HEPARIN-INDUCED ENDOTHELIAL-CELL CYTOSKELETAL REORGANIZATION - A POTENTIAL MECHANISM FOR VASCULAR RELAXATION

A potential mechanism for vascular relaxation. We have previously show n that heparin given subcutaneously on a daily basis lowers blood pres sure in hypertensive rat models, and that this blood pressure lowering effect is endothelium-dependent. The present study describes the effe cts of heparin on endothelial cell (EC) apical surface structures and cytoskeletal elements, namely, actin and vimentin as well as EC prolif erative activity. The EC line (CRL 1998) was cultured, treated with di fferent concentrations of heparin (0, 50, 100, 500 U/ml) for 4, 24 or 48 hours, and fixed for scanning electron microscopy (SEM), and immuno fluorescence microscopy (IFM) studies. Enzyme-linked immunosorbent ass ays (ELISA) and flow cytometric analysis were performed on EC monolaye rs treated with different concentrations of heparin for quantitative d etection of actin and vimentin. By SEM study the cell surface showed, generalized smoothing as a result of blunting of surface microvilli wi th increasing time of exposure and dosage of heparin. By IFM study, th e detectable actin signal within ECs became progressively reduced in b oth its cellular distribution and the apparent number of cells that re mained reactive. By 48 hr/500 U heparin, the actin signal was almost u ndetectable. Vimentin showed a moderate reduction in the cellular dist ribution of labeling. Quantitatively, actin was significantly reduced after the 24 hour treatment with a higher dose of heparin (500 U/ml), from a baseline optical density (OD) of 1.12 +/- 0.060 to 0.866 +/- 0. 008 (P < 0.0027). After 48 hours of treatment at both 100 U/ml and 500 U/ml heparin, actin was significantly reduced from a baseline OD of 1 .347 +/- 0.063 to 1.090 +/- 0.039 (P < 0.0039) and 0.844 +/- 0.074 (P < 0.008), respectively. How ever, vimentin was significantly reduced o nly after 48 hours of treatment with a high dose of heparin (500 U/ml) , from baseline OD 1.82 +/- 0.052 to 1.41 +/- 0.004 (P < 0.002). The f low cytometric findings were virtually identical to the ELISA data for actin and vimentin. These qualitative and quantitative changes in act in and vimentin are consistent with apparent smoothing and relaxation of the EC's apical surface. Labeling with the cell cycle marker MIB-1 (monoclonal antibody Ki-67), showed a progressive reduction in the obs erved intensity in heparin treated cells with substantially fewer cell s being positive. After a 48 hour treatment with heparin (500 U/ml), m ost ECs displayed only dim labeling of the nucleolus. This finding is consistent with an antiproliferative effect. Overall, these findings a re additive to our previous observations, and demonstrate that heparin causes EC cytoskeletal reorganization which is a potential mechanism for vascular relaxation.