Transplantation of cardiac progenitor cells ameliorates cardiac dysfunction after myocardial infarction in mice

Cardiac progenitor cells are a potential source of cell therapy for heart failure. Although recent studies have shown that transplantation of cardiac stem/progenitor cells improves function of infarcted hearts, the precise mechanisms of the improvement in function remain poorly understood. The present study demonstrates that transplantation of sheets of clonally expanded stem cell antigen 1–positive (Sca-1–positive) cells (CPCs) ameliorates cardiac dysfunction after myocardial infarction in mice. CPC efficiently differentiated into cardiomyocytes and secreted various cytokines, including soluble VCAM-1 (sVCAM-1). Secreted sVCAM-1 induced migration of endothelial cells and CPCs and prevented cardiomyocyte death from oxidative stress through activation of Akt, ERK, and p38 MAPK. Treatment with antibodies specific for very late antigen-4 (VLA-4), a receptor of sVCAM-1, abolished the effects of CPC-derived conditioned medium on cardiomyocytes and CPCs in vitro and inhibited angiogenesis, CPC migration, and survival in vivo, which led to attenuation of improved cardiac function following transplantation of CPC sheets. These results suggest that CPC transplantation improves cardiac function after myocardial infarction through cardiomyocyte differentiation and paracrine mechanisms mediated via the sVCAM-1/VLA-4 signaling pathway.

Accumulating evidence has suggested that myocardial regeneration is a promising therapy for various heart diseases (1). Recently, several groups, including our own, have reported that adult hearts contain cardiac stem/progenitor cells that can differentiate into functional cardiomyocytes in vitro and in vivo (2–6). Transplantation of cardiac stem/progenitor cells has been shown to improve cardiac function via newly formed cardiomyocytes and blood vessels (2, 7). On the other hand, it has been reported that when noncardiac stem cells are transplanted, paracrine factors play a major role in the improvement of cardiac function (8, 9). Several preclinical reports and clinical trials have demonstrated that intracoronary or intramyocardial injection of bone marrow–derived cells attenuates cardiac dysfunction following acute and chronic myocardial infarction (MI) (10–12). However, it is not known whether cardiac stem/progenitor cells are superior to other noncardiac stem/progenitor cells. Furthermore, it remains unclear to what extent paracrine effects or transdifferentiation of cardiac stem/progenitor cells contributes to beneficial effects on cardiac function.

Transplanted cells are the source of paracrine factors or newly formed cardiomyocytes, and the survival of grafted cells is a critical issue. The majority of the grafted cells have been reported to disappear within 1 week after transplantation when directly injected into ischemic hearts (9, 13), suggesting that alternative strategies to facilitate survival of grafted cells are required. We developed temperature-responsive culture dishes that were covalently grafted with the temperature-responsive polymer poly(N-isopropylacrylamide) (PIPAAm) (14). Lowering the temperature induces a rapid surface transition from hydrophobic (cell adhesive) to hydrophilic (non–cell adhesive), which results in the release of contiguous viable cell sheets with full preservation of cell-to-cell connections and adhesion proteins without using any enzymatic digestion (15). To date, cell sheet transplantations of skeletal myoblasts (16), mesenchymal stem cells derived from adipose tissue (17), and menstrual blood (18) have been reported to improve cardiac function in animal MI models. However, the precise mechanisms of the improvement, which include mutual interactions between host tissue and transplanted cells, remain poorly understood.

The present study demonstrates that transplanted cell sheets of clonally expanded stem cell antigen 1–positive (Sca-1–positive) cells (CPCs) differentiated into cardiomyocytes and vascular cells and prevented cardiac remodeling after MI. CPCs secreted soluble VCAM-1 (sVCAM-1), which facilitated engraftment and migration of CPCs from cell sheets into host myocardium and improved cardiac function after MI via angiogenic and cardioprotective effects mediated by paracrine mechanisms.

Establishment and character of CPCs. Since primary isolated Sca-1–positive cells, which were derived from adult murine hearts, consisted of several cell populations, including cardiac stem/progenitor cells, hematopoietic cells, and endothelial cells (ref. 4 and our unpublished observations), clonal cells were initially established from cardiac Sca-1–positive cells. A total of 10⁴ primary isolated Sca-1–positive cells derived from adult murine hearts were plated onto 10-cm culture dishes. After repeated limited dilutions, clonal cell lines were established. The efficiency of cloning was approximately 0.1%. The clonal cells were expanded for more than 500 population doublings (Figure 1A). Flow cytometric analysis revealed that almost 100% of cells expressed Sca-1, CD29, and CD44, approximately 20% of cells expressed CD34, and no cells expressed CD31, CD45, and c-kit throughout the culture passages (Table 1 and Supplemental Figure 1; supplemental material available online with this article; doi: 10.1172/JCI37456DS1), suggesting that the clonal cells were not homogenous. Under culture conditions of 80% confluency, the clonal cells expressed cardiac transcription factors, such as Nkx2.5 and GATA4 (Figure 1B), but not cardiac contractile proteins. These cell phenotypes remained unchanged throughout the culture passages. Gene profiles and cell-surface marker analyses revealed that CPCs were similar to primary isolated Sca-1–positive cells in analyses previously reported (4). Therefore, CPCs possessed features almost identical to those of intrinsic cardiac stem/progenitor cells. When CPCs were cultured under confluent conditions for 4 weeks, expression levels of Nkx2.5 and GATA4 were upregulated and expressions of myocyte enhancer factor 2C (MEF2C), atrial natriuretic peptide (ANP), β-myosin heavy chain (β-MHC), and sarcomeric α-actinin were detected at mRNA and protein levels (Figure 1, B and C). These results suggested that CPCs differentiated into immature cardiomyocytes in vitro. However, the cells did not exhibit spontaneous beating. To examine the cardiac differentiation potency of CPCs in vivo, 2.0 × 10⁶ CPCs labeled with red fluorescent protein (RFP) were injected directly into the infarcted myocardium within 5 minutes after left coronary artery ligation. At 4 weeks after transplantation, several GATA4-expressing RFP⁺ cells were recognized in the border areas and some RFP⁺ cells expressed sarcomeric α-actinin in a fine striated pattern, which suggested that CPCs differentiated into mature cardiomyocytes in vivo (Figure 1, D and E). However, transplanted CPC-derived RFP expression in the infarcted heart was very weak 1 week after transplantation compared with immediately after transplantation (Supplemental Figure 2). These results suggest that direct intramyocardial injection is not an ideal method for efficient engraftment.

Figure 1

Character of CPCs. (A) CPCs were expanded more than 500 population doublings (P.D.) over a 1-year period. (B and C) Cardiac mRNA and protein expressions in CPCs. (B) Left panels show RT-PCR. Noncontiguous lanes from the same gel were spliced together into a composite band. The thin white line indicates the spliced point. Right panels show Western blot. (C) Immunofluorescent images of CPCs 4 weeks after starting culture under confluent conditions. Scale bars: 100 μm. (D and E) Confocal microscopic images of the infarcted heart 4 weeks after direct injection of RFP⁺ CPCs. (D) GATA4-expressing RFP⁺ cells (arrowheads) were recognized in the infarcted region. (E) Some RFP⁺ cells (arrowheads) expressed sarcomeric α-actinin in the infarcted area. Scale bars: 5 μm.

Table 1

The percentage of cell-surface antigens

Cell sheet transplantation. To elucidate the functional benefits of CPC transplantation in the infarcted heart compared with effects of noncardiac stem/progenitor cells, cell sheet transplantation methods were utilized. RFP⁺ CPCs or adipose tissue–derived mesenchymal cells (ATMCs) isolated from GFP mice were cultured on temperature-responsive culture dishes at 37°C. The monolayered cell sheet was collected by decreasing the temperature at which it was cultured to 20°C. Final cell counts and areas of monolayered cell sheets prior to transplantation were 2.0 ± 0.2 × 10⁶ cells and 80.1 ± 3.3 mm² in CPCs and 1.9 ± 0.1 × 10⁶ cells and 79.3 ± 2.0 mm² in ATMCs, respectively (n = 5). The mice were randomly assigned into 3 groups: mice transplanted with monolayered CPCs (CPC group), mice transplanted with monolayered ATMCs (ATMC group), and mice that were not transplanted (MI group). Within 5 minutes after left coronary artery ligation, monolayered CPC or ATMC sheets were transplanted over the infarcted area and cardiac function was examined by echocardiography every week. Echocardiographic analysis revealed that LV diastolic dimension (LVDd) and LV systolic dimension (LVDs) were significantly decreased at 4 weeks and fractional shortening (FS) was markedly improved 3 weeks after transplantation in the CPC group compared with the MI and ATMC groups. These results suggested that transplantation of CPC sheets inhibited cardiac remodeling and improved cardiac function following MI (Figure 2A). Furthermore, LV end-diastolic pressure (LVEDP) and +dp/dt, as determined by catheter, were markedly improved in the CPC group compared with the remaining 2 groups at 4 weeks (Figure 2B). In contrast, in the ATMC group, LVDs was significantly smaller and FS was better 1 week after transplantation compared with the MI and CPC groups. However, these favorable effects were not observed 2 weeks after transplantation, and cardiac remodeling and dysfunction progressed in a manner similar to that in the MI group (Figure 2A). The fibrotic area, which was evaluated by Masson trichrome staining 4 weeks after transplantation, was significantly smaller in the CPC group compared with the other 2 groups (Figure 3A). At 1 week after transplantation, more vWF-positive blood vessels were observed in the border area of the ATMC group than in the MI and CPC groups (Figure 3B). At 4 weeks, a greater number of vessels were detected in the CPC group than in the MI and ATMC groups (Figure 3C). Furthermore, when lectin perfusion assay was performed at 4 weeks, more lectin-positive blood vessels were detected in the border area of the CPC group compared with the remaining 2 groups (Supplemental Figure 4). In contrast, there were few inflammatory cells in the border area of each group at 4 weeks (Supplemental Figure 5).

Figure 2

Effects of CPC sheet transplantation on cardiac function after MI. (A) Echocardiographic analysis. CPC sheet transplantation inhibited dilatation of LVDd and LVDs and improved FS 3 weeks later. ATMC transplantation inhibited dilatation of LVDs and FS reduction at 1 week, but not afterward. ^†P < 0.05 versus MI or MI plus CPCs (n = 10 per group). ^‡P < 0.01 versus MI or MI plus ATMCs (n = 10 per group). ^§P < 0.05 versus MI or MI plus ATMCs (n = 10 per group). (B) Catheterization analysis at 4 weeks after transplantation. CPC sheet transplantation improved LVEDP and +dp/dt compared with that in the MI or MI plus ATMC groups (n = 5). Data are shown as mean ± SEM.

Figure 3

Immunohistochemical analysis of transplanted hearts. (A) Masson trichrome staining. The fibrotic area at 4 weeks after transplantation was calculated and is shown in the graph (n = 6). Lower panels show representative images. Scale bars: 1 mm. (B and C) Endothelial cells were identified by immunohistochemical staining with anti-vWF Ab in the border zone of the infarcted hearts 1 week (B) and 4 weeks (C) after transplantation. Lower panels show representative images. The vessel number was quantified and is depicted in the graph (n = 6). HPF, high-power field. Scale bars: 100 μm. Data are shown as mean + SEM.

Cell survival and differentiation. Immunohistochemical analysis showed that many transplanted CPCs were present in the middle of the LV wall, including the normal and injured areas (normal area, 28.3 ± 9.7 cells/mm²; injured area, 235.9 ± 75.1 cells/mm²), after 4 weeks (Figure 4, A–C). This suggested that CPCs migrated from the epicardial cell sheet into the ventricular myocardium following transplantation. Conversely, GFP⁺ ATMCs were not observed in the myocardium (data not shown). Western blot analysis, using Abs against fluorescent proteins, revealed that approximately 20% of transplanted CPCs remained 4 weeks after transplantation, whereas only approximately 0.8% of transplanted ATMCs remained (Supplemental Figure 3). Furthermore, approximately 30% of RFP⁺ cells expressed sarcomeric α-actinin in a fine striated pattern, and some RFP⁺ cells also formed blood vessel structures (Figure 4, D–F). Because cardiomyocytes have the ability to fuse with surrounding noncardiomyocytes (19), the possibility that CPCs acquired cardiomyogenic features following fusion with existing cardiomyocytes was examined. When RFP⁺ CPC sheets were transplanted into hearts of GFP mice immediately following MI, approximately half of the α-actinin–expressing cells expressed GFP in injured areas 4 weeks after transplantation (Supplemental Figure 6, A, B, and E). In the normal areas, all of the α-actinin–expressing RFP⁺ cells expressed GFP (Supplemental Figure 6, C–E). To further ensure the occurrence of cell fusion, sheets of nonlabeled CPCs derived from male mice were transplanted into the infarcted hearts of female mice. At 4 weeks after transplantation, cells exhibiting a fine striated pattern possessed 3 X chromosomes and a Y chromosome in the nucleus (Supplemental Figure 7). These findings suggested that CPCs differentiated into cardiomyocytes via cell fusion–dependent and –independent mechanisms. Because approximately 20% of transplanted CPCs remained at 4 weeks (Supplemental Figure 3), approximately 4.0 × 10⁵ out of approximately 2.0 × 10⁶ transplanted CPCs were thought to survive and undergo engraftment. As approximately 30% of survived CPCs expressed cardiac contractile proteins (Figure 4, C and E), approximately 1.2 × 10⁵ CPCs were estimated to differentiate into cardiomyocytes. Since approximately half of the cardiac protein–expressing cells resulted from cell fusion with existing cardiomyocytes (Supplemental Figure 6E), CPC sheet transplantation was estimated to create approximately 0.6 × 10⁵ new cardiomyocytes in the entire heart. The number of cardiomyocytes in an adult murine heart has been estimated to be 3 × 10⁶ (20). Therefore, approximately 5% of cardiomyocytes were regenerated and might have contributed to improved cardiac function by CPC sheet transplantation.

Figure 4

Cell survival and differentiation of transplanted cells. (A) Fluorescent microscopic images of infarcted heart 4 weeks after RFP⁺ CPC sheet transplantation. Left sides of panels show endocardial area. Right sides of panels show epicardial area. Scale bars: 250 μm. (B) Confocal microscopic images of infarcted heart 4 weeks after CPC sheet transplantation (sarcomeric α-actinin, green; RFP, red; Topro, blue; yellow in merged images). Left panel shows normal area. Right panel shows injured area. Arrowheads indicate RFP⁺ cells. Scale bars: 5 μm. (C) Number of RFP⁺ cells were quantified and shown in the graph (n = 5). (D and E) Transplanted RFP⁺ cells expressed sarcomeric α-actinin in a fine striated pattern (D) and formed vessel structures around α-actinin–positive myocardium (E). Nuclei were stained with Topro. Arrowheads indicate vessel structures. Scale bars: 5 μm. (F) Percentages of α-actinin–positive cells or vessel structure–forming cells in existing RFP⁺ cells were calculated and shown in the graph (n = 5). Data are shown as mean + SEM.

Secretion of growth factors. Recent reports have suggested that cell transplantation improves cardiac function after MI through the release of humoral factors (8, 9). Analyses of conditioned medium (CM) from CPCs and ATMCs using a cytokine Ab array revealed that sVCAM-1 was more abundant in CPCs, while VEGF was dominantly expressed in ATMCs (Table 2). Western blot analysis of whole-cell lysates and ELISA of CM confirmed altered expressions of VCAM-1/sVCAM-1 and VEGF between CPCs and ATMCs (Figure 5A). VCAM-1 expression was almost identical among the 3 groups at 1 week. However, at 4 weeks after transplantation, expression levels remained high in the CPC group compared with the other 2 groups (Figure 5B). Time course of VCAM-1 expression was consistent with improved cardiac function (Figure 2A). Conversely, VEGF expression was significantly upregulated at 1 week in the ATMC group compared with the other groups. However, at 4 weeks, expression levels were similar among the 3 groups (Figure 5B). This was consistent with observations that transplanted ATMC sheets improved cardiac function at 1 week but not at 4 weeks (Figure 2A). The concentrations of sVCAM-1 and VEGF in peripheral blood remained unchanged in all groups 1 and 4 weeks after transplantation (Supplemental Figure 8).

Figure 5

Secreted factor–mediated angiogenesis. (A) Left panel shows Western blot analysis results using whole-cell lysates of cultured CPCs and ATMCs. Right panel shows the results of sVCAM-1 ELISA using CM from cultured CPCs and ATMCs (n = 3). (B) Western blot analysis results of VCAM-1 and VEGF expression in heart after MI. Normal heart was used as a control. Left panel shows representative images. Right panels show quantification results of VCAM-1 and VEGF expression (n = 3). (C) Scratch-wound assay. CPC-derived CM enhanced endothelial migration (n = 3). Lower panels show representative images (n = 3). Scale bars: 500 μm. (D) CPC-derived CM enhanced endothelial tube formation. Tube length was quantified and is shown in the graph (n = 3). Lower panels show representative images. Scale bars: 500 μm. miR, miRNA. Data are shown as mean + SEM.

Table 2

Results of cytokine Ab array

CPC-derived sVCAM-1–mediated angiogenesis and cardioprotective effects. CPC-derived CM and sVCAM-1 induced greater endothelial cell migration and tube formation compared with control medium (Figure 5, C and D). sVCAM-1–depleted CM, which was obtained from CPCs transfected with VCAM-1–specific microRNA (miRNA) plasmid vector (Supplemental Figure 9, A and B), induced significantly less endothelial cell migration (Figure 5C) and tube formation (Figure 5D) compared with CPC-derived CM. This suggested that angiogenic activity of CPC-derived CM was mediated at least in part by sVCAM-1. Subsequently, the protective effects of CPC-derived CM and sVCAM-1 on cardiomyocytes were analyzed. When cardiomyocytes were pretreated with CPC-derived CM or sVCAM-1, H₂O₂-induced damage of cardiomyocytes was significantly reduced (Figure 6A). The cardioprotective effects of CPC-derived CM were abolished by pretreatment of cardiomyocytes with Abs against very late antigen-4 (VLA-4, also known as α₄β₁ integrin), a principal coreceptor of sVCAM-1 (Figure 6A), or sVCAM-1–depleted CM (Figure 6B). These results suggested a crucial role for sVCAM-1/VLA-4 in cardiomyocyte survival.

Figure 6

sVCAM-1–mediated cardioprotective effects and CPC migration. (A and B) Cardiomyocyte viability following treatment with H₂O₂ was measured by MTT assay (n = 3). IgG isotype Abs were used as a control (A). (C) sVCAM-1 induced phosphorylation of FAK, Akt, ERK, and p38 MAPK in a dose-dependent manner. (D) Cardiomyocyte viability following treatment with H₂O₂ was measured by MTT assay (n = 4). SB, SB2035800; PD, PD98059. (E) CPC-derived CM induced phosphorylation of FAK, Akt, ERK, and p38 MAPK. Anti–VLA-4 Abs inhibited phosphorylation of FAK, Akt, and ERK induced by CPC-derived CM, but not phosphorylation of p38 MAPK. Arrow indicates appropriate size of phosphorylated p38 MAPK. (F) Cardiomyocyte viability following treatment with H₂O₂ was measured by MTT assay (n = 4). (G and I) CPC migration was measured using the scratch wound assay (n = 3). IgG isotype Ab was used as a control (G). (H) Anti–VLA-4 Abs inhibited phosphorylation of ERK and p38 MAPK of CPCs, but not Akt. Activity of p38 MAPK, but not Akt or ERK, was upregulated by sVCAM-1 treatment. Data are shown as mean + SEM.

Integrin-mediated signals influence cardioprotective effects of sVCAM-1. Integrin-mediated signaling induces cell migration and survival by activating various kinases, such as focal adhesion kinase (FAK), Akt, ERK1/2, and p38 MAPK (21, 22). CPC-derived CM and sVCAM-1 induced phosphorylation and activation of FAK, Akt, ERK, and p38 MAPK in neonatal rat cardiomyocytes (Figure 6, C and E). When cardiomyocytes were pretreated with inhibitors of Akt, PI3K (wortmannin), p38 MAPK (SB203580), or ERK (PD98059), the cardioprotective effects of CPC-derived CM and sVCAM-1 were significantly inhibited (Figure 6, D and F). When cardiomyocytes were pretreated with anti–VLA-4 Abs prior to culturing in CPC-derived CM, phosphorylation of FAK, Akt, and ERK but not p38 MAPK was inhibited (Figure 6E). This suggests that the protective effects of CPC-derived CM on cardiomyocytes were achieved through sVCAM-1/VLA-4–mediated activation of Akt and ERK as well as VLA-4–independent activation of p38 MAPK.

sVCAM-1–induced migration of CPCs. Because a large number of transplanted CPCs migrated from the epicardial cell sheet to the ventricular myocardium following transplantation (Figure 4A), the effects of CPC-derived CM and sVCAM-1 on CPC migration were analyzed. When treated with CPC-derived CM or sVCAM-1, CPC migration was promoted, and anti–VLA-4 Abs or sVCAM-1 depletion markedly inhibited CM-induced migration of CPCs (Figure 6G). When CPCs were treated with sVCAM-1, phospho–p38 MAPK expression was significantly increased. However, expression of phospho-Akt and phospho-ERK remained unchanged. Phosphorylation of p38 MAPK was significantly inhibited by anti–VLA-4 Ab treatment (Figure 6H), which suggested that CPCs activated p38 MAPK through VLA-4. SB203580 inhibited CPC-derived CM- and sVCAM-1–induced CPC migration compared with the control (Figure 6I). These findings suggest that CPCs secreted sVCAM-1 and induced CPC migration via the VLA-4/p38 MAPK signaling pathway. Moreover, when VCAM-1 expression was downregulated, CPC viability was significantly decreased and apoptosis increased (Supplemental Figure 10), suggesting that VCAM-1 might be important for CPC survival.

VLA-4 signaling plays a crucial role in the beneficial effects of CPC sheet transplantation. The present findings suggest that CPC-secreted sVCAM-1 induced angiogenesis as well as CPC migration and survival and protected cardiomyocytes via VLA-4 in vitro. Subsequently, VLA-4 signaling was analyzed to determine its role in improved cardiac function following transplantation of CPC sheets. Because echocardiographic analysis revealed improved cardiac function 3 weeks after transplantation (Figure 2A), i.p. injection of anti–VLA-4 Abs was performed daily from 2 to 3 weeks after CPC sheet transplantation. At 4 weeks after transplantation, injection of anti–VLA-4 Abs significantly attenuated the beneficial effects of CPC sheet transplantation on cardiac function, fibrosis, and angiogenesis (Figure 7, A–D). The number of RFP⁺ CPCs in the infarcted area was also markedly decreased following treatment with anti–VLA-4 Abs (Figure 7E). In contrast, anti–VLA-4 Ab treatment did not affect cardiac function, fibrotic area, or blood vessel number in nontransplanted MI mice (Supplemental Figure 11). These findings suggest that CPC sheet transplantation improved cardiac function of infarcted hearts through VLA-4–mediated angiogenesis as well as survival and migration of transplanted CPCs.

Figure 7

The roles of VLA-4 signaling on CPC sheet transplantation-mediated improved cardiac function. Analysis of cardiac function by echocardiography (A, n = 5) and catheterization (B, n = 5). Anti–VLA-4 Ab treatment inhibited the reduction of LVDd, LVDs, and LVEDP and the improvement of FS and +dp/dt by CPC sheet transplantation. Isotype Ab was used as a control. ^†P < 0.05 versus anti–VLA-4 Abs (n = 5 per group). ^‡P < 0.01 versus anti–VLA-4 Abs (n = 5 per group). (C) Masson trichrome staining. The fibrotic area 4 weeks after transplantation was calculated and is shown in the graph (n = 5). Anti–VLA-4 Ab treatment inhibited the reduction of fibrotic area following CPC sheet transplantation. Lower panels show representative images. Scale bars: 1 mm. (D) vWF staining. The number of vWF-positive vessels in the border area was counted and is shown in the graph (n = 5). Anti–VLA-4 Ab treatment inhibited the increased number of vessels in the border area following CPC sheet transplantation. Lower panels show representative images. Scale bars: 100 μm. Nuclei were stained with hematoxylin. (E) RFP staining. The number of RFP-positive cells (brown) was counted and is shown in the graph (n = 5). Anti–VLA-4 Ab treatment decreased the number of RFP⁺ cells in the infarcted area following CPC sheet transplantation. Lower panels show representative images. Nuclei were stained with hematoxylin. Scale bars: 100 μm. Data are shown as mean + SEM.

The present study reports that CPC sheet transplantation inhibited cardiac remodeling and restored cardiac function after MI by increasing the number of blood vessels and cardiomyocytes in the injured area. sVCAM-1 was identified as one of the dominant paracrine factors in CPCs and was shown to induce angiogenesis, cardioprotection, and CPC migration and survival through the VLA-4 signaling pathway. Therefore, sVCAM-1 plays a critical role in improved cardiac function following MI.

CPC transplantation restored cardiac function and angiogenic activity and prevented cardiac remodeling 4 weeks after transplantation. In contrast, ATMC transplantation attenuated cardiac dysfunction and enhanced angiogenesis transiently, and cardiac remodeling progressed at 4 weeks. These findings suggest varying cell survival rates (Supplemental Figure 3) and distinct protein expression profiles (Figure 5B) in CPCs and ATMCs in the transplanted areas.

Through direct comparison of the protein expression profiles of CPCs and ATMCs, sVCAM-1 was identified as one of the predominantly expressed CPC-derived paracrine factors. VCAM-1, a 110-kDa transmembrane glycoprotein, is detected in various cells, including endothelial and bone marrow stromal cells (23). A soluble form of VCAM-1 has been reported to be shed from VCAM-1 on the cell surface by proteases, including TNF-α–converting enzyme (24). TNF-α–converting enzyme has also been reported to be elevated in the myocardium in heart failure (25) and to be required for fetal murine cardiac development and modeling (26). sVCAM-1 induces migration of endothelial cells through VLA-4 (21, 27, 28). The present study demonstrates that CM from VCAM-1–knocked-down CPCs did not induce endothelial migration, tube formation, cardioprotection, or CPC migration. Anti–VLA-4 Ab treatment abolished the protective effects of CPC-derived CM on cardiomyocytes and migration of CPCs and attenuated improved cardiac function following CPC sheet transplantation. This suggests that sVCAM-1 is a major paracrine factor of cardioprotection. VLA-4 is an integrin dimer that is composed of CD49d (α₄) and CD29 (β₁). Although studies have shown that the β₁ integrin signaling cascade regulates migration, differentiation, and death of various types of cells, such as endothelial cells, cardiomyocytes, and epidermal and hematopoietic stem cells (29–31), the role of sVCAM-1–mediated VLA-4 signaling in stem/progenitor cells remains elusive.

CPC-derived CM and sVCAM-1 phosphorylated several integrin-related downstream signaling molecules, such as Akt, ERK, and p38 MAPK, in cardiomyocytes. Consistent with previous studies, which indicated that Akt and ERK are critical for growth and survival of cardiomyocytes (32, 33), this study showed that promotion of cardiomyocyte survival by CPC-derived CM and sVCAM-1 was regulated through VLA-4–mediated activation of Akt and ERK. Furthermore, migration of CPCs, almost all of which expressed CD29 (β₁ integrin; Supplemental Figure 1), was facilitated via the VLA-4/p38 MAPK signaling pathway. VCAM-1–knockout mice and α₄ integrin–null mice have been shown to exhibit embryonic lethality, which was partly attributed to impaired epicardium formation surrounding the ventricular and atrial chambers, which suggests that VCAM-1/α₄ integrin signaling is critical for heart development (34, 35). In the present study, anti–VLA-4 Ab treatment reduced survival of transplanted CPCs. Furthermore, when VCAM-1 of CPCs was downregulated by specific miRNA, CPC viability was reduced and apoptosis was increased (Supplemental Figure 10), which suggests that VCAM-1–mediated signaling is also important for CPC survival. Since transplanted CPCs are the sources of not only paracrine factors but also newly formed cardiomyocytes, VCAM-1–mediated paracrine effects might also contribute to the cardiomyogenesis of CPCs through improved engraftment. A recent study has suggested that adult cardiac stem cells express α₄ and β₁ integrin in the niches (36). However, the role of α₄ and β₁ integrins is not fully understood. Since adult mammalian cardiomyocytes have been reported to be refreshed by endogenous stem cells after injury (37), our results suggest that sVCAM-1 secreted from transplanted CPCs promoted migration and self renewal of not only transplanted CPCs, but also endogenous cardiac stem/progenitor cells, through α₄ and β₁ integrin.

VCAM-1 is known as an inflammatory mediator, and increased sVCAM-1 in plasma has been reported following acute MI (38). During acute-phase MI, infiltrating leukocytes release cytokines, which activate VCAM-1 expression and promote leukocyte transmigration. In the present study, increased VCAM-1 expression in myocardium was observed in each group 1 week after MI/transplantation, when many inflammatory cells were also observed (data not shown). At 4 weeks, however, VCAM-1 expression remained upregulated in the CPC group despite few inflammatory cells in the infarcted area (Supplemental Figure 5). Furthermore, peripheral blood concentrations of sVCAM-1 were similar between groups 1 and 4 weeks after transplantation, which suggested that VCAM-1 expression in the transplanted heart was derived from CPC sheets, rather than circulating cells in the peripheral blood. VCAM-1 and its receptor, VLA-4, are important for fusion between hematopoietic progenitor cells and cardiomyocytes (39), and Oh et al. have reported that approximately 50% of cardiac protein–expressing transplanted cells arise from fusion with existing cardiomyocytes (3), which suggests that VCAM-1 mediates fusion between CPCs and dormant cardiomyocytes.

The present study compared transplanted cell survival between cell sheet transplantation and direct cell injection (Supplemental Figures 2 and 3). At 1 week after cell sheet transplantation, approximately 40% of cells survived (Supplemental Figure 3), while only 10% of cells survived after direct cell injection (Supplemental Figure 2). Immediately following transplantation, RFP expression in the heart was similar between cell sheet transplantation and direct cell injection, which suggested that the initial transplantation efficiency was the same. These findings indicate that cell sheet transplantation was superior to direct injection into the myocardium.

Many reports have demonstrated that endogenous cardiac stem/progenitor cells or bone marrow–derived cells mobilize to the infarcted area after injury and recruit additional cells through a feedback mechanism (40, 41). As shown in Table 2, CPCs expressed several chemokines, including stromal cell–derived factor-1 (SDF-1), which recruits bone marrow–derived cells to the infarcted myocardium (42). Therefore, CPC sheet transplantation may induce migration of bone marrow–derived cells to the infarcted heart, thereby improving cardiac function. Recently, anti–α₄ integrin Ab treatment was shown to improve cardiac function 2 weeks after MI by inhibiting interactions between bone marrow cells and their niches and promoting bone marrow cell migration and vasculogenesis (43). In the present study, anti–VLA-4 Ab treatment significantly attenuated improved cardiac function and angiogenesis following CPC sheet transplantation. Moreover, anti–VLA-4 Ab treatment did not affect cardiac function, fibrotic area, or blood vessel number in nontransplanted MI mice (Supplemental Figure 11). These findings suggest that bone marrow cells from their niches do not significantly contribute to the beneficial effects of CPC sheet transplantation.

There were a few limitations to the present study. The CPCs used in the experiments exhibited gene expression patterns similar to those of freshly isolated cardiac Sca-1–positive cells. However, profiles associated with proliferation, migration, and cardiomyocyte differentiation may not be the same. HUVECs were employed in endothelial migration and tube formation assays in vitro, and there might be differences between HUVECs and cardiac endothelial cells. Nevertheless, CPC sheet-mediated transplantation might be superior to the combination of other cell sources and tissue engineering methods. Improved survival of transplanted CPCs, in combination with several growth factors (44, 45), or multilayered cell sheets (46) might improve the beneficial effects of CPC sheet transplantation.

The present study identified the VCAM-1/VLA-4 signaling pathway as an important mechanism for CPC transplantation–mediated improved cardiac function. However, other paracrine factors, including thymosin β4 (47), have also been reported to contribute to cardiac repair following MI. Therefore, multiple mechanisms and mutual crosstalk might be involved in cell sheet transplantation to improve cardiac function. It remains to be determined which mechanisms are the most important and should be improved.

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We thank R. Kobayashi, A. Furuyama, H. Nagao, and K. Yoshihara for their excellent technical assistance. We thank M. Okabe, Osaka University, for the GFP transgenic mice and Shunichi Morikawa, Tokyo Women’s Medical University, for his technical advice. This study was supported by a grant-in-aid for Scientific Research on Priority Areas and for Exploratory Research, Ministry of Education, Culture, Sports, Science and Technology: Health (to I. Komuro); the Cell Science Research Foundation and the Takeda Science Foundation (to T. Nagai); Health and Labour Sciences Research grants (to T. Shimizu); a grant-in-aid for Scientific Research, Developmental Scientific Research, and Scientific Research from the Ministry of Education, Science, Sports, and Culture; the Sakakibara Memorial Foundation; the Hisako Yamakawa Award and a Japan Heart Foundation/Novartis Grant for Research Award on Molecular and Cellular Cardiology, 2009, Takeda Science Foundation (to K. Matsuura).

Address correspondence to: Katsuhisa Matsuura, Department of Cardiology, Tokyo Women’s Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo, 162-8666, Japan. Phone: 81-3-3353-8111; Fax: 81-3-3356-0441; E-mail: mkatu2002@yahoo.co.jp. Or to: Issei Komuro, Department of Cardiovascular Science and Medicine, Chiba University Graduate School of Medicine, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan. Phone: 81-43-226-2097; Fax: 81-43-226-2096; E-mail: komuro-tky@umin.ac.jp.

Conflict of interest: The authors have declared that no conflict of interest exists.

Nonstandard abbreviations used: ATMC, adipose tissue–derived mesenchymal cell; CM, conditioned medium; CPC, clonally expanded Sca-1–positive cell; FAK, focal adhesion kinase; FS, fractional shortening; IMDM, Iscove’s Modified Dulbecco’s Medium; LVDd, LV diastolic dimension; LVDs, LV systolic dimension; LVEDP, LV end-diastolic pressure; MI, myocardial infarction; miRNA, microRNA; RFP, red fluorescent protein; Sca-1, stem cell antigen 1; sVCAM-1, soluble VCAM-1; VLA-4, very late antigen-4.

Reference information: J. Clin. Invest.119:2204–2217 (2009). doi:10.1172/JCI37456

1. Dimmeler, S., Zeiher, A.M., Schneider, M.D. 2005. Unchain my heart: the scientific foundations of cardiac repair. J. Clin. Invest. 115:572-583.
   View this article via: JCI PubMed Google Scholar
2. Beltrami, A.P., et al. 2003. Adult cardiac stem cells are multipotent and support myocardial regeneration. Cell. 114:763-776.
   View this article via: CrossRef PubMed Google Scholar
3. Oh, H., et al. 2003. Cardiac progenitor cells from adult myocardium: homing, differentiation, and fusion after infarction. Proc. Natl. Acad. Sci. U. S. A. 100:12313-12318.
   View this article via: CrossRef PubMed Google Scholar
4. Matsuura, K., et al. 2004. Adult cardiac Sca-1-positive cells differentiate into beating cardiomyocytes. J. Biol. Chem. 279:11384-11391.
   View this article via: CrossRef PubMed Google Scholar
5. Messina, E., et al. 2004. Isolation and expansion of adult cardiac stem cells from human and murine heart. Circ. Res. 95:911-921.
   View this article via: CrossRef PubMed Google Scholar
6. Laugwitz, K.L., et al. 2005. Postnatal isl1+ cardioblasts enter fully differentiated cardiomyocyte lineages. Nature. 433:647-653.
   View this article via: CrossRef PubMed Google Scholar
7. Smith, R.R., et al. 2007. Regenerative potential of cardiosphere-derived cells expanded from percutaneous endomyocardial biopsy specimens. Circulation. 115:896-908.
   View this article via: CrossRef PubMed Google Scholar
8. Mirotsou, M., et al. 2007. Secreted frizzled related protein 2 (Sfrp2) is the key Akt-mesenchymal stem cell-released paracrine factor mediating myocardial survival and repair. Proc. Natl. Acad. Sci. U. S. A. 104:1643-1648.
   View this article via: CrossRef PubMed Google Scholar
9. Cho, H.J., et al. 2007. Role of host tissues for sustained humoral effects after endothelial progenitor cell transplantation into the ischemic heart. J. Exp. Med. 204:3257-3269.
   View this article via: CrossRef PubMed Google Scholar
10. Schächinger, V., et al. 2006. Intracoronary bone marrow-derived progenitor cells in acute myocardial infarction. N. Engl. J. Med. 355:1210-1221.
    View this article via: CrossRef PubMed Google Scholar
11. Perin, E.C., et al. 2008. Comparison of intracoronary and transendocardial delivery of allogeneic mesenchymal cells in a canine model of acute myocardial infarction. J. Mol. Cell. Cardiol. 44:486-495.
    View this article via: CrossRef PubMed Google Scholar
12. Beeres, S.L., et al. 2007. Intramyocardial injection of autologous bone marrow mononuclear cells in patients with chronic myocardial infarction and severe left ventricular dysfunction. Am. J. Cardiol. 100:1094-1098.
    View this article via: CrossRef PubMed Google Scholar
13. Zhang, M., et al. 2001. Cardiomyocyte grafting for cardiac repair: graft cell death and anti-death strategies. J. Mol. Cell. Cardiol. 33:907-921.
    View this article via: CrossRef PubMed Google Scholar
14. Okano, T., et al. 1993. A novel recovery system for cultured cells using plasma-treated polystyrene dishes grafted with poly (N-isopropylacrylamide). J. Biomed. Mater. Res. 27:1243-1251.
    View this article via: CrossRef PubMed Google Scholar
15. Kushida, A., et al. 1999. Decrease in culture temperature releases monolayer endothelial cell sheets together with deposited fibronectin matrix from temperature-responsive culture surfaces. J. Biomed. Mater. Res. 45:355-362.
    View this article via: CrossRef PubMed Google Scholar
16. Memon, I.A., et al. 2005. Repair of impaired myocardium by means of implantation of engineered autologous myoblast sheets. J. Thorac. Cardiovasc. Surg. 130:1333-1341.
    View this article via: CrossRef PubMed Google Scholar
17. Miyahara, Y., et al. 2006. Monolayered mesenchymal stem cells repair scarred myocardium after myocardial infarction. Nat. Med. 12:459-465.
    View this article via: CrossRef PubMed Google Scholar
18. Hida, N., et al. 2008. Novel cardiac precursor-like cells from human menstrual blood-derived mesenchymal cells. Stem Cells. 26:1695-1704.
    View this article via: CrossRef PubMed Google Scholar
19. Matsuura, K., et al. 2004. Cardiomyocytes fuse with surrounding noncardiomyocytes and reenter the cell cycle. J. Cell. Biol. 167:351-363.
    View this article via: CrossRef PubMed Google Scholar
20. Limana, F., et al. 2002. bcl-2 overexpression promotes myocyte proliferation. Proc. Natl. Acad. Sci. U. S. A. 99:6257-6262.
    View this article via: CrossRef PubMed Google Scholar
21. Giancotti, F.G., Ruoslahti, E. 1999. Integrin signaling. Science. 285:1028-1032.
    View this article via: CrossRef PubMed Google Scholar
22. Nakao, S., Kuwano, T., Ishibashi, T., Kuwano, M., Ono, M. 2003. Synergistic effect of TNF-alpha in soluble VCAM-1-induced angiogenesis through alpha 4 integrins. J. Immunol. 170:5704-5711.
    View this article via: PubMed Google Scholar
23. Funk, P.E., Stephan, R.P., Witte, P.L. 1995. Vascular cell adhesion molecule 1-positive reticular cells express interleukin-7 and stem cell factor in the bone marrow. Blood. 86:2661-2671.
    View this article via: PubMed Google Scholar
24. Garton, K.J., et al. 2003. Stimulated shedding of vascular cell adhesion molecule 1 (VCAM-1) is mediated by tumor necrosis factor-alpha-converting enzyme (ADAM 17). J. Biol. Chem. 278:37459-37464.
    View this article via: CrossRef PubMed Google Scholar
25. Satoh, M., et al. 1999. Tumor necrosis factor-α-converting enzyme and tumor necrosis factor-α in human dilated cardiomyopathy. Circulation. 99:3260-3265.
    View this article via: PubMed Google Scholar
26. Shi, W., et al. 2003. TACE is required for fetal murine cardiac development and modeling. Dev. Biol. 261:371-380.
    View this article via: CrossRef PubMed Google Scholar
27. Koch, A.E., Halloran, M.M., Haskell, C.J., Shah, M.R., Polverini, P.J. 1995. Angiogenesis mediated by soluble forms of E-selectin and vascular cell adhesion molecule-1. Nature. 376:517-519.
    View this article via: CrossRef PubMed Google Scholar
28. Chan, B.M., Elices, M.J., Murphy, E., Hemler, M.E. 1992. Adhesion to vascular cell adhesion molecule 1 and fibronectin. Comparison of alpha 4 beta 1 (VLA-4) and alpha 4 beta 7 on the human B cell line JY. J. Biol. Chem. 267:8366-8370.
    View this article via: PubMed Google Scholar
29. Shai, S.Y., et al. 2002. Cardiac myocyte-specific excision of the beta1 integrin gene results in myocardial fibrosis and cardiac failure. Circ. Res. 90:458-464.
    View this article via: CrossRef PubMed Google Scholar
30. Watt, F.M. 2002. Role of integrins in regulating epidermal adhesion, growth and differentiation. EMBO J. 21:3919-3926.
    View this article via: CrossRef PubMed Google Scholar
31. Papayannopoulou, T. 2003. Bone marrow homing: the players, the playfield, and their evolving roles. Curr. Opin. Hematol. 10:214-219.
    View this article via: CrossRef PubMed Google Scholar
32. Muraski, J.A., et al. 2007. Pim-1 regulates cardiomyocyte survival downstream of Akt. Nat. Med. 13:1467-1475.
    View this article via: CrossRef PubMed Google Scholar
33. Purcell, N.H., et al. 2007. Genetic inhibition of cardiac ERK1/2 promotes stress-induced apoptosis and heart failure but has no effect on hypertrophy in vivo. Proc. Natl. Acad. Sci. U. S. A. 104:14074-14079.
    View this article via: CrossRef PubMed Google Scholar
34. Kwee, L., et al. 1995. Defective development of the embryonic and extraembryonic circulatory systems in vascular cell adhesion molecule (VCAM-1) deficient mice. Development. 121:489-503.
    View this article via: PubMed Google Scholar
35. Yang, J.T., Rayburn, H., Hynes, R.O. 1995. Cell adhesion events mediated by alpha 4 integrins are essential in placental and cardiac development. Development. 121:549-560.
    View this article via: PubMed Google Scholar
36. Urbanek, K., et al. 2006. Stem cell niches in the adult mouse heart. Proc. Natl. Acad. Sci. U. S. A. 103:9226-9231.
    View this article via: CrossRef PubMed Google Scholar
37. Hsieh, P.C., et al. 2007. Evidence from a genetic fate-mapping study that stem cells refresh adult mammalian cardiomyocytes after injury. Nat. Med. 13:970-974.
    View this article via: CrossRef PubMed Google Scholar
38. Tousoulis, D., et al. 2007. Differences in inflammatory and thrombotic markers between unstable angina and acute myocardial infarction. Int. J. Cardiol. 115:203-207.
    View this article via: CrossRef PubMed Google Scholar
39. Zhang, S., et al. 2007. Fusion of human hematopoietic progenitor cells and murine cardiomyocytes is mediated by alpha 4 beta 1 integrin/vascular cell adhesion molecule-1 interaction. Circ. Res. 100:693-702.
    View this article via: CrossRef PubMed Google Scholar
40. Wang, X., et al. 2006. The role of the sca-1+/CD31- cardiac progenitor cell population in postinfarction left ventricular remodeling. Stem Cells. 24:1779-1788.
    View this article via: CrossRef PubMed Google Scholar
41. Kogata, N., et al. 2006. Cardiac ischemia activates vascular endothelial cadherin promoter in both preexisting vascular cells and bone marrow cells involved in neovascularization. Circ. Res. 98:897-904.
    View this article via: CrossRef PubMed Google Scholar
42. Elmadbouh, I., et al. 2007. Ex vivo delivered stromal cell-derived factor-1alpha promotes stem cell homing and induces angiomyogenesis in the infarcted myocardium. J. Mol. Cell. Cardiol. 42:792-803.
    View this article via: CrossRef PubMed Google Scholar
43. Qin, G., et al. 2006. Functional disruption of alpha4 integrin mobilizes bone marrow-derived endothelial progenitors and augments ischemic neovascularization. J. Exp. Med. 203:153-163.
    View this article via: CrossRef PubMed Google Scholar
44. Urbanek, K., et al. 2005. Cardiac stem cells possess growth factor-receptor systems that after activation regenerate the infarcted myocardium, improving ventricular function and long-term survival. Circ. Res. 97:663-673.
    View this article via: CrossRef PubMed Google Scholar
45. Zhu, W., et al. 2008. IGFBP-4 is an inhibitor of canonical Wnt signalling required for cardiogenesis. Nature. 454:345-349.
    View this article via: CrossRef PubMed Google Scholar
46. Shimizu, T., et al. 2006. Polysurgery of cell sheet grafts overcomes diffusion limits to produce thick, vascularized myocardial tissues. FASEB J. 20:708-710.
    View this article via: PubMed Google Scholar
47. Bock-Marquette, I., Saxena, A., White, M.D., Dimaio, J.M., Srivastava, D. 2004. Thymosin beta4 activates integrin-linked kinase and promotes cardiac cell migration, survival and cardiac repair. Nature. 432:466-472.
    View this article via: CrossRef PubMed Google Scholar
48. Planat-Bénard, V., et al. 2004. Spontaneous cardiomyocyte differentiation from adipose tissue stroma cells. Circ. Res. 94:223-229.
    View this article via: PubMed Google Scholar