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

我们先前证实：a)在炎症性腹主动脉瘤(IAAA)患者中，CMV-IE主要且特异地表达于主动脉壁。(Lab Invest 1996a，Natural Med 1998)b)CMV-IE基因转移在体外和体内都能刺激血管平滑肌细胞的增殖。(Biochem BiPhys Common 1997)血管内皮生长因子在IAAA的主动脉壁中大量表达，将血管内皮生长因子基因转移到兔颈动脉不仅能刺激新生内膜的形成，还能刺激新生内膜中“漏水的、血管瘤样的”脆弱血管。在本研究中，我们尝试建立了在血管壁上特异性表达巨细胞病毒IE1或巨细胞病毒IE2的小鼠系，2)利用重组病毒载体…评价了血管形成的分子机制更多信息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，我们使用了小鼠前内皮素-1启动子。我们在F1代获得了2个CMV-IE1 TG株和5个CMV-IE2株系，目前正在进行基因表达鉴定。此时，在每只F1动物中都没有发现明显的表型。F2的建立和共表达CMV-IE1和-IE2的TG的产生现已被评估。2)“脆性”血管生成的分子机制：我们用重组仙台病毒(SeV)测试了VEGF165过表达的效果，并与FGF2基因转移进行了直接比较。肌肉注射SeV可显著促进FGF2的表达，在严重肢体缺血模型中，随着血液灌注量的增加和内源性VEGF表达的增强，对保肢有显著的治疗效果。相反，VEGF165的过度表达，在第一天比基线高5倍，也强烈地诱导肌肉中的内源性血管内皮生长因子，导致加速截肢而没有恢复血液灌流。有趣的是，VEGF165或FGF2治疗的缺血肢体的存活骨骼肌显示出相似的PCAM-1阳性血管密度。然而，在含有血管内皮生长因子的肌肉中，肌动蛋白阳性的细胞衬里显示的新生血管的成熟明显受到干扰。此外，体内抗血管内皮细胞生长因子中和抗体可完全减弱FGF2的治疗作用，表明内源性血管内皮细胞生长因子确实参与了FGF2的作用。这些结果提示，血管内皮生长因子在治疗肢体缺血中是必要的，但应精细调控以降低其表达。FGF2的治疗效果与内源性血管内皮生长因子的协调血管生成作用相关，为治疗性血管生成提供了重要的见解。较少

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