The effect of transfection of antisense cDNA for procollagen alpha 1 (IV) on stimulated proliferation in rat glomerular endothelial cells.

Abstract

Glomerular endothelial cells were stably transfected with a pMAMneo-Blue vector recombinant for procollagen alpha 1 (IV) cDNA in the sense (S) or antisense (AS) orientation utilizing a calcium phosphate precipitation technique. Cellular clones resistant to G418 antibiotic were selected and expanded for further analysis. Immunofluorescence microscopy demonstrated less Type IV collagen in the AS clones (1.0 +/- 0.3) than in control parent (P) and S clones (2.0 +/- 0.4) (P < 0.05). Western analysis showed that the AS clones synthesized 20 +/- 10% of the 205-kd alpha 1 (IV) chain of Type IV collagen compared with P cells (P < 0.05). As transfected clones demonstrated similar basal proliferation rates as control cells when cultured in 0.5% fetal calf serum (FCS), but failed to undergo fetal calf serum (FCS)-stimulated hyperplasia when grown on standard fibronectin-coated surfaces in 40% FCS (P < 0.05, compared with P- and S-transfected control cells). There were significant linear relationships between the presence of Type IV collagen as detected by either immunofluorescence microscopy or alpha 1 (IV) peptide chain quantitation by Western analysis and the ability of cells to undergo FCS-stimulated hyperplasia when grown on fibronectin (P < 0.05). Growth on a surface comprised of fibronectin plus Type IV collagen restored the capacity of AS transfected cells to respond to FCS stimulation (P < 0.001), but had no significant effect on the proliferative behavior of P or S cells. Measurements of AS RNA levels in these cells suggest that the inhibition of stimulated proliferation is determined by the presence of a threshold quantity of cellular AS RNA. These data demonstrate that Type IV collagen plays a critical role in conditioning glomerular endothelial cells to respond to proliferative stimuli.