additionally, increased peptide nanofiber density resulted in decreased protein diffusion (134, 135), suggesting a straight-forward strategy for controlling protein release kinetics. Recent in vitro studies have examined the release of functional proteins and cytokines, including lysozyme, bovine serum albumin, VEGF, and bone-derived neurotrophic factor, from similar nanofiber scaffolds and have shown slow and sustained release profiles over 2 to 3 weeks, with diffusion through nanofibers dependent primarily on protein size ( ). For this study, we investigated the release kinetics of human MMP-2 from the RAD16-II nanofibers. The data showed that NF scaffolds can be effectively used as a MMP-2 delivery vehicle, with a faster initial release burst slowing to a more steady release rate and reaching a cumulative release of 16% by day 3. These results are consistent with previous studies on protein release from hydrogel scaffolds (135), with not all of the incorporated MMP-2 released in vitro due to entrapment in the highly entangled nanofiber structure. However, after delivery to the myocardium the peptide nanofibers would be subjected to intensive cell migration, particularly by highly invasive cardiac fibroblasts, leading to structural deformation and disruption of the nanofiber network (164) and increased release of the incorporated protein into the surrounding tissue. This was observed in previous studies, 108

119 where retention of IGF-1 incorporated into NFs and injected into the myocardium of rats temporally decreased up to 28 days (111). Additionally, studies have shown that RAD16-II remained present 28 days after treatment in diabetic wounds (103) and after injection into the myocardium in mice and rats (101, 111) without significant immune response, allowing for slow and sustained protein release. Interestingly however, while the RAD16-II nanofibers clearly are effective as a protein delivery vehicle, no clear significant improvement in fibroblasts matrix remodeling response was seen with incorporation of exogenous MMP-2 into NFs. In fact, our data suggest that there may be actually a cell de-activation in the presence of exogenous MMP, resulting in overall less migration and remodeling. Indeed, for diabetic fibroblasts cultured in the NF scaffolds (without addition of MMP-2), there were significant trends of increased MMP-2 expression in the media, decreased collagen I deposition and decreased scaffold stiffness by day 14, indicating increased matrix remodeling. In contrast, although a significant remodeling response was still observed in diabetic cells cultured in NF+MMP scaffolds, as compared to Matrigel, there were no observed differences in these parameters in NF+MMP scaffolds with time, suggesting a more mild cell response. Therefore, the results from this study suggest that using the NFs alone to stimulate native MMP-2 expression may ultimately be the more suitable strategy to improve reparative matrix remodeling. Additionally, the use of nanofibers alone may prove to be of a better riskbenefit value by removing the need to add further complexity to cardiac tissue engineering strategies though the addition of an exogenous agent such as MMP-2 (194). Cardiac fibroblasts were investigated in this study as they represent two-thirds of total cardiac cells and are primarily responsible for extracellular matrix deposition homoeostasis (80, 81) and produce a full complement of MMPs (12, 126). In vitro studies on cardiac fibroblast 109

120 cultures in high glucose conditions have demonstrated reduced growth factor expression, reduced cellular migration, decreased MMP activation, and increased collagen synthesis (28-31). These observations suggest that diabetic conditions may lead to an impaired matrix remodeling response by cardiac fibroblasts via a MMP-related mechanism. The results of this study suggest that this impairment may be attenuated through stimulation of native MMP-2 expression in the RAD16-II peptide nanofiber scaffold, as relatively small differences in matrix remodeling response were seen between diabetic and wild type cardiac fibroblasts. The findings of this study may also have implications for the role of cardiac fibroblasts in revascularization of fibrotic heart tissue. Our previous studies (16) elucidated the chemical and mechanical regulation of capillary morphogenesis by fibroblasts and demonstrated that the nanofiber microenvironment supports endothelial-fibroblasts angiogenic interactions in vitro. Taken together, these results suggest that the nanofiber microenvironment can promote matrix remodeling without excessive stiffening (which may be caused by cell-mediated contraction seen in other scaffolds (17, 195)), while still maintaining a pro-angiogenic environment attractive for both endothelial cells and fibroblasts, thus emphasizing the potential of this material for cardiac tissue engineering applications. In summary, the findings from our study may contribute to developing of novel therapeutic strategies for the promotion of reparative matrix remodeling and cardiac regeneration in diabetic cardiomyopathy. DCM leads to excess collagen deposition, myocardial fibrosis, and cardiac hypertrophy (11, 20). The prevention and treatment of DCM is a clinically relevant and active research focus, with studies suggesting that glycemic control is beneficial early in myocardial dysfunction (22, 61), however late diagnoses of diabetes and/or DCM may limit this preventative measure. Neurohormonal antagonism has demonstrated preserved diastolic 110

121 function in the diabetic heart in animal models (22, 62), however, it has not yet been translated to clinical studies. While strategies such as these appear promising, there still exists a need for novel approaches for treatment of diabetic cardiomyopathy which may focus on alternative molecular mechanisms for the disease, including cardiac dysregulation of MMP-2 expression and activity (24-27). In this study, we observed increased MMP-2 concentration resulting from the nanofiber microenvironment improved in vitro matrix remodeling by cardiac fibroblasts, both wild type and diabetic. Furthermore, RAD16-II and similar nanofibers are easily administered and have been locally injected into the myocardium either alone (101) or as a protein delivery vehicle (102, 111, 112, 114). Therefore, the results of this study suggest that peptide nanofibers may be a uniquely suited cardiac tissue engineering substrate to increase local MMP-2 concentration in the diabetic heart, leading to therapeutic matrix remodeling and cardiac regeneration. 111

122 CHAPTER 6 Discussion and Conclusions Our long-term research goal is to develop a new cardiac tissue engineering approach to treat cardiovascular diseases by applying recent advances in nanobiotechnology to modify the microenvironment of heart muscle and promote cardiac regeneration. The central hypothesis of the dissertation research was that RAD16-II peptide nanofibers can be used as a microenvironment for a cardiac tissue engineering approach which promotes cardiac regeneration via revascularization and reparative matrix remodeling by cardiac fibroblasts. The studies discussed in Chapters 3, 4 and 5 validate this hypothesis through the investigation of the four aims outlined in Chapter 1. In this Chapter, the results and implications of these studies will be discussed, as well as their relation to each other and future strategies for cardiac tissue engineering and regeneration. The goal of Aim 1 was to elucidate the mechanisms for fibroblast-mediated temporal regulation of angiogenesis and matrix remodeling using a comprehensive approach and the culture system of RAD16-II nanofibers. This aim tested the hypothesis that fibroblasts temporally regulate capillary morphogenesis chemically via growth factor expression and mechanically via cell-mediated scaffold disruption, ECM deposition and remodeling. This study (16) was discussed in Chapter 3 and confirmed this hypothesis. The results suggest that at the early stages of the tissue repair process, fibroblasts may play a major role in capillary morphogenesis both by paracrine growth factor signaling as well as mechanical disruption of ECM to lead the way for the formation of endothelial cell networks. At the later stages, the role of fibroblasts as regulators of the mechanical microenvironment becomes more prominent, 112

123 as endothelial-fibroblast interactions appeared to help maintain the balance in ECM homeostasis, enhancing both MMP-2 and collagen I production and resulting in improved integrity and a more stable microenvironment. While our long term goal is to develop a cardiac tissue engineering approach to promote cardiac regeneration, in order to develop novel strategies to promote vascularization and remodeling in damaged myocardial tissue, it is necessary to first clearly understand the interactions between major cardiac cell types. Therefore, this in vitro study provided important and needed insight into the interactions between endothelial cells and fibroblasts and temporal role that fibroblasts play in capillary morphogenesis and matrix remodeling. This allows us to make educated inferences regarding cell-cell interactions in healing cardiac tissue in vivo and helps to design the most effective strategies for cardiac regeneration. Clearly, the findings of this study emphasize the importance of developing an optimal microenvironment which supports multiple cell types, cell-cell interactions and is permissive for cell migration for cardiac tissue engineering applications. Additionally, while this study used RAD16-II peptide nanofibers as a controlled microenvironment for the study of cell-cell interactions, it emphasized the potential use of peptide nanofibers as a biomaterial for cardiac tissue engineering strategies. Our study was in agreement with previous studies which have shown that synthetic RAD16-II peptide nanofibers provide a pro-angiogenic microenvironment, enhance capillary-like network formation in vitro without the addition of external growth factors and allow for long term study of cell-cell interactions (14-16). Therefore, while not the primary focus of the study, it was still demonstrated that peptide nanofibers provide an angiogenic environment which supports 113

124 multiple cardiac cell types and may be an appropriate biomaterial scaffold for use in future development of cardiac tissue engineering approaches. Finally, while this study focused on the interactions between wild type endothelial cells and fibroblasts, it also provides interesting insight into the clinical focus of the later aims, that of diabetes. Multiple studies have focused on effects of diabetes on keratinocytes, endothelial cells, fibroblasts, macrophages and smooth muscle cells (81, 172, 196, 197). Deficiencies in angiogenic responses have been observed, including decreased VEGF production, proliferation, migration and tubulogenesis (81, 173). The author of this dissertation is authoring a paper entitled Pro-angiogenic peptide nanofiber microenvironment restores angiogenic potential of diabetic endothelial cells (Hurley et al, in preparation) which was not included in this manuscript. The results from this study demonstrate that diabetic endothelial cells exhibit deficiencies in their angiogenic potential both in vitro and in vivo. However, this study also shows that peptide nanofibers have the potential to correct for these deficiencies, via increased cell proliferation, VEGF production, and capillary morphogenesis. This suggests that peptide nanofibers may be able to augment diabetic deficiencies in other cell types, including fibroblasts. Therefore, the results of Aim 1 and this study together suggests that co-culture of diabetic endothelial cells and diabetic fibroblasts in the peptide nanofiber microenvironment may enhance the angiogenic potential and provide important insight into cell-cell interactions in the diabetic heart that are essential for developing effective cardiac tissue engineering strategies for diabetic cardiac pathologies. The goal of Aim 2 was to determine the effects of matrix stiffness on cell-cell interactions and mechanical regulation of the microenvironment. This aim tested the hypothesis 114

125 that a more compliant nanofiber matrix would promote in vitro network formation and cell migration. Two concentrations of peptide nanofibers were investigated: stiff nanofibers (1.0%) to better represent the fibrotic microenvironment of the healing heart, and more compliant nanofibers (0.6%). It should be noted that while these peptide concentrations investigated were significantly less stiff than cardiac muscle ( kpa (19)), they do have considerable structural integrity for a hydrogel scaffold and represent a promising biomaterial for cardiac tissue engineering applications. The results of this study (16) showed that the compliant nanofibers allowed for coordinated migration of endothelial cells and fibroblasts as well as scaffold disruption. In contrast, in the stiff nanofibers, only the highly migratory fibroblasts appeared able to easily move through the scaffold, resulting in lower stiffness in the fibroblast constructs. Additionally, unpublished data from the author demonstrates that the more compliant nanofibers maintain this permissive environment up to day 14, with increased stiffness levels in all experimental groups indicating native ECM deposition by both endothelial cells and fibroblasts. In contrast, at day 14 the stiffer nanofibers exhibited decreased stiffness in the cocultures, again indicating the role of fibroblast migration in a stiffer scaffold. These results are in agreement with previously reported enhanced capillary morphogenesis in more compliant RAD16-II and other scaffolds (17, 18), and confirm that in the peptide nanofiber culture system matrix stiffness does affect cell behavior, with fibroblasts playing a more significant role in mechanical regulation in denser nanofibers. These results suggest that more compliant nanofibers better support migration of both endothelial cells and fibroblasts, allowing for a better balance between cell migration and ECM remodeling and may ultimately be better suited for improved angiogenesis and long term stability. This further contributes to the conclusion from Aim 1 that a successful cardiac tissue 115

126 engineering microenvironment needs to support cell-cell interactions and be permissive for cell migration. Together, the results from Aim 1 and 2 contribute important design parameters to consider in future development of cardiac tissue engineering approaches. Additionally, this emphasis on design parameters is important to take into consideration when designing microenvironments which need to be multifunctional. Studies demonstrate that a straight-forward strategy for controlling protein release kinetics is increasing peptide nanofiber density, resulting in decreased protein diffusion (134, 135). While this is clearly a simple method for controlling protein release from nanofibers, the results from Aim 2 indicate that a stiffer scaffold may not be as permissible for all cardiac cell types if the goal is also cell delivery or in vivo cell infiltration. Therefore, it is important to consider all potential applications and needs as well as advantages and disadvantages when designing the optimal biomaterial scaffold for cardiac regeneration. The goal of Aim 3 was to determine the effect of diabetes and mechanical strain on the reparative matrix remodeling response of cardiac fibroblasts in vitro. This aim tested two hypotheses: 1) diabetes will result in a diminished reparative matrix remodeling response by cardiac fibroblasts in vitro, with decreased MMP expression and increased extracellular matrix deposition, and 2) stimulation of cardiac fibroblasts with mechanical strain will result in enhanced matrix remodeling response and attenuate diabetes-induced phenotypic cell alterations. The results from this study were not in agreement with the hypotheses. Neither in vitro model of diabetic fibroblast phenotype resulted in complete diminished matrix remodeling response, with cells cultured in high glucose exhibiting only increased ECM deposition and cells harvested from diabetic animals exhibiting only decreased MMP expression. Additionally, the results also 116

127 demonstrated that application of cyclic strain results in limited improvements in matrix remodeling response, both by wild type fibroblasts and those of diabetic phenotype. Therefore, while mechanical stretch may prove to be an effective strategy to compensate for the dysregulation of matrix remodeling by diabetic cardiac fibroblasts, the results of Aim 3 do not support this observation in our particular experimental setup. Improved stimulation parameters such as increased magnitude and duration of strain may prove to be a more efficacious approach, resulting in an improved matrix remodeling response by cardiac fibroblasts. Importantly, Aim 3 investigates the matrix remodeling response of both wild type and diabetic fibroblasts in response to strain, and may therefore have potential therapeutic implications for many different cardiac pathologies, including coronary heart disease, infarction and diabetic cardiomyopathy. The results of Aim 3 are also important to take into consideration when developing potential strategies for in vitro development of engineered cardiac tissue. Studies have shown that engineered constructs exposed to electric field stimulation or mechanical stretch exhibit electromechanical properties approaching those in the native heart (53, 137, 138). Therefore exposure to environmental stimuli which occur in the healthy myocardium, such as mechanical stretch and electrical stimulation, may prove to be an effective approach to stimulate cardiac regeneration in vitro. These results from Aim 3 can be extended to the stimulation of more physiological environment of three-dimensional engineered tissue, with determination of appropriate stimulation parameters in a two-dimensional setting providing a possible basis for determination of stimulation parameters for tissue engineered grafts and ultimately for in vivo transplantation and incorporation. 117

128 The goal of Aim 4 was to quantify the effect of the nanofiber microenvironment on MMP2-mediated extracellular matrix remodeling by normal and diabetic cardiac fibroblasts in vitro. The results from this study helped to validate two of our hypotheses: 1) the peptide nanofiber microenvironment will attenuate matrix remodeling-related deficiencies observed in diabetic cardiac fibroblasts, and 2) the peptide nanofiber microenvironment will promote native MMP-2 expression and improve the matrix remodeling response by cardiac fibroblasts in vitro. The results of this study demonstrate that nanofibers support MMP-mediated temporal in vitro matrix remodeling by cardiac fibroblasts both wild type and diabetic. Importantly, no significant differences were observed between wild type and diabetic fibroblast remodeling response, indicating that the NF environment may attenuate diabetes-induced phenotypic cell alterations (28). Interestingly, while the results of this study support the hypothesis that peptide nanofiber delivery of exogenous MMP-2 would promote in vitro matrix remodeling by cardiac fibroblasts, it did not demonstrate that the effect was additive to that of nanofibers alone. While the RAD16- II nanofibers are an effective protein delivery vehicle, no clear improvement in fibroblast matrix remodeling response was seen by incorporation of exogenous MMP-2 into NFs. One potential explanation for this limited improvement may be the short half-life of MMP-2, particularly in an in vitro setting. A previous study demonstrates that approximately 50-60% of MMP-2 remains active at 20 hours at 37 C (198). This roughly correlates to our day 1 time point, and may explain the limited effect of exogenous MMP-2 delivery on matrix remodeling responses in our in vitro setting, as the protein does not remain present to enzymatically degrade the extracellular matrix and enhance the remodeling contributions by the resident fibroblasts. Therefore, the results from this in vitro study suggest that using the NFs alone to stimulate native MMP-2 118

129 expression by diabetic cardiac fibroblasts may ultimately be the more suitable strategy to improve reparative matrix remodeling. Additionally, the use of nanofibers alone may prove to be of a better risk-benefit value by removing the need to add further complexity to cardiac tissue engineering strategies though the addition of an exogenous agent such as MMP-2 (194). However, MMP-2 half-life is likely much longer in vivo in collagen-rich tissues (198) and the results from the proposed animal study outlined in Chapter 7 may likely result in a different conclusion, with delivery of exogenous MMP-2 proving more effective in vivo. One important issue to discuss regarding this strategy is its potential limitation to only the clinical conditions of diabetes and diabetic cardiomyopathy. Recent studies of diabetic human patients ( ) and in animal models (24-27, 62, 131, 132) suggest that MMP activity is decreased in diabetes, thus highlighting this particular mechanism as a novel therapeutic target. However, this may be one of the only cardiac pathology in which this impairment is observed. In fact, there is significant evidence to suggest that reduced angiogenesis and pathological matrix remodeling after myocardial infarction lead to further injury and that limiting myocardial remodeling via MMP inhibition may be a promising treatment approach (199, 200). Clearly, conditions with already increased levels of MMPs and severe pathological remodeling would not benefit from our investigative approach of promoting increased MMP concentration. However, the use of peptide nanofibers to improve native protein expression by cells and as a myocardial protein delivery vehicle can be extended well beyond the scope of this project, into the development of regenerative strategies for various cardiac pathologies as well as for other tissue engineering approaches. 119

130 For the studies performed in this dissertation, the author was directly responsible for all experiments, either through execution or supervision. All experiments were performed in triplicate or greater and repeated at least twice to result in a sufficient sample size (minimum of N=2, n=6). While all experiments were generally performed by the author, multiple experiments demonstrate repeatability and reproducibility in the data set. This can be seen by the standard deviation values, which are reasonable (±25%) throughout the experiments performed and generally have enough sensitivity to allow for clear determination of statistical significance (p<0.05). Additionally, many of the assays performed in this dissertation research are routinely performed in the laboratory by other researchers for alternate projects, and comparable data is obtained demonstrating further reproducibility in the experimental techniques and analyses. Whenever possible, analysis was performed by a blinded individual, often an undergraduate student working in the lab. This was the case for much of the counting performed for proliferation and apoptosis experiments, where the author would generally perform the staining and capture the fluorescent images and the blinded observer would identify and quantify the positive results. In the case of many assays, including ELISA and rheometry experiments, blinding was not an option due to the expertise needed to perform the experiments and therefore the author took as unbiased experimental approach as possible so as to not influence the outcome in any way. Finally, for capillary morphogenesis experiments, the data was independently analyzed using a custom MATLAB program to remove the researcher completely from the quantitation and analysis. In summary, the studies performed for this dissertation thesis help identify the role of fibroblasts in temporal regulation of the angiogenic process and in reparative matrix remodeling. 120

131 Additionally, the effects of diabetes on the matrix remodeling response by cardiac fibroblasts and potential compensatory tissue engineering strategies (i.e. mechanical stretch, peptide nanofiber microenvironment) were investigated as well. The culture system of RAD16-II nanofibers was utilized as both a controlled microenvironment and as a system which supports MMP2-mediated ECM remodeling by cardiac fibroblasts in vitro. The findings of these studies will contribute towards developing an optimal environment to enhance cardiac regeneration and for cardiac tissue engineering applications. 121

132 CHAPTER 7 Future Directions The research performed in this dissertation provides important insight into the role of fibroblasts in cardiac regeneration and the potential use of peptide nanofibers as a protein delivery tool and scaffold for cardiac tissue engineering. However, our knowledge in the subject matter remains incomplete and can always be advanced. Therefore, the following future directions and studies are proposed. In Aims 1 and 2, the mechanisms of temporal regulation of the angiogenic process by fibroblasts were investigated using a comprehensive approach and the culture system of RAD16- II nanofibers. Normal mammalian myocardium has a complex cellular organization, where the three major cell types are cardiomyocytes, endothelial cells and cardiac fibroblasts. These cells are arranged in an intricate spatial network and communicate constantly. The importance of cardiomyocyte-endothelial interactions in angiogenesis has been previously documented (101, 145) and the importance of endothelial-fibroblast interactions in angiogenesis was documented in Aims 1 and 2 (16). However, it is clear that in order to best develop novel strategies to promote vascularization, it is necessary to understand the interactions between all of the major cardiac cell types - cardiomyocytes, endothelial cells and fibroblasts. Therefore, cardiomyocytes should be incorporated into the culture system of RAD16-II nanofiber system with similar experimental design and response measures as in Aims 1 and 2. This multi-cell co-culture will more effectively mimic the cardiac tissue microenvironment and allow for further insight into 122

133 temporal regulation of the angiogenic process by all cardiac cell types, contributing towards creating the optimal environment for cardiac tissue engineering applications. Additionally, while Aims 1 and 2 provided important information regarding the role of fibroblasts in the capillary morphogenesis process, it should be noted that the relatively short 6 day in vitro experimental duration does not most accurately represent the extended timeframe of the in vivo healing process. A suggestion for future research would be to extend the timeframe to a minimum of 30 days to better mimic the normal wound healing response in cardiac tissue in vivo (79), both for the co-culture experiments of endothelial cells and fibroblasts, but also for any further studies of multi-cell co-culture including cardiomyocytes. Finally, one limitation noted for Aims 1 and 2 was use of dermal fibroblasts due to availability instead of the more physiologically appropriate cardiac fibroblasts. While similarities in dermal and cardiac wound healing do exist (168) and the results of the study are representative of endothelial-fibroblast interaction in cardiac tissue revascularization, the author recommends the use of cardiac fibroblasts for future studies. In Aim 3, the potential of environmental stimuli which occur in the healthy myocardium, such as mechanical stretch and electrical stimulation, was investigated as a strategy to stimulate cardiac regeneration. In particular, mechanical stretch was investigated for its potential to compensate for the dysregulation of matrix remodeling by diabetic cardiac fibroblasts. As previously discussed, the experimental parameters selected for the study did not result in a significant improvement in the matrix remodeling response. Therefore, one recommendation would be to repeat the experiments with increased strain parameters (minimum 10% stretch) and for a longer duration (> 1 day) to see if the response is more pronounced. Additionally, there 123

134 clearly remains much more to learn about the effect of mechanical stretch on all cardiac cell types both in culture and in engineered cardiac tissue, in particular with regards to the effect of diabetic phenotype in conjunction with cyclic strain. Therefore, similar studies regarding the effect of strain on the angiogenic response by endothelial cells with diabetic phenotype are currently being conducted by our group (Sheikh et al., in preparation). Additional studies on the effect of strain on the angiogenic and matrix remodeling response of co-cultures of endothelial cells and fibroblasts would also provide important insight on the potential benefits of stimulation with mechanical stretch. Finally, as the research group is interested in the use of peptide nanofibers to promote angiogenesis and matrix remodeling by endothelial cells and fibroblasts, experiments should be performed which investigate the use of nanofibers as a scaffold for cells during application of strain. The angiogenic and/or matrix remodeling response by cardiac cells resulting from nanofibers combined with mechanical strain would provide important implications for the possible stimulation of tissue engineered constructs prior to implantation. While Aim 4 focused on quantifying the effect of the nanofiber microenvironment on MMP2-mediated extracellular matrix remodeling by normal and diabetic cardiac fibroblasts in vitro, the in vivo component of the research remains to be completed. It is clear that impaired cardiac tissue remodeling is a major factor contributing to cardiac dysfunction in diabetic cardiomyopathy (11, 20, 22, 60). As previously discussed, recent studies have demonstrated that DCM is associated with reduced cardiac expression and activity of MMP-2, resulting in increased collagen accumulation and fibrosis in both diabetic human patients ( ) and animal models (24-27), and improving the tissue remodeling process may prove to be a novel treatment approach. Peptide nanofibers have been used effectively for local and controlled 124

135 protein delivery (102, 111, 112) in the myocardium and our results from Aim 4 thoroughly investigated the use of RAD16-II NFs to increase local MMP-2 concentration and stimulate the in vitro matrix remodeling response of cardiac fibroblasts, in particular diabetic fibroblasts. Therefore, future research should be focused on determining the effect of MMP-2-enriched microenvironment on ECM remodeling by cardiac fibroblasts in a rat model of diabetic cardiomyopathy (streptozotocin-induced type I diabetes). This study would test the hypothesis that increased MMP-2 concentration (native or exogenous) resulting from peptide nanofiber delivery to the cardiac microenvironment will result in improved myocardial remodeling in vivo, as compared to controls. Significant preliminary work has already been performed on this in vivo study by the author, including the validation of the diabetic animal model. Briefly, type I diabetes was induced in Sprague Dawley rats (n=6; Charles River) using streptozotocin (STZ) which results in β-cell apoptosis and stoppage of insulin production (201). The onset of diabetes was almost immediate, confirmed by serum glucose levels >450mg/dl at one week post injection. Six weeks was allowed for development of diabetic cardiomyopathy (24). Diabetic animals and age- and strain-matched wild type controls were sacrificed and heart tissue was harvested either for cardiac cell isolation (for use in Aims 3 and 4 and other related projects) or embedded in paraffin for histological analysis. Picosirius red staining of heart tissue sections demonstrated that cardiac fibrosis is evident in the diabetic heart at 6 weeks post STZ injection (Figure 11), validating both the use of the STZ type I diabetic animal model as well as the 6 week time frame for cardiac fibrosis and diabetic cardiomyopathy development. Briefly, the following experimental design is proposed for future in vivo studies investigating increased MMP-2 concentration (native or exogenous) resulting from peptide 125

136 nanofiber delivery to the cardiac microenvironment. Peptide nanofibers (RAD16-II, 1.0%) with or without human MMP-2 (100 ng/ml) would be injected into the left ventricle wall of STZ-rats (n=6 per group based on power analysis). Diabetic animals injected with PBS and sham surgeries would serve as additional controls (at least n=6 per group). Four weeks after treatments, animals woud be sacrificed. Response variables would include cardiac function assessment with echocardiography, immunostaining to quantify structural changes and ECM deposition, cellular infiltration, vascularization and inflammation, and ELISA/Western blot and gelatin zymography to determine collagen I and IV and MMP-2 expression and activity. Preliminary studies have already been performed with peptide nanofibers with or without exogenous MMP-2 injected into the myocardium of wild type rats (n=2; Figure 27a). These preliminary results indicate localized delivery and retention of nanofibers in the myocardium 3 days post injection (Figure 27b). Additionally, staining indicates the maintained presence of exogenous MMP-2 in the nanofibers 3 days post injection (Figure 27c). These preliminary results provide support for continued future in vivo studies. Figure 27. Myocardial injection of peptide nanofibers and exogenous MMP-2 in a wild type rat animal model. a) Harvested rat heart 3 days post injection. b) Hemotoxylin & Eoisin staining of myocardial tissue section. Yellow arrows indicate peptide nanofibers. 126