Lunan Zhang

Paracrine action accounts for marked protection of ischemic heart by Akt-modified mesenchymal stem cells

Paracrine action accounts for marked protection of ischemic heart by Akt-modified mesenchymal stem cells

Evidence supporting paracrine hypothesis for Akt-modified mesenchymal stem cell-mediated cardiac protection and functional improvement

We previously reported that intramyocardial injection of bone marrow‐derived mesenchymal stem cells overexpressing Akt (Akt‐MSCs) inhibits ventricular remodeling and restores cardiac function measured 2 wk after myocardial infarction. Here, we report that the functional improvement occurs in < 72 h. This early remarkable effect cannot be readily attributed to myocardial regeneration from the donor cells. Thus, we hypothesized that paracrine actions exerted by the cells through the release of soluble factors might be important mechanisms of tissue repair and functional improvement after injection of the Akt‐MSCs. Indeed, in the current study we demonstrate that conditioned medium from hypoxic Akt‐MSCs markedly inhibits hypoxia‐induced apoptosis and triggers vigorous spontaneous contraction of adult rat cardiomyocytes in vitro. When injected into infarcted hearts, the Akt‐MSC conditioned medium significantly limits infarct size and improves ventricular function relative to controls. Sup‐port to the paracrine hypothesis is provided by data showing that several genes, coding for factors (VEGF, FGF‐2, HGF, IGF‐I, and TB4) that are potential mediators of the effects exerted by the Akt‐MSC conditioned medium, are significantly up‐regulated in the Akt‐MSCs, particularly in response to hypoxia. Taken together, our data support Akt‐MSC‐mediated para‐crine mechanisms of myocardial protection and functional improvement.‐Gnecchi, M., He, H., Noiseux, N., Liang, O. D., Zhang, L., Morello, F., Mu, H., Melo, L. G., Pratt, R. E., Ingwall, J. S., Dzau, V. J. Evidence supporting paracrine hypothesis for Akt‐modified mes‐enchymal stem cell‐mediated cardiac protection and functional improvement. FASEB J. 20, 661–669 (2006)

MicroRNA-Mediated In Vitro and In Vivo Direct Reprogramming of Cardiac Fibroblasts to Cardiomyocytes

Rationale: Repopulation of the injured heart with new, functional cardiomyocytes remains a daunting challenge for cardiac regenerative medicine. An ideal therapeutic approach would involve an effective method at achieving direct conversion of injured areas to functional tissue in situ. Objective: The aim of this study was to develop a strategy that identified and evaluated the potential of specific micro (mi)RNAs capable of inducing reprogramming of cardiac fibroblasts directly to cardiomyocytes in vitro and in vivo. Methods and Results: Using a combinatorial strategy, we identified a combination of miRNAs 1, 133, 208, and 499 capable of inducing direct cellular reprogramming of fibroblasts to cardiomyocyte-like cells in vitro. Detailed studies of the reprogrammed cells demonstrated that a single transient transfection of the miRNAs can direct a switch in cell fate as documented by expression of mature cardiomyocyte markers, sarcomeric organization, and exhibition of spontaneous calcium flux characteristic of a cardiomyocyte-like phenotype. Interestingly, we also found that miRNA-mediated reprogramming was enhanced 10-fold on JAK inhibitor I treatment. Importantly, administration of miRNAs into ischemic mouse myocardium resulted in evidence of direct conversion of cardiac fibroblasts to cardiomyocytes in situ. Genetic tracing analysis using Fsp1Cre-traced fibroblasts from both cardiac and noncardiac cell sources strongly suggests that induced cells are most likely of fibroblastic origin. Conclusions: The findings from this study provide proof-of-concept that miRNAs have the capability of directly converting fibroblasts to a cardiomyocyte-like phenotype in vitro. Also of significance is that this is the first report of direct cardiac reprogramming in vivo. Our approach may have broad and important implications for therapeutic tissue regeneration in general.

Isolation and Transplantation of Autologous Circulating Endothelial Cells Into Denuded Vessels and Prosthetic Grafts Implications for Cell-Based Vascular Therapy

Background—Blood-borne endothelial cells originating from adult bone marrow were reported previously. These cells have the properties of an endothelial progenitor cell (EPC) and can be mobilized by cytokines and recruited to sites of neovascularization, where they differentiate into mature endothelial cells. Current protocols for isolation of EPCs from peripheral blood rely on enrichment and selection of CD34+ mononuclear cells. Methods and Results—In this report, we describe a streamlined method for the isolation and expansion of EPCs from peripheral blood and evaluate their therapeutic potential for autologous cell-based therapy of injured blood vessels and prosthetic grafts. A subset of unfractionated mononuclear cells exhibited the potential to differentiate in vitro into endothelial cells under selective growth conditions. The cells were efficiently transduced ex vivo by a retroviral vector expressing the LacZ reporter gene and could be expanded to yield sufficient numbers for therapeutic applications. Transplantation of these cells into balloon-injured carotid arteries and into bioprosthetic grafts in rabbits led to rapid endothelialization of the denuded vessels and graft segments, resulting in significant reduction in neointima deposition. Conclusions—We conclude that transplantation of EPCs may play a crucial role in reestablishing endothelial integrity in injured vessels, thereby inhibiting neointimal hyperplasia. These findings may have implications for novel and practical cell-based therapies for vascular disease.

Secreted frizzled related protein 2 (Sfrp2) is the key Akt-mesenchymal stem cell-released paracrine factor mediating myocardial survival and repair

Stem cell therapy has emerged as a promising tool for the treatment of a variety of diseases. Previously, we have shown that Akt-modified mesenchymal stem cells mediate tissue repair through paracrine mechanisms. Using a comprehensive functional genomic strategy, we show that secreted frizzled related protein 2 (Sfrp2) is the key stem cell paracrine factor that mediates myocardial survival and repair after ischemic injury. Sfrp2 is known to modulate Wnt signaling, and we demonstrate that cardiomyocytes treated with secreted frizzled related protein increase cellular β-catenin and up-regulate expression of antiapoptotic genes. These findings reveal the key role played by Sfrp2 in mediating the paracrine effects of Akt-mesenchymal stem cells on tissue repair and identify modulation of Wnt signaling as a therapeutic target for heart disease.

Inhibition of neointimal cell bcl-x expression induces apoptosis and regression of vascular disease

We postulated that activation of a genetic program that tonicaiiy inhibits intimal cell death is a necessary condition for the pathogenesis of vascular disease. Studies of vascular lesions in humans and animal models documented increased expression of the anti-apoptotic gene product Bcl-xL within intimal cells. Downregulation of intimal cell bcl-xL expression with the use of antisense oligonucleotides induced apoptosis and acute regression of vascular lesions. These findings indicate that apoptosis regulatory genes such as bcl-xL are critical determinants of intimal lesion formation and that targeted apoptosis may be a novel therapy for intimal vascular disease.

Cytokine-Induced Mobilization of Circulating Endothelial Progenitor Cells Enhances Repair of Injured Arteries

Background—The existence of circulating endothelial progenitor cells (CEPCs) has previously been documented. These cells can be mobilized by cytokines and are recruited to sites of injury, where they may participate in tissue repair. In the present study, we examined the hypothesis that mobilization of CEPCs by exogenous granulocyte-colony stimulating factor (G-CSF) enhances repair of injured arteries by facilitating reendothelialization and inhibiting neointima development. Methods and Results—Male rats were injected daily with 50 &mgr;g/kg recombinant human G-CSF or 0.9% NaCl SC for 8 days. On the fifth day of treatment, 1 mL of blood was collected for fluorescence-activated cell sorting analysis of mononuclear cells, and the animals underwent balloon angioplasty of the common carotid artery. The animals were killed at 2 or 4 weeks after injury, and the carotid arteries were harvested and processed for immunohistochemistry, scanning electron microscopy (SEM), and morphometric analysis of endothelialization and neointimal formation. G-CSF increased the number of circulating mononuclear cells that express endothelial cell lineage markers several-fold. SEM and immunohistochemical staining with the endothelial marker, platelet and endothelial cell adhesion molecule-1, showed rapid and nearly complete (>90%) reendothelialization of the denuded vessels in the G-CSF–treated animals compared with <20% in the control animals. Reendothelialization was paralleled by a decrease in inflammation in the vessel wall. Neointima thickness was reduced by ≈60% in the G-CSF–treated animals compared with control animals at 2 and 4 weeks after injury. Conclusion—We postulate that cytokine-induced mobilization of CEPCs may be a suitable therapeutic strategy for prevention of restenosis after revascularization procedures.

Enhanced Inhibition of Neointimal Hyperplasia by Genetically Engineered Endothelial Progenitor Cells

Background—Circulating endothelial progenitor cells (EPCs) have been reported previously. In this study, we examined the hypothesis that overexpression of vasculoprotective gene endothelial nitric oxide synthase (eNOS) and heme oxygenase-1 (HO-1) in EPCs enhances their ability to inhibit neointimal hyperplasia. Methods and Results—EPCs were isolated from rabbit peripheral blood, expanded in culture, and transduced with pseudotyped retroviral vectors expressing human eNOS (eNOS-EPCs), HO-1 (HO-1-EPCs), or green fluorescent protein (GFP-EPCs). Transduction efficiency of EPCs ex vivo was >90%. Four groups of rabbits (n=5 to 6 per group) were subjected to balloon angioplasty of the common carotid artery. Immediately after injury, ≈5×106 autologous eNOS-EPCs or HO-1-EPCs were transplanted into the injured vessel. Control animals received an equivalent number of GFP-EPCs or Ringer’s saline. Two weeks after transplantation, eNOS and HO-1 transgene transcripts and proteins were detected in the transduced rabbit vessels. Endothelialization was enhanced in the EPC-transplanted vessels independently of gene transfer. Neointimal thickening was significantly reduced in the GFP-EPC–treated vessels relative to the saline control. Neointima size was further reduced in vessels treated with eNOS-EPCs. Surprisingly, no additional reduction was seen in vessels treated with HO-1-EPCs relative to GFP-EPCs. Thrombosis occurred in ≈50% of the saline-treated vessels but was virtually absent in all EPC-transplanted vessels. Conclusions—We conclude that transplantation of autologous EPCs overexpressing eNOS in injured vessels enhances the vasculoprotective properties of the reconstituted endothelium, leading to inhibition of neointimal hyperplasia. This cell-based gene therapy strategy may be useful in treatment of vascular disease.

Intimal hyperplasia after vascular injury is inhibited by antisense cdk 2 kinase oligonucleotides.

The cell cycle regulatory enzyme, cdk (cyclin-dependent kinase) 2 kinase, is activated in the rat carotid artery after balloon angioplasty injury, and may mediate smooth muscle proliferation. To test the hypothesis that inhibition of the expression of this key enzyme can inhibit intimal hyperplasia, we studied the effect of antisense phosphorothioate oligodeoxynucleotides (ODN) against cdk 2 kinase administered by intraluminal delivery using hemagglutinating virus of Japan (HVJ)-liposome-mediated transfer. The specificity of antisense cdk 2 ODN was confirmed by the observation that mRNA level of cdk 2 kinase in injured vessels was markedly diminished by the antisense ODN treatment. At 2 wk after transfection, antisense cdk 2 ODN treatment (15 microM) resulted in a significant inhibition (60%) in neointima formation, compared with sense ODN-treated and untreated vessels. Since we have previously observed that cell division cycle 2 kinase mRNA was also activated after vascular injury, we administered the combination of antisense cdc 2 and cdk 2 ODN in this study. Antisense cdc 2 ODN alone (15 microM) only reduced intimal formation by 40%. Combined antisense treatment resulted in near complete inhibition of neointima formation. To understand the mechanism of the sustained effect of a single antisense ODN administration, we examined kinetics of ODN in the vessel wall. Using phosphorothioate FITC-labeled ODN, we transfected carotid artery using the HVJ-liposome method. Fluorescence localized immediately to the medial layer, and persisted up to 2 wk after transfection. These results demonstrate that a single intraluminal administration of antisense ODN directed to cell cycle regulatory genes (e.g., cdk 2 kinase) using the HVJ method can result in a sustained inhibition of neointima formation after balloon angioplasty in rat carotid injury model.

Evidence for direct local effect of angiotensin in vascular hypertrophy. In vivo gene transfer of angiotensin converting enzyme.

In vitro studies have demonstrated that angiotensin (Ang) II directly stimulates vascular smooth muscle cell (VSMC) growth. However, it is still unclear if Ang II exerts a direct effect on vascular hypertrophy in vivo independent of its effect on blood pressure. In vivo gene transfer provides the opportunity to assess the effects of increased activity of the vascular angiotensin system in the intact animal while avoiding an increase in circulating angiotensin or in blood pressure. Accordingly, we transfected the human angiotensin converting enzyme (ACE) vector into intact rat carotid arteries by the hemagglutinating virus of Japan-liposome method. 3 d after transfection, we detected increased ACE activity in the transfected artery. Immunohistochemistry localized immunoreactive ACE in the medial VSMC as well as in the intimal endothelial cells. The increase in vascular ACE activity was associated with a parallel increase in DNA synthesis as assessed by BrdU (bromo-deoxyuridine) index and vascular DNA content. This increase in DNA synthesis was abolished by the in vivo administration of an Ang II receptor-specific antagonist (DuP 753). Morphometry at 2 wk after transfection revealed an increase in the wall to lumen ratio of the ACE-transfected blood vessel as compared with control vector transfected vessels. This was accompanied by increases in protein and DNA contents without an increase in cell number. Local transfection of ACE vector did not result in systemic effects such as increased blood pressure, heart rate, or serum ACE activity. These morphological changes were abolished by the administration of the Ang II receptor antagonist. In this study, we used in vivo gene transfer to increase local expression of vascular angiotensin converting enzyme and provided proof that increased autocrine/paracrine angiotensin can directly cause vascular hypertrophy independent of systemic factors and hemodynamic effects. This approach has important potentials for defining the role of autocrine/paracrine substances in vascular biology and hypertension.