The effect of transfection of antisense cDNA for procollagen α1(IV) on stimulated proliferation in rat glomerular endothelial cells

Abstract

Glomerular endothelial cells were stably transfected with a pMAMneo-Blue vector recombinant for procollagen α1(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 α1(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 α1(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.