Ryohei Miyazaki

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.

Prolyl Hydroxylase Domain Protein 2 Plays a Critical Role in Diet-Induced Obesity and Glucose Intolerance

Background— Recent studies suggest that the oxygen-sensing pathway consisting of transcription factor hypoxia-inducible factor and prolyl hydroxylase domain proteins (PHDs) plays a critical role in glucose metabolism. However, the role of adipocyte PHD in the development of obesity has not been clarified. We examined whether deletion of PHD2, the main oxygen sensor, in adipocytes affects diet-induced obesity and associated metabolic abnormalities. Methods and Results— To delete PHD2 in adipocyte, PHD2-floxed mice were crossed with aP2-Cre transgenic mice (Phd2f/f/aP2-Cre). Phd2f/f/aP2-Cre mice were resistant to high-fat diet–induced obesity (36.7±1.7 versus 44.3±2.0 g in control; P<0.01) and showed better glucose tolerance and homeostasis model assessment–insulin resistance index than control mice (3.6±1.0 versus 11.1±2.1; P<0.01). The weight of white adipose tissue was lighter (epididymal fat, 758±35 versus 1208±507 mg in control; P<0.01) with a reduction in adipocyte size. Macrophage infiltration into white adipose tissue was also alleviated in Phd2f/f/aP2-Cre mice. Target genes of hypoxia-inducible factor, including glycolytic enzymes and adiponectin, were upregulated in adipocytes of Phd2f/f/aP2-Cre mice. Lipid content was decreased and uncoupling protein-1 expression was increased in brown adipose tissue of Phd2f/f/aP2-Cre mice. Knockdown of PHD2 in 3T3L1 adipocytes induced a decrease in the glucose level and an increase in the lactate level in the supernatant with upregulation of glycolytic enzymes and reduced lipid accumulation. Conclusions— PHD2 in adipose tissue plays a critical role in the development of diet-induced obesity and glucose intolerance. PHD2 might be a novel target molecule for the treatment of obesity and associated metabolic abnormalities.

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.

Inhibition of Prolyl Hydroxylase Domain-Containing Protein Downregulates Vascular Angiotensin II Type 1 Receptor

Inhibition of prolyl hydroxylase domain-containing protein (PHD) by hypoxia stabilizes hypoxia-inducible factor 1 and increases the expression of target genes, such as vascular endothelial growth factor. Although the systemic renin-angiotensin system is activated by hypoxia, the role of PHD in the regulation of the renin-angiotensin system remains unknown. We examined the effect of PHD inhibition on the expression of angiotensin II type 1 receptor (AT1R). Hypoxia, cobalt chloride, and dimethyloxalylglycine, all known to inhibit PHD, reduced AT1R expression in vascular smooth muscle cells. Knockdown of PHD2, a major isoform of PHDs, by RNA interference also reduced AT1R expression. Cobalt chloride diminished angiotensin II–induced extracellular signal–regulated kinase phosphorylation. Cobalt chloride decreased AT1R mRNA through transcriptional and posttranscriptional mechanisms. Oral administration of cobalt chloride (14 mg/kg per day) to C57BL/6J mice receiving angiotensin II infusion (490 ng/kg per minute) for 4 weeks significantly attenuated perivascular fibrosis of the coronary arteries without affecting blood pressure level. These data suggest that PHD inhibition may be beneficial for the treatment of cardiovascular diseases by inhibiting renin-angiotensin system via AT1R downregulation.

Acetylcholinesterase inhibitors attenuate angiogenesis.

Donepezil {(RS)-2-[(1-benzyl-4-piperidyl)methyl]-5,6-dimethoxy-2,3-dihydroinden-1-one} is a reversible acetylcholinesterase inhibitor and used for treatment of patients with AD (Alzheimers disease). Recent studies showed that treatment with donepezil reduced production of inflammatory cytokines in PBMCs (peripheral blood mononuclear cells). It was also reported that muscle-derived inflammatory cytokines play a critical role in neovascularization in a hindlimb ischaemia model. We sought to determine whether donepezil affects angiogenesis. A hindlimb ischaemia model was created by unilateral femoral artery ligation. Blood flow recovery examined by laser Doppler perfusion imaging and capillary density by immunohistochemical staining of CD31-positive cells in the ischaemic hindlimb were significantly decreased in donepezil- and physostigmine-treated mice compared with control mice after 2 weeks. Donepezil reduced expression of IL (interleukin)-1β and VEGF (vascular endothelial growth factor) in the ischaemic hindlimb. Intramuscular injections of IL-1β to the ischaemic hindlimb reversed the donepezil-induced VEGF down-regulation and the anti-angiogenic effect. Hypoxia induced IL-1β expression in C2C12 myoblast cells, which was inhibited by pre-incubation with ACh (acetylcholine) or LY294002, a PI3K (phosphoinositide 3-kinase) inhibitor. Donepezil inhibited phosphorylation of Akt [also known as PKB (protein kinase B)], a downstream kinase of PI3K, in the ischaemic hindlimb. These findings suggest that cholinergic stimulation by acetylcholinesterase inhibitors suppresses angiogenesis through inhibition of PI3K-mediated IL-1β induction, which is followed by reduction of VEGF expression. Acetylcholinesterase inhibitor may be a novel anti-angiogenic therapy.

Thiamylal sodium increased inflammation and the proliferation of vascular smooth muscle cells

Background Thiamylal sodium is a common anesthetic barbiturate prepared in alkaline solution for clinical use. There is no previously reported study on the effects of barbiturates on the inflammation and proliferation of vascular smooth muscle cells (VSMCs). Here, we examined the effects of clinical-grade thiamylal sodium solution (TSS) on the inflammation and proliferation of rat VSMCs. Methods Expression levels of interleukin (IL)-1α, IL-1β, IL-6, and toll-like receptors in rat VSMCs were detected by quantitative reverse transcription-polymerase chain reaction and microarray analyses. The production of IL-6 by cultured VSMCs or ex vivo-cultured rat aortic segments was detected in supernatants by enzyme-linked immunosorbent assay. VSMC proliferation and viability were determined by the water-soluble tetrazolium-1 assay and trypan blue staining, respectively. Results TSS increased expression of IL-1α, IL-6, and TLR4 in VSMCs in a dose-dependent manner, and reduced IL-1β expression. Ex vivo TSS stimulation of rat aorta also increased IL-6. Low concentrations of TSS enhanced VSMC proliferation, while high concentrations reduced both cell proliferation and viability. Expression of IL-1 receptor antagonist, which regulates cell proliferation, was not increased by TSS stimulation. Exposure of cells to the TSS additive, sodium carbonate, resulted in significant upregulation of IL-1α and IL-6 mRNA levels, to a greater extent than TSS. Conclusions TSS-induced proinflammatory cytokine production by VSMCs is caused by sodium carbonate. However, pure thiamylal sodium has an anti-inflammatory effect in VSMCs. TSS exposure to VSMCs may promote vascular inflammation, leading to the progression of atherosclerosis or in-stent restenosis, resulting in vessel bypass graft failure.