Date on Master's Thesis/Doctoral Dissertation

5-2014

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Physiology and Biophysics

Committee Chair

Tyagi, Suresh C.

Committee Co-Chair (if applicable)

Sen, Utpal

Committee Member

Roberts, Andrew M.

Committee Member

Joshua, Irving G.

Committee Member

Wead, William B.

Committee Member

Bratcher, Adrienne P.

Subject

Hypertension--Treatment; Aorta--Diseases

Abstract

Background: Hyperhomocysteinemia (HHcy) is prevalent in hypertensive patients 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 accumulation of collagen in the aorta resulting in stiffness and development of hypertension. Although the exact mechanism of extracellular matrix remodeling (ECM) is unclear, emerging evidence implicates epigenetic regulation involving DNA methylation. The purpose of the study was to investigate whether inhibition of DNA methylation reduces high blood pressure by regulating aortic ECM remodeling in HHcy. Methods: In the first set of experiments, we studied the effect of HHcy in causing epigenetic changes leading to aortic remodeling resulting in hypertension. Cystathionine beta synthase heterozygous knockout (CBS+/-) mice of C57BL/6J background were used as HHcy model and their Wild type (WT) littermates were used as controls. Hcy level in plasma was measured by HPLC-UV. Blood pressure was measured using tail-cuff method. The mRNA expression levels of ECM proteins and epigenetic modifications enzymes in the aorta were measured by RT-PCR. Global methylation level was measured by ELISA. In the second set of experiments, we studied effects of an epigenetic DNA methyltransferase inhibitor, 5-Aza-2’-deoxycytidine (Aza) in HHcy. For in vivo studies, we used 8-12 weeks old WT and CBS+/- mice and administered them with Aza for four weeks. During the treatment period, blood pressure was measured using tail-cuff method. Aorta wall thickness, lumen diameter and resistive index were measured before and after treatment using ultrasound. Aortic responses to vasoconstrictor and vasodilators were measured using tissue myobath. Collagen accumulation in the aortic walls was measured by Mason Trichrome and Picrosirius red staining. ECM proteases, Hcy metabolism enzymes and DNMT1 expression were measured using immunostaining. Global methylation levels were assessed using ELISA. For cell culture studies, mouse aortic smooth muscle cells were treated with Aza and Hcy for 48 hrs. MMP9 activity was measured using gelatin zymography and DNMT1, 3a and 3b expression was measured using immunoblotting. Results: In CBS+/- mice, increased plasma Hcy levels were associated with an increase in blood pressure. There was an increase in the mRNA levels of collagen and decrease in elastin in HHcy mice. Global hypermethylation was accompanied by an increased expression of methylation proteins in HHcy. Aza treatment normalized plasma SAM, SAH and Hcy levels and blood pressure in HHcy mice. Thoracic and abdominal aorta ultrasound revealed reduction in resistive index and wall-to-lumen ratio. Vascular response to vasoactive agents improved following Aza treatment in HHcy. Histology showed a marked reduction in collagen deposition in the aorta. Aza treatment decreased the expression of DNMT1, 3b, MMP9, TIMP1 and S-adenosyl homocysteine hydrolase (SAHH) and upregulated methylene tetrahydrofolate reductase (MTHFR) Conclusion: We conclude that reduction of DNA methylation in HHcy reduces adverse aortic remodeling to mitigate hypertension.

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