Tumor necrosis fractor-alpha-mediated proliferation of vascular smooth muscle cells involves cyclooxygenase

TNF stimulated PGI2 and TXA2 levels in a COX-2-dependent manner. We have previously established that TNF increases COX-2 mRNA and protein expression. The COX-2-selective inhibitors NS-398 and nimesulide and the TXA 2 receptor antagonist BMS 180,291 inhibited TNF-meditated increases in DNA content and cell number by ∼95%. TNF stimulated proliferation of VSMC cells by shortening duration of G1 phase of the cell cycle, which was reflected by the increased cell number in cultures and higher percentage of cells in S and/or G2/M phases of the cycle. This effect was abolished in the presence of NS-398, a selective COX-2 inhibitor, suggesting a COX-2-dependent mechanism contributed to TNF-mediated stimulation of cell proliferation. Addition of TNF did not affect protein to DNA ratio, measured by flow cytometry, suggesting that TNF does not induce VSMC hypertrophy. TNF-mediated modulation of VSMC hyperplasia was further studied in an in vitro model of cell injury, where an area of cells was mechanically removed from a confluent monolayer. Treatment with TNF for 48hr increased proliferation of cells in the injured area by ∼75%, an effect that was prevented in the presence of NS-398 (TNF+NS-398(non-injured area): 5.7+/-1.7; TNF+NS-398(injured-area): 7.4+/-0.8 (%cells S-phases], p<0.05) and effect that was prevented in the presence of NS-398 (TNF(non-injured area): 6.24+/-0.74; TNF(injured-area): 11.08□ 1 [%cells S-phases ], p<0.05). Complete wound healing in the area of injury was observed after addition of TNF for 72 hr; an effect prevented by NS-398. TNF-induced proliferation in the injured area had decreased by 72 hr; possibly related to contact inhibition in the healed area. These data suggest that TNF induces hyperplasia of normal VSMC as well as of VSMC in response to "injury" in vitro. The results obtained from the use of LSC in an in vitro model of injury further supports the contribution of COX-2 to VSMC proliferation induced by TNF.;TNF is known to stimulate both COX-2 and iNOS pathways. The interaction of this pathway was further studied with respect to VMSC proliferation. Indeed, in our system TNF induced a transient increase in VSMC COX-2 and iNOS mRNA accumulation without affecting COX-1 mRNA. COX-2 and iNOS protein expression also increased after challenge with TNF as did TNF-mediated PGI2 and TXA2 production. Pretreatment with aminoguanidine (AG), a selective iNOS inhibitor, attenuated TNF-mediated increases in PGI2 synthesis while TXA2 production and COX-2 protein expression were unaffected. NO donors increased COX-2 protein expression and PGI2 synthesis but had no effect on TXA2 production. Inhibition of NOS activity increased TNF-mediated proliferation of VSMC by approximately 23%, while NO-donors decreased cell number by about 50%. The antiproliferative effect of NO-donors was prevented when COX-2 activity was inhibited with NS-398, a selective COX-2 inhibitor. The data indicated that TNF induces VSMC hyperplasia, but not hypertrophy. Thus, the COX-2 dependent proliferative response of VSMC to TNF was modulated in a NO-dependent manner.