Semester

Summer

Date of Graduation

2023

Document Type

Dissertation

Degree Type

PhD

College

School of Pharmacy

Department

Pharmaceutical Sciences

Committee Chair

Mohammed A. Nayeem

Committee Member

Ahmed Hanif

Committee Member

Werner Geldenhuys

Committee Member

Salik Hussain

Committee Member

Grazyna D. Szklarz

Abstract

Coronary Artery Disease (CAD) is one of the leading causes of mortality worldwide, and endothelial dysfunction is a major precursor for cardiovascular diseases (CVDs). Arachidonic acids in the vascular endothelial membrane are metabolized by the cytochrome P450-epoxygenases (CYP-2Js and 2Cs) to produce epoxyeicosatrienoic acid (EETs). These anti-inflammatory metabolites or oxylipins have been shown to be cardio-protective against ischemic/reperfusion injury and mediate vasodilation through hyperpolarization of smooth muscle cells. However, the enzyme called soluble epoxide hydrolase (sEH), mainly hydrolyzes EETs to its inactive form (DHETs) and an increase in sEH upregulates Angiotensin-II type1-receptor and facilitates endothelial dysfunction.

we previously showed vascular endothelial overexpression of human-CYP2J2 enhances coronary reactive hyperemia (vascular relaxation) in Tie2-CYP2J2 Tr mice, and eNOS-/- mice had overexpression of CYP2J-epoxygenase with adenosine A2A receptor-induced enhance relaxation, but we did not see the response in CYP2J-epoxygenase knockout mice. Therefore, for our first study, we hypothesized that Cyp2j5-gene deletion affects acetylcholine- and NECA (adenosine)-induced relaxation and their response is partially inhibited by angiotensin-II (Ang-II) in mice. The data collected suggested that ACh-induced relaxation in Cyp2j5-/- mice may depend on nitric oxide (NO) but not on CYP-epoxygenases, and that adenosine (NECA)-induced different responses in male vs. female Cyp2j5-/- mice when Ang-II treated.

In addition, it has also been reported that the coronary reactive hyperemic response was reduced in adenosine A2A receptor-null (A2AAR-/-, sEH-overexpressed) mice, and it was reversed by the sEH inhibitor. However, this has not been tested in aortic vascular response, therefore, for our second study, we hypothesized that A2AAR-gene deletion in mice (A2AAR-/-) affects adenosine-induced vascular response by an increase in sEH and adenosine A1 receptor (A1AR) activities. A2AAR-/- mice showed an increase in sEH, AI AR, and CYP450-4A protein expression but a decrease in CYP450-2C compared to C57BL/6 mice. NECA (adenosine-analog) and CCPA (adenosine A1 receptor-agonist)-induced dose-dependent vascular response were tested with t-AUCB (sEH-inhibitor) and angiotensin-II (Ang-II) in A2AAR-/- vs. C57BL/6 mice. In A2AAR-/-, NECA and CCPA-induced increase in dose-dependent vasoconstriction compared to C57BL/6 mice. However, NECA and CCPA-induced dose-dependent vascular contraction in A2AAR-/- was reduced by t-AUCB with NECA. Similarly, dose-dependent vascular contraction in A2AAR-/- was reduced by t-AUCB with CCPA. Ang-II enhanced NECA and CCPA-induced dose-dependent vascular contraction in A2AAR-/- with NECA, and dose-dependent vascular contraction in A2AAR-/- was also enhanced by Ang-II with CCPA. This Ang-II-enhanced NECA and CCPA-induced dose-dependent vascular contraction was however reduced by t-AUCB in A2AAR-/-mice. Therefore, our data suggest that the dose-dependent vascular contraction in A2AAR-/- mice depends on the increase in sEH, A1AR, and CYP4A protein expression.

Exploring existing interactions between adenosine receptors and CYPs (cyp-epoxygenases) in vascular response could be beneficial as this provides an opportunity to discover potential diagnostics as well as identify possible therapeutic targets in the treatment of cardiovascular diseases.

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