Differentiation of coronary smooth muscle cells to a cell cycle-arrested hypertrophic growth status by a synthetic non-toxic heparin-mimicking compound

Abstract

Studies on the mode of action of basic fibroblast growth factor (bFGF) identified an essential role of heparan sulfate and heparin-like molecules in the formation of distinct bFGF-heparan sulfate–bFGF-receptor complexes that are required for bFGF-induced signal transduction. In coronary smooth muscle cells that express 6–8 ng bFGF mg −1 cell protein, the heparan sulfate chains of membrane-associated proteoheparan sulfate are implicated in bFGF signaling and thus are involved in the regulation of proliferation and differentiation of vascular smooth muscle cells. We studied the mode of action of a synthetic non-sulfated heparin-mimicking compound termed RG-13 577 (poly-4-hydroxyphenoxy acetic acid, Mr ∼5 kD) and found a dose-dependent antiproliferative effect that was characterized by a block of G 1/S-phase transition indicated by a marked (80%) reduction of [ 3H]thymidine incorporation at a concentration of 5 μg ml −1 RG-13 577. Cell cycle analysis showed a block of cell division in the G 1-phase. In response to RG-13 577 the cells were converted into a hypertrophic growth status within 72 h as judged from a doubling of the cellular protein content and measurement of cell and nucleus size. The increased cell protein content resulted from a de novo synthesis and was also associated with an increase in the incorporation of [ 35S]sulfate into cell-associated proteoglycans, including the proteoheparan sulfate coreceptor of bFGF. In contrast, the compound-induced G 1-phase arrest was associated with an extensive downregulation of the cellular and pericellular bFGF level. The reduced bFGF content was accompanied by downregulation of the bFGF signaling-involved protein kinase C-α and MAP kinase, abrogation of MAP kinase phosphorylation and overexpression of protein kinase C-γ. RG-13 577 failed to elicit apoptotic reactions at a concentration range of 0.5–10 μg ml −1 and its effect was reversible upon removal of the compound. It appears that RG-13 577 induces a phenotype transformation of coronary SMC into a metabolically active hypertrophic status that could promote repair processes after balloon angioplasty (PTCA) without stimulating cell proliferation. Development of non-toxic polyanionic compounds may provide an effective strategy to inhibit cell proliferation associated with restenosis following balloon angioplasty and coronary artery bypass surgery.