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

我们以前证明：a）CMV-IE主要特异性表达于炎性腹主动脉瘤（IAAA）患者的主动脉壁。(Lab Invest 1996 a，Nature Med 1998）B）CMV-IE基因转移在体外和体内刺激血管平滑肌细胞增殖。（Biochem Biophys Res Common 1997）c）血管内皮生长因子（VEGF）在IAAA的主动脉壁中大量表达，并且VEGF基因转移至兔颈动脉不仅刺激新生内膜形成，而且刺激新生内膜中的“渗漏和血管瘤样”脆弱血管。(Lab Invest 1996 b），然而，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）试图建立在血管壁特异性表达CMV-IE 1或CMV-IE 2的小鼠品系，2）使用重组病毒载体评估“脆弱”（或者，无功能）血管生成形成的分子机制。 ...更多信息 rs.1）CMV-IE转基因小鼠（TG）的建立：为了进行CMV-IE的血管特异性表达，我们使用鼠前内皮素原-1启动子。我们在F1代获得了2个CMV-IE 1 TG系和5个CMV-IE 2系，目前正在评估基因表达评估。此时，在每只F1动物中未发现明显的表型。现在评估F2的建立和TG共表达CMV-IE 1和-IE 2的产生。2）“脆弱”血管生成形成的分子机制：我们测试了使用重组仙台病毒（SeV）过表达VEGF 165的效果，与FGF 2基因转移直接比较。肌内注射SeV强烈增强FGF 2，在严重肢体缺血的小鼠模型中产生显著的保肢治疗效果，其中增加的血液灌注与增强的内源性VEGF表达相关。与此相反，VEGF 165-过表达，5倍高于基线的第1天，也强烈诱发内源性VEGF在肌肉中，导致加速截肢没有恢复血液灌注。有趣的是，VEGF 165或FGF 2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。平滑肌细胞肌动蛋白阳性细胞衬里提示新形成血管的成熟，然而，在VEGF的肌肉中明显受到干扰。此外，体内抗VEGF中和抗体完全减弱了FGF 2的治疗作用，因此表明内源性VEGF确实有助于FGF 2的作用。这些结果表明，VEGF是必要的，但应微妙地调节，以降低表达，以治疗缺血肢体。FGF 2的治疗作用与内源性VEGF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。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