Atsushi Iwakura

Estrogen-Mediated, Endothelial Nitric Oxide Synthase–Dependent Mobilization of Bone Marrow–Derived Endothelial Progenitor Cells Contributes to Reendothelialization After Arterial Injury

Background—We hypothesized that estrogen-induced acceleration of reendothelialization might be mediated in part by effects involving mobilization and incorporation of bone marrow–derived endothelial progenitor cells (EPCs). Methods and Results—Carotid injury was induced in ovariectomized wild-type mice receiving either 17&bgr;-estradiol or placebo. Estradiol treatment significantly accelerated reendothelialization of injured arterial segments within 7 days and resulted in a significant reduction of medial thickness 14 and 21 days after the injury. Significant increases in circulating EPCs 3 days after the injury were observed in the estradiol group compared with placebo-treated mice. These data were further supported by fluorescence-activated cell sorting analysis, which disclosed a significant increase in Sca-1/Flk-1–positive cells in estradiol versus control mice. To evaluate the effects of estradiol on bone marrow–derived EPC incorporation at sites of reendothelialization, carotid injury was established in ovariectomized wild-type mice transplanted with bone marrow from transgenic donors expressing &bgr;-galactosidase transcriptionally regulated by the Tie-2 promoter. Significantly greater numbers of X-gal–positive cells were observed at reendothelialized areas in the estradiol group 3 days after injury as compared with placebo. Fluorescent immunohistochemistry 14 days after the injury documented a marked increase in cells expressing both &bgr;-gal, indicating bone marrow origin and Tie-2 expression, and isolectin B4, also indicating endothelial lineage, in the estradiol group compared with control. In contrast, estradiol did not accelerate reendothelialization or augment EPC mobilization into the peripheral circulation after injury in endothelial nitric oxide synthase–deficient mice (eNOS−/−). Furthermore, estradiol exhibited direct stimulatory effects on EPC mitogenic and migration activity and inhibited EPC apoptosis. Conclusions—Estradiol accelerates reendothelialization and attenuates medial thickening after carotid injury in part by augmenting mobilization and proliferation of bone marrow–derived EPCs and their incorporation into the recovering endothelium at the site of injury.

Src blockade stabilizes a Flk/cadherin complex, reducing edema and tissue injury following myocardial infarction

Ischemia resulting from myocardial infarction (MI) promotes VEGF expression, leading to vascular permeability (VP) and edema, a process that we show here contributes to tissue injury throughout the ventricle. This permeability/edema can be assessed noninvasively by MRI and can be observed at the ultrastructural level as gaps between adjacent endothelial cells. Many of these gaps contain activated platelets adhering to exposed basement membrane, reducing vessel patency. Following MI, genetic or pharmacological blockade of Src preserves endothelial cell barrier function, suppressing VP and infarct volume, providing long-term improvement in cardiac function, fibrosis, and survival. To our surprise, an intravascular injection of VEGF into healthy animals, but not those deficient in Src, induced similar endothelial gaps, VP, platelet plugs, and some myocyte damage. Mechanistically, we show that quiescent blood vessels contain a complex involving Flk, VE-cadherin, and beta-catenin that is transiently disrupted by VEGF injection. Blockade of Src prevents disassociation of this complex with the same kinetics with which it prevents VEGF-mediated VP/edema. These findings define a molecular mechanism to account for the Src requirement in VEGF-mediated permeability and provide a basis for Src inhibition as a therapeutic option for patients with acute MI.

Sonic hedgehog myocardial gene therapy: tissue repair through transient reconstitution of embryonic signaling

Sonic hedgehog (Shh) is a crucial regulator of organ development during embryogenesis. We investigated whether intramyocardial gene transfer of naked DNA encoding human Shh (phShh) could promote a favorable effect on recovery from acute and chronic myocardial ischemia in adult animals, not only by promoting neovascularization, but by broader effects, consistent with the role of this morphogen in embryogenesis. After Shh gene transfer, the hedgehog pathway was upregulated in mammalian fibroblasts and cardiomyocytes. This resulted in preservation of left ventricular function in both acute and chronic myocardial ischemia by enhanced neovascularization, and reduced fibrosis and cardiac apoptosis. Shh gene transfer also enhanced the contribution of bone marrow–derived endothelial progenitor cells to myocardial neovascularization. These data suggest that Shh gene therapy may have considerable therapeutic potential in individuals with acute and chronic myocardial ischemia by triggering expression of multiple trophic factors and engendering tissue repair in the adult heart.

Endothelial Progenitor Cells Are Rapidly Recruited to Myocardium and Mediate Protective Effect of Ischemic Preconditioning via “Imported” Nitric Oxide Synthase Activity

Background—The function of bone marrow–derived endothelial progenitor cells (EPCs) in repair of ischemic tissue has been the subject of intense scrutiny, and the capacity of these cells to contribute significantly to new blood vessels remains controversial. The possibility that EPCs could act as reservoirs of cytokines has been implied by several observations; however, a specific role for cytokine delivery has not been identified. Methods and Results—We performed a series of experiments that revealed the rapid recruitment of EPCs to the myocardium by very short periods of ischemia, so-called ischemic preconditioning. The recruited EPCs express an array of potentially cardioprotective cytokines including nitric oxide synthase isoforms. Bone marrow transplantation studies, using donor marrow null for nitric oxide synthase isoforms, revealed that both endothelial and inducible nitric oxide synthase derived from bone marrow cells play essential roles in the cardioprotective effect that normally occurs after ischemic preconditioning. Conclusions—These findings provide novel insights about the role of bone marrow–derived cells in ischemic preconditioning and also reveal that distinct mechanisms regulate recovery after ischemia-reperfusion and chronic ischemic injury.

Estradiol Enhances Recovery After Myocardial Infarction by Augmenting Incorporation of Bone Marrow–Derived Endothelial Progenitor Cells Into Sites of Ischemia-Induced Neovascularization via Endothelial Nitric Oxide Synthase–Mediated Activation of Matrix Metalloproteinase-9

Background— Recent data have indicated that estradiol can modulate the kinetics of endothelial progenitor cells (EPCs) via endothelial nitric oxide synthase (eNOS)–dependent mechanisms. We hypothesized that estradiol could augment the incorporation of bone marrow (BM)–derived EPCs into sites of ischemia-induced neovascularization, resulting in protection from ischemic injury. Methods and Results— Myocardial infarction (MI) was induced by ligation of the left coronary artery in ovariectomized mice receiving either 17β-estradiol or placebo. Estradiol induced significant increases in circulating EPCs 2 and 3 weeks after MI in estradiol-treated animals, and capillary density was significantly greater in estradiol-treated animals. Greater numbers of BM-derived EPCs were observed at ischemic sites in estradiol-treated animals than in placebo-treated animals 1 and 4 weeks after MI. In eNOS-null mice, the effect of estradiol on mobilization of EPCs was lost, as was the functional improvement in recovery from acute myocardial ischemia. A decrease was found in matrix metalloproteinase-9 (MMP-9) expression in eNOS-null mice under basal and estradiol-stimulated conditions after MI, the mobilization of EPCs by estradiol was lost in MMP-9–null mice, and the functional benefit conferred by estradiol treatment after MI in wild-type mice was significantly attenuated. Conclusions— Estradiol preserves the integrity of ischemic tissue by augmenting the mobilization and incorporation of BM-derived EPCs into sites of neovascularization by eNOS-mediated augmentation of MMP-9 expression in the BM. Moreover, these data have broader implications with regard to our understanding of the role of EPCs in post-MI recovery and on the sex discrepancy in cardiac events.

Estrogen Receptors α and β Mediate Contribution of Bone Marrow–Derived Endothelial Progenitor Cells to Functional Recovery After Myocardial Infarction

Background— Estradiol (E2) modulates the kinetics of circulating endothelial progenitor cells (EPCs) and favorably affects neovascularization after ischemic injury. However, the roles of estrogen receptors &agr; (ER&agr;) and &bgr; (ER&bgr;) in EPC biology are largely unknown. Methods and Results— In response to E2, migration, tube formation, adhesion, and estrogen-responsive element–dependent gene transcription activities were severely impaired in EPCs obtained from ER&agr;-knockout mice (ER&agr;KO) and moderately impaired in ER&bgr;KO EPCs. The number of ER&agr;&Kgr;&Ogr; EPCs (42.4±1.5; P<0.001) and ER&bgr;KO EPCs (55.4±1.8; P= 0.03) incorporated into the ischemic border zone was reduced as compared with wild-type (WT) EPCs (72.5±1.3). In bone marrow transplantation (BMT) models, the number of mobilized endogenous EPCs in E2-treated mice was significantly reduced in ER&agr;KO BMT (WT mice transplanted with ER&agr;KO bone marrow) (2.03±0.18%; P= 0.004 versus WT BMT) and ER&bgr;KO BMT (2.62±0.07%; P= 0.02 versus WT) compared with WT BMT (2.87±0.13%) (WT to WT BMT as control) mice. Capillary density at the border zone of ischemic myocardium also was significantly reduced in ER&agr;KO BMT and ER&bgr;KO BMT compared with WT mice (WT BMT, 1718±75/mm2; ER&agr;KO BMT, 1107±48/mm2; ER&bgr;KO BMT, 1567±50/mm2). ER&agr; mRNA was expressed more abundantly on EPCs compared with ER&bgr;. Moreover, vascular endothelial growth factor was significantly downregulated on ER&agr;KO EPCs compared with WT EPCs both in vitro and in vivo. Conclusions— Both ER&agr; and ER&bgr; contribute to E2-mediated EPC activation and tissue incorporation and to preservation of cardiac function after myocardial infarction. ER&agr; plays a more prominent role in this process. Moreover, ER&agr; contributes to upregulation of vascular endothelial growth factor, revealing possible mechanisms of an effect of E2 on EPC biology. Finally, these data provide additional evidence of the importance of bone marrow–derived EPC phenotype in ischemic tissue repair.

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.

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 μg 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.

Estradiol-induced, endothelial progenitor cell-mediated neovascularization in male mice with hind-limb ischemia

Abstract We investigated whether administration of estradiol to male mice augments mobilization of bone marrow-derived endothelial progenitor cells (EPC) and incorporation into foci of neovascularization after hind-limb ischemia, thereby contributing to blood flow restoration. Mice were randomized and implanted with placebo pellets or pellets containing low-dose estradiol (0.39 mg) or high-dose estradiol (1.7 mg). Hind-limb ischemia was induced by unilateral resection of the left femoral artery 1 week after pellet implantation, then EPC mobilization and functional recovery was evaluated. EPC recruitment was assessed in mice transplanted with bone marrow from transgenic donors expressing β-galactosidase driven by the Tie-2 promoter. EPC culture assay performed 2 weeks after pellet implantation revealed a significantly greater (p < 0.05) number of circulating EPCs in the high-dose estradiol group than in the low-dose estradiol and placebo groups. At 3 and 4 weeks after induction of hind-limb ischemia, perfusion was significantly greater (p < 0.05) in high-dose estradiol mice than in mice implanted with the low-dose estradiol or placebo pellets. At 1 and 4 weeks after hind-limb ischemia surgery, more bone marrow-derived EPCs, identified as β-galactosidase-positive cells, were observed in ischemic regions from high-dose estradiol animals than in low-dose (p < 0.05) or placebo groups (p < 0.05). These results indicate that estradiol dose-dependently increases the levels of EPCs in peripheral blood in male animals, improves the recovery of blood flow, and decreases limb necrosis after hind-limb ischemia, and that this enhancement occurs, in part, through augmentation of EPC mobilization and greater incorporation of bone marrow-derived EPCs into foci of neovascularization.

ò-Adrenergic Receptor Signaling Regulates rAAV Transduction through Calcineurin in Heart Muscle Cells

Recombinant adeno-associated virus (rAAV)-based gene therapy represents a promising approach for the treatment of heart muscle diseases, but the molecular mechanisms that direct rAAV transduction remain unclear. Here we demonstrate that ?-adrenergic receptor stimulation with isoproterenol (ISO) markedly increased cardiomyocyte transduction of rAAV in vitro and in vivo. Conversely, chronic ?-adrenergic receptor downregulation significantly suppressed rAAV transduction. Pretreatment with calcium signaling cascade inhibitors including calcineurin inhibitory peptide (CNIP) strongly suppressed the positive effects of ISO on rAAV transduction. Additionally we document that ISO treatment led to a significant increase in double-stranded (ds) DNA synthesis of the rAAV genome and an increase in promoter activity. Moreover, stimulation with ISO did not affect rAAV transduction in calcineurin nullizygous mice. Collectively, we conclude that a calcium-dependent pathway regulates rAAV vector transduction at a number of stages that may include vector mobilization, conversion, and transcription activity. Modulating this pathway through ?-adrenergic signaling enhances rAAV-mediated gene delivery to cardiomyocytes, and may be valuable when considering therapeutic approaches for heart muscle diseases.