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

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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