Abstract 13625: High Glucose Enhances Vascular Responses to Thrombin via Protease-activated Receptor-4 Upregulation in Human Vascular Smooth Muscle Cells.
Diabetes is associated with vascular remodeling, inflammation and enhanced thrombin generation. Thrombin, in addition to its coagulatory actions, promotes vascular smooth muscle cell (SMC) mitogenesis and migration via protease-activated receptors (PAR)-1, PAR-3 and PAR−4. Here we investigated the effect of high glucose on PAR expression and cellular responsiveness to thrombin in human vascular SMC. Human saphenous vein SMC were incubated under normal (5.5 mmol/L) or high (25 mmol/L) glucose conditions. High glucose induced a rapid and sustained increase in PAR-4 mRNA (to 3.5±0.6 fold at 1.5h and to 2.7±0.4 fold at 96h; n=7, p<0.05) as determined by real-time PCR, while expression of PAR-1 and PAR-3 were unaffected. PAR-4 total protein levels, immunofluorescence and cell-surface expression were also increased at least 2 fold (n=5, p<0.05) over 48h treatment. Accordingly, high glucose pretreatment (48h) enhanced thrombin (3U/mL) induced intracellular calcium mobilization (to 4.2±0.18 fold of basal), SMC migration (to 5.5±1.2 fold) and TNFα transcription (to 2.6±0.40 fold) (all n≥3; p<0.05). High glucose also enhanced cellular responses to peptide agonist of PAR-4 (AYPGKF) but not of PAR-1 (TFLLRN). PAR-4 siRNA abolished the enhancement of thrombin stimulated SMC migration and TNFα transcription in cells exposed to high glucose. Exposure to high glucose increased PAR-4 luciferase activity by 3-fold (n=4; p<0.05) over 24h. Accordingly, high glucose regulation of PAR-4 was prevented by knockdown of protein kinase C-δ or NFκB. High glucose increased the nuclear transition of NFκB (maximal at 3h) followed by its PKC-dependent binding to the PAR-4 promoter in a chromatin immunoprecipitation assay. Immunohistochemistry of human diabetic atherosclerotic plaques or preliminary in-situ hybridization on human diabetic veins confirmed the expression of PAR-4 in diabetic vessels. In conclusion, these data show that high glucose treatment enhances cellular responses to thrombin through selective upregulation of PAR-4 in vascular SMC, which occurs via PKCδ and NFκB dependent signaling. The findings suggest that high glucose enhanced thrombin responses via PAR-4 upregulation may play an important role in diabetes associated vascular pathologies.
- © 2010 by American Heart Association, Inc.