Growth Inhibition of Bovine Pulmonary Artery Smooth Muscle Cells Following Long-Term Heparin Treatment

Heparin (HP) inhibits pulmonary artery smooth muscle cell (PASMC) growth in vitro and vascular remodeling in vivo. Barzu et al. (1994) suggested that the antiproliferative effect of HP on rat aortic smooth muscle cell in vitro diminishes with prolonged exposure to heparin. We exposed cultured bovine PASMC (BPASMC) to prolonged pretreatment with 20 mu g/ml of 0-hexanoylated HP from passages 3 to 13 and compared them to control (no pretreatment) cultures of identical passages. The pretreated BPASMC and control groups were growth arrested for 48 h, followed by treatment of 0-hexanoylated HP at different doses. On day 5, the growth inhibition of BPASMC was determined. The percent inhibition by 1 mu g/ml of 0-hexanoylated HP was 46 +/- 14% versus 62 +/- 13%, for control and pretreated BPASMC, respectively. At 10 mu g/ml the inhibition was 62 +/- 7% versus 84 +/- 6%. For 100 mu g/ml the inhibition increased to 92 +/- 5% versus 100% and at 200 mu g/ml the inhibition was 95 +/- 3% versus 100%. BPASMC (with or without preexposure to 0-hexanoylated HP), at passage 13, were sensitive to the growth inhibitory effect of 0-hexanoylated HP with no significant difference among the groups (95 +/- 3% inhibition vs. 100% for pretreated BPASMC). We found that 0-hexanoylated HP-induced necrosis as shown by flow cytometry and only minor apoptosis. Caspase-3 and PARP detection was insignificant between the groups. In summary, no cell subpopulation at long-term treatment exhibited resistance to 0-hexanoylated HP. The HP antiproliferative effect on SMC is potentially important in defining new approaches to the treatment of the remodeled vasculature of pulmonary hypertension. J. Cell. Physiol. 221: 603-608, 2009. (C) 2009 Wiley-Liss, Inc.