Advanced glycated end-products inhibit dilation through constitutive endothelial RAGE and Nox1/4 in rat isolated skeletal muscle arteries.

OBJECTIVE: This study investigated the actions of advanced glycated end-products (AGE), their receptors (RAGE), and NAD(P)H oxidase (Nox) subtypes 1, 2, and 4 on mechanisms of endothelium-dependent dilation of the rat cremaster muscle artery (CMA). METHODS: Immunofluorescence studies were used to examine expression of RAGE in rat arteries. ROS accumulation was measured using luminescence and fluorescence assays. Functional studies were performed using pressure myography. RESULTS: High levels of RAGE expression were shown in the endothelial cells of the CMA, compared with low endothelial expression in middle cerebral and mesenteric arteries and the aorta. Exogenous AGE (in vitro glycated bovine serum albumin) stimulated H2O2 accumulation in CMA, which was prevented by the RAGE antagonist FPS-ZM1, the NAD(P)H oxidase (Nox) inhibitor apocynin and inhibited by the Nox1/4 inhibitor setanaxib, but not the Nox2 inhibitor GSK2795039. In functional studies, AGE inhibited vasodilation of CMA stimulated by acetylcholine, sodium nitroprusside, and the BKCa activator NS1619, but not adenosine-induced dilation. FPS-ZM1, apocynin, and setanaxib prevented the inhibitory effects of AGE on responses to acetylcholine and NS-1619. CONCLUSION: These observations suggest RAGE are constitutively expressed in the endothelium of the rat CMA and may be activated by AGE to stimulate Nox1/4 and ROS formation with resulting inhibition of NO and BKCa-mediated endothelium-dependent dilation.

Endothelial cell Orai1 is essential for endothelium-dependent contraction of mouse carotid arteries in normotensive and hypertensive mice.

Endothelium-dependent contraction (EDC) exists in blood vessels of normotensive animals, but is exaggerated in hypertension. An early signal in EDC is cytosolic Ca2+ rise in endothelial cells. In this study we investigated the functional role of Orai1, a major endothelial cell Ca2+ entry channel, in EDC. Hypertension model was established in WT mice by intake of L-NNA in the drinking water (0.5 g/L) for 4 weeks or osmotic pump delivery of Ang II (1.5 mg·kg-1·d-1) for 2 weeks. In TRPC5 KO mice, the concentration of L-NNA and Ang II were increased to 1 g/L or 2 mg·kg-1·d-1, respectively. Arterial segments were prepared from carotid arteries and aortas, and EDC was elicited by acetylcholine in the presence of Nω-nitro-L-arginine methyl ester. We showed that low concentration of acetylcholine (3-30 nM) initiated relaxation in phenylephrine-precontracted carotid arteries of both normotensive and hypertensive mice, while high concentration of acetylcholine (0.1-2 µM) induced contraction. Application of selective Orai1 inhibitors AnCoA4 (100 µM) or YM58483 (400 nM) had no effect on ACh-induced relaxation but markedly reduced acetylcholine-induced EDC. We found that EDC was increased in hypertensive mice compared with that of normotensive mice, which was associated with increased Orai1 expression in endothelial cells of hypertensive mice. Compared to TRPC5 and TRPV4, which were also involved in EDC, endothelial cell Orai1 had relatively greater contribution to EDC than either TRPC5 or TRPV4 alone. We identified COX-2, followed by PGF2α, PGD2 and PGE2 as the downstream signals of Orai1/TRPC5/TRPV4. In conclusion, Orai1 coordinates together with TRPC5 and TRPV4 in endothelial cells to regulate EDC responses. This study demonstrates a novel function of Orai1 in EDC in both normotensive and hypertensive mice, thus providing a general scheme about the control of EDC by Ca2+-permeable channels.

Extract of Chenopodium album lowers blood pressure in rats through endothelium-dependent and -independent vasorelaxation.

OBJECTIVES: To investigate the antihypertensive effect of crude extract of Chenopodium album (Ca.Cr), based on its medicinal use in hypertension. METHODS: Ca.Cr and its fractions were tested in-vivo in normotensive anesthetized rats for blood pressure-lowering effect. In-vitro experiments were performed on isolated rat aortae to explore the vascular mechanism(s). RESULTS: In normotensive anesthetized rats, Ca.Cr produced a dose-dependent (1-300mg/kg) fall (30%mmHg) in mean arterial pressure (MAP). Among the fractions, nHexane was the most potent (46% fall). In rat aortic rings precontracted with phenylephrine (PE), Ca.Cr and its fractions (except Ca.Aq) produced endothelium-dependent vasorelaxation, which was partially reversed with endothelium removal and by pretreating intact aortic rings with L-NAME (10µM) and atropine (1µM). This relaxation to Ca.Cr and fractions (nHexane, ethylacetate and chloroform) was also eliminated with indomethacin pretreatment, however, it unmasked a vasoconstriction effect with Ca.Cr only. Surprisingly, the aqueous fraction produced a calcium sensitive strong vasoconstriction instead of vasorelaxation. The crude extract and its fractions (except Ca.Aq) also antagonized vasoconstriction induced with high K+ (80mM), suggesting calcium antagonistic effect. The aqueous fraction produced mild vasorelaxation against high K+. This effect was further confirmed when pretreatment of the aortic rings with different concentrations of crude extract and fractions suppressed CaCl2 concentration response curves, similar to verapamil. In acute toxicity test, Ca.Cr extract was found safe up to 5g/kg body weight in mice. CONCLUSION: These findings suggest that crude extract and fractions of C. album produced vasorelaxant effect through muscarinic receptors linked-NO pathway, prostaglandin (endothelium-dependent) and calcium antagonism (endothelium-independent), which explains the blood pressure lowering effect of C. album in rats.

Endothelial KIR2 channel dysfunction in aged cerebral parenchymal arterioles.

Aging is associated with cognitive decline via incompletely understood mechanisms. Cerebral microvascular dysfunction occurs in aging, particularly impaired endothelium-mediated dilation. Parenchymal arterioles are bottlenecks of the cerebral microcirculation, and dysfunction causes a mismatch in nutrient demand and delivery, leaving neurons at risk. Extracellular nucleotides elicit parenchymal arteriole dilation by activating endothelial purinergic receptors (P2Y), leading to opening of K+ channels, including inwardly-rectifying K+ channels (KIR2). These channels amplify hyperpolarizing signals, resulting in dilation. However, it remains unknown if endothelial P2Y and KIR2 signaling are altered in brain parenchymal arterioles during aging. We hypothesized that aging impairs endothelial P2Y and KIR2 function in parenchymal arterioles. We observed reduced dilation to the purinergic agonist 2-methyl-S-ADP (1 µM) in arterioles from Aged (>24-month-old) mice when compared to Young (4-6 months of age) despite similar hyperpolarization in endothelial cells tubes. No differences were observed in vasodilation or endothelial cell hyperpolarization to activation of small- and intermediate-conductance Ca2+-activated K+ channels (KCa2.3 / KCa3.1) by NS309. Hyperpolarization to 15 mM [K+]E was smaller in Aged than Young mice, despite a paradoxical increased dilation in Aged arterioles to 15 mM [K+]E that was unchanged by endothelium removal. KIR2 Inhibition attenuated vasodilatory responses to 15 mM [K+]E and 1 µM 2-me-S-ADP in both Young and Aged arterioles. Further, we observed a significant increase in myogenic tone in Aged parenchymal arterioles, which was not enhanced by endothelium removal. We conclude that aging impairs endothelial KIR2 channel function in the cerebral microcirculation with possible compensation by smooth muscle cells.

Involvement of nitric oxide synthase and reactive oxygen species in TRPA1-mediated cutaneous vasodilation in young and older adults.

OBJECTIVE: To investigate the nitric oxide synthase (NOS) and reactive oxygen species (ROS) contributions of the cutaneous vasodilator response to transient receptor potential ankyrin-1 channel (TRPA1) activation in young and older adults. MATERIALS AND METHODS: In sixteen young (20 ± 2 years, 8 females) and sixteen older adults (61 ± 5 years, 8 females), cutaneous vascular conductance normalized to maximum vasodilation (%CVCmax) was assessed at four dorsal forearm skin sites continuously perfused via microdialysis with: 1) vehicle solution (Control, 2 % dimethyl sulfoxide, 2 % Ringer, 96 % propylene glycol), 2) 10 mM Ascorbate (non-specific ROS inhibitor), 3) 10 mM L-NAME (non-specific NOS inhibitor), or 4) Ascorbate+L-NAME. The TRPA1 agonist cinnamaldehyde was co-administered at all sites [0 % (baseline), 2.9 %, 8.8 %, 26.4 %; ≥ 30 min per dose]. RESULTS: %CVCmax was not different between groups for Control, L-NAME, and Ascorbate (all p > 0.05). However, there were significant main dose effects for each site wherein %CVCmax was greater than baseline from 2.9 % to 26.4 % cinnamaldehyde for Control and Ascorbate, and at 26.4 % cinnamaldehyde for L-NAME and Ascorbate+L-NAME (all p < 0.05). For Ascorbate+L-NAME, there was a significant main group effect, wherein perfusion was 6 %CVCmax [95% CI: 2, 11, p < 0.05] greater in the older compared to the young group across all cinnamaldehyde doses. There was a significant main site effect for area under the curve wherein L-NAME and Ascorbate+L-NAME were lower than Control and Ascorbate across groups (all p < 0.05). CONCLUSION: The NOS-dependent cutaneous vasodilator response to TRPA1 activation is maintained in older adults, with no detectable contribution of ascorbate-sensitive ROS in either age group.

Increased cerebral vascular resistance underlies preserved cerebral blood flow in response to orthostasis in humans on a high-salt diet.

Cerebral blood flow autoregulation protects brain tissue from blood pressure variations and maintains cerebral perfusion pressure by changes in vascular resistance. High salt (HS) diet impairs endothelium-dependent vasodilation in many vascular beds, including cerebral microcirculation, and may affect vascular resistance. The aim of present study was to determine if 7-day HS diet affected the reactivity of middle cerebral artery (MCA) to orthostatic challenge in healthy human individuals, and if autoregulatory mechanisms and sympathetic neural regulation were involved in this phenomenon.Twenty-seven persons participated in study (F:21, M:6, age range 19-24). Participants consumed 7-day low-salt (LS) diet (< 2.3 g kitchen salt/day) and afterwards 7-day HS diet (> 11.2 g kitchen salt/day). Blood and urine analysis and anthropometric measurements were performed after each diet. Arterial blood pressure, heart rate and heart rate variability, and cerebral and systemic hemodynamic parameters were recorded simultaneously with transcranial Doppler ultrasound and The Task Force® Monitor in response to orthostatic test.Participants remained normotensive during HS diet. Following both, the LS and HS dietary protocols, mean cerebral blood flow (CBF), as well as the velocity time integral and diastolic blood pressure decreased, and cerebral pulsatility index increased after rising up. Importantly, cerebrovascular resistance significantly increased in response to orthostasis only after HS diet. Urine concentration of noradrenaline and vanillylmandelic acid, baroreflex sensitivity (BRS), and sympathetic neural control was significantly decreased in HS diet.Results suggest that CBF in response to orthostatic test was preserved in HS condition due to altered vascular reactivity of MCA, with increased cerebrovascular resistance and blunted BRS and sympathetic activity.

Phosphorylated PKM2 regulates endothelium-dependent vasodilation in diabetes. / ç£·é ¸åŒ–PKM2è°ƒæŽ§ç³–å°¿ç— å† çš®ä¾èµ–æ€§è¡€ç®¡èˆ’å¼ åŠŸèƒ½.

OBJECTIVES: Endothelium-dependent vasodilation dysfunction is the pathological basis of diabetic macroangiopathy. The utilization and adaptation of endothelial cells to high glucose determine the functional status of endothelial cells. Glycolysis pathway is the major energy source for endothelial cells. Abnormal glycolysis plays an important role in endothelium-dependent vasodilation dysfunction induced by high glucose. Pyruvate kinase isozyme type M2 (PKM2) is one of key enzymes in glycolysis pathway, phosphorylation of PKM2 can reduce the activity of pyruvate kinase and affect the glycolysis process of glucose. TEPP-46 can stabilize PKM2 in its tetramer form, reducing its dimer formation and phosphorylation. Using TEPP-46 as a tool drug to inhibit PKM2 phosphorylation, this study aims to explore the impact and potential mechanism of phosphorylated PKM2 (p-PKM2) on endothelial dependent vasodilation function in high glucose, and to provide a theoretical basis for finding new intervention targets for diabetic macroangiopathy. METHODS: The mice were divided into 3 groups: a wild-type (WT) group (a control group, C57BL/6 mice) and a db/db group (a diabetic group, db/db mice), which were treated with the sodium carboxymethyl cellulose solution (solvent) by gavage once a day, and a TEPP-46 group (a treatment group, db/db mice+TEPP-46), which was gavaged with TEPP-46 (30 mg/kg) and sodium carboxymethyl cellulose solution once a day. After 12 weeks of treatment, the levels of p-PKM2 and PKM2 protein in thoracic aortas, plasma nitric oxide (NO) level and endothelium-dependent vasodilation function of thoracic aortas were detected. High glucose (30 mmol/L) with or without TEPP-46 (10 µmol/L), mannitol incubating human umbilical vein endothelial cells (HUVECs) for 72 hours, respectively. The level of NO in supernatant, the content of NO in cells, and the levels of p-PKM2 and PKM2 protein were detected. Finally, the effect of TEPP-46 on endothelial nitric oxide synthase (eNOS) phosphorylation was detected at the cellular and animal levels. RESULTS: Compared with the control group, the levels of p-PKM2 in thoracic aortas of the diabetic group increased (P<0.05). The responsiveness of thoracic aortas in the diabetic group to acetylcholine (ACh) was 47% lower than that in the control group (P<0.05), and that in TEPP-46 treatment group was 28% higher than that in the diabetic group (P<0.05), while there was no statistically significant difference in the responsiveness of thoracic aortas to sodium nitroprusside (SNP). Compared with the control group, the plasma NO level of mice decreased in the diabetic group, while compared with the diabetic group, the phosphorylation of PKM2 in thoracic aortas decreased and the plasma NO level increased in the TEPP-46 group (both P<0.05). High glucose instead of mannitol induced the increase of PKM2 phosphorylation in HUVECs and reduced the level of NO in supernatant (both P<0.05). HUVECs incubated with TEPP-46 and high glucose reversed the reduction of NO production and secretion induced by high glucose while inhibiting PKM2 phosphorylation (both P<0.05). At the cellular and animal levels, TEPP-46 reversed the decrease of eNOS (ser1177) phosphorylation induced by high glucose (both P<0.05). CONCLUSIONS: p-PKM2 may be involved in the process of endothelium-dependent vasodilation dysfunction in Type 2 diabetes by inhibiting p-eNOS (ser1177)/NO pathway.

The effect of grape (Vitis vinifera) seed extract supplementation on flow-mediated dilation, blood pressure, and heart rate: A systematic review and meta-analysis of controlled trials with duration- and dose-response analysis.

The objective of this systematic review and meta-analysis of controlled trials was to assess the long-term effect of grape seed extract (GSE) supplementation on flow-mediated dilation (FMD), systolic blood pressure (SBP), diastolic blood pressure (DBP), and heart rate (HR) in adults. Web of Science, Scopus, Medline, Cochrane Library, and Google Scholar were searched up to May 24, 2021. Nineteen trials were included in this study. Weighted mean difference (WMD) and 95% confidence interval (CI) were calculated using a random-effects model. GSE supplementation significantly reduced DBP (WMD: -2.20 mmHg, 95% CI: -3.79 to -0.60, I2 = 88.8%) and HR (WMD: -1.25 bpm, 95% CI: -2.32 to -0.19, I2 = 59.5%) but had no significant effects on FMD (WMD: 1.02%, 95% CI: -0.62 to 2.66, I2 = 92.0%) and SBP (WMD: -3.55 mmHg, 95% CI: -7.59 to 0.49, I2 = 97.4%). Subgroup analysis revealed that the dose and duration of GSE administration and the characteristics of study participants could be sources of between-study heterogeneity. Significant non-linear relationships were found between DBP and the duration of GSE supplementation (P = 0.044) and its dose (P = 0.007). In conclusion, GSE may be beneficial for individuals with or at risk of cardiovascular disease because it may have hypotensive and HR-lowering properties.

Reduced Function of Endothelial Nitric Oxide and Hyperpolarization in Artery Grafts with Poor Runoff.

BACKGROUND: The endothelium regulates vascular tonus by releasing nitric oxide (endothelium-derived nitric oxide, EDNO) and hyperpolarizing factor (endothelium-derived hyperpolarizing factor, EDHF). In vein grafts with poor runoff, lack of function of these factors causes severe intimal hyperplasia. This study evaluated how the functions of EDNO and EDHF are altered in artery grafts under poor runoff conditions. MATERIALS AND METHODS: The right common carotid arteries of rabbits were excised and implanted in their original positions as autogenous grafts under normal runoff conditions ("nonoccluded grafts") or poor runoff conditions ("poor runoff grafts"). Histochemical changes, acetylcholine (ACh)-induced effects on endothelium-dependent relaxation and smooth muscle cell (SMC) hyperpolarization were examined. RESULTS: Both artery graft types displayed negligible intimal hyperplasia. In the absence and presence of an EDNO synthase inhibitor, ACh-induced relaxation was lower in grafts with poor runoff than in nonoccluded grafts. Furthermore, ACh-induced but not nonreceptor agonist A23187-induced SMC hyperpolarization was lower in the poor runoff graft group than in the nonoccluded graft group. CONCLUSIONS: Unlike in those in vein grafts, the functions of EDNO and EDHF in autogenous carotid artery grafts under poor runoff conditions were reduced but partly maintained. In such artery grafts, intimal hyperplasia caused by surgical operation was not present. These results may explain some of the mechanisms underlying the improved patency of artery grafts compared with vein grafts.

Adenosine mono-phosphate-activated protein kinase-mammalian target of rapamycin signaling participates in the protective effect of chronic intermittent hypobaric hypoxia on vascular endothelium of metabolic syndrome rats.

Our previous study demonstrated that chronic intermittent hypobaric hypoxia (CIHH) protects vascular endothelium function through ameliorating autophagy in mesenteric arteries of metabolic syndrome (MS) rats. This study aimed to investigate the role of adenosine mono-phosphate-activated protein kinase-mammalian target of rapamycin (AMPK-mTOR) signaling in CIHH effect. Six-week-old male Sprague-Dawley rats were divided into control (CON), MS model, CIHH treatment (CIHH), and MS + CIHH groups. Serum pro-inflammatory cytokines were measured. The endothelium dependent relaxation (EDR), endothelial ultrastructure and autophagosomes were observed in mesenteric arteries. The expression of phosphor (p)-AMPKα, p-mTOR, autophagy-related and endoplasmic reticulum stress-related proteins, p-endothelial nitric oxide synthase, and cathepsin D were assayed. In MS rats, pro-inflammatory cytokines were increased, EDR was attenuated, and endothelial integrity was impaired. In addition, the expression level of p-AMPKα and cathepsin D was down-regulated, but the level of p-mTOR was up-regulated. While in MS + CIHH rats, all aforementioned abnormalities were ameliorated, and the beneficial effect of CIHH was cancelled by AMPKα inhibitor. In conclusion, AMPK-mTOR signaling pathway participates in the protection of CIHH on vascular endothelium of MS rats.

Acute Exposure to Glycated Proteins Impaired in the Endothelium-Dependent Aortic Relaxation: A Matter of Oxidative Stress.

Chronically increased levels of high molecular weight advanced glycation end products (HMW-AGEs) are known to induce cardiovascular dysfunction. Whether an acute increase in HMW-AGE levels affects vascular function remains unknown. In this study, we examined whether acute exposure to HMW-AGEs disturbs aortic vasomotor function. Aortae were obtained from healthy male rats and were acutely pre-treated with HMW-AGEs in organ baths. Aortic relaxation responses to cumulative doses of acetylcholine (ACh), in the presence or absence of superoxide dismutase (SOD), were measured after precontraction with phenylephrine (PE). Furthermore, levels of 3-nitrotyrosine were evaluated on aortic paraffine sections. In our study, we show that acute exposure to HMW-AGEs significantly decreases the aortic relaxation response to ACh. SOD pre-treatment prevents acute HMW-AGEs-induced impairment by limiting superoxide formation. In conclusion, our data demonstrate that acute exposure to HMW-AGEs causes adverse vascular remodelling, characterised by disturbed vasomotor function due to increased oxidative stress. These results create opportunities for future research regarding the acute role of HMW-AGEs in cardiovascular dysfunction.

Lifelong voluntary aerobic exercise prevents age- and Western diet- induced vascular dysfunction, mitochondrial oxidative stress and inflammation in mice.

KEY POINTS: The results of the present study establish the temporal pattern of age-related vascular dysfunction across the adult lifespan in sedentary mice consuming a non-Western diet, and the underlying mechanisms The results demonstrate that consuming a Western diet accelerates and exacerbates vascular ageing across the lifespan in sedentary mice They also show that lifelong voluntary aerobic exercise has remarkable protective effects on vascular function throughout the lifespan, in the setting of ageing alone, as well as ageing compounded by Western diet consumption Overall, the results indicate that amelioration of mitochondrial oxidative stress and inflammation are key mechanisms underlying the voluntary aerobic exercise-associated preservation of vascular function across the lifespan in both the presence and absence of a Western dietary pattern ABSTRACT: Advancing age is the major risk factor for cardiovascular diseases, driven largely by vascular endothelial dysfunction (impaired endothelium-dependent dilatation, EDD) and aortic stiffening (increased aortic pulse wave velocity, aPWV). In humans, vascular ageing occurs in the presence of differences in diet and physical activity, but the interactive effects of these factors are unknown. We assessed carotid artery EDD and aPWV across the lifespan in mice consuming standard (normal) low-fat chow (NC) or a high-fat/high-sucrose Western diet (WD) in the absence (sedentary, SED) or presence (voluntary wheel running, VWR) of aerobic exercise. Ageing impaired nitric oxide-mediated EDD (peak EDD 88 ± 12% 6 months P = 0.003 vs. 59 ± 9% 27 months NC-SED), which was accelerated by WD (60 ± 18% 6 months WD-SED). In NC mice, aPWV increased 32% with age (423 ± 13 cm/s at 24 months P < 0.001 vs. 321 ± 12 cm/s at 6 months) and absolute values were an additional ∼10% higher at any age in WD mice (P = 0.042 vs. NC-SED). Increases in aPWV with age in NC and WD mice were associated with 30-65% increases in aortic intrinsic wall stiffness (6 vs. 19-27 months, P = 0.007). Lifelong aerobic exercise prevented age- and WD-related vascular dysfunction across the lifespan, and this protection appeared to be mediated by mitigation of vascular mitochondrial oxidative stress and inflammation. Our results depict the temporal impairment of vascular function over the lifespan in mice, acceleration and exacerbation of that dysfunction with WD consumption, the remarkable protective effects of voluntary aerobic exercise, and the underlying mechanisms.

Apigenin restores endothelial function by ameliorating oxidative stress, reverses aortic stiffening, and mitigates vascular inflammation with aging.

We assessed the efficacy of oral supplementation with the flavanoid apigenin on arterial function during aging and identified critical mechanisms of action. Young (6 mo) and old (27 mo) C57BL/6N mice (model of arterial aging) consumed drinking water containing vehicle (0.2% carboxymethylcellulose; 10 young and 7 old) or apigenin (0.5 mg/mL in vehicle; 10 young and 9 old) for 6 wk. In vehicle-treated animals, isolated carotid artery endothelium-dependent dilation (EDD), bioassay of endothelial function, was impaired in old versus young (70% ± 9% vs. 92% ± 1%, P < 0.0001) due to reduced nitric oxide (NO) bioavailability. Old mice had greater arterial reactive oxygen species (ROS) production and oxidative stress (higher nitrotyrosine) associated with greater nicotinamide adenine dinucleotide phosphate oxidase (oxidant enzyme) and lower superoxide dismutase 1 and 2 (antioxidant enzymes); ex vivo administration of Tempol (antioxidant) restored EDD to young levels, indicating ROS-mediated suppression of EDD. Old animals also had greater aortic stiffness as indicated by higher aortic pulse wave velocity (PWV, 434 ± 9 vs. 346 ± 5 cm/s, P < 0.0001) due to greater intrinsic aortic wall stiffness associated with lower elastin levels and higher collagen, advanced glycation end products (AGEs), and proinflammatory cytokine abundance. In old mice, apigenin restored EDD (96% ± 2%) by increasing NO bioavailability, normalized arterial ROS, oxidative stress, and antioxidant expression, and abolished ROS inhibition of EDD. Moreover, apigenin prevented foam cell formation in vitro (initiating step in atherosclerosis) and mitigated age-associated aortic stiffening (PWV 373 ± 5 cm/s) by normalizing aortic intrinsic wall stiffness, collagen, elastin, AGEs, and inflammation. Thus, apigenin is a promising therapeutic for arterial aging.NEW & NOTEWORTHY Our study provides novel evidence that oral apigenin supplementation can reverse two clinically important indicators of arterial dysfunction with age, namely, vascular endothelial dysfunction and large elastic artery stiffening, and prevents foam cell formation in an established cell culture model of early atherosclerosis. Importantly, our results provide extensive insight into the biological mechanisms of apigenin action, including increased nitric oxide bioavailability, normalization of age-related increases in arterial ROS production and oxidative stress, reversal of age-associated aortic intrinsic mechanical wall stiffening and adverse remodeling of the extracellular matrix, and suppression of vascular inflammation. Given that apigenin is commercially available as a dietary supplement in humans, these preclinical findings provide the experimental basis for future translational studies assessing the potential of apigenin to treat arterial dysfunction and reduce cardiovascular disease risk with aging.

Hydrogen sulfide-dependent microvascular vasodilation is improved following chronic sulfhydryl-donating antihypertensive pharmacotherapy in adults with hypertension.

Hypertension is characterized by systemic microvascular endothelial dysfunction, in part due to a functional absence of hydrogen sulfide (H2S)-mediated endothelium-dependent dilation. Treatment with a sulfhydryl-donating ACE inhibitor (SH-ACE inhibitor) improves endothelial function in preclinical models of hypertension. To date, no studies have directly assessed the effects of SH-ACE-inhibitor treatment on H2S-dependent vasodilation in humans with hypertension. We hypothesized that SH-ACE-inhibitor treatment would improve H2S-mediated endothelium-dependent vasodilation. Ten adults with hypertension [1 woman and 9 men; 56 ± 9 yr; systolic blood pressure (SBP): 141 ± 8.5 mmHg; diastolic blood pressure (DBP): 90.3 ± 6 mmHg] were treated (16 wk) with the SH-ACE-inhibitor captopril. Red blood cell flux (laser-Doppler flowmetry) was measured continuously during graded intradermal microdialysis perfusion of the endothelium-dependent agonist acetylcholine (ACh; 10-10 to 10-1 M) alone (control) and in combination with an inhibitor of enzymatic H2S production [10-3 M aminooxyacetate (AOAA)] preintervention and postintervention. Cutaneous vascular conductance (CVC; flux/mmHg) was calculated and normalized to the site-specific maximal CVC (0.028 M sodium nitroprusside and local heat to 43°C). Area under the curve was calculated using the trapezoid method. The 16-wk SH-ACE-inhibitor treatment resulted in a reduction of blood pressure (systolic BP: 129 ± 10 mmHg; diastolic BP: 81 ± 9 mmHg, both P < 0.05). Preintervention, inhibition of H2S production had no effect on ACh-induced vasodilation (316 ± 40 control vs. 322 ± 35 AU AOAA; P = 0.82). Captopril treatment improved ACh-induced vasodilation (316 ± 40 pre vs. 399 ± 55 AU post; P = 0.04) and increased the H2S-dependent component of ACh-induced vasodilation (pre: -6.6 ± 65.1 vs. post: 90.2 ± 148.3 AU, P = 0.04). These data suggest that SH-ACE-inhibitor antihypertensive treatment improves cutaneous microvascular endothelium-dependent vasodilation in adults with hypertension, in part via H2S-dependent mechanisms.NEW & NOTEWORTHY This is the first study to prospectively assess the effects of sulfhydryl antihypertensive treatment on microvascular endothelial function in adults with hypertension. Our data suggest that 16 wk of SH-ACE-inhibitor antihypertensive treatment improves cutaneous microvascular endothelium-dependent vasodilation in middle-aged adults with hypertension, in part via H2S-dependent mechanisms.

Hypoglycemia increases endothelial-dependent vasodilation through suppressing phosphorylation at Threonine 495/497 site of endothelial nitric oxide synthase.

OBJECTIVE: Phosphorylation plays an essential role in the regulation of endothelial nitric oxide synthase (eNOS) activity. However, the phosphorylation of eNOS under hypoglycemia and whether hypoglycemia changes eNOS activity is unknown. This paper aims to clarify the regulation of eNOS phosphorylation and its activity change under hypoglycemia. METHODS: Bovine aortic endothelial cells (BAECs) and Sprague-Dawley rats were treated with hypoglycemia, and the phosphorylation of eNOS was subjected to western blot. Blood nitric oxide (NO) concentration was determined by NO kit and endothelial-dependent vasodilation was detected by multi-wire myograph. RESULTS: In both BAECs and rats' thoracic aorta, hypoglycemia induced eNOS phosphorylation decrease specifically on Threonine (Thr) 497. Inhibition of ubiquitination of protein kinase C α subunit (PKCα) reverses the decrease of eNOS phosphorylation in hypoglycemia. Ubiquitinated PKCα can be reversed by AMPK knockdown. In rats, insulin induced hypoglycemia increased the concentration of NO in arterial blood, and progressively enhanced the endothelium-dependent vasodilation of the thoracic and mesenteric aorta. CONCLUSIONS: In vitro, the activation of AMPK may lead to the expression of PKCα by regulating ubiquitination, resulting in a decrease in the level of P-eNOS Thr497 phosphorylation under hypoglycemia. In vivo, insulin-induced hypoglycemia produces a beneficial cardiovascular effect on rats.

Type 2 diabetes impairs vascular responsiveness to nitric oxide, but not the venoarteriolar reflex or post-occlusive reactive hyperaemia in forearm skin.

The venoarteriolar reflex (VAR) is a local mechanism by which vasoconstriction is mediated in response to venous congestion. This response may minimize tissue overperfusion, preventing capillary damage and oedema. Post-occlusive reactive hyperaemia (PORH) is used to assess microvascular function by performing a brief local arterial occlusion resulting in a subsequent rapid transient vasodilation. In the current study, we hypothesized that type 2 diabetes (T2D) attenuates VAR and PORH responses in forearm skin in vivo. In 11 healthy older adults (Control, 58 ± 8 years) and 13 older adults with controlled T2D (62 ± 10 years), cutaneous blood flow measured by laser-Doppler flowmetry was monitored following a 3-min venous occlusion of 45 mm Hg that elicited the VAR, followed by a 3-min recovery period and then a 5-min arterial occlusion of 240 mm Hg that induced PORH. Finally, sodium nitroprusside, a nitric oxide donor, was administered to induce maximum vasodilation. VAR and PORH variables were similar between groups. By contrast, maximal cutaneous blood flow induced by sodium nitroprusside was lower in the T2D group. Taken together, our observations indicate that T2D impairs vascular smooth muscle responsiveness to nitric oxide, but not VAR and PORH in forearm skin.

HMGB1 impairs endothelium-dependent relaxation in diabetes through TLR4/eNOS pathway.

Endothelium-dependent relaxation (EDR) is an initial key step leading to various vascular complications in patients with diabetes. However, the underlying mechanism of EDR impairment in diabetes is not fully understood. Present study defined the role of high-mobility group protein (HMGB1) in EDR related to diabetes. Serum level of HMGB1 was increased in diabetic patients and in db/db mice. Serum HMGB1 level was also positively correlated with HbA1c and negatively correlated with nitric oxide (NO) in diabetic patients. Results from wire myograph showed that recombinant HMGB1 (rHMGB1) was capable of impairing EDR of aortas from wild-type (WT) mice by an eNOS-dependent mechanism. Consistently, HMGB1 inhibitor glycyrrhizin acid (GA) decreased the serum level of HMGB1 and rescued EDR impairment partly in db/db mice. Furthermore, rHMGB1 mediated EDR impairment was abolished in aortas of TLR4-/- mice. In addition, high-glucose-induced HMGB1 upregulation and secretion in endothelial cells. In conclusion, HMGB1 contributes to the EDR impairment through TLR4/eNOS pathway in the setting of diabetes. GA as the HMGB1 inhibitor could attenuate EDR impairment in an animal model of diabetes.

Omeprazole suppresses endothelial calcium response and eNOS Ser1177 phosphorylation in porcine aortic endothelial cells.

BACKGROUND: Although high doses of proton pump inhibitors can elicit an anticancer effect, this strategy may impair vascular biology. In particular, their effects on endothelial Ca2+ signaling and production of endothelium-derived relaxing factor (EDRF) are unknown. To this end, we investigated the effects of high dosages of omeprazole on endothelial Ca2+ responses and EDRF production in primary cultured porcine aortic endothelial cells. METHODS AND RESULTS: Omeprazole (10-1000 µM) suppressed both bradykinin (BK)- and thapsigargin-induced endothelial Ca2+ response in a dose-dependent manner. Furthermore, omeprazole slightly attenuated Ca2+ mobilization from the endoplasmic reticulum, whereas no inhibitory effects on endoplasmic reticulum Ca2+-ATPase were observed. Omeprazole decreased BK-induced phosphorylation of endothelial nitric oxide synthase (eNOS) at Ser1177 and tended to decrease BK-induced nitric oxide production. Production of prostaglandin I2 metabolites, especially 6-keto-prostaglandin 1α, also tended to be reduced by omeprazole. CONCLUSION: Our results are the first to indicate that high doses of omeprazole may suppress both store-operated Ca2+ channels and partially the G protein-coupled receptor/phospholipase C/inositol 1,4,5-triphosphate pathway, and decreased BK-induced, Ca2+-dependent phosphorylation of eNOS(Ser1177). Thus, high dosages of omeprazole impaired EDRF production by attenuating intracellular Ca2+ signaling.

Involvement of Gap Junctions in Acetylcholine-Induced Endothelium-Derived Hyperpolarization-Type Dilation of Retinal Arterioles in Rats.

An electrical communication between the endothelial and smooth muscle cells via gap junctions, which provides the signaling pathway known as endothelium-dependent hyperpolarization (EDH), plays a crucial role in controlling the vascular tone. In this study, we investigated the role of gap junctions in the acetylcholine (ACh)-induced EDH-type dilation of rat retinal arterioles in vivo. The dilator response was evaluated by measuring the diameter of retinal arterioles. Intravitreal injection of gap junction blockers (18ß-glycyrrhetinic acid and carbenoxolone) reduced the ACh-induced dilation of retinal arterioles. Moreover, the retinal arteriolar response to ACh was attenuated by 18ß-glycyrrhetinic acid under treatment with a combination of NG-nitro-L-arginine methyl ester (a nitric oxide (NO) synthase inhibitor; 30 mg/kg) and indomethacin (a cyclooxygenase inhibitor; 5 mg/kg). The NO- and prostaglandin-independent, EDH-related component of ACh-induced dilation of retinal arterioles was prevented by intravitreal injection of iberiotoxin, which inhibits large-conductance Ca2+-activated K+ channels. Furthermore, the combination of 18ß-glycyrrhetinic acid and iberiotoxin produced greater attenuation in the EDH-related response than that by the individual agent. Treatment with 18ß-glycyrrhetinic acid revealed no significant effect on NOR3 (an NO donor)-induced retinal vasodilator response. These results suggest that gap junctions contribute to the ACh-induced, EDH-type dilation of rat retinal arterioles in vivo.

Trimethylamine-N-oxide (TMAO) Selectively Disrupts Endothelium-Dependent Hyperpolarization-Type Relaxations in a Time-Dependent Manner in Rat Superior Mesenteric Artery.

The vascular action of trimethylamine-N-oxide (TMAO)-the gut microbiota-derived metabolite-in contributing cardiovascular disease is a controversial topic. A recent study has shown that acute exposure of TMAO at moderate concentrations inhibits endothelium-dependent hyperpolarization (EDH)-type relaxations selectively in rat isolated femoral arteries, but not in mesenteric arteries. Here we determined the efficacy of higher TMAO concentrations with longer exposure times on vascular reactivity in rat isolated superior mesenteric arteries. Acetylcholine-induced EDH-type relaxations were examined before and after incubation with TMAO (0.1-10 mM) at increasing exposure times (1-24 h). One- and 4-h-incubations with TMAO at 0.1-3 mM did not cause any change in EDH-type relaxations. However, when the incubation time was increased to 24 h, responses to acetylcholine were reduced in arteries incubated with 1-3 mM TMAO. In addition, at higher TMAO concentration (10 mM) the decrease in EDH relaxations could be detected both in 4-h- and 24-h-incubations. The EDH-relaxations were preserved in rings incubated with 10 mM TMAO for 24 h in the presence of SKA-31 (10 µM), the small (SKCa)- and intermediate (IKCa)-conductance calcium-activated potassium channel activator. Contractile responses to phenylephrine increased in arteries exposed to 10 mM TMAO for 24 h. Interestingly, nitric oxide (NO)-mediated relaxations remained unchanged in arteries treated for 24 h at any TMAO concentration. Our study revealed that TMAO selectively disrupted EDH-type relaxations time-dependently without interfering with NO-induced vasodilation in rat isolated mesenteric arteries. Disruption of these relaxations may help explain the causal role of elevated TMAO levels in certain vascular diseases.