Endothelium-dependent Vasorelaxation Research Articles

Alterations in triglyceride rich lipoproteins are related to endothelial dysfunction in metabolic syndrome

Our aim was to analyze the effect of circulating triglyceride rich lipoprotein (TRL) on endothelial function in metabolic syndrome (MetS). MethodsWe studied 40 patients with MetS (ATPIII), divided into those presenting normal endothelial function (n=19) and those with endothelial dysfunction (n=21) by means of the evaluation of pulse wave velocity, before and after brachial artery ischemia. In fasting serum we measured lipid and lipoprotein profile, insulin and glucose (HOMA-IR). Moreover, isolated TRL (d<1006g/l) were chemically characterized. In parallel, using randomly selected TRL from MetS patients with endothelial dysfunction (n=6) and MetS patients with normal endothelial function (n=6), the ability of TRL to inhibit ACh-induced vasorelaxation (10−9–10−5mM) on aortic rings previously pre-contracted by noradrenaline (10−8mM) was evaluated. ResultsInterestingly, TRL isolated from MetS patients presenting endothelial dysfunction showed triglyceride over-enrichment (59.1±4.8 vs. 54.1±4.7%; p=0.04), even after adjusting by potential confounders (p=0.05). In addition, while TRL resulting from both MetS groups significantly inhibited endothelium dependent vasorelaxation (p<0.001), TRL from MetS patients with endothelial dysfunction showed a strong tendency to a greater inhibition of vasorelaxation (p=0.06). Moreover, TRL-triglyceride (%) showed a strong tendency to correlate with the grade of vasorelaxation inhibition exerted by TRL (r=0.60; p=0.05). ConclusionThese results, taken together, would allow inferring for the first time that the predominance of triglyceride over-enriched TRL in circulation in MetS would induce endothelial dysfunction, contributing to the inherent cardiovascular risk of MetS.

N-methylnicotinamide protects against endothelial dysfunction and attenuates atherogenesis in apolipoprotein E-deficient mice.

Epidemiological studies have demonstrated that N-methylnicotinamide (MNA) may exert antithrombotic and anti-inflammatory effects on the endothelium. However, the exact role of MNA in endothelial function remains uncertain. Apolipoprotein E-deficient (apoE(-/-) ) mice fed with a high-fat, high-cholesterol diet (HCD) and human umbilical vein endothelial cells (HUVECs) were used to explore the role of MNA in endothelial function and its underlying mechanism. The endothelium-dependent vasorelaxation to acetylcholine in the aortas of low and high dose MNA-fed apoE(-/-) mice was improved by 24 and 36% (p < 0.05), respectively, compared with high-fat, HCD-fed control. MNA significantly increased nitric oxide/cyclic guanosinemonophosphate levels and decreased asymmetric dimethylarginine (ADMA) concentrations by induction of dimethylarginine dimethylaminohydrolase (DDAH)2 both in aorta and endothelial cells. Neither the activity nor the protein expression of DDAH1 was influenced upon MNA treatment. Then, DDAH2 depletion by RNA interference in HUVECs abolished the protective effect of MNA on endothelial function. Mechanically, this could be attributed to a direct modulation of the methylation level of DDAH2 gene promoter region by MNA. The present study reveals a novel mechanism through which MNA improves endothelial dysfunction and attenuates atherogenesis via the modulation of ADMA-DDAH axis.

Effects of acrylic resin monomers on porcine coronary artery reactivity

The purpose of the present investigation was to assess the reactivity of porcine coronary arteries under in vitro conditions following their exposure to methyl methacrylate (MMA) and hydroxyethyl methacrylate (HEMA) monomers. Confirming previous studies using rat aortas, both MMA and HEMA induced acute/direct relaxation of coronary ring preparations, which was partly dependent on the endothelium. With prolonged tissue exposure, both monomers caused time- and concentration-dependent inhibition of receptor-mediated contraction of the vascular smooth muscle caused by prostaglandin F2∝ (PGF2∝), with HEMA causing more inhibition than MMA. Hydroxyethyl methacrylate, but not MMA, also produced impairment of non-receptor-mediated contraction of the coronary smooth muscle induced by KCl. On the other hand, neither HEMA nor MMA altered relaxation of the smooth muscle produced by the direct-acting pharmacological agent, sodium nitroprusside (SNP). While exposure to HEMA impaired endothelium-dependent vasorelaxation caused by bradykinin (BK), MMA markedly enhanced this endothelial-mediated response of the arteries. The enhanced endothelial response produced by MMA was linked to nitric oxide (NO) release. In conclusion, with prolonged tissue exposure, MMA causes less pronounced effects/adverse consequences on coronary smooth muscle function relative to the effect of HEMA, while enhancing vasorelaxation associated with release of NO from the endothelium. Accordingly, MMA-containing resin materials appear to be safer for human applications than materials containing HEMA.

Geraniol improves endothelial function by inhibiting NOX-2 derived oxidative stress in high fat diet fed mice

Endothelial dysfunction occurs in obese patients and high-fat diet (HFD) fed experimental animals. While geraniol has been reported to ameliorate inflammation and oxidative stress, inhibit tumor cell proliferation, and improve atherosclerosis, its direct effect on endothelial function remains uncharacterized. The present study therefore investigated the effect of geraniol on endothelial function in HFD mice and its underlying mechanisms. C57 BL/6 mice were fed an HFD (n = 40) or a normal diet (n = 20) for 8 weeks. HFD fed mice then were randomized to intraperitoneal treatment with geraniol (n = 20) or vehicle (n = 20) for another 6 weeks. Acetylcholine (Ach)-induced endothelial dependent vasorelaxation was measured on wire myography; reactive oxygen species (ROS) generation was assessed by fluorescence imaging, and NADPH oxidases (NOXs) and adhesive molecules VCAM-1 and ICAM-1 protein expression by western blotting. Geraniol improved endothelial function in HFD fed mice, as evidenced by its: 1. restoring endothelial dependent vasorelaxation induced by Ach, and reversing increased VCAM-1 and ICAM-1 expression; 2. attenuating HFD induced increased serum TBARS and aortic ROS generation; and 3. downregulating aortic NOX-2 expression in both HFD fed mice and in palmitic acid treated endothelial cells. Geraniol therefore protects against endothelial dysfunction induced by HFD through reducing NOX-2 associated ROS generation.

Targeting miR-200c to Ameliorate Diabetes-Induced Endothelial Dysfunction.

Endothelial dysfunction is one of the earliest manifestations of diabetes that can lead to numerous debilitating micro- and macrovascular complications (1,2). Identifying factors that mediate or augment endothelial dysfunction in diabetes can lead to the development of much-needed therapies that target early stages of various vascular complications and thereby prevent further progression. Chronic endothelial dysfunction includes impaired endothelium-dependent vasorelaxation (EDR), increased inflammation, and dysregulation of vascular remodeling (1–3). Inactivation or reduced bioavailability of gasotransmitter nitric oxide (NO), also known as EDR factor, is a key factor involved in the development of vascular diseases such as hypertension and atherosclerosis (1,4), which are accelerated under diabetic conditions. NO is produced by endothelial NO synthase (eNOS), and several studies have demonstrated increased oxidative stress, eNOS uncoupling, and reduced NO bioavailability under diabetic conditions (1–4). In addition to NO, endothelial cells (ECs) also produce several prostanoids with vasodilator and vasoconstrictor activities (5) mainly via the actions of the cyclooygenase enzymes COX-1 and COX-2. A fine balance between the levels of these prostanoids contributes to normal EDR. COX-2 produces both prostacyclin (PGI2), which reduces platelet aggregation, and prostaglandin E2 (PGE2), which can reduce EDR. In diabetes, high glucose (HG)-induced oxidative stress and reactive oxygen species (ROS) increase the expression of COX-2 and prostaglandin H2 (PGH2), a common precursor of prostaglandins. However, HG-induced ROS inhibit further metabolism of PGH2 into the vasodilator PGI2 via inhibition of PGI …

Involvement of endothelium in the vasorelaxant effects of 3,4-DHPEA-EA and 3,4-DHPEA-EDA, two major functional bioactives in olive oil

The olive oil polyphenols 3,4-DHPEA-EA and 3,4-DHPEA-EDA displayed an endothelium-dependent vasorelaxant effect in rat aorta, starting at ~1 µM and abolished by NG-nitro-L-arginine (L-NA) or N-acetylcysteine, and an endothelium-independent vasorelaxant effect, starting at ~10 µM. Hydroxytyrosol only presented an endothelium-independent effect at 100 µM. DHPEA-EA and 3,4-DHPEA-EDA, but not hydroxytyrosol, also increased NO generation within endothelial cells. At higher concentrations, the three compounds reduced arginine-vasopressin-induced increase of cytosolic Ca2+ concentration ([Ca2+]c) in vascular myocytes. By UV-visible spectroscopy, we found that these polyphenols undergo autoxidative processes in organ-bath conditions. Thus, 3,4-DHPEA-EA and 3,4-DHPEA-EDA have an endothelium-dependent vasorelaxant effect caused by an enhanced NO production, probably through a redox mechanism within endothelial cells and an endothelium-independent vasorelaxant effect mediated by a reduction of agonist-induced [Ca2+]c increase in vascular myocytes. Bearing in mind the plasmatic concentrations of these polyphenols following dietary intake of olive oil, these effects could modulate vascular tone in vivo.

0294 : Glucuronide metabolites of hydroxytyrosol are responsible for the antioxidative properties of olive oil

Hydroxytyrosol (HT) is known to be a strong polyphenolic antioxidant of olive oil. However, its in vivo effects are controversial since intestinal absorption is low and it is highly metabolized into glucuronides (GC), suggesting that these metabolites could be responsible of HT biological activity in vivo . Our objectives were to study the direct effects of HT and its glucuronides on vascular function and the oxidative stress vascular protection on healthy and diabetic rats The vascular function was assessed on aortas from diabetic-High Fat Sucrose diet (HFS) and age-matched (22 weeks) control fed with a standard diet (SD) Wistar rats, in comparison to young (9 weeks) control Wistar rats (ySD). The endothelium-dependent and independent vasorelaxing effects of these compounds were assessed via incremental doses of HT and GC (10 -9 to 10 -4 M) and compared to Resveratrol (Res). Expression and phosphorylation of Ser 1177 eNOS were measured by Western blot. To evaluate the anti-oxidative effects of these polyphenols (100 μM, 5min), vascular function was assessed in presence/absence of t -BOOH (500μM, 30min). HT and GC did not show any endothelium-dependent and independent vasorelaxation effects in ySD and HFS rats while Res produced vasorelaxing effect at 10 -4 M in all groups of rats and no vasorelaxant effects were noticed in SD rats. The overall absence of vasorelaxing effect can be explained by the lack eNOS phosphorylation. As for the antioxidative properties, HT, GC and Res increased significantly endothelium-dependent relaxation in comparison of aorta rings treated only with t -BOOH (79.9%±9.2, 74.1%±8.6, 71.0%±8.2, 48.4%±5.3 respectively). In addition, co-incubation of HT/GC and Res/GC increased even more the vasorelaxation under pro-oxydant conditions. In diabetic HFS rats, (i) the incubation for 5min with HT and GC did not improve neither vasorelaxation nor the contraction; (ii) however, a prolonged 30 min incubation resulted in more pronounced and significant contractile (64.8%±12.4 and 95.6%±16.3, respectively) and endothelium-dependent relaxation effects (30%±7.5 and 58.1%±10.3 respectively) for GC in comparison to HT. In conclusion, GC seems to be responsible for the anti-oxidative properties of olive oil but further experimentations have to be realized concerning its possible interconversion in HT by β-glucuronidase. The author hereby declares no conflict of interest

0229 : Aerobic exercise training modulates microparticles miRNAs: link to improved endothelial function and regulation of inflammation?

Exercise training is known to stimulate vascular function and remodeling in a shear stress and inflammation dependent manner. Microparticles (MPs) released from vascular cells in response to shear stress, play a role in cell-cell crosstalk through carrying bioactive molecules such as miRNAs. Thus, the aim of our study was to explore whether exercise training impacts vascular wall cells and contributes to vascular function improvement through the modulation of inflammation molecules release. Therefore, we investigated the presence in MPs of 9 inflammation-regulatory miRNAs. A group of sedentary women (n=6, BMI<25Kg/m 2 ) recruited at F. Hached Hospital (Sousse, Tunisia) was enrolled in an 8-weeks training program. Vascular function was assessed by Laser Doppler Flowmetry, oxidant stress and systemic inflammatory marker (CRPus) and selected circulating cytokines were measured. Furthermore, before and after exercise training, circulating MPs quantification by flow cytometry and miRNAs content by real-time PCR, were realized. While exercise training improved significantly the endothelial-dependent vasorelaxation, decreased systemic inflammation and modified the profile of circulating cytokines, circulating MPs level and oxidant stress remained unchanged. The miRNAs profile revealed that 1/ miR155 and miR302a were not detected in MPs neither before nor after training program; 2/ miR21, miR150, miR320a, miR146a, miR124a, miR126 and miR223 were expressed in circulating MPs of sedentary women; and 3/ after training program, a significant increase of miR21, miR146a, miR124a, miR150 and miR223 content was observed while miR126 and miR223 remained unchanged. Our results highlight the role of MPs as vehicle for miRNAs and the importance of inflammation-regulatory miRNAs as effectors in the vascular wall in order to generate an optimal vascular function and to control inflammation. The author hereby declares no conflict of interest

SIRT3 Deficiency Induces Endothelial Insulin Resistance and Blunts Endothelial-Dependent Vasorelaxation in Mice and Human with Obesity.

Recent evidence implicates the critical role of Sirtuin 3 (SIRT3) in the development of many metabolic diseases, but the contribution of SIRT3 to vascular homeostasis remains largely unknown. The aim of this study was to investigate the role of SIRT3 in endothelial insulin resistance and vascular dysfunction in obesity. We found an impaired insulin-induced mesenteric vasorelaxation and concomitant reduced vascular SIRT3 expression in morbid obese human subjects compared with the non-obese subjects. Downregulation of SIRT3 in cultured human endothelial cells increased mitochondrial reactive oxygen species (mtROS) and impaired insulin signaling as evidenced by decreased phosphorylation of Akt and endothelial nitric oxide synthase and subsequent reduced nitric oxide (NO) release. In addition, obese mice induced by 24-week high-fat diet (HFD) displayed an impaired endothelium-dependent vasorelaxation to both insulin and acetylcholine, which was further exacerbated by the gene deletion of Sirt3. Scavenging of mtROS not only restored insulin-stimulated NO production in SIRT3 knockdown cells, but also improved insulin-induced vasorelaxation in SIRT3 knockout mice fed with HFD. Taken together, our findings suggest that SIRT3 positively regulates endothelial insulin sensitivity and show that SIRT3 deficiency and resultant increased mtROS contribute to vascular dysfunction in obesity.

Off-Target Vascular Effects of Cholesteryl Ester Transfer Protein Inhibitors Involve Redox-Sensitive and Signal Transducer and Activator of Transcription 3-Dependent Pathways.

Elevated blood pressure was an unexpected outcome in some cholesteryl ester transfer protein (CETP) inhibitor trials, possibly due to vascular effects of these drugs. We investigated whether CETP inhibitors (torcetrapib, dalcetrapib, anacetrapib) influence vascular function and explored the putative underlying molecular mechanisms. Resistance arteries and vascular smooth muscle cells (VSMC) from rats, which lack the CETP gene, were studied. CETP inhibitors increased phenylephrine-stimulated vascular contraction (logEC50 (:) 6.6 ± 0.1; 6.4 ± 0.06, and 6.2 ± 0.09 for torcetrapib, dalcetrapib, and anacetrapib, respectively, versus control 5.9 ± 0.05). Only torcetrapib reduced endothelium-dependent vasorelaxation. The CETP inhibitor effects were ameliorated by N-acetylcysteine (NAC), a reactive oxygen species (ROS) scavenger, and by S3I-201 [2-hydroxy-4-[[2-(4-methylphenyl)sulfonyloxyacetyl]amino]benzoic acid], a signal transducer and activator of transcription 3 (STAT3) inhibitor. CETP inhibitors increased the phosphorylation (2- to 3-fold) of vascular myosin light chain (MLC) and myosin phosphatase target subunit 1 (MYPT1) (procontractile proteins) and stimulated ROS production. CETP inhibitors increased the phosphorylation of STAT3 (by 3- to 4-fold), a transcription factor important in cell activation. Activation of MLC was reduced by NAC, GKT137831 [2-(2-chlorophenyl)-4-[3-(dimethylamino)phenyl]-5-methyl-1H-pyrazolo[4,3-c]pyridine-3,6-dione] (Nox1/4 inhibitor), and S3I-201. The phosphorylation of STAT3 was unaffected by NAC and GKT137831. CETP inhibitors did not influence activation of mitogen-activated proteins kinases (MAPK) or c-Src. Our data demonstrate that CETP inhibitors influence vascular function and contraction through redox-sensitive, STAT3-dependent, and MAPK-independent processes. These phenomena do not involve CETP because the CETP gene is absent in rodents. Findings from our study indicate that CETP inhibitors have vasoactive properties, which may contribute to the adverse cardiovascular effects of these drugs such as hypertension.

IDH2 deficiency impairs mitochondrial function in endothelial cells and endothelium-dependent vasomotor function

Mitochondrial NADP(+)-dependent isocitrate dehydrogenase (IDH2) plays an essential role protecting cells against oxidative stress-induced damage. A deficiency in IDH2 leads to mitochondrial dysfunction and the production of reactive oxygen species (ROS) in cardiomyocytes and cancer cells. However, the function of IDH2 in vascular endothelial cells is mostly unknown. In this study the effects of IDH2 deficiency on mitochondrial and vascular function were investigated in endothelial cells. IDH2 knockdown decreased the expression of mitochondrial oxidative phosphorylation (OXPHOS) complexes I, II and III, which lead to increased mitochondrial superoxide. In addition, the levels of fission and fusion proteins (Mfn-1, OPA-1, and Drp-1) were significantly altered and MnSOD expression also was decreased by IDH2 knockdown. Furthermore, knockdown of IDH2 decreased eNOS phosphorylation and nitric oxide (NO) concentration in endothelial cells. Interestingly, treatment with Mito-TEMPO, a mitochondrial-specific superoxide scavenger, recovered mitochondrial fission–fusion imbalance and blunted mitochondrial superoxide production, and reduced the IDH2 knockdown-induced decrease in MnSOD expression, eNOS phosphorylation and NO production in endothelial cells. Endothelium-dependent vasorelaxation was impaired, and the concentration of bioavailable NO decreased in the aortic ring in IDH2 knockout mice. These findings suggest that IDH2 deficiency induces endothelial dysfunction through the induction of dynamic mitochondrial changes and impairment in vascular function.

Linagliptin reduces effects of ET-1 and TLR2-mediated cerebrovascular hyperreactivity in diabetes

AimsThe anti-hyperglycemic agent linagliptin, a dipeptidyl peptidase-4 inhibitor, has been shown to reduce inflammation and improve endothelial cell function. In this study, we hypothesized that DPP-IV inhibition with linagliptin would improve impaired cerebral blood flow in diabetic rats through improved insulin-induced cerebrovascular relaxation and reversal of pathological cerebrovascular remodeling that subsequently leads to improvement of cognitive function. Main methodsMale type-2 diabetic Goto-Kakizaki (GK) and nondiabetic Wistar rats were treated with linagliptin, and ET-1 plasma levels and dose response curves to ET-1 (0.1–100nM) in basilar arteries were assessed. The impact of TLR2 antagonism on ET-1 mediated basilar contraction and endothelium-dependent relaxation to acetylcholine (ACh, 1nM–1M) in diabetic GK rats was examined with antibody directed against the TLR2 receptor (Santa Cruz, 5μg/mL). The expression of TLR2 in middle cerebral arteries (MCAs) from treated rats and in brain microvascular endothelial cells (BMVEC) treated with 100nM linagliptin was assessed. Key findingsLinagliptin lowered plasma ET-1 levels in diabetes, and reduced ET-1-induced vascular contraction. TLR2 antagonism in diabetic basilar arteries reduced ET-1-mediated cerebrovascular dysfunction and improved endothelium-dependent vasorelaxation. Linagliptin treatment in the BMVEC was able to reduce TLR2 expression in cells from both diabetic and nondiabetic rats. ConclusionsThese results suggest that inhibition of DPPIV using linagliptin improves the ET-1-mediated cerebrovascular dysfunction observed in diabetes through a reduction in ET-1 plasma levels and reduced cerebrovascular hyperreactivity. This effect is potentially a result of linagliptin causing a decrease in endothelial TLR2 expression and a subsequent increase in NO bioavailability.

Multidrug resistance associated protein-1 (MRP1) deficiency attenuates endothelial dysfunction in diabetes

AimThe multidrug resistance associated protein-1 (MRP1) is the main transporter of oxidized glutathione in endothelial cells, and blockade of MRP1 improves endothelial cell dysfunction induced by reactive oxygen species. We therefore investigated the role of MRP1 in hyperglycemia-induced endothelial dysfunction and ROS production. Methods and ResultsDiabetes was induced in 12week old male MRP1−/−- or corresponding FVB wild-type (wt) mice by injection of streptozotocin (50mg/kg for 5days). Eight weeks thereafter acetylcholine-induced endothelium-dependent vasorelaxation was blunted in aortic rings from diabetic wt mice (blood glucose levels >250mg/dl) compared with nondiabetic animals (Rmax 74±2% vs. 94±2%, p<0.001). However in aortae from diabetic mice lacking MRP1, endothelium-dependent vasorelaxation was only mildly impaired (Rmax 87±3%, p<0.001 vs. wt). Endothelium-independent relaxation induced by DEA-NONOate was not different among the groups. Streptozotocin-induced diabetes significantly increased aortic superoxide anion and hydrogen peroxide production in wild-type but not in MRP1−/− mice. Aortic levels of glutathione were significantly diminished in STZ-treated FVB mice, while preserved in MRP1−/− mice. Further, in cultured human aortic endothelial cells, high glucose levels (30mmol/l) over 5days significantly increased superoxide production which was inhibited by downregulation of MRP1 via siRNA. ConclusionsThese data indicate that MRP1 plays an important role for endothelial dysfunction and reactive oxygen species production in diabetes and under conditions of hyperglycemia. MRP1 therefore may represent a therapeutic target in treatment of diabetes induced vascular dysfunction.

Unraveling Interactions Between Anesthetics and the Endothelium: Update and Novel Insights.

The vascular endothelium is one of the largest organs in the body and consists of a single layer of highly specialized cells with site-specific morphology and functions. Endothelial cells play a vital role in the regulation of vascular tone in arterial, venous, microvascular, and lymphatic vascular beds. The endothelium also coordinates angiogenesis and controls cell adhesion, fluid homeostasis, and both innate and adaptive immunity. Fundamental research has shown that general and local anesthetics markedly modulate the biological activities of endothelial cells under aerobic and ischemia-reperfusion conditions, making the endothelium an important target of anesthetics in the cardiovascular system. Halogenated volatile anesthetics provide significant anti-inflammatory actions and protect the endothelium against ischemia-reperfusion injury, despite their inhibiting effects on endothelium-dependent vasorelaxation. They provide not only acute but also potential long-term, beneficial effects. Although many effects of IV anesthetics on endothelial function are controversial, or completely unexplored, propofol and opioids appear to have the most favorable profile with respect to the preservation of endothelial function. Some opioids and ketamine have stereoselective effects on the endothelium. Finally, there is experimental evidence to suggest important effects of anesthetics on the regulation of vascular permeability, proliferation of stem cells, including endothelial progenitor cells, and promotion or inhibition of tumor growth, potentially related to alterations in angiogenesis. However, most of these findings are from in vitro experiments and await confirmation in an in vivo setting. Thus, the clinical implications of these interactions remain uncertain.

Abstract 141: Endothelial TRPA1 Channel is Protective Against Ischemia and Hemorrhagic Strokes in Mice

The transient receptor potential ankyrin 1 (TRPA1) cation channel is more selective for Ca 2+ versus Na + ions (∼8:1). We showed that TRPA1 is located in the endothelium of cerebral arteries (eTRPA1) and that reactive oxygen species (ROS)-induced channel activity generates localized Ca 2+ signals (TRPA1 sparklets) that initiate endothelium-dependent hyperpolarization and vasorelaxation. Cerebrovascular disease is associated with increased ROS generation and oxidative stress. Thus, we hypothesized that eTRPA1 activity is protective against stroke. Tissue-specific deletion of eTRPA1 in mice was achieved using the Cre -recombinase (Tie2 promoter) / loxP system. Ischemic strokes were induced by permanent middle cerebral artery occlusion (pMCAO). Neurological function after pMCAO was assessed by a modified neurological severity scale, ranging from 0 to 4 (0: normal behavior; 1: failure to extend contralateral paw; 2: circling; 3: rolling to one side; 4: stupor). Deletion of eTRPA1 increased post-stroke (24 hours) neurological damage in eTRPA1 -/- versus WT mice, as shown by a higher severity score (2.6±0.4 vs 3.8±0.2, p&lt;0.05), and larger infarct size (%hemisphere infarcted: 45±7 vs 62±4%, p0.05, N=8), eTRPA1 -/- mice were significantly more susceptible to hemorrhagic strokes compared to WT. Kaplan-Meyer survival plots showed a higher survival rate for WT vs eTRPA1 -/- at day 10 (61 vs 54%), day 15 (44 vs 21%) and day 20 (22 vs 8%) (p&lt;0.05, N=25). All of the eTRPA1 -/- mice suffered hemorrhagic strokes by day 22, whereas some of the WT mice survived until day 52. Further, eTRPA1 -/- mice showed a higher number of hemorrhagic lesions in the cerebral cortex when compared to WT (12±4 vs 23±4 lesions per animal, p&lt;0.05, N=25 mice per group). These data suggest that eTRPA1 protects against major cerebrovascular accidents and may be considered as a therapeutic target for reducing cerebral infarct after ischemia and as a preventive agent against hemorrhagic strokes.

Role of growth hormone-releasing hormone in dyslipidemia associated with experimental type 1 diabetes

Dyslipidemia associated with triglyceride-rich lipoproteins (TRLs) represents an important residual risk factor for cardiovascular and chronic kidney disease in patients with type 1 diabetes (T1D). Levels of growth hormone (GH) are elevated in T1D, which aggravates both hyperglycemia and dyslipidemia. The hypothalamic growth hormone-releasing hormone (GHRH) regulates the release of GH by the pituitary but also exerts separate actions on peripheral GHRH receptors, the functional role of which remains elusive in T1D. In a rat model of streptozotocin (STZ)-induced T1D, GHRH receptor expression was found to be up-regulated in the distal small intestine, a tissue involved in chylomicron synthesis. Treatment of T1D rats with a GHRH antagonist, MIA-602, at a dose that did not affect plasma GH levels, significantly reduced TRL, as well as markers of renal injury, and improved endothelial-dependent vasorelaxation. Glucagon-like peptide 1 (GLP-1) reduces hyperglucagonemia and postprandial TRL, the latter in part through a decreased synthesis of apolipoprotein B-48 (ApoB-48) by intestinal cells. Although plasma GLP-1 levels were elevated in diabetic animals, this was accompanied by increased rather than reduced glucagon levels, suggesting impaired GLP-1 signaling. Treatment with MIA-602 normalized GLP-1 and glucagon to control levels in T1D rats. MIA-602 also decreased secretion of ApoB-48 from rat intestinal epithelial cells in response to oleic acid stimulation in vitro, in part through a GLP-1-dependent mechanism. Our findings support the hypothesis that antagonizing the signaling of GHRH in T1D may improve GLP-1 function in the small intestine, which, in turn, diminishes TRL and reduces renal and vascular complications.

Cardiovascular effects induced by northeastern Brazilian red wine: Role of nitric oxide and redox sensitive pathways

Red wines have been shown to protect the vascular system by increasing the endothelial formation of nitric oxide (NO). This study investigated the vasoactive properties of an alcohol-free Cabernet-Sauvignon northeastern Brazilian red wine – RIOSOL (RSCS) extract. The polyphenolic-rich RSCS contained 4.2 mg GAE/L of polyphenols, and its major compounds included quercetin, myricetin and kaempferol. In normotensive conscious rats, RSCS produced hypotension and tachycardia, which were attenuated by the endothelial NO synthase inhibitor (eNOS), L-NAME. In addition, after 2 weeks of oral treatment, RSCS reduced arterial pressure in L-NAME-treated hypertensive rats. RSCS caused NO-mediated relaxations in phenylephrine contracted mesenteric artery rings, and induced the formation of NO and superoxide anions and a redox-sensitive phosphorylation of Akt and eNOS in cultured endothelial cells. In conclusion, the findings indicate that an alcohol-free lyophilized RSCS induced hypotension and endothelium-dependent vasorelaxation as a consequence of the activation of the Akt-eNOS-NO pathway in a redox-sensitive manner.

Exercise training improves vascular mitochondrial function.

Exercise training is recognized to improve cardiac and skeletal muscle mitochondrial respiratory capacity; however, the impact of chronic exercise on vascular mitochondrial respiratory function is unknown. We hypothesized that exercise training concomitantly increases both vascular mitochondrial respiratory capacity and vascular function. Arteries from both sedentary (SED) and swim-trained (EX, 5 wk) mice were compared in terms of mitochondrial respiratory function, mitochondrial content, markers of mitochondrial biogenesis, redox balance, nitric oxide (NO) signaling, and vessel function. Mitochondrial complex I and complex I + II state 3 respiration and the respiratory control ratio (complex I + II state 3 respiration/complex I state 2 respiration) were greater in vessels from EX relative to SED mice, despite similar levels of arterial citrate synthase activity and mitochondrial DNA content. Furthermore, compared with the SED mice, arteries from EX mice displayed elevated transcript levels of peroxisome proliferative activated receptor-γ coactivator-1α and the downstream targets cytochrome c oxidase subunit IV isoform 1,isocitrate dehydrogenase(Idh)2, and Idh3a, increased manganese superoxide dismutase protein expression, increased endothelial NO synthase phosphorylation (Ser(1177)), and suppressed reactive oxygen species generation (all P< 0.05). Although there were no differences in EX and SED mice concerning endothelium-dependent and endothelium-independent vasorelaxation, phenylephrine-induced vasocontraction was blunted in vessels from EX compared with SED mice, and this effect was normalized by NOS inhibition. These training-induced increases in vascular mitochondrial respiratory capacity and evidence of improved redox balance, which may, at least in part, be attributable to elevated NO bioavailability, have the potential to protect against age- and disease-related challenges to arterial function.

Stimulation of calcium-sensing receptors induces endothelium-dependent vasorelaxations via nitric oxide production and activation of IKCa channels

Stimulation of vascular calcium-sensing receptors (CaSRs) is reported to induce both constrictions and relaxations. However, cellular mechanisms involved in these responses remain unclear. The present study investigates the effect of stimulating CaSRs on vascular contractility and focuses on the role of the endothelium, nitric oxide (NO) and K+ channels in these responses. In wire myography studies, increasing [Ca2+]o from 1mM to 6mM induced concentration-dependent relaxations of methoxamine pre-contracted rabbit mesenteric arteries. [Ca2+]o-induced relaxations were dependent on a functional endothelium, and were inhibited by the negative allosteric CaSR modulator Calhex-231. [Ca2+]o-induced relaxations were reduced by inhibitors of endothelial NO synthase, guanylate cyclase, and protein kinase G. CaSR activation also induced NO production in freshly isolated endothelial cells (ECs) in experiments using the fluorescent NO indicator DAF-FM. Pre-treatment with inhibitors of large (BKCa) and intermediate (IKCa) Ca2+-activated K+ channels (iberiotoxin and charybdotoxin), and Kv7 channels (linopirdine) also reduced [Ca2+]o-induced vasorelaxations. Increasing [Ca2+]o also activated IKCa currents in perforated-patch recordings of isolated mesenteric artery ECs. These findings indicate that stimulation of CaSRs induces endothelium-dependent vasorelaxations which are mediated by two separate pathways involving production of NO and activation of IKCa channels. NO stimulates PKG leading to BKCa activation in vascular smooth muscle cells, whereas IKCa activity contributes to endothelium-derived hyperpolarisations.

Progressive vascular remodelling, endothelial dysfunction and stiffness in mesenteric resistance arteries in a rodent model of chronic kidney disease

Chronic kidney disease (CKD) and hypertension are co-morbid conditions both associated with altered resistance artery structure, biomechanics and function. We examined these characteristics in mesenteric artery together with renal function and systolic blood pressure (SBP) changes in the Lewis polycystic kidney (LPK) rat model of CKD. Animals were studied at early (6-weeks), intermediate (12-weeks), and late (18-weeks) time-points (n=21), relative to age-matched Lewis controls (n=29). At 12 and 18-weeks, LPK arteries exhibited eutrophic and hypertrophic inward remodelling characterised by thickened medial smooth muscle, decreased lumen diameter, and unchanged or increased media cross-sectional area, respectively. At these later time points, endothelium-dependent vasorelaxation was also compromised, associated with impaired endothelium-dependent hyperpolarisation and reduced nitric oxide synthase activity. Stiffness, elastic-modulus/stress slopes and collagen/elastin ratios were increased in 6 and 18-week-old-LPK, in contrast to greater arterial compliance at 12weeks. Multiple linear regression analysis highlighted SBP as the main predictor of wall–lumen ratio (r=0.536, P<0.001 n=46 pairs). Concentration–response curves revealed increased sensitivity to phenylephrine but not potassium chloride in 18-week-LPK. Our results indicate that impairment in LPK resistance vasculature is evident at 6weeks, and worsens with hypertension and progression of renal disease.