Expression of antisense transcripts encoding an extracellular matrix protein by stably transfected vascular smooth...

The ability to selectively inhibit the expression of various extracellular matrix proteins would provide an approach towards elucidating not only the function of these molecules but allow some insight to be gained into the mechanisms by which these molecules influence various aspects of vascular smooth muscle cell behavior such as growth, migration, morphology, and extracellular matrix assembly. The present study explores the feasibility of employing an antisense RNA approach to study vascular smooth muscle extracellular matrix proteoglycan function. Vascular smooth muscle cells were transfected with a test plasmid designed to express an antisense transcript encoding an extracellular matrix protein. The test plasmid was constructed by inserting the cDNA encoding the majority of the rat chondrosarcoma extracellular matrix link protein in an antisense orientation relative to the rat metallothionein-I promoter. Several stably transfected cell lines were isolated and characterized with respects to link antisense transcript expression, morphology, growth, and smooth muscle cytoskeletal protein expression. Results from these studies have shown that vascular smooth muscle cells could be stably transfected and link antisense transcript could be expressed at high levels by these cells. In addition, the rat metallothionein-I promoter was found to be a strong inducible promoter in vascular smooth muscle cells. Cell biology characterization of the various transfected vascular smooth muscle cell lines generated in this study has shown that one type of transfected vascular smooth muscle cell line expressed cellular characteristics indicative of early passaged vascular smooth muscle cells while a second type of transfected cell line was morphologically similar to vascular smooth muscle cells recovered from neonatal rat aortas and mechanically injured arteries obtained from adult rats.(ABSTRACT TRUNCATED AT 250 WORDS)