Date on Master's Thesis/Doctoral Dissertation

8-2016

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Physiology and Biophysics

Degree Program

Physiology and Biophysics, PhD

Committee Chair

Tyagi, Suresh C

Committee Co-Chair (if applicable)

Tyagi, Neetu

Committee Member

Joshua, Irving G

Committee Member

Lominadze, David

Committee Member

Bratcher, Adrienne P

Author's Keywords

vascular remodeling; mitochondrial fission; mitochondrial apoptosis; vascular inflammation; innate immune response

Abstract

Background: Hyperhomocysteinemia (HHcy) has been observed to promote hypertension, but the mechanisms are unclear. Toll-like receptor 4 (TLR-4) is a cellular membrane protein that is ubiquitously expressed in all cell types of the vasculature. TLR-4 activation has been shown to promote inflammation that has been associated with pathogenesis of hypertension. In this study, we hypothesize that HHcy induces hypertension by TLR-4 activation that promotes inflammatory cytokine up-regulation (IL-1β, IL-6, TNF-α) and initiation of mitochondrial dysfunction leading to cell death and chronic vascular inflammation. Methods: To test this hypothesis, we used C57BL/6J mice (WT); Cystathionine-β-synthase deficient mice (CBS+/-) with genetic mild HHcy; C3H/HeJ (C3H) mice, with TLR-4 mutation and mice with combined genetic CBS enzyme deficiency and TLR-4 mutation (CBS+/-/C3H). Arterial blood pressure was measured using non- invasive tail- cuff method. Ultrasonography of the superior mesenteric artery was performed to assess resistance to blood flow and to measure wall/lumen ratio. Collagen deposition in the SMA was analyzed using Masson’s trichrome staining. The levels of oxidative stress markers and endothelial nitric oxide synthase (eNOS) were measured by western blotting and the expression of endothelial cell markers analyzed by immunohistochemistry (IHC). The proteins of mitochondrial dynamics were assessed by western blot, qPCR and IHC. The levels of inflammatory markers were analyzed by qPCR and IHC. Mitochondrial apoptosis protein expression was measured by western blot, qPCR and IHC. DNA fragmentation in the SMA was assessed by TUNEL assay. Results: Ultrasonography of the SMA detected an increase of wall-to-lumen ratio and resistance to blood flow in CBS+/- mice that was associated with arterial blood pressure elevation in the same mouse model detected by tail- cuff measurement. The SMA from CBS+/- mice expressed elevated markers of inflammation, oxidative stress, mitochondrial fission and mitochondrial apoptosis. The increased collagen accumulation was observed in the SMA of CBS+/- mouse model. However, all these changes were attenuated in CBS+/-/C3H mouse model. Conclusions: We conclude that HHcy promotes TLR-4- driven chronic vascular inflammation and mitochondria- mediated cell death inducing peripheral vascular remodeling and hypertension. TLR-4 mutation attenuates vascular inflammation and cell death that prevents vascular remodeling and suppresses hypertension.

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