Abstract 1663: Essential Role of Non-Sterol Pathway in the Proliferation of Vascular Smooth Muscle Cells: A Comparative Study on the Disruption of 3-Hydroxy-3-Methylglutaryl CoenzymeA Reductase or Squalene Synthase
The 3-hydroxy-3-methylglutaryl coenzymeA reductase (HMGCoAR) and squalene synthase (SS) are key enzymes for cholesterol biosynthesis in tissues. Although both HMGCoAR and SS are involved in the generation of cholesterol, a critical membrane component of caveolae, only HMGCoAR is involved in the generation of isoprenoids, which are used to activate small GTP-binding proteins including Ras and Rho. To determine the role of these enzymes in VSMC proliferation, we have generated mice homozygous for the floxed HMGCoAR and SS, respectively, and isolated VSMC from these animals. The cultured VSMC were infected with adenovirus expressing Cre recombinase (AxCAN-Cre) to delete HMG-CoAR or SS genes. The disruption of each gene was confirmed by Southern and Northerm blot analyses as well as the measurements of enzyme activities. The enzyme activities of HMG-CoAR or SS were reduced in floxed HMGCoAR VSMC or floxed SS VSMC which overexpressed Cre recombinase (HMGCoARKO or SSKO), respectively, compared with control VSMC infected with Ad-LacZ (1.85 vs. 18.92 or 2.53 vs. 8.08 pmol/min/mg protein). MTT assay were performed to determine viable cell numbers after incubation with MEM containing 10%(V/V) FCS for 48h. Optical Density (OD) at 570nm was significantly decreased by 44% (0.61±0.11 vs. 1.08±0.08, p<0.0001) in HMGCoARKO VSMC compared with control VSMC. In contrast, the OD values were not different between SSKO and control VSMC. The addition of mevalonate, farnesyl-pyrophosphate (FPP) or geranylgeranyl-pyrophasphate (GGPP) recovered the OD by 23% (p<0.0001), 31% (p<0.0001) or 12% (p<0.005) in HMGCoARKO VSMC, when compared to control VSMC, but the addition of squalene did not. These results indicate that the non-sterol pathway that provides the cells with isoprenoids for modification of small GTP-binding proteins is much more essential for the proliferation of VSMC than the sterol pathway that provides cell membrane with cholesterol.