Study On Mechanism Of VEGF Increasing Permeability Of Endothelial Cells To LDL

Vascular endothelial growth factor (VEGF) was initially identified as a vascular permeability factor. Afterwards study in this issue find that VEGF can enhance vascular permeability to a variety of biological molecules. Secretion of VEGF from foam cells and accumulation in human atherosclerotic lesions initiate the speculation on the probably involvement of VEGF in atherosclerosis(AS) formation and progress, but the precisely role of VEGF on AS is so far unknown. The experiments aim to study the effect of VEGF on vascular permeability to LDL which intimately correlate with AS in cultured endothelial cells(EC) monolayer and in co-cultured bilayer with smooth muscle cells(SMC) to investigate the probable role of VEGF on AS formation and progress.Endothelial extracellular matrix (ECM) play a important role in modulation of vascular permeability. Type IV collagen is a unique endothelial-specific ECM and matrix metalloproteinase-2 (MMP-2) is one of two special Type IV collagenase, which can effectively interfere in vascular permeability (VP). In our experiments we also aim to study the effect of VEGF on the production and activity of MMP-2 in EC to investigate the probable molecular mechanism that ECM component degradation resulted from alternation of MMP-2 production and activity involves in VP promoted by VEGF.Methods:LDL was conjugated to fluorescein isothiocyanate (FITC) and the fluoro -spectrophotometry was established to quantify the concentration of FITC-LDL; In cultured bovine endothelial cells (BAEC) monolayer and in co-cultured bilayer with bovine smooth muscle cells (BASMC), the effect of VEGF on vascular permeability was studied. Antagonistic action of drugs on VEGF was also studied in this model; Immunohistochemistry was applied to detect the expression of MMP-2 in endothelial cells; ELISA method was used to quantified the secretion of MMP-2 in culture supernatant from EC; MMP-2 activity in culture supernatant from EC was assayed by gelatin zymography method; The expression of MMP-2 mRNA in EC was measuredby Northern Blot analysis; The expression of membrane type metalloproteinase-1/MT1-MMP mRNA was measured by RT-PCR and Northern Blot analysis; 3H-L-proline incorporation was used to determine the collagen synthesis and radio-labeled base membrane with 3H-L-proline was used to detect degradation of collagen in base membrane.Results:(1) The establishment of conjugation LDL to FITC and quantification of FITC-LDL by fluorospectrophotometry. Our results show that the method have the advantage of simplification, feasibility, sensitivity, a perfect standard curve and with negligible perturbation from the culture medium;(2) The establishment of permeability assay in cultured BAEC monolayer or in co-cultured bilayer with BASMC based on PET membrane. BAEC were plated to PET membrane or simultaneously BAEC and BASMC were cultured on two profiles of PET membrane. The confluent monolayer of BAEC were identified with F-actin staining and observed under inverted phase-contrast microscope, further testified by assay of basal permeability, and the well without confluence was rejected. Meantime the directly action of FITC-LDL on confluent BAEC monolayer and on BAEC viability were also studied. The results show that at the highest concentration of 125μg/ml and the longest course of 6 hours in our experiment, no obvious injuries on BAEC monolayer and BAEC viability was observed;(3) VEGF dose-dependently increased endothelial monolayer permeability to FITC-LDL. Significant effect was observed at concentration of 50μg.L-1, the permeability ratio risen from 19.07±1.13% to 22.46±1.59%(P<0.05); the effect become more significant at concentration of 100μg.L-1, the permeability ratio reached to 27.13±2.27%(P<0.01); no more potent effect was observed at higher concentration of 200μg.L-1.(4) Imperatorin (10 and 100 μmol.L~{-1}) significantly inhibited the effect of 100μg.L~{-1} VEGF, the permeability ratio decreased from 27.04±2.20% to 22.78±1.37%(P<0.05) and 20.83±1.31%(P<0.01) respect...