Role of human cytomegalovirus infection and the expression of immediate early gene products (CMV-IE) in the pathogenesis of vascular lesion formations

我们之前证明过；a) CMV-IE在炎症性腹主动脉瘤（IAAA）患者的主动脉壁中特异表达。（Lab Invest 1996a, Nature Med 1998）b) CMV-IE基因转染刺激血管平滑肌细胞体外和体内增殖。（Biochem Biophys Res Common 1997）c)血管内皮生长因子（Vascular endothelial growth factor， VEGF）在IAAA的主动脉壁中大量表达，VEGF基因转移到家兔颈动脉中，不仅刺激了新内膜的形成，还刺激了新内膜中“渗漏的、血管瘤样的”脆弱血管。（Lab Invest 1996b），然而，目前还没有研究评估CMV-IE在血管壁炎症和血管生成事件中的确切作用。在本研究中，我们1)尝试建立在血管壁上特异性表达CMV-IE1或CMV-IE2的小鼠细胞系，2)利用重组病毒载体评估“脆弱”（或无功能）血管生成的分子机制。更多内容1)CMV-IE转基因小鼠（TG）的建立：为了实现CMV-IE的血管特异性表达，我们使用了小鼠内皮素-1前原启动子。我们在F1代获得了2株CMV-IE1 TG和5株CMV-IE2，目前正在进行基因表达评估。此时，在每个F1动物中没有发现明显的表型。现在评估F2的建立和TG共表达CMV-IE1和-IE2的产生。2)“脆弱”血管生成形成的分子机制：我们使用重组仙台病毒（SeV）测试了VEGF165过表达的效果，并直接与FGF2基因转移进行了比较。肌内注射SeV可显著提高FGF2，在小鼠重度肢体缺血模型中，血液灌注增加与内源性VEGF表达增强相关，对保肢具有显著的治疗作用。相反，vegf165过表达，在第1天比基线高5倍，也强烈诱发肌肉内源性VEGF，导致截肢加速，但血液灌注没有恢复。有趣的是，VEGF165或fgf2处理的缺血肢体的活骨骼肌显示相似的pcam -1阳性血管密度。然而，平滑肌细胞肌动蛋白阳性细胞壁提示新形成血管的成熟在有VEGF的肌肉中明显受到干扰。此外，体内抗VEGF中和抗体完全降低了FGF2的治疗效果，这表明内源性VEGF确实有助于FGF2的作用。这些结果表明，VEGF是必需的，但应精心调节至低表达，以治疗缺血肢体。FGF2的治疗效果与内源性VEGF的血管生成效应一致，为治疗性血管生成提供了重要的见解。少

We previously demonstrated that ;a) CMV-IE is expressed dominantly and specifically in the aortic walls in patients with inflammatory abdominal aortic aneurysms (IAAA). (Lab Invest 1996a, Nature Med 1998)b) CMV-IE gene transfer stimulates vascular smooth muscle cell proliferation in vitro and in vivo. (Biochem Biophys Res Common 1997)c) Vascular endothelial growth factor (VEGF) is abundantly expressed in the aortic wall of IAAA, and VEGF gene transfer to rabbit carotid arteries stimulated not only neointimal formation, but also "leaky, and angioma-like" fragile vessels in the neointima. (Lab Invest 1996b), however, there has been no study assessing the exact roles of CMV-IE in the inflammatory and angiogenic events in vessel wall.In the present study, we 1) tried to establish mouse lines expressing CMV-IE1 or CMV-IE2 specifically in vessel wall, and 2) assessed the molecular mechanisms of formation of "fragile" (alternatively, non-functional ) angiogenesis using recombinant viral vecto … More rs.1) Establishment of CMV-IE transgenic mice (TG) :To perform vessel specific expression of CMV-IE, we used murine preproendothelin-1 promoter. We obtained 2 lines of CMV-IE1 TG and 5 lines of CMV-IE2 in F1 generation, and gene expression assessments are now under evaluation. At this time, no apparent phenotype was found in each F1 animal. Establishment of F2 and generation of TG co-expressing CMV-IE1 and -IE2 are now assessed.2) Molecular mechanisms of formation of "fragile" angiogenesis :We tested effects of overexpression of VEGF165 using recombinant Sendai virus (SeV), as directly compared to FGF2 gene transfer. Intramuscular injection of SeV strongly boosted FGF2, resulting in significant therapeutic effects for limb salvage with increased blood perfusion associated with enhanced endogenous VEGF expression in murine models of critical limb ischemia. Incontrast, VEGF165-overexpression, 5-times higher than that of baseline on day 1, also strongly evoked endogenous VEGF in muscles, resulting in an accelerated limb amputation without recovery of blood perfusion. Interestingly, viable skeletal muscles of either VEGF165- or FGF2-treated ischemic limbs showed similar PCAM-1-positive vessel densities. Maturation of newly formed vessels suggested by smooth muscle cell actin-positive cell lining, however, was significantly disturbed in muscles with VEGF. Further, therapeutic effects of FGF2 were completely diminished by anti-VEGF neutralizing antibody in vivo, thus indicationg that endogenous VEGF does contribute to the effect of FGF2. These results suggest that VEGF is necessary, but should be delicately regulated to lower expression to treat ischemic limb. The therapeutic effect of FGF2, associated with the harmonized angiogenic effects seen with endogenous VEGF, provides important insights into therapeutic angiogenesis. Less