Abstract
Chitosan and heparin-based hydrogels possess potent properties such as air permeability, absorption and water retention. Our group has previously reported heparin-loaded, freeze-dried porous hydrogels, using chitosan/PVA and variable concentrations of PCL. Significant angiogenic effect of these hydrogels was observed. In this follow-up study, we have investigated the underlying molecular mechanisms of increased angiogenesis due to these heparin-releasing chitosan-based hydrogels, in human umbilical vein endothelial cells (HUVECs). Cells were cultured in complete ECM media and supplemented with 10% FBS and 1% Penicillin-Streptomycin. HUVECs were treated with heparin-releasing chitosan-based hydrogels in different concentrations (H4, H8 and H16) for 1, 3 and 7 days. RNA was extracted using trizol and quantified by nanodrop. Primers were designed for the target genes including VEGF, VEGFR, Ap-1, BCL-2, HIF-1, BAX, IL-1 and TNF-α. Gene expression analysis were performed using PCR. Gel electrophoresis was used to visualize results. MTT and DCFH-DA assays were performed to check cytotoxicity and reactive oxygen species (ROS) respectively. Hydrogels H8, had significantly high effect on the expression of VEGF and VEGFR, as compared to H4 and H16. At day 1, most of the target genes showed higher expression. HIF-1 showed high expression at day one, with a gradual decrease at day 3 and 4. We observed no expression of AP-1 with hydrogels H4, H16 and control, but a high expression with hydrogels H8, at day 1 and 3. BCL-2, IL-1and TNF-α also revealed high expression with hydrogels H8. A low expression of BAX was observed in all cases. Our study demonstrates high expression of genes with H4 probably due to high concentration of heparin. Similarly, at day 1, the high expression is attributed to the fact that these hydrogels release maximum heparin in the first 24 h.