Yukio Tsurumi

Vascular Endothelial Growth Factor/Vascular Permeability Factor Enhances Vascular Permeability Via Nitric Oxide and Prostacyclin

BACKGROUND Vascular endothelial growth factor (VEGF), an endothelial cell mitogen that promotes angiogenesis, was initially identified as a vascular permeability factor (VPF). Abundant evidence suggests that angiogenesis is preceded and/or accompanied by enhanced microvascular permeability. The mechanism by which VEGF/VPF increases vascular permeability (VP), however, has remained enigmatic. Accordingly, we used an in vivo assay of VP (Miles assay) to study the putative mediators of VEGF/VPF-induced permeability. METHODS AND RESULTS VEGF/VPF and positive controls (platelet-activating factor [PAF], histamine, and bradykinin) all increased vascular permeability. Prior administration of the tyrosine kinase inhibitors genistein or herbimycin A prevented VEGF/VPF-induced permeability. Placenta growth factor, which binds to Flt-1/VEGF-R1 but not Flk-1/KDR/VEGF-R2 receptor tyrosine kinase, failed to increase permeability. Other growth factors such as basic fibroblast growth factor (FGF), acidic FGF, platelet-derived growth factor-BB, transforming growth factor-beta, scatter factor, and granulocyte macrophage-colony stimulating factor (8 to 128 ng) failed to increase permeability. VEGF/VPF-induced permeability was significantly attenuated by the nitric oxide (NO) synthase inhibitors N(omega)-nitro-L-arginine (10 mg/kg) or N(omega)-nitro-L-arginine methyl ester (20 mg/kg) and the cyclooxygenase inhibitor indomethacin (5 mg/kg). The inactive enantiomer N(omega)-nitro-D-arginine methyl ester (20 mg/kg) did not inhibit VEGF/VPF-induced permeability. In vitro studies confirmed that VEGF/VPF stimulates synthesis of NO and prostaglandin metabolites in microvascular endothelial cells. Finally, NO donors and the prostacyclin analogue taprostene administered together but not alone reproduced the increase in permeability observed with VEGF/VPF. CONCLUSIONS These results implicate NO and prostacyclin produced by the interaction of VEGF/VPF with its Flk-1/KDR/VEGF-R2 receptor as mediators of VEGF/VPF-induced vascular permeability. Moreover, this property appears unique to VEGF/VPF among angiogenic cytokines.

Direct Intramuscular Gene Transfer of Naked DNA Encoding Vascular Endothelial Growth Factor Augments Collateral Development and Tissue Perfusion

BACKGROUND Striated muscle has been shown to be capable of taking up and expressing foreign genes transferred in the form of naked plasmid DNA, although typically with a low level of gene expression. In the case of genes that encode secreted proteins, however, low transfection efficiency may not preclude bio-activity of the secreted gene product. Accordingly, we investigated the hypothesis that intramuscular (IM) gene therapy with naked plasmid DNA encoding vascular endothelial growth factor (VEGF) could augment collateral development and tissue perfusion in an animal model of hindlimb ischemia. METHODS AND RESULTS Ten days after ischemia was induced in one rabbit hindlimb, 500 micrograms of phVEGF165, or the reporter gene LacZ, was injected IM into the ischemic hindlimb muscles. Thirty days later, angiographically recognizable collateral vessels and histologically identifiable capillaries were increased in VEGF transfectants compared with controls. This augmented vascularity improved perfusion to the ischemic limb, documented by a superior calf blood pressure ratio for phVEGF165 (0.85 +/- 0.05) versus controls (0.64 +/- 0.05, P < .01), improved blood flow in the ischemic limb (measured with an intra-arterial Doppler wire) at rest (phVEGF165 = 21.3 +/- 3.9 mL/min, control = 14.6 +/- 1.6 mL/min, P < .01) and after a vasodilator (phVEGF165 = 54.2 +/- 12.0 mL/min, control = 37.3 +/- 8.9 mL/min, P < .01) and increased microspheres in the adductor (phVEGF165 = 4.3 +/- 1.6 mL.min-1.100 g of tissue-1, control = 2.9 +/- 1.2 mL.min-1.100 g of tissue-1, P < .05) and gastrocnemius (phVEGF165 = 3.9 +/- 1.0 mL.min-1.100 g of tissue-1, control = 2.8 +/- 1.4 mL.min-1.100 g of tissue-1, P < .05) muscles of the ischemic limb. CONCLUSIONS Ischemic skeletal muscle represents a promising target for gene therapy with naked plasmid DNA. IM transfection of genes encoding angiogenic cytokines, particularly those that are naturally secreted by intact cells, may constitute an alternative treatment strategy for patients with extensive peripheral vascular disease in whom the use of intravascular catheter-based gene transfer is compromised and/or prohibited.

RECIPROCAL RELATION BETWEEN VEGF AND NO IN THE REGULATION OF ENDOTHELIAL INTEGRITY

Balloon angioplasty disrupts the protective endothelial lining of the arterial wall, rendering arteries susceptible to thrombosis and intimal thickening. We show here that vascular endothelial growth factor (VEGF), an endothelial cell mitogen, is upregulated in medial smooth muscle cells of the arterial wall in response to balloon injury. Both protein kinase C (PKC) and tyrosine kinase pp60src mediate augmented VEGF expression. In contrast, nitric oxide (NO) donors inhibit PKC-induced VEGF upregulation by interfering with binding of the transcription factor activator protein-1 (AP-1) to the VEGF promoter. Inhibition of VEGF promoter activation suggests that NO secreted by a restored endothelium functions as the negative feedback mechanism that downregulates VEGF expression to basal levels. Administration of a neutralizing VEGF antibody impaired reendothelialization following balloon injury performed in vivo. These findings establish a reciprocal relation between VEGF and NO in the endogenous regulation of endothelial integrity following arterial injury.

Accelerated Restitution of Endothelial Integrity and Endothelium-Dependent Function After phVEGF165 Gene Transfer

BACKGROUND Delinquent reendothelialization (rET) has been shown to have a permissive, if not facilitatory, impact on smooth muscle cell proliferation. This inverse relation has been attributed to certain functions of the endothelium, including barrier regulation of permeability, thrombogenicity, and leukocyte adherence, as well as production of growth-inhibitory molecules. Accordingly, the present investigation was designed to test the hypothesis that an endothelial cell (EC) mitogen could serve as the basis for a novel gene therapy strategy designed to facilitate EC regeneration, reduce neointimal thickening, and promote recovery of EC dysfunction after balloon injury. METHODS AND RESULTS New Zealand White rabbits underwent simultaneous balloon injury and gene transfer of one femoral artery with phVEGF165, encoding the 165-amino acid isoform of vascular endothelial growth factor (VEGF), or pGSVLacZ. In each animal transfected with phVEGF165 or pGSVLacZ, the contralateral femoral artery was also subjected to balloon injury but not to gene transfer. For pGSVLacZ, rET remained incomplete at 4 weeks after transfection; in contrast, phVEGF165 produced prompt rET, which was 95% complete by 1 week. Furthermore, rET in the contralateral, balloon-injured, nontransfected limb of the VEGF group was similarly accelerated. Consequently, intimal thickening was diminished, thrombotic occlusion was less frequent, and recovery of EC-dependent vasomotor reactivity was accelerated in VEGF transfectants compared with control animals. A similar benefit was observed for the contralateral, balloon-injured, nontransfected limb. CONCLUSIONS Catheter-mediated, site-specific arterial gene transfer of phVEGF165 can accelerate rET at local and remote sites, leading to inhibition of neointimal thickening, reduction in thrombogenicity, and restoration of endothelium-dependent vasomotor reactivity. These findings support the notion that gene transfer encoding for an EC-specific mitogen may be useful for preventing the complications, including restenosis, of balloon angioplasty.

Coronary Pressure Measurement After Stenting Predicts Adverse Events at Follow-Up A Multicenter Registry

Background—Coronary stenting is associated with a restenosis rate of 15% to 20% at 6-month follow-up, despite optimum angiographic stent implantation. In this multicenter registry, we investigated the relation between optimum physiological stent implantation as assessed by poststent fractional flow reserve (FFR) and outcome at 6 months. Methods and Results—In 750 patients, coronary pressure measurement at maximum hyperemia was performed after angiographically apparently satisfactory stent implantation. Poststenting FFR was calculated and related to major adverse events (including need for repeat target vessel revascularization) at 6 months. In 76 patients (10.2%), at least 1 adverse event occurred. Five patients died, 19 experienced myocardial infarction, and 52 underwent at least 1 repeat target vessel revascularization. By multivariate analysis, FFR immediately after stenting was the most significant independent variable related to all types of events. In 36% of the patients, FFR normalized (>0.95), and event rate was 4.9% in that group. In 32% of the patients, poststent FFR was between 0.90 and 0.95, and event rate was 6.2%. In 32% of patients, poststent FFR was <0.90, and event rate was 20.3%. In 6% of the patients, FFR was <0.80, and event rate was 29.5% (P <0.001). Conclusions—FFR after stenting is a strong independent predictor of outcome at 6 months.

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.

Telmisartan induces proliferation of human endothelial progenitor cells via PPARγ-dependent PI3K/Akt pathway

OBJECTIVE Although recent clinical trials have suggested that angiotensin II type 1 receptor blockers (ARBs) reduced cardiovascular events, the precise mechanisms involved are still unknown. Telmisartan, an ARB, has recently been identified as a ligand of peroxisome proliferator-activated receptor-gamma (PPARgamma). On the other hand, since endothelial progenitor cells (EPCs) are thought to play a critical role in ischemic diseases, we investigated effects of telmisartan on proliferation of EPCs. METHODS AND RESULTS Human peripheral blood mononuclear cells were isolated from healthy volunteers, and cultured on fibronectin-coated dishes in the presence or absence of telmisartan. Four days after starting culture, adherent cells were collected, and equal numbers of cells were reseeded into methylcellulose medium with or without telmisartan. In the presence of telmisartan, numbers of colonies increased in a dose-dependent manner. DiI-AcLDL uptake and lectin and CD31, CD34 staining revealed that these colonies were EPCs. Increase in colony number by treatment with telmisartan was absolutely inhibited when cultured with a specific inhibitor of PPARgamma. In addition, we observed that specific inhibitors of phosphoinositide-3 kinase (PI3K) abolished telmisartan-stimulated increase of monocytic EPC-like cells and telmisartan induced phosphorylation of Akt. Furthermore, mRNA expression of p21 was downregulated in a dose dependent manner, suggesting that growth inductive effects of telmisartan might be regulated by the PI3K/Akt and p21 signaling pathway. CONCLUSIONS These findings suggest that telmisartan might contribute to endothelial integrity and vasculogenesis in ischemic regions by increasing numbers of EPCs.

Sudden Cardiac Death and Left Ventricular Ejection Fraction During Long-Term Follow-up After Acute Myocardial Infarction in the Primary Percutaneous Coronary Intervention Era. Results from the HIJAMI-II Registry

Objective: To determine the incidence of sudden cardiac death (SCD) according to left ventricular ejection fraction (LVEF) in survivors of myocardial infarction (MI) in the primary percutaneous coronary intervention (PCI) era. Design: A multicentre observational prospective registered cohort study. Setting: 18 medical centres in Japan. Patients: 4122 consecutive patients (mean age 66 (SD 12) years, 73.7% male) with acute MI, who were discharged alive. Main outcome measures: The primary end-point was SCD, and a secondary end-point was death from any cause. Results: Patients were categorised into three groups: LVEF >40% (n = 3416), LVEF ⩽40% and >30% (n = 507) and LVEF ⩽30% (n = 199). Among all patients, 77.8% received PCI and 3.7% received coronary artery bypass graft surgery. During an average follow-up of 4.1 years, SCD was 1.2% and mortality was 13.1%. Patients with LVEF ⩽30% and LVEF ⩽40% and >30% were at increased risk for SCD (HR 5.99, 95% CI 2.73 to 13.14, p<0.001, HR 3.37, 95% CI 1.74 to 6.50, p<0.001, respectively), and mortality (HR 3.85, 95% CI 2.96 to 5.00, p<0.001, HR 2.06, 95% CI 1.66 to 2.57, p<0.001, respectively), compared to patients with LVEF >40%. Kaplan-Meier estimates of SCD in patients with LVEF ⩽30% were 2.9%, 5.1% and 5.1% at 1, 3 and 5 years, respectively. Conclusion: There is a low incidence of SCD in survivors of MI in the primary PCI era, although LVEF is a predictor of increased risk for SCD.

Influence of plasma lipoprotein(a) levels on coronary vasomotor response to acetylcholine

OBJECTIVES This study was undertaken to examine the influence of plasma lipoprotein(a) [Lp(a)] levels on coronary endothelial vasomotor function. BACKGROUND Epidemiologic studies have demonstrated a direct relation between elevated plasma levels of Lp(a) and increased risk of coronary artery disease. Well recognized coronary risk factors are known to affect endothelium-dependent vasomotion; however, the influence of Lp(a) on coronary vasomotor function has not been determined. METHODS We used quantitative coronary angiography to measure left anterior descending coronary artery diameter changes produced by intracoronary acetylcholine and isosorbide dinitrate in 30 patients with angiographically normal coronary arteries. Plasma Lp(a) levels were determined with enzyme-linked immunosorbent assay. RESULTS Vasomotor response to acetylcholine ranged from +13% to -47% in the proximal, from +23% to -53% in the middle and from +13% to -56% in the distal segment of the left anterior descending coronary artery. According to univariate linear regression analysis, Lp(a) had a significant inverse correlation with vasomotor response to acetylcholine: r = 0.47, p < 0.01 in the proximal; r = -0.61, p < 0.001 in the middle; and r = -0.52, p < 0.01 in the distal segment of the left anterior descending coronary artery. By multiple stepwise regression analysis, plasma Lp(a) was the significant predictor of vasomotion in response to acetylcholine in all tested segments (p < 0.01). CONCLUSIONS Elevated Lp(a) levels were associated with impairment of endothelium-dependent vasodilation even when atherosclerotic lesions were not recognizable by angiography. This finding suggests that elevated plasma levels of Lp(a) cause endothelial dysfunction and may contribute in part to later development of atherosclerosis, as shown in epidemiologic studies.

Obstructive sleep apnea is associated with greater thoracic aortic size.

To the Editor: Obstructive sleep apnea (OSA), characterized by partial or complete occlusion of the pharynx during sleep, results in persistent inspiratory effort and interruption of airflow. During each episode of apnea, OSA patients develop increased transmural pressure in the aortic wall.