Vascular endothelial growth factor synergistically enhances induction of E-selectin by tumor necrosis factor-alpha.

OBJECTIVE: The regulation of endothelial cell adhesion molecules (CAMs) by vascular endothelial growth factor (VEGF) was investigated in cell cultures and in a rabbit model of atherogenic neointima formation. METHODS AND RESULTS: VEGF regulation of vascular CAM-1 (vascular cell adhesion molecule), intercellular CAM-1 (intercellular adhesion molecule), and E-selectin were investigated in human umbilical vein endothelial cells using quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, and flow cytometry, and in the rabbit collar model of atherogenic macrophage accumulation by immunostaining. VEGF alone caused no significant induction of vascular cell adhesion molecule-1, intercellular adhesion molecule-1, or E-selectin compared with tumor necrosis factor-alpha. In both hypercholesterolemic and normal rabbits, adenoviral VEGF-A165 expression caused no increase in endothelial vascular cell adhesion molecule-1 or E-selectin. In contrast, pretreatment of human umbilical vein endothelial cells with VEGF significantly increased E-selectin expression induced by tumor necrosis factor-alpha, compared with tumor necrosis factor-alpha alone, whereas vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 were unaffected. VEGF similarly enhanced IL-1beta-induced E-selectin upregulation. VEGF also synergistically increased tumor necrosis factor-alpha-induced E-selectin mRNA and shedding of soluble E-selectin. Synergistic upregulation of E-selectin expression by VEGF was mediated via VEGF receptor-2 and calcineurin signaling. CONCLUSIONS: VEGF alone does not activate endothelium to induce CAM expression; instead, VEGF "primes" endothelial cells, sensitizing them to cytokines leading to heightened selective pro-inflammatory responses, including upregulation of E-selectin.

Apolipoprotein E (apoE) isoforms differentially induce nitric oxide production in endothelial cells.

Although apolipoprotein E3 (apoE3) is atheroprotective, two common isoforms, apoE2 and apoE4, produce recessive and dominant hyperlipidaemias, respectively. Using a fluorescent assay, we report herein that apoE3 particles secreted from recombinant cells stimulate more nitric oxide release in cultured human EA.hy926 endothelial cells than apoE2 or apoE4 (141% more than controls vs. 61 or 11%). Phosphatidylinositol (PI) 3-kinase inhibitors suppressed the apoE effect, while apoE receptor 2 (apoER2) was tyrosine phosphorylated. We conclude that apoE stimulates endothelial nitric oxide release in an isoform-dependent manner, and propose that tyrosine phosphorylation of apoER2 initiates PI3-kinase signalling and activation of nitric oxide synthase.

Acute regression of advanced and retardation of early aortic atheroma in immunocompetent apolipoprotein-E (apoE) deficient mice by administration of a second generation [E1(-), E3(-), polymerase(-)] adenovirus vector expressing human apoE.

Apolipoprotein E (apoE) is a 34 kDa glycoprotein with multiple actions that help protect against the development of atherosclerosis. Here, we have assessed the atheroprotective potential of an [E1(-), E3(-), polymerase(-)] adenovirus vector expressing human apoE, comparing intramuscular and intravenous (liver-directed) injections in hypercholesterolaemic apoE-deficient mice (apoE(-/-)). Intramuscular injections resulted in low expression of apoE and afforded no protection against atherogenesis. In contrast, 3 and 7 days after intravenous injections into young (6-8-week-old) apoE(-/-) mice, plasma levels of apoE were elevated and were accompanied by reductions in plasma cholesterol and normalization of lipoprotein profiles. Thereafter, plasma apoE was still detectable up to day 70, but gradually declined, although no humoral immune response was evoked, and there was a return to dyslipoproteinaemia. High levels of the vector genome were still present in livers of treated animals at 70 days, implying that decrease in apoE expression was due to cellular shutdown of the cytomegalovirus promoter. Importantly, liver-directed apoE gene transfer to these young mice retarded progression of atherosclerosis by 38% (treated, 8.21 +/- 1.05%; untreated, 13.26 +/- 0.98%, P < 0.05), during the 70 day study period. Moreover, when 10-month-old apoE(-/-) mice with advanced atherosclerosis were treated with the adenovirus vector, there was clear regression of aortic lesion area by 1 month [24.3 +/- 1.7% compared to 40.7 +/- 2.6% in baseline controls (P < 0.002)]. We conclude that the stability of the adenovirus vector genome in the livers of intravenously treated animals provides an ideal platform to evaluate liver-specific promoters for sustained transgene expression and control of atherosclerotic lesion pathology.