Hypoxia-inducible factor-1 α deletion in myeloid lineage attenuates hypoxia-induced pulmonary hypertension.

Hypoxemia is seen in patients with pulmonary hypertension and hypoxic pulmonary vasoconstriction worsens their clinical condition. However, vasoconstriction is not the only aspect through which hypoxia induces the progression to pulmonary hypertension. Hypoxia-inducible factor-1α (HIF-1α) is a transcription factor responding to hypoxic conditions by regulating hundreds of genes involved in angiogenesis, erythropoiesis, inflammation, and proliferation. We sought to determine the contribution of HIF-1α in myeloid lineage cells to the pulmonary vascular response to chronic exposure to hypoxia. We generated myeloid-specific HIF-1α knockout (MyeHIF1KO) mice by using Cre-lox P system, and exposed them to hypoxic conditions for 3 weeks to induce pulmonary hypertension. Macrophages from MyeHIF1KO and control mice were used for western blotting, RT-qPCR, chemotaxis assay, and ATP assay. MyeHIF1KO mice exposed to hypoxia for 3 weeks exhibited a significant reduction in the right ventricular systolic pressure accompanied by a decrease in the ratio of the right ventricular weight to left ventricular weight, muscularization of the small pulmonary arteries, and infiltration of macrophages into the lung and right ventricle compared with control mice. HIF-1α-deficient peritoneal macrophages showed less migration toward monocyte chemoattractant protein-1 and a decrease in intracellular ATP levels. These results indicate that HIF-1α in macrophages contributes to the progression of pulmonary vascular remodeling and pulmonary hypertension induced by chronic exposure to hypoxic conditions. The inhibition of myeloid-specific HIF-1α may be a novel therapeutic strategy for the treatment of pulmonary hypertension.

Suppression of Abdominal Aortic Aneurysm Formation in Mice by Teneligliptin, a Dipeptidyl Peptidase-4 Inhibitor.

AIM: Dipeptidyl peptidase-4 (DPP-4) inhibitors lower blood glucose levels through inhibition of incretin degradation, which stimulates insulin secretion. Recent studies reported that DPP-4 inhibitors suppressed atherogenesis in apolipoprotein E-knockout (ApoEKO) mice. In this study, we investigated whether teneligliptin, a DPP-4 inhibitor, affects the development of abdominal aortic aneurysms (AAA) in ApoEKO mice. METHODS: ApoEKO mice were fed a high-fat diet (HFD) and infused with angiotensin (Ang) II by osmotic mini pumps for 4 weeks to induce AAA with (DPP-4i group) or without (control group) teneligliptin administered orally from 1 week before HFD and Ang II infusion to the end of the experiment. Confluent rat vascular smooth muscle cells (VSMCs) were serum-starved for 48 hours, then incubated with or without teneligliptin for another 24 hours and stimulated with Ang II. RESULTS: Treatment with teneligliptin significantly reduced the AAA formation rate (30.7% vs. 71.4% vs. control, P＜0.05), aortic dilatation (1.32±0.09 mm vs. 1.76±0.18 mm in the control, P＜0.05) and severity score (0.75±0.28 vs. 1.91±0.4 in the control, P＜0.05). Elastin degradation grade was also attenuated in DPP-4i group (2.83±0.17 vs. 3.45±0.16 in the control, P＜0.05). The number of macrophages infiltrating into the abdominal aorta was decreased in the DPP-4i group (51.8± 29.8/section vs. 219.5±78.5/section in the control, P＜0.05). Teneligliptin attenuated Ang II-induced phosphorylation of extracellular signal-regulated kinase (ERK) and Akt, and mRNA expression of monocyte chemoattractant protein-1 in VSMCs. CONCLUSION: Treatment with teneligliptin suppressed AAA formation in ApoEKO mice with HFD and Ang II infusion. Suppression of macrophage infiltration by teneligliptin may be involved in the inhibition of AAA formation.

A case of coronary subclavian vertebral steal syndrome successfully treated with stenting to the stenosis of left subclavian artery.

Coronary subclavian vertebral steal syndrome (CSVSS) is a rare but important complication of coronary artery bypass graft surgery (CABG) when an internal mammary artery (IMA) is used. This syndrome is defined as a retrograde flow from coronary artery via the IMA and the vertebral artery to the subclavian artery due to a proximal subclavian artery stenosis. We describe a case of a 64-year-old female who underwent CABG, complaining of dyspnea and chest pain by exercise of left arm, and dizziness when she turned her face to the left. Her blood pressure was 113/69 mmHg in the left arm and 137/84 mmHg in the right arm. Coronary angiography revealed retrograde flow from the left anterior descending (LAD) artery to the left IMA. Aortography showed that the ostium of the left subclavian artery had a severe stenosis and that the left vertebral artery was visualized retrogradely. Thereby, the diagnosis of CSVSS was made. The stenosis of the left subclavian artery was successfully treated with a percutaneous transluminal angioplasty and stent implantation, resulting in the restoration of antegrade flow from the left IMA to the LAD artery and from the left subclavian artery to the left vertebral artery. She was discharged with no chest pain and dizziness. .

PPARγ Agonists Attenuate Palmitate-Induced ER Stress through Up-Regulation of SCD-1 in Macrophages.

BACKGROUND: Clinical trials have shown that treatment of patients with type 2 diabetes with pioglitazone, a peroxisome proliferator-activated receptor (PPAR)γ agonist, reduces cardiovascular events. However, the effect of PPARγ agonists on endoplasmic reticulum (ER) stress that plays an important role in the progression of atherosclerosis has not been determined. We sought to determine the effect of PPARγ agonists on ER stress induced by palmitate, the most abundant saturated fatty acid in the serum. METHODS AND RESULTS: Protein expression of ER stress marker was evaluated by Western blot analysis and stearoyl-CoA desaturase1 (SCD-1) mRNA expression was evaluated by qRT-PCR. Macrophage apoptosis was detected by flowcytometry. Pioglitazone and rosiglitazone reduced palmitate-induced phosphorylation of PERK, a marker of ER stress, in RAW264.7, a murine macrophage cell line. Pioglitazone also suppressed palmitate-induced apoptosis in association with inhibition of CHOP expression, JNK phosphorylation and cleavage of caspase-3. These effects of pioglitazone were reversed by GW9662, a PPARγ antagonist, indicating that PPARγ is involved in this process. PPARγ agonists increased expression of SCD-1 that introduces a double bond on the acyl chain of long-chain fatty acid. 4-(2-Chlorophenoxy)-N-(3-(3-methylcarbamoyl)phenyl)piperidine-1-carboxamide, an inhibitor of SCD-1, abolished the anti-ER stress and anti-apoptotic effects of pioglitazone. These results suggest that PPARγ agonists attenuate palmitate-induced ER stress and apoptosis through SCD-1 induction. Up-regulation of SCD-1 may contribute to the reduction of cardiovascular events by treatment with PPARγ agonists.

A case of Kounis syndrome associated with transcatheter arterial chemoembolization for hepatocellular carcinoma.

Kounis syndrome, which is known as allergic angina and allergic myocardial infarction today, was described as the coexistence of acute coronary syndrome with allergic reactions in 1991 by Kounis and Zavras. We report a case of a 79-year-old man with hypertension, hepatocellular carcinoma (HCC), and no allergic history. He had received transcatheter arterial chemoembolization (TACE) for treatment of HCC five times without allergic reactions. At the sixth time of TACE, he presented an anaphylactic reaction such as systemic erythema and severe arterial hypotension. Simultaneously, he complained of anterior chest pain and electrocardiogram showed significant ST segment elevation in inferior leads, indicating inferior myocardial infarction. Emergency coronary angiography, however, did not demonstrate any organic stenoses or occluded lesions of the coronary arteries. We made the diagnosis of Kounis syndrome associated with TACE. Although Kounis syndrome is a rare condition, physicians should be aware of possible co-occurrence of anaphylactic reactions and acute coronary syndrome. .

Novel roles of hypoxia response system in glucose metabolism and obesity.

Oxygen is essential for ATP production in mitochondria through oxidative phosphorylation. Metazoans are equipped with the hypoxia response system that includes hypoxia-inducible factor (HIF), prolyl hydroxylase domain protein (PHD), and von Hippel-Lindau ubiquitin ligase system to combat or adapt hypoxic conditions. PHD is an oxygen-sensing enzyme that is responsible for HIF-α hydroxylation and subsequent proteasomal degradation at normoxic conditions. In hypoxic conditions, PHD activity is inhibited and transcriptional activity of HIF is increased, resulting in the induction of a broad range of genes that are involved in glucose metabolism, angiogenesis, and erythropoiesis. A worldwide epidemic of obesity, a critical risk factor for diabetes and cardiovascular diseases, has led to intense studies on adipose tissue biology, which revealed that adipose tissue functions as an endocrine organ that affects the whole body. Recent studies also suggest that inflammation and hypoxia of adipose tissue that occur as adipose tissue mass expands play an important role in the development of insulin resistance, in which PHD/HIF pathway is critically involved. The PHD/HIF pathway may be an attractive and potential target for the treatment of obesity and associated diseases.

Suppression of abdominal aortic aneurysm formation by inhibition of prolyl hydroxylase domain protein through attenuation of inflammation and extracellular matrix disruption.

In the present study we sought to determine the effect of CoCl2, an inhibitor of PHD (prolyl hydroxylase domain protein), on the development of AAA (abdominal aortic aneurysm). AAA was induced in C57BL/6 mice by periaortic application of CaCl2 (AAA group). NaCl (0.9%)-treated mice were used as a sham control (SHAM group). Mice were treated with 0.05% CoCl2 in the drinking water (AAA/CoCl2 group). At 1 and 6 weeks after the operation, aortic tissue was excised for further examination. After 6 weeks of CaCl2 treatment, aortic diameter and macrophage infiltration into the aortic adventitia were increased in the AAA group compared with the SHAM group. Treatment with CoCl2 reduced the aneurysmal size and macrophage infiltration compared with the AAA group. Aortic expression of inflammatory cytokines and MCP-1 (monocyte chemoattractant protein-1) and the activities of MMP-9 (matrix metalloproteinase-9) and MMP-2 were enhanced in the AAA group and attenuated in the AAA/CoCl2 group. Expression of cytokines and the activities of MMPs were already increased after 1 week of CaCl2 treatment, but were suppressed by CoCl2 treatment in association with reduced NF-κB (nuclear factor κB) phosphorylation. Treatment with CoCl2 in mice prevented the development of CaCl2-induced AAA in association with reduced inflammation and ECM (extracellular matrix) disruption. The results of the present study suggest that PHD plays a critical role in the development of AAA and that there is a therapeutic potential for PHD inhibitors in the prevention of AAA development.

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 (Phd2(f/f)/aP2-Cre). Phd2(f/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 Phd2(f/f)/aP2-Cre mice. Target genes of hypoxia-inducible factor, including glycolytic enzymes and adiponectin, were upregulated in adipocytes of Phd2(f/f)/aP2-Cre mice. Lipid content was decreased and uncoupling protein-1 expression was increased in brown adipose tissue of Phd2(f/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.

Chitinase inhibition promotes atherosclerosis in hyperlipidemic mice.

Chitinase 1 (CHIT1) is secreted by activated macrophages. Chitinase activity is raised in atherosclerotic patient sera and is present in atherosclerotic plaque. However, the role of CHIT1 in atherosclerosis is unknown. Preliminary studies of atherosclerosis in cynomolgous monkeys revealed CHIT1 to be closely correlated with areas of macrophage infiltration. Thus, we investigated the effects of a chitinase inhibitor, allosamidin, on macrophage function in vitro and on atherosclerotic development in vivo. In RAW264.7 cells, allosamidin elevated monocyte chemoattractant protein 1 and tumor necrosis factor alpha expression, and increased activator protein 1 and nuclear factor-κB transcriptional activity. Although inducible nitric oxide synthase, IL-6, and IL-1ß expression were increased, Arg1 expression was decreased by chitinase inhibition, suggesting that suppression of CHIT1 activity polarizes macrophages into a M1 phenotype. Allosamidin decreased scavenger receptor AI, CD36, ABCA1, and ABCG1 expression which led to suppression of cholesterol uptake and apolipoprotein AI-mediated cholesterol efflux in macrophages. These effects were confirmed with CHIT1 siRNA transfection and CHIT1 plasmid transfection experiments in primary macrophages. Apolipoprotein E-deficient hyperlipidemic mice treated for 6 weeks with constant administration of allosamidin and fed an atherogenic diet showed aggravated atherosclerotic lesion formation. These data suggest that CHIT1 exerts protective effects against atherosclerosis by suppressing inflammatory responses and polarizing macrophages toward an M2 phenotype, and promoting lipid uptake and cholesterol efflux in macrophages.

Resveratrol attenuates angiotensin II-induced senescence of vascular smooth muscle cells.

Resveratrol (3,5,4'-trihydroxystilbene), a polyphenol abundant in red wine, is known to extend the life span of diverse species. On the contrary, it was reported that angiotensin (Ang) II enhances senescence of vascular smooth muscle cells (VSMCs). We, therefore, examined whether resveratrol attenuates Ang II-induced senescence of VSMC. Senescence-associated ß-galactosidase (SA ß-gal) assay showed that Ang II induced senescence of VSMC. The Ang II-induced senescence was inhibited by losartan, an Ang II type 1 receptor (AT1R) antagonist but not by PD123319, Ang II type 2 receptor antagonist, indicating that AT1R is responsible for the induction of senescence. Resveratrol suppressed Ang II-induced senescence of VSMC in a dose-dependent manner. In addition, resveratrol suppressed Ang II-induced induction of p53 and its downstream target gene p21, both of which play an important role in the induction of senescence. Resveratrol suppressed senescence of VSMC possibly through inhibition of AT1R-dependent induction of p53/p21. Suppression of p53 induction may be involved in the longevity by resveratrol.

Beraprost sodium, a stable prostacyclin analogue, improves insulin resistance in high-fat diet-induced obese mice.

Obesity induces hypertrophy of adipocyte resulting in production of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and monocyte chemoattractant protein 1 (MCP1 (CCL2)). These cytokines play an important role in the development of insulin resistance. Beraprost sodium (BPS), a prostaglandin I2 analogue, is reported to attenuate inflammation. In this study, we examined the effect of BPS on glucose metabolism in mice fed a high-fat diet (HFD). Four-week-old C57/B6 male mice were fed a HFD for 12 weeks (HFD group) and the treatment group received oral BPS (300 µg/kg per day) for the same period. Then, glucose metabolism, histological changes, and gene expression of white adipose tissue (WAT) were examined. Body weight was increased, and glucose intolerance and insulin resistance were developed in the HFD group. Treatment with BPS improved glucose tolerance and insulin action without body weight change. Histological analysis of WAT showed an increase in the size of adipocyte and macrophage infiltration in the HFD group, which was attenuated by BPS treatment. BPS reduced HFD-induced expression of MCP1 and TNF-α in WAT. BPS also attenuated hepatic steatosis induced by the HFD. These results suggest that BPS improved glucose intolerance possibly through suppression of inflammatory cytokines in WAT. BPS may be beneficial for the treatment of obesity-associated glucose intolerance.

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 (Alzheimer's 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)-1ß and VEGF (vascular endothelial growth factor) in the ischaemic hindlimb. Intramuscular injections of IL-1ß to the ischaemic hindlimb reversed the donepezil-induced VEGF down-regulation and the anti-angiogenic effect. Hypoxia induced IL-1ß 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-1ß induction, which is followed by reduction of VEGF expression. Acetylcholinesterase inhibitor may be a novel anti-angiogenic therapy.

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.

Acetylcholinesterase inhibitors attenuate atherogenesis in apolipoprotein E-knockout mice.

OBJECTIVE: Donepezil, a reversible acetylcholinesterase inhibitor, improves cognitive function of Alzheimer's 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 prolyl hydroxylase domain-containing protein suppressed lipopolysaccharide-induced TNF-alpha expression.

OBJECTIVE: Prolyl hydroxylase domain-containing proteins (PHDs) play pivotal roles in oxygen-sensing system through the regulation of alpha-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 alpha (TNF-alpha) induction in macrophages. METHODS AND RESULTS: Northern blot analysis and ELISA revealed that LPS-induced TNF-alpha upregulation was strongly suppressed by PHD inhibitors, dimethyloxallyl glycine (DMOG), and TM6008 in RAW264.7 macrophages. DMOG suppressed LPS-induced TNF-alpha upregulation in HIF-1alpha-depleted cells and HIF-1alpha overexpression failed to suppress the induction of TNF-alpha. DMOG rather suppressed LPS-induced NF-kappaB transcriptional activity. Downregulation of Phd1 or Phd2 mRNA by RNA interference partially attenuated LPS-induced TNF-alpha induction. DMOG also inhibited LPS-induced TNF-alpha production in peritoneal macrophages as well as human macrophages. CONCLUSIONS: PHD inhibition by DMOG or RNA interference inhibited LPS-induced TNF-alpha upregulation in macrophages possibly through NF-kappaB inhibition, which is independent of HIF-1alpha 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.

Improvement of neovascularization capacity of bone marrow mononuclear cells from diabetic mice by ex vivo pretreatment with resveratrol.

Implantation of bone marrow-derived mononuclear cells (BMMCs) is known to accelerate blood flow recovery in a hindlimb ischemia model in mice. However, the neovascularization capacity of BMMCs from diabetic mice is impaired. Resveratrol, a natural polyphenolic compound abundant in red wine, is known to extend the lifespan of high cholesterol-fed mice. We tested whether resveratrol improves the neovascularization capacity of BMMCs from diabetic mice. Diabetes was induced by the injection of streptozotocin into C57B/6 mice. BMMCs from normal mice and diabetic mice were implanted into the ischemic limb induced by ligation of the unilateral femoral artery. Blood flow recovery measured by the laser Doppler method was significantly decreased in mice that received BMMCs from diabetic mice compared with BMMCs from normal mice. However, ex vivo treatment of BMMCs from diabetic mice, but not from normal mice, with resveratrol for 30 min significantly improved blood flow recovery. Capillary density measured by PECAM-1 positive cells was significantly increased in mice that received either normal BMMCs or diabetic BMMCs treated with resveratrol. Treatment of BMMCs from diabetic mice with resveratrol increased mRNA expression of vascular endothelial growth factor and endothelial nitric oxide synthase and decreased production of reactive oxygen species. Resveratrol improved the impaired neovascularization capacity of BMMCs derived from diabetic mice. The effects of resveratrol may be due to a reduction of oxidative stress and an induction of angiogenic factors. Resveratrol may be beneficial by improving the neovascularization capacity of BMMCs in patients with diabetes mellitus.

CREB-mediated IL-6 expression is required for 15(S)-hydroxyeicosatetraenoic acid-induced vascular smooth muscle cell migration.

OBJECTIVE: Migration of vascular smooth muscle cells (VSMCs) from media to intima is a key event in the pathophysiology of atherosclerosis and restenosis. The lipoxygenase products of polyunsaturated fatty acids (PUFA) were shown to play a role in these diseases. cAMP response element binding protein (CREB) has been implicated in the regulation of VSMC growth and motility in response to thrombin and angiotensin II. The aim of the present study was to test the role of CREB in an oxidized lipid molecule, 15(S)-HETE-induced VSMC migration and neointima formation. METHODS AND RESULTS: 15(S)-HETE stimulated VSMC migration in CREB-dependent manner, as measured by the modified Boyden chamber method. Blockade of MEK1, JNK1, or p38MAPK inhibited 15(S)-HETE-induced CREB phosphorylation and VSMC migration. 15(S)-HETE induced expression and secretion of interleukin-6 (IL-6), as analyzed by RT-PCR and ELISA, respectively. Neutralizing anti-IL-6 antibodies blocked 15(S)-HETE-induced VSMC migration. Dominant-negative mutant-mediated blockade of ERK1/2, JNK1, p38MAPK, or CREB suppressed 15(S)-HETE-induced IL-6 expression in VSMCs. Serial 5' deletions and site-directed mutagenesis of IL-6 promoter along with chromatin immunoprecipitation using anti-CREB antibodies showed that cAMP response element is essential for 15(S)-HETE-induced IL-6 expression. Dominant-negative CREB also suppressed balloon injury-induced IL-6 expression, SMC migration from media to intimal region, and neointima formation. Adenovirus-mediated transduction of 15-lipoxygenase 2 (15-LOX2) caused increased production of 15-HETE in VSMCs and enhanced IL-6 expression, SMC migration from media to intimal region, and neointima formation in response to arterial injury. CONCLUSIONS: The above results suggest a role for 15-LOX2-15-HETE in the regulation of VSMC migration and neointima formation involving CREB-mediated IL-6 expression.

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.

Inhibition of tumor necrosis factor-alpha-induced interleukin-6 expression by telmisartan through cross-talk of peroxisome proliferator-activated receptor-gamma with nuclear factor kappaB and CCAAT/enhancer-binding protein-beta.

Telmisartan, an angiotensin II type 1 receptor antagonist, was reported to be a partial agonist of peroxisome proliferator-activated receptor-gamma. Although peroxisome proliferator-activated receptor-gamma 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-alpha (TNF-alpha). Telmisartan decreased TNF-alpha-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-gamma antagonist, peroxisome proliferator-activated receptor-gamma may be involved in the process. Telmisartan suppressed IL-6 gene promoter activity induced by TNF-alpha. Deletion analysis suggested that the DNA segment between -150 bp and -27 bp of the IL-6 gene promoter that contains nuclear factor kappaB and CCAAT/enhancer-binding protein-beta sites was responsible for telmisartan suppression. Telmisartan attenuated TNF-alpha-induced nuclear factor kappaB- and CCAAT/enhancer-binding protein-beta-dependent gene transcription and DNA binding. Telmisartan also attenuated serum IL-6 level in TNF-alpha-infused mice and IL-6 production from rat aorta stimulated with TNF-alpha ex vivo. These data suggest that telmisartan may attenuate inflammatory process induced by TNF-alpha in addition to the blockade of angiotensin II type 1 receptor. Because both TNF-alpha 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.