Date of Award

3-30-2021

Document Type

Doctoral Dissertation - Open Access

Degree Name

Doctor of Philosophy

Department

Pharmacology

First Advisor

Michal L. Schwartzman

Abstract

20-hydroxyeicosatetraenoic acid (20-HETE) is the ω-hydroxylation product of arachidonic acid catalyzed by CYP4A and 4F enzymes. 20-HETE is a vasoactive eicosanoid of the microcirculation exhibiting effects on both vascular smooth muscle cells (VSMC) and endothelial cells (EC). In VSMCs, 20-HETE’s bioactions include the stimulation of contraction, migration, and growth. In ECs, elevated 20-HETE is associated with reduced nitric oxide (NO) bioavailability, increased angiotensin converting enzyme (ACE) expression, and the promotion of inflammation. Recently in our laboratory, we identified GPR75 as a novel target of 20-HETE that promotes changes in blood pressure and vascular function. The aim of this study is to assess the consequences of VSMC-targeted overexpression of Cyp4a12, the primary 20-HETE producing enzyme in mice, on blood pressure, vascular function, and vascular remodeling. Moreover, we looked to examine whether the administration of a 20-HETE receptor antagonist reverses the vascular phenotype associated with elevations in 20-HETE. Mice with VSMC-specific overexpression of Cyp4a12 (Myh11-4a12) and their littermate controls (WT) were generated by crossbreeding Cyp4a12-flox mice (gifted by Dr. Schunck) with Myh11-Cre mice. Myh11-4a12 were administered AAA (10 mg.kg-1.day-1), a 20-HETE receptor antagonist, in the drinking water (vehicle). At the end of the experiments (12 days), renal interlobar arteries (RIA) and mesenteric arteries (MA) were harvested for the assessment of 20-HETE levels by LC-MS/MS, and vascular contractility, vasodilation, and remodeling using wire and pressure myography.

The Myh11-4a12 mice showed higher Cyp4a levels in MA compared to WT mice (6.5±0.71 vs 3.4±0.70; Cyp4a/β-actin p increased 20-HETE levels in the MA (3334±891 vs. 545±197 pg/mg protein; p HETE levels were not different (117±9 vs. 93±2 pg/mL; p=0.18) when compared to WT. Myh11-4a12 mice displayed higher SBP compared to WT mice (145±2 vs. 127±2 mmHg; Myh11-4a12 mice (124±2 mmHg vs. 147±4 mmHg, treated Myh11-4a12 mice displayed a higher media to lumen (M:L) ratio (0.277±0.025 vs 0.163 ± 0.009; (15114±1871 vs 10560±641 μm2; Moreover, RIA from Myh11-4a12 mice on AAA exhibit a lower M:L ratio compared to Myh11-4a12 mice on vehicle (0.215 ± 0.013, not different (14268 ± 1259 μm2). Higher constrictor responsiveness to phenylephrine (EC50: 1.63x10-7 ± 3.75x10-8 vs 5.00x10-7 ± 0.7.85x10-8 M, response at 10-4 M: 65 ± 1 vs 83 ± 2 % relaxation, from Myh11-4a12 mice compared to WT. Treatment of Myh11-4a12 mice with AAA diminished the constrictor responsiveness to phenylephrine (EC50 of 4.22x10-7± 4.55x10-8 M, acetylcholine (92 ± 2 % relaxation, phosphorylation of myosin light chain compared to WT mice (1.60 ± 0.23 vs 1.00 ± 0.06, Ser19 p-MLC/total MLC fold change, exhibited a decrease in myosin light chain phosphorylation compared to Myh11-4a12 mice on vehicle (0.94 ± 0.11, Ser19 p-MLC/total MLC fold change, p<0.05). Upstream of myosin light chain is Rho kinase (ROCK). Expression of ROCK1 was assessed in RIA and Myh11-4a12 mice had increased ROCK1 expression compared to WT mice (1.60 ± 0.21 vs 1.00 ± 0.18, ROCK1/β-tubulin fold change, ROCK1/β-tubulin fold change). Pressure myography was performed to assess 20-HETE receptor antagonism on ROCK activity. In RIA from WT mice, 20-HETE administration promoted a greater myogenic response and both AAA and ROCK inhibitor (Y-27632) inhibited the 20-HETE response in relation to the myogenic tone.

The results of this study suggest that VSMC specific overproduction of Cyp4a12 and 20-HETE promotes increase in blood pressure and changes in vascular reactivity. 20-HETE receptor antagonism was capable of reversing the vascular pathology in mice with elevated 20-HETE presumably through inhibition of ROCK activity.

Additional Files
Thesis permission for 4.12.2021.pdf (104 kB)
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