IN-VIVO HYPEROXIC EXPOSURE INCREASES CULTURED LUNG FIBROBLAST PROLIFERATION AND C-HA-RAS EXPRESSION

Exposure to hyperoxia has been demonstrated to alter the cell number o f lung fibroblasts in vivo The precise mechanism of lung fibroblast pr oliferation after hyperoxic exposure has not been elucidated, however, We examined the growth characteristics of lung fibroblasts isolated f rom 21-day-old rats exposed to air or 100% O-2 for 8 days. Cell prolif eration was assessed by hemocytometry, [H-3]thymidine incorporation, a nd fractional labeling with the thymidine analog bromodeoxyuridine. Un der all conditions: tested, fibroblasts isolated from O-2-exposed rats grew more rapidly than those from air-exposed rats. Conditioned mediu m from fibroblasts isolated from hyperoxia-exposed rats failed to incr ease the [H-3]thymidine incorporation of control cells to that observe d in cells isolated from hyperoxia-exposed animals, suggesting that an autocrine growth factor was not responsible for the excess proliferat ion. Sensitivity to exogenous growth factors was assessed by measuring the response to increasing concentrations of insulin-like growth fact or-1 (IGF-1). Relative to 1% fetal bovine serum (FBS), concentrations of IGF-1 between 3 and 30 ng/ml significantly increased the [H-3]thymi dine incorporation of fibroblasts derived from hyperoxic animals, wher eas control cells were unresponsive to IGF-1 stimulation. The apparent sensitivity to IGF-1 led us to assess the effect of in vivo hyperoxic exposure on the expression of c-Ha-ras, which encodes a membrane-boun d, GTP-binding/hydrolyzing protein essential for progression through G (1) in the cell cycle. ras mRNA levels in quiescent, control cells wer e minimal but increased following serum stimulation. The c-Ha-ras expr ession of lung fibroblasts from hyperoxia-exposed animals, on the othe r hand, was substantial in quiescent cells and remained high after ser um exposure. When normalized to levels of 7S cytoplasmic RNA, growth-a rrested fibroblasts from O-2-exposed rats displayed a 38% greater expr ession of c-Ha-ras message compared with fibroblasts from air-exposed rats (P = 0.0013). The level of c-Ha-ras mRNA correlated positively wi th DNA synthesis in response to 10% FBS (r = 0.70, P < 0.05). We concl ude that in vivo hyperoxic exposure increases the proliferation and c- Ha-ras expression of cultured lung fibroblasts. The precise relationsh ip between these findings remains to be elucidated.