Porcine vascular smooth muscle cells immortalized with SV40 ori-defective DNA: characteristics of cell growth and collagen synthesis.

Abstract

A cell line derived from medial smooth muscle cells (SMC) was established from the porcine coronary artery by transfection with ori-defective simian virus 40 plasmid DNA (SV40 DNA). The characteristics of transfected cells (SV40-SMC) such as cell growth, collagen and non-collagen syntheses were investigated. SV40-SMC expressed SV40 large T antigen, c-myc and c-myb encoded proteins in the nuclei. SV40-SMC demonstrated a 'hills and valleys'-like arrangement in overconfluence and actin filaments upon immunofluorescent staining. Under electron microscopic observation, SV40-SMC had larger amounts of synthetic organelles and smaller amounts of filament bundles than those of SMC. SV40-SMC demonstrated three times higher growth activity and 4.4 times greater cellular density than SMC. Smooth muscle cells did not grow in media containing 5% plasma derived serum (PDS) instead of normal serum, whereas SV40-SMC proliferated in this medium. SV40-SMC did not grow in soft agar gel, while HeLa S3 cells, a cell line of human cervical carcinoma, formed colonies in this gel. By immunofluorescent (IF) staining, collagen phenotypes I, III, IV and V were detected in both SV40-SMC and SMC. However, protein synthesis including collagen and non-collagen was higher in SV40-SMC than in the control sample. It was concluded that SV40-SMC were a continuous cell line for vascular SMC regarding morphological characteristics, and demonstrated a higher growth activity, with increased collagen and non-collagen syntheses. This cell line is useful for the investigation of atherogenesis in relation to a proliferation of SMC and an accumulation of extracellular matrices in vascular intima.