Figure 1.

Heparanase knock down and overexpression modulates endothelial inhibition of vascular smooth muscle cell (vSMC) proliferation. (a) Expression of heparanase identified by Western blotting in cells transfected with a vector expressing siRNA specific for mammalian heparanase (siHPA). Four siHPA were evaluated and the one that most effectively reduced heparanase was used in all subsequent experiments. A scrambled sequence (pRS) served as control. (b) Immunocytochemically identified heparanase (Bar = 10 μm) in cells transfected with pHPA, siHPA or pRS. (c) Quantification of cellular staining for heparanase in transfected cells. Values are based on measurement of intensity of the average of ten cells from five independent experiments (n=5). ^*Statistically significant difference from control group (p < 0.05). (d) Measurement of heparanase’s effect on total glycosaminoglycans and heparan sulfate in endothelial cells. Endothelial cell HSPGs were metabolically labeled using ³H-glucosamine. Cell lysates and conditioned media were then analyzed for glycosaminoglycan content using ion exchange chromatography. Heparanase overexpression caused a reduction in cellular and soluble HSPGs in endothelial cells. Conversely, reduction of heparanase expression in endothelial cells by siRNA led to an increase in both cellular and soluble HSPGs. Plots are the average of three independent analyses. Black line = pRS; red line = pHPA; blue line = siHPA. (e) Heparanase regulates endothelial inhibition of vSMC proliferation. Conditioned media from endothelial cells that were transfected with pcDNA, pHPA, siHPA or pRS vectors was harvested and applied to cultures of vSMCs. Bars represent the relative growth of vSMC in the presence of conditioned media of endothelial cells transfected with the indicated vectors as measured by 3H-thymidine incorporation. ^*Statistically significant difference between groups (p < 0.05).