The proposed study will test the hypothesis that vascular expression of COX and release of prostaglandins (PGs) are suppressed by cytochrome P450-derived epoxyeicosatrienoic acids (EETs), as a function of estrogen-dependent downregulation of soluble epoxide hydrolase (sEH), the enzyme responsible for the degradation of EETs, leading to the potentiation of endothelial function, via an enhanced EET-mediated shear stress-induced dilation (SSID) in vessels with NO dysfunction. Studies will be conducted on arteries isolated from three animal models: 1) eNOS-KO mice to be used as a genomic model of NO deficiency, and 2) mice chronically fed with high fructose (HF), as a model of metabolic syndrome; both animals represent the gender-specific phenotype of SSID, characterized as the PG-mediation in males and EET-mediated greater response in females, and 3) sEH-KO mice, they have increased circulating and tissue EETs. Specific Am 1 is to determine the effect of in vivo increased circulating and vascular EETs on the PG-mediated SSID, via feeding male eNOS-KO and HF mice with sEH inhibitors for 4-6 weeks. Blood pressure will be monitored. SSID, endothelial mediators (PGs or EETs) and expression of COXs and CYP2C29 in arteries will be assessed.
Specific Aim 2 is designed to explore the mechanisms responsible for the EET-induced inhibition of COX/PG by the in vitro incubation of vessels isolated from male HF and eNOS-KO mice with EETs for 24-48 hours. SSID will be evaluated and the switch of endothelial mediators (from PGs to EETs) will be revealed. Specifically, roles of transcription factors, such as PPARs in the EET-dependent inhibition of COX/PGs will be clarified by using specific siRNAs or pharmacologic inhibitors.
Specific Aim 3 aims to demonstrate that estrogen-dependent downregulation of sEH is responsible for the potentiation of female phenotype of endothelial function. Gender difference in vascular expression of sEH will be identified, and the specific role of estrogen and its receptors (ERs) in the response will be clarified by ovariectomy (OV) with and without estrogen replacement, and applying specific inhibitors of ERs. Particularly, estrogen-dependent epigenetic regulation of Ephx2 gene by DNA methylation to silence sEH expression will be characterized.
Specific Aim 4 will explore the mechanisms by which, the deletion of Ephx2 gene (sEH-KO) potentiates endothelial function and improves blood pressure regulation. Specifically, the absence of, or diminishing the gender difference in the regulation of endothelial function of sEH-KO mice will be clarified. Moreover, distinct compensatory mechanisms in response to HF-induced impairment of NO synthesis in sEH-KO and WT mice will be elucidated via dynamically following up vascular interactions of NO, EETs and PGs, as a function of fructose feeding period. In summary, this project will provide mechanistically-based evidence for the actions of estrogen that mimic effects of sEH inhibitors to initiate an EET-dependent potentiation of cardiovascular function and benefit to the control of blood pressure.
The endothelium is the inner layer of blood vessels. Endothelial cells release vasoactive factors (agents) when they are stimulated by estrogen or shear stress. Estrogen(s) is a female hormone(s) that is primarily produced by the female reproductive system, such as the ovaries. Shear stress is the mechanical force on the endothelium when blood flows through the vessels. The agents released from endothelial cells can dilate (enlarge) the vessels, leading to greater blood supply to organs/tissues and then, are metabolized (degradation) by specific enzymes. When the metabolizing enzymes are inhibited, actions of the vasoactive agents become strengthened. Thus, this project is to study how estrogen potentiates the function of the vasoactive agents released from the endothelium, via suppressing their degradation in different animal models that display pathological characteristics of human diseases, such as diabetes and high blood pressure.
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