Selective inhibition of histone deacetylase 8 improves vascular hypertrophy, relaxation, and inflammation in angiotensin II hypertensive mice

Abstract

Background: The dysregulation of histone deacetylase (HDAC) protein expression or its enzyme activity is implicated in a variety of diseases. Cardiac HDAC6 and HDAC8 enzyme activity induced by deoxycorticosterone acetate (DOCA) hypertension was attenuated by sodium valproate, a pan-HDAC inhibitor. However, the HDAC6-selective inhibitor, tubastatin A, did not attenuate angiotensin II-induced hypertension. The purpose of this study was to investigate whether PCI34051, an HDAC8-selective inhibitor, can modulate angiotensin II-induced hypertension and its regulatory mechanism.

Methods: An angiotensin II-regulated mouse model was used in this study. Animals received vehicle or PCI34051 (3 mg·kg - ¹·day- ¹) via intraperitoneal injection. Systolic blood pressure was measured by the tail-cuff method. Blood vessel thickness was measured following hematoxylin and eosin staining, VCAM-1 immunohistochemistry was performed in the aortas, and mRNA expression of renin-angiotensin system components, inflammation markers, and NADPH oxidase (Nox) was determined by RT-PCR. The effect of PCI34051 on vasorelaxation was studied in rat aortic rings, and its effect on nitric oxide (NO) production was determined using DAF-FM DA, a fluorescent dye, in human umbilical vascular endothelial cells (HUVECs).

Results: PCI34051 administration reduced systolic blood pressure via downregulation of angiotensin II receptor type 1 (AT1) mRNA expression. PCI34051 treatment attenuated vascular hypertrophy by decreasing E2F3 and GATA6 mRNA expression. Vascular relaxation after PCI34051 treatment was more dependent on vascular endothelial cells and it was blocked by an NO synthase (NOS) inhibitor. In addition, NO production increased in HUVECs after PCI34051 treatment; this was decreased by the NOS inhibitor. The expression of inflammatory molecules and adhesion molecules VCAM-1 and ICAM-1 decreased in the aortas of angiotensin II-infused mice after PCI34051 administration. However, PCI34051 did not affect Nox or its regulatory subunits.

Conclusions: PCI34051 lowered high blood pressure through modulation of arterial remodeling, vasoconstriction, and inflammation in an angiotensin II-induced hypertension model. We suggest that HDAC8 could be a potential therapeutic target for hypertension.

Keywords: Arterial remodeling; Hypertension; Inflammation; PCI34051; Vascular relaxation.

Fig. 1

PCI34051 decreases blood pressure and AT1 mRNA expression in Ang II-infused mice. a Representative western blot images of HDAC8. VSMCs were treated with vehicle or PCI34051 at different concentrations for 24 h. b HDAC8 protein was quantified using densitometry. * P < 0.05 versus vehicle-treated VSMCs. c Systolic blood pressures in the experimental groups at 14th day after Ang II infusion. *** P < 0.001 versus sham group; ## P< 0.01 versus Ang II-infused group. d and e Aortic AT1 (d) and ACE1 (e) mRNA expression levels were normalized to GAPDH and relative amounts were calculated. Data are presented as mean ± SEM (n = 8 per group). *** P < 0.001 versus sham-treated group; # P < 0.05 versus Ang II-infused group; NS indicates not significant

Fig. 2

PCI34051 decreases vascular hypertrophy and expression of cell growth-related genes in Ang II-infused mice. a Representative images of H&E-stained aorta sections. Scale bar = 50 μm. b Aortic wall thickness was measured using NIS Elements Software (n = 8 per group). *** P < 0.001 versus sham group; ### P < 0.001 versus Ang II-infused group. E2F3 (c) and GATA6 (d) mRNA expression levels were normalized to GAPDH and relative amounts were calculated. Data are presented as mean ± SEM (n = 8 per group). * P < 0.05 and ** P < 0.01 versus sham-treated group; # P < 0.05 versus Ang II-infused group

Fig. 3

PCI34051 increases vascular relaxation in rat aortic rings and NO production in HUVECs. a Relaxation of aortic rings in response to PCI34051 was assessed in endothelium-intact and endothelium-denuded aortas. Relaxation is expressed as the percentage of the maximal contractile response to U46619. Data are presented as mean ± SEM (n = 4 per group). b Endothelium-intact rat aortic rings were pretreated with N^G-nitro-L-arginine methyl ester (L- NAME, 10 μM or 100 μM) or vehicle for 30 min. When vascular contractions induced by U46619 (30 nM) reached a plateau, PCI34051 was added cumulatively to elicit relaxation. Data are presented as mean ± SEM (n = 4 per group). c HUVECs were treated with PCI34051 (1 μM) in the presence or absence of L-NAME (250 μM) for 24 h and then labeled with a fluorescent NO indicator, DAF-FM DA (2.5 μM), for 30 min. Three independent experiments were conducted and representative images are shown. Scale bar = 100 μm

Fig. 4

PCI34051 suppresses proinflammatory cytokines in aortic tissues of Ang II- infused mice TNF-α (a), IL-1β (b), MCP-1 (c), iNOS (d), IL-6 (e), and Cox-2 (f) mRNA expression in the aorta were normalized to GAPDH, and the relative expression was quantified. Data are presented as mean ± SEM (n = 8 per group). * P < 0.05, ** P < 0.01, and *** P < 0.001 versus sham-treated group; # P < 0.05 versus Ang II-infused group; NS indicates not significant

Fig. 5

PCI34051 inhibits the expression of adhesion molecules in aortic tissues of Ang II-infused mice VCAM-1 (a) and ICAM-1 (b) aortic mRNA expression levels were normalized to GAPDH and relative amounts were calculated. Data are presented as mean ± SEM (n = 8 per group). ** P < 0.01 versus sham-treated group; # P < 0.05 versus Ang II-infused group. c VCAM-1 expression was assessed by immunohistochemistry. Arrows indicate the expression of VCAM-1 in the aorta (brown). Scale bar = 50 μm. The lower Ang II-infused group image shows a negative control.

Fig. 6

PCI34051 does not affect Nox isoforms or Nox subunits in Ang II-infused mice. Aortic mRNA expression levels of Nox1 (a), Nox2 (b), and Nox4 (c), and Nox subunits p22phox (d) and p47phox (e), were normalized to GAPDH and relative amounts were calculated. Data are presented as mean ± SEM (n = 8 per group). * P < 0.05 and ** P < 0.01 versus sham-treated group; NS indicates not significant