HDAC9 over-expression increases vascular ET-1 and TLR4 mRNA abundance

Histone deactylases (HDACs) are classical epigenetic modifiers that deacetylate lysine residues on histones but also deacetylate non‐histone proteins. Previous studies suggest HDAC9 is a causative and disease‐relevant gene in stroke and coronary artery disease. Single nucleotide polymorphisms in the HDAC9 gene are associated with an increased risk of cardiovascular disease. As such, inhibitors of HDAC9 are suggested to be vasoprotective. However, the HDAC9 dependent mechanisms leading to vascular dysfunction remain elusive. Endothelial dysfunction is an early initiator of vascular disease. We previously found that vascular tissues with increased HDAC9 expression also showed increased endothelin‐1 (ET‐1) and toll‐like receptor 4 (TLR4). Thus, we hypothesized that HDAC9 directly increases ET‐1 and/or TLR4 expression in vascular tissue. Thoracic aortae and renal vessels were isolated from adult (12 weeks, male and female) HDAC9 over‐expressing mice (HDAC9‐OE) to measure ET‐1 and TLR4 expression. In general, HDAC9‐OE mice showed a 4‐fold increase in HDAC9 expression in the aorta and a 3‐fold upregulation in the renal vessels. RNA expression studies from aortic tissue revealed that both ET‐1 and TLR4 were increased in HDAC9‐OE mice compared to controls (relative mRNA; ET‐1: control 1.00 ±0.99 vs HDAC9‐OE 1.89 ±0.31; TLR4: control 1.00 ±0.78 vs HDAC9‐OE 10.89 ±4.59, n=2–10). Further, renal vessels showed a 5‐fold increase in ET‐1 expression (relative mRNA 1.00 ±0.87 vs 5.53 ±0.64, n=2–10) whereas TLR4 expression was unchanged in HDAC9‐OE mice compared to controls (relative mRNA 1.00 ±0.43 vs 0.96 ±0.18, n=2–10). Previous in vitro studies showed that over‐expression of HDAC9 in transfected mouse aortic endothelial cells upregulates ET‐1 expression supporting our in vivo results. In contrast, plasma of HDAC9‐OE mice showed no difference in circulating ET‐1 levels (control 0.51 ±0.1065 pg/ml vs HDAC9‐OE 0.40 ±0.02655 pg/ml, n=2–10). Therefore, our results indicate that HDAC9 over‐expression in the aorta induces expression of ET‐1 and TLR4 however in renal vessels HDAC9 over‐expression appears to only regulate ET‐1 and not TLR4. The HDAC9 dependent molecular mechanisms leading to ET‐1 expression may be tissue specific and will be investigated further.