Heparin-induced endothelial cell cytoskeletal reorganization: a potential mechanism for vascular relaxation

We have previously shown that heparin given subcutaneously on a daily basis lowers blood pressure in hypertensive rat models, and that this blood pressure lowering effect is endothelium-dependent. The present study describes the effects of heparin on endothelial cell (EC) apical surface structures and cytoskeletal elements, namely, actin and vimentin as well as EC proliferative activity. The EC line (CRL 1998) was cultured, treated with different concentrations of heparin (0, 50, 100, 500 U/ml) for 4, 24 or 48 hours, and fixed for scanning electron microscopy (SEM), and immunofluorescence microscopy (IFM) studies. Enzyme-linked immunosorbent assays (ELISA) and flow cytometric analysis were performed on EC monolayers treated with different concentrations of heparin for quantitative detection of actin and vimentin. By SEM study the cell surface showed generalized smoothing as a result of blunting of surface microvilli with increasing time of exposure and dosage of heparin. By IFM study, the detectable actin signal within ECs became progressively reduced in both its cellular distribution and the apparent number of cells that remained reactive. By 48 hr/500 U heparin, the actin signal was almost undetectable. Vimentin showed a moderate reduction in the cellular distribution 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. However, vimentin was significantly reduced only 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 flow cytometric findings were virtually identical to the ELISA data for actin and vimentin. These qualitative and quantitative changes in actin 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 observed intensity in heparin treated cells with substantially fewer cells being positive. After a 48 hour treatment with heparin (500 U/ml), most ECs displayed only dim labeling of the nucleolus. This finding is consistent with an antiproliferative effect. Overall, these findings are additive to our previous observations, and demonstrate that heparin causes EC cytoskeletal reorganization which is a potential mechanism for vascular relaxation.