THE EFFECT OF TRANSFECTION OF ANTISENSE CDNA FOR PROCOLLAGEN ALPHA-1(IV) ON STIMULATED PROLIFERATION IN RAT GLOMERULAR ENDOTHELIAL-CELLS

Glomerular endothelial cells were stably transfected with a pMAMneo-Bl ue vector recombinant for procollagen alpha 1(IV) cDNA in the sense (S ) or antisense (AS) orientation utilizing a calcium phosphate precipit ation technique. Cellular clones resistant to G418 antibiotic were sel ected 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-k d alpha 1(IV) chain of Type IV collagen compared with P cells (P < 0.0 5). AS transfected clones demonstrated similar basal proliferation rat es as control cells when cultured in 0.5% fetal calf serum (FCS), but failed to undergo fetal calf serum (FCS)-stimulated hyperplasia when g rown on standard fibronectin-coated surfaces in 40% FCS (P < 0.05, com pared with P- and S-transfected control cells). There were significant linear relationships between the presence of Type IV collagen as dete cted by either immunofluorescence microscopy or al(IV) peptide chain q uantitation by Western analysis and the ability of cells to undergo FC S-stimulated hyperplasia when grown on fibronectin (P < 0.05). Growth on a surface comprised of fibronectin plus Type IV collagen restored t he capacity of AS transfected cells to respond to FCS stimulation (P < 0.001), but had no significant effect on the proliferative behavior o f P or S cells. Measurements of AS RNA levels in these cells suggest t hat the inhibition of stimulated proliferation is determined by the pr esence of a threshold quantity of cellular AS RNA. These data demonstr ate that Type IV collagen plays a critical role in conditioning glomer ular endothelial cells to respond to proliferative stimuli.