Luis G. Melo

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)

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.

Targeted migration of mesenchymal stem cells modified with CXCR4 gene to infarcted myocardium improves cardiac performance

With the goal of devising a non-invasive cell therapy for cardiac repair that may be well tolerated by patients with myocardial infarction (MI), this study evaluated the efficacy of intravenous infusion of genetically modified mesenchymal stem cells (MSCs) overexpressing CXC chemokine receptor 4 (CXCR4). CXCR4 is the cognate receptor for stromal-derived factor-1 (SDF-1), a chemokine required for homing of progenitor cells to ischemic tissues. In this study, retrovirally transduced MSCs constitutively expressing CXCR4 (CXCR4-MSCs) were delivered intravenously 24 hours after coronary occlusion/reperfusion in rats. When compared with untransduced MSCs, CXCR4-MSCs homed in toward the infarct region of the myocardium in greater numbers. In the CXCR4-MSC-treated animals, echocardiographic imaging 30 days after MI showed a decrease in anterior wall thinning and good preservation of left ventricular (LV) chamber dimensions, whereas the animals treated with saline or unmodified MSCs showed significant remodeling. Histochemical analysis showed a decrease in collagen I/III ratio in the infarcted wall of CXCR4-MSC-treated animals, thereby suggesting improved chamber compliance. Assessment revealed post-MI recovery of LV function in the CXCR4-MSC-treated animals, whereas LV function remained depressed in the saline and MSC-treated animals. In summary, intravenous delivery of genetically modified MSCs expressing CXCR4 may be a useful, non-invasive, and safe therapeutic strategy for post-infarction myocardial repair.

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.

Bone Marrow-Derived Mesenchymal Stem Cells: Isolation, Expansion, Characterization, Viral Transduction, and Production of Conditioned Medium

Mesenchymal stem cells (MSCs) are defined as self-renewing and multipotent cells capable of differentiating into multiple cell types, including osteocytes, chondrocytes, adipocytes, hepatocytes, myocytes, neurons, and cardiomyocytes. MSCs were originally isolated from the bone marrow stroma but they have recently been identified also in other tissues, such as fat, epidermis, and cord blood. Several methods have been used for MSC isolation. The most common method is based on the ability of the MSCs to selectively adhere to plastic surfaces. Phenotypic characterization of MSCs is usually carried out using immunocytochemical detection or fluorescence-activated cell sorting (FACS) analysis of cell surface molecule expression. However, the lack of specific markers renders the characterization of MSCs difficult and sometimes ambiguous. MSCs posses remarkable expansion potential in culture and are highly amenable to genetic modification with various viral vectors rendering them optimal vehicles for cell-based gene therapy. Most importantly, MSC plasticity and the possibility to use them as autologous cells render MSCs suitable for cell therapy and tissue engineering. Furthermore, it is known that MSCs produce and secrete a great variety of cytokines and chemokines that play beneficial paracrine actions when MSCs are used for tissue repair. In this chapter, we describe methods for isolation, ex vivo expansion, phenotypic characterization, and viral infection of MSCs from mouse bone marrow. We also describe a method for preparation of conditioned and concentrated conditioned medium from MSCs. The conditioned medium can be easily tested both in vitro and in vivo when a particular paracrine effect (i.e., cytoprotection) is hypothesized to be an important mechanism of action of the MSCs and/or screened to identify a target paracrine/autocrine mediator.

Heme oxygenase-1 (HO-1) inhibits postmyocardial infarct remodeling and restores ventricular function

We reported previously that predelivery of the anti‐oxidant gene heme oxygenase‐1 (HO‐1) to the heart by adeno associated virus (AAV) markedly reduces injury after acute myocardial infarction (MI). However, the effect of HO‐1 gene delivery on postinfarction recovery has not been investigated. In the current study, we assessed the effect of HO‐1 gene delivery on post‐MI left ventricle (LV) remodeling and function using echocardiographic imaging and histomorphometric approaches. Two groups of Sprague‐Dawley rats were injected with 4 × 1011 particles of AAV‐LacZ (control) or AAV‐hHO‐1 in the LV wall. Eight wk after gene transfer, the animals were subjected to 30 min of ischemia by ligation of left anterior descending artery (LAD) followed by reperfusion. Echocardiographic measurements were obtained in a blinded fashion prior and at 1.5 and 3 months after I/R. Ejection fraction (EF) was reduced by 13% and 40% in the HO‐1 and LacZ groups, respectively at 1.5 months after MI. Three months after MI, EF recovered fully in the HO‐1, but only partially in the LacZ‐treated animals. Post‐MI LV dimensions were markedly increased and the anterior wall was markedly thinned in the LacZtreated animals compared with the HO‐1‐treated animals. Significant myocardial scarring and fibrosis were observed in the LacZ‐group in association with elevated levels of interstitial collagen I and III and MMP‐2 activity. Post‐MI myofibroblast accumulation was reduced in the HO‐1‐treated animals, and retroviral over‐expression of HO‐1 reduced proliferation of isolated cardiac fibroblasts. Our data indicate that rAAV‐HO‐1 gene transfer markedly reduces fibrosis and ventricular remodeling and restores LV function and chamber dimensions after myocardial infarction.—Liu, X., Pachori, A. S., Ward, C. A., Davis, J. P., Gnecchi, M., Kong, D., Zhang, L., Murduck, J., Yet, S.‐F., Perrella, M. A., Pratt, R. E., Dzau, V. J., Melo, L. G. Heme oxygenase‐1 (HO‐1) inhibits postmyocardial infarct remodeling and restores ventricular function. FASEB J. 20, 207–216 (2006)

Early Beneficial Effects of Bone Marrow Derived Mesenchymal Stem Cells Overexpressing Akt on Cardiac Metabolism after Myocardial Infarction

Administration of mesenchymal stem cells (MSCs) is an effective therapy to repair cardiac damage after myocardial infarction (MI) in experimental models. However, the mechanisms of action still need to be elucidated. Our group has recently suggested that MSCs mediate their therapeutic effects primarily via paracrine cytoprotective action. Furthermore, we have shown that MSCs overexpressing Akt1 (Akt‐MSCs) exert even greater cytoprotection than unmodified MSCs. So far, little has been reported on the metabolic characteristics of infarcted hearts treated with stem cells. Here, we hypothesize that Akt‐MSC administration may influence the metabolic processes involved in cardiac adaptation and repair after MI. MI was performed in rats randomized in four groups: sham group and animals treated with control MSCs, Akt‐MSCs, or phosphate‐buffered saline (PBS). High energy metabolism and basal 2‐deoxy‐glucose (2‐DG) uptake were evaluated on isolated hearts using phosphorus‐31 nuclear magnetic resonance spectroscopy at 72 hours and 2 weeks after MI. Treatment with Akt‐MSCs spared phosphocreatine stores and significantly limited the increase in 2‐DG uptake in the residual intact myocardium compared with the PBS‐ or the MSC‐treated animals. Furthermore, Akt‐MSC‐treated hearts had normal pH, whereas low pH was measured in the PBS and MSC groups. Correlative analysis indicated that functional recovery after MI was inversely related to the rate of 2‐DG uptake. We conclude that administration of MSCs overexpressing Akt at the time of infarction results in preservation of normal metabolism and pH in the surviving myocardium. STEM CELLS 2009;27:971–979

Protection of Human Vascular Smooth Muscle Cells From H2O2-Induced Apoptosis Through Functional Codependence Between HO-1 and AKT

Objective—Oxidative stress (OS) induces smooth muscle cell apoptosis in the atherosclerotic plaque, leading to plaque instability and rupture. Heme oxygenase-1 (HO-1) exerts cytoprotective effects in the vessel wall. Recent evidence suggests that PKB/Akt may modulate HO-1 activity. This study examined the role of Akt in mediating the cytoprotective effects of HO-1 in OS-induced apoptosis of human aortic smooth muscle cells (HASMCs). Methods and Results—HASMCs were transduced with retroviral vectors expressing HO-1, Akt, or GFP and exposed to H2O2. Cell viability was assessed by MTT assay. OS was determined by CM-H2DCFDA fluorescence, and apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL), caspase-3 activity, and Bcl-2/Bad levels. Mitochondrial membrane potential (&Dgr;&PSgr;m) was assessed by fluorescence-activated cell sorter (FACS) using JC-1. HO-1 reduced H2O2-induced OS and apoptosis. Akt knockdown removed the protective effect of HO-1 on &Dgr;&PSgr;m during exposure to H2O2. Conversely, HO-1 knockdown removed the protective effect of Akt on &Dgr;&PSgr;m. Inhibition of PI3K-Akt reduced induction of HO-1 protein expression by H2O2 and blocked its anti-apoptotic effects. The Akt-mediated upregulation of HO-1 was dependent on activation of HO-1 promoter by Nrf2. Conclusion—HO-1 and Akt exert codependent cytoprotective effects against OS-induced apoptosis in HASMCs. These findings may have implications for the design of novel therapeutic strategies for plaque stabilization.