The effect of von hippel-lindau gene transfer on human vascular smooth muscle cell proliferation and apoptosis.
Von Hippel-Lindau (VHL) gene is a tumor suppressor gene that plays a genome "gatekeeper'' role and controls several downstream effector genes. We have previously demonstrated that both in vivo and in vitro adenovirus-mediated gene transfer of tumor suppressor genes into the vascular endothelium is effective in decreasing neointimal hyperplasia and abnormal cell proliferation. The degree of apoptosis induced by these genes is critical in mediating the in vivo responses to gene therapy and the maintenance of the crucial balance between cell death and viability. Since VHL gene is known to regulate vascular endothelial growth factor (VEGF) as well as other angiogenic factors, it may exhibit a greater potential in the attenuation of vascular disorders in comparison to other tumor suppressor genes. This study focused on whether adenovirus-mediated VHL gene transfer into human vascular smooth muscle cells has an effect on cell proliferation and induction of apoptosis. Human aortic smooth muscle cells (HASMC) were grown as monolayers and transfected with varying titers of adenovirus containing the VHL cDNA (AdVHL). The negative controls were adenovirus containing green fluorescent protein (AdGFP), vector alone (AdNull), and virus-free infection medium. Adenovirus encoding wild-type p53 (Adp53) was used as positive control. Cell viability and proliferation were determined by using trypan blue exclusion and MTS-based CellTiter 96 AQ Proliferation Assay. Apoptosis was evaluated by TUNEL assay, morphologic changes, and nucleosomal DNA degradation. Following AdVHL transfection HASMCs demonstrated a dose-dependent decrease in viability as compared to negative controls (p < 0.05). AdVHL-transfected cells exhibited a decrease in their proliferative ability by 40.21 +/-1.66 (SEM)%. In cultures transfected with the positive control, Adp53, the cell viability as well as proliferation was highly reduced (p < 0.001). AdGFP and AdNull did not increase HASMC apoptosis above baseline levels. The cells exposed to adenoviruses expressing tumor suppressor genes underwent apoptosis, with Adp53 demonstrating a very high magnitude of cell death (75.27 +/-3.52 [SEM]%). AdVHL expression caused 45.36 +/-2.55 (SEM)% apoptosis in HASMC. Recombinant adenovirus-mediated VHL expression is efficacious in limiting vascular smooth muscle cell growth in vitro. Overexpression of VHL suppresses HASMC proliferation and regulates apoptosis. Further experiments are indicated to examine whether VHL may be a useful adjunct in limiting myointimal hyperplasia.
Jacob
Theresa
T
Division of Vascular Surgery, Department of Surgery, Maimonides Medical Center, Brooklyn, NY 11219, USA.
Ascher
Enrico
E
Hingorani
Anil
A
Kallakuri
Shreedhar
S
D000256:Adenoviridae; D017209:Apoptosis / Q000235:genetics*; D018929:Cell Culture Techniques; D016923:Cell Death / Q000235:genetics; D049109:Cell Proliferation; D002470:Cell Survival / Q000235:genetics; D004730:Endothelium, Vascular / Q000166:cytology / Q000502:physiology; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D009131:Muscle, Smooth, Vascular / Q000166:cytology* / Q000502:physiology; D025521:Tumor Suppressor Proteins / Q000235:genetics*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics*; D051794:Von Hippel-Lindau Tumor Suppressor Protein
D025521:Tumor Suppressor Proteins; D044767:Ubiquitin-Protein Ligases; D051794:Von Hippel-Lindau Tumor Suppressor Protein; C497446:VHL protein, human