M. M. Kassem1,2, D. Brusch1,2, K. G. Maier1,2, V. Gahtan1,2 1State University Of New York Upstate Medical University,Vascular And Endovascular Services,Syracuse, NY, USA 2Syracuse VA Medical Center,Syracuse, NY, USA
Introduction: Thrombospondins (TSPs) are matricellular glycoproteins expressed in response to vascular injury and are mediators of arterial remodeling. While TSP-1 and TSP-2 induce vascular smooth muscle cell (VSMC) proliferation, TSP-5 does not. However, all three TSPs promote VSMC migration. Previously we showed that TSP-1,-2 and -5 alter gene expression associated with arterial remodeling in VSMCs. The current study assesses the expression of these genes at the protein level. Our previous work has shown TSP-1, -2 and -5 all downregulate IL-8 gene expression while they increase ANGPTL-4 gene expression. Furthermore, both TSP-1 and -2 upregulate the PDGF-BB gene, while only TSP-1 increased TGF β1 gene expression. We hypothesized that protein expression of by all three TSPs would mirror the effects on the genes in VSMCs.
Methods: Quiescent Human VSMCs were exposed to TSP-1,-2 or -5 (20 μg/ml) or serum free media (SFM), for 24 hours. Cell lysates were used to determine TSP-1, -2 and -5 expression. The culture media was used to detect secreted proteins (TGF-β1, PDGF-BB, ANGPTL-4, IL-8). TSP-1, -2, -5 and ANGPTL-4 protein expression were measured by Western blot. IL-8, PDGF-BB and TGF-β1 protein secretion were determined by ELISA. Statistical analysis was performed by ANOVA and t-test. P< 0.05 was considered significant.
Results: The table lists proteins for each treatment as upregulated, downregulated or went from undetectable to detectable. TSP-2 was found to be a significant self-promoter and it upregulates ANGPTL-4 25 fold. While the effect of TSP-1 on autoregulation was inconclusive, TSP-1 upregulates secretion of ANGPTL-4, PDGF-BB and TGF-β1. TSP-5 wasn’t detected in TSP-1, -2 and SFM treated cells.
Conclusion: Substantial differences exist in VSMC protein expression patterns following exposure to the thrombospondins. This study confirms our previous gene expression data in VSMCs treated with TSP-1,-2 and -5. These findings suggest that TSP-1 promotes vascular remodeling, in part, by increasing ANGPTL-4, PDGF-BB and TGF-β1 secretion. Additionally, TSP-2 upregulates TSP-1 and -2 expression and increases secretion of PDGF-BB and ANGPTL-4. TSP-5 may be protective against arterial remodeling as it up regulates its own expression and does not upregulate PDGF-BB. These findings increase our understanding of the mechanisms by which TSPs differentially regulate VSMC function.
