Kentaro Jujo

Endothelial progenitor cells in neovascularization of infarcted myocardium.

Historically, revascularization of ischemic tissue was believed to occur through the migration and proliferation of endothelial cells in nearby tissues; however, evidence accumulated in recent years indicates that a subpopulation of adult, peripheral-blood cells, collectively referred to as endothelial progenitor cells (EPCs), can differentiate into mature endothelial cells. After ischemic insult, EPCs are believed to home to sites of neovascularization, where they contribute to vascular regeneration by forming a structural component of capillaries and by secreting angiogenic factors; new evidence indicates that EPCs can also differentiate into cardiomyocytes and smooth-muscle cells. These insights into the molecular and cellular processes of tissue formation suggest that cardiac function may be preserved after myocardial infarction by transplanting EPCs into ischemic heart tissue, thereby enhancing vascular and myocardial recovery. This therapeutic strategy has been effective in animal models of ischemic disorders, and results from randomized clinical trials suggest that cell-based strategies may be safe and feasible for treatment of myocardial infarction in humans and have provided early evidence of efficacy. However, the scarcity of EPCs in the peripheral blood and evidence that several disease states reduce EPC number and/or function have prompted the development of several strategies to overcome these limitations, such as the administration of genetically modified EPCs that overexpress angiogenic growth factors. To optimize therapeutic outcomes, researchers must continue to refine methods of EPC purification, expansion, and administration, and to develop techniques that overcome the intrinsic scarcity and phenotypic deficiencies of EPCs.

CXCR4 blockade augments bone marrow progenitor cell recruitment to the neovasculature and reduces mortality after myocardial infarction

We hypothesized that a small molecule CXCR4 antagonist, AMD3100 (AMD), could augment the mobilization of bone marrow (BM)-derived endothelial progenitor cells (EPCs), thereby enhancing neovascularization and functional recovery after myocardial infarction. Single-dose AMD injection administered after the onset of myocardial infarction increased circulating EPC counts and myocardial vascularity, reduced fibrosis, and improved cardiac function and survival. In mice transplanted with traceable BM cells, AMD increased BM-derived cell incorporation in the ischemic border zone. In contrast, continuous infusion of AMD, although increasing EPCs in the circulation, worsened outcome by blocking EPC incorporation. In addition to its effects as a CXCR4 antagonist, AMD also up-regulated VEGF and matrix metalloproteinase 9 (MMP-9) expression, and the benefits of AMD were not observed in the absence of MMP-9 expression in the BM. These findings suggest that AMD3100 preserves cardiac function after myocardial infarction by enhancing BM-EPC–mediated neovascularization, and that these benefits require MMP-9 expression in the BM, but not in the ischemic region. Our results indicate that AMD3100 could be a potentially useful therapy for the treatment of myocardial infarction.

Reduction in Hexokinase II Levels Results in Decreased Cardiac Function and Altered Remodeling After Ischemia/Reperfusion Injury

Rationale: Cardiomyocytes switch substrate utilization from fatty acid to glucose under ischemic conditions; however, it is unknown how perturbations in glycolytic enzymes affect cardiac response to ischemia/reperfusion (I/R). Hexokinase (HK)II is a HK isoform that is expressed in the heart and can bind to the mitochondrial outer membrane. Objective: We sought to define how HKII and its binding to mitochondria play a role in cardiac response and remodeling after I/R. Methods and Results: We first showed that HKII levels and its binding to mitochondria are reduced 2 days after I/R. We then subjected the hearts of wild-type and heterozygote HKII knockout (HKII+/−) mice to I/R by coronary ligation. At baseline, HKII+/− mice have normal cardiac function; however, they display lower systolic function after I/R compared to wild-type animals. The mechanism appears to be through an increase in cardiomyocyte death and fibrosis and a reduction in angiogenesis; the latter is through a decrease in hypoxia-inducible factor–dependent pathway signaling in cardiomyocytes. HKII mitochondrial binding is also critical for cardiomyocyte survival, because its displacement in tissue culture with a synthetic peptide increases cell death. Our results also suggest that HKII may be important for the remodeling of the viable cardiac tissue because its modulation in vitro alters cellular energy levels, O2 consumption, and contractility. Conclusions: These results suggest that reduction in HKII levels causes altered remodeling of the heart in I/R by increasing cell death and fibrosis and reducing angiogenesis and that mitochondrial binding is needed for protection of cardiomyocytes.

CXCR4 Antagonist AMD3100 Accelerates Impaired Wound Healing in Diabetic Mice

The antagonism of CXC-chemokine receptor 4 (CXCR4) with AMD3100 improves cardiac performance after myocardial infarction by augmenting the recruitment of endothelial progenitor cells (EPCs) from the bone marrow to the regenerating vasculature. We investigated whether AMD3100 may accelerate diabetes-impaired wound healing through a similar mechanism. Skin wounds were made on the backs of leptin-receptor–deficient mice and treated with AMD3100 or saline. Fourteen days after treatment, wound closure was significantly more complete in AMD3100-treated mice (AMD3100: 87.0±2.6%, Saline: 33.1±1.8%; P<0.0001) and was accompanied by greater collagen-fiber formation, capillary density, smooth-muscle-containing vessel density, and monocyte/macrophage infiltration. On day 7 after treatment, AMD3100 was associated with higher circulating EPC and macrophage counts and with significantly upregulated mRNA levels of stromal-cell–derived factor 1 and platelet-derived growth-factor B in the wound bed. AMD3100 also promoted macrophage proliferation and phagocytosis and the migration and proliferation of diabetic mouse primary dermal fibroblasts and 3T3 fibroblasts, which express very little CXCR4. In conclusion, a single topical application of AMD3100 promoted wound healing in diabetic mice by increasing cytokine production, mobilizing bone-marrow EPCs, and enhancing the activity of fibroblasts and monocytes/macrophages, thereby increasing both angiogenesis and vasculogenesis. Not all of the AMD3100-mediated effects evolved through CXCR4 antagonism.

CXC-Chemokine Receptor 4 Antagonist AMD3100 Promotes Cardiac Functional Recovery After Ischemia/Reperfusion Injury via Endothelial Nitric Oxide Synthase–Dependent Mechanism

Background— CXC-chemokine receptor 4 (CXCR4) regulates the retention of stem/progenitor cells in the bone marrow (BM), and the CXCR4 antagonist AMD3100 improves recovery from coronary ligation injury by mobilizing stem/progenitor cells from the BM to the peripheral blood. Thus, we investigated whether AMD3100 also improves recovery from ischemia/reperfusion injury, which more closely mimics myocardial infarction in patients, because blood flow is only temporarily obstructed. Methods and Results— Mice were treated with single subcutaneous injections of AMD3100 (5 mg/kg) or saline after ischemia/reperfusion injury. Three days later, histological measurements of the ratio of infarct area to area at risk were smaller in AMD3100-treated mice than in mice administered saline, and echocardiographic measurements of left ventricular function were greater in the AMD3100-treated mice at week 4. CXCR4+ cells were mobilized for just 1 day in both groups, but the mobilization of sca1+/flk1+ cells endured for 7 days in AMD3100-treated mice compared with just 1 day in the saline-treated mice. AMD3100 upregulated BM levels of endothelial nitric oxide synthase (eNOS) and 2 targets of eNOS signaling, matrix metalloproteinase-9 and soluble Kit ligand. Furthermore, the loss of BM eNOS expression abolished the benefit of AMD3100 on sca1+/flk1+ cell mobilization without altering the mobilization of CXCR4+ cells, and the cardioprotective effects of AMD3100 were retained in eNOS-knockout mice that had been transplanted with BM from wild-type mice but not in wild-type mice with eNOS-knockout BM. Conclusions— AMD3100 prolongs BM progenitor mobilization and improves recovery from ischemia/reperfusion injury, and these benefits appear to occur through a previously unidentified link between AMD3100 and BM eNOS expression.

The Hedgehog Transcription Factor Gli3 Modulates Angiogenesis

Rationale: The Gli transcription factors are mediators of Hedgehog (Hh) signaling and have been shown to play critical roles during embryogenesis. Previously, we have demonstrated that the Hh pathway is reactivated by ischemia in adult mammals, and that this pathway can be stimulated for therapeutic benefit; however, the specific roles of the Gli transcription factors during ischemia-induced Hh signaling have not been elucidated. Objective: To investigate the role of Gli3 in ischemic tissue repair. Methods and Results: Gli3-haploinsufficient (Gli3+/−) mice and their wild-type littermates were physiologically similar in the absence of ischemia; however, histological assessments of capillary density and echocardiographic measurements of left ventricular ejection fractions were reduced in Gli3+/− mice compared to wild-type mice after surgically induced myocardial infarction, and fibrosis was increased. Gli3-deficient mice also displayed reduced capillary density after induction of hindlimb ischemia and an impaired angiogenic response to vascular endothelial growth factor in the corneal angiogenesis model. In endothelial cells, adenovirus-mediated overexpression of Gli3 promoted migration (modified Boyden chamber), small interfering RNA–mediated downregulation of Gli3 delayed tube formation (Matrigel), and Western analyses identified increases in Akt phosphorylation, extracellular signal-regulated kinase (ERK)1/2 activation, and c-Fos expression; however, promoter–reporter assays indicated that Gli3 overexpression does not modulate Gli-dependent transcription. Furthermore, the induction of endothelial cell migration by Gli3 was dependent on Akt and ERK1/2 activation. Conclusions: Collectively, these observations indicate that Gli3 contributes to vessel growth under both ischemic and nonischemic conditions and provide the first evidence that Gli3 regulates angiogenesis and endothelial cell activity in adult mammals.

Sonic-hedgehog–induced functional recovery after myocardial infarction is enhanced by AMD3100-mediated progenitor-cell mobilization

OBJECTIVES This study was designed to compare the effectiveness of Sonic hedgehog (Shh) gene transfer, AMD3100-induced progenitor-cell mobilization, and Shh-AMD3100 combination therapy for treatment of surgically induced myocardial infarction (MI) in mice. BACKGROUND Shh gene transfer improves myocardial recovery by up-regulating angiogenic genes and enhancing the incorporation of bone marrow-derived progenitor cells (BMPCs) in infarcted myocardium. Here, we investigated whether the effectiveness of Shh gene therapy could be improved with AMD3100-induced progenitor-cell mobilization. METHODS Gene expression and cell function were evaluated in cells cultured with medium collected from fibroblasts transfected with plasmids encoding human Shh (phShh). MI was induced in wild-type mice, in matrix metalloproteinase (MMP)-9 knockout mice, and in mice transplanted with bone marrow that expressed green-fluorescent protein. Mice were treated with 100 μg of phShh (administered intramyocardially), 5 mg/kg of AMD3100 (administered subcutaneously), or both; cardiac function was evaluated echocardiographically, and fibrosis, capillary density, and BMPC incorporation were evaluated immunohistochemically. RESULTS phShh increased vascular endothelial growth factor and stromal cell-derived factor 1 expression in fibroblasts; the medium from phShh-transfected fibroblasts increased endothelial-cell migration and the migration, proliferation, and tube formation of BMPCs. Combination therapy enhanced cardiac functional recovery (i.e., left ventricular ejection fraction) in wild-type mice, but not in MMP-9 knockout mice, and was associated with less fibrosis, greater capillary density and smooth muscle-containing vessel density, and enhanced BMPC incorporation. CONCLUSIONS Combination therapy consisting of intramyocardial Shh gene transfer and AMD3100-induced progenitor-cell mobilization improves cardiac functional recovery after MI and is superior to either individual treatment for promoting therapeutic neovascularization.

Improved Culture-Based Isolation of Differentiating Endothelial Progenitor Cells from Mouse Bone Marrow Mononuclear Cells

Numerous endothelial progenitor cell (EPC)-related investigations have been performed in mouse experiments. However, defined characteristics of mouse cultured EPC have not been examined. We focused on fast versus slow adherent cell population in bone marrow mononuclear cells (BMMNCs) in culture and examined their characteristics. After 24 h-culture of BMMNCs, attached (AT) cells and floating (FL) cells were further cultured in endothelial differentiation medium separately. Immunological and molecular analyses exhibited more endothelial-like and less monocyte/macrophage-like characteristics in FL cells compared with AT cells. FL cells formed thick/stable tube and hypoxia or shear stress overload further enhanced these endothelial-like features with increased angiogenic cytokine/growth factor mRNA expressions. Finally, FL cells exhibited therapeutic potential in a mouse myocardial infarction model showing the specific local recruitment to ischemic border zone and tissue preservation. These findings suggest that slow adherent (FL) but not fast attached (AT) BMMNCs in culture are EPC-rich population in mouse.

Randomized pilot trial comparing tolvaptan with furosemide on renal and neurohumoral effects in acute heart failure

Loop diuretics are first‐line medications for congestive heart failure (CHF); however, they are associated with serious adverse effects, including decreased renal function, and sympathetic nervous and renin–angiotensin system activation. We tested whether tolvaptan, a vasopressin V2‐receptor antagonist, could reduce unfavourable furosemide‐induced effects during CHF treatment.

Estradiol promotes neural stem cell differentiation into endothelial lineage and angiogenesis in injured peripheral nerve

Neural stem cells (NSCs) differentiate into endothelial cells (ECs) and neuronal cells. Estradiol (E2) is known to exhibit proangiogenic effects on ischemic tissues via EC activation. Therefore, we hypothesized that E2 can promote the therapeutic potential of NSC transplantation for injured nerve repair via the differentiation of NSCs into ECs during neovascularization. NSCs isolated from newborn mouse brains were transplanted into injured sciatic nerves with (NSC/E2 group) or without E2-conjugated gelatin hydrogel (E2 group). The NSC/E2 group exhibited the greatest recovery in motor nerve conduction velocity, voltage amplitude, and exercise tolerance. Histological analyses revealed increased intraneural vascularity and blood perfusion as well as striking NSC recruitment to the neovasculature in the injured nerves in the NSC/E2 group. In vitro, E2 enhanced the NSC migration and proliferation inhibiting apoptosis. Fluorescence-activated cell sorting analysis also revealed that E2 significantly increased the percentage of CD31 in NSCs, and the effect of E2 was completely neutralized by the estrogen receptor antagonist ICI. The combination of E2 administration and NSC transplantation cooperatively improved the functional recovery of injured peripheral nerves, at least in part, via E2-associated NSC differentiation into ECs. These findings provide a novel mechanistic insight into both NSC biology and the biological effects of endogenous E2.