Endothelial FGF receptor signaling accelerates atherosclerosis.

Members of the fibroblast growth factor (FGF) family have been clinically applied to the treatment of ischemic diseases because of their strong angiogenic actions. Although tissue ischemia is predominantly caused by atherosclerosis, the roles of endothelial FGF receptors (FGF-Rs) in atherosclerosis remain obscure. We generated endothelial cell (EC)-targeted constitutively active FGF-R2-overexpressing mice, using the Tie2 promoter (Tie2-FGF-R2-Tg), and crossed them with apolipoprotein E (ApoE)-deficient mice (ApoE-KO) to generate Tie2-FGF-R2-Tg/ApoE-deficient mice (Tie2-FGF-R2-Tg/ApoE-KO). After being fed a Western diet for 8 wk, the Tie2-FGF-R2-Tg/ApoE-KO demonstrated 2.0-fold greater atherosclerotic lesion area on the luminal surfaces of the aortas than the ApoE-KO (P < 0.01). The level of p21(Cip1) protein, a cell cycle inhibitor, in the FGF-R2-overexpressing EC was 2.5-fold greater than that in the wild-type (WT) EC at the baseline (P < 0.01). FGF-R2 overexpression in the EC resulted in increased expression of VCAM-1 and ICAM-1, acceleration of apoptosis, and decreased proliferative activity, all of which were normalized by small interfering RNA (siRNA)-mediated knockdown of p21(Cip1) (75% reduction in protein level, P < 0.01). Furthermore, the expression of PDGF-B and Egr-1, a PDGF/p21(Cip1)-inducible transcription factor, in the aortic endothelium of Tie2-FGF-R2-Tg/ApoE-KO was significantly greater than that in ApoE-KO. The proliferation of vascular smooth muscle cells in the aortic media of Tie2-FGF-R2-Tg/ApoE-KO was 2.0-fold higher than that in ApoE-KO (P < 0.01). Thus our study reveals that endothelial FGF-R2 signaling aggravates atherosclerosis by promoting p21(Cip1)-mediated EC dysfunction and cautions against the use of FGF for therapeutic angiogenesis in the setting of atherosclerosis.

Endothelium-targeted overexpression of constitutively active FGF receptor induces cardioprotection in mice myocardial infarction.

Fibroblast growth factor receptor (FGFR) is expressed in a variety of cells and is involved in their proliferation/migration/survival. To elucidate FGFR-mediated specific action of vascular endothelial cells (ECs) on myocardial ischemia, we generated endothelium-targeted transgenic mice overexpressing constitutively active FGFR2 using Tie2 promoter (FGFR2-Tg). Infarct size, vessel formation and blood perfusion were significantly improved 28 days after myocardial infarction (MI) in FGFR2-Tg, compared with wild-type mice. Aortic ECs isolated from FGFR-Tg showed a marked increase in migratory capacity and tube formation. These in vitro angiogenic activities were blocked by PI3-kinase inhibitor. Whereas, parameters obtained from echocardiography were already improved at three days after MI. Cardiomyocyte apoptosis at the ischemic border zone was decreased in FGFR2-Tg (32.1%, p < 0.05) and cardiac mRNA expression of FGF2 (basic FGF) was also up-regulated (142%, p < 0.05) at 3 days after MI. 1% oxygen-mediated apoptosis was significantly inhibited in FGFR2-Tg-ECs and this inhibition was abolished by PI3-kinase inhibitor. FGFR2-Tg-ECs exposed to 1% oxygen exhibited enhanced phosphorylation of 416-Tyr-Src, 473-Ser-Akt, and HIF1alpha accumulation. The production of FGF2 was enhanced 2.1-fold in FGFR-Tg-ECs under 1% oxygen via the Src/Akt/HIF1alpha pathway, which induced the peri-vessel migration of vascular smooth muscle cells (VSMCs) and anti-apoptotic effects on VSMCs and cardiomyocytes. FGF receptor signaling in ECs promoted migration, survival and autocrine production of FGF2, leading to reduced infarct size, which is associated with anti-apoptotic action in the early stage and with enhanced angiogenesis in the late stage after MI.

Circulating endothelial progenitor cells decreased in patients with sclerodermatous chronic graft-versus-host disease.

Chronic graft-versus-host disease (cGVHD) is a common late complication of allogeneic stem cell transplantation (allo-SCT). Some cGVHD patients develop skin lesions, and the skin lesions in sclerodermatous cGVHD (s-cGVHD) patients resemble those in progressive systemic sclerosis (PSS), which is characterized by impaired production of circulating endothelial progenitor cells (EPCs). We investigated, retrospectively, whether low EPC production may promote the development of sclerodermatous lesions in cGVHD. Peripheral blood (PB) was obtained from 14 healthy volunteers and 27 allo-SCT patients. Five patients developed s-cGVHD. CD34(+) cells were purified by using the magnetic cell-sorting separation system, and the CD34(+)/CD133(+)/vascular endothelial growth factor (VEGF) receptor-2(+) EPCs were quantified. The endothelial cell colony-formation potential was evaluated. Serum VEGF and basic fibroblast growth factor (b-FGF) concentrations were measured by ELISA. The s-cGVHD patients had significantly lower median circulating EPCs frequencies than non-s-cGVHD patients or control (145 of 20 mL [interquartial range-IQR 107-193] versus 1083.5 [IQR 669.3-2151]; P = .0023, and versus 1530.5 [IQR 961.3-2158]; P = .0012, respectively). They also had impaired median endothelial-forming ability compared to non-s-cGVHD patients or controls (3.8 [IQR 1.0-4.3] versus 12.8 [IQR 8.8-28.8], and versus 26.4 [IQR 23.6-30.6], respectively; P = .0012). Their VEGF and b-FGF serum levels were also higher than in controls. In conclusion, s-cGVHD patients show findings consistent with those seen in PSS with impaired vasculogenesis that may limit blood perfusion and may contribute to the development of sclerodermatous lesions.

Central role of calcium-dependent tyrosine kinase PYK2 in endothelial nitric oxide synthase-mediated angiogenic response and vascular function.

BACKGROUND: The involvement of Ca2+-dependent tyrosine kinase PYK2 in the Akt/endothelial NO synthase pathway remains to be determined. METHODS AND RESULTS: Blood flow recovery and neovessel formation after hind-limb ischemia were impaired in PYK2-/- mice with reduced mobilization of endothelial progenitors. Vascular endothelial growth factor (VEGF)-mediated cytoplasmic Ca2+ mobilization and Ca2+-independent Akt activation were markedly decreased in the PYK2-deficient aortic endothelial cells, whereas the Ca2+-independent AMP-activated protein kinase/protein kinase-A pathway that phosphorylates endothelial NO synthase was not impaired. Acetylcholine-mediated aortic vasorelaxation and cGMP production were significantly decreased. Vascular endothelial growth factor-dependent migration, tube formation, and actin cytoskeletal reorganization associated with Rac1 activation were inhibited in PYK2-deficient endothelial cells. PI3-kinase is associated with vascular endothelial growth factor-induced PYK2/Src complex, and inhibition of Src blocked Akt activation. The vascular endothelial growth factor-mediated Src association with PLCgamma1 and phosphorylation of 783Tyr-PLCgamma1 also were abolished by PYK2 deficiency. CONCLUSION: These findings demonstrate that PYK2 is closely involved in receptor- or ischemia-activated signaling events via Src/PLCgamma1 and Src/PI3-kinase/Akt pathways, leading to endothelial NO synthase phosphorylation, and thus modulates endothelial NO synthase-mediated vasoactive function and angiogenic response.

Intracoronary transplantation of non-expanded peripheral blood-derived mononuclear cells promotes improvement of cardiac function in patients with acute myocardial infarction.

BACKGROUND: Transplantation of non-expanded peripheral blood mononuclear cells (PBMNCs) enhances neovessel formation in ischemic myocardium and limbs by releasing angiogenic factors. This study was designed to examine whether intracoronary transplantation of PBMNCs improves cardiac function after acute myocardial infarction (AMI). METHODS AND RESULTS: After successful percutaneous coronary intervention (PCI) for a ST-elevation AMI with occlusion of proximal left anterior descending coronary artery within 24 h, patients received an intracoronary infusion of PBMNCs within 5 days after PCI (PBMNC group). PBMNCs were obtained from patients by COBE spectra-apheresis and concentrated to 10 ml, 3.3 ml of which was infused via over-the-wire catheter. The global left ventricular ejection fraction (LVEF) change from baseline to 6 months followup in th ePBMNC group that underwent standard PCI for similar AMI [corrected]. The primary endpoint was the global left ventricular ejection fraction (LVEF) change from baseline to 6 months' follow-up. The data showed that the absolute increase in LVEF was 7.4% in the control group and 13.4% (p=0.037 vs control) in the PBMNC group. Cell therapy resulted in a greater tendency of DeltaRegional ejection fraction (EF) or significant improvement in the wall motion score index and Tc-99m-tetrofosmin perfusion defect score associated with the infarct area, compared with controls. Moreover, intracoronary administration of PBMNCs did not exacerbate either left ventricular (LV) end-diastolic and end-systolic volume expansion or high-risk arrhythmia, without any adverse clinical events. CONCLUSION: Intracoronary infusion of non-expanded PBMNCs promotes improvement of LV systolic function. This less invasive and more feasible approach to collecting endothelial progenitor cells may provide a novel therapeutic option for improving cardiac function after AMI.

Spironolactone modulates expressions of cardiac mineralocorticoid receptor and 11beta-hydroxysteroid dehydrogenase 2 and prevents ventricular remodeling in post-infarct rat hearts.

Aldosterone antagonists have been reported to prevent ventricular remodeling after myocardial infarction (MI) via their action to extracellular matrix (ECM). However, it remains largely unknown whether aldosterone antagonists attenuate myocyte loss in the remodeling process. The present study examined whether spironolactone prevents myocyte apoptosis and improves post-infarct ventricular remodeling in rats. MI was achieved by permanent occlusion of the left coronary artery. Administration of spironolactone (100 mg/kg/day) was started immediately after MI. Sprague-Dawley rats were divided into four groups: 1) sham, 2) spironolactone-treated sham, 3) untreated MI, 4) spironolactone-treated MI. Echocardiographic parameters (left ventricular [LV] diastolic dimension [LVDd], fractional shortening [%FS]), hemodynamic parameters (LV systolic pressure [LVSP], LV end-diastolic pressure [LVEDP], dP/dt(max) and dP/dt(min)) and collagen accumulation quantitated by Masson's Trichrome staining were significantly improved in the spironolactone-treated MI group on the 14th day, compared with the untreated MI group. Moreover, the percentage of apoptotic myocytes evaluated by terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL) assay was significantly lower in the spironolactone-treated MI group on the 2nd (3.54% vs. 5.79% in untreated MI group), 7th (0.65% vs. 1.37% in untreated MI group) and 14th days (0.11% vs. 0.16% in untreated MI group). Real time reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that the expression of mineralocorticoid receptor (MR) mRNA and that of 11beta-hydroxysteroid dehydrogenase 2 (11beta-HSD2) mRNA, which is known to confer aldosterone selectivity on MR, were upregulated in the untreated MI group, and that spironolactone significantly suppressed the expression of these genes. Moreover, spironolactone significantly inhibited aldosterone-induced apoptosis in cultured rat cardiac myocytes in a dose-dependent fashion. Our study demonstrates that, in addition to their effect on ECM, aldosterone antagonists inhibit myocyte apoptosis and prevent post-infarct ventricular remodeling by modulating the expression levels of MR and 11beta-HSD2, which are enhanced in the remodeling heart.

Erythropoietin-mobilized endothelial progenitors enhance reendothelialization via Akt-endothelial nitric oxide synthase activation and prevent neointimal hyperplasia.

We investigated whether the mobilization of endothelial progenitor cells (EPCs) by exogenous erythropoietin (Epo) promotes the repair of injured endothelium. Recombinant human Epo was injected (1000 IU/kg for the initial 3 days) after wire injury of the femoral artery of mice. Neointimal formation was inhibited by Epo to 48% of the control (P<0.05) in an NO-dependent manner. Epo induced a 1.4-fold increase in reendothelialized area of day 14 denuded vessels, 55% of which was derived from bone marrow (BM) cells. Epo increased the circulating Sca-1(+)/Flk-1(+) EPCs (2.0-fold, P<0.05) with endothelial properties NO dependently. BM replacement by GFP- or beta-galactosidase-overexpressing cells showed that Epo stimulated both differentiation of BM-derived EPCs and proliferation of resident ECs. BM-derived ECs increased 2.2- to 2.7-fold (P<0.05) in the Epo-induced neoendothelium, where the expression of Epo receptor was upregulated. Epo induced Akt/eNOS phosphorylation and NO synthesis on EPCs and exerted an antiapoptotic action on wire-injured arteries. In conclusion, Epo treatment inhibits the neointimal hyperplasia after arterial injury in an NO-dependent manner by acting on the injured vessels and mobilizing EPCs to the neo-endothelium.

Nicorandil regulates Bcl-2 family proteins and protects cardiac myocytes against hypoxia-induced apoptosis.

Nicorandil has been shown to inhibit myocyte apoptosis by opening of mitochondrial ATP-sensitive potassium (mitoK(ATP)) channels and nitrate-like effect against oxidative stress. However, the detailed mechanism of nicorandil-mediated cardioprotection under hypoxic conditions remains to be largely unknown. The present study examined whether nicorandil can inhibit apoptosis via regulation of Bcl-2 family proteins in hypoxic myocytes. Neonatal rat cardiac myocytes were exposed to hypoxia for 7 hours. Hypoxia-induced myocyte apoptosis (13.9+/-0.9%) under glucose-rich conditions. Myocyte apoptosis was accompanied by loss of mitochondrial membrane potential (Deltapsi(m)), cytochrome c release from mitochondria into cytosol, and activation of caspase-3. Hypoxia also significantly increased Bax and decreased Bcl-2 mRNA and protein expression, thereby increasing Bax/Bcl-2 ratio. Nicorandil 100 micromol/l significantly decreased the percentage of apoptotic myocytes (7.2+/-0.5%) by inhibiting loss of Deltapsi(m) and translocation of cytochrome c. These effects of nicorandil were partially but significantly inhibited by cotreatment of either 500 micromol/l 5-hydroxydecanoate, a selective mitoK(ATP) channel antagonist, or 10 micromol/l 1H-[1,2,4]oxidazolo[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylate cyclase. Moreover, nicorandil significantly inhibited the hypoxia-induced changes in Bax and Bcl-2 expression, and concomitant increased Bax and decreased Bcl-2 immunoreactivity in mitochondria. These effects of nicorandil in Bax and Bcl-2 expression were significantly blunted by cotreatment of ODQ and 5-HD, respectively. Cotreatment of KT5823, an inhibitor of protein kinase G, significantly blocked the effect of nicorandil on Bax expression and 8-bromo-cyclic guanosine 3',5' monophosphate (8-bromo-cGMP), a cGMP analog, mimicked the effect of nicorandil on Bax expression. The present study demonstrates that nicorandil regulates Bcl-2 family proteins via opening of mitoK(ATP) channels and nitric oxide-cGMP signaling and inhibits hypoxia-induced mitochondrial death pathway.