HEPARIN DECREASES THE RATE OF PROLIFERATION OF RAT VASCULAR SMOOTH-MUSCLE CELLS BY RELEASING TRANSFORMING GROWTH-FACTOR BETA-LIKE ACTIVITY FROM SERUM

Objectives: Various heparins have been reported to inhibit the prolife ration of vascular smooth muscle cells (VSMCs). The effects of eight c hemically distinct heparins on the cell cycle and differentiation of p rimary and passaged cultures of rat aortic VSMCs have been characteris ed and the mechanism of heparin action investigated. Methods: VSMCs fr om adult rat aorta were prepared by enzyme dispersion and stimulated t o enter the cell cycle with 10% serum in the presence or absence of he parin. Progressions through S phase and M phase were measured by [H-3] -thymidine incorporation and cell counting respectively. Flow cytometr y was used to confirm the effects of heparin on VSMC cell cycle progre ssion. The effect of heparin on VSMC differentiation was investigated by analysing smooth muscle specific myosin heavy chain content of the cells after heparin treatment. Results: Eight heparins at concentratio ns between 5 mu g.ml(-1) and 100 mu g.ml(-1) partially inhibited VSMC proliferation (27% to 76% 96 hours after addition of heparin), but did not affect the entry of the cells into S phase. Flow cytometry confir med that VSMC populations in the presence of heparin contained signifi cantly (p < 0.005) more cells in the G(2)/M phase of the cell cycle th an control populations. Heparin also blocked the dedifferentiation of primary cultures of VSMCs stimulated by serum. These effects of hepari n were completely reversed by the presence of a neutralising antiserum to transforming growth factor beta (TGF beta) and heparin attached to agarose beads was as effective as free heparin as a growth inhibitor of VSMCs. Conclusions: Heparins of varying molecular weight and antico agulent properties all partially inhibited VSMC proliferation predomin antly by extending the G(2)/M phase of the cell cycle. Heparin also in hibited dedifferentiation of primary cultures of VSMCs. Heparin (<100 mu g.ml(-1)) acted extracellularly to release TGF beta from serum, whi ch accounted for the effects of heparin on proliferation and different iation.