title: Heparin decreases the rate of proliferation of rat vascular smooth muscle cells by releasing transforming growth factor beta-like activity from serum.
abstract: OBJECTIVE
  Various heparins have been reported to inhibit the proliferation of vascular smooth muscle cells (VSMCs). The effects of eight chemically distinct heparins on the cell cycle and differentiation of primary and passaged cultures of rat aortic VSMCs have been characterised and the mechanism of heparin action investigated.
  
  
  METHODS
  VSMCs from adult rat aorta were prepared by enzyme dispersion and stimulated to enter the cell cycle with 10% serum in the presence or absence of heparin. Progressions through S phase and M phase were measured by [3H]-thymidine incorporation and cell counting respectively. Flow cytometry was used to confirm the effects of heparin on VSMC cell cycle progression. 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 concentrations between 5 micrograms.ml-1 and 100 micrograms.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 confirmed that VSMC populations in the presence of heparin contained significantly (p < 0.005) more cells in the G2/M phase of the cell cycle than control populations. Heparin also blocked the dedifferentiation of primary cultures of VSMCs stimulated by serum. These effects of heparin 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 anticoagulant properties all partially inhibited VSMC proliferation predominantly by extending the G2/M phase of the cell cycle. Heparin also inhibited dedifferentiation of primary cultures of VSMCs. Heparin (< 100 micrograms.ml-1) acted extracellularly to release TGF beta from serum, which accounted for the effects of heparin on proliferation and differentiation.
authors.affiliation: Department of Biochemistry, University of Cambridge, United Kingdom.
authors.forename: D J
authors.initials: DJ
authors.lastname: Grainger
authors.forename: C M
authors.initials: CM
authors.lastname: Witchell
authors.forename: J V
authors.initials: JV
authors.lastname: Watson
authors.forename: J C
authors.initials: JC
authors.lastname: Metcalfe
authors.forename: P L
authors.initials: PL
authors.lastname: Weissberg
mesh_terms: D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D003470:Culture Media; D005434:Flow Cytometry; D006493:Heparin / Q000494:pharmacology*; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects*; D051381:Rats; D016212:Transforming Growth Factor beta / Q000097:blood*
chemical_list: D003470:Culture Media; D016212:Transforming Growth Factor beta; D006493:Heparin