Minimally modified low-density lipoprotein upregulates mouse mesenteric arterial 5-HT1B receptor in vivo via activation of the JAK2/STAT3 pathway.

OBJECTIVES: To explore whether minimally modified low-density lipoprotein (mmLDL) upregulates mesenteric arterial 5-hydroxytryptamine 1B (5-HT1B) receptor expression by activating the JAK2/STAT3 signaling pathway. METHODS: Mice were randomly divided into the following groups: the normal saline (NS), LDL, mmLDL, mmLDL+galiellactone (GL, a JAK2/STAT3 pathway inhibitor), and mmLDL+DMSO groups. The dose-response curve of mesenteric arterial ring constriction after administration of 5-carboxamidotryptamine (5-CT), an agonist of 5-HT1B, was recorded with a microvascular tensiometer. JAK2, p-JAK2, STAT3, p-STAT3, and 5-HT1B receptor protein expression levels were determined by Western blotting. 5-HT1B receptor mRNA levels were measured by RT-PCR. 5-HT1B receptor protein expression was determined by immunofluorescence. RESULTS: Injection of mmLDL into the tail vein significantly increased the contractile dose-response curve after 5-CT stimulation, as the Emax was 82.15 ±â€¯6.15% in the NS group and 171.88 ±â€¯5.78% in the mmLDL group (P < 0.01); significantly elevated 5-HT1B receptor mRNA and protein expression levels; and significantly increased p-JAK2 and p-STAT3 protein expression levels. After intraperitoneal injection of GL, the vasoconstrictive response was significantly reduced compared with that in the mmLDL group, as the Emax was decreased to 97.14 ±â€¯1.20% (P < 0.01); 5-HT1B receptor mRNA and protein expression levels were significantly reduced; STAT3 phosphorylation and p-JAK2 and p-STAT3 protein expression were not significantly changed; and 5-HT1B receptor expression was altered via inhibition of p-STAT3 binding to DNA, which suppressed transcription. CONCLUSIONS: mmLDL can upregulate 5-HT1B receptor expression in mouse mesenteric arteries by activating the JAK2/STAT3 signaling pathway.

Osteoprotegerin regulates vascular function through syndecan-1 and NADPH oxidase-derived reactive oxygen species.

Osteogenic factors, such as osteoprotegerin (OPG), are protective against vascular calcification. However, OPG is also positively associated with cardiovascular damage, particularly in pulmonary hypertension, possibly through processes beyond effects on calcification. In the present study, we focused on calcification-independent vascular effects of OPG through activation of syndecan-1 and NADPH oxidases (Noxs) 1 and 4. Isolated resistance arteries from Wistar-Kyoto (WKY) rats, exposed to exogenous OPG, studied by myography exhibited endothelial and smooth muscle dysfunction. OPG decreased nitric oxide (NO) production, eNOS activation and increased reactive oxygen species (ROS) production in endothelial cells. In VSMCs, OPG increased ROS production, H2O2/peroxynitrite levels and activation of Rho kinase and myosin light chain. OPG vascular and redox effects were also inhibited by the syndecan-1 inhibitor synstatin (SSNT). Additionally, heparinase and chondroitinase abolished OPG effects on VSMCs-ROS production, confirming syndecan-1 as OPG molecular partner and suggesting that OPG binds to heparan/chondroitin sulphate chains of syndecan-1. OPG-induced ROS production was abrogated by NoxA1ds (Nox1 inhibitor) and GKT137831 (dual Nox1/Nox4 inhibitor). Tempol (SOD mimetic) inhibited vascular dysfunction induced by OPG. In addition, we studied arteries from Nox1 and Nox4 knockout (KO) mice. Nox1 and Nox4 KO abrogated OPG-induced vascular dysfunction. Vascular dysfunction elicited by OPG is mediated by a complex signalling cascade involving syndecan-1, Nox1 and Nox4. Our data identify novel molecular mechanisms beyond calcification for OPG, which may underlie vascular injurious effects of osteogenic factors in conditions such as hypertension and/or diabetes.

Opioids Cause Sex-Specific Vascular Changes via Cofilin-Extracellular Signal-Regulated Kinase Signaling: Female Mice Present Higher Risk of Developing Morphine-Induced Vascular Dysfunction than Male Mice.

Recent studies have shown that chronic use of prescription or illicit opioids leads to an increased risk of cardiovascular events and pulmonary arterial hypertension. Indices of vascular age and arterial stiffness are also shown to be increased in opioid-dependent patients, with the effects being more marked in women. There are currently no studies investigating sex-specific vascular dysfunction in opioid use, and the mechanisms leading to opioid-induced vascular damage remain unknown. We hypothesized that exposure to exogenous opioids causes sex-specific vascular remodeling that will be more pronounced in female. Acknowledging the emerging roles of cofilins and extracellular signal-regulated kinases (ERKs) in mediating actin dynamics, we investigated the effects of morphine on these molecules. Twenty-four hour exposure to morphine increased inactivated cofilin and activated ERKs in resistance arteries from female mice, which may promote stress fiber over-assembly. We also performed continuous intraluminal infusion of morphine in pressurized resistance arteries from male and female mice using culture pressure myographs. We observed that morphine reduced the vascular diameter in resistance arteries from female, but not male mice. These results have significant implications for the previously unexplored role of exogenous opioids as a modifiable cardiovascular risk factor, especially in women.

Streptococcal Exotoxin Streptolysin O Causes Vascular Endothelial Dysfunction Through PKCß Activation.

Streptolysin O (SLO) is produced by common hemolytic streptococci that cause a wide range of diseases from pharyngitis to life-threatening necrotizing fasciitis and toxic shock syndrome. Although the importance of SLO in invasive hemolytic streptococcus infection has been well demonstrated, the role of circulating SLO in noninvasive infection remains unclear. The aim of this study was to characterize the pharmacological effect of SLO on vascular functions, focusing on cellular signaling pathways. In control Wistar rats, SLO treatment (1-1000 ng/ml) impaired acetylcholine-induced endothelial-dependent relaxation in the aorta and second-order mesenteric artery in a dose-dependent manner without any effects on sodium nitroprusside-induced endothelium-independent relaxation or agonist-induced contractions. SLO also increased phosphorylation of the endothelial NO synthase (eNOS) inhibitory site at Thr495 in the aorta. Pharmacological analysis indicated that either endothelial dysfunction or eNOS phosphorylation was mediated by protein kinase Cß (PKCß), but not by the p38 mitogen-activated protein kinase pathway. Consistent with this, SLO increased phosphorylation levels of protein kinase C substrates in the aorta. In vivo study of control Wistar rats indicated that intravenous administration of SLO did not change basal blood pressure but significantly counteracted the acetylcholine-induced decrease in blood pressure. Interestingly, plasma anti-SLO IgG levels were significantly higher in 10- to 15-week-old spontaneously hypertensive rats compared with age-matched control rats (P < 0.05). These findings demonstrated that SLO causes vascular endothelial dysfunction, which is mediated by PKCß-induced phosphorylation of the eNOS inhibitory site. SIGNIFICANCE STATEMENT: This study showed for the first time that in vitro exposure of vascular tissues to SLO impairs endothelial function, an effect that is mediated by protein kinase C ß-induced phosphorylation of the endothelial NO synthase inhibitory site. Intravenous administration of SLO in control and hypertensive rats blunted the acetylcholine-induced decrease in blood pressure, providing evidence for a possible role of SLO in dysregulation of blood pressure.

Vascular effects of disrupting endothelial mTORC1 signaling in obesity.

The mechanistic target of rapamycin complex 1 (mTORC1) signaling complex is emerging as a critical regulator of cardiovascular function with alterations in this pathway implicated in cardiovascular diseases. In this study, we used animal models and human tissues to examine the role of vascular mTORC1 signaling in the endothelial dysfunction associated with obesity. In mice, obesity induced by high-fat/high-sucrose diet feeding for ∼2 mo resulted in aortic endothelial dysfunction without appreciable changes in vascular mTORC1 signaling. On the other hand, chronic high-fat diet feeding (45% or 60% kcal: ∼9 mo) in mice resulted in endothelial dysfunction associated with elevated vascular mTORC1 signaling. Endothelial cells and visceral adipose vessels isolated from obese humans display a trend toward elevated mTORC1 signaling. Surprisingly, genetic disruption of endothelial mTORC1 signaling through constitutive or tamoxifen inducible deletion of endothelial Raptor (critical subunit of mTORC1) did not prevent or rescue the endothelial dysfunction associated with high-fat diet feeding in mice. Endothelial mTORC1 deficiency also failed to reverse the endothelial dysfunction evoked by a high-fat/high-sucrose diet in mice. Taken together, these data show increased vascular mTORC1 signaling in obesity, but this vascular mTORC1 activation appears not to be required for the development of endothelial impairment in obesity.

Blue laser-induced selective vasorelaxation by the activation of NOSs.

Phototherapy has been tried for treating cardiovascular diseases. In particular, ultraviolet and blue visible lights were suggested to be useful due to their nitric oxide (NO)-production ability in the skin. However, the effects of blue light on the arterial contractility are controversial. Here, we hypothesized that appropriate protocol of blue laser can induce selective vasorelaxation by activating vasodilating signaling molecules in arteries. Using organ chamber arterial mechanics, NO assay, Matrigel assay, and microarray, we showed that a 200-Hz, 300-µs, 445-nm pulsed-laser (total energy of 600 mJ; spot size 4 mm) induced selective vasorelaxation, without vasocontraction in rat mesenteric arteries. The laser stimulation increased NO production in the cord blood-endothelial progenitor cells (CB-EPCs). Both the laser-induced vasorelaxation and NO production were inhibited by a non-selective, pan-NO synthase inhibitor, L-NG-Nitro arginine methyl ester. Microarray study in CB-EPCs suggested up-regulation of cryptochrome (CRY)2 as well as NO synthase (NOS)1 and NOSTRIN (NOS trafficking) by the laser. In conclusion, this study suggests that the 445-nm blue puled-laser can induce vasorelaxation possibly via the CRY photoreceptors and NOSs activation. The blue laser-therapy would be useful for treating systemic hypertension as well as improving local blood flow depending on the area of irradiation.

Calcitriol Supplementation Ameliorates Microvascular Endothelial Dysfunction in Vitamin D-Deficient Diabetic Rats by Upregulating the Vascular eNOS Protein Expression and Reducing Oxidative Stress.

Diabetes mellitus contributes to macro- and microvascular complications, leading to adverse cardiovascular events. This study examined the effects of vitamin D deficiency on the vascular function and tissue oxidative status in the microcirculation of diabetic rats and to determine whether these effects can be reversed with calcitriol (active vitamin D metabolite) supplementation. Streptozotocin-induced diabetic rats were fed for 10 weeks with control diet (DC) or vitamin D-deficient diet without (DD) or with oral calcitriol supplementation (0.15 µg/kg) in the last four weeks (DDS) (10 rats each group). A nondiabetic rat group that received control diet was also included (NR). After 10 weeks, rats were sacrificed; mesenteric arterial rings with and without endothelium were studied using wire myograph. Western blotting of the mesenteric arterial tissue was performed to determine the protein expression of endothelial nitric oxide synthase (eNOS) enzyme. Antioxidant enzyme superoxide dismutase (SOD) activity and oxidative stress marker malondialdehyde (MDA) levels in the mesenteric arterial tissue were also measured. The DC group had significantly lower acetylcholine-induced relaxation and augmented endothelium-dependent contraction, with reduced eNOS expression, compared to NR rats. In mesenteric arteries of DD, acetylcholine-induced endothelium-dependent and sodium nitroprusside-induced endothelium-independent relaxations were lower than those in DC. Calcitriol supplementation in DDS restored endothelium-dependent relaxation. Mesenteric artery endothelium-dependent contraction of DD was greater than DC; it was not affected by calcitriol supplementation. The eNOS protein expression and SOD activity were significantly lower while MDA levels were greater in DD compared to DC; these effects were not observed in DDS that received calcitriol supplementation. In conclusion, vitamin D deficiency causes eNOS downregulation and oxidative stress, thereby impairing the vascular function and posing an additional risk for microvascular complications in diabetes. Calcitriol supplementation to diabetics with vitamin D deficiency could potentially be useful in the management of or as an adjunct to diabetes-related cardiovascular complications.

GTP-cyclohydrolase deficiency induced peripheral and deep microcirculation dysfunction with age.

The present study assessed the impact of impaired tetrahydrobiopterin (BH4) production on vasoreactivity from conduit and small arteries along the vascular tree as seen during aging. For this purpose, the mutant hyperphenylalaninemic mouse (hph-1) was used. This model is reported to be deficient in GTP cyclohydrolase I, a rate limiting enzyme in BH4 biosynthesis. BH4 is a key regulator of vascular homeostasis by regulating the nitric oxide synthase 3 (NOS3) activity. In GTP-CH deficient mice, the aortic BH4 levels were decreased, by -77% in 12 week-middle-aged mice (young) and by -83% in 35-45 week-middle-aged mice (middle-aged). In young hph-1, the mesenteric artery ability to respond to flow was slightly reduced by 9%. Aging induced huge modification in many vascular functions. In middle-aged hph-1, we observed a decrease in aortic cGMP levels, biomarker of NO availability (-46%), in flow-mediated vasodilation of mesenteric artery (-31%), in coronary hyperemia response measured in isolated heart following transient ischemia (-27%) and in cutaneous microcirculation dilation in response to acetylcholine assessed in vivo by laser-doppler technic (-69%). In parallel, the endothelium-dependent relaxation in response to acetylcholine in conduit blood vessel, measured on isolated aorta rings, was unchanged in hph-1 mice whatever the age. Our findings demonstrate that in middle-aged GTP-CH depleted mice, the reduction of BH4 was characterized by an alteration of microcirculation dilatory properties observed in various parts of the vascular tree. Large conduit blood vessels vasoreactivity, ie aorta, was unaltered even in middle-aged mice emphasizing the main BH4-deletion impact on the microcirculation.

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.

Disrupted eNOS activity and expression account for vasodilator dysfunction in different stage of sepsis.

AIMS: Sepsis is a severe endothelial dysfunction syndrome. The role of endothelial nitric oxide synthase (eNOS) in endothelial dysfunction induced by sepsis is controversial. To explore the role of eNOS in vascular dysfunction. MAIN METHODS: The effect of sepsis on vasodilation and eNOS levels was examined in septic mouse arteries and in cell models. KEY FINDINGS: In early sepsis mouse arteries, endothelium-dependent relaxation decreased and phosphorylation of the inhibitory Thr495 site in endothelial nitric oxide synthase increased. Mechanically, the phosphorylation of endothelial nitric oxide synthase at Thr497 in bovine aortic endothelial cells occurred in a protein kinase C-α dependent manner. In late sepsis, both nitric oxide-dependent relaxation responses and endothelial nitric oxide synthase levels were decreased in septic mice arteries. Endothelial nitric oxide synthase levels expression levels decreased in tumor necrosis factor-α-treated human umbilical vein endothelial cells and this could be prevented by the ubiquitin proteasome inhibitor (MG-132). MG-132 could reverse the decrease in endothelial nitric oxide synthase expression and improve nitric oxide-dependent vasodilator dysfunction in septic mice arteries. SIGNIFICANCE: These data indicate that vasodilator dysfunction is induced by the increased phosphorylation of endothelial nitric oxide synthase in early sepsis and its degradation in late sepsis.

Pregnancy decreases O-GlcNAc-modified proteins in systemic arteries of normotensive and spontaneously hypertensive rats.

AIM: We determined the role played by O-linked N-acetylglucosamine (O-GlcNAc) of proteins in systemic arteries during late pregnancy in normotensive and hypertensive rats. MAIN METHODS: O-GlcNAc levels and O-GlcNAc modification of endothelial nitric oxide synthase (eNOS) were determined in aorta (conductance vessel) and mesenteric arteries (resistance vessels) of non-pregnant (NP) and pregnant (P) Wistar rats and spontaneously hypertensive rats (SHR). Vascular O-GlcNAc-modified proteins, O-GlcNAcase (OGA) and O-GlcNAc transferase (OGT) expression, and OGA activity were analyzed. Concentration-response to phenylephrine (PE) curves were constructed for arteries with and without endothelium. Arteries were treated with vehicle or PugNAc (OGA inhibitor, 100 µmol/L) in the presence of L-NAME (NOS inhibitor, 100 µmol/L). KEY FINDINGS: The content of vascular O-GlcNAc-modified proteins was lower, OGT and OGA expression did not change, and OGA activity was higher in arteries of P-Wistar rats and P-SHR compared to arteries of NP-groups. Reactivity to PE increased in arteries of P-Wistar rats treated with PugNAc compared to vehicle. O-GlcNAcylation of eNOS decreased in P-SHR compared to NP-SHR. PugNAc partially inhibited the effects of endothelium removal and L-NAME on reactivity to PE in arteries of P-Wistar rats. However, PugNAc did not alter reactivity to PE in arteries of P-SHR. Our data showed that pregnancy decreased the content of vascular O-GlcNAc-modified proteins. SIGNIFICANCE: Increased OGA activity and decreased O-GlcNAc modification of eNOS boosts eNOS activity in arteries of P-Wistar rats. In P-SHR, altered OGA activity may lower the content of O-GlcNAc-modified proteins, but decreased OGT activity seems a potential mechanism to reduce glycosylation.

Cadmium exposure activates NADPH oxidase, renin-angiotensin system and cyclooxygenase 2 pathways in arteries, inducing hypertension and vascular damage.

Exposure to high concentrations of cadmium (Cd), widely used in many industries and found in air, food and contaminated water, is not uncommon. Cd damages the cardiovascular system, but the vascular mechanisms involved are not fully understood. This study investigated the mechanisms involved in cardiovascular damage after exposure to high Cd concentrations. Three-month-old male Wistar rats were treated intraperitoneally for 14 days with distilled water (Untreated group) or 1 mg/kg cadmium chloride (Cd group). We investigated the systolic blood pressure (SBP) and vascular reactivity of mesenteric resistance arteries (MRA) and the aorta by analysing contractile and relaxation responses in the absence and presence of the endothelium; we also evaluated pathways involved in vascular tone regulation. Superoxide anion production, COX-2 protein expression and in situ detection of COX-2, AT-1, and NOX-1 were evaluated. Oxidative status, creatinine level and angiotensin-converting enzyme (ACE) activity in plasma were also evaluated. Fourteen-day exposure to a high Cd concentration induced hypertension associated with vascular dysfunction in MRA and the aorta. In both vessels, there was increased participation of cyclooxygenase 2 (COX2), angiotensin II type 1 (AT1) receptor and NOX1. MRA also presented endothelial dysfunction, denoted by impaired acetylcholine-mediated relaxation. All vascular changes were accompanied by increased reactive oxygen species production and COX2, NOX1 and AT1 receptor expression in vascular tissue. Overall, high Cd concentrations induced cardiovascular damage: hypertension, endothelial dysfunction and vascular damage in conductance and resistance arteries, NADPH oxidase, renin-angiotensin system and COX2 pathway activation.

Progesterone promotes endothelial nitric oxide synthase expression through enhancing nuclear progesterone receptor-SP-1 formation.

Progesterone exerts antihypertensive actions partially by modulating endothelial nitric oxide synthase (eNOS) activity. Here, we aimed to investigate the effects and mechanisms of progesterone on eNOS expression. First, human umbilical vein endothelial cells (HUVECs) were exposed to progesterone and then the eNOS transcription factor specificity protein-1 (SP-1) and progesterone receptor (PRA/B) expression were assessed by Western blotting and qRT-PCR. The interaction between SP-1 and PRA/B was next determined through coimmunoprecipitation assay. The chromatin immunoprecipitation assay and luciferase assay were used to investigate the relationship of PRA/B, SP-1, and eNOS promoter. At last, rats were intraperitoneally injected with progesterone receptor antagonist RU-486, and then the expression of eNOS and vasodilation function in thoracic aorta and mesenteric artery were measured. The results showed that progesterone could increase eNOS expression in HUVECs. Further study showed that progesterone increased PRA-SP-1 complex formation and facilitated PRA/B and SP-1 binding to eNOS promoter. Mutating SP-1 or PR-binding motif on eNOS promoter abolished the effect of progesterone on eNOS gene transcription. We also observed that progesterone receptor antagonist RU-486 reduced eNOS expression and impaired vasodilation in rats. Those results suggest that progesterone modulates eNOS expression through promoting PRA-SP-1 complex formation, and progesterone antagonist attenuates eNOS expression, leading to the loss of vascular relaxation.NEW & NOTEWORTHY Progesterone directly upregulated endothelial nitric oxide synthase (eNOS) expression in human endothelial cells. Progesterone augmented eNOS promoter activity through a progesterone receptor A- and specificity protein-1-dependent manner. Antagonism of the progesterone receptor reduced eNOS expression and impaired vasodilation in rats.

Electrical stimulation of the carotid sinus lowers arterial pressure and improves heart rate variability in L-NAME hypertensive conscious rats.

We evaluated the effects of long-term (48 h) electrical stimulation of the carotid sinus (CS) in hypertensive rats. L-NAME-treated (10 days) Wistar rats were implanted with a catheter in the femoral artery and a miniaturized electrical stimulator attached to electrodes positioned around the left CS, encompassing the CS nerve. One day after implantation, arterial pressure (AP) was directly recorded in conscious animals for 60 min. Square pulses (1 ms, 3 V, 30 Hz) were applied intermittently (20/20 s ON/OFF) to the CS for 48 h. After the end of stimulation, AP was recorded again. Nonstimulated rats (control group) and rats without electrodes around the CS (sham-operated) were also studied. Next, the animals were decapitated, and segments of mesenteric resistance arteries were removed to study vascular function. After the stimulation period, AP was 16 ± 5 mmHg lower in the stimulated group, whereas sham-operated and control rats showed similar AP between the first and second recording periods. Heart rate variability (HRV) evaluated using time and frequency domain tools and a nonlinear approach (symbolic analysis) suggested that hypertensive rats with electrodes around the CS, stimulated or not, exhibited a shift in cardiac sympathovagal balance towards parasympathetic tone. The relaxation response to acetylcholine in endothelium-intact mesenteric arteries was enhanced in rats that underwent CS stimulation for 48 h. In conclusion, long-term CS stimulation is effective in reducing AP levels, improving HRV and increasing mesenteric vascular relaxation in L-NAME hypertensive rats. Moreover, only the presence of electrodes around the CS is effective in eliciting changes in HRV similar to those observed in stimulated rats.

Secreted Monocyte miR-27a, via Mesenteric Arterial Mas Receptor-eNOS Pathway, Causes Hypertension.

BACKGROUND: Essential hypertension is associated with increased plasma concentrations of extracellular vesicles (EVs). We aimed to determine the role of monocyte miR-27a in EVs on arterial Mas receptor expression, and its involvement in the pathogenesis of hypertension. METHODS: THP-1 cells were transfected with miR-27a mimic and miR-27a inhibitor, and EVs were collected. Mas receptor expression and endothelial nitric oxide synthase (eNOS) phosphorylation were determined by immunoblotting. Sprague-Dawley (SD) rats received EVs via tail-vein injection. Blood pressure (BP) was measured with the tail-cuff method. The vasodilatory response of mesenteric arteries was measured using a small vessel myograph. RESULTS: EVs from THP-1 cells increased rat BP by impairing Ang-(1-7)-mediated vasodilation in mesenteric arteries, which was further exaggerated by EVs from lipopolysaccharides-treated THP-1 cells. As the receptor and key signaling of Ang-(1-7), next experiments found that Mas receptor expression and eNOS phosphorylation were decreased in mesenteric arteries from EVs-treated SD rats. Screening studies found miR-27a in EVs may be involved in this process. Through transfection with miR-27a inhibitor or miR-27a mimic, we found that miR-27a downregulates Mas receptor expression in endothelial cells. Injection of EVs from miR-27a-transfected HEK-293 cells decreased Mas receptor and eNOS phosphorylation in mesenteric arteries, impaired Ang-(1-7)-mediated vasodilation and increased BP. Earlier effects were reversed using cells with downregulation of miR-27 in EVs. CONCLUSIONS: Monocyte miR-27a in EVs decreases Mas receptor expression and eNOS phosphorylation in endothelium, impairs Ang-(1-7)-mediated vasodilation, and causes hypertension. Understanding the contributions of EVs in the pathogenesis of hypertension may facilitate their use as a diagnostic biomarker.

Long-lasting blood pressure lowering effects of nitrite are NO-independent and mediated by hydrogen peroxide, persulfides, and oxidation of protein kinase G1α redox signalling.

AIMS: Under hypoxic conditions, nitrite (NO2-) can be reduced to nitric oxide (NO) eliciting vasorelaxation. However, nitrite also exerts vasorelaxant effects of potential therapeutic relevance under normal physiological conditions via undetermined mechanisms. We, therefore, sought to investigate the mechanism(s) by which nitrite regulates the vascular system in normoxia and, specifically, whether the biological effects are a result of NO generation (as in hypoxia) or mediated via alternative mechanisms involving classical downstream targets of NO [e.g. effects on protein kinase G1α (PKG1α)]. METHODS AND RESULTS: Ex vivo myography revealed that, unlike in thoracic aorta (conduit vessels), the vasorelaxant effects of nitrite in mesenteric resistance vessels from wild-type (WT) mice were NO-independent. Oxidants such as H2O2 promote disulfide formation of PKG1α, resulting in NO- cyclic guanosine monophosphate (cGMP) independent kinase activation. To explore whether the microvascular effects of nitrite were associated with PKG1α oxidation, we used a Cys42Ser PKG1α knock-in (C42S PKG1α KI; 'redox-dead') mouse that cannot transduce oxidant signals. Resistance vessels from these C42S PKG1α KI mice were markedly less responsive to nitrite-induced vasodilation. Intraperitoneal (i.p.) bolus application of nitrite in conscious WT mice induced a rapid yet transient increase in plasma nitrite and cGMP concentrations followed by prolonged hypotensive effects, as assessed using in vivo telemetry. In the C42S PKG1α KI mice, the blood pressure lowering effects of nitrite were lower compared to WT. Increased H2O2 concentrations were detected in WT resistance vessel tissue challenged with nitrite. Consistent with this, increased cysteine and glutathione persulfide levels were detected in these vessels by mass spectrometry, matching the temporal profile of nitrite's effects on H2O2 and blood pressure. CONCLUSION: Under physiological conditions, nitrite induces a delayed and long-lasting blood pressure lowering effect, which is NO-independent and occurs via a new redox mechanism involving H2O2, persulfides, and PKG1α oxidation/activation. Targeting this novel pathway may provide new prospects for anti-hypertensive therapy.

Chronic Treatment With Acetylcholinesterase Inhibitors Attenuates Vascular Dysfunction in Spontaneously Hypertensive Rats.

BACKGROUND: Acetylcholinesterase inhibition prevents autonomic imbalance, reduces inflammation, and attenuates the development of hypertension. Considering that vascular dysfunction is a crucial feature of arterial hypertension, we investigated the effects of chronic administration of acetylcholinesterase inhibitors-pyridostigmine or donepezil-on vascular reactivity of spontaneously hypertensive rats (SHR). METHODS: Endothelium-dependent relaxant responses to acetylcholine (ACh) and contractile responses induced by electric field stimulation (EFS) and alpha-adrenergic agonist were studied in mesenteric resistance arteries from SHR and Wistar Kyoto rats. SHR were treated for 16 weeks with vehicle, pyridostigmine (1.5 mg/kg/day) or donepezil (1.4 mg/kg/day). RESULTS: Pyridostigmine and donepezil decreased the vasoconstrictor responses to EFS, which were increased in vehicle-treated SHR. Acetylcholinesterase inhibition increased the modulatory effects of nitric oxide (NO) on SHR vascular reactivity, that is, N(ω)-nitro-(L)-arginine methyl ester (L-NAME) increased EFS-induced contractions and reduced ACh-induced relaxation, with more significant effects in pyridostigmine- and donepezil-treated SHR. The acetylcholinesterase inhibitors also decreased vascular reactive oxygen species levels. CONCLUSIONS: This study demonstrates for the first time that long-term administration of acetylcholinesterase inhibitors, pyridostigmine or donepezil, attenuates vascular reactivity dysfunction in SHR by decreasing reactive oxygen species generation and increasing NO bioavailability; possibly via increased endothelial NO synthase activity, and inhibition of NADPH oxidase activity.

Effects of obesity on insulin: insulin-like growth factor 1 hybrid receptor expression and Akt phosphorylation in conduit and resistance arteries.

Insulin and insulin-like growth factor-1 stimulate specific responses in arteries, which may be disrupted by diet-induced obesity. We examined (1) temporal effects of high-fat diet compared to low-fat diet in mice on insulin receptor, insulin-like growth factor-1 receptor, insulin receptor/insulin-like growth factor-1 receptor hybrid receptor expression and insulin/insulin-like growth factor-1-mediated Akt phosphorylation in aorta; and (2) effects of high-fat diet on insulin and insulin-like growth factor-1-mediated Akt phosphorylation and vascular tone in resistance arteries. Medium-term high-fat diet (5 weeks) decreased insulin-like growth factor-1 receptor expression and increased hybrid expression (~30%) only. After long-term (16 weeks) high-fat diet, insulin receptor expression was reduced by ~30%, insulin-like growth factor-1 receptor expression decreased a further ~40% and hybrid expression increased a further ~60%. Independent correlates of hybrid receptor expression were high-fat diet, duration of high-fat diet and plasma insulin-like growth factor-1 (all p < 0.05). In aorta, insulin was a more potent activator of Akt than insulin-like growth factor-1, whereas in resistance arteries, insulin-like growth factor-1 was more potent than insulin. High-fat diet blunted insulin-mediated vasorelaxation ( p < 0.01) but had no effect on insulin-like growth factor-1-mediated vasorelaxation in resistance arteries. Our findings support the possibility that hybrid receptor level is influenced by nutritional and metabolic cues. Moreover, vessel-dependent effects of insulin and insulin-like growth factor-1 on vascular tone and Akt activation may have implications in treating obesity-related vascular disease.

Deoxycholylglycine, a conjugated secondary bile acid, reduces vascular tone by attenuating Ca2+ sensitivity via rho kinase pathway.

Patients with cirrhosis have reduced systemic vascular resistance and elevated circulating bile acids (BAs). Previously, we showed that secondary conjugated BAs impair vascular tone by reducing vascular smooth muscle cell (VSMC) Ca2+ influx. In this study, we investigated the effect of deoxycholylglycine (DCG), on Ca2+ sensitivity in reducing vascular tone. First, we evaluated the effects of DCG on U46619- and phorbol-myristate-acetate (PMA)-induced vasoconstriction. DCG reduced U46619-induced vascular tone but failed to reduce PMA-induced vasoconstriction. Then, by utilizing varied combinations of diltiazem (voltage-dependent Ca2+ channel [VDCC] inhibitor), Y27632 (RhoA kinase [ROCK] inhibitor) and chelerythrine (PKC inhibitor) for the effect of DCG on U46619-induced vasoconstriction, we ascertained that DCG inhibits VDCC and ROCK pathway with no effect on PKC. We further assessed the effect of DCG on ROCK pathway. In ß-escin-permeabilized arteries, DCG reduced high-dose Ca2+- and GTPγS (a ROCK activator)-induced vasoconstriction. In rat vascular smooth muscle cells (VSMCs), DCG reduced U46619-induced phosphorylation of myosin light chain subunit (MLC20) and myosin phosphatase target subunit-1 (MYPT1). In permeabilized VSMCs, DCG reduced Ca2+- and GTPγS-mediated MLC20 and MYPT1 phosphorylation, and further, reduced GTPγS-mediated membrane translocation of RhoA. In VSMCs, long-term treatment with DCG had no effect on ROCK2 and RhoA expression. In conclusion, DCG attenuates vascular Ca2+ sensitivity and tone via inhibiting ROCK pathway. These results enhance our understanding of BAs-mediated regulation of vascular tone and provide a platform to develop new treatment strategies to reduce arterial dysfunction in cirrhosis.

Protein tyrosine phosphatase 1B inactivation limits aging-associated heart failure in mice.

We have previously shown that protein tyrosine phosphatase 1B (PTP1B) inactivation in mice [PTP1B-deficient (PTP1B-/-) mice] improves left ventricular (LV) angiogenesis, perfusion, remodeling, and function and limits endothelial dysfunction after myocardial infarction. However, whether PTP1B inactivation slows aging-associated cardiovascular dysfunction remains unknown. Wild-type (WT) and PTP1B-/- mice were allowed to age until 18 mo. Compared with old WT mice, in which aging increased the LV mRNA expression of PTP1B, old PTP1B-/- mice had 1) reduced cardiac hypertrophy with decreased LV mRNA levels of hypertrophic markers and atrial and brain natriuretic peptides, 2) lower LV fibrosis (collagen: 16 ± 3% in WT mice and 5 ± 3% in PTP1B-/- mice, P < 0.001) with decreased mRNA levels of transforming growth-factor-ß1 and matrix metalloproteinase-2, and 3) higher LV capillary density and lower LV mRNA level of hypoxic inducible factor-1α, which was associated over time with a higher rate of proangiogenic M2 type macrophages and a stable LV mRNA level of VEGF receptor-2. Echocardiography revealed an age-dependent LV increase in end-diastolic volume in WT mice together with alterations of fractional shortening and diastole (transmitral Doppler E-to-A wave ratio). Invasive hemodynamics showed better LV systolic contractility and better diastolic compliance in old PTP1B-/- mice (LV end-systolic pressure-volume relation: 13.9 ± 0.9 in WT mice and 18.4 ± 1.6 in PTP1B-/- mice; LV end-diastolic pressure-volume relation: 5.1 ± 0.8 mmHg/relative volume unit in WT mice and 1.2 ± 0.3 mmHg/relative volume unit in PTP1B-/- mice, P < 0.05). In addition, old PTP1B-/- mice displayed a reduced amount of LV reactive oxygen species. Finally, in isolated resistance mesenteric arteries, PTP1B inactivation reduced aging-associated endothelial dysfunction (flow-mediated dilatation: -0.4 ± 2.1% in WT mice and 8.2 ± 2.8% in PTP1B-/- mice, P < 0.05). We conclude that PTP1B inactivation slows aging-associated LV remodeling and dysfunction and reduces endothelial dysfunction in mesenteric arteries. NEW & NOTEWORTHY The present study shows that protein tyrosine phosphatase 1B inactivation in aged mice improves left ventricular systolic and diastolic function associated with reduced adverse cardiac remodeling (hypertrophy, fibrosis, and capillary rarefaction) and limits vascular endothelial dysfunction. This suggests that protein tyrosine phosphatase 1B inhibition could be an interesting treatment approach in age-related cardiovascular dysfunction.