Functional disruption of alpha4 integrin mobilizes bone marrow-derived endothelial progenitors and augments ischemic neovascularization.

The cell surface receptor alpha4 integrin plays a critical role in the homing, engraftment, and maintenance of hematopoietic progenitor cells (HPCs) in the bone marrow (BM). Down-regulation or functional blockade of alpha4 integrin or its ligand vascular cell adhesion molecule-1 mobilizes long-term HPCs. We investigated the role of alpha4 integrin in the mobilization and homing of BM endothelial progenitor cells (EPCs). EPCs with endothelial colony-forming activity in the BM are exclusively alpha4 integrin-expressing cells. In vivo, a single dose of anti-alpha4 integrin antibody resulted in increased circulating EPC counts for 3 d. In hindlimb ischemia and myocardial infarction, systemically administered anti-alpha4 integrin antibody increased recruitment and incorporation of BM EPCs in newly formed vasculature and improved functional blood flow recovery and tissue preservation. Interestingly, BM EPCs that had been preblocked with anti-alpha4 integrin ex vivo or collected from alpha4 integrin-deficient mice incorporated as well as control cells into the neovasculature in ischemic sites, suggesting that alpha4 integrin may be dispensable or play a redundant role in EPC homing to ischemic tissue. These data indicate that functional disruption of alpha4 integrin may represent a potential angiogenic therapy for ischemic disease by increasing the available circulating supply of EPCs.

Analysis of angiogenesis induced by local IGF-1 expression after myocardial infarction using microSPECT-CT imaging.

Insulin-like growth factor-1 (IGF-1) has been found to exert favorable effects on angiogenesis in prior animal studies. This study explored the long-term effect of IGF-1 on angiogenesis using microSPECT-CT in infarcted rat hearts after delivering human IGF-1 gene by adeno-associated virus (AAV). Myocardial infarction (MI) was induced in Sprague-Dawley rats by ligation of the proximal anterior coronary artery and a total of 10(11) AAV-CMV-lacZ (control) or IGF-1 vectors were injected around the peri-infarct area. IGF-1 expression by AAV stably transduced heart muscle for up to 16 weeks post-MI and immunohistochemistry revealed a remarkable increase in capillary density. A (99m)Tc-labeled RGD peptide (NC100692, GE Healthcare) was used to assess temporal and regional alpha(v) integrin activation. Rats were injected with NC100692 followed by (201)Tl chloride and in vivo microSPECT-CT imaging was performed. After imaging, hearts were excised and cut for quantitative gamma-well counting (GWC). NC100692 retention was significantly increased in hypoperfused regions of both lacZ and IGF-1 rats at 4 and 16 weeks post-MI. Significantly higher activation of alpha(v) integrin was observed in IGF-1 rats at 4 weeks after treatment compared with control group, although the activation was lower in the IGF-1 group at 16 weeks. Local IGF-1 gene delivery by AAV can render a sustained transduction and improve cardiac function post-MI. IGF-1 expression contributes to enhanced alpha(v) integrin activation which is linked to angiogenesis. MicroSPECT-CT imaging with (99m)Tc-NC100692 and quantitative GWC successfully assessed differences in alpha(v) integrin activation between IGF-1-treated and control animals post-MI.

The cytoskeletal protein ezrin regulates EC proliferation and angiogenesis via TNF-alpha-induced transcriptional repression of cyclin A.

TNF-alpha modulates EC proliferation and thereby plays a central role in new blood vessel formation in physiologic and pathologic circumstances. TNF-alpha is known to downregulate cyclin A, a key cell cycle regulatory protein, but little else is known about how TNF-alpha modulates EC cell cycle and angiogenesis. Using primary ECs, we show that ezrin, previously considered to act primarily as a cytoskeletal protein and in cytoplasmic signaling, is a TNF-alpha-induced transcriptional repressor. TNF-alpha exposure leads to Rho kinase-mediated phosphorylation of ezrin, which translocates to the nucleus and binds to cell cycle homology region repressor elements within the cyclin A promoter. Overexpression of dominant-negative ezrin blocks TNF-alpha-induced modulation of ezrin function and rescues cyclin A expression and EC proliferation. In vivo, blockade of ezrin leads to enhanced transplanted EC proliferation and angiogenesis in a mouse hind limb ischemia model. These observations suggest that TNF-alpha regulates angiogenesis via Rho kinase induction of a transcriptional repressor function of the cytoskeletal protein ezrin and that ezrin may represent a suitable therapeutic target for processes dependent on EC proliferation.

CD34-positive cells exhibit increased potency and safety for therapeutic neovascularization after myocardial infarction compared with total mononuclear cells.

BACKGROUND: We compared the therapeutic potential of purified mobilized human CD34+ cells with that of mobilized total mononuclear cells (tMNCs) for the preservation/recovery of myocardial tissue integrity and function after myocardial infarction (MI). METHODS AND RESULTS: CD34+ cells were purified from peripheral blood tMNCs of healthy volunteers by magnetic cell sorting after a 5-day administration of granulocyte colony-stimulating factor. Phosphate-buffered saline (PBS), 5x10(5) CD34+ cells/kg, 5x10(5) tMNCs/kg (low-dose MNCs [loMNCs]), or a higher dose of tMNCs (hiMNCs) containing 5x10(5) CD34+ cells/kg was transplanted intramyocardially 10 minutes after the induction of MI in athymic nude rats. Hematoxylin and eosin staining revealed that moderate to severe hemorrhagic MI on day 3 was more frequent in the hiMNC group than in the PBS and CD34+ cell groups. Immunostaining for human-specific CD45 revealed abundant distribution of hematopoietic/inflammatory cells derived from transplanted cells in the ischemic myocardium of the hiMNC group. Capillary density on day 28 was significantly greater in the CD34+ cell group (721.1+/-19.9 per 1 mm2) than in the PBS, loMNC, and hiMNC groups (384.7+/-11.0, 372.5+/-14.1, and 497.5+/-24.0 per 1 mm2) (P<0.01). Percent fibrosis area on day 28 was less in the CD34(+) cell group (15.6+/-0.9%) than in the PBS, loMNC, and hiMNC groups (26.3+/-1.2%, 27.5+/-1.8%, and 22.2+/-1.8%) (P<0.05). Echocardiographic fractional shortening on day 28 was significantly higher in the CD34+ cell group (30.3+/-0.9%) than in the PBS, loMNC, and hiMNC groups (22.7+/-1.5%, 23.4+/-1.1%, and 24.9+/-1.7%; P<0.05). Echocardiographic regional wall motion score was better preserved in the CD34+ cell group (21.8+/-0.5) than in the PBS, loMNC, and hiMNC groups (25.4+/-0.4, 24.9+/-0.4, and 24.1+/-0.6; P<0.05). CONCLUSIONS: CD34+ cells exhibit superior efficacy for preserving myocardial integrity and function after MI than unselected circulating MNCs.

Synergistic effect of combined intramyocardial CD34+ cells and VEGF2 gene therapy after MI.

Previous studies have shown that local angiogenic gene therapy acts, in part, by recruiting endothelial progenitor cells (EPCs) to ischemic tissue. Recent data indicate that patients with the most severe vascular disease may have insufficient or deficient EPCs and the poorest response to angiogenic therapy. Accordingly, we hypothesized that combining human CD34(+) cell implantation with local vascular endothelial growth factor 2 (phVEGF2) gene therapy might overcome these deficiencies. The addition of VEGF2 to EPC cultures resulted in significant and dose-dependent decreases in EPC apoptosis. Phosphorylated Akt (p-Akt) was increased in VEGF2-treated EPCs. In vivo, myocardial infarction (MI) was induced by ligation of the left anterior descending coronary artery in 34 immunodeficient rats. The animals were then randomized to one of four treatment groups: cell therapy alone with human CD34(+) cells; VEGF2 gene therapy alone; combination therapy with CD34(+) cells plus phVEGF2; or CD34(-) cells and 50 microg empty plasmid. Four weeks after MI, animals treated with combination therapy showed improved fractional shortening, increased capillary density, and reduced infarct size compared with the other three groups. Combination therapy was also associated with an increased number of circulating EPCs 1 week after MI. Combined subtherapeutic doses of cell and gene therapy result in a significant therapeutic effect compared to monotherapy. This approach may overcome therapeutic failures (e.g. inability of certain patients to mobilize sufficient EPCs) and may also offer safety advantages by allowing lower dosing strategies.

Synergistic effect of bone marrow mobilization and vascular endothelial growth factor-2 gene therapy in myocardial ischemia.

BACKGROUND: We performed a series of investigations to test the hypothesis that combining angiogenic gene therapy and cytokine (CK)-induced endothelial progenitor cell mobilization would be superior to either strategy alone for treatment of chronic myocardial ischemia. METHODS AND RESULTS: A swine model of chronic myocardial ischemia and a murine model of acute myocardial infarction were used in this study. In both models, animals were randomly assigned to 1 of 4 treatment groups: Combo group, intramyocardial vascular endothelial growth factor (VEGF)-2 gene transfer plus subcutaneous injection of CKs; VEGF-2, VEGF-2 gene transfer plus saline subcutaneously injected; CK, empty vector transfer plus CKs; and control, empty vector plus subcutaneous saline. Acute myocardial infarction was also induced in wild-type mice 4 weeks after bone marrow transplantation from enhanced green fluorescent protein transgenic mice to permit observation of bone marrow-derived cells in the myocardium after acute myocardial infarction. In chronic myocardial ischemia, combination therapy resulted in superior improvement in all indexes of perfusion and function compared with all other treatment groups. In the bone marrow transplant mice, double immunofluorescent staining revealed that the combination of CK-induced mobilization and local VEGF-2 gene transfer resulted in a significant increase in the number of bone marrow-derived cells incorporating into the neovasculature, indicating that recruitment and/or retention of bone marrow-derived progenitors was enhanced by mobilization and that local VEGF-2 gene transfer can provide signals for recruitment or incorporation of circulating progenitor cells. CONCLUSIONS: Mobilization of endothelial progenitor cells with cytokines potentiates VEGF-2 gene therapy for myocardial ischemia and enhances bone marrow cell incorporation into ischemic myocardium.

Autologous transplantation of endothelial progenitor cells genetically modified by adeno-associated viral vector delivering insulin-like growth factor-1 gene after myocardial infarction.

The regenerative potential of bone marrow-derived endothelial progenitor cells (EPCs) has been adapted for the treatment of myocardial and limb ischemia via ex vivo expansion. We sought to enhance EPC function by the efficient genetic modification of EPCs in a rat model of myocardial infarction. Peripheral blood EPCs were expanded and transduced, using adeno-associated virus (AAV). AAV-mediated EPC transduction efficacy was 23 ± 1.2%, which was improved by 4.0- to 7.2-fold after pretreatment with the tyrosine kinase inhibitor genistein. Adult rats (n = 7 in each group) underwent myocardial infarction by left anterior descending coronary artery occlusion, and received autologous EPCs transduced by AAV-IGF-1 or AAV-lacZ into the periinfarct area. Echocardiography demonstrated that cardiac function in the IGF-1-EPC group was significantly improved compared with the lacZ-EPC control group 12 weeks after myocardial infarction. In addition, IGF-1-expressing EPCs led to reduced cardiac apoptosis, increased cardiomyocyte proliferation, and increased numbers of capillaries in the periinfarct area. AAV expression was limited to the targeted heart region only. Pretreatment with genistein markedly improved AAV transduction of EPCs. IGF-1-expressing EPCs exhibit favorable cell-protective effects with tissue-limited expression in rat heart postinfarction.

Long-term stable expression of human growth hormone by rAAV promotes myocardial protection post-myocardial infarction.

Recombinant adeno-associated virus (rAAV)-based gene therapy represents a promising approach for the treatment of heart diseases. It has been shown that growth hormone (GH) exerts a favorable effect on cardiovascular function in clinical and animal studies. This study explores a chronic stage after myocardial infarction and the potential therapeutic effects of delivering a human GH gene by rAAV following coronary artery ligation in Sprague-Dawley rats. rAAV vectors stably transduced heart muscle for up to 22 weeks after myocardial infarction (MI). Overexpression of GH via rAAV vectors significantly improved not only cardiac function but also LV pathologic remodeling was attenuated post-MI compared to the control rAAV-lacZ injected group. rAAV-mediated expression of GH also resulted in a significant induction of several angiogenic genes such as eNOS, VEGF and bFGF in rat hearts. Immunohistochemistry revealed an increase in capillary density and proliferation of cells and a decrease in the number of TUNEL-positive cardiomyocytes in the rAAV-GH group. Based on these data, we conclude that rAAV-mediated GH delivery can render a long-term transduction in the infarcted heart and improve cardiac function through promoting angiogenesis and proliferation of cells and protecting cardiomyocytes from ischemia-induced apoptosis.