Candidate genes and mechanisms for 2-methoxyestradiol-mediated vasoprotection

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, cyclin-dependent 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 (PPARγ 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 1α. 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.

Figure 1

Inhibitory effects of 2-ME on serum-induced activation of pathways regulating cell cycle. Panel-A depicts the microarray analysis of transcriptional changes in HASMCs treated with 2-ME (3 μmol/L) for 30-hours. Expression levels are depicted as color scale from red (up-regulation) to blue (down-regulation). Gene Set Enrichment Analysis (GSEA) revealed down-regulation of key genes known to induce cytokinesis. Panel-B shows Western-blots and bar graph (changes in optical density; mean±SEM) depicting the significant inhibitory effects of 2-ME on cyclin-D1and cyclin-B1 expression and on cdk4 and cdk6 activity in HASMCs treated with 2-ME. Panel-C illustrates representative photomicrographs and bar graph depicting the inhibitory effects of 2-ME on tubulin polymerization in proliferating HASMCs. The tubulin polymerization experiments were conducted in triplicate. Western blotting and cdk activity data are presented as mean±SEM (n=3). *p<0.05 versus vehicle-treated control. Arrows indicate the genes for which protein expression was confirmed.

Figure 2

Stimulatory effects of 2-ME on prostaglandin synthesis in HASMCs. Panel-A depicts the microarray analysis of transcriptional changes in HASMCs treated with 2-ME (3μmol/L) for 30 hours. Expression levels are depicted as color scale from red (up-regulation) to blue (down-regulation). Gene Set Enrichment Analysis (GSEA) revealed induction of key proteins known to facilitate prostaglandin synthesis. Panel-B shows the significant stimulatory effects of 2-ME on COX-2 protein expression in HASMCs treated with 2-ME. Data are presented as mean±SEM (n=3). *p<0.05 versus vehicle-treated control. Arrows indicate the genes for which protein expression was confirmed.

Figure 3

Inhibitory effects of 2-ME on pathways regulating cholesterol synthesis. Panel-A depicts the microarray analysis of transcriptional changes in HASMCs treated with 2-ME (3μmol/L) for 30-hours. Expression levels are depicted as color scale from red (up-regulation) to blue (down-regulation). Gene Set Enrichment Analysis (GSEA) revealed down-regulation of key genes known to facilitate cholesterol synthesis. Panel-B shows the significant inhibitory effects of 2-ME on circulating levels of cholesterol, progesterone and testosterone in male rats treated for 14-days with 2-ME. Data for cholesterol levels represent mean±SEM (n=7 placebo; and n=7 2-ME treated). *p<0.05 versus vehicle-treated control.

Figure 4

Stimulatory effects of 2-ME on plaque-associated regulatory molecules in HASMCs. Panel-A depicts the transcriptional changes in HASMCs treated with 2-ME (3μmol/L) for 30-hours. Expression levels are depicted as color scale from red (up-regulation) to blue (down-regulation). Gene Set Enrichment Analysis (GSEA) revealed up-regulation of key plaque-associated transcripts. Panel-B shows the significant stimulatory effects of 2-ME on MMP-1 expression in HASMCs treated with 2-ME. HASMCs treated with LPS served as a positive control. Data for microarray analysis and Western-blotting represent mean±SEM (n=3). *p<0.05 versus vehicle-treated control. Arrows indicate the genes for which protein expression was confirmed.

Figure 5

Inhibitory effects of 2-ME on pathways down-regulated by rosiglitazone in HASMCs. Panel-A depicts the structural similarity between 2-ME and PPARγ ligand rosiglitazone. Panel-B depicts the microarray analysis of transcriptional changes in HASMCs treated with 2-ME (3μmol/L) for 30-hours. Expression levels are depicted as color scale from red (up-regulation) to blue (down-regulation). Gene Set Enrichment Analysis (GSEA) revealed down-regulation of key genes known to be down-regulated by rosiglitazone. Panel-C demonstrates the stimulatory effects of 2-ME on PPARδ, PPARγ and PPARα using a transactivation assay. Panel-D depicts representative photomicrographs and Western-blots demonstrating the inhibitory effects of 1μmol/L 2-ME on hypoxia (Hx) induced HIF-1α expression in HASMCs. The bar graph represents the densitometeric analysis of Western-blots (mean±SEM) from three separate experiments. Data for microarray analysis and Western-blotting represent mean±SEM (n=3). The immunostaining experiments were conducted in quadruplicate. *p<0.05 versus HASMCs treated with hypoxia (Hx). Normoxia (Nx). Arrows indicate the genes for which protein expression was confirmed.

Figure 6

Depicts the attenuation by PPARγ antagonist GW9662 of the concentration-dependent inhibitory effects of 2-ME (0.001–10μmol/L; left-panels) or rosiglitazone (0.1–100μmol/L; right-panels) on 2.5%FCS-induced cell-number (A) and collagen ([³H]proline-incorporation) (B) in HASMCs. Also shown are the effects of NS398 on 2-ME-induced inhibition of cell number and DNA-synthesis (C). Data represent mean±SEM from 3 separate experiments conducted in triplicates. *p<0.05 significant reversal of the inhibitory effects of 2-ME or rosiglitazone by GW9662.