Trichostatin A Modulates Angiotensin II-induced Vasoconstriction and Blood Pressure Via Inhibition of p66shc Activation

Abstract

Histone deacetylase (HDAC) has been recognized as a potentially useful therapeutic target for cardiovascular disorders. However, the effect of the HDAC inhibitor, trichostatin A (TSA), on vasoreactivity and hypertension remains unknown. We performed aortic coarctation at the inter-renal level in rats in order to create a hypertensive rat model. Hypertension induced by abdominal aortic coarctation was significantly suppressed by chronic treatment with TSA (0.5 mg/kg/day for 7 days). Nicotinamide adenine dinucleotide phosphate-driven reactive oxygen species production was also reduced in the aortas of TSA-treated aortic coarctation rats. The vasoconstriction induced by angiotensin II (Ang II, 100 nM) was inhibited by TSA in both endothelium-intact and endothelium-denuded rat aortas, suggesting that TSA has mainly acted in vascular smooth muscle cells (VSMCs). In cultured rat aortic VSMCs, Ang II increased p66shc phosphorylation, which was inhibited by the Ang II receptor type I (AT1R) inhibitor, valsartan (10 µM), but not by the AT2R inhibitor, PD123319. TSA (1~10 µM) inhibited Ang II-induced p66shc phosphorylation in VSMCs and in HEK293T cells expressing AT1R. Taken together, these results suggest that TSA treatment inhibited vasoconstriction and hypertension via inhibition of Ang II-induced phosphorylation of p66shc through AT1R.

Keywords: Angiotensin II; Angiotensin receptor type I; Hypertension; Trichostatin A; p66shc.

Fig. 1. Chronic treatment of trichostatin A (TSA) inhibited aortic coarctation-induced hypertension. (A) Effect of chronic TSA treatment on blood pressure in sham-operated and aortic coarctation rats. Animals were treated for 7 days by subcutaneously administration of vehicle (DMSO) or TSA (0.5 mg/kg/day). Bars represent the mean±S.E.M. (n=4 or 5) for systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial pressure (MAP), and pulse pressure (PP). (B) Superoxide production in aortas of the abdominal aortic coarctation and sham-operated rats was determined by a lucigenin chemiluminescence assay. Bars represent the mean±S.E.M. (n=5 or 6). Basal and nicotinamide adenine dinucleotide phosphate (NADPH)-driven superoxide were measured in the aortas of the rats. NADPH (0.1 mM) was added to measure NADPH oxidase activity. Sham, sham-operated group; AC, aortic coarctation; TSA, TSA-treated sham-operated group; AC+TSA, TSA-treated aortic coarctation group. ^*p<0.05 (vs. sham group). ^#p<0.05 (vs. aortic coarctation group).

Fig. 2. Trichostatin A (TSA) inhibited angiotensin II (Ang II)-induced vasoconstriction in rat aorta. Vasoconstriction was evoked by Ang II treatment at doses between 1 and 100 nM in aortic rings with intact (A) or rubbed endothelium (B). Contraction was expressed as a percentage of the pre-contracted tension obtained with a high K⁺ (60 mM) solution. Each bar represents the mean±S.E.M. (n=6). Control: DMSO vehicle. ^*p<0.05 (vs. control), ^**p< 0.01 (vs. control).

Fig. 3. Angiotensin II evoked p66shc phosphorylation in vascular smooth muscle cells. Cells were treated first with DMSO (control) or the indicated concentrations of valsartan (angiotensin II receptor type 1 inhibitor) (A), or PD123319 (angiotensin II receptor type 2 inhibitor) (B) for 30 min, and subsequently with 100 nM Ang II. Cell lysates were immunoblotted for phospho-p66shc, Shc, and actin. Densitometric scanning was performed to quantify the phosphop66shc/p66shc levels. Bars represent the mean±S.E.M. (n=4). ^**p<0.01 (vs. control), ^#p<0.01 (vs. only angiotensin II-treated cells).

Fig. 4. Trichostatin A (TSA) inhibited angiotensin II-induced p66shc phosphorylation in vascular smooth muscle cells. Cells were exposed to 100 nM angiotensin II for 30 min, in the presence or absence of TSA. Western blotting was performed for phospho-p66shc, Shc, and actin. Bars represent the mean phospho-p66shc/p66shc levels±S.E.M. (n=3). ^**p<0.01 (vs. control), ^#p<0.01 (vs. only angiotensin II-treated cells).

Fig. 5. Trichostatin A (TSA) inhibited angiotensin II-induced p66shc phosphorylation in HEK293T cells, ectopically expressing angiotensin II receptor type 1. (A) Fluorescence images of EGFP-tagged angiotensin II receptor type 1 or type 2 (×400 magnification). HEK293T cells were transfected with EGFP (upper), EGFP-tagged angiotensin II receptor type 1 (middle) or type 2 (lower). The plasma membrane was stained using CellMask Deep Red solution. (B) Cells expressing angiotensin II receptor type 1 were pretreated with either DMSO (control) or the indicated concentrations of TSA for 30 min. The cells were then either left untreated (DMSO), treated with TSA alone (controls), or exposed to 100 nM angiotensin II in the presence of TSA for 30 min. Western blotting was performed for phospho-p66shc and Shc. Bars represent the mean phospho-p66shc/p66shc levels±S.E.M. (n=3). ^**p<0.01 (vs. control), ^*p<0.05 (vs. control), ^#p<0.01 (vs. only angiotensin II-treated cells).