Abstract
Due to the injury to the artery from PCI or surgical stenting, neointimal hyperplasia and restenosis are induced by the secreted growth factors and cytokines. These growth factors and cytokines promote phenotype switch in VSMCs which results in inhibition of VSMC differentiation, induction of cell proliferation and migration. Restenosis, which has an incidence rate of about 10% in using DES, leads to ischemia and myocardial infarction. Recently, different epigenetic mechanisms have been identified which regulate different cellular pathways and enhance the pathological progression of the disease.
To identify different molecular mechanisms and the effector proteins involved in promoting vascular restenosis and intimal hyperplasia, we carried out pre-clinical studies using Yucatan microswine as large mammals that were fed with atherogenic diet. Angioplasty was performed in the LCX. After completion of the study, tissues were harvested for immunofluorescence and immunohistochemical staining to check for in vivo expression of epigenetic and downstream mediators. To identify the underlying molecular mechanisms, primary VSMCs were isolated from the coronary arteries and cultured for in vitro studies. After transfecting the cultured VSMCs with siRNA specific for HDAC2, HDAC10, and SOCS3, the cells were stimulated with IGF-1 and TNF-α together, or with PDGF-BB. The primary VSMCs were also transfected with the microRNA miR-490-3p. The transfected cells were treated with DNMT1 inhibitor (5-Aza-Cytidine) and HDAC2 inhibitor (Romidepsin). MTT and scratch wound assays were used to measure VSMCs proliferation and migration. Finally, the expression of epigenetic markers and downstream regulatory proteins were detected by western blot analysis.
Results show increased expression of HDAC2 during intimal hyperplasia. Subsequent inhibition of HDAC2 expression was found to result in DNMT1 inhibition, which presumably was involved in promoting VSMC proliferation, migration and phenotype switch. HDAC10 and HDAC2 were found to co-express during intimal hyperplasia, and inhibition of HDAC10 was found to represses the expression of HDAC2 and DNMT1in VSMCs. Further, SOCS3 expression was found to inhibit the cell proliferation enhancer, Galectin-3, through HDAC10 and DNMT1 inhibition.
Future studies using HDAC10 and HDAC2 double knockout mice with carotid artery ligation will be helpful in unraveling the molecular mechanisms that regulate HDAC10-HDAC2-DNMT1 pathway associated with the development of neointimal hyperplasia. Moreover, co-localization studies and protein immunoprecipitation assays would prove possible molecular interactions between HDAC10 and HDAC2. Development of HDAC10 specific inhibitor would help in identifying the role of HDAC10 in neointimal hyperplasia and restenosis.