Non-neuronal cell-derived acetylcholine, a key modulator of the vascular endothelial function in health and disease.

Vascular endothelial cells play an important role in regulating peripheral circulation by modulating arterial tone in the microvasculature. Elevated intracellular Ca2+ levels are required in endothelial cells to induce smooth muscle relaxation via endothelium-dependent mechanisms such as nitric oxide production, prostacyclin, and endothelial cell hyperpolarization. It is well established that exogenous administration of acetylcholine can increase intracellular Ca2+ concentrations, followed by endothelium-dependent vasodilation. Although endogenous acetylcholine's regulation of vascular tone remains debatable, recent studies have reported that endogenously derived acetylcholine, but not neuronal cell-derived acetylcholine, is a key modulator of endothelial cell function. In this minireview, we summarize the current knowledge of the non-neuronal cholinergic system (NNCS) in vascular function, particularly vascular endothelial cell function, which contributes to blood pressure regulation. We also discuss the possible pathophysiological impact of endothelial NNCS, which may induce the development of vascular diseases due to endothelial dysfunction, and the potential of endothelial NNCS as a novel therapeutic target for endothelial dysfunction in the early stages of metabolic syndrome, diabetes, and hypertension.

Absorbed Bioactive Compounds Replicate Guanxin II-Induced Endothelium-Associated in/ex vivo Vasodilation.

OBJECTIVE: To develop an interference-free and rapid method to elucidate Guanxin II (GX II)'s representative vasodilator absorbed bioactive compounds (ABCs) among enormous phytochemicals. METHODS: The contents of ferulic acid, tanshinol, and hydroxysafflor yellow A (FTA) in GX II/rat serum after the oral administration of GX II (30 g/kg) were detected using ultra-performance liquid chromatography-mass spectrometry. Totally 18 rats were randomly assigned to the control group (0.9% normal saline), GX II (30 g/kg) and FTA (5, 28 and 77 mg/kg) by random number table method. Diastolic coronary flow velocity-time integral (VTI), i.e., coronary flow or coronary flow-mediated dilation (CFMD), and endothelium-intact vascular tension of isolated aortic rings were measured. After 12 h of exposure to blank medium or 0.5 mmol/L H2O2, endothelial cells (ECs) were treated with post-dose GX II of supernatant from deproteinized serum (PGSDS, 300 µL PGSDS per 1 mL of culture medium) or FTA (237, 1539, and 1510 mg/mL) for 10 min as control, H2O2, PGSDS and FTA groups. Nitric oxide (NO), vascular endothelial growth factor (VEGF), endothelin-1 (ET-1), superoxide dismutase (SOD), malondialdehyde (MDA) and phosphorylated phosphoinositide 3 kinase (p-PI3K), phosphorylated protein kinase B (p-AKT), phosphorylated endothelial nitric oxide synthase (p-eNOS) were analyzed. PGSDS was developed as a GX II proxy of ex vivo herbal crude extracts. RESULTS: PGSDS effectively eliminates false responses caused by crude GX II preparations. When doses equaled the contents in GX II/its post-dose serum, FTA accounted for 98.17% of GX II -added CFMD and 92.99% of PGSDS-reduced vascular tension. In ECs, FTA/PGSDS was found to have significant antioxidant (lower MDA and higher SOD, P<0.01) and endothelial function-protective (lower VEGF, ET-1, P<0.01) effects. The increases in aortic relaxation, endothelial NO levels and phosphorylated PI3K/Akt/eNOS protein induced by FTA/PGSDS were markedly abolished by NG-nitro-L-arginine methyl ester (L-NA, eNOS inhibitor) and wortmannin (PI3K/AKT inhibitor), respectively, indicating an endothelium-dependent vasodilation via the PI3K/AKT-eNOS pathway (P<0.01). CONCLUSION: This study provides a strategy for rapidly and precisely elucidating GX II's representative in/ex vivo cardioprotective absorbed bioactive compounds (ABCs)-FTA, suggesting its potential in advancing precision ethnomedicine.

Infrared light elicits endothelium-dependent vasodilation in isolated occipital arteries of the rat via soluble guanylyl cyclase-dependent mechanisms.

The left and right occipital arteries provide blood supply to afferent cell bodies in the ipsilateral nodose and petrosal ganglia. This supply is free of an effective blood-ganglion barrier, so changes in occipital artery blood flow directly affect the access of circulating factors to the afferent cell bodies. The application of infrared (IR) light to modulate neural and other cell processes has yielded information about basic biological processes within tissues and is gaining traction as a potential therapy for a variety of disease processes. To address whether IR can directly modulate vascular function, we performed wire myography studies to determine the actions of IR on occipital arteries isolated from male Sprague-Dawley rats. Based on our previous research that functionally-important differences exist between occipital artery segments close to their origin at the external carotid artery (ECA) and those closer to the nodose ganglion, the occipital arteries were dissected into two segments, one closer to the ECA and the other closer to the nodose ganglion. Segments were constricted with 5-hydroxytryptamine to a level equal to 50% of the maximal response generated by the application of a high (80 mM) concentration of K+ ions. The direct application of pulsed IR (1,460 nm) for 5 s produced a rapid vasodilation in occipital arteries that was significantly more pronounced in segments closest to the ECA, although the ECA itself was minimally responsive. The vasodilation remained for a substantial time (at least 120 s) after cessation of IR application. The vasodilation during and following cessation of the IR application was markedly diminished in occipital arteries denuded of the endothelium. In addition, the vasodilation elicited by IR in endothelium-intact occipital arteries was substantially reduced in the presence of a selective inhibitor of the nitric oxide-sensitive guanylate cyclase, 1H-[1,2,4]oxadiazolo [4,3-a]quinoxalin-1-one (ODQ). It appears that IR causes endothelium-dependent, nitric-oxide-mediated vasodilation in the occipital arteries of the rat. The ability of IR to generate rapid and sustained vasodilation may provide new therapeutic approaches for restoring or improving blood flow to targeted tissues.

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.

DL-3-n-butylphthalide improves the endothelium-dependent vasodilation in high-fat diet-fed ApoE-/- mice via suppressing inflammation, endothelial necroptosis and apoptosis.

Impaired endothelium-dependent vasodilation in atherosclerosis is a high-risk factor for myocardial infarction and ischemic stroke, and inflammation, necroptosis and apoptosis contribute to endothelial dysfunction in atherosclerosis. Although DL-3-n-butylphthalide (NBP) has been widely used in treating ischemic stroke, its effect on endothelium-dependent vasodilation remains unknown. This study aims to explore whether NBP is able to improve endothelium-dependent vasodilation in atherosclerosis and the underlying mechanisms. Male ApoE-/- mice were fed with a high-fat diet (HFD) for 9-16 weeks to establish a model of atherosclerosis. NBP were given to the mice after eating HFD for 6 weeks and atorvastatin served as a positive control. The endothelium-dependent vasodilation, the blood flow velocity, the atherosclerotic lesion area, the serum levels of lipids, inflammatory cytokines and necroptosis-relevant proteins (RIPK1, RIPK3 and MLKL), and the endothelial necroptosis and apoptosis within the aorta were measured. Human umbilical vein endothelial cells (HUVECs) were incubated with oxidized low-density lipoprotein (ox-LDL) for 48 h to mimic endothelial injury in atherosclerosis, lactate dehydrogenase release, the ratio of necroptosis and apoptosis and the expression of necroptosis-relevant proteins were examined. Similar to atorvastatin, NBP improves endothelium-dependent vasodilation, decreases aortic flow velocity and reduces atherosclerotic lesion area in HFD-fed ApoE-/- mice, concomitant with a reduction in serum lipids, inflammatory cytokines and necroptosis-relevant proteins, and endothelial necroptosis and apoptosis. Consistently, NBP inhibited necroptosis and apoptosis in ox-LDL-treated HUVECs. Based on these observations, we conclude that NBP exerts beneficial effects on improving the endothelium-dependent vasodilation in atherosclerosis via suppressing inflammation, endothelial necroptosis and apoptosis.

Galanin receptors modulate cutaneous vasodilation elicited by whole-body and local heating but not thermal sweating in young adults.

Galanin receptor subtypes GAL1, GAL2, and GAL3 are involved in several biological functions. We hypothesized that 1) GAL3 receptor activation contributes to sweating but limits cutaneous vasodilation induced by whole-body and local heating without a contribution of GAL2; and 2) GAL1 receptor activation attenuates both sweating and cutaneous vasodilation during whole-body heating. Young adults underwent whole-body (n = 12, 6 females) and local (n = 10, 4 females) heating. Forearm sweat rate (ventilated capsule) and cutaneous vascular conductance (CVC; ratio of laser-Doppler blood flow to mean arterial pressure) were assessed during whole-body heating (water-perfusion suit circulated with warm (35 °C) water), while CVC was also assessed by local forearm heating (from 33 °C to 39 °C and elevated to 42 °C thereafter; each level of heating maintained for ∼30 min). Sweat rate and CVC were evaluated at four intradermal microdialysis forearm sites treated with either 1) 5% dimethyl sulfoxide (control), 2) M40, a non-selective GAL1 and GAL2 receptor antagonist, 3) M871 to selectively antagonize GAL2 receptor, or 4) SNAP398299 to selectively antagonize GAL3 receptor. Sweating was not modulated by any GAL receptor antagonist (P > 0.169), whereas only M40 reduced CVC (P ≤ 0.003) relative to control during whole-body heating. Relative to control, SNAP398299 augmented the initial and sustained increase in CVC during local heating to 39 °C, and the transient increase at 42 °C (P ≤ 0.028). We confirmed that while none of the galanin receptors modulate sweating during whole-body heating, GAL1 receptors mediate cutaneous vasodilation. Further, GAL3 receptors blunt cutaneous vasodilation during local heating.

Serum, interstitial and sweat ATP in humans exposed to heat stress: Insights into roles of ATP in the heat loss responses.

Hyperthermia increases intravascular adenosine triphosphate (ATP) and is associated with greater hyperthermia-induced cutaneous vasodilation. Hyperthermia may also increase skin interstitial fluid ATP thereby activating cutaneous vascular smooth muscle cells and sweat glands. We evaluated the hypothesis that whole-body heating would increase skin interstitial fluid ATP, and this response would be associated with an increase in cutaneous vasodilation and sweating. Nineteen (8 females) young adults underwent whole-body heating using a water-perfusion suit to increase core temperature by ~1°C during which time cutaneous vascular conductance (CVC, ratio of laser-Doppler blood flow to mean arterial pressure) and sweat rate (ventilated capsule technique) were measured at four forearm skin sites to minimize between-site variations. Dialysate from the skin sites were collected via intradermal microdialysis. Heating increased serum ATP, CVC, and sweat rate (all p ≤ 0.031). However, heating did not modulate dialysate ATP (median, baseline vs. end-heating: 2.38 vs. 2.70 nmol/ml) (p = 0.068), though the effect size was moderate (Cohen's d = 0.566). While the heating-induced increase in CVC was not correlated with changes in serum ATP (r = 0.439, p = 0.060), we observed a negative correlation (rs = -0.555, p = 0.017) between dialysate ATP and CVC. We did not observe a significant correlation between the heating-induced sweating and serum, dialysate, or sweat ATP (rs = 0.091 to -0.322, all p ≥ 0.222). Altogether, we showed that passive heating increases ATP in blood and possibly skin interstitial fluid, with the latter potentially blunting cutaneous vasodilation. However, ATP does not appear to modulate sweating.

The Role of Rehabilitation in Arterial Function Properties of Convalescent COVID-19 Patients.

Coronavirus disease (COVID-19) is a respiratory disease, although arterial function involvement has been documented. We assess the impact of a post-acute COVID-19 rehabilitation program on endothelium-dependent vasodilation and arterial wall properties. We enrolled 60 convalescent patients from COVID-19 and one-month post-acute disease, who were randomized at a 1:1 ratio in a 3-month cardiopulmonary rehabilitation program (study group) or not (control group). Endothelium-dependent vasodilation was evaluated by flow-mediated dilation (FMD), and arterial wall properties were evaluated by carotid-femoral pulse wave velocity (cf-PWV) and augmentation index (AIx) at 1 month and at 4 months post-acute disease. FMD was significantly improved in both the study (6.2 ± 1.8% vs. 8.6 ± 2.4%, p < 0.001) and control groups (5.9 ± 2.2% vs. 6.6 ± 1.8%, p = 0.009), but the improvement was significantly higher in the study group (rehabilitation) (p < 0.001). PWV was improved in the study group (8.2 ± 1.3 m/s vs. 6.6 ± 1.0 m/s, p < 0.001) but not in the control group (8.9 ± 1.8 m/s vs. 8.8 ± 1.9 m/s, p = 0.74). Similarly, AIx was improved in the study group (25.9 ± 9.8% vs. 21.1 ± 9.3%, p < 0.001) but not in the control group (27.6 ± 9.2% vs. 26.2 ± 9.8 m/s, p = 0.15). Convalescent COVID-19 subjects of the study group (rehabilitation) with increased serum levels of circulating IL-6 had a greater reduction in FMD. Conclusively, a 3-month cardiopulmonary post-acute COVID-19 rehabilitation program improves recovery of endothelium-dependent vasodilation and arteriosclerosis.

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.

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.

Phosphorylated PKM2 regulates endothelium-dependent vasodilation in diabetes / 中南大学学报(医学版)

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.

Silybin induces endothelium-dependent vasodilation via TRPV4 channels in mouse mesenteric arteries.

Silybin is a flavonolignan extracted from the seeds of Silybum marianum that has been used as a dietary supplement for treating hepatic diseases and components of metabolic syndrome such as diabetes, obesity and hypertension. Transient receptor potential vanilloid 4 (TRPV4) channels are Ca2+-permeable, nonselective cation channels that regulate vascular endothelial function and blood flow. However, the relationship between silybin and TRPV4 channels in small mesenteric arteries remains unknown. In our study, we carried out a molecular docking experiment by using Discovery Studio v3.5 to predict the binding of silybin to TRPV4. Activation of TRPV4 with silybin was detected via intracellular Ca2+ concentration ([Ca2+]i) measurement and patch clamp experiments. The molecular docking results showed that silybin was likely to bind to the ankyrin repeat domain of TPRV4. [Ca2+]i measurements in mesenteric arterial endothelial cells (MAECs) and TRPV4-overexpressing HEK293 (TRPV4-HEK293) cells demonstrated that silybin induced Ca2+ influx by activating TRPV4 channels. The patch clamp experiments indicated that in TRPV4-HEK293 cells, silybin induced TRPV4-mediated cation currents. In addition, in high-salt-induced hypertensive mice, oral administration of silybin decreased systolic blood pressure (SBP) and significantly improved the arterial dilatory response to acetylcholine. Our findings provide the first evidence that silybin could induce mesenteric endothelium-dependent vasodilation and reduce blood pressure in high-salt-induced hypertensive mice via TRPV4 channels, thereby revealing the potential effect of silybin on preventing endothelial dysfunction-related cardiovascular diseases.

Angiotensin II-induced endothelial dysfunction: Impact of sex, genetic background, and rho kinase.

The renin-angiotensin system (RAS) contributes to vascular disease with multiple cardiovascular risk factors including hypertension. As a major effector within the RAS, angiotensin II (Ang II) activates diverse signaling mechanisms that affect vascular biology. Despite the impact of such vascular pathophysiology, our understanding of the effects of Ang II in relation to the function of endothelial cells is incomplete. Because genetic background and biological sex can be determinants of vascular disease, we performed studies examining the direct effects of Ang II using carotid arteries from male and female mice on two genetic backgrounds, C57BL/6J and FVB/NJ. Although FVB/NJ mice are much less susceptible to atherosclerosis than C57BL/6J, the effects of Ang II on endothelial cells in FVB/NJ are poorly defined. Overnight incubation of isolated arteries with Ang II (10 nmol/L), impaired endothelial function in both strains and sexes by approximately one-half (p < 0.05). To examine the potential mechanistic contribution of Rho kinase (ROCK), we treated arteries with SLX-2119, an inhibitor with high selectivity for ROCK2. In both male and female mice of both strains, SLX-2119 largely restored endothelial function to normal, compared to vessels treated with vehicle. Thus, Ang II-induced endothelial dysfunction was observed in both FVB/NJ and C57BL/6J mice. This effect was sex-independent. In all groups, effects of Ang II were reversed by inhibition of ROCK2 with SLX-2119. These studies provide the first evidence that ROCK2 may be a key contributor to Ang II-induced endothelial dysfunction in both sexes and in mouse strains that differ in relation to other major aspects of vascular disease.

Cardiovascular risk in patients receiving antihypertensive drug treatment from the perspective of endothelial function.

Blood-pressure-lowering therapy with antihypertensive drugs can reduce the risk of cardiovascular morbidity and mortality in patients with hypertension. However, patients treated with antihypertensive drugs generally have a worse prognosis than untreated individuals. Consistent with the results obtained from epidemiological studies, a clinical study showed that endothelial function was impaired more in treated patients with hypertension than in untreated individuals with the same blood pressure level, suggesting that blood-pressure-lowering therapy with currently available antihypertensive drugs cannot restore endothelial function to the level of that in untreated individuals. Several mechanisms of endothelial dysfunction in treated patients are postulated: irreversible damage to the endothelium caused by higher cumulative elevated blood pressure exposure over time; the persistence of the primary causes of hypertension even after the initiation of antihypertensive drug treatment, including an activated renin-angiotensin-aldosterone system, oxidative stress, and inflammation; and higher global cardiovascular risk related not only to conventional cardiovascular risk factors but also to undetectable nonconventional risk factors. Lifestyle modifications/nonpharmacological interventions should be strongly recommended for both untreated and treated individuals with hypertension. Lifestyle modifications/nonpharmacological interventions may directly correct the primary causes of hypertension, which can improve endothelial function and consequently reduce cardiovascular risk regardless of the use or nonuse of antihypertensive drugs.

Inhibition of mitochondrial reactive oxygen species improves coronary endothelial function after cardioplegic hypoxia/reoxygenation.

OBJECTIVE: Cardioplegic ischemia-reperfusion and diabetes mellitus are correlated with coronary endothelial dysfunction and inactivation of small conductance calcium-activated potassium channels. Increased reactive oxidative species, such as mitochondrial reactive oxidative species, may contribute to oxidative injury. Thus, we hypothesized that inhibition of mitochondrial reactive oxidative species may protect coronary small conductance calcium-activated potassium channels and endothelial function against cardioplegic ischemia-reperfusion-induced injury. METHODS: Small coronary arteries and endothelial cells from the hearts of mice with and without diabetes mellitus were isolated and examined by using a cardioplegic hypoxia and reoxygenation model to determine whether the mitochondria-targeted antioxidant Mito-Tempo could protect against coronary endothelial and small conductance calcium-activated potassium channel dysfunction. The microvessels or mouse heart endothelial cells were treated with or without Mito-Tempo (0-10 µM) 5 minutes before and during cardioplegic hypoxia and reoxygenation. Microvascular function was assessed in vitro by vessel myography. K+ currents of mouse heart endothelial cells were measured by whole-cell patch clamp. The levels of intracellular cytosolic free calcium (Ca2+) concentration, mitochondrial reactive oxidative species, and small conductance calcium-activated potassium protein expression of mouse heart endothelial cells were measured by Rhod-2 fluorescence staining, MitoSox, and Western blotting, respectively. RESULTS: Cardioplegic hypoxia and reoxygenation significantly attenuated endothelial small conductance calcium-activated potassium channel activity, caused calcium overload, and increased mitochondrial reactive oxidative species of mouse heart endothelial cells in both the nondiabetic and diabetes mellitus groups. In addition, treating mouse heart endothelial cells with Mito-Tempo (10 µM) reduced cardioplegic hypoxia and reoxygenation-induced Ca2+ and mitochondrial reactive oxidative species overload in both the nondiabetic and diabetes mellitus groups, respectively (P < .05). Treatment with Mito-Tempo (10 µM) significantly enhanced coronary relaxation responses to adenosine 5'-diphosphate and NS309 (P < .05), and endothelial small conductance calcium-activated potassium channel currents in both the nondiabetic and diabetes mellitus groups (P < .05). CONCLUSIONS: Administration of Mito-Tempo improves endothelial function and small conductance calcium-activated potassium channel activity, which may contribute to its enhancement of endothelium-dependent vasorelaxation after cardioplegic hypoxia and reoxygenation.

Na+-K+-ATPase plays a major role in mediating cutaneous thermal hyperemia achieved by local skin heating to 39°C.

Na+-K+-ATPase is integrally involved in mediating cutaneous vasodilation during an exercise-heat stress, which includes an interactive role with nitric oxide synthase (NOS). Here, we assessed if Na+-K+-ATPase also contributes to cutaneous thermal hyperemia induced by local skin heating, which is commonly used to assess cutaneous endothelium-dependent vasodilation. Furthermore, we assessed the extent to which NOS contributes to this response. Cutaneous vascular conductance (CVC) was measured continuously at four forearm skin sites in 11 young adults (4 women). After baseline measurement, local skin temperature was increased from 33°C to 39°C to induce cutaneous thermal hyperemia. Once a plateau in CVC was achieved, each skin site was continuously perfused via intradermal microdialysis with either: 1) lactated Ringer solution (control), 2) 6 mM ouabain, a Na+-K+-ATPase inhibitor, 3) 20 mM l-NAME, a NOS inhibitor, or 4) a combination of both. Relative to the control site, CVC during the plateau phase of cutaneous thermal hyperemia (∼50% max) was reduced by the lone inhibition of Na+-K+-ATPase (-19 ± 8% max, P = 0.038) and NOS (-32 ± 4% max, P < 0.001), as well as the combined inhibition of both (-37 ± 9% max, P < 0.001). The magnitude of reduction was similar between NOS inhibition alone and combined inhibition (P = 1.000). The administration of both Na+-K+-ATPase and NOS inhibitors fully abolished the plateau of CVC with values returning to preheating baseline values (P = 0.439). We show that Na+-K+-ATPase contributes to cutaneous thermal hyperemia during local skin heating to 39°C, and this response is partially mediated by NOS.NEW & NOTEWORTHY Cutaneous thermal hyperemia during local skin heating to 39°C, which is highly dependent on nitric oxide synthase (NOS), is frequently used to assess endothelium-dependent cutaneous vasodilation. We showed that Na+-K+-ATPase mediates the regulation of cutaneous thermal hyperemia partly via NOS-dependent mechanisms although a component of the Na+-K+-ATPase modulation of cutaneous thermal hyperemia is NOS independent. Thus, as with NOS, Na+-K+-ATPase may be important in the regulation of cutaneous endothelial vascular function.

Characterization of endothelium-dependent and -independent processes in occipital artery of the rat: relevance to control of blood flow to nodose sensory cells.

Circulating factors access cell bodies of vagal afferents in nodose ganglia (NG) via the occipital artery (OA). Constrictor responses of OA segments closer in origin from the external carotid artery (ECA) differ from segments closer to NG. Our objective was to determine the role of endothelium in this differential vasoreactivity in rat OA segments. Vasoreactivity of OA segments (proximal segments closer to ECA, distal segments closer to NG) was examined in wire myographs. We evaluated 1) vasoconstrictor effects of 5-hydroxytryptamine (5-HT) in intact and endothelium-denuded OA segments in absence/presence of soluble guanylate cyclase (SGC) inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), 2) vasodilator responses elicited by the endothelium dependent vasodilator, acetylcholine (ACh), in intact or endothelium-denuded OA segments in absence/presence of ODQ, and 3) vasodilator responses elicited by NO-donor MAHMA NONOate, in intact OA segments in absence/presence of ODQ. Intact distal OA responded more to 5-HT than intact proximal OA. Endothelium denudation increased 5-HT potency in both OA segments, especially proximal OA. ODQ increased maximal responses of 5-HT in both segments, particularly proximal OA. ACh similarly relaxed both OA segments, effects abolished by endothelial denudation and attenuated by ODQ. MAHMA NONOate elicited transient vasodilation in both segments. Effects of ODQ against ACh were segment dependent whereas those against MAHMA NONOate were not. The endothelium regulates OA responsiveness in a segment-dependent fashion. Endothelial cells at the OA-ECA junction more strongly influence vascular tone than those closer to NG. Differential endothelial regulation of OA tone may play a role in controlling blood flow and access of circulating factors to NG.NEW & NOTEWORTHY This study demonstrates that the endothelium-dependent regulation of smooth muscle tone of occipital arteries is segment-dependent. Endothelial cells at the occipital artery-external carotid artery junction (entryway of blood flow to the nodose ganglia) more strongly influence vascular tone than those closer to the nodose ganglia. This differential endothelial regulation of occipital artery tone may control blood flow and access of circulating factors to the nodose ganglia.

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.

Volume Elastic Modulus, Vascular Function, and Vascular Structure in Patients with Cardiovascular Risk Factors.

AIMS: Volume elastic modulus (VE), an index of arterial elasticity, and arterial diameter of the brachial artery can be automatically measured by a newly developed oscillometric device. We investigated the associations of VE with flow-mediated vasodilation (FMD), an index of endothelium-dependent vasodilation, nitroglycerine-induced vasodilation (NID), an index of endothelium-independent vasodilation, and intima-media thickness (IMT) of the brachial artery and association of oscillometrically measured brachial artery diameter with ultrasonographically measured brachial artery diameter in patients with cardiovascular risk factors. METHODS: Oscillometric measurements of VE and brachial artery diameter and ultrasound measurements of brachial artery diameter, FMD, NID, and IMT of the brachial artery were performed in 50 patients with cardiovascular risk factors. RESULTS: The mean values were 2.1±0.4 mmHg/% for VE, 0.31±0.05 mm for brachial IMT, 4.48±0.70 mm for oscillometric brachial artery diameter, and 4.30±0.55 mm for ultrasound brachial artery diameter. VE significantly correlated with brachial IMT (r=0.51, P＜0.001), whereas there was no significant correlation of VE with FMD (r=-0.08, P=0.58) or NID (r=0.07, P=0.61). Multivariate analysis revealed that VE was significantly associated with brachial IMT (ß=0.33, P=0.04). Oscillometric brachial artery diameter significantly correlated with ultrasound brachial artery diameter (r=0.79, P＜0.001). The Bland-Altman plot showed good agreement between oscillometric brachial artery diameter and ultrasound brachial artery diameter (mean difference, -0.17 mm; limits of agreement, -1.03 mm to 0.69 mm). CONCLUSIONS: In patients with cardiovascular risk factors, VE may represent atherosclerotic structural alterations of the vascular wall but not vascular function. The accuracy of oscillometric measurement of brachial artery diameter is acceptable.

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.