Objectives Diabetic patients continued to develop vascular complications despite achieving normal glucose level, suggesting metabolic memory which was associated with histone H3 lysine (H3K9) methylation through gene repression. Here we hypothesised that H3K9 di-methylation (H3K9me2) might be involved in vascular remodelling under diabetic condition; and histone demethylase KDM3A, an important regulator for H3K9me2, might play a key role in these pathologies.
Methods To generate diabetic model, male SD rats were fed with 60% fat diet for 4 weeks followed by intraperitoneal injection with 40mg/kg streptozotocin for another 2 weeks. The carotid arteries from diabetic rats (glucose 15-33 mM) were injured by balloon catheter to induced neointimal hyperplasia, and transfected with adenovirus encoding KDM3A gene or control adenovirus (Ad-GFP) for KDM3A overexpression. A KDM3A siRNA was delivered by lentivirus to down-regulate KDM3A level; the control lentivirus is Lenti-NC. Vascular smooth muscle cells (VSMCs) were isolated from thoracic aorta of SD rats. Microarray analysis was performed to identify altered gene profile in carotid arteries after balloon injury. Chip assay was used to evaluate the H3K9me2 level at the promoter region of certain genes.
Results H3K9me2 level was decreased by 51.3% in carotid arteries from diabetic rats compared to normal rats, which was associated with 1.32-fold increase of KDM3A mRNA during neointimal formation. After 28-day balloon injury, the area of neointimal to media (I/M) ratio was further enhanced by KDM3A overexpression compared with Ad-GFP control group (1.58 ± 0.30 vs. 0.86 ± 0.13, p < 0.05). However, KDM3A knockdown significantly decreased I/M ratio as compared to Lenti-NC (0.64 ± 0.18 vs. 1.45 ± 0.19, p < 0.05). Microarray analysis revealed ROCK2- and AGTR1-dependent vascular remodelling pathways was mediated by KDM3A after balloon injury at 7 days. KDM3A, ROCK2 and AGTR1 were up-regulated 4.17-fold, 1.57-fold, 4.14-fold, respectively, in KDM3A overexpression group as compared to Ad-GFP; whereas KDM3A knockdown reduced the expression of these 3 genes by 42.5%, 21.3%, 28.5%, respectively (p < 0.05 vs. Lenti-NC). The KDM3A effects were further verified in VSMCs. HG (30mM)- and Ang II (1μM)-stimulated proliferation and migration of VSMCs was further enhanced in KDM3A overexpression group. However, the PCNA level, a proliferation index, was markedly blunted by KDM3A knockdown compared with Lenti-NC group (0.20 ± 0.02 vs. 0.15 ± 0.03, p < 0.05). Similar inhibitory effect of migration was also observed after KDM3A knockdown. Western blot indicated HG-induced expression of ROCK2 and downstream target p-MYPT-1 was further enhanced by KDM3A overexpression, but oppositely abolished by 41.4% and 31.9%, respectively, after knocking-down KDM3A (p < 0.05 vs. Lenti-NC). AGTR1 levels was also pronouncedly elevated after KDM3A overexpression, but blocked by 52.8% after KDM3A knockdown under 1μM Ang II stimulation (p < 0.05 vs. Lenti-NC); the downstream target ERK1/2 activity changed in the same tendency. More importantly, KDM3A knockdown increases H3K9m2 in the proximal promoter region of ROCK2 and AGTR1 genes in cultured VSMCs by Chip assay (p < 0.05 vs. Lenti-NC).
Conclusions H3K9me2 may be a major underlying mechanism for vascular remolding in diabetes, and histone demethylase KDM3A may represent a promising therapeutic approach for the prevention of coronary artery disease combined with diabetes.