TISSUE-SPECIFIC STEM CELLS

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1 TISSUE-SPECIFIC STEM CELLS Complement Component 3 is Necessary to Preserve Myocardium and Myocardial Function in Chronic Myocardial Infarction MARCIN WYSOCZYNSKI, a MITESH SOLANKI, a SYLWIA BORKOWSKA, b PATRICK VAN HOOSE, a KENNETH R. BRITTIAN, a SUMANTH D. PRABHU, a,c MARIUSZ Z. RATAJCZAK, b GREGG ROKOSH a Key Words. Complement component C3 Mobilization Myocardial infarction Myocardial regeneration Stem cells a Institute of Molecular Cardiology and b James Graham Brown Cancer Center, University of Louisville, Louisville, Kentucky, USA; c Division of Cardiovascular Disease, University of Alabama- Birmingham, Birmingham, Alabama, USA Correspondence: Gregg Rokosh, Ph.D., Institute of Molecular Cardiology, University of Louisville, 580 South Preston Street Rm 304C, Louisville, Kentucky 40202, USA. Telephone: ; Fax: ; gregg.rokosh@louisville.edu Received August 16, 2013; accepted for publication April 4, 2014; first published online in STEM CELLS EXPRESS May 8, VC AlphaMed Press /2014/$30.00/ /stem.1743 ABSTRACT Activation of the complement cascade (CC) with myocardial infarction (MI) acutely initiates immune cell infiltration, membrane attack complex formation on injured myocytes, and exacerbates myocardial injury. Recent studies implicate the CC in mobilization of stem/progenitor cells and tissue regeneration. Its role in chronic MI is unknown. Here, we consider complement component C3, in the chronic response to MI. C3 knockout (KO) mice were studied after permanent coronary artery ligation. C3 deficiency exacerbated myocardial dysfunction 28 days after MI compared to WT with further impaired systolic function and LV dilation despite similar infarct size 24 hours post-mi. Morphometric analysis 28 days post-mi showed C3 KO mice had more scar tissue with less viable myocardium within the infarct zone which correlated with decreased c-kit pos cardiac stem/progenitor cells (CPSC), decreased proliferating Ki67 pos CSPCs and decreased formation of new BrdU pos /a-sarcomeric actin pos myocytes, and increased apoptosis compared to WT. Decreased CSPCs and increased apoptosis were evident 7 days post-mi in C3 KO hearts. The inflammatory response with MI was attenuated in the C3 KO and was accompanied by attenuated hematopoietic, pluripotent, and cardiac stem/progenitor cell mobilization into the peripheral blood 72 hours post-mi. These results are the first to demonstrate that CC, through C3, contributes to myocardial preservation and regeneration in response to chronic MI. Responses in the C3 KO infer that C3 activation in response to MI expands the resident CSPC population, increases new myocyte formation, increases and preserves myocardium, inflammatory response, and bone marrow stem/progenitor cell mobilization to preserve myocardial function. STEM CELLS 2014;32: INTRODUCTION The complement cascade (CC), integral to first line host defense and innate immunity, plays a significant role in the immune response subsequent to myocardial infarction (MI). After MI, myocardial tissue damage serves as a potent CC activator which then contributes to tissue damage by promoting infiltration of immune cells and formation of the membrane attack complex (MAC) on the host cells within the ischemic region [1]. Necrotic cells expose intracellular antigens leading to robust activation of CC through classical or alternative pathways [1]. Necrotic cells release proteolytic enzymes that can directly cleave and activate complement component C3 and C5 without activation of classical or alternative pathways [2, 3]. C3 and C5 cleavage fragments contribute to initiation of the immune response in ischemia/ reperfusion injury by chemoattraction of immune cells and MAC formation on cells within the ischemic region [4 6]. CC becomes activated acutely within hours of MI [7]. Hence, it has been thought to be implicated in immune response initiation and to exacerbate injury in infarcted mice. In experimental models of MI, complement depletion by cobra venom and blocking complement component C5 activation with a monoclonal antibody masking its cleavage site have been shown to be beneficial reducing injury size [5, 8, 9]. However, these studies focused on the CC in acute MI models without considering its role in chronic scar formation, remodeling, or regeneration. Clinically, trials testing CC blockade at C5 to prevent MAC formation and chemoattraction of neutrophils (NE) in patients suffering with myocardial infarction were equivocal as no clear benefit was observed in long-term follow-up including survival and left ventricle (LV) systolic function [10 13]. These results suggest the CC may be more complex than initially perceived. STEM CELLS 2014;32: VC AlphaMed Press 2014

2 Wysoczynski, Solanki, Borkowska et al Recent evidence suggests that components of the CC may serve novel functions that modulate diverse regenerative processes, such as cell survival, growth, differentiation, and trafficking of the stem/progenitor cells (mobilization/homing). Complement has recently been implicated as a mediator of lens and limb regeneration in lower vertebrates and brain, liver, bone, and bone marrow (BM) in rodents [14 21]. Early studies of liver regeneration established that classical pathway activation through C3a and C5a and respective receptors, C3aR and C5aR, is necessary. Regeneration in mice with targeted deletion of C3, C5, C3aR, or C5aR was compromised demonstrating that cleavage products C3a and C5a were necessary for the response. Complement initiates the priming phase of liver regeneration that requires proliferative and growth signals to replace lost mass. C3a and C5a also increase hepatocyte survival during regeneration [16, 17, 21]. In the brain, complement contributes to neurogenesis in both the basal state and in response to ischemia (middle cerebral artery occlusion) [22]. Neurogenesis in C3 knockout (KO) mice was impaired in the ischemic area and subventricular zone. C3a promoted neural differentiation of progenitors and accentuated stromal cellderived factor 1a stimulated migration and mitogen activated protein kinase signaling [20]. CC activation in BM was necessary for stress-induced BM hematopoiesis but not homeostasis. Regeneration of BM hematopoiesis after hematopoietic stem/ progenitor cell (HSPC) transplantation was impaired in C3, C5, C3aR, and C5aR KO mice and was also associated with impaired HSPC homing [18, 23 28]. Recent studies suggest that CC might favor bone formation where C3a and C5a influence bone cell migration, osteoblast osteoclast interaction, and modulation of the inflammatory response by osteoblasts [19]. In this study, we examine the effect of C3 on the chronic response to MI that includes scar formation, remodeling, and regeneration. Our studies show that LV function is impaired and progression toward heart failure is accelerated in C3 KO mice. Increased scar with decreased viable myocardium and fewer new myocytes in the C3 KO suggests regeneration was impaired. Impaired inflammatory response and mobilization of BM stem/progenitor cells and reduced proliferative response of c-kit pos resident cardiac stem cells in infarcted hearts of C3 KO mice contribute to attenuated regeneration. These findings provide the first evidence that, in addition to responding to acute injury, the complement system is involved in the chronic response to MI, which includes preservation and regeneration of the myocardium. MATERIALS AND METHODS A full Methods section is available in the Supporting Information. Murine Model of MI All procedures were conducted under the approval of the University of Louisville IACUC in accordance with the NIH Guide for the Care and Use of Laboratory Animals (DHHS publication No. [NIH]85-23, rev. 1996) as previously described [29]. Female C57BL/6 (wild type (WT), Sham n 5 7, MI n 5 9) and C3 KO (Jackson Laboratories, Bar Harbor, ME, org; Stock , Sham n 5 6, MI n 5 8) mice (mice were bred and used at weeks when available) were anesthetized and the coronary artery occlusion or sham surgery performed as previously described [29]. Mortality of WT and C3 KO was 22.7% and 31.6%, respectively. Mice were injected daily with 5-bromo-2 -deoxycytidine (BrdC) (80 mg/kg, i.p.) beginning the day of operation until sacrifice. Group assignment was randomized at the time of surgery. In vivo cardiac function was assessed immediately prior to sacrifice 28 days after ligation by echocardiography as previously described [29, 30]. Echocardiography In vivo cardiac function was assessed immediately prior to sacrifice 28 days after ligation by echocardiography as previously described [29, 30]. M-mode, 2D, and Doppler echocardiography (Vevo 770, Visual Sonics, Toronto, ON, CAN, MHz linear array transducer, 120 Hz frame rate) analysis provided LV end-systolic or diastolic short axis or long axis areas (ESA, EDA, LVALS, and LVALD), shortaxis end-systolic (ES) and end-diastolic (ED) diameter (D) and wall thickness (WT) and long-axis end-systolic and enddiastolic volume (ESV and EDV). LV systolic function was measured as ejection fraction (EF) and fractional shortening (FS). Complement C5 activation Complement C5 activation was measured as the formation of the cleavage product C5b-9 at indicated time points after MI or sham operation by ELISA (Kamiya Biomedical Company, Seattle, WA, Histology After final echocardiography and peripheral blood (PB) collection, hearts were harvested in diastole with saturated KCl and CdCl (100 mmol/l) injected through the apex into the LV cavity. An additional group of WT (WT, Sham n 5 5, MI n 5 6) and C3 KO (WT, Sham 5 6, MI 5 6) mice were infarcted as above and processed for c-kit and terminal deoxynucleotidyl transferase dutp nick end labaling (TUNEL) staining. Hearts were fixed in formalin, sectioned, and histology and morphometry performed as previously described [29, 31]. Mobilization and Blood Counts WT and C3 KO mice without surgery, after sham surgery, and MI (n for each group at 72 hours, Fig. 7; n for each group at each time point for time course, Supporting Information Fig. 7) were bled from the retro-orbital plexus for complete blood counts (WBC, NE, and LY, Hemavet) and flow cytometry before and 24, 48, 72 hours and 7 days after MI. Colony Forming Unit-Granulocytes/Macrophage Assay Red blood cells from WT and C3 KO mice before and 24, 48, 72, 96 hours, and 7 days after sham surgery or MI (n for each group, Fig. 7B; Supporting Information Fig. 7A) were lysed and nucleated cells used for colony forming unit-granulocytes/macrophage (CFU-GM) assays. Real-time Reverse Transcriptase Polymerase Chain Reaction and Reverse Transcriptase Polymerase Chain Reaction Total mrna was isolated from the white blood cell (WBC) fraction from WT and C3 KO mice before and 72 hours after sham surgery or MI (n 5 6 8) to measure Oct-4, Nanog, Rex-1, Rif1, Dppa1, Gata-4, and NKX2.5, and b2-microglobulin VC AlphaMed Press 2014

3 2504 The Complement Cascade in Chronic Myocardial Infarction Figure 1. Echocardiographic assessment of left ventricle (LV) function, volumes and diameters. C3 deficiency attenuates cardiac function and accentuates myocardial injury after coronary artery occlusion. C3 KO mice and their wild type controls underwent coronary artery ligation or sham operation. LV function, EF (A), FS(B), ESA, EDA, LVALS, and LVALD (C F) were assessed 28 days after surgery in vivo by echocardiography in Sham WT (n 5 7), Sham C3 KO (n 5 6), WT MI wild type (n 5 9), and C3 KO MI (n 5 8). Values are the mean 6 SEM. *, p <.05 vs. WT MI. Abbreviations: C3 KO, C3 knock out; EF, ejection fraction; ESA, end-systolic area short axis; EDA, enddiastolic area short axis; FS, fractional shortening; LVALS, left ventricle area long axis (end-systolic); LVALD, left ventricle area long axis (end-diastolic); MI, myocardial infarction; WT, wild type. mrna levels by quantitative reverse transcriptase polymerase chain reaction (qrt-pcr) and RT-PCR. Statistical Analysis All data are expressed as the mean 6 SEM. Statistical analysis of normally distributed data (Kolmogorov-Smirnov) was performed by unpaired T test. Non-normal data were analyzed by the Mann-Whitney test. A value of p <.05 was considered significant. RESULTS C3 Deficiency Exacerbates Myocardial Dysfunction After Coronary Artery Ligation Complement system activation with myocardial injury has characteristically been associated with exacerbated injury VC AlphaMed Press 2014 when studied in acute ischemia reperfusion MI models without further follow up to determine impact on post-mi remodeling and contribution to failure [7, 8, 10, 11, 13]. Planimetric analysis of heart sections from WT and C3 KO mice 24 hours post-mi indicated no significant differences in infarct size ( % of LV WT vs % of LV C3KO, Supporting Information Fig. 1). Thus, C3 deficiency had no effect on infarct size acutely. Despite C3 deletion, C5 activation 48 hours after MI remained similar to that in WT mice (Supporting Information Fig. 2). In chronic studies, LV function was evaluated by echocardiography 4 weeks after infarction. No differences in LV systolic function measured by ejection fraction (EF) and fractional shortening (FS), LV end-systolic or diastolic short axis or long axis areas (ESA, EDA, LVALS, and LVALD) or LV geometry evaluated by LV inner diameter in systole and diastole (LVIDD, LVISD) and EDV, ESV were observed between sham operated WT and C3 KO mice (Fig. 1A 1F, STEM CELLS

4 Wysoczynski, Solanki, Borkowska et al Figure 2. Morphometric analysis and assessment of left ventricle (LV) hypertrophy. C3 deficiency increases myocardial injury and accentuates LV hypertrophy after MI. Scar and viable myocardium in the risk region were determined in Masson s trichrome stained LV sections 28 days after surgery. Myocyte cross-sectional area was measured in sections stained with FITC-conjugated WGA. (A): Representative Masson s trichrome stained heart short axis sections. Scar as percent of LV was increased in C3 KO hearts after infarction (B) as viable myocardium in the risk region was decreased (C). Expansion index in hearts of C3KO mice after infarction was elevated compared with ligated hearts of wild type animals (D). Myocyte cross-sectional area in FITC-labeled WGA stained sections was significantly increased in both risk and remote regions in C3 KO hearts after MI (E). Representative FITC-labeled WGA stained cross-sections (F). Scale bars 5 50 mm. Values are the mean 6 SEM. Sham WT (n 5 7), Sham C3 KO (n 5 6), WT MI wild type (n 5 9), and C3 KO MI (n 5 8). *, p <.05 vs. WT MI. Abbreviations: C3 KO, C3 knock out; DAPI, 4,6-diamidino-2-phenylindole; MI, myocardial infarction; WGA, wheat germ agglutinin; WT, wild type; a-sa, a-sarcomeric actin. Supporting Fig. 3A 3D). However, function in infarcted C3 KO mice was significantly impaired compared to WT with decreased EF and FS (Fig. 1A, 1B). This was associated with exacerbated dilation in C3 KO hearts compared to WT with increased ESA, EDA, LVALS, and LVALD (Fig. 1C 1F) and LVEDV, LVESV, LVIDD, and LVISD (Supporting Information Fig. 3A 3D). Thus, chronic myocardial remodeling after MI is exacerbated in C3 KO mice despite the absence of an effect on acute MI. C3 Deficiency Increases Scar Size and Remodeling The effect of C3 deficiency on LV dilation, remodeling, and failure after MI was further characterized, in Masson s VC AlphaMed Press 2014

5 2506 The Complement Cascade in Chronic Myocardial Infarction trichrome stained sections from WT and C3 KO hearts after MI or sham surgery (Fig. 2A). Morphometric analysis at sacrifice 4 weeks after MI demonstrated C3 deficiency significantly increased myocardial scar compared to WT (Fig. 2B). The increase in scar was accompanied by a decrease in viable myocardium within the risk region (Fig. 2C). Morphometry confirmed echocardiography data showing increased expansion index (Fig. 2D), thinned infarct wall, and exacerbated myocyte hypertrophy in risk and remote regions in the C3 KO compared to WT (Supporting Information Fig. 4, Fig. 2E, 2F). To examine the mechanisms by which C3 deficiency could impair the response to injury leading to LV dilatation and dysfunction, capillary density was assessed as an index of the heart s capacity to respond to increased stress [29]. Myocardial capillary density in risk and remote regions was measured in sections stained with the endothelial specific FITC-isolectin B4. Capillary density was similar in sham C3 KO and WT mice. Capillary density in WT hearts 28 days post-mi decreased progressively in remote and risk areas. This decrease was exacerbated in the infarcted C3 KO hearts with a significant decrease in the risk area a smaller insignificant decline in the remote area (Fig. 3A, 3B). The further decline in capillary density in the infarcted C3 KO risk area suggests neovascularization may be impaired and could contribute to exacerbation of MI. Heart failure is associated with an increase in apoptotic cell death that contributes to myocyte loss and decreased functional capacity. C3 deficiency had no effect on apoptosis (assessed by TUNEL staining) in the sham-operated group at the 7- or 28-day time points. Apoptosis was significantly increased in the C3 KO LV after MI compared to WT LV (Fig. 3C 3E). TUNEL positive cells in C3 KO LV after MI were found only in the risk region with no observed difference in the remote region (Supporting Information Fig. 5A, 5B). Significant TUNEL staining was seen in myocytes in border zone and risk regions 7 days after MI that then decreased substantially by 28 days and accounted for a small fraction of the total apoptotic cells (yellow arrow highlights an apoptotic myocyte, Fig. 3E; Supporting Information Fig. 5C). Increased apoptosis in the risk region would contribute to decreased viable myocardium in the risk region and impair regenerative processes to further aggravate injury. Figure 3. C3 deficiency decreases capillary density and increases apoptosis in infarcted mice. Capillaries in risk and remote regions of the left ventricle (LV) were labeled with FITC-conjugated isolectin B4 and counted. (A): Quantitative analysis of capillary density demonstrating decreased risk region capillary density in C3 KO mice after MI. (B): Representative fluorescent confocal images of FITC-isolectin stained crosssections from the border zone of wild type and C3 KO mice after MI or anterior LV after sham operation. Apoptotic cell death was assessed in WT and C3 KO hearts 7 (C) and 28 (D) days after MI and sham operation by counting the total number of TUNEL 1 cells in risk and remote regions. (C, D): TUNEL staining and apoptotic cell death is increased in the LV of C3 KO hearts after 7 and 28 days after MI. (E): Representative fluorescent confocal images of TUNEL stained apoptotic nuclei from WT and C3KO mice after MI or sham operation. Arrows denote apoptotic nuclei. (F): Representative image of TUNEL 1 myocyte undergoing apoptosis 28 days post-mi. Scale bars 5 50 mm. Values are the mean- 6 SEM (n 5 6 8). *, p <.05 vs. WT MI; N.S., not significant. Abbreviations: C3 KO, C3 knock out; DAPI, 4,6-diamidino-2-phenylindole; MI, myocardial infarction; TUNEL, terminal deoxynucleotidyl transferase dutp nick end labaling; WT, wild type; asa, a-sarcomeric actin. VC AlphaMed Press 2014 Reduced c-kit pos Cardiac Stem/Progenitor Cells and Impaired Regeneration in C3 KO Mice The heart has a limited capacity to regenerate new myocardium that may depend on interplay between peripheral c-kitpos cell populations resident in the BM and c-kit pos resident cardiac stem/progenitor cells (CSPC) [32 35]. Complement activation is known to affect hematopoietic stem and progenitor cell mobilization from the BM, however, the role of complement in mobilization post-mi is unknown. The decrease in capillary density in the risk region of C3 KO mice post-mi suggests altered angiogenesis/neovasculogenesis and implicates mobilization of peripheral endothelial progenitor cell (EPC) populations. After MI, the number of endogenous c-kit pos CSPCs increase within the first few days postinjury and remain elevated for several weeks [33]. Resident c-kit pos CSPCs in heart sections from WT and C3 KO mice 4 weeks after MI or sham operation were quantitated after identification by immunofluorescent confocal microscopy as previously STEM CELLS

6 Wysoczynski, Solanki, Borkowska et al Figure 4. Reduced numbers of CSPCs in hearts of C3 KO mice after infarction. c-kit 1 CSPCs in risk and remote regions of LV sections after MI or sham surgery were counted after detection by immunofluorescent staining and confocal microscopy. (A): Confocal microscopy images of c-kit 1 CSPCs in nonischemic region of LV section. (B): Cluster of c-kit 1 CSPCs in infarcted region of LV. (C, D): Number of c-kit 1 CSPCs were reduced in C3 KO mice LV compared to wild type controls 7 and 28 days, respectively, after MI. (E): Number of c-kit 1 CSPCs were lower in both risk and remote regions in C3 KO mice after MI. Values are the mean 6 SEM (n 5 6 8). *, p <.05 vs. WT MI. Abbreviations: CSPCs, cardiac stem/progenitor cells; C3 KO, C3 knock out; DAPI, 4 6-diamidino-2-phenylindole; LV, left ventricle; MI, myocardial infarction; WT, wild type; a-sa, a-sarcomeric actin. described [29]. These cells were characterized to be negative for the hematopoietic and lymphoid markers CD34 and CD45, supporting the identification of these cells as cardiac resident and excluding mast cells that express CD34. In sham operated mice low levels of c-kit pos CSPCs were identified with no differences observed between WT and C3 KO hearts at 7 and 28 days post-mi. C3 deletion also had no effect on the number of Iba-1 pos macrophages in remote, border, or infarct zones. This macrophage population did not contribute to the c-kit pos cell population with only approximately 0.002% of Iba-1 pos macrophages staining positively for c-kit (data not shown). In WT hearts, the number of c-kit pos CSPCs was markedly increased 7 days after MI and these levels declined significantly by 28 days post-mi (Fig. 4C 4E). In C3 KO hearts, the number of CSPCs was significantly reduced 7 and 28 days post-mi compared to those in WT mice (Fig. 4C 4E). Notably, C3 KO hearts levels were only slightly elevated and did not reach significance over sham operated C3 KO mice (less than twofold) (Fig. 4A 4E). Thus, the increase in c-kit pos CSPCs in the LV after MI was significantly attenuated in C3 KO mice 7 and 28 days after MI. Furthermore, the number of c-kit pos CSPCs in C3 KO LV after MI was reduced in both remote and risk regions (Fig. 4E). To begin to delineate the mechanism by which C3 deletion affected c-kit pos CSPC numbers we first examined whether the rate of c-kit pos CSPC proliferation was affected. c-kit pos VC AlphaMed Press 2014

7 2508 The Complement Cascade in Chronic Myocardial Infarction Figure 5. Impaired proliferation of CSPCs in hearts of C3 KO mice after infarction. c-kit 1 CSPC proliferation was determined by Ki67 staining of heart sections of C3 KO and WT mice after infarction or sham operation. (A): Confocal microscope image of proliferating c- kit 1 Ki67 1 CSPCs in nonischemic region. (B): Images of proliferating (Ki67 1 ) and quiescent (Ki67 2 ) c-kit expressing CSPCs in infarcted region of LV. (C): Reduced number of proliferating CSPCs in LV sections of C3 KO mice after MI. (D): Reduced proliferation of c-kit 1 CSPCs in both risk and remote regions in heart sections of C3 KO mice. Values are the mean 6 SEM (n 5 6 8). *, p <.05 vs. WT MI. Abbreviations: CSPC, cardiac stem/progenitor cells; DAPI, 4 6-diamidino-2-phenylindole; MI, myocardial infarction; WT, wild type; asa, a- sarcomeric actin. CSPC proliferation in LV sections was measured by staining with the proliferation/mitosis marker, Ki67 (Fig. 5A, 5B). Quantitative analysis of c-kit pos Ki67 pos cells in LV sections from sham operated mice showed no differences between WT and C3 KOs. c-kit pos Ki67 pos CSPCs were increased post-mi, however the level to which proliferating CSPCs increased in C3 KO hearts was significantly less than that in WT (Fig. 5C). The reduction of c-kit pos Ki67 pos cells in C3 KO hearts was consistently found in both the remote and risk regions (Fig. 5D). VC AlphaMed Press 2014 Thus, these findings indicate that c-kit pos CSPC proliferation was reduced in C3 KO hearts. c-kit pos CSPCs have the ability to proliferate and differentiate to cardiac myocyte precursors and adult myocytes [32 35]. Adult myocytes were considered terminally differentiated and did not divide implying that any new myocytes with an adult phenotype were of a stem/progenitor cell origin. However, recently, adult myocytes have been shown to divide [36]. Thus differentiating a sarcomeric actin (asa pos ) and dividing (BrdU pos ) STEM CELLS

8 Wysoczynski, Solanki, Borkowska et al Figure 6. Diminished cardiomyogenesis in C3 KO mice after MI. Newly formed myocytes, BrdU 1 asa 1 in LV sections of infarcted WT and C3 KO mice were counted after immunofluorescent staining and confocal microscopy. BrdU 1 asa 1 myocytes in the risk region (A) are shown in confocal microscopic images. Sections are counterstained with cardiac myocyte specific transcription factors GATA4 (B) and Nkx2.5 (C) and images acquired from the border zone to verify BrdU-labeled nuclei are of myocyte origin. Scale bars 5 20 mm. (D): Quantitative analysis of BrdU 1 asa 1 myoyctes in risk and remote regions of LV sections from WT and C3 KO mice after MI. Arrows denote BrdU 1 asa 1 myocytes. Values are the mean 6 SEM (n 5 6 8). *, p <.05 vs. WT MI. Abbreviations: BrdU, 5-bromo-2 -deoxyuridine; C3 KO, C3 knock out; DAPI, 4,6-diamidino-2-phenylindole; WT, wild type; asa, a sarcomeric actin. CSPCs which were small and of an immature phenotype (Fig. 6A; Supporting Information Fig. 5A, 5B) and large mature adult myocytes labeled with BrdU (Fig. 6A; Supporting Information Fig. 5C, 5D) were identified as newly formed BrdU pos asa pos myocytes [32, 36]. To further verify the BrdU labeled nuclei associated with asa pos cardiomyocytes were of cardiac origin, sections were stained for cardiac-specific transcription factors GATA4 and Nkx2.5. In Figure 6B and 6C, the BrdU labeling of myocyte nuclei are counterstained with both GATA4 and Nkx2.5 further demonstrating BrdU labeling as a consequence of DNA replication occurs in cardiomyocytes. The number of newly formed myocytes was determined by counting BrdU pos asa pos cells in WT and C3 KO LV sections after MI and sham surgery (Fig. 6A, 6D; Supporting Information Fig. 5). We found that in LV sections of infarcted C3 KO mice the number of newly formed myocytes was significantly reduced compared to WT. BrdUpos asa pos cells were reduced in both risk and remote regions (Fig. 6D). These data indicate that the regenerative response in C3 deficient mice is diminished and correlates with impaired LV function measured by echocardiography and LV remodeling. Impaired Mobilization of BM Stem/Progenitor Cells and Inflammatory Response in C3 KO Mice As noted above, complement activation affects mobilization but whether it participates in MI induced mobilization of BM stem/progenitor cells is unknown. Mobilization after infarction VC AlphaMed Press 2014

9 2510 The Complement Cascade in Chronic Myocardial Infarction Figure 7. Reduced mobilization of bone marrow stem/progenitor cells in C3 KO mice after MI. Mobilization of bone marrow cells 72 hours after MI and sham operation was determined in peripheral blood of WT and C3 KO mice. (A): Flow cytometry gating strategy for determination of Lin 2 Sca-1 1 c-kit 1 hematopoietic and Lin 2 CD45 2 Sca-1 1 or Lin 2 CD45 2 c-kit 1 nonhematopoietic stem cells. (B): Number of circulating CFU-GMs, Lin 2 Sca-1 1 c-kit 1 (SKL), Lin 2 CD45 2 Sca-1 1, and Lin 2 CD45 2 c-kit 1 was reduced in C3 KO mice after MI. Reduced mrna level for cardiac (Nkx2.5 and GATA4) (C) and pluripotent specific markers (Oct4, Nanog, Dppa3, and Rex-1) (D) in peripheral blood cells of C3 KO mice after MI, evaluated by real time polymerase chain reaction and marker specific primers. The number of WBC, NE, and LY in peripheral blood was measured before and 24, 48, 72 hours, and 7 days post-mi (E). Values are the mean 6 SEM (n 5 6 8). *, p <.05 vs. WT MI. Abbreviations: CFU-GM, colony forming unit-granulocytes/macrophage; C3 KO, C3 knock out; LY, lymphocytes; MI, myocardial infarction; NE, neutrophils; WT, wild type; WBC, white blood cells. VC AlphaMed Press 2014 STEM CELLS

10 Wysoczynski, Solanki, Borkowska et al in C3 KO mice did not correlate with size of the initial injury. Maximal mobilization of BM hematopoietic stem/progenitor cells occurs hours after MI as shown by the number of circulating clonogenic CFU-GM progenitors and early circulating Sca-1 pos c-kit pos Lin neg cells (SKL cells) hematopoietic stem cells as shown by flow cytometry (Supporting Information Fig. 6A, 6B). Notably, CFU-GM and SKL numbers were significantly reduced in C3 KO mice compared to WT from 48 to 96 hours after MI, whereas levels were significantly increased in WT mice (Supporting Information Fig. 6A, 6B; Fig. 7A, 7B). Importantly, the degree of BM mobilization in patients after MI correlates with long-term outcome measured by LV function [37]. Thus reduced SKL mobilization would contribute to exacerbated injury by attenuating regeneration. In addition to being a source of hematopoietic stem/progenitor cells, BM is also a source of pluripotent very small embryonic like stem cells (VSEL cells) and tissue committed stem/progenitor cells including cardiac and endothelial progenitors [38, 39]. These, cells are also mobilized in response to tissue injury including MI [38, 40, 41] and have been associated with myocardial regeneration [34, 42]. We used flow cytometry to evaluate the number of circulating Lin neg CD45 neg c-kit pos and Lin neg CD45 neg Sca-1 pos tissue committed and pluripotent stem cells (Fig. 7A). Similar to hematopoietic stem cells, populations of Lin neg CD45 neg c-kit pos and Linneg CD45 neg Sca-1 pos cells are robustly mobilized at 72 hours post- MI in WT mice. Notably, mobilization of these populations was also significantly reduced in infarcted C3 deficient mice (Fig. 7A, 7B). These results demonstrate that C3 is an important factor triggering mobilization of hematopoietic, VSEL, and tissuecommitted BM stem/progenitor cells after MI. Stem and progenitor cells mobilized after MI are enriched in subpopulations that express pluripotent and lineage specific factors that are considered important for tissue regeneration [38]. To confirm the enrichment of PB with VSELs and cardiac progenitor cells after MI, we measured mrna expression levels of cardiac specific transcription factors GATA-4 and Nkx2.5 (Fig. 7C) and pluripotency factors Oct-4, Nanog, Rex-1, Rif1, and Dppa3 (Fig. 7D in PB-derived cells harvested 72 hours after coronary ligation by qrt- PCR. We found that PB of WT mice after infarction was indeed enriched in cells containing mrna for these markers compared to sham operated animals. Notably, mrna levels for both pluripotency and cardiac transcription factors in PB were significantly reduced in infarcted C3 KO mice compared to WT (Fig. 7C, 7D). Complement is a first line contributor to innate immunity and its activation results in a strong inflammatory response and both complement and the inflammatory are activated with myocardial injury. C3 contribution to the inflammatory response was assessed by measuring WBC, NE, and lymphocytes (LY) in PB before and 24, 48, 72 hours, and 7 days after MI (Fig. 7E). WT mice responded to MI with increased WBCs, NEs, and LYs at 24 hours, which remained elevated through 72 hours and returned toward baseline levels by 7 days. In contrast, the increase in WBCs, NEs, and LYs with MI was blocked in C3 KO mice demonstrating C3 contributes significantly to the MI induced inflammatory response. DISCUSSION Our findings provide evidence of a novel role for complement in the chronic response to myocardial injury in which complement component C3 activation facilitates myocardial preservation and regeneration. Targeted C3 deletion in mice impairs the chronic response to MI leading to exacerbated dysfunction and remodeling, LV dilation, increased scar, and decreased viable myocardium within the scar. Importantly, these effects occurred despite full activation of C5 demonstrating C3 specifically plays an important role in the response to MI. These changes are accompanied by a constellation of contributing effects: increased apoptosis, decreased capillary density, and increased hypertrophy. The increase in resident c- kit pos CSPCs that has been shown to play a significant role in the myocardial response to injury was significantly attenuated at early and late time points in C3 KO hearts. The attenuated c-kit pos CSPC response in the C3 KO was due in part to decreased proliferation. Decreased total new and cycling c-kitpos CSPCs was consequently associated with decreased generation of new myocytes. This attenuated activation and expansion of c-kit pos CSPCs in the C3 KO was accompanied by reduced mobilization of distinct BM stem/progenitor populations after MI. In addition, C3 deletion significantly attenuated the MI inflammatory response. Thus this is the first report to demonstrate that C3 activation is necessary for mobilization of stem cell populations that activate resident c-kit pos CSPCs which may then contribute to formation of new myocardium in the chronic response to myocardial injury. This study in a chronic MI model is particularly relevant as complement activation has typically been shown to exacerbate myocardial injury [3, 43 48]. The absence of a significant effect on infarct size measured 24 hours post-mi in C3 KO hearts compared to WT, although suggesting C3 does not play a role in the initial extent of injury, implicates C5 as contributing significantly to the initial response as its activation with MI was unaffected by C3 deficiency. Interestingly, the similarity of acute injury in WT and C3 KO mouse occurred despite a significant attenuation of the inflammatory response. It may be interesting to postulate that the decrease in inflammatory response counterbalances attenuated stem cell mobilization. This is also significant as it can thus be stated that the effects observed throughout the chronic response to injury were not due to differences in acute injury. The eventual further deterioration of function and remodeling in the C3 KO heart after MI is likely a consequence of the absence of C3 influence at earlier time points. Attenuated mobilization, CSPC recruitment and expansion in the risk region, and increased apoptosis at earlier time points clearly would contribute to the overall outcome at 28 days. This timing is relevant as blockade of CC activation is a therapeutic approach to limit MI. This approach primarily considers the immune and inflammatory response, however, our studies clearly demonstrate that the CC server beneficial functions. However, chronic injury in the infarcted C3 KO hearts measured 4 weeks post-mi was exacerbated compared to WT hearts with increased LV dysfunction and dilatation implying further progression towards heart failure. Changes in key indices, increased myocyte hypertrophy, decreased capillary density, and increased apoptosis contribute to this dysfunction and dilatation. EPC mobilization after MI [49] has been associated with increased neovascularization [50], increased EPC recruitment, and improved function [51, 52]. Whereas the role of complement in HSPC retention and mobilization in BM is increasingly understood, its impact on these actions and in VC AlphaMed Press 2014

11 2512 The Complement Cascade in Chronic Myocardial Infarction particular for those of EPCs in the context of MI is unknown. However, as SKL mobilization was attenuated in the C3 KO after MI, EPC mobilization may also be impaired. Impaired EPC mobilization may contribute to decreased capillary density seen in the C3 KO post MI. Morphometric analysis of heart sections from C3 KOs indicated less viable myocardium in the risk region. This was correlated with impaired regeneration measured by formation of new myocytes indicated by BrdU pos asa pos immunofluorescent staining. The reduced number of new myocytes was associated with decreased c-kit pos CSPCs in the LV of C3 KO mice 7 days and 4 weeks after MI. This relationship between the number of activated c-kit pos CSPCs, the regenerative potential, and improved functional outcome, has been reported by others [29, 31 33]. The decreased number of CSPCs was likely a consequence of several factors, increased apoptosis, reduced proliferation of CSPCs and attenuated mobilization of stem/progenitors from the periphery and engraftment into infarcted C3 KO hearts. The impaired mobilization in the C3 KO is an important factor that contributes to several consequences of regeneration. Recently, c-kit pos BM cells were found to be important for increases in new myocytes and EPCs also home and engraft to injured myocardium [34, 53]. Thus, attenuated mobilization would limit c-kit pos CSPC expansion, activation, and formation of new myocardium. Taken together these findings demonstrate C3 is a key factor that promotes myocardial preservation and regeneration after MI. Initial studies in acute MI models demonstrated that blocking CC activation was beneficial and reduced the extent of injury [5, 9]. Studies focused on the C5 component, which upon cleavage, generates anaphylatoxin C5a and C5b initiating formation of the MAC on the surface of cells within the infarcted region. The strategy of blocking C5 activation with monoclonal antibodies that mask the cleavage site was successful in experimental models of myocardial ischemia/reperfusion in mice, rats and pigs [5, 8, 54]. Systemic C5 inhibition in clinical studies has resulted in mixed results. Pexelizumab (C5 blocking antibody), tested in COMPLY, COMMA, PRIMO- CABG, and APEX-AMI trials did not show significant decreases in infarct size [10 13]. These findings raised concerns whether complement blockade at C5 activation is sufficient to reduce injury and further therapies should focus on blocking directly C5a and MAC formation or upstream components of the CC. Experimental models of MI in mice using a C3aR antagonist have shown no beneficial effect on the size of the injury measured acutely [8]. C5 activation after MI in the C3 KO demonstrates the importance of alternate C5 convertaseindependent pathways, such as thrombin activation with MI [55]. Our studies in C3 KO mice with permanent coronary ligation confirmed that C3 also has no effect on the size of the injury measured acutely 24 hours after MI. In the past decade, several studies have shown the myocardium has the capacity to regenerate with the creation of new myocytes, disproving the paradigm that the myocardium is post mitotic and unable to regenerate. It is now clear the heart has the ability to regenerate with some discordance regarding the rate with which this occurs [34, 56]. Regeneration depends on the presence of c-kit pos resident CSPCs that, in response to injury, have the ability to proliferate and differentiate into cells expressing adult myocyte markers (amhc, ctni, asa) [31, 32]. C3 KO mice 4 weeks post-mi have more VC AlphaMed Press 2014 scar tissue in the LV and less viable myocardium in the risk region. The role of C3 in this remodeling process can be a consequence of increased myocyte survival in the infarct and increased regenerative response with formation of new myocardium including vasculature and myocytes. Based on our findings C3 would contribute to both functions to preserve myocardium. We found increased apoptosis in heart sections of C3 KO mice that demonstrates C3 plays a protective rather than detrimental role in chronic MI. In support of this observation it has been shown that both C3a and C5a increase survival of mesenchymal stem cells in response to hydrogen peroxide treatment [57] and we also find that the C3aR is present and functional in CSPCs. We also evaluated the role of C3 in formation of new myocytes. We found that C3 KO hearts after MI have fewer newly formed BrdU pos asa pos myocytes at 4 weeks post MI in the ischemic zone than in WT. This provides support for C3 involvement in regeneration of heart muscle in addition to increased survival. A role for C3 in tissue regeneration has also been reported in liver and brain models of injury [15, 16, 21, 22, 58]. Importantly, C3a receptors, are present in myocytes, fibroblasts and CSPCs and functional in CSPCs providing the opportunity for C3a to act directly on myocardium. These findings constitute a novel and beneficial role of C3 in the response to myocardial injury. After MI, the number of c-kit pos CSPCs in the heart rapidly increases. However, the molecular mechanism responsible for expansion of the pool of resident stem cells in response to injury is poorly understood. Studies have implicated cells mobilized from the BM or transplanted to play a role in activating resident c-kit pos CSPCs however the mechanism responsible for this is unknown [34]. Our finding that the number of c-kit pos CSPCs 4 weeks after MI were significantly attenuated in C3 KO mice compared to WT to an extent where they were not significantly increased compared to the Sham KO mice, suggests C3 is a significant factor in regulating c-kit pos CSPC activation. This difference in c-kit pos CSPCs in C3 KO versus WT hearts is partly attributed to decreased proliferation defined by the lower number of c-kit pos Ki67 pos CSPCs. Thus the action of C3 on CSPC proliferation after MI is an important link between activation of complement and expansion of c-kit pos CSPCs. The ability of BM stem cells to facilitate regeneration of the heart after MI has been demonstrated in animal models and in several clinical studies [59]. BM is a source of not only hematopoietic but also pluripotent (VSELs), cardiac, endothelial, and mesenchymal stem/progenitor cells that have therapeutic potential for regenerating infarcted myocardium [38, 39, 53, 60]. These cells are mobilized from the BM into PB in response to MI as demonstrated in murine models and in patients [38, 40, 41, 61]. Importantly, the degree of BM stem cell mobilization post-mi is positively correlated with LV function in patients [37]. We find that optimal mobilization of BM hematopoietic stem/progenitor cells after permanent coronary artery ligation in WT mice occurs 72 hours after surgery. Within this time frame mobilization of hematopoietic, pluripotent, and cardiac stem cells is severely attenuated in C3 KO mice compared to WT controls demonstrating that C3 is necessary for mobilization of BM cells after MI. We have previously shown that G-CSF and AMD3100 activate the CC where it then promotes mobilization [23, 24, 62 64]. Thus a role for C3 in myocardial regeneration can be derived from its effect STEM CELLS

12 Wysoczynski, Solanki, Borkowska et al on stem/progenitor cell mobilization. The subsequent involvement in myocardial regeneration is, however, controversial. Both direct and indirect actions of these different stem cell populations on myocardial regeneration and CSPC activation have been reported. Direct effects, cardiomyogenesis, have been demonstrated for Sca1 or cardiac side population [12, 65, 66] VSELS and SKL cells [60]. Importantly, we show here for the first time evidence that C3 promotes mobilization of several types of BM derived stem/progenitor cells in response to MI. The attenuated inflammatory response is concordant with attenuated stem cell mobilization highlighting the importance of C3 activation for both. The relative contribution of C3 and C5 to injury may be illustrated here where acutely attenuated C3 has no effect on C5 activation yet results in similar extent of injury. This suggests C5 may play the predominant role and is justifiably a therapeutic target. The relative contribution of inflammatory response to injury versus remodeling and regeneration is complex as several subpopulations of inflammatory cells including Gr1 low monocytes will contribute to the healing phase with myofibroblast recruitment, collagen production, and angiogenesis [67]. Thus attenuated contribution of this Gr1 low population to late phase responses associated with healing may contribute to the attenuated response to MI in the C3 KO. CONCLUSION In conclusion, our findings demonstrate that C3 deficiency has no effect on acute injury after permanent LAD ligation, however, C3 is necessary to preserve myocardial function and myocardium and prevent adverse remodeling in a chronic MI model. C3 contributes to myocardial survival and to CSPC activation as C3 KO hearts have reduced numbers of c-kit pos CSPCs due to increased apoptosis and reduced proliferation after MI. Mobilization of hematopoietic, VSEL, and tissuecommitted BM stem/progenitor cells after MI was also found to require C3 providing an alternate mechanism that can contribute to the formation of new BrdU pos asa pos myocytes and myocardial regeneration. Dysfunction and adverse remodeling in the C3 KO despite an attenuated inflammatory response suggests important components of the healing phase may play a role in the chronic phase of C3 action. All together, these data indicate that C3 is an important factor in regeneration and preservation of myocardium and myocardial function in response to MI. These findings provide the first evidence that the complement system is important for the mobilization and activation of cardiac stem/progenitor cells that promote myocardial regeneration after MI. ACKNOWLEDGMENTS This work was supported by NIH grants RO1HL and 2RO1HL to G.R., 2RO1DK and RO1HL to M.Z.R., and AHA 13SDG to M.W. AUTHOR CONTRIBUTIONS M.W. and G.R.: concept and design, financial support, data analysis and interpretation, manuscript writing, and final approval of manuscript; M.S., S.B., P.v.H., K.R.B., and S.D.P.: collection and assembly of data; M.Z.R.: concept and design. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST The authors indicate no potential conflicts of interest. REFERENCES 1 Arumugam TV, Magnus T, Woodruff TM et al. Complement mediators in ischemiareperfusion injury. Clin Chim Acta 2006;374: Amara U, Rittirsch D, Flierl M et al. Interaction between the coagulation and complement system. Adv Exp Med Biol 2008; 632: Hill JH, Ward PA. The phlogistic role of C3 leukotactic fragments in myocardial infarcts of rats. J Exp Med 1971;133: Crawford MH, Grover FL, Kolb WP et al. Complement and neutrophil activation in the pathogenesis of ischemic myocardial injury. Circulation 1988;78: Vakeva AP, Agah A, Rollins SA et al. 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