Estradiol improves cardiovascular function through up-regulation of SOD2 on vascular wall

Epidemiological studies have shown that estrogens have protective effects in cardiovascular diseases, even though the results from human clinical trials remain controversial, while most of the animal experiments confirmed this effect, but the detailed mechanism remains unclear. In this study, we found that estradiol (E2) treatment significantly increases the expression of mitochondrial superoxide dismutase (SOD2) in mice and in vitro in human aorta endothelial cells. Further investigation shows that E2 up-regulates SOD2 through tethering of estrogen receptor (ER) to Sp1 and the increased binding of Sp1 to GC-box on the SOD2 promoter, where ERα responses E2-mediated gene activation, and ERβ maintains basal gene expression level. The E2/ER-mediated SOD2 up-regulation results in minimized ROS generation, which highly favors healthy cardiovascular function. Gene therapy through lentivirus-carried endothelium-specific delivery to the vascular wall in high-fat diet (HFT) mice shows that the SOD2 expression in endothelial cells normalizes E2 deficiency-induced ROS generation with ameliorated mitochondrial dysfunction and vascular damage, while SOD2 knockdown worsens the problem despite the presence of E2, indicating that E2-induced SOD2 expression plays an important vasculoprotective role. To our knowledge, this is the first report for the mechanism by which E2 improves cardiovascular function through up-regulation of SOD2 in endothelial cells. In turn, this suggests a novel gene therapy through lentivirus-carried gene delivery to vascular wall for E2 deficiency-induced cardiovascular damage in postmenopausal women

Perinatal testosterone exposure potentiates vascular dysfunction by ERβ suppression in endothelial progenitor cells

<div><p>Recent clinical cohort study shows that testosterone therapy increases cardiovascular diseases in men with low testosterone levels, excessive circulating androgen levels may play a detrimental role in the vascular system, while the potential mechanism and effect of testosterone exposure on the vascular function in offspring is still unknown. Our preliminary results showed that perinatal testosterone exposure in mice induces estrogen receptor β (ERβ) suppression in endothelial progenitor cells (EPCs) in offspring but not mothers, while estradiol (E2) had no effect. Further investigation showed that ERβ suppression is due to perinatal testosterone exposure-induced epigenetic changes with altered DNA methylation on the ERβ promoter. During aging, EPCs with ERβ suppression mobilize to the vascular wall, differentiate into ERβ-suppressed mouse endothelial cells (MECs) with downregulated expression of SOD2 (mitochondrial superoxide dismutase) and ERRα (estrogen-related receptor α). This results in reactive oxygen species (ROS) generation and DNA damage, and the dysfunction of mitochondria and fatty acid metabolism, subsequently potentiating vascular dysfunction. Bone marrow transplantation of EPCs that overexpressed with either ERβ or a SIRT1 single mutant SIRT1-C152(D) that could modulate SIRT1 phosphorylation significantly ameliorated vascular dysfunction, while ERβ knockdown worsened the problem. We conclude that perinatal testosterone exposure potentiates vascular dysfunction through ERβ suppression in EPCs.</p></div

Bone marrow transplantation with ERβ overexpression in EPCs restores perinatal testosterone exposure-induced vascular dysfunction in male old offspring, while ERβ knockdown in EPCs worsens the problem.

<p>(a-c) The treated mice were given a bolus dose of 2mCi of ¹⁴C-OA through oral gavage, and the blood and tissues, including the heart, aorta and liver, were dissected for analysis of total radioactivity. (a) The in vivo ¹⁴C-OA uptake from the heart and aorta in 2h, n = 6. (b) The in vivo ¹⁴C-OA uptake from liver in 2h, n = 6. (c) The in vivo ¹⁴C-OA uptake in plasma in 1h, n = 7. (c) (d-g) The plasma was collected from treated mice for analysis of total cholesterol, n = 9 (d); triglyceride, n = 8 (e); LDL cholesterol, n = 11 (f); and HDL cholesterol, n = 12 (g). (h,i) The aortas were dissected from treated mice for vessel tension analysis. The rings were pre-constricted with phenylephrine, and the acetylcholine (Ach, 10⁻¹⁰–10⁻⁴ mol/l) was injected at the plateau of the phenylephrine-induced contraction. (h) The 10⁻⁴ mol/l Ach-induced aorta ring relaxation, n = 8–11; (i) The Ach-induced aorta ring relaxation curves. (j) The treated mice were used to measure the mean of systolic blood pressure, n = 10. *, <i>P</i><0.05, vs CTL group; ¶, <i>P</i><0.05, vs DHT group. Results are expressed as mean ± SEM.</p

Perinatal testosterone exposure potentiates vascular dysfunction in old male offspring (20 months old).

<p>(a-c) The treated male offspring were given a bolus dose of 2mCi of ¹⁴C-OA through oral gavage, and the blood and tissues, including the heart, aorta and liver, were dissected for analysis of total radioactivity. (a) The in vivo ¹⁴C-OA uptake from the heart and aorta in 2h, n = 8. (b) The in vivo ¹⁴C-OA uptake from liver in 2h, n = 7. (c) The in vivo ¹⁴C-OA uptake in plasma in 1h, n = 6. (d-g) The plasma was collected from treated male offspring for analysis of total cholesterol, n = 10 (d); triglyceride, n = 10 (e); LDL cholesterol, n = 12 (f); and HDL cholesterol, n = 11 (g). (h,i) The aortas were dissected from treated mice for vessel tension analysis. The rings were pre-constricted with phenylephrine, and the acetylcholine (Ach, 10⁻¹⁰–10⁻⁴ mol/l) was injected at the plateau of the phenylephrine-induced contraction. (h) The 10⁻⁴ mol/l Ach-induced aorta ring relaxation, n = 9–12; (i) The Ach-induced aorta ring relaxation curves. (j) The treated mice were used to measure the mean of systolic blood pressure, n = 11. *, <i>P</i><0.05, vs CTL group. Results are expressed as mean ± SEM.</p

Perinatal testosterone exposure suppresses ERβ expression and its target genes in EPCs in young offspring (2 months old).

<p>2-month old female mice were exposed to 5mg of 60-day release hormone pellets that contained either dihydrotestosterone (DHT) alone, estradiol (E2) alone, combined DHT and E2 (DHT/E2), or controlled vehicle (CTL) during a 7-week perinatal period. The mothers were sacrificed to measure the plasma hormone levels, and the MNCs (including EPCs and non-EPCs) were isolated from either the bone marrow or peripheral blood for analysis of gene expression. The male offspring was also sacrificed at 2 months old for isolation of MNCs (including EPCs and non-EPCs) for further analysis. (a) Dihydrotestosterone (DHT) level in plasma from mothers, n = 8. (b) The estradiol (E2) level in plasma from mothers, n = 8. (c) The ERβ mRNA in BM-derived MNCs from mothers, n = 7. (d) The ERβ mRNA in Circulating MNCs from mothers, n = 7. (e)The ERβ mRNA in BM-derived MNCs from male offspring, n = 6. (f) The ERβ mRNA in Circulating MNCs from male offspring, n = 6. (g) The mRNA levels in BM-derived EPCs from male offspring, n = 7. (h) The mRNA levels in Circulating EPCs from male offspring, n = 7. *, <i>P</i><0.05, vs CTL group; ¶, <i>P</i><0.05, vs DHT group; #, <i>P</i><0.05, vs DHT/E2 group. Results are expressed as mean ± SEM.</p

Perinatal testosterone exposure induces ERβ suppression in EPCs through increased methylation on ERβ promoter, while E2 has no effect.

<p>The EPCs were isolated from 2-month old male offspring for in vitro cell culture analysis. (a) The representative bands for ERβ methylation in EPCs from male offspring. (b) DNA methylation on ERβ by real-time PCR based methylation specific PCR (MSP) analysis in EPCs, n = 4. (c) ChIP analysis on ERβ promoter in BM-derived EPCs, n = 5. (d) ChIP analysis on ERβ promoter in Circulating EPCs, n = 5. *, <i>P</i><0.05, vs CTL group; ¶, <i>P</i><0.05, vs DHT group. Results are expressed as mean ± SEM.</p

Perinatal testosterone exposure induces ERβ suppression and its target genes from both circulating EPCs and mouse endothelial cells (MECs) in old male offspring (20 months old).

<p>(a-c). The EPCs were isolated from treated male offspring for further analysis. (a) mRNA level by qPCR, n = 4. (b) Protein level by western blotting, n = 4. (c) The representative bands for (b). (d) The MECs were isolated from the aorta using Laser Capture Microdissection (LCM) techniques to measure mRNA level by qPCR, n = 5. (e,f) The MECs were isolated from the heart and cultured in vitro for protein analysis using western blotting. (e) Protein level by Western blotting, n = 4. (f) Representative bands for (e). *, <i>P</i><0.05, vs CTL group. Results are expressed as mean ± SEM.</p

Perinatal testosterone exposure induces ROS generation and DNA damage, and dysfunction of mitochondria and fatty acid metabolism in both circulating EPCs and MECs in old male offspring (20 months old).

<p>The MECs from treated mice were isolated from the hearts during 20 months of age for in vitro culture analysis. (a) ROS formation, n = 6. (b) Quantitation of 3-nitrotyrosine (3-NT) formation, n = 4. (c) Representative γH2AX western blotting band. (d) Quantitation of γH2AX formation for (c), n = 5. (e) Mitochondrial DNA copies, n = 4. (f) Intracellular ATP levels, n = 5. (g) Representative western blotting band for OXPHOS proteins. (h) Quantitation of OXPHOS proteins for (g), n = 5. (i) In vitro ¹⁴C-OA fatty acid uptake, n = 4. (j) The in vitro palmitate oxidation rate, n = 4. *, <i>P</i><0.05, vs CTL group; ¶, <i>P</i><0.05, vs DHT group. Results are expressed as mean ± SEM.</p