Effect of transforming growth factor-β1, fibronectin and plasminogen activator inhibitor on migration of bovine aortic endothelial cells

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1 Yale University EliScholar A Digital Platform for Scholarly Publishing at Yale Yale Medicine Thesis Digital Library School of Medicine 1996 Effect of transforming growth factor-β1, fibronectin and plasminogen activator inhibitor on migration of bovine aortic endothelial cells Christine E. Brozowski Yale University Follow this and additional works at: Recommended Citation Brozowski, Christine E., "Effect of transforming growth factor-β1, fibronectin and plasminogen activator inhibitor on migration of bovine aortic endothelial cells" (1996). Yale Medicine Thesis Digital Library This Open Access Thesis is brought to you for free and open access by the School of Medicine at EliScholar A Digital Platform for Scholarly Publishing at Yale. It has been accepted for inclusion in Yale Medicine Thesis Digital Library by an authorized administrator of EliScholar A Digital Platform for Scholarly Publishing at Yale. For more information, please contact elischolar@yale.edu.

2 YALE MEDICAL LIBRARY EFFECT-OS : fibrone GROWTH FACT ACTIVA PLASM inogli OVINE.AORTIC' ENDOTHEUAi CHRISTINE

3 YALE UNIVERSITY CUSHING/WHITNEY MEDICAL LIBRARY

4 Digitized by the Internet Archive in 2017 with funding from The National Endowment for the Humanities and the Arcadia Fund

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10 Effect of Transforming Growth Factor-pl, Fibronectin and Plasminogen Activator Inhibitor on Migration of Bovine Aortic Endothelial Cells. A Thesis Submitted to the Yale University School of Medicine in Partial Fulfillment of the Requirements for the Degree of Doctor in Medicine by Christine E. Brozowski May 1996

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12 Abstract EFFECT OF TRANSFORMING PLASMINOGEN ACTIVATOR ENDOTHELIAL A. CELLS. Madri). of Medicine, Restenosis and procedures lead to of New Haven, CT. the Pathology, largest and formation proliferation, of have influence The of this transforming growth factor University, School of endarterectomy trauma of a have cells investigation (TGF-(3l), and was the Later of studies 50ug/ml which was in using a significantly our resulted a student's decrease laboratory in to effect (Fn), of and TGF- (31 decreased migration by 28% significant not shown on migration of bovine were did the endothelial fibronectin (PAI-2) cell and been Fibronectin decreased migration by 19% +. 4%. loug/ml by neointima. muscle 3%. (p<0.05) caused synthesis, factors muscle plasminogen activator inhibitor aortic endothelial cells. Joseph smooth matrix Growth smooth target on and on by Vascular development depends endothelium. BOVINE AORTIC (Sponsored Yale AND in deundation injury which may the migration, presence FIBRONECTIN complication angioplasty. hyperplasia an Brozowski such as these result Neointimal cells. Christine catheter FACTOR-(3l, INHIBITOR ON MIGRATION OF Department remains balloon GROWTH using decreased statistically significant. Both values t-test. migration higher migration (6% levels of + 10% PAI-2 at ± 4%). of + PAI-2 3%, TGF-(3l has been shown to

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14 be present in injury in vivo. migration and migration. vascular significant increases the rate is mediated this by sites of increases the likelihood smooth suggests that modulation of the of effects of vascular endothelial the presence of TGF-(3l at fibronectin can partially mimic migration, at TGF- (31 decreases the rate of Therefore, lesion quantities muscle the TGF-pl the TGF- Pi effect the extracellular on cell site of a restenosis. of cell on Since BAEC migration matrix. The effect of TGF- Pi on endothelial cell migration may also be due to increased expression of PAI-2 PAI or decreased upa expression, decreases BAEC migration. as

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18 2 Introduction Balloon angioplasty, endarterectomy, and synthetic grafting are therapeutic interventions currently performed to alleviate the effects of atherosclerosis. It has been estimated that over 350,000 coronary angioplasty procedures were performed in the United States in Restenosis remains the most important limitation of angioplasty, with studies suggesting that up to 50% of patients will develop restenoses within one year, with up to 20% of patients necessitating a repeat operation within 6 months.2,3 Procedures such as angioplasty result in endothelial denudation injury to the blood vessel wall Stenosing intimal lesions may develop from intimal hyperplasia which narrows the vessel lumen, and can compromise blood flow. This ultimately results in a failure of the reconstruction.4 The formation of a neointima after vascular injury depends on smooth muscle cell proliferation and migration and the synthesis of a new extracellular matrix.4 The normal tunica intima is composed of endothelial cells and smooth muscle cells.5 Endothelial cells line the vascular system, forming a nonthrombogenic surface for blood flow and covering smooth muscle cells. Smooth muscle cells compose the media of large vessels, maintain vessel wall integrity, control vascular tone and influence endothelial cell behavior. Following denudation injury, medial smooth muscle cells proliferate and migrate rapidly into the intima where they synthesize components which may narrow the vessel lumen. Endothelial cells migrate more slowly from the edges of the wound to reconstitute the damaged endothelial cell layer and reestablish vessel wall integrity, critical to maintaining lumen patency.6 Stimuli

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20 3 which retard migration of endothelial cells and enhance the migration of smooth muscle cells lead to the development of stenotic lesions at the site of vascular injury.7 Transforming growth factor (TGF-(3l), a component of platelet a granules, is synthesized and secreted by endothelial and smooth muscle cells.7 TGF-[3l regulates critical cell functions including growth, differentiation, activation, and migration.8 Specifically, TGF-(3l has been shown to be present in significant quantities at sites of vascular injury in vivo, and markedly inhibits endothelial cell migration and vessel wall repair following injury.9 It has been suggested that the TGF-(3l mediated decrease in endothelial cell migration is associated with increased expression of fibronectin (Fn) and plasminogen activator inhibitor (PAI-2)8. This report documents the effect of transforming growth factor (TGF-(31), fibronectin (Fn), and plasminogen activator inhibitor (PAI-2) on migration of bovine aortic endothelial cells as an in vitro model of re-endothelialization and repair of denudation injury such as that produced by balloon catheter angioplasty. Methods Bovine aortic endothelial cells (BAEC) were isolated and cultured with DMEM and 10% fetal calf serum (FCS). The cells were seeded into the middle of a steel fence with a known area and allowed to attach to the underlying type I collagen matrix below, and grow to confluency for 4-5 hours. The fence was then removed. With the loss of contact inhibition, cell migration radially outward was initiated. The cells were covered in media alone or with media plus

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22 4 exogenous transforming growth factor (TGF-[3l) at a final concentration of 0.5 ng/ml, plasminogen activator inhibitor (PAI-2) at a final concentration of 10 ug/ml, fibronectin (Fn) at a final concentration of 50 ug/ml, Aprotinin at 50 ug/ml, or EACA at 50 ug/ml. The cells migrated radially outward for six days, and were fed with additional media plus the appropriate growth factors at day three. Migrations were stopped 6 days post initiation of treatment. The cultures were then washed in PBS and fixed with 10% Buffered Formalin. Cells were stained with Harris' hematoxylin to evaluate cell migration. Petri dishes with stained cells were placed on an overhead projector to assess surface area covered by the cells. The surface areas were transferred to paper and a computer graphics tablet was used to quantify surface areas obtained by overhead projection. These surface areas were compared with known values of the initial fence area to obtain the net increase in surface area. Maximal response was obtained using DMEM media and 10% FCS only, thus, this was defined as the control with 100% relative migration. Migration in the presence of growth factors (TGF-(3l, Fn, PAI-2, EACA and Aprotinin) was compared and normalized to this value. Bar graphs were generated from mean values. The differences in means were analyzed using a student's t-test. Statistical significance was assumed for P<0.05. Results Treatment with TGF-pl decreased the distance of migration of BAEC on a type 1 collagen matrix by 28% + 3% compared to control. Fibronectin showed a 19% + 4% inhibition of

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24 5 migration distance. These were significant (p<0.05) using a student's t-test. In contrast, treatments with EACA, Aprotinin and EACA & Aprotinin did not show a significant decrease in migration compared to the control group. EACA and Aprotinin are non-specific protease inhibitors. These results suggest that non-specific protease inhibitors do not decrease the migration of endothelial cells. See Figure 1. PAI-2 at loug/ml showed only a 6% ± 4% decrease in migration which was not statistically significant. In later BAEC migration studies in our laboratory, however, PAI-2 concentration was increased to 50ug/ml with a significant decrease in migration of 10% + 3%. In additional studies, Petzelbauer et al demonstrated that stable over-expression of PAI-1 protein in BAEC using retroviral transduction resulted in a significant inhibition of BAEC migration. The g resultant BAEC migration was found to decrease 23% ± 2%. Discussion Repair of arterial injury produced by balloon angioplasty or atherectomy leads to the development of an intimal thickening and a subsequent narrowing of the vessel lumen.10 Neointimal formation depends on smooth muscle cell proliferation, migration, and matrix synthesis, and the presence/absence of an endothelium4 The two major cell types of the vessel wall, the smooth muscle cell and the endothelial cell, respond differently to injury. Medial SMCs begin to migrate from the media into the intima. Many cells proliferate, but up to one half do not divide 4 Once in the intima, SMCs continue to proliferate and form a thick layer.

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26 % Inhibition of Migration 40 -i n=6 10 n=8 - T 0 IF -10 o 1 c o O 1 CC LL o c LL CVJ < CL < o < LU c 'c o Q_ < _c 'c Q. < + < O < LU Treatment Figure 1

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28 6 The resulting neointima is further enlarged by extracellular matrix components synthesized by the smooth muscle cells. Endothelial cells, on the other hand, migrate more slowly from around the area of denudation to reconstitute the damaged endothelial cell layer. The cells at the edge of the wound divide and migrate inward, but the central portion of large injuries may remain uncovered.4'11 Importantly, proliferation of intimal smooth muscle cells ceases earlier in areas covered by regenerating endothelium than in regions lacking an endothelium.4 Primary lesions differ from restenotic lesions in that primary lesions consist of well-organized collagen and ground substance, and are hypocellular, while restenotic lesions have foci of hypercellularity consisting of proliferative vascular smooth muscle cells with associated matrix proteins.12 These smooth muscle cells have an unusual elongated shape with processes extending from the cells and are accompanied by significant matrix deposition composing 5060% of lesion size between the cells.12 Advanced atherosclerotic lesions show progressive development of small denuded regions. This loss of continuity of the endothelium in the evolution of atherosclerosis suggests that some property of the atheromatous wall or blood interacting with the wall is responsible for the inhibition of endothelial regeneration.1' Both platelet deposition and growth factor release have been suggested as important events initiating the cellular growth response after injury.12,14 A variety of growth factors have been described in vascular smooth muscle cells and/or atherosclerotic tissue, and have, therefore, been inferred to play a role in the development of restenosis lesions. These include the transforming growth factor-(3s, platelet-derived growth factors and receptors, fibroblast growth

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30 7 factors, and insulin-like growth factors.15 Platelet-derived growth factor (PDGF) has been found to exert little effect on smooth muscle cell replication, but markedly influences the ability of SMCs to migrate into the intima.16 TGF-(3l is a major component of platelet releasates and is synthesized and secreted by endothelial cells and smooth muscle cells.7 TGF-(3l has been shown to inhibit proliferation of endothelial cells, and to retard epithelial wound healing in vitro.17 TGF-(3l influences matrix production and protease inhibitors, both of which affect migration of endothelial cells. Additionally, TGF-(3l has been demonstrated to stimulate the proliferation of smooth muscle cells and the production of elastin and proteoglycan, common features of atherosclerotic plaques.15 TGF-(3l has been shown to be present in significant quantities at sites of vascular injury in vivo.8,10,18 In addition to decreasing endothelial cell migration, TGF-(3l has been documented to increase vascular smooth muscle cell migration.19 Stimuli which enhance migration of medial smooth muscle cells into the intima where they can synthesize matrix components leading to the development of a stenotic lesion at the site of vascular injury may result in the eventual occlusion of the vessel lumen.6 Because TGF-(3l decreases the rate of endothelial cell migration and increases the rate of smooth muscle cell migration, the presence of TGF-(3l at the site of a vascular lesion increases the likelihood of restenosis. Additional steps involved in restenosis include the release of growth factors, proteoglycan deposition and release of growth factors, proteoglycan deposition and extracellular matrix remodeling.2,20 Potential inhibitors at each step could be used as preventative therapy.

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32 TGF-(3l influences matrix production and protease inhibitors, both of which affect migration of endothelial cells. TGF-(3l has been observed to down-regulate the expression of upa activity in endothelial cells.21 Endothelial cells express tissue-type plasminogen activator (tpa), urokinase-like plasminogen activator (upa) and plasminogen activator inhibitor (PA1). upa interaction with the upa receptor has been associated with an increase in endothelial cell motility.8 PAI-2 is a protease inhibitor which is specific for upa receptor sites, and decreases BAEC migration. TGF-(3l decreases the expression of upa and subsequently lessens the rate of migration of endothelial cells. Thus, the TGF-(5l effect on BAEC migration may be due to the combined effects of increased expression of PA1, decreased upa expression, and influencing the extracellular matrix (increased fibronectin deposition). In other studies in our laboratory, Petzelbauer et al showed that unlike endothelial cells, bovine aortic smooth muscle cells exhibited an increased rate of migration in response to the addition of exogenous TGF-(3l, fibronectin or PAI-2, or by the stable over-expression of PAI-1 protein. Endothelial cell replication and migration are triggered by damage to the vessel wall These cells migrate from the leading edge of a lesion to reestablish the endothelial monolayer, and repair the vessel wall It has been shown that TGF-(3 inhibits both endothelial replication and migration, thus decreasing endothelial regeneration.13 Heimark,13 et al demonstrated that TGF-(3 transiently decreases DNA synthesis in endothelial cells. Specifically, TGF-(3 delays the entry of regenerating endothelial cells into the S phase. Once cells have passed into S or G2, however, they are no longer responsive to TGF-(3.

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34 9 Increased TGF- 3l gene expression has been found in rat carotid arteries after balloon catheter injury.8,10 In studies by Nikol,15 et al, human atherosclerotic tissue specimens were examined for TGF-(3l mrna. Primary atherosclerotic lesions showed slightly, but not significantly, higher TGF-(3l expression than non-atherosclerotic tissue. TGF-(3l was maximally expressed in restenotic tissues. TGF-(3l expression in restenotic atherosclerotic tissues was significantly greater than that found in primary atherosclerotic tissue, consistent with the hypothesis that increased TGF-(3l mrna expression is related to smooth muscle cell proliferation.15 These results suggest a role for TGF-(3l in the development of restenosis lesions. Neointimal formation requires the synthesis of a new extracellular matrix. Major constituents of matrices produced by intimal SMCs include fibronectin, elastin and type I collagen.10 The addition of exogenous TGF-(3l to arterial SMC in vitro has been shown to stimulate extracellular protein synthesis.10 TGF-(3l has been shown to increase fibronectin production in endothelial cells, smooth muscle cells and fibroblasts.8,22 Fibronectin promotes rapid attachment of bovine aortic endothelial cells, but little migration.21 BAECs treated with TGF(31 have shown increased fibronectin mrna levels, and synthesis and deposition of fibronectin8,19. Increased mrna levels of fibronectin and collagen a2(i) have been found to correlate with the onset of neointimal formation.10 In this paper we have shown that the addition of exogenous TGF-(3l or fibronectin decreases BAEC migration. Fibronectin can partially mimic the effects of TGF-(3l on BAEC migration

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36 10 suggesting that the TGF-(3l effect on migration is mediated by modulation of the synthetic matrix. TGF-(3l alters matrix production by increasing fibronectin deposition, increasing PAI expression, and decreasing upa expression.8 The majority of medical therapies designed to prevent restenosis are aimed at reducing smooth muscle cell migration as the primary cause of restenoses pathology. However, enhancement of endothelial cell regeneration and decreased matrix production are other potential targets to consider as proliferation of smooth muscle cells ceases earlier in areas covered by an regenerating endothelium, and as the matrix composes a large part of the lesion volume. Conclusion This paper has demonstrated that TGF-(3l and fibronectin inhibit migration of bovine aortic endothelial cells. Other studies in our laboratory have shown that the addition of exogenous PAI-2 in high concentration (50ug/ml) also decreased BAEC migration, and that PAI & TGF(31, and Fn & TGF-(3l were not additive. Therefore, we surmise that TGF-(3l alters matrix production by increasing fibronectin deposition, increasing PAI-2 expression and decreasing upa expression. Conversely, TGF-(3l has been shown to enhance migration in smooth muscle cells. Previous studies have shown that the most intimal smooth muscle cell proliferation occurs in the region last covered by regenerating endothelium.24,25 Therefore, stimuli which retard the migration of endothelial cells and enhance the migration of smooth muscle cells lead to the development of stenotic lesions at the site of vascular injury. Consequently, reducing the

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38 11 TGF-pl burden at the site of vascular lesion may decrease the incidence of restenosis following vascular injury.

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40 12 References 1. Faxon, D.P., and J.W. Currier, Prevention of post-ptca Restenosis. Annals of the New York Academy of Sciences, 748:419-27, Nobuyoshi, M., Kimura, T., Noksaka, H., Mioka, S., Veno, K., Yokoi, H., Hamasaki, N., Horiuchi, H., and H. Ohishi, Restenosis After Successful Percutaneus Transluminal Coronary Angioplasty: Serial Angiographic Follow-up of 229 Patients. Journal of the American College of Cardiology, 12(3):616-23, Serruys, P.W., Luijten, H.E., Beatt, K.J., Geuskens, R., de Feyter, P.J., van den Brand, M., Reiber, J.H., ten Katen, H.J., van Es, G.A., and P.G. Hugenholtz, Incidence of Restenosis After Successful Coronary Angioplasty: A Time-Related Phenomenon. A Quantitative Angiographic Study in 342 Consecutive Patients at 1, 2, 3, and 4 Months. Circulation, 77(2): , Clowes, A.W., and M.A. Reidy, Prevention of Stenosis After Vascular Reconstruction: Pharmacologic Control of Intimal Hyperplasia - A Review. Journal of Vascular Surgery, 13(6):885-91, Bondjers G., Glukhora M., Hansson G.K., Postnov, Y.V., Reidy M.A., and S.M. Schwartz, Hypertension and Atherosclerosis. Cause and Effect, or Two Effects with One Unknown Cause? Circulation, 84(6Suppl):V12-16, Bell, L., Luthringer, D.J., Madri, J.A., and S.L. Warren, Autocrine Angiotensin System Regulation of Bovine Aortic Endothelial Cell Migration and Plasminogen Activator Involves Modulation of Proto-oncogene pp60c-src Expression. Journal of Clinical Investigation, 89: , Madri, J.A., and L. Bell, Vascular Cell Responses to Injury: Modulation by Extracellular Matrix and Soluble Factors. Cell Interactions in Atherosclerosis, ed. H. Robenek and N.J. Severs Petzelbauer, E., Springhom, J.P., Tucker, A.M., and J.A. Madri, The Role of Plasminogen Activator Inhibitor in the Reciprocal Regulation of Bovine Aortic Endothelial and Smooth Muscle Cell Migration by TGF-(3l. American Journal of Pathology In Revision Madri, J.A., Reidy, M.A., Kocher, O., and L. Bell, Endothelial Cell Behavior After Denudation Injury Is Modulated by Transforming Growth Factor-(3l and Fibronectin. Laboratory Investigation, 60 (6): , Majesky, M.W., Lindner, V., Twardzik, D.R., Schwartz, S.M., and M.A. Reidy, Production of Transforming Growth Factor (Jl during Repair of Arterial Injury. Clinical Investigation, 88: , Journal of

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42 Reidy, M.A., Clowes, A.W., and S.M. Schwartz, Endothelial Regeneration: V. Inhibition of Endothelial Regrowth in Arteries of Rat and Rabbit. Laboratory Investigation, 49(5): , Isner, J.M., Vascular Remodeling: Honey, I Think I Shrunk the Artery. Circulation, 89(6): , Heimark, R.L., Twardzik, D.R., and S.M. Schwartz, Inhibition of Endothelial Regeneration by Type-Beta Transforming Growth Factor from Platelets. Science, 233, , Wilcox, J.N., Molecular Biology: Insight into the Causes and Prevention of Restenosis After Arterial Intervention. American Journal of Cardiology, 72:88E-95E, Nikol, S., Isner, J.M., Pickering, G., Kearney, M., Leclerc, G., and L. Weir, Expression of Transforming Growth Factor-(3l Is Increased in Human Vascular Restenosis Lesions. Journal of Clinical Investigation, 10: , Reidy, M.A., Fingerle, J., and V. Lindner, Factors Controlling the Development of Arterial Lesions After Injury. Circulation, 86 (SuppL III): 43-III-46, Biro, S., Yu, Z.X., and W. Casscells. Fibroblast and Transforming Growth Factors in Endothelial Wound Healing in vitro. Journal of the American College of Cardiology, 17:24A (Abstr.), Reidy, M.A., Factors Controlling Smooth-Muscle Cell Proliferation. Archives of Pathology & Laboratory Medicine, 116(12): , Madri, J.A., Bell, L., and J.R. Merwin, Modulation of Vascular Cell Behavior by Transforming Growth Factors (3. Molecular Reproduction and Development, 32: , Forrester, J.S., Fishbein, M., Helfant, R., and J. Fagin, A Paradigm for Restenosis Based on Cell Biology: Clues for the Development of New Preventive Therapies. Journal of the American College of Cardiology, 17: , Sankar, S., Mahooti-Brooks, N., Centrella, M., McCarthy, T.L., and J.A. Madri, Expression of Transforming Growth Factor Beta Type HI Receptor in Vascular Endothelial Cells Increases Their Responsiveness to Transforming Growth Factor (32. Biological Chemistry, 270(22): , Journal of

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44 Madri. J.A., Pratt, B.M., and J. Yannariello-Brown, Matrix-Driven Cell Size Change Modulates Aortic Endothelial Cell Proliferation and Sheet Migration. American Journal of Pathology, 132(1): 18-27, Pratt, B.M., Form, D., and J.A. Madri, Endothelial Cell-Extracellular Interactions. Annals of the New York Academy of Sciences, 460: , Matrix 24. Clowes, A.W., Reidy, M.A., and M.A. Clowes, Kinetics of Cellular Proliferation after Arterial Injury: I. Smooth Muscle Growth in the Absence of Endothelium. Laboratory Investigation, 49(3): , Fishman, J.A., Ryan, G.B., and M.J. Kamovsky, Endothelial Regeneration in the Rat Carotid Artery and the Significance of Endothelial Denudation in the Pathogenesis of Myointimal Thickening, Laboratory Investigation 32: , 1975.

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48 HARVEY CUSHING / JOHN HAY WHITNEY MEDICAL LIBRARY MANUSCRIPT THESES Unpublished theses submitted for the Master's and Doctor's degrees and deposited in the Medical Library are to be used only with due regard to the rights of the authors. Bibliographical references may be noted, but passages must not be copied without permission of the authors, and without proper credi being given in subsequent written or published work. This thesis by has been used by the following persons, whose signatures attest their acceptance of the above restrictions. NAME AND ADDRESS DATE

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