Aging vs. Estrogen Loss: Effects on Inflammatory Gene/Protein expression and EETs.

摘要

Introduction: Large clinical trials on humans showed estrogen administered to postmenopausal women increased the risk of cardiovascular disease, despite an abundance of evidence to the contrary in animal studies. Timing, however, may be a major cause of this effect, as estrogen is usually administered immediately after ovariectomy (Ovx) in animal studies, but many years post menopause in humans. A critical component of many cardiovascular diseases is inflammation; both estrogen loss, caused by menopause, and aging have inflammatory consequences. Epoxyeicosatrienoic acids (EETs) are anti-inflammatory molecules synthesized by various cytochrome P450 (CYP) enzymes from arachidonic acid. EETs are in the third (Cytochrome P450) pathway of arachidonic acid metabolism, others being cyclooxygenases and lipoxygenases. Vascular function is maintained to a large degree by the actions of endothelial progenitor cells (EPCs), which are often harmed by inflammation.;Methods: Adult (6 mo) and aged (22 mo) Norway Brown rats were ovariectomized (Ovx); rats from each group immediately received 17-β-estradiol replacement (OP or Early), while one group of aged rats received replacement 9 wk post-ovx (Late). An additional group of sham-operated aged rats was used. Microarrays were used to analyze left ventricles for inflammatory genes, with key genes analyzed by Western blotting. Mass spectrometry was used to measure levels of EETs and their metabolites, dihydroxyeicosatrienoic acids (DHETs) in the aorta and serum, while Western blotting was used to identify levels of CYP and soluble epoxide hydrolase (SEH) proteins. Cultured EPCs were treated with an inhibitor of SEH, EETs or DHETs to analyze wound healing.;Results: Timing Hypothesis—Expression of proinflammatory genes CD11b, MIP-1β, STAT3, EMAP II, and fibronectin were found to be significantly increased in the Late replacement group, while decreases in Cx3cr1 seen in the early group were abolished in the Late group. Notably, Late replacement had significantly increased levels of TNF-α and iNOS protein. This suggests that the deleterious effects of delayed estrogen administration seen in human studies may be caused by increased expression of inflammatory genes.;EETs—Levels of CYP 2C2, CYP 2C6, and CYP 2J2, the principal CYPs responsible for EETs synthesis, as well as soluble epoxide hydrolase (sEH), which metabolizes EETS to DHETs, were determined via western blot. Overall CYP levels decreased with age, though CYP 2C6 increased in the liver. sEH was increased in the kidney with estrogen replacement. Despite protein changes, no differences were measured in plasma or aortic tissue levels of EETs. However, plasma 14,15 DHET was increased in aged Ovx, and 5,6 DHET in adult OP. Neither aging nor estrogen loss decreased the anti-inflammatory EETs in the cardiovascular system.;Wound Healing—Treatment of EPCs had no significant effect on wound healing, requiring further study.