Intracoronary Administration of Cardiac Progenitor Cells Alleviates Left Ventricular Dysfunction in Rats With a 30-Day-Old Infarction
Background— Administration of cardiac progenitor cells (CPCs) 4 hours after reperfusion ameliorates left ventricular function in rats with acute myocardial infarction (MI). Clinically, however, this approach is not feasible, because expansion of autologous CPCs after acute MI requires several weeks. Therefore, we sought to determine whether CPCs are beneficial in the more clinically relevant setting of an old MI (scar).
Methods and Results— One month after coronary occlusion/reperfusion, rats received an intracoronary infusion of vehicle or enhanced green fluorescent protein–labeled CPCs. Thirty-five days later, CPC-treated rats exhibited more viable myocardium in the risk region, less fibrosis in the noninfarcted region, and improved left ventricular function. Cells that stained positive for enhanced green fluorescent protein that expressed cardiomyocyte, endothelial, and vascular smooth muscle cell markers were observed only in 7 of 17 treated rats and occupied only 2.6% and 1.1% of the risk and noninfarcted regions, respectively. Transplantation of CPCs was associated with increased proliferation and expression of cardiac proteins by endogenous CPCs.
Conclusions— Intracoronary administration of CPCs in the setting of an old MI produces beneficial structural and functional effects. Although exogenous CPCs can differentiate into new cardiac cells, this mechanism is not sufficient to explain the benefits, which suggests paracrine effects; among these, the present data identify activation of endogenous CPCs. This is the first report that CPCs are beneficial in the setting of an old MI when given by intracoronary infusion, the most widely applicable therapeutic approach in patients. Furthermore, this is the first evidence that exogenous CPC administration activates endogenous CPCs. These results open the door to new therapeutic applications for the use of autologous CPCs in patients with old MI and chronic ischemic cardiomyopathy.
Received April 9, 2009; accepted November 6, 2009.
Cell-based therapies have the potential to alleviate left ventricular (LV) dysfunction and remodeling after acute myocardial infarction (MI) in experimental animal models1 and in humans.2,3 Among the various cells used, c-kit–positive (c-kitpos) cardiac progenitor cells (CPCs) are attractive because they normally reside in the heart and presumably are responsible for replenishing the pool of cardiac myocytes and coronary vessels under normal conditions.4–6 The practical utility of CPCs is further supported by the fact that these cells can be isolated from small fragments of cardiac tissue and expanded for subsequent autologous administration.5,6
Clinical Perspective on p 305
Transplantation of autologous or syngeneic CPCs has been found to be effective in limiting LV dysfunction and remodeling in rodent models of acute MI, both in the setting of a permanent coronary occlusion4–6 and in that of a transient occlusion followed by reperfusion.7 Clinically, however, this approach would be problematic, because administration of autologous CPCs to patients with acute MI would necessitate an endomyocardial biopsy followed by isolation and expansion of c-kitpos cells from the tissue fragment, all of which would require weeks, so that CPC transplantation would have to be postponed until after the acute phase of the infarct. By the time a sufficient number of autologous CPCs would be ready for therapeutic use, the inflammatory response to acute injury would have abated, and a scar would have formed. At this chronic stage, the local myocardial environment is very different from that of an acute MI, because the expression of the cytokines and adhesion molecules that are upregulated during the acute phase and play an important role in the chemotaxis, homing, and differentiation of stem cells8–11 is absent or markedly diminished. For example, CPCs are known to express CXCR-4, the receptor for stromal-derived factor-1,7 a cytokine that plays a pivotal role in orchestrating the chemotaxis, homing, and differentiation of various progenitor/stem cells.8,10,11 The expression of stromal-derived factor-1 in the myocardium increases markedly after ischemia/reperfusion, peaking at 1 day, but then resolves by 7 days.8,10,11 The migration, survival, and differentiation of CPCs are also modulated by hepatocyte growth factor and insulin-like growth factor-1,5,6,12,13 both of which are upregulated shortly after ischemia/reperfusion but subsequently decline.14,15 In view of the marked downregulation of stromal-derived factor-1, hepatocyte growth factor, insulin-like growth factor-1, and other signals in the setting of a mature scar, it is unclear whether transplantation of CPCs at this stage would still be effective.
In a recent study of rats with subacute MI (20-day-old infarcts), CPC administration was found to result in tissue regeneration and LV functional improvement.6 In that investigation, however, as in most previous studies,4,5 CPCs were injected directly into the myocardium, an approach that would be impractical for widespread use in patients. Furthermore, the study6 was performed in the setting of a permanent coronary occlusion, which differs importantly from the contemporary clinical situation, in which the occluded artery is usually recanalized.16 Recently, Johnston et al17 have reported salubrious effects of intracoronary infusion of cardiosphere-derived cells in pigs with old MI; however, studies with intracoronary infusion of a selected population of c-kitpos cells (CPCs) have not been reported. Accordingly, the goal of the present investigation was to determine whether administration of CPCs is effective in regenerating cardiac tissue and alleviating postinfarction LV remodeling and dysfunction when these cells are given by intracoronary infusion (the most broadly applicable clinical strategy) in the setting of an old MI (scar) produced by a temporary coronary occlusion followed by reperfusion. To directly compare the results of the present investigation with those of our previous study in the setting of an acute MI,7 we used a similar rat model and the same dose of CPCs as in that study.7
Detailed methodology is provided in the online-only Data Supplement.
Exclusions and Gross Measurements
Exclusions are detailed in the online-only Data Supplement. Intracoronary injection of CPCs resulted in uniform distribution of these cells throughout the LV (online-only Data Supplement Figure IX).
As expected, both of the groups subjected to infarction exhibited LV hypertrophy and dilatation compared with normal rats (Table). There were no significant differences between the vehicle- and CPC-treated groups with respect to body weight, LV weight, LV length, or LV volume (determined directly at postmortem examination), although LV volume tended to be lower in CPC-treated rats (−12%, P=NS).
Echocardiographic and Hemodynamic Measurements
At the time of treatment (30 days after MI), thickening fraction in the infarcted (anterior) LV wall, LV fractional shortening, and LV ejection fraction were markedly decreased vis-á-vis baseline (Figures 1A and 1B; online-only Data Supplement Table II), which indicates the development of severe post-MI dysfunction; all echocardiographic parameters were virtually indistinguishable between the vehicle- and CPC-treated groups, which demonstrates that the degree of regional and global LV functional deterioration at the time of treatment was similar. As expected, in the vehicle group, the infarcted wall thickening fraction, fractional shortening, and ejection fraction continued to deteriorate during the ensuing 35 days; in contrast, in the CPC-treated group, the infarcted wall thickening fraction increased significantly, and fractional shortening and ejection fraction remained stable (Figure 1B). As a consequence, at 35 days, all 3 parameters were significantly higher in the CPC-treated than in the vehicle group (Figure 1B). Thus, transplantation of CPCs improved both regional and global LV function.
The hemodynamic studies performed just before euthanasia (35 days after treatment) led to similar conclusions. Compared with normal age-matched rats not subjected to surgery, all LV functional parameters were markedly depressed in both groups of infarcted rats, but the deterioration was less in the treated than in the vehicle group (Figure 2; online-only Data Supplement Table III). This was the case not only for load-dependent (LV end-diastolic pressure, LV dP/dtmax, and LV ejection fraction) but also for load-independent (end-systolic elastance, preload-activated maximal power, and preload recruitable stroke work; Figure 2) indices of LV systolic function. Thus, 2 independent methods of functional assessment (echocardiography and hemodynamic studies) consistently demonstrated that intracoronary CPC infusion improved LV systolic performance.
Morphometric and Histological Analysis
As illustrated in Figure 3A, the amount of viable tissue within the risk region was greater in CPC-treated rats than in vehicle-treated rats. In each heart, a detailed quantitative analysis was performed on 2 sections (1 from each of 2 midventricular slices; online-only Data Supplement Figure X); the results are summarized in online-only Data Supplement Table IV. Although the circumferential extent of the scar and the size of the risk region were indistinguishable between the 2 groups, the amount of viable myocardium within the risk region was 25% greater (P<0.01) and the thickness of the anterior (infarcted) wall 24% greater (P<0.05) in CPC-treated than in vehicle-treated rats (Figure 3B; online-only Data Supplement Table IV), which suggests that the infusion of CPCs resulted in formation of new myocardium in the infarcted region. The LV expansion index was also reduced (P<0.05) in the CPC-treated group (Figure 3B). The hemodynamic parameters at the time of euthanasia were significantly related to the amount of viable myocardium in the risk region (r=0.44 to 0.69, P<0.05 to 0.01; online-only Data Supplement Figure XI), which suggests that the functional improvement noted in CPC-treated rats was accounted for in part by the greater proportion of viable tissue in the risk region.
Although the median myocyte cross-sectional area was slightly smaller (P<0.05) in the risk region in the CPC-treated group than in the vehicle-treated group, no difference was observed in the noninfarcted region (online-only Data Supplement Figure XII-B and XII-C). These data are consistent with the gross measurements (vide supra) and indicate that transplantation of CPCs did not affect hypertrophy in the noninfarcted region, although a small effect was noted in the risk region.
In both groups, collagen content in the noninfarcted region (posterior wall) was increased compared with normal hearts, but the increase was significantly less (−33%, P<0.01) in CPC-treated rats (Figure 4C). This reduced collagen deposition in the noninfarcted myocardium may have contributed at least in part to the functional benefits of CPC therapy.
Detection of Transplanted CPCs and Their Progeny
Surprisingly, despite careful analysis of 4 LV slices per heart, performed on 7 to 10 histological sections in each slice at 40- to 80-μm intervals, cells that stained positive for enhanced green fluorescent protein (EGFPpos cells) were found in only 7 of the 17 CPC-treated rats. In these 7 hearts, EGFPpos cells were relatively rare, accounting for only 2.6±1.1% of the area of the risk region and 1.1±0.4% of the noninfarcted region (Figure 5). These areas of EGFP positivity were calculated to correspond to 0.51±0.21×106 EGFPpos cells/heart in the risk region and 1.05±0.43×106 EGFPpos cells/heart in the noninfarcted region (Figure 5B). Real-time polymerase chain reaction detection of the EGFP cDNA marker in transplanted CPC genomic DNA essentially confirmed the immunohistochemical findings (online-only Data Supplement Figure XIII-A). Thus, at 35 days after intracoronary infusion of CPCs, the transplanted cells (or their progeny) were present in a minority of the hearts, and in those, they occupied a small fraction of the myocardium.
Differentiation of Transplanted CPCs
A significant fraction of the transplanted EGFPpos cells expressed cardiac-specific proteins, ie, α-sarcomeric actin, major histocompatibility complex, α-actinin, and troponin I (Figure 5C; online-only Data Supplement Figures XIV and XV), which suggests differentiation toward a cardiomyocytic lineage; for example, 36.0±1.7% of total EGFPpos cells in the risk region and 25.0±3.9% in the noninfarcted region were positive for α-sarcomeric actin (Figure 5D). However, because most of the EGFPpos/α-sarcomeric actin–positive cells were small and did not exhibit the typical morphology and sarcomeric structure of mature cardiac myocytes (Figure 5C), it remains unclear whether these cells were genuine differentiating myocytes. In addition, microvessels containing EGFPpos cells that expressed von Willebrand factor, platelet and endothelial cell adhesion molecule, or α-smooth muscle actin were found in the risk region (online-only Data Supplement Figures XVI, XVII, and XVIII), which suggests differentiation into endothelial and vascular smooth muscle lineages. These observations are compatible with a possible cardiac and vascular specification of transplanted CPCs resulting in formation of immature myocytes and new coronary vessels.
Relationship Between Salubrious Effects of CPC Infusion and Presence of Transplanted CPCs at 35 Days
When the 7 CPC-treated hearts that exhibited EGFPpos cells and the 10 hearts that displayed no EGFP positivity were examined separately (online-only Data Supplement Figure XIX), the amount of viable myocardium in the risk region was increased significantly in both subsets relative to vehicle-treated hearts (online-only Data Supplement Figure XIX-A). The anterior LV wall thickness was increased significantly only in the EGFP-negative (EGFPneg) hearts, and the LV expansion index was similar between the 2 subgroups. Echocardiographic (online-only Data Supplement Figure XIX-B) and hemodynamic (online-only Data Supplement Figure XIX-C) analyses revealed no consistent pattern favoring 1 subgroup over the other; both EGFPpos and EGFPneg hearts exhibited evidence of functional improvement. Together, these data suggest that the beneficial effects of exogenous CPCs do not depend on the presence of these cells or their progeny at 35 days after infusion.
Cellular Proliferation After Transplantation
Tissue bromodeoxyuridine (BrdU) incorporation (BrdU was given in the last 2 weeks of life) was significantly greater in CPC-treated hearts than in vehicle-treated hearts (Figure 6B) both in the 7 CPC-treated rats that exhibited EGFPpos cells and in the 10 CPC-treated hearts that did not (Figure 6C). The higher content of BrdU-positive (BrdUpos) cells in CPC-treated hearts was generalized, being equally apparent in the risk and noninfarcted regions (Figures 6B and 6C). In the 7 CPC-treated hearts that exhibited EGFPpos cells, most of the BrdU incorporation was accounted for by EGFPpos cells: Approximately two thirds of the BrdUpos cells expressed EGFP, and conversely, approximately one third of the EGFPpos cells were also positive for BrdU (Figure 6D). The analysis of Ki67-positive cells (presented in online-only Data Supplement Figure XX) yielded similar findings. Taken together, the BrdU and Ki67 data indicate that administration of CPCs was associated with increased cellular proliferative activity in the heart, and although most of this activity was accounted for by the transplanted CPCs, other cells were also involved.
Transplanted CPCs Proliferate and Activate Proliferation of Endogenous c-kitpos Cells
To determine whether endogenous CPCs (c-kitpos/EGFPneg cells) were among the cells activated by exogenous CPC administration, LV sections were stained with antibodies against c-kit (Figure 7; online-only Data Supplement Figure XXI). Relative to normal hearts, in the infarcted hearts (both vehicle and CPC treated), there was a dramatic increase (>10-fold) in the total number of c-kitpos cells and in the number of c-kitpos/BrdUpos cells (ie, new c-kitpos cells that were formed in the last 2 weeks of life; Figure 7B). The increase in c-kitpos cells was observed both in the risk region and in the noninfarcted region, although it was more pronounced in the former (Figure 7C). Rather than absolute numbers, however, probably a better measure of c-kitpos cell proliferation is given by the percentage of the total c-kitpos cell population that was newly formed in the last 2 weeks of life (c-kitpos/BrdUpos cells). This percentage was essentially nil in normal hearts but averaged ≈40% to 50% in the infarcted hearts (both vehicle and CPC treated; Figure 7D), which indicates marked proliferation of endogenous CPCs in response to infarction; importantly, this percentage was significantly (P<0.01) higher in CPC-treated than in vehicle-treated hearts, both in those with and in those without EGFPpos cells (Figure 7D). (In the 7 hearts with EGFPpos cells, this difference cannot be ascribed to the transplanted CPCs, because in these hearts, c-kitpos cells were measured only in areas that did not contain EGFPpos cells; in these hearts, almost half of the c-kitpos cells were EGFPneg [online-only Data Supplement Figure XXII].) The fact that the percentage of c-kitpos/BrdUpos cells was increased in CPC-treated hearts, even in those with no EGFPpos cells, suggests activation of endogenous CPC proliferation by the exogenous CPCs.
A similar pattern was observed for Ki67, although in this case, the percentage of c-kitpos cells that were Ki67 positive was similar in the 2 subsets of CPC-treated hearts and in the vehicle-treated hearts (online-only Data Supplement Figure XXI). (Unlike BrdU incorporation, which reflects the cumulative number of DNA replications over 2 weeks, Ki67 expression provides a “snapshot” of the number of cycling cells at the time of euthanasia.) There was a significant correlation between the myocardial content of c-kitpos cells and the amount of viable myocardium in the risk region (Figure 7E), which suggests that c-kitpos cell proliferation contributes to myocardial repair.
To gain insights into the effect of CPC transplantation on the commitment of endogenous CPCs to the cardiac lineage, a quantitative analysis of c-kitpos/EGFPneg cells (endogenous CPCs) expressing the cardiac transcription factor Nkx2.5 and the sarcomeric protein major histocompatibility complex was performed (Figures 8A and 8B). The number of endogenous CPCs (c-kitpos/EGFPneg cells) that expressed Nkx2.5 and major histocompatibility complex was higher in CPC-treated than in vehicle-treated hearts, both in the risk and in the noninfarcted regions; the magnitude of this effect, however, varied between EGFPneg and EGFPpos hearts (Figure 8B). Because of the presence of clusters of EGFPpos cells in CPC-treated EGFPpos hearts, cell density was higher in these hearts than in CPC-treated EGFPneg hearts and control hearts; consequently, the magnitude of the increase in endogenous CPCs in EGFPpos hearts was greater when the number of cells was normalized to area (mm2) rather than to number of nuclei (Figure 8B). The number of BrdUpos cells (new cells formed in the last 2 weeks of life) that expressed α-sarcomeric actin was also increased in the CPC-treated hearts (Figures 8C and 8⇓D), which suggests that the increase in cardiac-committed endogenous CPCs was associated with increased formation of new myocytes. However, the absolute number of BrdUpos/α-sarcomeric actinpos cells was small (ranging from 20 to 206 cells/mm2), and newly formed (BrdUpos) cells with a mature myocyte phenotype and organized sarcomeric structure were extremely rare. Because BrdU was administered only in the last 2 weeks of life, definitive quantitative conclusions on the effect of CPC transplantation on formation of new myocytes cannot be made.
We have previously found that in rats with acute MI, intracoronary administration of syngeneic CPCs early (4 hours) after reperfusion was effective in regenerating cardiac tissue and ameliorating LV remodeling and dysfunction.7 Clinically, however, the application of this paradigm to patients with acute MI would be problematic, because expansion of autologous CPCs from endomyocardial biopsy samples requires weeks. By the time autologous CPCs are ready for therapy, the infarct is a scar, and inflammation has resolved. It is unclear whether infusion of CPCs would be beneficial at this stage, ie, in the setting of a scar, in which the expression of growth factors and adhesion molecules is markedly diminished or even absent. The present study was undertaken to address this important translational issue. We used a transient coronary occlusion followed by reperfusion (as opposed to a permanent coronary occlusion) because in current practice, most patients with acute MI are reperfused, either spontaneously or iatrogenically. A model of a 30-day-old reperfused MI was selected because by this time, the acute inflammatory response in the rat has resolved, and the formation of the scar is complete.18
Our salient findings can be summarized as follows: (1) Intracoronary infusion of syngeneic CPCs at 30 days after MI resulted, 35 days later, in improved LV structure, as manifested by a greater content of viable myocardium in the risk region, increased thickness of the infarcted LV wall, reduced fibrosis in the noninfarcted region, and attenuated LV expansion index; (2) these salubrious effects were associated with improved regional and global LV function, as demonstrated by 2 independent methods (echocardiography and hemodynamic studies) that used both load-dependent and -independent indices; (3) in contrast, transplantation of CPCs did not alleviate LV dilation; (4) the improvement in global LV function can be accounted for, in part, by the increased amount of viable tissue in the infarcted region; (5) although transplanted CPCs gave rise to new cells expressing markers specific for cardiac myocytes, endothelial cells, and vascular smooth muscle cells, this phenomenon cannot explain the improvement in LV function entirely, because it was inconsistent (occurring only in a minority of the treated animals) and of small magnitude; (6) transplantation of exogenous CPCs promoted proliferation of endogenous cells, including endogenous CPCs, both in the risk and in the noninfarcted regions; and (7) transplantation of exogenous CPCs was also associated with an increase in the number of endogenous CPCs that exhibited apparent cardiogenic commitment, both in the risk and the noninfarcted regions.
Taken together, these results demonstrate that intracoronary administration of CPCs in the setting of a myocardial scar produces beneficial structural and functional effects within a relatively short time frame (35 days) and that the observed benefits cannot be entirely accounted for by direct differentiation of exogenous CPCs into new cardiac cells (although such differentiation can occur), which suggests that paracrine mechanisms are likely to play an important role. The present quantitative analysis of cardiac-committed c-kitpos cells supports the concept that 1 such paracrine mechanism is the activation of endogenous CPCs. Previous studies4–7 have demonstrated salutary effects of CPCs when these cells were given immediately after MI, either by intramyocardial injection4–6 or intracoronary infusion.7 However, to the best of our knowledge, this is the first report that CPCs are beneficial in the setting of an old MI when given by the intracoronary route, the most widely applicable therapeutic approach in patients. Furthermore, this is the first evidence that exogenous CPC administration activates endogenous CPCs.19
An elegant study by Johnston et al17 has recently been reported in which pigs with a 4-week-old MI were given a different form of cell therapy, autologous cardiosphere-derived cells produced from endomyocardial biopsy samples.17 In that study, selective intracoronary infusion of cardiosphere-derived cells into the territory of myocardium subjected to ischemia/reperfusion 4 weeks earlier resulted in engraftment, formation of mature cardiac cells, reduction in relative infarct size, and improvement in LV remodeling and hemodynamic function 8 weeks later. Despite the use of different cells, the present results agree with these findings.
Effect of CPCs on Cardiac Structure and Function
The present results indicate that transplantation of CPCs exerts important salutary effects on post-MI LV dysfunction even after the healing process is completed. Global LV systolic function was improved significantly, as documented by 2 independent methods (echocardiography and hemodynamic studies) and by a variety of parameters, both load dependent and independent (Figure 2). Regional function in the infarcted region was also ameliorated (Figure 1), presumably as a result of the greater proportion of viable tissue in this region (Figure 3) and/or of favorable metabolic adaptations in the surviving myocardium.20 Although LV dimensions did not differ significantly between vehicle-treated and CPC-treated hearts (Table; online-only Data Supplement Table IV), the scarred region was thicker in the treated group (Figure 3), which again may reflect increased viable myocardium content (Figure 3). The LV expansion index, which quantifies both the degree of LV dilation and the degree of infarct wall thinning,21 was reduced significantly by CPCs (Figure 3). Another salutary effect of CPC infusion was a reduction in collagen content in the noninfarcted region (Figure 4), which constitutes not only a marker of reduced cardiac injury but also a potential mechanism for improved cardiac performance. The myocyte cross-sectional area in the noninfarcted region was not affected by CPC transplantation (online-only Data Supplement Figure XII); this, together with the similarity in LV weight between the 2 groups (Table), indicates no effect on LV hypertrophy. The smaller myocyte cross-sectional area in the infarcted region of CPC-treated hearts (online-only Data Supplement Figure XII) may reflect either an attenuation of the hypertrophic response in this region or the formation of new myocytes (presumably smaller than mature cells).
Previous studies of CPCs in models of acute MI4–7 have shown a more robust engraftment and differentiation of the transplanted cells than in the present study. The reasons for this difference are unclear but (in addition to the considerations outlined below for the chronic MI model) may relate to the difference in models (acute versus old MI) and the scarcity of proinflammatory substances that facilitate homing of transplanted CPCs in the present model (old MI with scar). In a recent study in rats,6 intramyocardial injection of CPCs on the border of the infarct at 20 days after MI resulted in regeneration of cardiac tissue (≈40% of the scar was replaced with new myocardium), attenuation of LV dilation, and alleviation of LV dysfunction. The present results are consistent with these observations in the sense that CPCs attenuated LV dysfunction. However, in contrast to that study,6 evidence of extensive regeneration of cardiac tissue by exogenous CPCs was not observed here: EGFPpos cells (ie, transplanted CPCs or their progeny) were found only in 7 of 17 treated rats and accounted for only 2.6% of the tissue in the risk region (Figure 5); furthermore, mature EGFPpos myocytes were observed only rarely (online-only Data Supplement Figures XIV and XV).
The reason for this apparent discrepancy is unclear. The major differences between the 2 studies are intramyocardial injection versus intracoronary infusion of CPCs, permanent coronary occlusion6 versus transient occlusion followed by reperfusion, and longer follow-up in the present investigation (35 versus 20 days). One possible reason for the low yield of EGFPpos cells in the present study is the immunogenic potential of this foreign protein, which potentially may induce a T-cell immune response against the labeled cells,22 such that cells labeled with EGFP may actively engraft but subsequently be cleared by the immune system.22,23 The magnitude of immunologic rejection of EGFP-carrying cells remains controversial and is most likely context dependent.22–24 Whether this phenomenon is operative in the system used in the present study remains to be determined. An additional variable that may have influenced the assessment of myocardial regeneration by EGFP-tagged CPCs involves the insertion of the lentiviral integrant in repressive regions of the genome25; transgene silencing would interfere with the recognition of labeled cells by immunocytochemistry, although the transgene should still be detectable by polymerase chain reaction.
Mechanism of the Beneficial Effects of CPCs
The mechanism for the beneficial effects of CPCs in the present study remains unclear. Coronary vessel density was not affected by CPCs (online-only Data Supplement Figure XXIII). The percentage of terminal dUTP nick end-labeling–positive nuclei did not differ between the 2 groups at 35 days after treatment (online-only Data Supplement Figure XXIV), although we did not assess apoptosis at earlier time points. Differentiation of transplanted CPCs into cardiac cells that resulted in regeneration of dead myocardium is unlikely to have been a major factor. Despite a detailed pathological analysis of 28 to 40 sections per heart, we were unable to find any EGFPpos cells in 10 of the 17 CPC-treated hearts, and this was supported by real-time polymerase chain reaction (online-only Data Supplement Figure XIII). In the remaining 7 hearts, EGFPpos cells accounted for only ≈2.6% of the risk region and ≈1.1% of the noninfarcted region (Figure 5), values that were insufficient to account for the functional improvement. Although we did observe colocalization of EGFP with cardiac-specific proteins (Figure 5; online-only Data Supplement Figures XIV and XV), these cells were usually small and did not exhibit the characteristic morphology of adult cardiac myocytes (eg, shape, sarcomeres; Figure 5). Moreover, in the CPC-treated group, the increase in viable tissue within the infarcted region and the improvement in systolic LV performance were observed not only in the subset of 7 hearts that had EGFPpos cells but also in the subset of 10 hearts that did not (online-only Data Supplement Figure XIX). Taken together, these findings indicate that direct formation of new parenchyma from the transplanted CPCs was not the major reason for the benefits observed, which implies the existence of other mechanisms.
Among these, the most plausible would appear to be the secretion of cytokines/growth factors by CPCs, which resulted in paracrine actions on endogenous cells that may have persisted even after the transplanted CPCs had disappeared, a phenomenon that has been reported to occur with various stem/progenitor cells.26,27 CPCs have been shown to express hepatocyte growth factor and insulin-like growth factor-1, which may exert important actions on both the surrounding myocardium and the resident endogenous CPCs.28 The mitigation of myocardial fibrosis in the noninfarcted region (Figure 4) could be 1 manifestation of such a paracrine action of transplanted CPCs. Another could be the activation of endogenous (EGFPneg) cells, such as c-kitpos CPCs, which in turn would generate new cardiac cells. This possibility is supported by several observations: (1) The content of BrdUpos and Ki67-positive cells was increased in both the infarcted and noninfarcted regions of CPC-treated hearts (Figure 6; online-only Data Supplement Figure XX), which indicates that the infusion of CPCs was associated with cellular proliferation; (2) although in the CPC-treated hearts that exhibited EGFPpos cells, most of this proliferative activity was accounted for by the transplanted (EGFPpos) cells or their progeny (Figure 6D), BrdU incorporation was also increased in CPC-treated EGFPneg hearts (Figure 6C), which indicates that the exogenous CPCs induced proliferation of endogenous cells; (3) more directly, CPC-treated hearts contained more c-kitpos cells than vehicle-treated hearts, independent of the presence of EGFPpos cells (Figure 7; the increased content of c-kitpos cells in EGFPpos hearts shown in Figure 7 cannot be accounted for by the transplanted EGFPpos cells, because in these hearts, c-kitpos cells were deliberately counted in areas devoid of EGFPpos cells); (4) the percentage of total c-kitpos cells that were new (ie, formed in the last 2 weeks of life), as detected by BrdU incorporation, was increased significantly in CPC-treated hearts, even in those with no EGFPpos cells (Figure 7D); (5) the amount of viable tissue in the risk region was augmented by CPC infusion even in hearts devoid of EGFPpos cells (online-only Data Supplement Figure XIX-A), which suggests formation of new myocytes from endogenous CPCs; (6) the amount of viable tissue in the risk region correlated significantly with the number of c-kitpos cells (Figure 7E); (7) more directly, the number of c-kitpos/EGFPneg cells that expressed markers of cardiomyogenic differentiation (Nkx2.5 and major histocompatibility complex) was increased in CPC-treated hearts, both in the risk and in the noninfarcted regions (Figures 8A and 8B), which suggests an increase in the number of endogenous CPCs committed to cardiac lineage; and (8) the number of new (BrdU labeled) α-sarcomeric actinpos cells was also increased in CPC-treated hearts (Figures 8C and 8D), which suggests increased formation of new myocytes.
The most straightforward and plausible interpretation of all of these facts is that exogenous CPCs promoted proliferation of endogenous CPCs, which resulted in formation of new cardiac cells. Robust quantification of this phenomenon, however, is limited by the inability to track endogenous CPCs effectively through commitment and loss of c-kit expression after CPC treatment. Although the present quantitative analysis revealed that the absolute number of new (BrdUpos) α-sarcomeric actinpos cells was small (20 to 206 cells/mm2; Figure 8D), and although we rarely observed BrdU labeling in cells with an adult myocyte phenotype, it should be kept in mind that BrdU was administered only in the last 2 weeks of life; thus, the magnitude of total new myocyte formation cannot be ascertained from the present data.
This is the first study to demonstrate that intracoronary delivery of CPCs improves LV structure and function even when implemented as late as 1 month after MI, when the infarcted tissue has been replaced by a scar. This is also the first study to provide evidence that infusion of exogenous CPCs promotes the recruitment of endogenous CPCs and that this phenomenon persists for at least 5 weeks after CPC transplantation. The present findings suggest potential new therapeutic applications for the use of autologous CPCs in the large population of patients with old MI and chronic ischemic cardiomyopathy.
We gratefully acknowledge Qiuli Bi for expert technical assistance during the course of these studies and Maiying Kong for expert consultation on statistical analysis.
Sources of Funding
This study was supported in part by National Institutes of Health grants R01-HL-68088, HL-70897, HL-76794, HL-78825, HL-55757, HL-74351, and HL-91202.
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Heart failure after myocardial infarction is one of the most prevalent causes of morbidity and mortality worldwide. Although pharmaceutical and interventional therapies have greatly ameliorated this problem, new more effective approaches are urgently needed. The discovery of resident cardiac progenitor cells (CPCs) in the adult mammalian heart has sparked hope for development of an “ideal” cell type for cardiac reparative/regenerative therapy. These resident CPCs have been shown to commit to a myocardial lineage and can be harvested from the heart and expanded in culture. Previous in vivo studies have shown that administration of CPCs to rats with acute myocardial infarction results in tissue regeneration and left ventricular functional improvement. We sought to extend these findings by determining whether intracoronary infusion of CPCs (the most clinically relevant strategy) can improve function in the setting of an old myocardial infarction (scar). We found that intracoronary infusion of CPCs into a scar produces beneficial structural and functional effects, and that although exogenous CPCs can differentiate into new cardiac cells, this mechanism is not sufficient to explain all of the observed benefits, which suggests paracrine effects. Infusion of exogenous CPCs activated endogenous CPCs, which suggests that this may be a major mechanism for the benefit. These data support the potential therapeutic utility of CPCs in patients with old myocardial infarction.
The online-only Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.109.871905/DC1.
↵*The first 2 authors contributed equally to this work.
Guest Editor for this article was Elizabeth Murphy, PhD.