Altered expression of the natriuretic peptide system in genetically modified heme oxygenase-1 mice treated with high dietary salt.

Heme oxygenase-1 (HO-1) has been well established as a cytoprotective molecule, and has been shown to exert cardioprotective effects in both hypertension and cardiac hypertrophy. However, the precise mechanism of the cardioprotective effect of HO-1 has yet to be fully elucidated. With the natriuretic peptide system (NPS) as a key player in cardiovascular homeostasis and tissue dynamics, we sought to examine the effect of high dietary salt treatment in genetic models of HO-1 expression, and assessed the expression of the NPS in the left ventricle (LV), to determine if the effects of altered HO-1 expression may be due to modified levels of the NPS. Age-matched 12-week old male HO-1 knockout (HO-1(-/-)) and HO-1 cardiomyocyte-specific transgenic overexpressing (HO-1(Tg)) mice were treated with either normal salt (NS; 0.8%) or high salt (HS; 8.0%) chow for 5 weeks. LV mRNA expression was determined using quantitative real-time PCR. ANP peptide level was measured in the LV and plasma using radioimmunoassay, and LV cyclic 3'-5' guanosine monophosphate level was measured using an enzyme immunoassay kit. HO-1(-/-) fed HS diet had significantly higher left ventricle-to-body weight ratio (LV/BW) compared to HO-1(+/+) mice fed NS diet. HO-1(-/-) mice had significantly reduced expression of the NPS compared to controls, and these mice did not exhibit a salt-induced increase in ANP expression. HS treatment had no noticeable effect on LV/BW in HO-1(Tg) mice compared to controls. HO-1(Tg) mice had significantly higher ANP and BNP expression compared to controls. There were no differences in LV cGMP levels among all genotypes and dietary treatments. HO-1 ablation resulted in significantly lower mRNA expression of the NPS, whereas HO-1 overexpression resulted in higher mRNA expression of the NPS. Both were substantiated by peptide levels as measured by RIA. These data indicate that the detrimental effect of reduced HO-1 expression and the cardioprotective effect of increased HO-1 expression may be due, in part, to altered expression of the NPS.

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.

Heme oxygenase-1 inhibits pro-oxidant induced hypertrophy in HL-1 cardiomyocytes.

AIMS: Reactive oxygen species (ROS) activate multiple signaling pathways involved in cardiac hypertrophy. Since HO-1 exerts potent antioxidant effects, we hypothesized that this enzyme inhibits ROS-induced cardiomyocyte hypertrophy. METHODS: HL-1 cardiomyocytes were transduced with an adenovirus constitutively expressing HO-1 (AdHO-1) to increase basal HO-1 expression and then exposed to 200 microM hydrogen peroxide (H2O2). Hypertrophy was measured using 3H-leucine incorporation, planar morphometry and cell-size by forward-scatter flow-cytometry. The pro-oxidant effect of H2O2 was assessed by redox sensitive fluorophores. Inducing intracellular redox imbalance resulted in cardiomyocyte hypertrophy through transactivation of nuclear factor kappa B (NF-kappaB). RESULTS: Pre-emptive HO-1 overexpression attenuated the redox imbalance and reduced hypertrophic indices. This is the first time that HO-1 has directly been shown to inhibit oxidant-induced cardiomyocyte hypertrophy by a NF-kappaB-dependent mechanism. CONCLUSION: These results demonstrate that HO-1 inhibits pro-oxidant induced cardiomyocyte hypertrophy and suggest that HO-1 may yield therapeutic potential in treatment of.

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.

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.

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. Support 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 paracrine mechanisms of myocardial protection and functional improvement.

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 x 10(11) 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 LacZ-treated 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 overexpression 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.

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 (delta psi(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 delta psi(m) during exposure to H2O2. Conversely, HO-1 knockdown removed the protective effect of Akt on delta psi(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. CONCLUSIONS: 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.

Endothelial progenitor cell and mesenchymal stem cell isolation, characterization, viral transduction.

Endothelial progenitor cells (EPCs) and mesenchymal stem cells (MSCs) have emerged as potentially useful substrates for neovascularization and tissue repair and bioengineering. EPCs are a heterogeneous group of endothelial cell precursors originating in the hematopoietic compartment of the bone marrow. MSCs are a rare population of fibroblast-like cells derived from the bone marrow stroma, constituting approximately 0.001-0.01% of the nucleated cells in the marrow. Both cells types have been isolated from the bone marrow. In addition, EPC can be isolated from peripheral blood as well as the spleen, and MSC has also been isolated from peripheral adipose tissue. Several approaches have been used for the isolation of EPC and MSC, including density centrifugation and magnetic bead selection. Phenotypic characterization of both cell types is carried out using immunohistochemical detection and fluorescence-activated cell sorting analysis of cell-surface molecule expression. However, the lack of specific markers for each cell type renders their characterization difficult and ambiguous. In this chapter, we describe the methods that we use routinely for isolation, characterization, and genetic modification of EPC and MSC from human, rabbit, and mouse peripheral blood and bone marrow.

Assessment of hypoxia and TNF-alpha response by a vector with HRE and NF-kappaB response elements.

Hypoxia and inflammatory cytokine activation (H&I) are common processes in many acute and chronic diseases. Thus, a single vector that responds to both hypoxia and inflammatory cytokines, such as TNF-alpha, is useful for assesing the severity of such diseases. Adaptation to hypoxia is regulated primarily by hypoxia inducible transcription factor (HIF alpha) nuclear proteins that engage genes containing a hypoxia response element (HRE). Inflammation activates a multitude of cytokines, including TNF-alpha, that invariably modulate activation of the nuclear factor kappa B (NF-kB) transcription factor. We constructed a vector that encompassed both a hypoxia response element (HRE), and a NF-kappaB responsive element. We show that this vector was functionally responsive to both hypoxia and TNF-alpha, in vitro and in vivo. Thus, this vector might be suitable for the detection and assessment of hypoxia or TNF-alpha.

Genetic therapies for cardiovascular diseases.

Recent advances in understanding the molecular and cellular basis of cardiovascular diseases, together with the availability of tools for genetic manipulation of the cardiovascular system, offer possibilities for new treatments. Gene therapies have demonstrated potential usefulness for treating complex cardiovascular diseases, such as hypertension, atherosclerosis and myocardial ischemia, in various animal models. Some of these experimental therapies are now undergoing clinical evaluation in patients with cardiovascular disease. However, the successful transition of these therapies into mainstream clinical practice awaits further improvements to vector platforms and delivery tools and the documentation of clinical feasibility, safety and efficacy through multi-center randomized trials.

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.

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 microg/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 approximately 60% in the G-CSF-treated animals compared with control animals at 2 and 4 weeks after injury. CONCLUSIONS: 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, approximately 5x10(6) 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 approximately 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.

Gene therapy strategy for long-term myocardial protection using adeno-associated virus-mediated delivery of heme oxygenase gene.

BACKGROUND: Ischemia and oxidative stress are the leading mechanisms for tissue injury. An ideal strategy for preventive/protective therapy would be to develop an approach that could confer long-term transgene expression and, consequently, tissue protection from repeated ischemia/reperfusion injury with a single administration of a therapeutic gene. In the present study, we used recombinant adeno-associated virus (rAAV) as a vector for direct delivery of the cytoprotective gene heme oxygenase-1 (HO-1) into the rat myocardium, with the purpose of evaluating this strategy as a therapeutic approach for long-term protection from ischemia-induced myocardial injury. METHODS AND RESULTS: Human HO-1 gene (hHO-1) was delivered to normal rat hearts by intramyocardial injection. AAV-mediated transfer of the hHO-1 gene 8 weeks before acute coronary artery ligation and release led to a dramatic reduction (>75%) in left ventricular myocardial infarction. The reduction in infarct size was accompanied by decreases in myocardial lipid peroxidation and in proapoptotic Bax and proinflammatory interleukin-1beta protein abundance, concomitant with an increase in antiapoptotic Bcl-2 protein level. This suggested that the transgene exerts its cardioprotective effects in part by reducing oxidative stress and associated inflammation and apoptotic cell death. CONCLUSIONS: This study documents the beneficial therapeutic effect of rAAV-mediated transfer, before myocardial injury, of a cytoprotective gene that confers long-term myocardial protection from ischemia/reperfusion injury. Our data suggest that this novel "pre-event" gene transfer approach may provide sustained tissue protection from future repeated episodes of injury and may be beneficial as preventive therapy for patients with or at risk of developing coronary ischemic events.

Gene and cell-based therapies for heart disease.

Heart disease remains the prevalent cause of premature death and accounts for a significant proportion of all hospital admissions. Recent developments in understanding the molecular mechanisms of myocardial disease have led to the identification of new therapeutic targets, and the availability of vectors with enhanced myocardial tropism offers the opportunity for the design of gene therapies for both protection and rescue of the myocardium. Genetic therapies have been devised to treat complex diseases such as myocardial ischemia, heart failure, and inherited myopathies in various animal models. Some of these experimental therapies have made a successful transition to clinical trial and are being considered for use in human patients. The recent isolation of endothelial and cardiomyocyte precursor cells from adult bone marrow may permit the design of strategies for repair of the damaged heart. Cell-based therapies may have potential application in neovascularization and regeneration of ischemic and infarcted myocardium, in blood vessel reconstruction, and in bioengineering of artificial organs and prostheses. We expect that advances in the field will lead to the development of safer and more efficient vectors. The advent of genomic screening technology should allow the identification of novel therapeutic targets and facilitate the detection of disease-causing polymorphisms that may lead to the design of individualized gene and cell-based therapies.

Endothelium-targeted gene and cell-based therapies for cardiovascular disease.

Most common cardiovascular diseases are accompanied by endothelial dysfunction. Because of its predominant role in the pathogenesis of cardiovascular disease, the vascular endothelium is an attractive therapeutic target. The identification of promoter sequences capable of rendering endothelial-specific transgene expression together with the recent development of vectors with enhanced tropism for endothelium may offer opportunities for the design of new strategies for modulation of endothelial function. Such strategies may be useful in the treatment of chronic diseases such as hypertension, atherosclerosis, and ischemic artery disease, as well as in acute myocardial infarction and during open heart surgery for prevention of ischemia and reperfusion (I/R)-induced injury. The recent identification of putative endothelial progenitor cells in peripheral blood may allow the design of autologous cell-based strategies for neovascularization of ischemic tissues and for the repair of injured blood vessels and bioengineering of vascular prosthesis. "Proof-of-concept" for some of these strategies has been established in animal models of cardiovascular disease. However the successful translation of these novel strategies into clinical application will require further developments in vector and delivery technologies. Further characterization of the processes involved in mobilization, migration, homing, and incorporation of endothelial progenitor cells into the target tissues is necessary, and the optimal conditions for therapeutic application of these cells need to be defined and standardized.

Heme oxygenase-1 overexpression exacerbates heart failure with aging and pressure overload but is protective against isoproterenol-induced cardiomyopathy in mice.

INTRODUCTION: Heme oxygenase-1 (HO-1) is a cytoprotective enzyme induced by stress. Heart failure is a condition of chronic stress-induced remodeling and is often accompanied by comorbidities such as age and hypertension. HO-1 is known to be protective in the setting of acute myocardial infarction. The role of HO-1 in heart failure is not known, particularly in the setting of pressure overload. METHODS: Mice with alpha-myosin heavy chain restricted expression of HO-1 were aged for 1 year. In addition, mice underwent transverse aortic constriction (TAC) or were infused with isoproterenol (ISO) to induce heart failure. RESULTS: HO-1 transgenic mice developed spontaneous heart failure after 1 year compared to their wild-type littermates and showed accelerated cardiac dysfunction 2 weeks following TAC. Wild-type mice undergoing pressure overload demonstrated extensive interstitial fibrosis that was prevented by HO-1 overexpression, yet HO-1 transgenic mice had reduced capillary density, contractile reserve, and elevated end-diastolic pressure. However, HO-1 transgenic mice had significantly attenuated ISO-induced cardiac dysfunction, interstitial fibrosis, and hypertrophy compared to control. Isolated cardiomyocytes from HO-1 transgenic mice treated with ISO did not show evidence of hypercontracture/necrosis and had reduced NADH oxidase activity. CONCLUSIONS: HO-1 is an effective mechanism for reducing acute myocardial stress such as excess beta-adrenergic activity. However, in our age and pressure overload models, HO-1 showed detrimental rather than therapeutic effects in the development of heart failure.

Ex vivo Akt/HO-1 gene therapy to human endothelial progenitor cells enhances myocardial infarction recovery.

The aim of this study was to evaluate the overexpression of genes central to cell survival and angiogenesis to enhance the function of human late outgrowth endothelial progenitor cells (EPCs) and their utility for infarct recovery. Ischemic myocardial injury creates a hostile microenvironment, which is characterized by hypoxia, oxidative stress, and inflammation. The infarct microenvironment prevents adhesion, survival, and integration of cell transplants that promote neovascularization. EPCs are dysfunctional as a result of risk factors in cardiovascular patients. Protein kinase B (Akt) and heme-oxygenase-1 (HO-1) are intracellular proteins that play an important role in angiogenesis and cell survival. Late outgrowth EPCs transduced ex vivo with Akt and HO-1 demonstrate improved adhesion to extracellular matrix, improved migration toward human cardiomyocytes, and an improved paracrine profile under stress. Enhanced late outgrowth EPCs reduce the tumor necrosis factor-α (TNF-α) burden both in vitro and in vivo, attenuating nuclear factor-κB (NF-κB) activity and promoting cell survival. Akt and HO-1 enhance late outgrowth EPC neovascularization, resulting in improved cardiac performance and reduced negative remodeling after myocardial infarction in nude mice. Alteration of the infarct microenvironment through gene modification of human late outgrowth EPCs enhances the function and integration of transplanted cells for restoration of cardiac function.