Epigenetic regulation of aortic remodeling in hyperhomocysteinemia

Abstract

Hyperhomocysteinemia (HHcy) is prevalent in patients with hypertension and is an independent risk factor for aortic pathologies. HHcy is known to cause an imbalance between matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs), leading to the accumulation of collagen in the aorta and resulting in stiffness and development of hypertension. Although the exact mechanism of extracellular matrix (ECM) remodeling is unclear, emerging evidence implicates epigenetic regulation involving DNA methylation. Our purpose was to investigate whether 5-aza-2'-deoxycytidine (Aza), a DNA methyltransferase (DNMT1) inhibitor, reduces high blood pressure (BP) by regulating aortic ECM remodeling in HHcy. Wild-type and cystathionine β-synthase (CBS)(+/-) HHcy mice were treated with Aza (0.5 mg/kg body weight). In HHcy mice, Aza treatment normalized the plasma homocysteine (Hcy) level and BP. Thoracic and abdominal aorta ultrasound revealed a reduction in the resistive index and wall-to-lumen ratio. Vascular response to phenylephrine, acetylcholine, and sodium nitroprusside improved after Aza in HHcy mice. Histology showed a marked reduction in collagen deposition in the aorta. Aza treatment decreased the expression of DNMT1, MMP9, TIMP1, and S-adenosyl homocysteine hydrolase (SAHH) and upregulated methylene tetrahydrofolate reductase (MTHFR). We conclude that reduction of DNA methylation by Aza in HHcy reduces adverse aortic remodeling to mitigate hypertension.

Keywords: 5-aza-2′-deoxycytidine; DNA methylation; extracellular matrix.

Figure 1.

Physiological parameters. A, B) Body (A) and heart (B) weights of 10-wk-old male WT and CBS^+/− mice (n=6). C) Plasma Hcy levels from all groups (n=4). *P < 0.05 vs. WT and WT + Aza; ^†P < 0. 05 vs. CBS.

Figure 2.

BP was measured by the tail cuff method. Line graphs represent systolic BP (A), diastolic BP (B), and mean BP (C). Values are expressed as means ± se (n=4). *P < 0.05 vs. WT and WT + Aza; ^†P < 0.05 vs. CBS.

Figure 3.

A) B-mode ultrasound image of ascending aorta. Wall thickness (mm) and lumen diameter (mm) were measured by Vevo 2100 (Visual Sonics Vevo Ultra Imaging System). B) Wall-to-lumen ratio of the ascending aorta. C) Pulse wave mode images of ascending aorta. D) RI provides an estimate of the resistance offered by the ascending aorta and can be calculated as (PSV − EDV)/EDV. Bars represent means ± sem (n=4). *P < 0.05 vs. WT and WT + Aza; ^†P < 0.05 vs. CBS.

Figure 4.

A) B-mode ultrasound image of abdominal aorta. Lumen diameter (mm) was measured by Vevo 2100 (Visual Sonics Vevo Ultra Imaging System). B) Lumen diameter of the abdominal aorta. C) Pulse wave mode images of abdominal aorta. D) RI provides an estimate of the resistance offered by the abdominal aorta and can be calculated as (PSV − EDV)/EDV. Bars represent means ± sem (n=4). *P < 0.05 vs. WT and WT + Aza; ^†P < 0.05 vs. CBS.

Figure 5.

Aorta was extracted from experimental mice (WT, WT+Aza, CBS, and CBS+Aza) according to the protocol described in Materials and Methods. Aortic rings were mounted in a myobath containing Krebs solution. The rings were treated with Phe (A) and Ach (B) in a dose-dependent manner and SNP (C), as described in Materials and Methods (means±se; n = 4). *P < 0.05 vs. WT and WT+Aza; ^†P < 0.05 vs. CBS.

Figure 6.

Mason trichrome staining. A) Collagen is stained dark blue. (B) Mean ± sem percentage intensity of blue staining in the aorta (n=4). Original image, ×20. *P < 0.05 vs. WT and WT + Aza; ^†P < 0.05 vs. CBS.

Figure 7.

A) Picrosirius red staining for type I and III collagen (yellow and green represent type I and type III collagen, respectively). B, C) Mean ± sem percentage intensity of yellow (B) and green (C) in aorta. Original image, ×20. *P < 0.05 vs. WT and WT + Aza; ^†P < 0.05 vs. CBS.

Figure 8.

Immunohistochemistry. A, C) Protein expression of SAHH, Hcy, and MTHFR (A) and MMP9 and TIMP1 (C) was measured. Expression of MTHFR and SAHH is shown as green fluorescence; Hcy, MMP9, and TIMP1 as red fluorescence. B, D) Bar graph representations of the immunohistochemistry images. The y axis represents the percentage change in mean ± sem intensity (n=4). Original image: Hcy, ×20; SAHH, MTHFR, MMP9, and TIMP1, ×10. *P < 0.05 vs. WT, WT + Aza, and CBS + Aza; ^†P < 0.05 vs. WT, WT + Aza, and CBS+Aza; ^ψP < 0.05 vs. WT and WT + Aza; ^‡P < 0.05 vs. WT and WT + Aza.

Figure 9.

Immunohistochemistry. A) Protein expression of DNMT1 is shown as green fluorescence. B) Bar graph representation of DNMT1 expression. The y axis represents the percentage change in mean ± sem intensity (n=4). Original image: ×10. *P < 0.05 vs. WT, WT + Aza, and CBS + Aza.

Figure 10.

Overall methylation analysis was measured using ELISA. Bar graphs represent mean ± sem percentage of 5-mC (n=4). *P < 0.05 vs. WT and WT + Aza; ^†P < 0.05 vs. CBS.

Figure 11.

Schematic representation of epigenetic regulation of aortic remodeling in hyperhomocysteinemia.