血管石灰化形成の新たな実験モデルの構築

学位の種別:課程博士University of Tokyo(東京大学

Testosterone Deficiency Accelerates Neuronal and Vascular Aging of SAMP8 Mice: Protective Role of eNOS and SIRT1

Oxidative stress and atherosclerosis-related vascular disorders are risk factors for cognitive decline with aging. In a small clinical study in men, testosterone improved cognitive function; however, it is unknown how testosterone ameliorates the pathogenesis of cognitive decline with aging. Here, we investigated whether the cognitive decline in senescence-accelerated mouse prone 8 (SAMP8), which exhibits cognitive impairment and hypogonadism, could be reversed by testosterone, and the mechanism by which testosterone inhibits cognitive decline. We found that treatment with testosterone ameliorated cognitive function and inhibited senescence of hippocampal vascular endothelial cells of SAMP8. Notably, SAMP8 showed enhancement of oxidative stress in the hippocampus. We observed that an NAD+-dependent deacetylase, SIRT1, played an important role in the protective effect of testosterone against oxidative stress-induced endothelial senescence. Testosterone increased eNOS activity and subsequently induced SIRT1 expression. SIRT1 inhibited endothelial senescence via up-regulation of eNOS. Finally, we showed, using co-culture system, that senescent endothelial cells promoted neuronal senescence through humoral factors. Our results suggest a critical role of testosterone and SIRT1 in the prevention of vascular and neuronal aging

Senescent endothelial cells of hippocampus are decreased by treatment with DHT.

<p><b>A.</b> Oxidative stress level was measured by detection of carbonyl groups introduced into proteins. <b>B.</b> Acetyl-choline concentration was measured by a colorimetric method. <b>C.</b> SA-βgal-stained endothelial cells and SIRT1 expression in CA3 area of hippocampus in SAMR1 and SAMP8 with or without DHT treatment. Immunofluorescent staining for SIRT1 (green), PECAM-1 (red), and DAPI (blue). <b>D.</b> Number of SA-βgal-stained endothelial cells in CA3 area of hippocampus in SAMR1 and SAMP8 with or without DHT treatment. <b>E.</b> Expression of SIRT1, PECAM-1, and β-actin was analyzed using cerebral micro vascular cells. <b>F.</b> Escape latency of SAMR1 (N = 10) and SAMP8 mice (N = 10). Male mice were treated daily for 2 weeks with DHT (500 µg s.c) and L-NAME (20 mg/kg gavage) before trials. <b>G.</b> Escape latency of SAMR1 (N = 5) and SAMP8 mice (N = 5). Male mice were treated daily for 2 weeks with DHT (500 µg s.c) and L-VNIO (5 mg/kg IP) before trials. (*p<0.05, n.s: not significant).</p

Supplementation of testosterone improves cognitive function in SAMP8 mice.

<p><b>A.</b> Escape latency and plasma testosterone level of male SAMR1 (N = 10) and SAMP8 mice (N = 10) at 18 months of age. These mice were implanted subcutaneously with a placebo or a 21-day-release 2.5 mg testosterone pellet in the dorsal neck. <b>B.</b> Number of SA-βgal-stained Leydig cells in testes in SAMR1 and SAMP8. Arrows indicate Leydig cells. Representative SA-βgal-stained testes from SAMR1 and SAMP8. <b>C.</b> Escape latency of castrated SAMR1 (upper, N = 5) and recipient SAMP8 (lower, N = 5). Observation (0–10 weeks) was started from 3 weeks after operation. <b>D.</b> SIRT1 expression in hippocampus of SAMP8 with or without DHT treatment. Immunofluorescent staining for SIRT1 (green) and DAPI (blue). <b>E.</b> Expression of AR in SAMR1 and SAMP8 brains. (*p<0.05).</p

Testosterone inhibits oxidative stress-induced endothelial senescence through eNOS/SIRT1.

<p><b>A.</b> Testosterone inhibited SA-βgal activity and senescent morphological appearance induced by hydrogen peroxide (100 µmol/L). <b>B.</b> Expression of eNOS, SIRT1, and PAI-1 in hydrogen peroxide (100 µmol/L)-treated HUVEC under treatment with DHT or testosterone. <b>C.</b> Overexpression of SIRT1 and DHT reduced SA-βgal activity. eNOS expression was increased by overexpression of SIRT1, and DHT increased phosphorylation of eNOS (Ser1177). <b>D.</b> SIRT1 inhibition by siRNA or sirtinol (100 µmol/L) abrogated the effect of testosterone on SA-βgal activity. <b>E.</b> Treatment with testosterone or DHT increased eNOS activity. <b>F.</b> eNOS inhibition by siRNA or L-NAME (10 mM) abrogated the effect of testosterone on SA-βgal activity. <b>G.</b> Treatment with L-NAME decreased SIRT1 expression in DHT-treated HUVEC. (*p<0.05, N = 3).</p

Testosterone deficiency causes senescence of hippocampus and cognitive impairment in SAMP8 mice.

<p><b>A.</b> Body weight, appearance, and plasma testosterone level of male SAMR1 and SAMP8 mice at 12 weeks of age. <b>B.</b> Escape latency of SAMR1 (N = 10) and SAMP8 mice (N = 10). Male mice were treated daily for 2 weeks with DHT (500 µg s.c) before trials. Swim speed during quadrant test on day 10. <b>C.</b> Total distance and the ratio of central/total distance were measured in open field tests. <b>D.</b> Number of amyloid ß plaques, pyramidal cells, and SA-ßgal-positive cells in CA1 and CA3 areas of hippocampus in SAMR1 and SAMP8. (*p<0.05, n.s: not significant).</p

Oxidative stressed-induced endothelial cell senescence promotes adjacent neuronal cell senescence.

<p><b>A.</b> Co-culture cell culture dish. <b>B.</b> Number of SA-βgal-stained MHC and senescent appearance of MHC were increased, and acetyl-choline concentration was decreased by co-culture with senescent endothelial cells. Senescent MHC are indicated by arrows. <b>C.</b> Expression of SIRT1, PAI-1, p53, and β-actin in MHC co-cultured with senescent endothelial cells. <b>D.</b> Expression of IL-6, IL-8, MCP-1, and TNF-α in endothelial cells were analyzed by RT-PCR. <b>E.</b> The number of SA-βgal-stained MHC was decreased by treatment with testosterone in both MHC and HUVEC (MHC, testosterone (+)), or HUVEC (MHC, testosterone (−)) alone. <b>F.</b> Resveratrol decreased the number of SA-βgal-stained MHC co-cultured with senescent endothelial cells. (*p<0.05, N = 3). <b>G.</b> Hypothetical signal transduction pathways of testosterone in endothelial cells.</p