Candidate Genes and Mechanisms for 2-Methoxyestradiol–Mediated Vasoprotection

Abstract

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...

2-Methoxyestradiol blocks the RhoA/ROCK1 pathway in human aortic smooth muscle cells

“…Our previous studies using microarray analysis showed that 2-ME modulates multiple mechanisms that can affect tissue factor expression. Specifically, 2-ME induces COX2 expression, activates PPAR␣ and -␥, inhibits 3-hydroxyl-3-methylglutaryl coenzyme A (HMG-CoA), blocks squalene epoxidase and mevalonate (diphospho) decarboxylase (which convert mevalonic acid to isopentenyl-PP and squalene), and inhibits NF-B and phosphatidylinositol 3-kinase (6,7,23). Moreover, 2-ME inhibits MAPK, a known activator of tissue factor (21).…”

“…Taken together, these observations indicate that RhoA acts as a microtubuledependent signal in cytokinesis. It is known that 2-ME disrupts tubulin polymerization (4), and our previous microarray experiment shows a downregulation by 2-ME of genes necessary for mitotic spindle assembly (6). Thus, it is conceivable that 2-ME inhibits the activation of the RhoA/ROCK1 pathway via its microtubule disrupting action and thereby blocks cytokinesis (Fig.…”

Considerations of Sex and Gender Differences in Preclinical and Clinical Trials

“…Rosiglitazone used to treat type II diabetes also was suspended by the EMEA and restricted by the United States Federal Drug Administration because of increased cardiovascular risk (Hancox 2011). Rosiglitazone acts through peroxisome proliferator-activated receptor g (PPAR g), a receptor which is also modulated by metabolites of estrogen (Barchiesi et al 2010). Rosiglitazone may also inhibit estrogen synthesis in ovarian tissue which could indirectly as well as directly affect bone resorption (Harslof et al 2011;Seto-Young et al 2011).…”

Differential Gene Expression Profile in Essential Hypertension

“…We mainly use peripheral blood samples [45] in human and vascular smooth muscle cell (VSMC) [46,47] , adrenal [44] , heart [2,48,49] and kidney [2,44,[53][54][55][56]58] in animal models.…”