Local Activation or Implantation of Cardiac Progenitor Cells Rescues Scarred Infarcted Myocardium Improving Cardiac Function
Ischemic heart disease is characterized chronically by a healed infarct, foci of myocardial scarring, cavitary dilation, and impaired ventricular performance. These alterations can only be reversed by replacement of scarred tissue with functionally competent myocardium. We tested whether cardiac progenitor cells (CPCs) implanted in proximity of healed infarcts or resident CPCs stimulated locally by hepatocyte growth factor and insulin-like growth factor-1 invade the scarred myocardium and generate myocytes and coronary vessels improving the hemodynamics of the infarcted heart. Hepatocyte growth factor is a powerful chemoattractant of CPCs, and insulin-like growth factor-1 promotes their proliferation and survival. Injection of CPCs or growth factors led to the replacement of ≈42% of the scar with newly formed myocardium, attenuated ventricular dilation and prevented the chronic decline in function of the infarcted heart. Cardiac repair was mediated by the ability of CPCs to synthesize matrix metalloproteinases that degraded collagen proteins, forming tunnels within the fibrotic tissue during their migration across the scarred myocardium. New myocytes had a 2n karyotype and possessed 2 sex chromosomes, excluding cell fusion. Clinically, CPCs represent an ideal candidate cell for cardiac repair in patients with chronic heart failure. CPCs may be isolated from myocardial biopsies and, following their expansion in vitro, administered back to the same patients avoiding the adverse effects associated with the use of nonautologous cells. Alternatively, growth factors may be delivered locally to stimulate resident CPCs and promote myocardial regeneration. These forms of treatments could be repeated over time to reduce progressively tissue scarring and expand the working myocardium.
Coronary artery disease, hypertension, idiopathic dilated cardiomyopathy, and the unsuccessful repair of a valvular defect lead with time to severe ventricular dysfunction. Cavitary dilation is a common characteristic but the phenotypic architecture and loading of the heart vary significantly among these conditions. If we consider the evolution of the postinfarcted heart, the size of the infarct is not an infallible predictor of the short-, mid-, and long-term outcome of the disease. Negative remodeling and accumulation of damage in the spared myocardium may become critical determinants of cardiac decompensation and its progression to terminal failure.1 The number of events differs in the patient population and segmental losses of myocardium are by far more complex to reconstitute than small foci of injury or scattered myocyte death across the wall. Although areas of spontaneous regeneration have been found,2 these regions are minute and do not reduce significantly infarct size. Myocyte formation occurs acutely3 and chronically,4 but the addition of new cells is mostly restricted to the viable myocardium.
The identification of cardiac progenitor cells (CPCs) in the adult heart5–10 has challenged the generally accepted but never proven paradigm that the heart is a postmitotic organ but has not answered the question why CPCs do not reach spontaneously an infarcted area and rebuild the lost myocardium completely or in part. This is not a particular behavior of the heart but a common defect of proliferating organs. In all cases, occlusion of a supplying artery results in infarcts and scar formation in the skin, intestine, liver, spleen, brain, and bone marrow.1 Although the administration of CPCs locally or their activation by growth factors (GFs) regenerates infarcted myocardium acutely,5,8,11 whether the same protocols can rescue old scarred infarcts remains an important unanswered question. This possibility would make cellular therapy more relevant to the management of chronic human heart failure.
Therefore, in this study, rats with a healed myocardial infarct were treated with implantation of clonogenic CPCs or with intramyocardial delivery of hepatocyte (H)GF and insulin-like (I)GF-1. These GFs were used because CPCs express c-Met and IGF-1 receptors and HGF is a powerful chemoattractant of CPCs, whereas IGF-1 promotes their division and survival.8,11
Materials and Methods
Adult rats were injected with CPCs or GFs 20 days after infarction and scar formation. Hearts were evaluated 20 days later to establish the efficacy of these interventions. Experimental protocols are described in the expanded Materials and Methods section at http://circres.ahajournals.org.
Myocardial infarction was produced in rats and 20 days later animals were randomly injected with enhanced green fluorescent protein (EGFP)-tagged clonogenic CPCs (CPC-treated), HGF and IGF-1 (GF-treated), or saline (untreated). Rats were exposed to 5-bromodeoxyuridine (BrdUrd) to detect newly formed myocardial structures. In GF-treated rats, BrdUrd labeling identified regenerated myocytes and vessels, whereas in CPC-treated animals, EGFP and BrdUrd were both used to recognize the CPC progeny. Animals were euthanized 20 days after treatment.
Using fluorescein-conjugated HGF, we have previously shown that fluorescence intensity is higher adjacent to the infarct than in the distant myocardium up to 1 hour after its injection, preserving the expected chemotactic gradient between these 2 regions of the damaged heart. Most importantly, HGF quantity measured by ELISA 24 hours after infarction was confirmed to be higher in the border than in the remote myocardium.11 Additionally, the effects of HGF and IGF-1 on CPCs were measured biochemically; the autocatalytic phosphorylation of c-Met and IGF-1R, phospho-Akt, phospho–insulin receptor substrate-1, and phospho–focal adhesion kinase increased in CPCs isolated from the infarcted heart 12, 24, and 72 hours following coronary ligation and GF administration.11
Only animals with infarcts involving more than 45% of left ventricular (LV) myocytes were studied to establish the effects of CPCs and GFs on infarcts associated with LV failure.12 Infarct size averaged 67% in all animals (Figure I in the online data supplement). In treated rats, the infarcted region showed a thicker wall with a mixture of scarred and new myocardium. Myocardial regeneration was absent in untreated infarcts; the LV wall was mostly replaced by collagen bundles (Figure 1A through 1C). Infarct thickness was 0.26±0.05, 0.52±0.11, and 0.45±0.08 mm in untreated, CPC-treated, and GF-treated rats, respectively. The increased thickness in treated rats was significant (P<0.001).
Regeneration accounted for 50 and 56 mm3 of myocardium, which resulted in a 40±12% and 44±5% recovery of the scar in CPC- and GF-treated rats, respectively. Additionally, the injection of CPCs reduced infarct size by 39%, from 69% to 42%, whereas GFs decreased infarct dimension by 43%, from 67% to 39% (supplemental Figure I).
Cardiac Anatomy and Ventricular Function
Large myocardial infarcts resulted in a 2.2-fold increase in LV cavitary volume and a 56% decrease in ventricular mass/chamber volume ratio. Treatment with CPCs or GFs attenuated ventricular dilation by ≈21% and the change in ventricular mass/chamber volume ratio by ≈34% (Figure 2A). Ejection fraction (EF) was obtained 2 days before treatment in 18-day-old infarcts and 14 days after treatment in 34-day-old infarcts. At 18 days, EF was decreased in all animals (Figure 2B). The absence of contraction in the infarcted region was apparent and persisted in untreated infarcts at 34 days (supplemental Figure II). Conversely, contraction reappeared in treated infarcts. From 18 to 34 days, treated animals showed an increase in EF, whereas EF was further reduced in untreated rats (Figure 2B); the latter was dictated partly by progressive ventricular dilation typical of this model.12 Hemodynamically, treated infarcts showed a lower increase in LV end-diastolic pressure and a lower decrease in LV systolic pressure, LV developed pressure, and dP/dt. These factors, together with the attenuation in ventricular dilation, resulted in a relative reduction in diastolic stress (Figure 2C).
The new myocardium consisted of myocytes and vessels which collectively comprised ≈90% of the regenerated tissue (supplemental Figure III). CPCs led to the formation of a larger number of arterioles, whereas the number of capillaries and myocytes was comparable in treated hearts. The number of new myocytes markedly exceeded the number of myocytes lost but they had volumes varying from ≈300 to ≈10 000 μm3 (supplemental Figure III). Myocyte regeneration was confirmed by FACS analysis of cells isolated by enzymatic digestion from the scarred region of treated and untreated hearts (Figure 3A through 3C; supplemental Figure IV). EGFP-positive/α-sarcomeric actin (α-SA)–positive myocytes and BrdUrd-positive/α-SA–positive myocytes obtained from infarcted hearts injected respectively with CPCs and GFs constituted ≈26% of the cell pool. Conversely, in untreated infarcts, BrdUrd-positive/α-SA–positive myocytes represented 1% of the cell population. Several BrdUrd-positive/α-SA–negative cells reflected replicating fibroblasts. This population of fibroblasts was markedly decreased in treated infarcts.
Myocyte Phenotype and Mechanics
New myocytes with a volume of 2000 μm3 or larger comprised ≈40% of the population; they expressed cardiac myosin heavy chain, α-SA, α-actinin, and troponin I. Connexin 43 and N-cadherin were present between new myocytes and preexisting and regenerated myocytes (Figure 3D; supplemental Figure V). To determine whether small myocytes represented dividing amplifying cells, Ki67 was evaluated; ≈10% of myocytes were Ki67-positive. They corresponded to the fraction of replicating cells at euthanasia. Additionally, ≈83% of myocytes were BrdUrd-positive (Figure 3E), indicating that myocytes were continuously added over the 20-day period to the developing myocardium. Apoptosis comprised ≈0.4% of new myocytes.
To evaluate whether the newly formed myocytes actively contributed to ventricular performance, myocytes were enzymatically dissociated from the regenerated and surviving myocardium and their mechanical properties were determined. This analysis was restricted to hearts (n=4) treated with EGFP-positive CPCs. The detection of EGFP by fluorescence microscopy in freshly isolated myocytes was confirmed by immunolabeling and confocal microscopy of formalin-fixed cells (not shown). EGFP-positive myocytes and EGFP-negative myocytes had a volume of ≈7500 and 57 000 μm3, respectively. CPC-derived myocytes contracted in response to electric stimulation and exhibited higher cell shortening than resident cells (Figure 4A). Newly formed smaller myocytes embedded within the scar did not tolerate well enzymatic digestion; they lacked an intact membrane and could not be used in this analysis. Therefore, these results have this inherent limitation.
We then determined whether the regenerated myocytes were the product of cell fusion; measurements of DNA content in mono- and binucleated myocytes showed diploid DNA content per nucleus (Figure 4B) excluding polyploidization or fusion. Higher DNA values were found only in Ki67-positive cycling myocytes. Because female rats were used and endogenous CPCs were mobilized by GFs or female CPCs were locally injected, the number of X chromosomes was evaluated. In all cases, at most 2 X chromosomes were found in newly formed myocytes further excluding fusion events (Figure 4C).
To determine the response of the surviving myocardium, tissue volume composition, myocyte cell volume, arteriole and capillary density, BrdUrd labeling of myocytes and endothelial cells (ECs), and Ki67 expression in myocytes were measured. The proportion of myocytes, coronary vessels, and other interstitial structures was comparable in untreated and treated infarcts (supplemental Figure VI). Moreover, myocyte volume increased 70%, 53%, and 49% in untreated, CPC-treated, and GF-treated infarcts, respectively. However, these differences were not significant. The decrease in arteriole and capillary density was also similar in untreated and treated infarcts (Figure 5).
BrdUrd-positive myocytes and ECs and Ki67 labeling of myocytes were comparable in all infarcted hearts (Figure 5). However, the presence of similar levels of myocyte and EC formation in treated hearts with decreased loading conditions suggests that the injected CPCs may have exerted a paracrine effect activating resident CPCs. A similar mechanism of growth has previously been shown.13
Invasive Properties of CPCs
EGFP-tagged CPCs were injected in proximity of chronic infarcts and their ability to spread across the scar was evaluated ex vivo by 2-photon microscopy 4 and 24 hours after cell implantation. The coronary vasculature was perfused with rhodamine-labeled dextran, and collagen was detected by second harmonic generation.14 At 4 and 24 hours after cell delivery, microscopic fields were examined continuously for 5 to 7 hours to measure the movement of EGFP-positive cells. CPCs migrated through collagen bundles and accumulated within the scar. Whereas single EGFP-positive CPCs were detected at 4 hours, clusters of moving cells were present at 24 hours (Figure 6A through 6E), suggesting that these cells possessed the ability to permeate scarred myocardium.
EGFP-positive CPCs moved at a speed of 14±4 μm/h, and there were 520±360 cells/mm3 of scarred myocardium. Corresponding values in 12 to 24 hours acute infarcts were 21±5 μm/h and 2300±600 cells/mm3 of infarcted myocardium (Figure 6F through 6J).
To study the effects of GFs, a lentivirus-carrying EGFP was injected 20 days after infarction and 2 days later GFs were given. After 4 hours, the translocation of cells from the border to the infarct was analyzed for 5 to 10 hours. EGFP-labeled cells entered the scar and moved within the scar (not shown). EGFP-positive cells were characterized by confocal microscopy; they corresponded to c-kit–positive CPCs. Nonmoving EGFP-positive cells represented fibroblasts, ECs, and myocytes. EGFP-tagged CPCs whether locally injected or activated by GFs created tunnels surrounded by thick bundles of collagen type 1 and type III (not shown).
In chronic infarcts, GF-activated CPCs moved at 30±12 μm/h, and there were 270±150 cells/mm3 of scarred myocardium. Corresponding values in 12 to 24 hours acute infarcts were 62±23 μm/h and 450±230 cells/mm3 of infarcted myocardium.
Matrix metalloproteinases (MMPs) degrade the extracellular matrix and play a key role in invasive growth, suggesting that intracellular or membrane-bound MMPs favor CPC translocation. Importantly, HGF increases the function of MMP-2 and MMP-9 in CPCs.11 Therefore, the expression of MMP-2, MMP-9, and MMP-14 was evaluated in samples of infarcted scarred myocardium 1, 2, and 3 days after the injection of CPCs. MMP-2 and MMP-9 cleave interstitial collagen and MMP-14 belongs to the group of membrane-bound MMPs, which digest several interstitial proteins and activate other MMPs.15,16 The catalytic activity of MMPs is blocked by the tissue inhibitors of MMPs (TIMPs); TIMP-4 is highly expressed in the adult heart.17
In the presence of CPCs, MMP-2, MMP-9, and MMP-14 were significantly increased and TIMP-4 was markedly decreased (Figure 7A and supplemental Figure VII). Importantly, the activity of MMP-9 was 10-fold higher in CPC-treated than in untreated infarcted hearts (Figure 7B and 7C). However, MMP-2 activity was similar in the 2 groups of rats. Additionally, MMP-14 was not evaluated in this assay because it does not possess gelatinase activity. Because MMP-14 activates MMP-2 and MMP-9, we cannot exclude that MMP-14 contributed to the enhanced function of MMP-9.
Cytokine and GF Profile
In an attempt to identify the signals involved in the migration and homing of CPCs in the infarcted heart and determine whether substantial differences existed between acute and chronic infarcts, a cytokine and GF array was performed (supplemental Table I). In comparison with sham-operated rats, the scarred myocardium in untreated chronic infarcts contained higher quantities of the cell adhesion molecule soluble intercellular adhesion molecule (sICAM)-1, the chemo-attractant CXC chemokine ligand (CXCL)7, basic fibroblast (bF)GF, and the MMP inhibitor TIMP-1 (Figure 8). The latter promotes cell division, opposes apoptosis, and participates in vessel remodeling.18 Conversely, the inhibitor of cytokine synthesis interleukin-10 was downregulated. Untreated chronic infarcts also had greater levels of sICAM-1 and bFGF than untreated acute infarcts. However, the acutely infarcted myocardium possessed significantly higher amounts of proteins involved in the early phases of the inflammatory response (cytokine-induced neutrophil chemoattractant [CINC]-1, CINC-2α/β, tumor necrosis factor-α) together with chemokines associated with acute tissue injury (LPS-induced CXC chemokine [LIX], macrophage inflammatory protein-3α). Interferon-γ–inducible protein (IP)-10 was more abundant in untreated acute than in untreated chronic infarcts (Figure 8). This CXC3 receptor ligand is implicated in vessel homeostasis and repair.19
With cell treatment, there was an upregulation of several cytokines and GFs in the scarred myocardium including CINC-3, platelet-derived GF receptor α, IP-10, LIX, L-selectin, TIMP-1, and the member of the epidermal growth factor family amphiregulin. Cell treatment in acute infarcts increased a variety of chemokines that, with the exception of IP-10, differed from those in chronic infarcts (Figure 8).
The results of the present study demonstrate that CPCs locally activated by GFs or injected directly in proximity of a healed infarct can rescue nearly 45% of the infarct by replacing fibrotic tissue with functionally competent myocardium. Myocardial regeneration protects the infarcted heart from the progressive increase in cavitary dilation, decrease in wall thickness, and deterioration in ventricular function with time. These findings, together with previous observations in the acutely infarcted heart,5,8,10,12 strongly suggest that CPCs are a powerful form of cell therapy for ischemic cardiomyopathy. CPCs are effective whether administered intramyocardially,5,10 via the coronary route,20 or activated in situ with HGF and IGF-1, which trigger their growth and mobilization shortly after an ischemic event8,11 and, as shown here, chronically at the completion of healing. CPCs migrate through the myocardial interstitium reaching areas of necrotic and scarred myocardium, where they home, divide, and differentiate into myocytes and vascular structures. From a clinical perspective, CPCs appear to represent an ideal candidate for cardiac repair in patients with chronic heart failure in which discrete areas of damage are present in combination with multiple foci of replacement fibrosis across the ventricular wall.1 Potentially, CPCs may be isolated from endomyocardial biopsy or surgical samples and, following their expansion in vitro, administrated back to patients, avoiding the inevitable and threatening adverse effects of rejection and other complications with nonautologous transplantation. Alternatively, GFs can be delivered locally to stimulate resident CPCs and promote myocardial regeneration. Importantly, these strategies may be repeated to reduce further myocardial scarring and expand the working myocardium.
Experimental and clinical studies have documented that bone marrow progenitor cells (BMPCs), administered acutely and chronically after infarction, improve ventricular function in animals and humans.21 Based on these initial observations, double-blind clinical trials have been conducted and, with 1 exception,22 the actual efficacy of BMPCs in human heart failure has been shown.23,24 These findings show rather convincingly that BMPCs have a potential therapeutic effect on the infarcted heart, but the mechanism involved remains unclear. The ability of BMPCs to transdifferentiate and form cardiomyocytes and coronary vessels has been controversial, and data in favor and against this possibility have been reported.1 Recently, 4 laboratories have addressed this issue in a collective manner and provided strong evidence in support of the notion of BMPC plasticity and the therapeutic import of BMPCs for myocardial regeneration.25 The implementation of BMPCs in patients has a 6-year history and any other form of cell therapy will have to be compared with that of BMPCs. This will be inevitable for CPCs as well.
The identification of CPCs has challenged the generally accepted but never proven paradigm that the heart is a postmitotic organ but has not answered the question why CPCs do not reach spontaneously an infarcted area rebuilding completely or in part the lost myocardium. This was in the past and continues today to be interpreted as a growth limitation of the adult heart. Conversely, the present results document that there is a significant pool of functionally competent CPCs located in the surviving myocardium adjacent to healed infarcts that, following activation with GFs, infiltrate the scarred tissue and generate a remarkable number of cardiomyocytes and coronary vessels. Similarly, the injection of adult CPCs leads rapidly to extensive myocardial regeneration. CPCs are programmed to acquire the myocyte, vascular smooth muscle cell, and EC lineages and, in contrast to BMPCs, do not have to transdifferentiate to give rise to cardiac cell categories. Transdifferentiation is a time-consuming process that necessitates the reorganization of chromatin through activation and silencing of specific transcription factors together with epigenetic changes.26 Nevertheless, BMPCs are easily accessible, and this is a critical factor clinically.
Based on the present results, the possibility is advanced that CPCs may be superior to BMPCs for myocardial regeneration. However, control animal studies addressing this question have yet to be performed. CPCs are well equipped to sustain the physiological turnover of myocytes, smooth muscle cells, and ECs of the adult heart but are not programmed to sense distant damage, translocate to this site and activate regenerative growth in response to injury. This limitation, together with the death of CPCs in the ischemic area,8,11 precludes an effective reconstitution of the infarcted myocardium. Importantly, this is not a unique deficiency or a peculiar behavior of the heart but a common deficiency of proliferating organs; artery occlusion results in tissue necrosis and scarring in the skin, intestine, liver, spleen, kidney, brain, and bone marrow, regardless of whether they are self-renewing organs regulated by a stem cell compartment.1
Observations here indicate that myocardial scarring interferes with the migration and engraftment of locally injected or GF-activated CPCs. Acute infarcts are more amenable to CPC translocation and homing, providing a milieu in which CPCs rapidly accumulate and generate a committed progeny. In spite of the less favorable environment dictated by collagen deposition chronically after infarction, CPCs retained the ability to infiltrate the scar, digest part of the connective tissue, and form cardiomyocytes and coronary vessels. The comparable effects on myocardial regeneration obtained by local CPC delivery and GF activation of resident CPCs may be explained by the pattern of migration of these cells within the scar; the higher number of cells found with intramyocardial injection of CPCs was compensated by the faster speed of migration of GF-activated CPCs. When these 2 variables are considered, ie, speed and cell number, the accumulation of cells under these conditions is remarkably similar. However, this was not the case in acute infarcts, in which cell infiltration was more efficient following CPC implantation than after CPC activation by GFs.
Whether these results have implications for the treatment of chronic human heart failure is difficult to predict. Risk factor such as aging and diabetes are frequently present in patients with ischemic cardiomyopathy, and they have profound negative consequences on the number and function of CPCs.27 However, functionally competent human CPCs have been isolated from a variety of patients undergoing open-heart surgery,10 and the activation of CPCs by GFs in senescent rats with severe ventricular decompensation reverses the cardiac phenotype and prolongs lifespan.28 Collectively, these findings raise the possibility that the strategies used here in an animal model may be relevant to the management of human heart failure.
CPCs and Cytokine Milieu
The invasion of the scarred tissue by CPCs appeared to be mediated by enhanced activity of MMP-9 and possibly MMP-14. MMP-9 is critical for the recruitment of bone marrow stem cells and their mobilization from quiescent to proliferative niches,29 and a similar mechanism may be operative in the translocation to the chronically infarcted heart of GF-treated resident CPCs or delivered CPCs. The upregulation of MMP-9 expression and activity in CPCs is dependent on HGF.11 Additionally, stromal cell–derived factor (SDF)-1, which is highly expressed in myocytes and ECs after ischemic injury, acts on MMP-9 and promotes the differentiation of CPCs into vascular cells and cardiomyocytes.14 The lack of increase in MMP-2 activity observed here may favor the stability of SDF-1, which is degraded by this protease.30
Importantly, with respect to the intact heart, the content of cytokines and GFs differed in the scarred myocardium. The enhanced expression of the chemotactic factors sICAM-1, CXCL7, and bFGF in chronic infarcts may have created a condition facilitating migration and homing of CPCs.31 Additionally, the presence of sICAM-1, CXCL7, and TIMP-1 in the scar could have promoted CPC mobilization, EC migration and differentiation, and vessel formation.32 The increases in CXCL7 and TIMP-1 were restricted to chronic infarcts. As expected, the acutely infarcted myocardium displayed a cytokine profile that reflected an inflammatory response and tissue reaction to acute injury. However, the higher levels of sICAM-1 and bFGF within the scar are particularly relevant for myocardial regeneration because sICAM-1 may have created a niche structure that supported the engraftment of CPCs and bFGF is critical for the differentiation of resident CPCs into myocytes.33
The analysis of cytokine and GF expression in acute and chronic infarcted hearts offered the possibility to establish whether the delivery of CPCs participated in the synthesis and secretion of soluble proteins which exerted an autocrine or paracrine effect on myocardial regeneration. The presence of CPCs in the scar attenuated, at least in part, the differences in cytokine profile with acute infarcts by increasing the quantities of LIX, which is involved in stem cell maintenance and proliferation,34 and IP-10, which modulates vessel homeostasis and growth.19 Thus, acute and chronic myocardial damage lead to the expression of genes that create a microenvironment that conditions the activation, migration, and growth of CPCs, ultimately controlling the regenerative response of the pathological heart.
Sources of Funding
This work was supported by NIH grants. J.F.-M. was supported by the Foundation for Science and Technology, Ministry of Science, Technology and Higher Education (Portugal).
This manuscript was sent to James T. Willerson, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
Original received January 22, 2008; resubmission received April 29, 2008; revised resubmission received May 24, 2008; accepted May 29, 2008.
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