Dongfen Chen

Nitric oxide synthase modulates angiogenesis in response to tissue ischemia.

We tested the hypothesis that endothelial nitric oxide synthase (eNOS) modulates angiogenesis in two animal models in which therapeutic angiogenesis has been characterized as a compensatory response to tissue ischemia. We first administered L-arginine, previously shown to augment endogenous production of NO, to normal rabbits with operatively induced hindlimb ischemia. Angiogenesis in the ischemic hindlimb was significantly improved by dietary supplementation with L-arginine, compared to placebo-treated controls; angiographically evident vascularity in the ischemic limb, hemodynamic indices of limb perfusion, capillary density, and vasomotor reactivity in the collateral vessel-dependent ischemic limb were all improved by oral L-arginine supplementation. A murine model of operatively induced hindlimb ischemia was used to investigate the impact of targeted disruption of the gene encoding for ENOS on angiogenesis. Angiogenesis in the ischemic hindlimb was significantly impaired in eNOS-/- mice versus wild-type controls evaluated by either laser Doppler flow analysis or capillary density measurement. Impaired angiogenesis in eNOS-/- mice was not improved by administration of vascular endothelial growth factor (VEGF), suggesting that eNOS acts downstream from VEGF. Thus, (a) eNOS is a downstream mediator for in vivo angiogenesis, and (b) promoting eNOS activity by L-arginine supplementation accelerates in vivo angiogenesis. These findings suggest that defective endothelial NO synthesis may limit angiogenesis in patients with endothelial dysfunction related to atherosclerosis, and that oral L-arginine supplementation constitutes a potential therapeutic strategy for accelerating angiogenesis in patients with advanced vascular obstruction.

Age-Dependent Impairment of Angiogenesis

BACKGROUND The effect of aging on angiogenesis in ischemic vascular disease has not been studied. Accordingly, we investigated the hypothesis that angiogenesis is impaired as a function of age. METHODS AND RESULTS Forty days after the resection of 1 femoral artery, collateral vessel development was significantly impaired in old (aged 4 to 5 years; n=7) versus young (aged 6 to 8 months; n=6) New Zealand White (NZW) rabbits on the basis of reduced hindlimb perfusion (ischemic: normal blood pressure ratio=0.58+/-0.05 versus 0.77+/-0.06; P<0.005), reduced number of angiographically visible vessels (angiographic score=0.48+/-0.05 versus 0.70+/-0.05; P<0.01), and lower capillary density in the ischemic limb (130.3+/-5.8/mm2 versus 171.4+/-9.5/mm2; P<0.001). Angiogenesis was also impaired in old (aged 2 years) versus young (aged 12 weeks) mice as shown by reduced hindlimb perfusion (measured by laser Doppler imaging) and lower capillary density (353.0+/-14.3/mm2 versus 713.3+/-63.4/mm2; P<0.01). Impaired angiogenesis in old animals was the result of impaired endothelial function (lower basal NO release and decreased vasodilation in response to acetylcholine) and a lower expression of vascular endothelial growth factor (VEGF) in ischemic tissues (by Northern blot, Western blot, and immunohistochemistry). When recombinant VEGF protein was administered to young and old rabbits, both groups exhibited a significant and similar increase in blood pressure ratio, angiographic score, and capillary density. CONCLUSIONS Angiogenesis responsible for collateral development in limb ischemia is impaired with aging; responsible mechanisms include age-related endothelial dysfunction and reduced VEGF expression. Advanced age, however, does not preclude augmentation of collateral vessel development in response to exogenous angiogenic cytokines.

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.

Rescue of Diabetes-Related Impairment of Angiogenesis by Intramuscular Gene Therapy with Adeno-VEGF

Diabetes is a major risk factor for coronary and peripheral artery diseases. Although diabetic patients often present with advanced forms of these diseases, it is not known whether the compensatory mechanisms to vascular ischemia are affected in this condition. Accordingly, we sought to determine whether diabetes could: 1) impair the development of new collateral vessel formation in response to tissue ischemia and 2) inhibit cytokine-induced therapeutic neovascularization. Hindlimb ischemia was created by femoral artery ligation in nonobese diabetic mice (NOD mice, n = 20) and in control C57 mice (n = 20). Hindlimb perfusion was evaluated by serial laser Doppler studies after the surgery. In NOD mice, measurement of the Doppler flow ratio between the ischemic and the normal limb indicated that restoration of perfusion in the ischemic hindlimb was significantly impaired. At day 14 after surgery, Doppler flow ratio in the NOD mice was 0.49+/-0.04 versus 0.73+/-0.06 for the C57 mice (P< or =0.005). This impairment in blood flow recovery persisted throughout the duration of the study with Doppler flow ratio values at day 35 of 0.50+/-0.05 versus 0.90+/-0.07 in the NOD and C57 mice, respectively (P< or =0.001). CD31 immunostaining confirmed the laser Doppler data by showing a significant reduction in capillary density in the NOD mice at 35 days after surgery (302+/-4 capillaries/mm2 versus 782+/-78 in C57 mice (P< or =0.005). The reduction in neovascularization in the NOD mice was the result of a lower level of vascular endothelial growth factor (VEGF) in the ischemic tissues, as assessed by Northern blot, Western blot and immunohistochemistry. The central role of VEGF was confirmed by showing that normal levels of neovascularization (compared with C57) could be achieved in NOD mice that had been supplemented for this growth factor via intramuscular injection of an adenoviral vector encoding for VEGF. We conclude that 1) diabetes impairs endogenous neovascularization of ischemic tissues; 2) the impairment in new blood vessel formation results from reduced expression of VEGF; and 3) cytokine supplementation achieved by intramuscular adeno-VEGF gene transfer restores neovascularization in a mouse model of diabetes.

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.

Potentiated Angiogenic Effect of Scatter Factor/Hepatocyte Growth Factor via Induction of Vascular Endothelial Growth Factor The Case for Paracrine Amplification of Angiogenesis

BACKGROUND Scatter factor/hepatocyte growth factor (SF/HGF) is a pleiotropic growth factor that stimulates proliferation and migration of endothelial cells (ECs) via the c-Met receptor, present on ECs as well as other cell types, including smooth muscle cells (SMCs). We studied the effects of recombinant human (rh) SF/HGF in vitro and in vivo in a rabbit model of hindlimb ischemia. We further compared these effects with those of recombinant human vascular endothelial growth factor (rhVEGF165), an EC-specific mitogen. METHODS AND RESULTS In vitro, rhSF/HGF and rhVEGF165 exhibited similar effects on proliferation and migration of ECs. When both cytokines were administered together, the result was an additive effect on EC proliferation and a synergistic effect on EC migration. Application of rhSF/HGF to cultures of human SMCs resulted in the induction of VEGF mRNA and protein. In vivo, administration of rhSF/HGF (500 microg x 3) was associated with significant improvements in collateral formation (P<.001) and regional blood flow (P<.0005) and with a significant reduction in muscle atrophy (P<.0001). These effects were significantly more pronounced than those of rhVEGF165 administered according to the same protocol (P<.05). Neither remote angiogenesis nor other pathological sequelae were observed with either rhSF/HGF or rhVEGF165. CONCLUSIONS The pleiotropic effects of certain growth factors may potentiate angiogenesis via a combination of direct effects on EC proliferation and migration and indirect effects that result in the generation of other potent EC mitogens from non-EC populations. The synergistic effects demonstrated when SF/HGF and VEGF are administered together in vitro may be reproduced in vivo by SF/HGF-induced upregulation of VEGF in vascular SMCs.

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.

Passivation of Metallic Stents After Arterial Gene Transfer of phVEGF165Inhibits Thrombus Formation and Intimal Thickening

OBJECTIVES This study sought to test the hypothesis that direct gene transfer of an endothelial cell mitogen could passivate metallic stents by accelerating endothelialization of the prosthesis. BACKGROUND Thrombosis and restenosis comprise the principal clinical manifestations of compromised biocompatibility of endovascular stents. Previous studies have demonstrated that endothelial recovery at sites of balloon injury is a critical determinant of consequent intimal thickening and mural thrombus. We therefore investigated the potential for an endothelial cell mitogen delivered as plasmid DNA to optimize stent biocompatibility. METHODS Naked plasmid DNA encoding vascular endothelial growth factor (VEGF)/vascular permeability factor (VPF) (phVEGF165) was delivered locally using a hydrogel-coated balloon angioplasty catheter to 16 rabbit iliac arteries in which metallic stents had been placed at the site of balloon injury; the contralateral iliac artery of each rabbit was balloon injured and stented but not transfected. RESULTS Stent endothelialization was accelerated by phVEGF165 gene transfer (87.38 +/- 5.06% vs. 33.13 +/- 9.73% [mean +/- SEM] of the planimetered stent surface in the treated vs. contralateral limb, p = 0.005). This was associated with a significant reduction in mural thrombus (3.7 +/- 2.4% vs. 32.7 +/- 9.7%, p = 0.01) at day 7 and intimal thickening (maximal intimal area 0.61 +/- 0.09 vs. 1.44 +/- 0.12 mm2, p < 0.0001) at day 28. No benefit was observed from pCMV-luciferase in 14 similarly instrumented control rabbits. CONCLUSIONS These findings indicate that arterial gene transfer of naked plasmid DNA encoding for an endothelial cell mitogen may successfully passivate endovascular stents by accelerating stent endothelialization, thereby reducing in-stent thrombus and obstruction due to intimal thickening.

Divergence of Angiogenic and Vascular Permeability Signaling by VEGF Inhibition of Protein Kinase C Suppresses VEGF-Induced Angiogenesis, but Promotes VEGF-Induced, NO-Dependent Vascular Permeability

Vascular endothelial growth factor (VEGF) promotes angiogenesis by a variety of mechanisms including stimulation of endothelial cell proliferation and migration and increasing vascular permeability. Although its mitogenic activity is mediated primarily by the &bgr;2-isoforms of protein kinase C (PKC), little is known about the signaling pathways transducing its other physiological properties. Accordingly, we used a novel inhibitor molecule to examine the role of PKC isoforms &agr; and &bgr; in mediating VEGF-induced angiogenesis and vascular permeability. Because conventional inhibitors of PKC, such as staurosporine or calphostin C, also inhibit a variety of other protein kinases, we used a novel compound to specifically inhibit PKC. A myristoylated peptide, which mimics the pseudosubstrate motif of PKC-&agr; and -&bgr; subtypes, has been shown to be a highly selective and cell-permeable inhibitor of PKC. Blocking led, as expected, to abrogation of VEGF-induced endothelial cell proliferation in vitro. In vivo, VEGF-induced angiogenesis was impaired by myristoylated peptide. Surprisingly, selective inhibition of PKC induced vascular permeability in vivo via a NO-dependent mechanism. Moreover, PKC inhibition led to a 6.4-fold induction of NO synthase (NOS) activity in endothelial cells. Our findings demonstrate that activation of PKC is a major signaling pathway required for VEGF-induced proliferation and angiogenesis, whereas vascular permeability was enhanced by blocking PKC. Inhibition of calcium-dependent PKC by itself led to induction of NOS. Although NOS is a downstream target for VEGF-induced angiogenesis, its induction by PKC inhibition was not sufficient to promote neovascularization. These results reveal that angiogenesis and vascular permeability induced by VEGF are mediated by mechanisms which ultimately diverge.