Modulation of extracellular matrix proteins and the influence of fucoidan on cell proliferation of smooth muscle cells

Abstract

l)otntrgk.~tm.s.st~ .I, D-4400 Miitister, F.R. G. Cellular glycoconjugates serve as specific informationbearing components involved in scveral biological functions such as cell adhesion, cell growth or cell differentiation. One group of molecules which perform such functions are proteoglycans. owing to their versatility and capacity for multiple interactions with other molecules. Recent studies have indicatcd that heparin or heparin-like molecules are potent inhibitors of smooth muscle cell proliferation in vivo and it1 vitm [ I, 2). During our investigations on the biological role and cellular organization of thrombospondin in arterial wall cells, we observed, in addition to heparin and heparan sulphatc, a strong influence of fucoidan, a sulphated fucopolysaccharide of the brown marine algae Fircirs vesicirlo.su.s, on this extracellular matrix protein. This prompted us to invcstigatc in more detail the intlucncc of this sulphated polysaccharide on several biological functions of smooth muscle cells. Porcine aortic smooth muscle cells were grown by the cxplant method and cultured as described previously 131. For cell proliferation studies, cells were sparsely plated in Dulbecco's modified Eagle's medium (DMEM) + 10% (v/v) fetal calf serum (FCS) and placed after 24 h in DMEM + 0.5% (v/v) FCS for 72 h. Cell growth was initiated by replacing this medium with DMEM+ 10% (v/v) FCS containing I pCi [3H]thymidine/ml in the presence or absence of fucoidan (Sigma, Munich, F.R.G.) or other polysaccharides. Inhibition of cell proliferation was calculated on the basis of [3H]thymidine incorporation into DNA. For the analysis of the influence of fucoidan on cellular and secreted proteins, confluent smooth muscle cells were pretreated with fucoidan (100 pg/ml) for 24 h and mctabolically labelled with [Yjlmethionine. Cellular and secreted proteins were precipitated with trichloroacetic acid ( 1 O'%, v/v) and scparated by SDS/PAGE (7.5'%, w/v) under reducing conditions. lmmunoprecipitation of radioactively labelled proteins was performed as previously dcscribed. using a polyclonal antibody raised against porcine platelet thrombospondin 14) and a polyclonal antibody of commercial source for immunoprccipitation of fibronectin. Fucoidan is capable of inhibiting smooth muscle cell proliferation with SO%, inhibition at a concentration of 5- I0 pglrnl. In contrast to fucoidan, hcparan sulphate from calf aortic tissue or dextran sulphate (5000 kDa) needed a five times higher concentration to achieve a similar antiproliferative effect. Heparin of commcrcial source was even less active ( I50 pg/ml). Sulphation of the native fucoidan molecule was found to be necessary for the expression of antiproliferative activity. Thc effect of fucoidan on smooth muscle ccll growth was connected with substantial altera