Endothelium-dependent Vasorelaxation Research Articles

Transient Receptor Potential Vanilloid 4 Channel Deficiency Aggravates Tubular Damage after Acute Renal Ischaemia Reperfusion

Transient receptor potential vanilloid 4 (TRPV4) cation channels are functional in all renal vascular segments and mediate endothelium-dependent vasorelaxation. Moreover, they are expressed in distinct parts of the tubular system and activated by cell swelling. Ischaemia/reperfusion injury (IRI) is characterized by tubular injury and endothelial dysfunction. Therefore, we hypothesised a putative organ protective role of TRPV4 in acute renal IRI. IRI was induced in TRPV4 deficient (Trpv4 KO) and wild–type (WT) control mice by clipping the left renal pedicle after right–sided nephrectomy. Serum creatinine level was higher in Trpv4 KO mice 6 and 24 hours after ischaemia compared to WT mice. Detailed histological analysis revealed that IRI caused aggravated renal tubular damage in Trpv4 KO mice, especially in the renal cortex. Immunohistological and functional assessment confirmed TRPV4 expression in proximal tubular cells. Furthermore, the tubular damage could be attributed to enhanced necrosis rather than apoptosis. Surprisingly, the percentage of infiltrating granulocytes and macrophages were comparable in IRI–damaged kidneys of Trpv4 KO and WT mice. The present results suggest a renoprotective role of TRPV4 during acute renal IRI. Further studies using cell–specific TRPV4 deficient mice are needed to clarify cellular mechanisms of TRPV4 in IRI.

Gly-Val-Arg, an angiotensin-I-converting enzyme inhibitory tripeptide ameliorates hypertension on spontaneously hypertensive rats

Abstract The tripeptide GVR was previously shown to inhibit ACE and was able to reduce systolic blood pressure in SHRs within 6 h after administration. To further analyse the potential of tripeptide GVR in lowering blood pressure, a long term study was carried out where the tripeptide was orally administered to SHRs for 21 days. Elevated systolic blood pressure was significantly reduced when the peptide was administered at 50 mg/kg body weight and 100 mg/kg body weight. Body weight of the SHRs did not change significantly between the studied groups and the histopathological findings indicated that there were no abnormalities in liver and kidney tissues. From the acute toxicity analysis of this tripeptide, using the Up and Down method revealed that the peptide was non-toxic to SHRs. Tripeptide GVR demonstrated endothelium-dependent vasorelaxation activity using thoracic aortas from SD rats in which vasorelaxation was not observed in denuded aortas. The metabolomic analysis carried out from the sera of SHRs demonstrated changes on metabolites associated with pathways related to renin angiotensin system. As for proteomic analysis, differentially expressed proteins detected were mainly proteins related with inflammation. Based on the results, tripeptide GVR has a potential to ameliorate the elevated blood pressure in SHRs.

Stachydrine protects eNOS uncoupling and ameliorates endothelial dysfunction induced by homocysteine

BackgroundHyperhomocysteinemia (HHcy) is an independent risk factor for cardiovascular diseases (CVDs). Stachydrine (STA) is an active component in Chinese motherwort Leonurus heterophyllus sweet, which has been widely used for gynecological and cardiovascular disorders. This study is aimed to examine the effects of STA on homocysteine (Hcy)-induced endothelial dysfunction.MethodsThe effects of STA on vascular relaxation in rat thoracic aortas (TA), mesenteric arteries (MA) and renal arteries (RA) were measured by using Multi Myograph System. The levels of nitric oxide (NO), tetrahydrobiopterin (BH4) and guanosine 3′, 5′ cyclic monophosphate (cGMP) were determined. Endothelial nitric oxide synthase (eNOS) dimers and monomers were assayed by using Western blotting. GTP cyclohydrolase 1 (GTPCH1) and dihydrofolate reductase (DHFR) expressions were measured by using quantitative reverse transcriptase-PCR (qRT-PCR) and Western blotting.ResultsSTA effectively blocked Hcy-induced impairment of endothelium-dependent vasorelaxation in rat TA, MA and RA. STA-elicited arterial relaxations were reduced by NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME) or the NO-sensitive guanylyl cyclase inhibitor 1H- [1, 2, 4] Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), but not by inducible iNOS inhibitor 1400 W nor the nonselective COX inhibitor indomethacin. Hcy caused eNOS uncoupling and decreases in NO, cGMP and BH4, which were attenuated by STA. Moreover, STA prevented decreases of GTPCH1 and DHFR levels in Hcy-treated BAECs.ConclusionWe demonstrated that STA effectively reversed the Hcy-induced endothelial dysfunction and prevented eNOS uncoupling by increasing the expression of GTPCH1 and DHFR. These results revealed a novel mechanism by which STA exerts its beneficial vascular effects.

Fenugreek (Trigonella Foenum-Graecum) Seed Flour and Diosgenin Preserve Endothelium-Dependent Arterial Relaxation in a Rat Model of Early-Stage Metabolic Syndrome.

Fenugreek is a common herb possessing several bioactive components including diosgenin. Here, dietary fenugreek seed flour and diosgenin were evaluated on a model of endothelium-dependent vasorelaxation by abdominal aortas isolated from rats receiving high-fat, high-sugar diet (HFHSD). 60 male Wistar rats were randomized into six groups: (i) negative control getting conventional rat feed regimen; (ii) positive control receiving HFHSD; (iii) a test group fed 2 g/kg bw/day fenugreek seed flour (containing 10 mg/kg bw/day diosgenin) + HFHSD; (iv) three test groups fed 1, 10 and 50 mg/kg bw/day diosgenin + HFHSD. Alimentary treatments were carried out for six weeks. The abdominal aortas were isolated, and 2 mm wide rings were sectioned off and mounted at a resting tension of 10 mN in organ baths containing Krebs solution (36 °C) exposed to 95% O2 and 5% CO2. After 60-min incubation, a norepinephrine concentration-response (E/c) curve was generated to determine their half-maximal effective concentration (EC50) value. After 60-min wash-out, a pre-contraction with norepinephrine EC50 was made, followed by an acetylcholine E/c curve. Plasma glutathione levels, glutathione-handling enzyme activities and blood antioxidant capacities were also determined. HFHSD significantly decreased the dilatory response to acetylcholine and increased plasma glutathione levels and these effects were significantly reversed by fenugreek seed flour, 10 and 50 mg/kg bw/day diosgenin. Both fenugreek and diosgenin treatments prevent HFHSD-induced endothelial dysfunction and redox changes. As fenugreek treatment was more effective at lower acetylcholine concentrations than diosgenin treatments, components of fenugreek other than diosgenin may contribute to the beneficial effects of dietary fenugreek seed flour.

Acidosis potentiates endothelium-dependent vasorelaxation and gap junction communication in the superior mesenteric artery

Extracellular pH is an important physiological determinant of vascular tone that is normally maintained within 7.35–7.45. Any change outside this range leads to severe pathological repercussions. We investigated the unknown effects of extracellular acidosis on relaxation in the superior mesenteric artery (SMA) of goat. SMA rings were employed to maintain isometric contractions at extracellular pH (pHo) 7.4 and 6.8. We analyzed the effect of acidosis (pHo 6.8) compared to physiological pH (pHo 7.4) on three signaling mediators of endothelium-dependent hyperpolarization: nitric oxide (NO), prostaglandin I2 (PGI2), and myoendothelial gap junctions (MEGJ). NO and cyclic guanosine monophosphate (cGMP) levels were compared between normal and acidic pH. Quantitative real-time PCR (qPCR) studies determined the change in expression of vascular connexin (Cx), Cx37, Cx40, and Cx43. Under acidosis, acetyl choline-induced relaxation was augmented in an endothelium-dependent manner via eNOS-NO-cGMP signaling. Conversely, at normal pH, acetyl choline-induced vasorelaxation was mediated primarily via COX-PGI2 pathway. The functional activity of MEGJ was increased under acidosis as evident from increased sensitivity of connexin blockers and upregulated gene and protein expression of connexins. In conclusion, acetyl choline-induced augmented vasorelaxation under acidosis is mediated by NOS-NO-cGMP, with a partial role of MEGJ as EDH mediators in the SMA. Present data suggest a novel role of connexin as therapeutic targets to attenuate the detrimental effect of acidosis on vascular tone.

Nitric oxide-sensitive guanylyl cyclase stimulation improves experimental heart failure with preserved ejection fraction.

Heart failure with preserved ejection fraction (HFpEF) can arise from cardiac and vascular remodeling processes following long-lasting hypertension. Efficacy of common HF therapeutics is unsatisfactory in HFpEF. Evidence suggests that stimulators of the nitric oxide-sensitive soluble guanylyl cyclase (NOsGC) could be of use here. We aimed to characterize the complex cardiovascular effects of NOsGC stimulation using NO-independent stimulator BAY 41-8543 in a double-transgenic rat (dTGR) model of HFpEF. We show a drastically improved survival rate of treated dTGR. We observed less cardiac fibrosis, macrophage infiltration, and gap junction remodeling in treated dTGR. Microarray analysis revealed that treatment of dTGR corrected the dysregulateion of cardiac genes associated with fibrosis, inflammation, apoptosis, oxidative stress, and ion channel function toward an expression profile similar to healthy controls. Treatment reduced systemic blood pressure levels and improved endothelium-dependent vasorelaxation of resistance vessels. Further comprehensive in vivo phenotyping showed an improved diastolic cardiac function, improved hemodynamics, and less susceptibility to ventricular arrhythmias. Short-term BAY 41-8543 application in isolated untreated transgenic hearts with structural remodeling significantly reduced the occurrence of ventricular arrhythmias, suggesting a direct nongenomic role of NOsGC stimulation on excitation. Thus, NOsGC stimulation was highly effective in improving several HFpEF facets in this animal model, underscoring its potential value for patients.

Angiotensin-(1-7) and Alamandine on Experimental Models of Hypertension and Atherosclerosis.

The purpose of this review was to summarize the current knowledge on the role of angiotensin-(1-7) [Ang-(1-7)] and alamandine in experimental hypertension and atherosclerosis. The renin-angiotensin system (RAS) is a very complex system, composed of a cascade of enzymes, peptides, and receptors, known to be involved in the pathogenesis of hypertension and atherosclerosis. Ang-(1-7), identified and characterized in 1987, and alamandine, discovered 16years after, are the newest two main effector molecules from the RAS, protecting the vascular system against hypertension and atherosclerosis. While the beneficial effects of Ang-(1-7) have been widely studied in several experimental models of hypertension, much less studies were performed in experimental models of atherosclerosis. Alamandine has shown similar vascular effects to Ang-(1-7), namely, endothelial-dependent vasorelaxation mediated by nitric oxide and hypotensive effects in experimental hypertension. There are few studies on the effects of alamandine on atherosclerosis.

Testosterone to estrogen conversion is not responsible for the vasodilating effects of testosterone ex vivo.

Men have a higher incidence of cardiovascular disease, but poorer vasodilatation than women. However, testosterone exerts vasodilating actions ex vivo. We aimed to determine if reactive oxygen species (ROS) produced in vivo could cause 'eNOS uncoupling' that accounts for the disparity between in vivo and ex vivo results. Ovariectomized SHR and WKY rats were divided into 3 groups: untreated, estradiol benzoate (EB) treated, and testosterone propionate (TP) treated. EB and TP rats were treated for 8 weeks, and blood pressure, serum estrogen, progesterone, and testosterone were measured. Rats were euthanized and aorta samples were taken for examination of nitric oxide, phosphorylated eNOS (p-eNOS), H2O2, gr91phos, and pAkt. Mesenteric arterial rings were used in myographic studies of endothelium dependent and independent vasorelaxation. The influence of testosterone added to the bathing solution of rings from testosterone-supplemented rats with/without an eNOS inhibitor, with/without blockade of androgen or estrogen receptors, and with/without an inhibitor of gp91phox was examined. Treatment with testosterone for 8 weeks did not change endothelium-dependent relaxation in response to acetylcholine in the presence or absence of the eNOS inhibitor L-NAME, or in the presence or absence of blockade of the androgen receptors, estrogen receptors, or gp91phox. No change in nitric oxide, p-eNOS, pAkt, or gp91phos of the aorta was noted. A significant increase in H2O2 was seen in testosterone-supplemented SHR rats, but this was not accompanied by eNOS uncoupling. These results suggest that conversion of testosterone to estrogen is not responsible for its vasodilating effects seen ex vivo.

Small dense HDLs display potent vasorelaxing activity, reflecting their elevated content of sphingosine-1-phosphate

The functional heterogeneity of HDL is attributed to its diverse bioactive components. We evaluated whether the vasodilatory effects of HDL differed across HDL subpopulations, reflecting their distinct molecular composition. The capacity of five major HDL subfractions to counteract the inhibitory effects of oxidized LDL on acetylcholine-induced vasodilation was tested in a rabbit aortic rings model. NO production, an essential pathway in endothelium-dependent vasorelaxation, was studied in simian vacuolating virus 40-transformed murine endothelial cells (SVECs). Small dense HDL3 subfractions displayed potent vasorelaxing activity (up to +31% vs. baseline, P < 0.05); in contrast, large light HDL2 did not induce aortic-ring relaxation when compared on a total protein basis. HDL3 particles were enriched with sphingosine-1-phosphate (S1P) (up to 3-fold vs. HDL2), with the highest content in HDL3b and -3c that concomitantly revealed the strongest vasorelaxing properties. NO generation was enhanced by HDL3c in SVECs (1.5-fold, P < 0.01), a phenomenon that was blocked by the S1P receptor antagonist, VPC 23019. S1P-enriched reconstituted HDL (rHDL) was a 1.8-fold (P < 0.01) more potent vasorelaxant than control rHDL in aortic rings. Small dense HDL3 particles displayed potent protective effects against oxidative stress-associated endothelium dysfunction, potentially reflecting their elevated content of S1P that might facilitate interaction with S1P receptors and ensuing NO generation.

Potentiation of endothelium-dependent vasorelaxation of mesenteric arteries from spontaneously hypertensive rats by gemigliptin, a dipeptidyl peptidase-4 inhibitor class of anti-diabetic drug.

Dipeptidyl peptidase4 (DPP4) inhibitors such as gemigliptin are anti-diabetic drugs elevating plasma concentration of incretins such as GLP-1. In addition to the DPP4 inhibition, gemigliptin might directly improve the functions of vessels under pathological conditions. To test this hypothesis, we investigated whether the acetylcholine-induced endothelium dependent relaxation (ACh-EDR) of mesenteric arteries (MA) are altered by gemigliptin pretreatment in Spontaneous Hypertensive Rats (SHR) and in Wistar-Kyoto rats (WKY) under hyperglycemia-like conditions (HG; 2 hr incubation with 50 mM glucose). ACh-EDR of WKY was reduced by the HG condition, which was significantly recovered by 1 µM gemigliptin while not by saxagliptin and sitagliptin up to 10 µM. The ACh-EDR of SHR MA was also improved by 1 µM gemigliptin while similar recovery was observed with higher concentration (10 µM) of saxagliptin and sitagliptin. The facilitation of ACh-EDR by gemigliptin in SHR was not observed under pretreatment with NOS inhibitor, L-NAME. In the endotheliumdenuded MA of SHR, sodium nitroprusside induced dose-dependent relaxation was not affected by gemigliptin. The ACh-EDR in WKY was decreased by treatment with 30 µM pyrogallol, a superoxide generator, which was not prevented by gemigliptin. Exendin-4, a GLP-1 analogue, could not enhance the ACh-EDR in SHR MA. The present results of ex vivo study suggest that gemigliptin enhances the NOS-mediated EDR of the HG-treated MA as well as the MA from SHR via GLP-1 receptor independent mechanism.

Vascular mechanisms underlying the hypotensive effect of Rumex acetosa

Context: Rumex acetosa L. (Polygonaceae) is well known in traditional medicine for its therapeutic efficacy as an antihypertensive.Objective: The study investigates antihypertensive potential of crude methanol extract (Ra.Cr) and fractions of Rumex acetosa in normotensive and hypertensive rat models and probes the underlying vascular mechanisms.Materials and methods: Ra.Cr and its fractions were tested in vivo on normotensive and hypertensive Sprague-Dawley rats under anaesthesia for blood pressure lowering effect. In vitro experiments on rat and Oryctolagus cuniculus rabbit aortae were employed to probe the underlying vasorelaxant mechanism.Results: In normotensive rats under anaesthesia, Ra.Cr caused fall in MAP (40 mmHg) at 50 mg/kg with % fall of 27.88 ± 4.55. Among the fractions tested, aqueous fraction was more potent at the dose of 50 mg/kg with % fall of 45.63 ± 2.84. In hypertensive rats under similar conditions, extract and fractions showed antihypertensive effect at same doses while aqueous fraction being more potent, exhibited 68.53 ± 4.45% fall in MAP (70 mmHg). In isolated rat aortic rings precontracted with phenylephrine (PE), Ra.Cr and fractions induced endothelium-dependent vasorelaxation, which was partially blocked in presence of l-NAME, indomethacin and atropine. In isolated rabbit aortic rings pre-contracted with PE and K+-(80 mM), Ra.Cr induced vasorelaxation and shifted Ca2+ concentration–response curves to the right and suppressed PE peak formation, similar to verapamil, in Ca2+-free medium.Discussion and conclusions: The data indicate that l-NAME and atropine-sensitive endothelial-derived NO and COX enzyme inhibitors and Ca2+ entry blocking-mediated vasodilator effect of the extract explain its antihypertensive potential.

Acute Hyperbaric Oxygenation, Contrary to Intermittent Hyperbaric Oxygenation, Adversely Affects Vasorelaxation in Healthy Sprague-Dawley Rats due to Increased Oxidative Stress.

The present study was aimed at assessing endothelium-dependent vasorelaxation, at measuring superoxide production in the aorta and femoral artery, and at determining antioxidative enzyme expression and activity in aortas of male Sprague-Dawley rats (N = 135), randomized to an A-HBO2 group exposed to a single hyperbaric oxygenation session (120′ of 100% O2 at 2.0 bars), a 24H-HBO2 group (single session, examined 24 h after exposure), a 4D-HBO2 group (4 consecutive days of single sessions), and a CTRL group (untreated group). Vasorelaxation of aortic rings in response to acetylcholine (AChIR) and to reduced pO2 (HIR) was tested in vitro in the absence/presence of NOS inhibitor L-NAME and superoxide scavenger TEMPOL. eNOS, iNOS, antioxidative enzyme, and NADPH oxidase mRNA expression was assessed by qPCR. Serum oxidative stress markers and enzyme activity were assessed by spectrometry, and superoxide production was determined by DHE fluorescence. Impaired AChIR and HIR in the A-HBO2 group were restored by TEMPOL. L-NAME inhibited AChIR in all groups. Serum oxidative stress and superoxide production were increased in the A-HBO2 group compared to all other groups. The mRNA expression of iNOS was decreased in the A-HBO2 and 24H-HBO2 groups while SOD1 and 3 and NADPH oxidase were increased in the 4D-HBO2 group. The expression and activity of catalase and glutathione peroxidase were increased in the 4D-HBO2 group as well. AChIR was NO dependent. Acute HBO2 transiently impaired vasorelaxation due to increased oxidative stress. Vasorelaxation was restored and oxidative stress was normalized 24 h after the treatment.

Endothelial dysfunction occurs independently of adipose tissue inflammation and insulin resistance in ovariectomized Yucatan miniature-swine

ABSTRACTIn rodents, experimentally-induced ovarian hormone deficiency increases adiposity and adipose tissue (AT) inflammation, which is thought to contribute to insulin resistance and increased cardiovascular disease risk. However, whether this occurs in a translationally-relevant large animal model remains unknown. Herein, we tested the hypothesis that ovariectomy would promote visceral and perivascular AT (PVAT) inflammation, as well as subsequent insulin resistance and peripheral vascular dysfunction in female swine. At sexual maturity (7 months of age), female Yucatan mini-swine either remained intact (control, n = 9) or were ovariectomized (OVX, n = 7). All pigs were fed standard chow (15–20 g/kg), and were euthanized 6 months post-surgery. Uterine mass and plasma estradiol levels were decreased by ∼10-fold and 2-fold, respectively, in OVX compared to control pigs. Body mass, glucose homeostasis, and markers of insulin resistance were not different between control and OVX pigs; however, OVX animals exhibited greater plasma triglycerides and triglyceride:HDL ratio. Ovariectomy enhanced visceral adipocyte expansion, although this was not accompanied by brachial artery PVAT adipocyte expansion, AT inflammation in either depot, or increased systemic inflammation assessed by plasma C-reactive protein concentrations. Despite the lack of AT inflammation and insulin resistance, OVX pigs exhibited depressed brachial artery endothelial-dependent vasorelaxation, which was rescued with blockade of endothelin receptor A. Together, these findings indicate that in female Yucatan mini-swine, increased AT inflammation and insulin resistance are not required for loss of ovarian hormones to induce endothelial dysfunction.

Abstract 21290: Adipose Tissue Factors and the Adipokine Resistin-Induce Vascular Endothelial Dysfunction via Arginase Upregulation

Introduction: Obesity is a major contributor to cardiovascular disorders, for which endothelial cell (EC) dysfunction is a hallmark. Obesity-induced EC dysfunction has been linked to dysregulation of the visceral adipose tissue (VAT) resulting in increased secretion of the pro-inflammatory cytokine resistin and alterations in endothelial nitric oxide (NO) producing capacity. However, mechanisms underlying this pathology are not well understood. Hypothesis: Resistin impairs vascular EC function via upregulation of endothelial arginase1 (ARG1) expression and activity, thus limiting L-arginine availability for NO synthesis. Methods: To determine the effect of obesity on EC function, conditioned media prepared from VAT (VAT CM) of high fat diet-induced obese mice, VAT CM from lean mice or resistin (100 ηg/ml) were used to treat mouse aortic endothelial cells (MAEC) and aortic rings for 24 hr. Results: VAT CM from obese mice increased endothelial ARG1 mRNA by 34% and decreased NO level by 32% (p&lt;0.05), vs VAT CM from lean mice. Aortic rings incubated with obese VAT CM markedly reduced endothelium-dependent vasorelaxation (EDV) to acetylcholine. Moreover, resistin levels were 143% higher in obese VAT CM compared to lean VAT CM. In MAEC, resistin increased ARG1 mRNA by 30% and decreased NO production by 39% (p&lt;0.05). Co-treatment with the specific ARG inhibitor (ABH, 100 μM) or supplementing EC with L-arginine increased NO to near normal levels. Incubation of aortic rings with resistin also impaired EDV. This dysfunction was largely prevented by co-treatment with ABH or in aortic rings from EC specific-ARG1 knockout mice. The resistin-induced increase in ARG1 expression in MAEC is attributed to Toll-like receptor 4 (TLR4)-induced activation of p38 MAPK pathway as co-treatment with specific TLR4 inhibitor (CLI-095) inhibited p38 activation and ARG1 expression (34% and 33%, respectively, p&lt;0.05) and increased NO production by 38% (p&lt;0.05). Additionally, co-treatment with a specific p38 inhibitor prevented resistin-induced increase in endothelial ARG1 expression. Conclusions: VAT factors and particularly resistin induce vascular endothelial dysfunction via elevated arginase expression/activity. Support : NIH EY11766 &amp; HL70215 &amp; AHA fellowship

165 - Effect of Nitrite and N-acetylcysteine Treatment on Blood Pressure, Arterial Stiffness and Vascular Function in Spontaneously Hypertensive Rats

Introduction Reduced nitric oxide (NO) bioavailability and increased generation of reactive oxygen species and inflammation with advancing age play a major role in the development of cardiovascular disease (CVD) risk factors of hypertension, endothelial dysfunction and arterial stiffness. Therefore, increase in NO bioavailability by supplementing inorganic nitrite and improve in antioxidant defense system by supplementing N-acetylcysteine (NAC) could protect against the CVD. Hypertension is highly prevalent in a population and cause endothelial dysfunction and arterial stiffness. Hence spontaneously hypertensive rats (SHR) were used in this study as a model for hypertension-mediated endothelial dysfunction and arterial stiffness. Hypothesis Treatment of SHR with nitrite or NAC will reduce blood pressure, arterial stiffness, and endothelial dysfunction. Methods SHR (18 weeks) and normotensive Wistar Kyoto (WKY) rats were divided into 4 groups: 1) WKY, 2) SHR-cont, 3) SHR-nitrite and 4) SHR-NAC. Group 3 rats received nitrite (60 mg/L) in drinking water and group 4 rats received NAC pH 7.2 (1.2 g/L) for 3 months. Mean arterial pressure (MAP; tail-cuff method) and pulse wave velocity (PWV, a measure of arterial stiffness) were measured. Aortic endothelial function was determined with a wire myograph. Results MAP & PWV of SHR-cont were 1.8-fold and 1.6-fold higher compared with WKY respectively. MAP was modestly decreased in nitrite and NAC groups compared with that of SHR-cont. PWV did not decrease significantly with NAC or nitrite treatment. Endothelium dependent vasorelaxation was significantly decreased in SHR-cont compared with WKY. This decreased vasorelaxation was restored to WKY level in both nitrite and NAC groups. Conclusion Nitrite and NAC treatments completely protected against the hypertension induced endothelium dysfunction in SHR. Nitrite and NAC appears to be very promising dietary components to protect against age-associated endothelial dysfunction.

Tetramethylpyrazine suppresses angiotensin II-induced soluble epoxide hydrolase expression in coronary endothelium via anti-ER stress mechanism

Activation of soluble epoxide hydrolase (sEH) is associated with endothelial dysfunction in hypertension, though the underlying mechanisms are inadequately understood and the role of endoplasmic reticulum (ER) stress is yet to be studied in detail. Tetramethylpyrazine (TMP), a major bioactive ingredient of Chinese herb Chuanxiong, is well-known for its cardiovascular benefits. Nevertheless, whether TMP may protect vascular endothelium from ER stress and whether regulation of sEH is involved remain unknown. This study aimed at investigating the role of ER stress in angiotensin-II (Ang-II)-induced sEH dysregulation and elucidating the significance of ER stress regulation in the vasoprotective effect of TMP. Porcine primary coronary artery endothelial cells (PCECs) were used for western blot, ELISA, and reverse-transcription PCR analysis. Porcine coronary arteries were assessed in a myograph for endothelial dilator function. Ang-II induced expression of ER stress molecules in PCECs meanwhile enhanced sEH expression and decreased 11,12-EET. Exposure of PCECs to the chemical ER stress inducer tunicamycin also increased sEH expression. Inhibition of ER stress suppressed sEH upregulation, resulting in an increase of 11,12-EET. The impairment of endothelium-dependent vasorelaxation induced by Ang-II or tunicamycin was ameliorated by inhibitors of ER stress or sEH. TMP showed comparable inhibitory effect to ER stress inhibitors on the expression of ER stress molecules, the dysregulation of sEH/EET, and the impairment of endothelial dilator function. We demonstrated that ER stress mediates Ang-II-induced sEH upregulation in coronary endothelium. TMP has potent anti-ER stress capacity through which TMP normalizes sEH expression and confers protective effect against Ang-II on endothelial function of coronary arteries.

Omega-3 Fatty Acids Modulate TRPV4 Function through Plasma Membrane Remodeling

Dietary consumption of ω-3 polyunsaturated fatty acids (PUFAs), present in fish oils, is known to improve the vascular response, but their molecular targets remain largely unknown. Activation of the TRPV4 channel has been implicated in endothelium-dependent vasorelaxation. Here, we studied the contribution of ω-3 PUFAs to TRPV4 function by precisely manipulating the fatty acid content in Caenorhabditis elegans. By genetically depriving the worms of PUFAs, we determined that the metabolism of ω-3 fatty acids is required for TRPV4 activity. Functional, lipid metabolome, and biophysical analyses demonstrated that ω-3 PUFAs enhance TRPV4 function in human endothelial cells and support the hypothesis that lipid metabolism and membrane remodeling regulate cell reactivity. We propose a model whereby the eicosanoid's epoxide group location increases membrane fluidity and influences the endothelial cell response by increasing TRPV4 channel activity. ω-3 PUFA-like molecules might be viable antihypertensive agents for targeting TRPV4 to reduce systemic blood pressure.

Molecular and functional characterization of the endothelial ATP-sensitive potassium channel

ATP-sensitive potassium (KATP) channels are widely expressed in the cardiovascular system, where they regulate a range of biological activities by linking cellular metabolism with membrane excitability. KATP channels in vascular smooth muscle have a well-defined role in regulating vascular tone. KATP channels are also thought to be expressed in vascular endothelial cells, but their presence and function in this context are less clear. As a result, we aimed to investigate the molecular composition and physiological role of endothelial KATP channels. We first generated mice with an endothelial specific deletion of the channel subunit Kir6.1 (eKO) using cre-loxP technology. Data from qRT-PCR, patch clamp, ex vivo coronary perfusion Langendorff heart experiments, and endothelial cell Ca2+ imaging comparing eKO and wild-type mice show that Kir6.1-containing KATP channels are indeed present in vascular endothelium. An increase in intracellular [Ca2+], which is central to changes in endothelial function such as mediator release, at least partly contributes to the endothelium-dependent vasorelaxation induced by the KATP channel opener pinacidil. The absence of Kir6.1 did not elevate basal coronary perfusion pressure in eKO mice. However, vasorelaxation was impaired during hypoxia in the coronary circulation, and this resulted in greater cardiac injury during ischemia-reperfusion. The response to adenosine receptor stimulation was impaired in eKO mice in single cells in patch clamp recordings and in the intact coronary circulation. Our data support the existence of an endothelial KATP channel that contains Kir6.1, is involved in vascular reactivity in the coronary circulation, and has a protective role in ischemia reperfusion.

The cAMP effectors PKA and Epac activate endothelial NO synthase through PI3K/Akt pathway in human endothelial cells

3′,5′-Cyclic adenosine monophosphate (cAMP) exerts an endothelium-dependent vasorelaxant action by stimulating endothelial NO synthase (eNOS) activity, and the subsequent NO release, through cAMP protein kinase (PKA) and exchange protein directly activated by cAMP (Epac) activation in endothelial cells. Here, we have investigated the mechanism by which the cAMP-Epac/PKA pathway activates eNOS. cAMP-elevating agents (forskolin and dibutyryl-cAMP) and the joint activation of PKA (6-Bnz-cAMP) and Epac (8-pCPT-2′-O-Me-cAMP) increased cytoplasmic Ca2+ concentration ([Ca2+]c) in ≤30% of fura-2-loaded isolated human umbilical vein endothelial cells (HUVEC). However, these drugs did not modify [Ca2+]c in fluo-4-loaded HUVEC monolayers. In DAF-2-loaded HUVEC monolayers, forskolin, PKA and Epac activators significantly increased NO release, and the forskolin effect was reduced by inhibition of PKA (Rp-cAMPs), Epac (ESI-09), eNOS (L-NAME) or phosphoinositide 3-kinase (PI3K; LY-294,002). On the other hand, inhibition of CaMKII (KN-93), AMPK (Compound C), or total absence of Ca2+, was without effect. In Western blot experiments, Serine 1177 phosphorylated-eNOS was significantly increased in HUVEC by cAMP-elevating agents and PKA or Epac activators. In isolated rat aortic rings LY-294,002, but not KN-93 or Compound C, significantly reduced the vasorelaxant effects of forskolin in the presence of endothelium. Our results suggest that Epac and PKA activate eNOS via Ser 1177 phosphorylation by activating the PI3K/Akt pathway, and independently of AMPK or CaMKII activation or [Ca2+]c increase. This action explains, in part, the endothelium-dependent vasorelaxant effect of cAMP.

Obesity-induced vascular dysfunction and arterial stiffening requires endothelial cell arginase 1.

Elevation of arginase activity has been linked to vascular dysfunction in diabetes and hypertension by a mechanism involving decreased nitric oxide (NO) bioavailability due to L-arginine depletion. Excessive arginase activity also can drive L-arginine metabolism towards the production of ornithine, polyamines, and proline, promoting proliferation of vascular smooth muscle cells and collagen formation, leading to perivascular fibrosis. We hypothesized that there is a specific involvement of arginase 1 expression within the vascular endothelial cells in this pathology. To test this proposition, we used models of type 2 diabetes and metabolic syndrome. Studies were performed using wild type (WT), endothelial-specific arginase 1 knockout (EC-A1-/-) and littermate controls(A1con) mice fed high fat-high sucrose (HFHS) or normal diet (ND) for 6 months and isolated vessels exposed to palmitate-high glucose (PA/HG) media. Some WT mice or isolated vessels were treated with an arginase inhibitor, ABH [2-(S)-amino-6-boronohexanoic acid. In WT mice, the HFHS diet promoted increases in body weight, fasting blood glucose, and post-prandial insulin levels along with arterial stiffening and fibrosis, elevated blood pressure, decreased plasma levels of L-arginine, and elevated L-ornithine. The HFHS diet or PA/HG treatment also induced increases in vascular arginase activity along with oxidative stress, reduced vascular NO levels, and impaired endothelial-dependent vasorelaxation. All of these effects except obesity and hypercholesterolemia were prevented or significantly reduced by endothelial-specific deletion of arginase 1 or ABH treatment. Vascular dysfunctions in diet-induced obesity are prevented by deletion of arginase 1 in vascular endothelial cells or arginase inhibition. These findings indicate that upregulation of arginase 1 expression/activity in vascular endothelial cells has an integral role in diet-induced cardiovascular dysfunction and metabolic syndrome.