Kikuo Kasai

Metformin Inhibits Cytokine-Induced Nuclear Factor κB Activation Via AMP-Activated Protein Kinase Activation in Vascular Endothelial Cells

AMP-activated protein kinase (AMPK) is tightly regulated by the cellular AMP:ATP ratio and plays a central role in regulation of energy homeostasis and metabolic stress. Metformin has been shown to activate AMPK. We hypothesized that metformin may prevent nuclear factor &kgr;B (NF-&kgr;B) activation in endothelial cells exposed to inflammatory cytokines. Metformin was observed to activate AMPK, as well as its downstream target, phosphoacetyl coenzyme A carboxylase, in human umbilical vein endothelial cells (HUVECs). Metformin also dose-dependently inhibited tumor necrosis factor (TNF)-α–induced NF-&kgr;B activation and TNF-α–induced I&kgr;B kinase activity. Furthermore, metformin attenuated the TNF-α–induced gene expression of various proinflammatory and cell adhesion molecules, such as vascular cell adhesion molecule-1, E-selectin, intercellular adhesion molecule-1, and monocyte chemoattractant protein-1, in HUVECs. A pharmacological activator of AMPK, 5-amino-4-imidazole carboxamide riboside (AICAR), dose-dependently inhibited TNF-α- and interleukin-1β–induced NF-&kgr;B reporter gene expression. AICAR also suppressed the TNF-α- and interleukin-1β–induced gene expression of vascular cell adhesion molecule-1, E-selectin, intercellular adhesion molecule-1, and monocyte chemoattractant protein-1 in HUVECs. The small interfering RNA for AMPKα1 attenuated metformin or AICAR–induced inhibition of NF-&kgr;B activation by TNF-α, suggesting a possible role of AMPK in the regulation of cell inflammation. In light of these findings, we suggest that metformin attenuates the cytokine-induced expression of proinflammatory and adhesion molecule genes by inhibiting NF-&kgr;B activation via AMPK activation. Thus, it might be useful to target AMPK signaling in future efforts to prevent atherogenic and inflammatory vascular disease.

Globular adiponectin upregulates nitric oxide production in vascular endothelial cells

Aims/hypothesisAdiponectin, also called ACRP30, is a novel adipose tissue-specific protein that has been shown to improve insulin sensitivity and to exert anti-atherogenic effects. It is known that knockout mice lacking endothelial NO synthase (eNOS) develop hypertension, insulin resistance, hyperlipidaemia, and show augmented ischaemia-reperfusion damage. Thus, we examined whether globular adiponectin activates eNOS to produce NO.MethodsTo analyze NO production in bovine aortic endothelial cells (BAE), NOx (nitrite and nitrate) was measured in the medium with an automated NO detector/high-performance liquid chromatography system. eNOS activation was assessed by phosphorylation of the enzyme and its activity was evaluated by citrulline synthesis in human umbilical vein endothelial cells (HUVEC). eNOS mRNA and protein expressions in HUVEC were evaluated by Realtime PCR and Western blot analysis.ResultsGobular adiponectin increased NO production in BAE. It also caused eNOS phosphorylation and potentiated eNOS activity in HUVEC. In addition, globular adiponectin up-regulated the eNOS gene to increase protein expression in HUVEC.Conclusion/interpretationGlobular adiponectin increases NO production through two mechanisms, namely, by activation of eNOS enzyme activity and via an increase in eNOS expression. Activation and up-regulation of eNOS could explain some of the observed vasoprotective properties of globular adiponectin, as well as its beneficial effects on the cardiovascular system.

Vascular smooth muscle cell activation by C-reactive protein

OBJECTIVE C-reactive protein (CRP) is an important cardiovascular risk factor. Although the role of CRP has been implicated in atherogenesis, its direct effects on vascular cells are poorly defined. METHODS We investigated the responses to CRP in vascular smooth muscle cells (VSMC). RESULTS The present study shows that CRP induces parallel activation of the redox-responsive transcription factors NF-kappa B (NF-kappaB) and AP-1 and increases the activity of the MAP kinases (MAPKs), extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38MAPK, in VSMC. C-reactive protein increased the expression of early response genes, c-fos and c-jun and inflammatory genes, monocyte chemoattractant peptide (MCP-1) and interleukin-6 (IL-6). When VSMC were incubated with CRP, the inducible nitric oxide synthase (iNOS) promoter was activated. CRP alone was a weak inducer of NO production in VSMC as measured by determining nitrite levels, and interferon-gamma alone was totally ineffective, whereas CRP plus interferon-gamma was a powerful stimulus. This synergy for NO production corresponded to the results of iNOS mRNA expression analyzed by Northern blotting. The NF-kappaB activation caused by CRP was inhibited by 15-deoxy-12,14-prostaglandin J2 and the PPARgamma activators, rosiglitazone and pioglitazone. Fluvastatin and cerivastatin also reduced the activation of NF-kappaB by CRP. CONCLUSIONS CRP causes NF-kappaB activation which could lead to the induction of MCP-1, IL-6, and iNOS gene expression. CRP also activates the MAPK-->c-Fos/cJun-->AP-1 pathway. Thus, CRP may play a role in atherogenesis by activating VSMC.

RETRACTED ARTICLE: A glucagon-like peptide-1 (GLP-1) analogue, liraglutide, upregulates nitric oxide production and exerts anti-inflammatory action in endothelial cells

Aims/hypothesisGlucagon-like peptide-1 (GLP-1), a member of the proglucagon-derived peptide family, was seen to exert favourable actions on cardiovascular function in preclinical and clinical studies. The mechanisms through which GLP-1 modulates cardiovascular function are complex and incompletely understood. We thus investigated whether the GLP-1 analogue, liraglutide, which is an acylated GLP-1, has protective effects on vascular endothelial cells.MethodsNitrite and nitrate were measured in medium with an automated nitric oxide detector. Endothelial nitric oxide synthase (eNOS) activation was assessed by evaluating the phosphorylation status of the enzyme and evaluating eNOS activity by citrulline synthesis. Nuclear factor κB (NF-κB) activation was assessed by reporter gene assay.ResultsLiraglutide dose-dependently increased nitric oxide production in HUVECs. It also caused eNOS phosphorylation, potentiated eNOS activity and restored the cytokine-induced downregulation of eNOS (also known as NOS3) mRNA levels, which is dependent on NF-κB activation. We therefore examined the effect of liraglutide on TNFα-induced NF-κB activation and NF-κB-dependent expression of proinflammatory genes. Liraglutide dose-dependently inhibited NF-κB activation and TNFα-induced IκB degradation. It also reduced TNFα-induced MCP-1 (also known as CCL2), VCAM1, ICAM1 and E-selectin mRNA expression. Liraglutide-induced enhancement of nitric oxide production and suppression of NF-κB activation were attenuated by the AMP-activated protein kinase (AMPK) inhibitor compound C or AMPK (also known as PRKAA1) small interfering RNA. Indeed, liraglutide induced phosphorylation of AMPK, which occurs through a signalling pathway independent of cyclic AMP.Conclusions/interpretationLiraglutide exerts an anti-inflammatory effect on vascular endothelial cells by increasing nitric oxide production and suppressing NF-κB activation, partly at least through AMPK activation. These effects may explain some of the observed vasoprotective properties of liraglutide, as well as its beneficial effects on the cardiovascular system.

HMG-CoA Reductase Inhibitor Increases GTP Cyclohydrolase I mRNA and Tetrahydrobiopterin in Vascular Endothelial Cells

Objective—Endothelial nitric oxide synthase (eNOS) activity is supported by tetrahydrobiopterin (BH4), which appears to be important for generating protective NO but decreases uncoupling formation of superoxide. We investigated the effects of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, or statins, in terms of BH4 metabolism in human umbilical vein endothelial cells (HUVECs). Methods and Results—We measured the mRNA levels of GTP cyclohydrolase I (GTPCH), the rate-limiting enzyme in the first step of de novo BH4 synthesis, by real-time polymerase chain reaction. The mRNA of GTPCH, as well as of eNOS, was upregulated in HUVECs treated with cerivastatin. This increase was time and dose dependent. Fluvastatin was also observed to enhance GTPCH and eNOS mRNA levels. In parallel with this observation, cerivastatin increased intracellular BH4. Incubating HUVECs with tumor necrosis factor (TNF-&agr;) was observed to increase GTPCH mRNA while decreasing eNOS mRNA. In the presence of cerivastatin, the TNF-&agr;–mediated increase in GTPCH mRNA was enhanced, and the TNF-&agr;–mediated decrease in eNOS mRNA was attenuated. Cerivastatin increased the stability of eNOS mRNA. However, it did not alter the stability of GTPCH mRNA but increased GTPCH gene transcription, as shown by nuclear run-on assays. Preteatment of HUVECs with the selective GTPCH inhibitor, 2,4-diamino-6-hydroxypyrimidine, caused a decrease in intracellular BH4 and decreased citrulline formation after stimulation with ionomycin. Furthermore, the potentiating effect of cerivastatin was decreased by limiting the cellular availability of BH4. Conclusions—Our data demonstrate that statins elevate GTPCH mRNA, thereby increasing BH4 levels in vascular endothelial cells. In addition to augmenting eNOS expression, statins potentiate GTPCH gene expression and BH4 synthesis, thereby increasing NO production and preventing relative shortages of BH4.

RETRACTED ARTICLE: Angiotensin-II-induced oxidative stress elicits hypoadiponectinaemia in rats

Aims/hypothesisHypertension, endothelial dysfunction and insulin resistance are associated conditions that share oxidative stress and vascular inflammation as common features. Adiponectin is an abundant plasma adipokine that plays a physiological role in modulating lipid metabolism and exerts a potent anti-inflammatory activity. We hypothesised that adiponectin levels decrease in response to oxidative stress and that this may promote the development of hypertension, endothelial dysfunction and insulin resistance.MethodsRats were infused with angiotensin II (AngII) or its vehicle, either alone or in combination with tempo1 (4-hydroxy-2,2,6,6-tetramethyl piperidinoxyl), a membrane-permeable metal-independent superoxide dismutase mimetic, or tetrahydrobiopterin (BH4), one of the most potent naturally occurring reducing agents and an essential cofactor for nitric oxide synthase activity. Heart rate, systolic blood pressure, body weight and serum levels of adiponectin were measured on day 7 of treatment, and then the animals were killed. Vessel tone and superoxide production were measured ex vivo in thoracic vascular rings. The expression of adiponectin mRNA in adipose tissue was assessed by Northern blotting, and in 3T3-L1 adipocytes exposed to H2O2 by real-time PCR. The expression of NAD(P)H oxidase subunit mRNAs in the rats was assessed by RT-PCR and real-time PCR.ResultsHypertension and endothelial dysfunction were induced in rats by infusion of AngII and reversed by administration of tempol. Plasma concentrations of adiponectin and adipose tissue levels of adiponectin mRNA were decreased in AngII-infused rats, and this effect was prevented by cotreatment with tempol or BH4. The production of superoxide anions (O2−) was significantly increased in the aortae of AngII-treated rats, and this increase was prevented by the administration of tempol or BH4. Levels of mRNAs that encode NAD(P)H oxidase components, including p22phox, gp91phox, p47phox and Rac1, were similarly increased in adipose tissue, aortae and hearts of AngII-infused rats. Cotreatment of rats with tempol or BH4 reversed AngII-induced increases in NAD(P)H oxidase subunit mRNAs. Fully differentiated 3T3-L1 adipocytes, also exhibited diminished adiponectin mRNA levels when exposed to low concentrations of H2O2.Conclusions/interpretationOur results demonstrate that AngII-induced oxidative stress and endothelial dysfunction are accompanied by a decrease in adiponectin gene expression. Since antioxidants were observed to prevent the actions of AngII, and H2O2 on its own suppressed adiponectin expression, we conclude that adiponectin gene expression is negatively modulated by oxidative stress. Plasma adiponectin levels may provide a useful indicator of oxidative stress in vivo, and suppressed levels may contribute to the proinflammatory and metabolic derangements associated with type 2 diabetes, coronary artery disease and the metabolic syndrome.

High-glucose-induced nuclear factor κB activation in vascular smooth muscle cells

Objective: Vascular smooth muscle cell (VSMC) dysfunction plays a role in diabetic macrovasculopathy. This dysfunction may be caused or exacerbated by expression of many of genes potently activated by the transcriptional factor nuclear factor κB (NF-κB). We have examined whether culture of VSMCs under high glucose conditions to stimulate the diabetic state can lead to the activation of NF-κB. Methods: NF-κB activation was assessed in VSMCs stably transfected with a cis -reporter plasmid containing the NF-κB binding sites. Results: Within 3-h incubation, high glucose (27.5 or 55 mmol/l) alone induced an increase in NF-κB activity in VSMCs; this increase was mimicked by mannitol given to deliver the same osmolar stress to the cells. High glucose or mannitol also enhanced TNFα-stimulated NF-κB activity. Incubation with high glucose for 48 h followed by stimulation with TNFα led to a marked potentiation of NF-κB activation compared with normoglycemic (5.5 mmol/l) VSMCs exposed to TNFα, while mannitol attenuated this effect. A 48-h incubation with high glucose substantially reduced glutathione (GSH) levels compared with normoglycemic VSMCs, whereas mannitol significantly increased GSH levels. An antioxidant N-acetyl-l-cysteine and a selective protein kinase C (PKC) inhibitor GF109203X significantly suppressed the TNFα-induced NF-κB activation, and abrogated potentiation of TNFα-induced NF-κB activity caused by high glucose (27.5 mmol/l). Conclusion: These results suggest that acutely high glucose causes alterations in osmolarity leading to activation of NF-κB, but that exposure to high glucose for more prolonged times causes changes in antioxidant defences and activation of PKC, which potentiates cytokine activation of NF-κB. Further definition of these pathways will help to delineate important signals mediating the aberrant behavior of VSMCs under hyperglycemic/diabetic conditions.

Effect of glucose and insulin on immunoreactive endothelin-1 release from cultured porcine aortic endothelial cells

We measured the release of immunoreactive endothelin-1 (IR-ET-1) by cultured porcine aortic endothelial cells under normoglycemic (5.5 mmol/L) and hyperglycemic (27.5 and 55 mmol/L) conditions. Compared with cells incubated in the presence of a normal glucose concentration, cells incubated in 27.5 mmol/L glucose medium released 52% less IR-ET-1, and those incubated in 55 mmol/L glucose medium released 54% less IR-ET-1. The observed effects of elevated glucose on IR-ET-1 release were both sugar-specific and not due to increased osmolarity. Fetal calf serum (FCS)-stimulated IR-ET-1 release in the presence of elevated glucose was also less than that in the presence of a normal glucose concentration. In addition, the effects of two hormones, insulin and insulin-like growth factor 1 (IGF-1), on IR-ET-1 release were examined. Both insulin and IGF-1 dose-dependently stimulated IR-ET-1 release. Twenty micrograms/mL insulin and 10(-8) mol/L IGF-1 increased IR-ET-1 release by 38% and by 44%, respectively. These results indicate that hyperglycemic condition results in reduction of IR-ET-1 release from cultured porcine aortic endothelial cells and that insulin and IGF-1 stimulate its release. The possible relevance of these observations to physiological regulation of ET-1 release in vivo and pathological processes in diabetes remains to be established.

Cationic amino acid transporter gene expression in cultured vascular smooth muscle cells and in rats

Immunostimulants trigger vascular smooth muscle cells (VSMC) to express the inducible isoform of NO synthase (iNOS) and increased arginine transport activity. Although arginine transport in VSMC is considered to be mediated via the y+ system, we show here that rat VSMC in culture express the cat-1 gene transcript as well as an alternatively spliced transcript of the cat-2 gene. An RT-PCR cloning sequence strategy was used to identify a 141-base nucleotide sequence encoding the low-affinity domain of alternatively spliced CAT-2A and a 138-base nucleotide sequence encoding the high-affinity domain of CAT-2B in VSMC activated with lipopolysaccharide (LPS) in combination with interferon-gamma (IFN). With this sequence as a probe, Northern analyses showed that CAT-1 mRNA and CAT-2B mRNA are constitutively present in VSMC, and the expression of both mRNAs was rapidly stimulated by treatment with LPS-IFN, peaked within 4 h, and decayed to basal levels within 6 h after LPS-IFN. CAT-2A mRNA was not detectable in unstimulated or stimulated VSMC. Arginine transporter activity significantly increased 4-10 h after LPS-IFN. iNOS activity was reduced to almost zero in the absence of extracellular arginine uptake via system y+. Induction of arginine transport seems to be a prerequisite to the enhanced synthesis of NO in VSMC. Moreover, this work demonstrates tissue expression of CAT mRNAs with use of a model of LPS injection in rats. RT-PCR shows that the expression of CAT-1 and CAT-2B mRNA in the lung, heart, and kidney is increased by LPS administration to rats, whereas CAT-2A mRNA is abundantly expressed in the liver independent of LPS treatment. These findings suggest that together CAT-1 and CAT-2B play an important role in providing substrate for high-output NO synthesis in vitro as well as in vivo and implicate a coordinated regulation of intracellular iNOS enzyme activity with membrane arginine transport.

Troglitazone Upregulates Nitric Oxide Synthesis in Vascular Smooth Muscle Cells

We investigated the effects of troglitazone on cytokine-stimulated nitric oxide (NO) production in cultured rat vascular smooth muscle cells (VSMC). The increase in NO formation caused by interleukin-1alpha (IL-1) was enhanced by troglitazone in a concentration-dependent manner. Bacterial lipopolysaccharide-stimulated NO synthesis was also increased by troglitazone. The combinations of IL-1, tumor necrosis factor-alpha, or lipopolysaccharide with interferon-gamma (IFN) were strong stimuli for induction of NO synthesis in VSMC, which were further potentiated by the presence of troglitazone. When troglitazone was added at increasing intervals after the stimulation of VSMC with IL-1, the enhancement in NO production decreased as the interval lengthened, suggesting that troglitazone alters NO synthase (NOS) expression by VSMC rather than having a direct affect on VSMC NOS activity. Troglitazone had no effect on IL-1-elicited or IL-1/IFN-elicited nuclear factor-kappaB activity in VSMC. Troglitazone inhibited the degradation of cytokine-induced NOS mRNA. Thus troglitazone appears to enhance IL-1-induced NOS mRNA levels by prolonging its half-life rather than activating its transcription, which is nuclear factor -kappaB-dependent. No expression of peroxisome proliferator-activated receptor-gamma (PPARgamma) was detected in VSMC, and 15-deoxy-D12,14 prostaglandin J2, the natural ligand for the PPARgamma, did not resemble the effect of troglitazone on IL-1-induced NO synthesis. These results indicate that troglitazone upregulates cytokine-stimulated NO synthesis in VSMC through PPARgamma-independent mechanisms. Considering its inhibitory effects on the action of numerous growth factors on VSMC, the direct vascular effects of troglitazone shown in this study may have important implications for prevention of restenosis and possibly atherosclerosis.