Keita Inanaga

SIRT1, a Longevity Gene, Downregulates Angiotensin II Type 1 Receptor Expression in Vascular Smooth Muscle Cells

Objective—Resveratrol (3,5,4′-trihydroxystilbene), a polyphenol found in red wine, is known to activate sirtuin1 (SIRT1), a longevity gene. Previous studies have demonstrated that resveratrol extends the life span of diverse species through activation of SIRT1. It was also reported that inhibition of angiotensin II function by angiotensin II type I receptor (AT1R) antagonist prolonged rat life span. We, therefore, hypothesized that resveratrol may inhibit the renin-angiontein system and examined whether resveratrol affects AT1R expression in vascular smooth muscle cells (VSMCs). Methods and Results—Northern and Western blot analysis revealed that resveratrol significantly decreased the expression of AT1R at mRNA and protein levels in a dose- and time-dependent manner. Overexpression of SIRT1 reduced AT1R expression whereas nicotinamide, an inhibitor of SIRT1, increased AT1R expression and reversed the resveratrol-induced AT1R downregulation. AT1R gene promoter activity was decreased by resveratrol, but resveratrol did not affect the AT1R mRNA stability. Deletion analysis showed that the most proximal region of AT1R gene promoter containing Sp1 site is responsible for downregulation. Administration of resveratrol suppressed AT1R expression in the mouse aorta and blunted angiotensin II–induced hypertension. Conclusion—Resveratrol suppressed AT1R expression through SIRT1 activation both in vivo and in vitro. The inhibition of the renin-angiotensin system may contribute, at least in part, to the resveratrol-induced longevity and antiatherogenic effect of resveratrol.

Inhibition of Prolyl Hydroxylase Domain-Containing Protein Suppressed Lipopolysaccharide-Induced TNF-α Expression

Objective—Prolyl hydroxylase domain-containing proteins (PHDs) play pivotal roles in oxygen-sensing system through the regulation of &agr;-subunit of hypoxia-inducible factor (HIF), a key transcription factor governing a large set of gene expression to adapt hypoxia. Although tissue hypoxia plays an essential role in maintaining inflammation, the role of PHDs in the inflammatory responses has not been clearly determined. Here, we investigated the role of PHDs in lipopolysaccharide (LPS)-induced tumor necrosis factor &agr; (TNF-&agr;) induction in macrophages. Methods and Results—Northern blot analysis and ELISA revealed that LPS-induced TNF-&agr; upregulation was strongly suppressed by PHD inhibitors, dimethyloxallyl glycine (DMOG), and TM6008 in RAW264.7 macrophages. DMOG suppressed LPS-induced TNF-&agr; upregulation in HIF-1&agr;–depleted cells and HIF-1&agr; overexpression failed to suppress the induction of TNF-&agr;. DMOG rather suppressed LPS-induced NF-&kgr;B transcriptional activity. Downregulation of Phd1 or Phd2 mRNA by RNA interference partially attenuated LPS-induced TNF-&agr; induction. DMOG also inhibited LPS-induced TNF-&agr; production in peritoneal macrophages as well as human macrophages. Conclusions—PHD inhibition by DMOG or RNA interference inhibited LPS-induced TNF-&agr; upregulation in macrophages possibly through NF-&kgr;B inhibition, which is independent of HIF-1&agr; accumulation. This study suggests that PHDs are positive regulators of LPS-induced inflammatory process, and therefore inhibition of PHD may be a novel strategy for the treatment of inflammatory diseases.

Inhibition of Tumor Necrosis Factor-α–Induced Interleukin-6 Expression by Telmisartan Through Cross-Talk of Peroxisome Proliferator-Activated Receptor-γ With Nuclear Factor κB and CCAAT/Enhancer-Binding Protein-β

Telmisartan, an angiotensin II type 1 receptor antagonist, was reported to be a partial agonist of peroxisome proliferator-activated receptor-γ. Although peroxisome proliferator-activated receptor-γ activators have been shown to have an anti-inflammatory effect, such as inhibition of cytokine production, it has not been determined whether telmisartan has such effects. We examined whether telmisartan inhibits expression of interleukin-6 (IL-6), a proinflammatory cytokine, in vascular smooth muscle cells. Telmisartan, but not valsartan, attenuated IL-6 mRNA expression induced by tumor necrosis factor-α (TNF-α). Telmisartan decreased TNF-α–induced IL-6 mRNA and protein expression in a dose-dependent manner. Because suppression of IL-6 mRNA expression was prevented by pretreatment with GW9662, a specific peroxisome proliferator-activated receptor-γ antagonist, peroxisome proliferator-activated receptor-γ may be involved in the process. Telmisartan suppressed IL-6 gene promoter activity induced by TNF-α. Deletion analysis suggested that the DNA segment between −150 bp and −27 bp of the IL-6 gene promoter that contains nuclear factor &kgr;B and CCAAT/enhancer-binding protein-β sites was responsible for telmisartan suppression. Telmisartan attenuated TNF-α–induced nuclear factor &kgr;B– and CCAAT/enhancer-binding protein-β–dependent gene transcription and DNA binding. Telmisartan also attenuated serum IL-6 level in TNF-α–infused mice and IL-6 production from rat aorta stimulated with TNF-α ex vivo. These data suggest that telmisartan may attenuate inflammatory process induced by TNF-α in addition to the blockade of angiotensin II type 1 receptor. Because both TNF-α and angiotensin II play important roles in atherogenesis through enhancement of vascular inflammation, telmisartan may be beneficial for treatment of not only hypertension but also vascular inflammatory change.

Resveratrol attenuates angiotensin II-induced interleukin-6 expression and perivascular fibrosis

Recent studies have shown that resveratrol (3,5,4′-trihydroxystilbene), a polyphenolic compound found in grapes and red wine, has various beneficial effects on cardiovascular diseases and prolongs the life span of mice fed a high-fat diet. We hypothesized that resveratrol may attenuate vascular inflammatory response induced by angiotensin (Ang) II. We examined the effect of resveratrol on Ang II-induced interleukin (IL)-6 expression in vascular smooth muscle cells (VSMCs). Resveratrol significantly attenuated Ang II-induced IL-6 mRNA expression and IL-6 protein in the supernatant of VSMC in a dose-dependent manner. Resveratrol suppressed the IL-6 gene promoter activity. Resveratrol inhibited the Ang II-induced cAMP-response element-binding protein and nuclear factor-kappa B activity, which are critical for Ang II-induced IL-6 gene activation. An increase in the serum concentration of IL-6 induced by Ang II infusion was attenuated by an oral administration of resveratrol. Resveratrol also inhibited Ang II-induced hypertension and perivascular fibrosis of the heart. Although hydralazine reduced blood pressure level equal to resveratrol, it did not reduce the Ang II-induced IL-6 production and perivascular fibrosis. These data suggest that the inhibition of Ang II-induced vascular inflammation and high blood pressure by resveratrol may contribute, at least in part, to the anti-atherogenic effects of resveratrol.

Liver X Receptor Activator Downregulates Angiotensin II Type 1 Receptor Expression Through Dephosphorylation of Sp1

Atherosclerosis is considered to be a combined disorder of lipid metabolism and chronic inflammation. Recent studies have reported that liver X receptors (LXRs) are involved in lipid metabolism and inflammation and that LXR agonists inhibit atherogenesis. In contrast, angiotensin II is well known to accelerate atherogenesis through activation of the angiotensin II type 1 receptor (AT1R). To better understand the mechanism of LXR on the prevention of atherogenesis, we examined whether activation of LXR affects AT1R expression in vascular smooth muscle cells. T0901317, a synthetic LXR ligand, decreased AT1R mRNA and protein expression with a peak reduction at 6 hours and 12 hours of incubation, respectively. A well-established ligand of LXR, 22-(R)-hydroxycholesterol, also suppressed AT1R expression. The downregulation of AT1R by T0901317 required de novo protein synthesis. AT1R gene promoter activity measured by luciferase assay revealed that the DNA segment between −61 bp and +25 bp was sufficient for downregulation. Luciferase construct with a mutation in Sp1 binding site located in this segment lost its response to T0901317. T0901317 decreased Sp1 serine phosphorylation. Although preincubation of vascular smooth muscle cells with T0901317 for 30 minutes had no effect on angiotensin II–induced extracellular signal–regulated kinase phosphorylation, phosphorylation of extracellular signal–regulated kinase by angiotensin II was markedly suppressed after 6 hours of preincubation. These results indicate that the suppression of AT1R may be one of the important mechanisms by which LXR ligands exert antiatherogenic effects.

Acetylcholinesterase inhibitors attenuate atherogenesis in apolipoprotein E-knockout mice

OBJECTIVE Donepezil, a reversible acetylcholinesterase inhibitor, improves cognitive function of Alzheimers disease. Stimulation of cholinergic system was reported to improve long-term survival of rats with chronic heart failure and to attenuate inflammatory response in mice with lipopolysaccharide-induced sepsis. We sought to determine whether the pharmacological stimulation of cholinergic system by donepezil reduces atherogenesis in apolipoprotein (Apo) E-knockout (KO) mice. METHODS AND RESULTS Male ApoE-KO mice (10-week-old) were fed a high-fat diet and received infusion of angiotensin (Ang) II (490 ng/kg/day). Donepezil or physostigmine was administered for 4 weeks. Oral administration of donepezil (5 mg/kg/day) or infusion of physostigmine (2 mg/kg/day) significantly attenuated atherogenesis (Oil Red O-positive area) without significant changes in heart rate, blood pressure and total cholesterol levels. Administration of donepezil suppressed expression of monocyte chemoattractant protein-1 and tumor necrosis factor-α, NADPH oxidase activity and production of reactive oxygen species in the aorta. CONCLUSION The present study revealed novel anti-oxidative and anti-atherosclerotic effects of pharmacological stimulation of cholinergic system by donepezil. Donepezil may be used as a novel therapeutics for the atherosclerotic cardiovascular diseases.

Inhibition of MDM2 attenuates neointimal hyperplasia via suppression of vascular proliferation and inflammation

AIMS Tumour protein p53 plays an important role in the vascular remodelling process as well as in oncogenesis. p53 is negatively regulated by murine double minute 2 (MDM2). A recently developed MDM2 inhibitor, nutlin-3, is a non-genotoxic activator of the p53 pathway. So far, the effect of MDM2 inhibition on vascular remodelling has not been elucidated. We therefore investigated the effect of nutlin-3 on neointima formation. METHODS AND RESULTS Nutlin-3 up-regulated p53 and its downstream target p21 in vascular smooth muscle cells (VSMCs). DNA synthesis assay and flow cytometric analysis revealed that nutlin-3 inhibited platelet-derived growth factor (PDGF)-induced VSMC proliferation by cell cycle arrest. This inhibitory effect was abrogated in p53-siRNA-transfected VSMCs. Furthermore, nutlin-3 inhibited PDGF-stimulated VSMC migration. Treatment with nutlin-3 attenuated neointimal hyperplasia at 28 days after vascular injury in mice, associated with up-regulation of p53 and p21. BrdU incorporation was decreased at 14 days after injury in nutlin-3-treated mice. TUNEL assay showed that nutlin-3 did not exaggerate apoptosis of the injured vessels. Infiltration of macrophages and T-lymphocytes and mRNA expression of chemokine (C-C motif) ligand-5, interleukin-6, and intercellular adhesion molecule-1 were decreased in the injured vessels of nutlin-3-treated mice. Nutlin-3 suppressed NF-κB activation in VSMCs, but not in p53-siRNA-transfected VSMCs. CONCLUSIONS The MDM2 antagonist nutlin-3 inhibits VSMC proliferation, migration, and NF-κB activation, and also attenuates neointimal hyperplasia after vascular injury in mice, which is associated with suppression of vascular cell proliferation and an inflammatory response. Targeting MDM2 might be a potential therapeutic strategy for the treatment of vascular proliferative diseases.

Inducible cAMP Early Repressor Inhibits Growth of Vascular Smooth Muscle Cell

Objective—The role of inducible cAMP early repressor (ICER), a transcriptional repressor, in the vascular remodeling process has not been determined. We examined whether ICER affects growth of vascular smooth muscle cells (VSMCs). Methods and Results—Semi-quantitative RT-PCR and Western blot analysis showed that expression of ICER was increased in beraprost (a prostaglandin I2 analogue)-stimulated VSMCs in a time- and dose-dependent manner. The induction of ICER was inhibited by pretreatment with H89, a protein kinase A (PKA) inhibitor, suggesting that PKA mediates the induction of ICER expression. Beraprost suppressed platelet-derived growth factor–induced thymidine incorporation in VSMCs, which was reversed by transfection of short interfering RNA for ICER, not by scramble RNA. Overexpression of ICER by an adenovirus vector attenuated neointimal formation (intima/media ratio) by 50% compared with overexpression of LacZ. The number of terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling–positive cells was increased and the number of Ki-67–positive cells was decreased in ICER-transduced artery. Conclusion—These results suggest that ICER induces apoptosis and inhibits proliferation of VSMCs, and plays a critical role in beraprost-mediated suppression of VSMC proliferation. ICER may be an important endogenous inhibitor of vascular proliferation.

Inhibition of Balloon Injury-Induced Neointimal Formation by Olmesartan and Pravastatin in Rats with Insulin Resistance

The combined effect of an angiotensin II type 1 receptor blocker and a 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor on vascular lesion formation in the insulin-resistant state has not been examined. We tested whether or not combined treatment is superior to single-drug treatment for inhibiting vascular lesion formation in insulin-resistant rats. The rats were maintained on a fructose-rich diet for 4 weeks and then treated with olmesartan (1 mg/kg/day) and/or pravastatin (10 mg/kg/day) for 3 weeks. After 1 week of drug treatment, balloon injury of the carotid arteries was performed. Two weeks later, the injured arteries were harvested for morphometry and immunostaining. Olmesartan and pravastatin each modestly attenuated neointimal formation without significant changes in blood pressure or serum lipid levels. The combination of olmesartan and pravastatin significantly suppressed the neointimal formation compared with either monotherapy. The number of terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling (TUNEL)−positive cells was increased by olmesartan but not by pravastatin. Olmesartan and pravastatin each decreased the number of Ki-67−positive cells, which indicates cell proliferation, to the same extent. The combined treatment increased the number of TUNEL-positive cells but did not affect the number of Ki-67−positive cells. The combined treatment decreased the insulin level and increased the number of circulating endothelial progenitor cells. These results suggest that the combination of olmesartan and pravastatin is beneficial for the treatment of vascular diseases in the insulin-resistant state independently of blood pressure or cholesterol levels.