Abstract-2-Methoxyestradiol (2-ME; estradiol metabolite) inhibits vascular smooth muscle cell (VSMC) growth and protects against atherosclerosis and vascular injury; however, the mechanisms by which 2-ME induces these actions remain obscure. To assess the impact of 2-ME on biochemical pathways regulating VSMC biology, we used high-density oligonucleotide microarrays to identify differentially expressed genes in cultured human female aortic VSMCs treated with 2-ME acutely (4 hours) or long term (30 hours). Both single gene analysis and Gene Set Enrichment Analysis revealed 2-ME-induced downregulation of genes involved in mitotic spindle assembly and function in VSMCs. Also, Gene Set Enrichment Analysis identified effects of 2-ME on genes regulating cell-cycle progression, cell migration/adhesion, vasorelaxation, inflammation, and cholesterol metabolism. Transcriptional changes were associated with changes in protein expression, including inhibition of cyclin D1, cyclin B1, cyclindependent kinase 6, cyclin-dependent kinase 4, tubulin polymerization, cholesterol and steroid synthesis, and upregulation of cyclooxygenase 2 and matrix metalloproteinase 1. Microarray data suggested that 2-ME may activate peroxisome proliferator-activated receptors (PPARs) in VSMCs, and 2-ME has structural similarities with rosiglitazone (PPAR␥ agonist). However, our finding of weak activation and lack of binding of 2-ME to PPARs suggests that 2-ME may modulate PPAR-associated genes via indirect mechanisms, potentially involving cyclooxygenase 2. Indeed, the antimitogenic effects of 2-ME at concentrations that do not inhibit tubulin polymerization were blocked by the PPAR antagonist GW9662 and the cyclooxygenase 2 inhibitor NS398. Finally, we demonstrated that 2-ME inhibited hypoxia-inducible factor 1␣. Identification of candidate genes that are positively or negatively regulated by 2-ME provides important leads to investigate and better understand the mechanisms by which 2-ME induces its vasoprotective actions. (Hypertension. 2010;56:964-972.)Key Words: 2-methoxyestradiol Ⅲ PPAR Ⅲ vascular smooth muscle cells Ⅲ microarray analysis A s with cancer, abnormal cell growth plays a key role in cardiovascular diseases. For example, vascular smooth muscle cell (VSMC) proliferation is involved in vascular remodeling that occurs at sites of atherosclerosis, hypertension-induced vascular changes, and injury-induced restenosis. 1 Hence, drugs capable of inhibiting VSMC growth by targeting key mitogenic mechanisms are effective in protecting against cardiovascular disease. 2 Thus, it is not surprising that various antimitotic therapies used in cancer also protect against vascular proliferative disorders. 3 2-Methoxyestradiol (2-ME) is an endogenous metabolite of estradiol with no affinity for estrogen receptors that exerts anticarcinogenic effects by inhibiting growth of cancer cells and neovascularization of tumors and is in phase II clinical trials for cancer. 4,5 Consistent with the notion that anticancer drugs may be useful in cardiovascular d...

Abstract-Endothelial progenitor cells (EPCs) repair damaged endothelium and promote capillary formation, processes involving receptor tyrosine kinases (RTKs) and heme oxygenase 1 (HO-1). Because estradiol augments vascular repair, we hypothesize that estradiol increases EPC proliferation and capillary formation via RTK activation and induction of HO-1. Physiological concentrations of estradiol (10 nmol/L) increased EPC-induced capillary sprout and lumen formation in matrigel/fibrin/collagen systems. Propyl-pyrazole-triol (PPT; 100 nmol/L; estrogen receptor [ER]-␣ agonist), but not diarylpropionitrile (ER-␤ agonist), mimicked the stimulatory effects of estradiol on capillary formation, and methyl-piperidino-pyrazole (ER-␣ antagonist) abolished the effects of estradiol and PPT. Three different RTK activators (vascular endothelial growth factor, hepatocyte growth factor, and stromal derived growth factor 1) mimicked the capillary-stimulating effects of estradiol and PPT. SU5416 (RTK inhibitor) blocked the stimulatory effects of estradiol and PPT on capillary formation. Estradiol increased HO-1 expression by 2-to 3-fold, an effect blocked by SU5416, and PPT mimicked the effects of estradiol on HO-1. The ability of estradiol to enhance capillary formation, increase expression of HO-1, and augment phosphorylation of extracellular signal-regulated kinase 1/2, Akt, and vascular endothelial growth factor receptor 2 was mimicked by its cell-impermeable analog BSA estradiol. Actinomycin (transcription inhibitor) did not alter the effects of estradiol on RTK activity or vascular endothelial growth factor secretion. We conclude that estradiol via ER-␣ promotes EPC-mediated capillary formation by a mechanism that involves nongenomic activation of RTKs and HO-1 activation. Estradiol in particular and ER-␣ agonists in general may promote healing of injured vascular beds by promoting EPC activity leading to more rapid endothelial recovery and capillary formation after injury. (Hypertension. 2010;56:397-404.)

Jackson EK, Dubey RK. 2-Methoxyestradiol, an endogenous 17␤-estradiol metabolite, inhibits microglial proliferation and activation via an estrogen receptor-independent mechanism. Am J Physiol Endocrinol Metab 310: E313-E322, 2016. First published January 5, 2016 doi:10.1152 doi:10. /ajpendo.00418.2015 inhibits microglia proliferation. 2-Methoxyestradiol (2-ME) is an endogenous metabolite of estradiol with little affinity for estrogen receptors (ERs). We hypothesize that 2-ME inhibits microglial proliferation and activation and contributes to estradiol's inhibitory effects on microglia. We compared the effects of estradiol, 2-hydroxyestradiol [2-OE; estradiol metabolite produced by cytochrome P450 (CYP450)], and 2-ME [formed by catechol-O-methyltransferase (COMT) acting upon 2-OE] on microglial (BV2 cells) DNA synthesis, cell proliferation, activation, and phagocytosis. 2-ME and 2-OE were approximately three-and 10-fold, respectively, more potent than estradiol in inhibiting microglia DNA synthesis. The antimitogenic effects of estradiol were reduced by pharmacological inhibitors of CYP450 and COMT. Inhibition of COMT blocked the conversion of 2-OE to 2-ME and the antimitogenic effects of 2-OE but not 2-ME. Microglia expressed ER␤ and GPR30 but not ER␣. 2,3-Bis(4-hydroxyphenyl)-propionitrile (ER␤ agonist), but not 4,4=,4==-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (ER␣ agonist) or G1 (GPR30 agonist), inhibited microglial proliferation. The antiproliferative effects of estradiol, but not 2-OE or 2-ME, were partially reversed by ICI-182,780 (ER␣/␤ antagonist) but not by 1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1H-pyrazole (ER␣ antagonist) or G15 (GPR30 antagonist). Lipopolysaccharide increased microglia iNOS and COX-2 expression and phagocytosing activity of microglia; these effects were inhibited by 2-ME. We conclude that in microglia, 2-ME inhibits proliferation, proinflammatory responses, and phagocytosis. 2-ME partially mediates the effects of estradiol via ER-independent mechanisms involving sequential metabolism of estradiol to 2-OE and 2-ME. 2-ME could be of potential therapeutic use in postischemic stroke injuries. Interindividual differences in estradiol metabolism might affect the individual's ability to recover from stroke. stroke; microglia; 17␤-estradiol; 2-hydroxyestradiol; 2-methoxyestradiol

The purpose of the present study was to investigate whether the size of the opening in the zona pellucida (ZP) of human single pronuclear (1PN) oocytes made by laser and partial zona dissection (PZD) techniques might interfere with the survival and subsequent development to blastocyst stage upon vitrification and warming. Moreover, the viability of these blastocysts was evaluated by comparing their total cell number (TCN) to the TCN of blastocysts developed from control non-vitrified zona-intact 1PN oocytes. Prior to vitrification, a total of 97 and 88 1PN oocytes were subjected to polar body biopsy using laser-assisted and PZD techniques, respectively. The size of ZP opening made by laser and PZD techniques did not interfere with survival (94.8% and 95.4%) or development to the blastocyst stage (27.8% and 26.1%). However, the TCN of laser-derived blastocysts was significantly lower than the TCN of blastocysts developed from non-vitrified control 1PN oocytes (48.7 ± 3.4 versus 70.8 ± 7.1, P < 0.028). The vitrification protocol used here is thus revealed to be an effective method for cryopreservation of 1PN oocytes following polar body biopsy. However, the viability of blastocysts developed from laser-treated 1PN oocytes seems to be negatively affected by this method of biopsy.