Beneficial effect of the oligomerized polyphenol oligonol on high glucose-induced changes in eNOS phosphorylation and dephosphorylation in endothelial cells

Background and purpose:

Hyperglycaemia is known to reduce nitric oxide (NO) bioavailability by modulating endothelial NO synthase (eNOS) activity, and polyphenols are believed to have cardiovascular benefit. One possible mechanism could be through interaction with eNOS.

Experimental approach:

The effects of the oligomerized polyphenol oligonol on eNOS phosphorylation status and activity were examined in porcine aortic endothelial cells cultured in high glucose concentrations.

Key results:

Exposure to high glucose concentrations strongly inhibited eNOS phosphorylation at Ser-1177 and dephosphorylation at Thr-495 in bradykinin (BK)-stimulated cells. These inhibitory effects of high glucose were significantly prevented by treatment with oligonol. Akt and p38 mitogen-activated protein kinase (MAPK) were activated in BK-stimulated cells. High glucose inhibited Akt activation but enhanced p38 MAPK activation, both of which were reversed by oligonol treatment. The phosphatidylinositol 3-kinase inhibitor wortmannin blocked the reversal by oligonol of phosphorylation at Ser-1177, but not dephosphorylation at Thr-495, in BK-stimulated cells exposed to high glucose. The effect of oligonol on BK dephosphorylation under high glucose was mimicked by protein kinase C (PKC) ε-neutralizing peptides. These data suggest that the effects of oligonol on high glucose-induced attenuation of eNOS Ser-1177 phosphorylation and Thr-495 dephosphorylation may be regulated by Akt activation and PKCε inhibition respectively. Oligonol also prevented high glucose-induced attenuation of BK-stimulated NO production.

Conclusions and implications:

Oligonol prevented the impairment of eNOS activity induced by high glucose through reversing altered eNOS phosphorylation status. This mechanism may underlie the beneficial cardiovascular health effects of this oligomerized polyphenol.     

Long-lasting partnership between insulin resistance and endothelial dysfunction: role of metabolic memory

Background and Purpose

The persistence of deleterious effects of hyperglycaemia even after glucose normalization is referred to as ‘metabolic memory’. However, similar persistent effects of the metabolic consequences of a high fat diet (HFD) have not been described.

Experimental Approach

Rats were given a normal pellet diet (NPD) or a HFD for 3 months. The animals from the HFD group were then returned to the NPD to observe the long-term effects of insulin resistance. Endothelial dysfunction was assessed by carbachol-mediated vasorelaxation and eNOS phosphorylation.

Key Results

As expected, HFD consumption resulted in insulin resistance and endothelial dysfunction. Phosphorylation of eNOS at S1177 was decreased in HFD rats, compared with that in the NPD group. Rats on 3 months of HFD showed glucose intolerance and impaired insulin sensitivity and were then switched back to NPD (REV group). Levels of cholesterol and triglyceride, and adiposity returned to normal in REV rats. However, endothelium-dependent vascular responses to carbachol which were impaired in HFD rats, continued to be impaired in REV rats. Similarly, decreased eNOS phosphorylation after HFD was not improved after 1 or 6 months of REV.

Conclusions and Implications

Our data indicate that returning to NPD did not improve the insulin sensitivity or the endothelial dysfunction induced by HFD. Although some biochemical parameters responsible for insulin resistance and endothelial dysfunction were normalized, molecular and vascular abnormalities, involving NO, persisted for several months, highlighting the long-lasting effects of metabolic memory.     

Oleanolic acid induces relaxation and calcium-independent release of endothelium-derived nitric oxide

Background and purpose:

The present study investigated the mechanisms by which oleanolic acid, a component of olive oil, increases release of nitric oxide (NO).

Experimental approach:

Measurements of isometric tension, NO concentration, or endothelial cell calcium were made in rat isolated mesenteric arteries. Immunoblotting for endothelial NOS (eNOS) and Akt kinase were performed in primary cultures of human umbilical vein endothelial cells (HUVECs).

Key results:

Oleanolic acid (3–30 μM) evoked endothelium-dependent relaxations in noradrenaline-contracted rat superior and small mesenteric arteries. In rat superior mesenteric arteries, oleanolic acid induced simultaneous increases in NO concentration and relaxation, and these responses were inhibited by an inhibitor of NOS, asymmetric dimethyl-L-arginine (300 μM) and by the NO scavenger, oxyhaemoglobin (10 μM). Oleanolic acid-evoked NO increases were not reduced in Ca²⁺-free solution and in the presence of an inhibitor of endoplasmic reticulum calcium-ATPase, thapsigargin (1 μM). Oleanolic acid evoked relaxation without changes in endothelial cell calcium, but decreased smooth muscle calcium in arterial segments. Oleanolic acid failed to increase calcium in HUVECs, but increased time-dependently phosphorylation of Akt kinase at Serine⁴⁷³ (Akt-Ser⁴⁷³) and eNOS at Serine¹¹⁷⁷ (eNOS-Ser¹¹⁷⁷), which was attenuated by inhibitors of phosphoinositide-3-kinase.

Conclusions and implications:

This study provides direct evidence that a component of olive oil, oleanolic acid, activated endothelium-dependent release of NO and decreased smooth muscle cell calcium followed by relaxation. The oleanolic acid-evoked endothelium-derived NO release was independent of endothelial cell calcium and involved phosphoinositide-3-kinase-dependent phosphorylation of Akt-Ser⁴⁷³ followed by phosphorylation of eNOS-Ser¹¹⁷⁷.     

Raloxifene protects endothelial cell function against oxidative stress

Background and purpose:

Maintaining a delicate balance between the generation of nitric oxide (NO) and removal of reactive oxygen species (ROS) within the vascular wall is crucial to the physiological regulation of vascular tone. Increased production of ROS reduces the effect and/or bioavailability of NO, leading to an impaired endothelial function. This study tested the hypothesis that raloxifene, a selective oestrogen receptor modulator, can prevent endothelial dysfunction under oxidative stress.

Experimental approach:

Changes in isometric tension were measured in rat aortic rings. The content of cyclic GMP in aortic tissue was determined by radioimmunoassay. Phosphorylation of endothelial NOS (eNOS) and Akt was assayed by Western blot analysis.

Key results:

In rings with endothelium, ACh-induced relaxations were attenuated by a ROS-generating reaction (hypoxanthine plus xanthine oxidase, HXXO). The impaired relaxations were ameliorated by acute treatment with raloxifene. HXXO suppressed the ACh-stimulated increase in cyclic GMP levels; this effect was antagonized by raloxifene. The improved endothelial function by raloxifene was abolished by ICI 182,780, and by wortmannin or {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002. Raloxifene also protected endothelial cell function against H₂O₂. Raloxifene increased the phosphorylation of eNOS at Ser-1177 and Akt at Ser-473; this effect was blocked by ICI 182,780. Finally, raloxifene was not directly involved in scavenging ROS, and neither inhibited the activity of xanthine oxidase nor stimulated that of superoxide dismutase.

Conclusion and implications:

Raloxifene is effective against oxidative stress-induced endothelial dysfunction in vitro through an ICI 182,780-sensitive mechanism that involves the increased phosphorylation and activity of Akt and eNOS in rat aortae.     

Glucagon-like peptide-1 activates endothelial nitric oxide synthase in human umbilical vein endothelial cells

Aim:

To investigate the effects of glucagon-like peptide-1 (GLP-1) on endothelial NO synthase (eNOS) in human umbilical vein endothelial cells (HUVECs), and elucidate whether GLP-1 receptor (GLP-1R) and GLP-1(9–36) are involved in these effects.

Methods:

HUVECs were used. The activity of eNOS was measured with NOS assay kit. Phosphorylated and total eNOS proteins were detected using Western blot analysis. The level of eNOS mRNA was quantified with real-time RT-PCR.

Results:

Incubation of HUVECs with GLP-1 (50–5000 pmol/L) for 30 min significantly increased the activity of eNOS. Incubation of HUVECs with GLP-1 (500–5000 pmol/L) for 5 or 10 min increased eNOS phosphorylated at ser-1177. Incubation with GLP-1 (5000 pmol/L) for 48 h elevated the level of eNOS protein, did not affect the level of eNOS mRNA. GLP-1R agonists exenatide and GLP-1(9–36) at the concentration of 5000 pmol/L increased the activity, phosphorylation and protein level of eNOS. GLP-1R antagonist exendin(9–39) or DPP-4 inhibitor sitagliptin, which abolished GLP-1(9–36) formation, at the concentration of 5000 pmol/L partially blocked the effects of GLP-1 on eNOS.

Conclusion:

GLP-1 upregulated the activity and protein expression of eNOS in HUVECs through the GLP-1R-dependent and GLP-1(9–36)-related pathways. GLP-1 may prevent or delay the formation of atherosclerosis in diabetes mellitus by improving the function of eNOS.     

Reversal of SIN-1-induced eNOS dysfunction by the spin trap,DMPO, in bovine aortic endothelial cells via eNOS phosphorylation

Background and Purpose

Nitric oxide (NO) derived from eNOS is mostly responsible for the maintenance of vascular homeostasis and its decreased bioavailability is characteristic of reactive oxygen species (ROS)-induced endothelial dysfunction (ED). Because 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), a commonly used spin trap, can control intracellular nitroso-redox balance by scavenging ROS and donating NO, it was employed as a cardioprotective agent against ED but the mechanism of its protection is still not clear. This study elucidated the mechanism of protection by DMPO against SIN-1-induced oxidative injury to bovine aortic endothelial cells (BAEC).

Experimental Approach

BAEC were treated with SIN-1, as a source of peroxynitrite anion (ONOO⁻), and then incubated with DMPO. Cytotoxicity following SIN-1 alone and cytoprotection by adding DMPO was assessed by MTT assay. Levels of ROS and NO generation from HEK293 cells transfected with wild-type and mutant eNOS cDNAs, tetrahydrobiopterin bioavailability, eNOS activity, eNOS and Akt kinase phosphorylation were measured.

Key Results

Post-treatment of cells with DMPO attenuated SIN-1-mediated cytotoxicity and ROS generation, restoration of NO levels via increased in eNOS activity and phospho-eNOS levels. Treatment with DMPO alone significantly increased NO levels and induced phosphorylation of eNOS Ser¹¹⁷⁹ via Akt kinase. Transfection studies with wild-type and mutant human eNOS confirmed the dual role of eNOS as a producer of superoxide anion (O₂⁻) with SIN-1 treatment, and a producer of NO in the presence of DMPO.

Conclusion and Implications

Post-treatment with DMPO of oxidatively challenged cells reversed eNOS dysfunction and could have pharmacological implications in the treatment of cardiovascular diseases.     

Pyridoxine inhibits endothelial NOS uncoupling induced by oxidized low-density lipoprotein via the PKCα signalling pathway in human umbilical vein endothelial cells

BACKGROUND AND PURPOSE

One key mechanism for endothelial dysfunction is endothelial NOS (eNOS) uncoupling, whereby eNOS generates superoxide (O₂^•−) rather than NO. We explored the effect of pyridoxine on eNOS uncoupling induced by oxidized low-density lipoprotein (ox-LDL) in human umbilical vein endothelial cells (HUVECs) and the potential molecular mechanism.

EXPERIMENTAL APPROACH

HUVECs were incubated with ox-LDL with/without pyridoxine, N^G-nitro-L-arginine methylester (L-NAME), chelerythrine chloride (CHCI) or apocynin. Endothelial O₂^•− was measured using lucigenin chemiluminescence, and O₂^•−-sensitive fluorescent dye dihydroethidium (DHE). NO levels were measured by chemiluminescence, PepTag Assay for non-radioactive detection of PKC activity, depletion of PKCα and p47phox by siRNA silencing and the states of phospho-eNOS Thr495, total-eNOS, phospho-PKCα/βII, total PKC, phospho-PKCα, total PKCα and p47phox were measured by Western blot.

KEY RESULTS

Ox-LDL significantly increased O₂^•− production and reduced NO levels released from HUVECs; an effect reversed by eNOS inhibitor, L-NAME. Pyridoxine pretreatment significantly inhibited ox-LDL-induced O₂^•− generation and preserved NO levels. Pyridoxine also prevented the ox-LDL-induced reduction in phospho-eNOS Thr495 and PKC activity. These protective effects of pyridoxine were abolished by the PKC inhibitor, CHCI, or siRNA silencing of PKCα. However, depletion of p47phox or treatment with the NADPH oxidase inhibitor, apocynin, had no influence on these effects. Also, cytosol p47phox expression was unchanged by the different treatments.

CONCLUSIONS AND IMPLICATIONS

Pyridoxine mitigated eNOS uncoupling induced by ox-LDL. This protectant effect was related to phosphorylation of eNOS Thr495 stimulated by PKCα, not via NADPH oxidase. These results provide support for the use of pyridoxine in ox-LDL-related vascular endothelial dysfunction.     

Chronic administration of BMS309403 improves endothelial function in apolipoprotein E-deficient mice and in cultured human endothelial cells

BACKGROUND AND PURPOSE

Adipocyte fatty acid-binding protein (A-FABP) is up-regulated in regenerated endothelial cells and modulates inflammatory responses in macrophages. Endothelial dysfunction accompanying regeneration is accelerated by hyperlipidaemia. Here, we investigate the contribution of A-FABP to the pathogenesis of endothelial dysfunction in the aorta of apolipoprotein E-deficient (ApoE^−/−) mice and in cultured human endothelial cells.

EXPERIMENTAL APPROACH

A-FABP was measured in aortae of ApoE^−/−mice and human endothelial cells by RT-PCR, immunostaining and immunoblotting. Total and phosphorylated forms of endothelial nitric oxide synthase (eNOS) were measured by immunoblotting. Changes in isometric tension were measured in rings of mice aortae

KEY RESULTS

A-FABP was expressed in aortic endothelium of ApoE^−/− mice aged 12 weeks and older, but not at 8 weeks or in C57 wild-type mice. Reduced endothelium-dependent relaxations to acetylcholine, UK14304 (selective α₂-adrenoceptor agonist) and {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 (calcium ionophore) and decreased protein presence of phosphorylated and total eNOS were observed in aortae of 18 week-old ApoE^−/− mice compared with age-matched controls. A 6 week treatment with the A-FABP inhibitor, BMS309403, started in 12 week-old mice, improved endothelial function, phosphorylated and total eNOS and reduced plasma triglyceride levels but did not affect endothelium-independent relaxations. The beneficial effect of BMS309403 on UK14304-induced relaxations was attenuated by Pertussis toxin. In cultured human microvascular endothelial cells, lipid-induced A-FABP expression was associated with reduced phosphorylated eNOS and NO production and was reversed by BMS309403.

CONCLUSIONS AND IMPLICATIONS

Elevated expression of A-FABP in endothelial cells contributes to their dysfunction both in vivo and in vitro.     

The novel ATP-sensitive potassium channel opener iptakalim prevents insulin resistance associated with hypertension via restoring endothelial function

Aim:

To investigate the effects of iptakalim on endothelial dysfunction induced by insulin resistance (IR) and to determine whether iptakalim improved IR associated with hypertension in fructose-fed rats (FFRs) and spontaneously hypertensive rats (SHRs).

Methods:

Human umbilical vein endothelial cells (HUVECs) were used for in vitro study. The levels of endothelial vasoactive mediators and eNOS protein expression were determined using radioimmunoassays, ELISAs, colorimetric assays or Western blotting. Sprague-Dawley rats were fed with a high-fructose diet. In both FFRs and SHRs, tail-cuff method was used to measure systolic blood pressure (SBP), and hyperinsulinemic- euglycemic clamp was used to evaluate IR states.

Results:

(1) Cultured HUVECs incubated with the PI3-kinase inhibitor wortmannin (50 nmol/L) and insulin (100 nmol/L) induced endothelial dysfunction characterized by significantly reduced release of NO and expression of eNOS protein, and significantly increased production of ET-1. Pretreatment with iptakalim (0.1–10 μmol/L) could prevent the endothelial dysfunction. (2) In FFRs, the levels of SBP, fasting plasma glucose and insulin were significantly elevated, whereas the glucose infusion rate (GIR) and insulin sensitive index (ISI) were significantly decreased, and the endothelium-dependent vascular relaxation response to ACh was impaired. These changes could be prevented by oral administration of iptakalim (1, 3, or 9 mg·kg⁻¹·d⁻¹, for 4 weeks). The imbalance between serum NO and ET-1 was also ameliorated by iptakalim. (3) In 2–4 month-old SHRs (IR was established at the age of 4 months), oral administration of iptakalim (1, 3, or 9 mg·kg⁻¹·d⁻¹, for 8 weeks) significantly ameliorated hypertension and increased the GIR to the normal level.

Conclusion:

These results demonstrate that iptakalim could protect against IR-induced endothelial dysfunction, and ameliorate IR associated with hypertension, possibly via restoring the balance between NO and ET-1 signaling.     

PPARβ activation restores the high glucose-induced impairment of insulin signalling in endothelial cells

BACKGROUND AND PURPOSE

PPARβ enhances insulin sensitivity in adipocytes and skeletal muscle cells, but its effects on insulin signalling in endothelial cells are not known. We analysed the effects of the PPARβ/δ (PPARβ) agonists, GW0742 and L165041, on impaired insulin signalling induced by high glucose in HUVECs and aortic and mesenteric arteries from diabetic rats.

EXPERIMENTAL APPROACH

Insulin-stimulated NO production, Akt-Ser⁴⁷³ and eNOS-Ser¹¹⁷⁷ phosphorylation, and reactive oxygen species (ROS) production were studied in HUVECs incubated in low- or high-glucose medium. Insulin-stimulated relaxations and protein phosphorylation in vessels from streptozotocin (STZ)-induced diabetic rats were also analysed.

KEY RESULTS

HUVECs incubated in high-glucose medium showed a significant reduction in insulin-stimulated production of NO. High glucose also reduced insulin-induced Akt-Ser⁴⁷³ and eNOS-Ser¹¹⁷⁷ phosphorylation, increased IRS-1-Ser⁶³⁶ and ERK1/2-Thr¹⁸³-Tyr¹⁸⁵ phosphorylation and increased ROS production. The co-incubation with the PPARβ agonists GW0742 or L165041 prevented all these effects induced by high glucose. In turn, the effects induced by the agonists were suppressed when HUVEC were also incubated with the PPARβ antagonist GSK0660, the pyruvate dehydrogenase kinase (PDK)4 inhibitor dichloroacetate or after knockdown of both PPARβ and PDK4 with siRNA. The ERK1/2 inhibitor PD98059, ROS scavenger catalase, inhibitor of complex II thenoyltrifluoroacetone or uncoupler of oxidative phosphorylation, carbonyl cyanide m-chlorophenylhydrazone, also prevented glucose-induced insulin resistance. In STZ diabetic rats, oral GW0742 also improved insulin signalling and the impaired NO-mediated vascular relaxation.

CONCLUSION AND IMPLICATIONS

PPARβ activation in vitro and in vivo restores the endothelial function, preserving the insulin-Akt-eNOS pathway impaired by high glucose, at least in part, through PDK4 activation.     

Effects of angiotensin receptor blockers on endothelial nitric oxide release: the role of eNOS variants

AIM

Angiotensin II receptor blockers (ARBs) improve endothelial cell (EC)-dependent vasodilation in patients with hypertension through suppression of angiotensin II type 1 receptors but may have additional and differential effects on endothelial nitric oxide (NO) synthase (eNOS) function. To investigate this question, we tested the effects of various ARBs on NO release in ECs from multiple donors, including those with eNOS genetic variants linked to higher cardiovascular risk.

METHODS

The effects of ARBs (losartan, olmesartan, telmisartan, valsartan), at 1 µm, on NO release were measured with nanosensors in human umbilical vein ECs obtained from 18 donors. NO release was stimulated with calcium ionophore (1 µm) and its maximal concentration was correlated with eNOS variants. The eNOS variants were determined by a single nucleotide polymorphism in the promoter region (T-786C) and in the exon 7 (G894T), linked to changes in NO metabolism.

RESULTS

All of the ARBs caused an increase in NO release as compared with untreated samples (P < 0.01, n = 4–5 in all eNOS variants). However, maximal NO production was differentially influenced by eNOS genotype. Olmesartan increased maximal NO release by 30%, which was significantly greater (P < 0.01, n = 4–5 in all eNOS variants) than increases observed with other ARBs.

CONCLUSIONS

The ARBs differentially enhanced NO release in ECs in a manner influenced by eNOS single nucleotide polymorphisms. These findings provide new insights into the effects of ARBs on EC-dependent vasodilation and eNOS function.     

The prolactin family hormones regulate vascular tone through NO and prostacyclin production in isolated rat aortic rings

Aim:

Prolactin family hormones include growth hormone, placental lactogen and prolactin, which are able to regulate angiogenesis via NO and prostaglandins. However, their effects on vascular tone are not fully understood. The aim of this study was to evaluate the effects of prolactin family hormones on rat vascular tone in vitro.

Methods:

Aortic rings were prepared from adult male rats and precontracted with phenylephrine, then treated with the hormones and drugs. The tension was measured with isometric force displacement transducer connected to a polygraph. NO production and prostacyclin release in physiological solution was determined. Cultured rat aortic endothelial cells (RAECs) were treated with the hormones and drugs, and the phosphorylation of eNOS at serine 1177 was assessed using Western bolt analysis.

Results:

Administration of growth hormone or placental lactogen (0.01–100 nmol/L) induced endothelium-dependent vasodilation. Both the hormones significantly increased the phosphorylation of eNOS in RAECs and NO level in physiological solution. Preincubation with L-NAME blocked growth hormone- or placental lactogen-induced vasodilation and NO production. Preincubation with an antibody against growth hormone receptors blocked growth hormone- and placental lactogen-induced vasodilation. Addition of a single dose of prolactin (0.01 nmol/L) induced sustained vessel relaxation, whereas multiple doses of prolactin induced a biphasic contraction-relaxation effect. The vascular effects of prolactin depended on endothelium. Prolactin significantly increased the level of prostacyclin I₂ in physiological solution. Preincubation with indomethacin or an antibody against prolactin receptors blocked prolactin-induced vasodilation.

Conclusion:

The prolactin family hormones regulate rat vascular tone, selectively promoting either relaxation or contraction of vascular smooth muscle via activation of either growth hormone receptors or prolactin receptors within the endothelium.     

Acute activation of eNOS by statins involves scavenger receptor-B1, G protein subunit Gi, phospholipase C and calcium influx

Background and purpose:

Statins (HMG CoA reductase inhibitors) have beneficial effects independent of reducing cholesterol synthesis and this includes their ability to acutely activate endothelial nitric oxide synthase (eNOS). The mechanism by which this occurs is largely unknown and thus we characterized the pathways by which statins activate NOS, including involvement of scavenger receptor-B1 (SR-B1), which is expressed in endothelial cells and maintains cholesterol concentrations.

Experimental approach:

Nitric oxide production was monitored in bovine aortic endothelial cells (BAECs) exposed to lovastatin (LOV) or pravastatin (PRA) for 10–20 min, alone or following pre-exposure to the end product of HMG-CoA reductase (mevalonate), G protein inhibitors (pertussis/cholera toxins), phospholipase C (PLC) inhibitor (U-73122), or intracellular and extracellular calcium chelators – BAPTA-AM and EGTA (respectively), or a function blocking antibody to SR-B1.

Key results:

Both statins increased NO production in a rapid, dose-dependent and HMG-CoA reductase-independent manner. Inhibiting Gi protein or PLC almost completely blocked statin-induced NO generation. Additionally, removing extracellular calcium inhibited statin-induced NO production. COS-7 cells co-transfected with eNOS and SR-B1 increased NO production when exposed to LOV or high-density lipoprotein (HDL), an agonist of SR-B1. These effects were not observed in COS-7 cells with eNOS alone or co-transfected with bradykinin receptor 2, indicating specificity for SR-B1. Further, pretreatment of BAEC with blocking antibody for SR-B1 blocked NO responses to statins and HDL.

Conclusions and implications:

LOV and PRA acutely activate eNOS through pathways that include the cell surface receptor SR-B1, Gi protein, phosholipase C and entry of extracellular calcium into endothelial cells.     

The peroxisome proliferator-activated receptor-α (PPAR-α) agonist,AVE8134, attenuates the progression of heart failure and increases survival in rats

Aim:

To investigate the efficacy of the peroxisome proliferator-activated receptor-α (PPARα) agonist, AVE8134, in cellular and experimental models of cardiac dysfunction and heart failure.

Methods:

In Sprague Dawley rats with permanent ligation of the left coronary artery (post-MI), AVE8134 was compared to the PPARγ agonist rosiglitazone and in a second study to the ACE inhibitor ramipril. In DOCA-salt sensitive rats, efficacy of AVE8134 on cardiac hypertrophy and fibrosis was investigated. Finally, AVE8134 was administered to old spontaneously hypertensive rats (SHR) at a non-blood pressure lowering dose with survival as endpoint. In cellular models, we studied AVE8134 on hypertrophy in rat cardiomyocytes, nitric oxide signaling in human endothelial cells (HUVEC) and LDL-uptake in human MonoMac-6 cells.

Results:

In post-MI rats, AVE8134 dose-dependently improved cardiac output, myocardial contractility and relaxation and reduced lung and left ventricular weight and fibrosis. In contrast, rosiglitazone exacerbated cardiac dysfunction. Treatment at AVE8134 decreased plasma proBNP and arginine and increased plasma citrulline and urinary NOx/creatinine ratio. In DOCA rats, AVE8134 prevented development of high blood pressure, myocardial hypertrophy and cardiac fibrosis, and ameliorated endothelial dysfunction. Compound treatment increased cardiac protein expression and phosphorylation of eNOS. In old SHR, treatment with a low dose of AVE8134 improved cardiac and vascular function and increased life expectancy without lowering blood pressure. AVE8134 reduced phenylephrine-induced hypertrophy in adult rat cardiomyocytes. In HUVEC, Ser-1177-eNOS phosphorylation but not eNOS expression was increased. In monocytes, AVE8134 increased the expression of CD36 and the macrophage scavenger receptor 1, resulting in enhanced uptake of oxidized LDL.

Conclusion:

The PPARα agonist AVE8134 prevents post-MI myocardial hypertrophy, fibrosis and cardiac dysfunction. AVE8134 has beneficial effects against hypertension-induced organ damages, resulting in decreased mortality. The compound exerts its protective properties by a direct effect on cardiomyocyte hypertrophy, but also indirectly via monocyte signaling and increased endothelial NO production.     

Alagebrium attenuates acute methylglyoxal-induced glucose intolerance in Sprague-Dawley rats

Background and purpose:

Alagebrium is a breaker of cross-links in advanced glycation endproducts. However, the acute effects of alagebrium on methylglyoxal (MG), a major precursor of advanced glycation endproducts have not been reported. MG is a highly reactive endogenous metabolite, and its levels are elevated in diabetic patients. We investigated whether alagebrium attenuated the acute effects of exogenous MG on plasma MG levels, glucose tolerance and distribution of administered MG in different organs in Sprague-Dawley rats.

Experimental approach:

We measured MG levels (by HPLC), glucose tolerance, adipose tissue glucose uptake, GLUT4, insulin receptor and insulin receptor substrate 1 (IRS-1) protein expression, and phosporylated IRS-1 in rats treated with MG at doses of either 17.25 mg·kg⁻¹ i.p. (MG-17 i.p.) or 50 mg·kg⁻¹ i.v. (MG-50 i.v.) with or without alagebrium, 100 mg·kg⁻¹ i.p.

Key results:

Alagebrium attenuated the increased MG levels in the plasma, aorta, heart, kidney, liver, lung and urine after MG administration. In MG-treated rats, glucose tolerance was impaired, plasma insulin levels were higher and insulin-stimulated glucose uptake by adipose tissue was reduced, relative to the corresponding control groups. In rats treated with MG-50 i.v., GLUT4 protein expression and IRS-1 tyrosine phosphorylation were decreased. Alagebrium pretreatment attenuated these effects of MG. In an in vitro assay, alagebrium reduced the amount of detectable MG.

Conclusions and implications:

Alagebrium acutely attenuated MG-induced glucose intolerance, suggesting a possible preventive role for alagebrium against the harmful effects of MG.     

The gastrointestinal peptide obestatin induces vascular relaxation via specific activation of endothelium-dependent NO signalling

BACKGROUND AND PURPOSE

Obestatin is a recently discovered gastrointestinal peptide with established metabolic actions, which is linked to diabetes and may exert cardiovascular benefits. Here we aimed to investigate the specific effects of obestatin on vascular relaxation.

EXPERIMENTAL APPROACH

Cumulative relaxation responses to obestatin peptides were assessed in rat isolated aorta and mesenteric artery (n≥ 8) in the presence and absence of selective inhibitors. Complementary studies were performed in cultured bovine aortic endothelial cells (BAEC).

KEY RESULTS

Obestatin peptides elicited concentration-dependent relaxation in both aorta and mesenteric artery. Responses to full-length obestatin(1–23) were greater than those to obestatin(1–10) and obestatin(11–23). Obestatin(1–23)-induced relaxation was attenuated by endothelial denudation, l-NAME (NOS inhibitor), high extracellular K⁺, GDP-β-S (G-protein inhibitor), MDL-12,330A (adenylate cyclase inhibitor), wortmannin (PI3K inhibitor), KN-93 (CaMKII inhibitor), ODQ (guanylate cyclase inhibitor) and iberiotoxin (BK_Ca blocker), suggesting that it is mediated by an endothelium-dependent NO signalling cascade involving an adenylate cyclase-linked GPCR, PI3K/PKB, Ca²⁺-dependent eNOS activation, soluble guanylate cyclase and modulation of vascular smooth muscle K⁺. Supporting data from BAEC indicated that nitrite production, intracellular Ca²⁺ and PKB phosphorylation were increased after exposure to obestatin(1–23). Relaxations to obestatin(1–23) were unaltered by inhibitors of candidate endothelium-derived hyperpolarizing factors (EDHFs) and combined SK_Ca/IK_Ca blockade, suggesting that EDHF-mediated pathways were not involved.

CONCLUSIONS AND IMPLICATIONS

Obestatin produces significant vascular relaxation via specific activation of endothelium-dependent NO signalling. These actions may be important in normal regulation of vascular function and are clearly relevant to diabetes, a condition characterized by endothelial dysfunction and cardiovascular complications.     

Decreased production of neuronal NOS-derived hydrogen peroxide contributes to endothelial dysfunction in atherosclerosis

BACKGROUND AND PURPOSE

Reduced NO availability has been described as a key mechanism responsible for endothelial dysfunction in atherosclerosis. We previously reported that neuronal NOS (nNOS)-derived H₂O₂ is an important endothelium-derived relaxant factor in the mouse aorta. The role of H₂O₂ and nNOS in endothelial dysfunction in atherosclerosis remains undetermined. We hypothesized that a decrease in nNOS-derived H₂O₂ contributes to the impaired vasodilatation in apolipoprotein E-deficient mice (ApoE^−/−).

EXPERIMENTAL APPROACH

Changes in isometric tension were recorded on a myograph; simultaneously, NO and H₂O₂ were measured using carbon microsensors. Antisense oligodeoxynucleotides were used to knockdown eNOS and nNOS in vivo. Western blot and confocal microscopy were used to analyse the expression and localization of NOS isoforms.

KEY RESULTS

Aortas from ApoE^−/− mice showed impaired vasodilatation paralleled by decreased NO and H₂O₂ production. Inhibition of nNOS with L-Arg^NO2-L-Dbu, knockdown of nNOS and catalase, which decomposes H₂O₂ into oxygen and water, decreased ACh-induced relaxation by half, produced a small diminution of NO production and abolished H₂O₂ in wild-type animals, but had no effect in ApoE^−/− mice. Confocal microscopy showed increased nNOS immunostaining in endothelial cells of ApoE^−/− mice. However, ACh stimulation of vessels resulted in less phosphorylation on Ser852 in ApoE^−/− mice.

CONCLUSIONS AND IMPLICATIONS

Our data show that endothelial nNOS-derived H₂O₂ production is impaired and contributes to endothelial dysfunction in ApoE^−/− aorta. The present study provides a new mechanism for endothelial dysfunction in atherosclerosis and may represent a novel target to elaborate the therapeutic strategy for vascular atherosclerosis.     

Inhibition of matrix metalloproteinase-2 improves endothelial function and prevents hypertension in insulin-resistant rats

BACKGROUND AND PURPOSE

Insulin resistance is often found to be associated with high blood pressure. We propose that in insulin-resistant hypertension, endothelial dysfunction is the consequence of increased activity of vascular MMP-2. As MMP-2 proteolytically cleaves a number of extracellular matrix proteins, we hypothesized that MMP-2 impairs endothelial function by proteolytic degradation of endothelial NOS (eNOS) or its cofactor, heat shock protein 90 (HSP90).

EXPERIMENTAL APPROACH

We tested our hypothesis in bovine coronary artery endothelial cells and fructose-fed hypertensive rats (FHR), a model of acquired systolic hypertension and insulin resistance.

KEY RESULTS

Treatment of FHRs with the MMP inhibitor doxycycline, preserved endothelial function as well as prevented the development of hypertension, suggesting that MMPs impair endothelial function. Furthermore, incubating endothelial cells in vitro with a recombinant MMP-2 decreased NO production in a dose-dependent manner. Using substrate cleavage assays and immunofluorescence microscopy studies, we found that MMP-2 not only cleaves and degrades HSP90, an eNOS cofactor but also co-localizes with both eNOS and HSP90 in endothelial cells, suggesting that MMPs functionally interact with the eNOS system. Treatment of FHRs with doxycycline attenuated the decrease in eNOS and HSP90 expression but did not improve insulin sensitivity.

CONCLUSIONS AND IMPLICATIONS

Our data suggest that increased activity of MMP-2 in FHRs impairs endothelial function and promotes hypertension. Inhibition of MMP-2 could be a potential therapeutic strategy for the management of hypertension.