Published online before print October 23, 2008, doi: 10.1161/CIRCRESAHA.108.186742
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© 2008 American Heart Association, Inc.
Cellular Biology |
IGF-1–Overexpressing Mesenchymal Stem Cells Accelerate Bone Marrow Stem Cell Mobilization via Paracrine Activation of SDF-1
/CXCR4 Signaling to Promote Myocardial Repair
From the Department of Pathology and Laboratory Medicine, University of Cincinnati, Ohio.
Correspondence to Prof Muhammad Ashraf, Department of Pathology and Laboratory of Medicine, 231 Albert Sabin Way, University of Cincinnati, Cincinnati, OH 45267-0529. E-mail muhammad.ashraf{at}uc.edu
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Abstract |
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We hypothesized that mesenchymal stem cells (MSCs) overexpressing insulin-like growth factor (IGF)-1 showed improved survival and engraftment in the infarcted heart and promoted stem cell recruitment through paracrine release of stromal cell–derived factor (SDF)-1
. Rat bone marrow–derived MSCs were used as nontransduced (NormMSCs) or transduced with adenoviral-null vector (NullMSCs) or vector encoding for IGF-1 (IGF-1MSCs). IGF-1MSCs secreted higher IGF-1 until 12 days of observation (P<0.001 versus NullMSCs). Molecular studies revealed activation of phosphoinositide 3-kinase, Akt, and Bcl.xL and inhibition of glycogen synthase kinase 3β besides release of SDF-1 in parallel with IGF-1 expression in IGF-1MSCs. For in vivo studies, 70 µL of DMEM without cells (group 1) or containing 1.5x106 NullMSCs (group 2) or IGF-1MSCs (group 3) were implanted intramyocardially in a female rat model of permanent coronary artery occlusion. One week later, immunoblot on rat heart tissue (n=4 per group) showed elevated myocardial IGF-1 and phospho-Akt in group 3 and higher survival of IGF-1MSCs (P<0.06 versus NullMSCs) (n=6 per group). SDF-1 was increased in group 3 animal hearts (20-fold versus group 2), with massive mobilization and homing of ckit+, MDR1+, CD31+, and CD34+ cells into the infarcted heart. Infarction size was significantly reduced in cell transplanted groups compared with the control. Confocal imaging after immunostaining for myosin heavy chain, actinin, connexin-43, and von Willebrand factor VIII showed extensive angiomyogenesis in the infarcted heart. Indices of left ventricular function, including ejection fraction and fractional shortening, were improved in group 3 as compared with group 1 (P<0.05). In conclusion, the strategy of IGF-1 transgene expression induced massive stem cell mobilization via SDF-1 signaling and culminated in extensive angiomyogenesis in the infarcted heart.
Key Words: IGF-1 • heart • myocardial infarction • SDF-1 • stem cells
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Insulin-like growth factor (IGF)-1/IGF-1R ligand/receptor signaling has multifunctional activities including growth, development, and differentiation of cardiac muscle.1 Intrinsically, altered IGF-1 level is observed during different pathological conditions, thus implying its role in tissue protection and repair process.2,3 The main function of IGF-1 in the heart is to stimulate cardiac growth and contractility.4 Indeed, there are reports showing left ventricular (LV) hypertrophy caused by IGF-1 administration.5 However, IGF-1 has distinct beneficial effects on cardiomyocytes including their survival and proliferation.6 Cardiac-specific overexpression of IGF-1 attenuates dilated cardiomyopathy in tropomodulin-overexpressing transgenic mice.7 The therapeutic effects of cardiac-specific IGF-1 overexpression were signified by improved indices of cardiac structure and function.8 The benefits of IGF-1 therapy on the heart have also been achieved by IGF-1 protein delivery.9 Long-term systemic administration of IGF-1 improved cardiac function despite showing signs of cellular hypertrophy.10 A comparative study of IGF-1 gene and protein delivery clearly shows superior functional effects of gene delivery approach.11 Significant progress has been made for enhanced and localized overexpression of IGF-1 in the heart using both viral and nonviral vectors.12,13 IGF-1 gene delivery has also been combined with other growth factors and with heart cell therapy to promote donor cell survival, engraftment, and differentiation as a part of the multimodal therapy approach.14–17 These studies, however, do not provide much information on the role of IGF-1 to engage host-derived stem and progenitor cells in the cardiac repair process. We have previously shown that bone marrow (BM)-derived stem cells release paracrine factors at the site of the cell graft.18 Indeed, their genetic modification with therapeutic genes accentuates their ability to release these factors. We hypothesized that IGF-1 has a pivotal role in attracting stem cells to the injured heart and their differentiation via release of paracrine factors, besides activating molecular pathways of cell survival. We observed that ex vivo overexpression of IGF-1 resulted in elevated SDF-1
level, a potent chemoattractant of stem cells, both in vitro and after transplantation in the infarcted heart. Our results imply vital consequences of IGF-1 gene delivery and clearly support our rationale that IGF-1 overexpression in the heart accelerates stem cell migration and cardiac regeneration. |
Materials and Methods |
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MSCs transduced with Ad-Null vector without a therapeutic gene (NullMSCs) or transduced with human IGF-1 (hGF-1) transgene (IGF-1MSCs) were characterized in vitro and were later injected in a rat heart model of acute myocardial infarction (MI).19,20 Experimental protocols are described in the expanded Materials and Methods, available in the online data supplement at http://circres.ahajournals.org.
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Results |
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In Vitro Studies
Characterization of IGF-1MSCs and Molecular Studies
More than 99% of IGF-1MSCs overexpressed hIGF-1 (Figure 1A), which was
200-fold higher as compared with other groups of cells (supplemental Figure I, A). These results were further confirmed by Western blotting (Figure 1B). The hIGF-1 protein secretion from IGF-1MSCs was sustained for at least 12 days of observation. ELISA performed on the conditioned medium (CM) from IGF-1MSCs (IGF-1CM) showed cumulative peak level secretion of 110 ng/mL hIGF-1 between 6 to 9 days from the start of the transduction procedure. Only negligible amounts of IGF-1 were observed in the CM from NullMSCs (NullCM) and CM obtained from NormMSCs (IGF-1CM versus all other groups, P<0.05 on all time points) (supplemental Figure I, B).
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IGF-1 Overexpression Incurs Cytoprotection
The autocrine and paracrine bioactivity of hIGF-1 released from IGF-1MSCs in terms of cytoprotection was determined under oxygen-glucose deprivation (OGD). There was obvious morphological damage to NullMSCs under OGD, which was evident from their shrunken and wrinkled appearance (Figure 2, A1) as compared with NullMSCs without OGD (Figure 2, A2). IGF-1 overexpression reversed these changes (Figure 2, A3). Moreover, the morphological damage was accompanied by higher cell death in NullMSCs under OGD as compared with IGF-1MSCs (P<0.01 versus IGF-1MSCs) by lactate dehydrogenase (LDH) leakage studies (Figure 2, A4). The results were further confirmed by TUNEL staining (Figure 2, A5 through A8). The number of TUNEL+ cells was significantly higher in NullMSCs (Figure 2, A6) as compared with IGF-1MSCs (Figure 2, A7) under OGD using MSCs under normoxia (NormMSCs) as controls (Figure 2, A5). Figure 2, A8, is the graphical representation of the results (P<0.01 versus IGF-1MSCs). We also observed that human umbilical vein endothelial cells (HUVECs) (supplemental Figure II, A1) and cardiomyocytes (supplemental Figure II, B1) treated with NullCM showed shrinkage and morphological distortions after 8 hours of OGD as compared with their counterparts grown under normoxia (supplemental Figure II, A2 and B2). On the contrary, treatment of HUVECs and cardiomyocytes with IGF-1CM substantially preserved their morphological integrity (supplemental Figure II, A3 and B3). The percentage cell death in HUVECs and cardiomyocytes treated with IGF-1CM was lower as compared with the cells treated with NullCM (IGF-1CM versus NullCM P<0.001) (supplemental Figure II, A4 and B4). The poor resistance of HUVECs and cardiomyocytes cultured in NullCM under OGD was consistent with the observation that NullMSCs did not show IGF-1 expression (Figure 1).
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Molecular Signaling and Growth Factor Expression in IGF-1MSCs
Although multiple signaling pathways have been reported downstream of IGF-1/IGF-1R interaction, phosphoinositide 3-kinase (PI3K)/Akt signaling plays a major role in cytoprotection. IGF-1/IGF1R interaction activates PI3K to the cell membrane that, in turn, activates the Akt kinase (supplemental Figure II, C), thus activating its downstream substrates such as Bcl.xL and inhibition of glycogen synthase kinase (GSK)3β in IGF-1MSCs.21
The release of paracrine factors by BM cells is one of the contributing factors in cardiac function improvement.18 Our RT-PCR data showed that IGF-1 overexpression accentuated the release of various secretable growth factors, including hepatocyte growth factor, basic fibroblast growth factor, vascular endothelial growth factor (VEGF), and SDF-1 (supplemental Figure II, D). Western blot showed that SDF-1 protein expression was elevated in IGF-1MSCs as compared with other groups of cells (supplemental Figure II, E). The migratory response of NormMSCs (Figure 2, B1 through B4) and HUVECs (Figure 2, C1 through C4) to IGF-1CM was higher (P<0.001) as compared with NullCM and basal DMEM. The migratory response of NormMSCs to IGF-1CM was significantly impaired by prior treatment of the cells with 5 to 10 µg/mL AMD-3100, which is a well-known CXCR4 antagonist (supplemental Figure II, F1 through F4
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In Vivo Studies
All animals treated with DMEM (group 1), NullMSCs (group 2), and IGF-1MSCs (group 3) survived full length of the experiment. The animals were harvested at 7 days (n=10 per group) and 4 weeks (n=12 per group) after cell engraftment for molecular and histological studies.
Growth Factor Expression and Molecular Signaling in the Rat Heart
The heart tissue samples on day 7 showed continuous overexpression of hIGF-1 in group 3, whereas negligible hIGF-1 expression was detected in NullMSC transplanted group 2 animal hearts (supplemental Figure II, G). Concomitantly, elevated SDF-1 expression was also observed subsequent to hIGF-1 overexpression in group 3 animal hearts as compared with group 2 (P<0.001) (supplemental Figure II, G). Consistent with our in vitro studies, phospho-Akt (pAkt) protein expression in group 3 hearts was elevated as compared with group 2 (supplemental Figure II, H). These data demonstrated that IGF-1 gene overexpression activated critical survival signaling not only in IGF-1MSCs but also in the infarcted heart as a result of paracrine release of IGF-1 which contributed toward reduced cell apoptosis (Figure 3A).
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Donor Cell Survival Postengraftment
Real-time PCR for rat sry gene confirmed an extensive survival of the donor cells in the cell transplanted groups 2 and 3 (Figure 3B). Although the difference between group 2 and 3 was statistically insignificant (P=0.06), the survival of cells in IGF-1MSC group 3 was higher. Fluorescence in situ hybridization results further confirmed these findings (Figure 3C). By 4 weeks after intramyocardial engraftment, the cells were found incorporated predominantly into the center and border zones of the infarcted heart. In the periinfarct region, some of the male donor cell nuclei were seen integrated into muscle fibers, indicating their myogenic differentiation (Figure 3D).
Angiomyogenesis and Mobilization of Stem Cells
Immunofluorescence studies demonstrated that PKH26-labeled donor cells (red) were positive for -sarcomeric actinin (green), as indicated by colocalization of red and green fluorescence signals (supplemental Figure III, A1 through A4). Immunostaining for connexin-43, together with cardiac actin showed that the neofibers also developed intercellular communications (Figure 4, A1 through A4). In the cell-treated animal hearts, the neofibers showed markedly higher staining for connexin-43 as compared with the hearts treated with DMEM. Further confirmation of myogenic differentiation of the engrafted cells was achieved by immunofluorescence studies for myosin heavy chain slow isoform (MHC slow isoform) (supplemental Figure IV). These results vividly show the ability of MSCs to engraft in the ischemic heart and differentiate to adopt cardiac phenotype. Incidentally, engraftment and myogenic differentiation of IGF-1MSCs was conspicuously higher as compared with NullMSCs. Ultrastructure studies duplicated these findings and showed extensive presence of differentiating cells in the center of the infarct and periinfarct regions. The differentiating cells showed a spectrum of morphological changes that were characteristics of developing myofibers. Figure 4, B1, shows a cluster of cells undergoing myogenic differentiation in the infarct area. Figure 4, B2 through B4, shows magnified images of individual cells from the cluster. The cells in their earlier stage of differentiation showed nascent sarcomeric striations and contained focal aggregations of electron-dense material, suggestive of developing Z-discs (Figure 4, B3; green arrows). Some regions of the differentiating cells were seen having increasing amounts of myofibrils with a corresponding decrease in ribosomes and abundant mitochondria. They had sarcomeres that were out of registry as compared with the most mature cells (Figure 4; green arrows). Adjacent differentiating cells had intimately apposed, interdigitating cell membranes, identified with electron-dense membrane structures, suggestive of intermediate adherens and gap junctions (Figure 4, B3 and B4; blue arrows).
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Another important feature of our study was extensive presence of ckit+ (Figure 5, A1 through A3), MDR-1+ (Figure 5, B1 through B3), CD31+ (Figure 5C), and CD34+ (Figure 5D) cells in the infarcted myocardium. Their number was significantly higher in group 3 as compared with group 2. Immunofluorescence studies for myogenic markers also showed extensive presence of cells expressing MHC slow isoform in IGF-1MSCs transplanted hearts (supplemental Figure V). These cells lacked our fluorescent label (PKH26 red fluorescence), which indicated that they have been mobilized into the region of cell transplantation. Most of these cells appeared to be in their nascent phase of differentiation, appearing to be rounded or oblong (supplemental Figure V, A4, white arrows) and lacked typical striations of neofibers; however, they had started to express myogenic marker protein.
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Host Myocyte Protection and Infarct Size Reduction
Islands of the host myofibers were identified in the LV scar tissue under bright field microscopy after hematoxylin/eosin staining. These islands of myofibers were mainly located in the periphery of larger blood vessels in all animal groups. As compared with group 1 that rarely showed surviving myocytes within the center of the scar tissue (supplemental Figure VI, A), more myocyte islands were observed in the center of the infarct in the cell-transplanted groups (supplemental Figure VI, B and C). The hearts injected with IGF-1MSCs showed higher propensity of independent myocyte islands in the scar tissue. Similar observations were made during ultrastructural studies, which confirmed the presence of residual host myocardial islands in the infarcted heart (supplemental Figure VI, D and E). Gross histological examination revealed that infarct size expansion was substantially attenuated in cell treatment groups as compared with group 1 (Figure 6A through 6C). The percentage of infarct size in group 1 exhibited transmural scar tissue occupying 46.3±1.4% of the LV (Figure 6D). However, infarct size was reduced to 38.51±1.7% in group 2 (P=0.03 versus group 1) and 32.76±4.1% in group 3 (P=0.008 versus group 1; P=0.31 versus group 2), respectively.
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IGF-1 Overexpression and Angiogenesis
We next tested whether gene therapy–mediated overexpression of hIGF-1–modulated angiogenic response in the infarcted heart. Supplemental Figure VII shows the representative immunostaining for von Willebrand factor VIII in different animal groups. Blood vessel density at 4 weeks after their respective treatment was significantly different between group 1 (supplemental Figure VII, A and B) as compared with group 2 (supplemental Figure VII, C and D) and group 3 (supplemental Figure VII, E and F) in the infarct and periinfarct regions. Engraftment of IGF-1MSCs in group 3 gave the highest number of blood vessels per surface area (0.155 mm2) at high-power microscopic field (x40) in both infarct (37.7±1.8; P<0.01 versus group 1) and periinfarct areas (64.18±2.3; P<0.001 versus group 1) as compared with group 2 was 30.1±2 (P=0.01 versus group 1) and 53.8±3 (P<0.001 versus group 1), respectively (Figure 7). Blood vessel density was the lowest in DMEM group 1 animals in infarct (28.5±2.2) and periinfarct areas (41.8±2.1).
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Preservation of the LV Heart Function and Dimensions
Echocardiography at 4 weeks after coronary artery ligation showed that DMEM group 1 animals exhibited impaired contractility and increased LV dilation. Comparatively, the cell-transplanted groups showed more preserved LV contractile function and LV dimensions. Four weeks after their respective treatment, highest LV ejection fraction (LVEF) (63.05±1.37%) (Figure 8A) and LV fractional shortening (LVFS) (28.54±0.92%) (Figure 8B) were observed in the IGF-1MSC group 3. These were significantly higher as compared with group 2 (52.98±2.18%, P<0.001; 22.49±1.13%, P<0.001), respectively. Group 1 showed severely impaired LV contractile function with LVFS (17.51±1.54%) and LVEF (43.29±3.17%). Engraftment of IGF-1MSCs reduced the progression of LV cavity dilation and wall thinning measured in end diastole as compared with groups 1 and 2. The left ventricular end-diastolic dimension (LVEDd) and left ventricular end-systolic dimension (LVEDs) in group 1 were 7.36±0.15 and 6.07±0.17 mm, respectively (supplemental Table III). There was a limited increment of LVEDd and LVEDs in group 2 (6.93±0.16 mm, P=0.06; 5.35±0.1 mm, P<0.001) and group 3 (6.79± 0.11 mm, P=0.009; 4.84±0.09 mm, P<0.001) as compared with group 1. Moreover, anterior wall thickness at end diastole (AWTd) and end systole (AWTs) were best maintained in group 3 (1.1±0.04, P=0.059; 1.39±0.05 mm, P<0.001, respectively), followed by group 2 (1.07± 0.07 mm, P=0.164; 1.21±0.05 mm, P=0.002, respectively). DMEM treatment resulted in significant reduction of AWTd (0.94±0.08) and AWTs (0.91±0.06) in group 1 as compared with group 3. LV anterior wall thickness was significantly recovered in group 3 as compared with group 1. These results show that the indices of cardiac function and LV remodeling were significantly improved after IGF-1MSC transplantation in the infarcted heart.
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Discussion |
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The short biological half-life (
15 minutes) and smaller molecular size of IGF-1 warrants a steady source of IGF-1 input to ensure its nonfluctuating and consistent levels in the biological system. Given that IGF-1 protein therapy has limitations, we combined MSCs transplantation with hIGF-1 gene delivery by intramyocardial injection of IGF-1MSCs. IGF-1MSCs continued to secrete hIGF-1 protein until 7 days of observation postengraftment in the heart. Previous studies have shown that IGF-1R expression is elevated as a part of the intrinsic repair mechanism after MI.6 Transplantation of IGF-1MSCs during acute phase of MI in the present study ensured optimal participation of IGF-1/IGF1R system for enhanced cardiac repair. The important findings of our study are: (1) IGF-1MSCs survived better under anoxia as compared with NullMSCs; (2) release of SDF1- from IGF-1MSCs contributed to massive stem cell mobilization; (3) extensive presence of newly formed muscle fibers and blood vessels were observed after IGF-1MSC transplantation; and (4) localized IGF-1 overexpression significantly preserved LV wall thickness and contractile function.
PI3K/Akt Signaling and Cell Survival
Donor cell attrition is a crucial factor in their successful engraftment. Various remedial measures have been adopted to overcome this problem especially during acute phase of engraftment.20,22 Combining heart cell therapy with therapeutic gene delivery is more exciting in terms of donor cell survival and differentiation. We have previously shown that concomitant overexpression of angiopoietin-1 and Akt in MSCs promoted their survival in the infarcted heart.19 IGF-1 gene delivery to this end would be more advantageous because of its cardioprotective and cardiomyogenic activities.23 It is important to note that IGF-1/IGF-1R system has a wide distribution in the heart on myocytes, cardiac progenitor cells (CPCs), and cardiac fibroblasts, and its activation regulates many functions such as telomerase activity, hinders replicative senescence, and preserves functionally competent CPCs.6,24 We observed that preconditioning of Sca-1+ cells with IGF-1 enhanced their paracrine activity and the preconditioned cells showed multiple fold increase in IGF-1 overexpression (our unpublished data, 2008). However, IGF-1 overexpression was transient and disappeared in a few days. Transplantation of MSCs overexpressing hIGF-1 in the present study yielded more persistent and nonfluctuating supply of hIGF-1 in the heart at the site of the cell graft for a longer time duration as compared with the preconditioned cell engraftment.
IGF-1 exerts its biological effects by binding to its transmembrane receptors and activates Akt phosphorylation via PI3K signaling and serves as an essential mediator of IGF-1 signaling.21 The improved survival of IGF-1MSCs under OGD in the present study is attributed to IGF-1/IGF-1R interaction. Moreover, expression of CXCR4 on MSCs (supplemental Figure VIII), and its interaction between the elevated SDF-1 level subsequent to hIGF-1 overexpression also contributed to the antiapoptotic effects of hIGF-1 overexpression.
Besides its role in cell survival, pAkt negatively regulates kinase activity of GSK3β (a negative regulator of cell growth). These intracellular changes including inactivation of GSK-3β are associated with the expression of muscle specific proteins and potentiate myogenesis, in addition to their pivotal role in cell survival. Extensive myogenic response in our study is attributed to these molecular events which showed significantly higher pGSK3β in the IGF-1MSCs as compared with NullMSCs. More so, pAkt and Bcl.xL showed higher level of expression in group 3 animal hearts as compared with group 2. Incidentally, these molecular changes correlated well with reduced number of TUNEL+ cells in the infarcted heart and noticeably higher cell survival in group 3 as compared with group 2.
Paracrine Activation of SDF-1/CXCR4 Signaling and Stem Cell Mobilization
Besides having multilineage potential, MSCs can also secrete a plethora of angiogenic and mitogenic cytokines and growth factors in normoxic culture conditions, and their release increases significantly in response to anoxia.18 Secretion of paracrine factors by other cell types, including endothelial progenitor cells and cardiomyocytes, has also been reported.25,26 The presence of paracrine pathway in cardiomyocytes is important to maintain normal cardiac function through release of VEGF.25 During in vitro studies, the cytoprotective effect accorded by paracrine factors in CM from MSCs on cardiomyocytes was evident from their higher resistance to cell death on subsequent exposure to apoptotic stimulus as compared with control cardiomyocytes grown in nonconditioned culture medium.27 The expression of these bioactive molecules provides an alternative and/or supportive mechanism for BM cells during tissue repair.28,29 Genetic modification of BM cells for Akt overexpression accentuated the release of bioactive molecules, which acted in paracrine fashion to exert cytoprotective and ionotropic effects.30 Our results showed increased pAkt in IGF-1MSCs with a concomitant increase in SDF-1, whereas NullMSCs with low level pAkt showed minimal SDF-1 expression. SDF1-/CXCR4 ligand/receptor system plays a significant role in mobilization of stem cells.31 SDF-1 binds to CXCR4 receptor and modulates several biological functions including increased cell growth, proliferation, antiapoptosis, and emigrational and transcriptional activation. Additionally, SDF-1 is also a "retention factor" and ensures retention of the mobilized CXCR4+ cells for long enough to ensure their participation in repair process.32 Given the crucial role of SDF-1 in stem cell mobilization and retention, SDF-1 is also produced in response to myocardial ischemia. The elevated secretion of the intrinsic SDF-1 is, however, transient and drops back to normal within 4 to 6 days after ischemia.33 We have previously shown that transgenic overexpression of SDF-1 in the heart supports the declining intrinsic SDF-1 levels for extended duration and provides a cue for stem cells to mobilize and home into the infarcted heart.34 SDF-1 levels remained significantly high in the infarcted heart after transplantation of IGF-1MSCs until 7 days of observation. Our results showed a higher presence of mobilized cells in group 3 as compared with group 2. In agreement with a recent report that CPCs express CXCR4, it is highly likely that the mobilized cells observed in our study also occurred from the resident population of progenitor cells in the heart, as well as originating from BM.35 Similarly, there are reports that cardiomyocytes express CXCR4 receptors.36 It is, therefore, likely that the elevated SDF-1 promoted cardiomyocyte survival via interaction with CXCR4 (supplemental Figure VIII, C) and contributed to reduced TUNEL positivity in group 3.
IGF-1 Overexpression and Myocardial Angiomyogenesis
IGF-1 itself is a less potent inducer of angiogenic response as compared with other angiogenic growth factors such as basic fibroblast growth factor and VEGF. In some cases, the proangiogenic activity of IGF-1 is attributed to an associated VEGF upregulation.37 Considering a possible role for biologically active VEGF release following IGF-1 overexpression, Yau et al concomitantly delivered both the genes to the infarcted heart to seek possible synergism for antiapoptotic and myogenic effects.17 IGF-1 is a potent activator of endothelial cell migration and a powerful mitogen and antiapoptotic factor for vascular smooth muscle cells. These biological effects of IGF-1 are mediated by differential IGF-1 signaling pathways and require both PI3K and extracellular signal-regulated kinase. Our results show increased levels of various growth factors from IGF-1MSCs including VEGF, which is in harmony with the previous reports. The higher expression of multiple growth factors associated with IGF-1 gene delivery consequently led to homing of CD31+ and CD34+ cells and a greater degree of angiogenic response in the heart. The emigrational response of HUVECs to IGF-1CM in vitro may be attributed to presence of VEGF in IGF-1CM.
Improved Cardiac Function
The cell-based delivery of IGF-1 led to improved indices of LV contractile function, besides attenuation of remodeling indices, including LV wall thinning and dilatation. The significance of IGF-1 in this recovery process may be realized from the subsequent autocrine and paracrine activation of PI3K/Akt signaling, enhanced cell survival, release of growth factors including SDF-1 associated with IGF-1 overexpression, stem cell mobilization, and angiomyogenesis. The combined effect of these possible contributory factors was significantly lower in NullMSCs, thus suggestive of their dependence on IGF-1 overexpression. It is, however, difficult to delineate the independent role of each one of the contributory factors toward the improvement in cardiac function in general and that of the mobilized cells in particular.
In summary, IGF-1MSCs served as a reservoir of IGF-1, which acted in autocrine and paracrine fashion to activate survival signaling in MSCs and the host myocytes. IGF-1 also promoted multiple growth factor expression, including SDF-1 and VEGF, which stimulated BM and endothelial progenitor cell mobilization to the ischemic myocardium. The accelerated mobilization of stem cells resulted in extensive neovascularization and myogenesis in the infarcted heart. These results highlight the biological implications of IGF-1 overexpression on stem cell mobilization through SDF-1/CXCR4 interaction.
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Acknowledgments |
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Sources of Funding
This work was supported by NIH grants R37-HL074272, HL-080686, and HL087246 (to M.A.) and HL087288 and HL089535 (to H.Kh.H.).
Disclosures
None.
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Footnotes |
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Original received July 23, 2008; resubmission received September 3, 2008; revised resubmission received October 8, 2008; accepted October 9, 2008.
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References |
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