Mesenteric Resistance Arteries Research Articles

Alteration of Piezo1 signaling in type 2 diabetic mice: focus on endothelium and BKCa channel.

Piezo1 mechanosensitive ion channel plays a important role in vascular physiology and disease. This study aimed to elucidate the altered signaling elicited by Piezo1 activation in the arteries of type 2 diabetes. Ten- to 12-week-old male C57BL/6 (control) and type 2 diabetic mice (db-/db-) were used. The second-order mesenteric arteries (~ 150 μm) were used for isometric tension experiments. Western blot analysis and immunofluorescence staining were performed to observe protein expression. Piezo1 was significantly decreased in mesenteric arteries of type 2 diabetic mice compared to control mice, as analyzed by western blot and immunofluorescence staining. Piezo1 agonist, Yoda1, concentration-dependently induced relaxation of mesenteric arteries in both groups. Interestingly, the relaxation response was significantly greater in control mice than in db-/db- mice. The removal of endothelium reduced relaxation responses induced by Yoda1, which was greater in control mice than db-/db- mice. Furthermore, the relaxation response was reduced by pre-treatment with various types of K+ channel blockers in endothelium-intact arteries in control mice. In endothelium-denuded arteries, pre-incubation with charybdotoxin, an Ca2+-activated K+ channel (BKCa channel) blocker, significantly attenuated Yoda1-induced relaxation in db-/db- mice, while there was no effect in control mice. Co-immunofluorescence staining showed co-localization of Piezo1 and BKCa channel was more pronounced in db-/db- mice than in control mice. These results indicate that the vascular responses induced by Piezo1 activation are different in the mesenteric resistance arteries in type 2 diabetic mice.

Protein disulfide isomerase-mediated transcriptional upregulation of Nox1 contributes to vascular dysfunction in hypertension.

Nox1 signaling is a causal key element in arterial hypertension. Recently, we identified protein disulfide isomerase A1 (PDI) as a novel regulatory protein that regulates Nox1 signaling in VSMCs. Spontaneously hypertensive rats (SHR) have increased levels of PDI in mesenteric resistance arteries compared with Wistar controls; however, its consequences remain unclear. Herein, we investigated the role of PDI in mediating Nox1 transcriptional upregulation and its effects on vascular dysfunction in hypertension. We demonstrate that PDI contributes to the development of hypertension via enhanced transcriptional upregulation of Nox1 in vascular smooth muscle cells (VSMCs). We show for the first time that PDI sulfenylation by hydrogen peroxide contributes to EGFR activation in hypertension via increased shedding of epidermal growth factor-like ligands. PDI also increases intracellular calcium levels, and contractile responses induced by ANG II. PDI silencing or pharmacological inhibition in VSMCs significantly decreases EGFR activation and Nox1 transcription. Overexpression of PDI in VSMCs enhances ANG II-induced EGFR activation and ATF1 translocation to the nucleus. Mechanistically, PDI increases ATF1-induced Nox1 transcription and enhances the contractile responses to ANG II. Herein we show that ATF1 binding to Nox1 transcription putative regulatory regions is augmented by PDI. Altogether, we provide evidence that HB-EGF in SHR resistance vessels promotes the nuclear translocation of ATF1, under the control of PDI, and thereby induces Nox1 gene expression and increases vascular reactivity. Thus, PDI acts as a thiol redox-dependent enhancer of vascular dysfunction in hypertension and could represent a novel therapeutic target for the treatment of this disease.

LRRC8A-VRAC modulates the contractile response by managing the superoxide anion influx in diabetic vasculogenic erectile dysfunction

Introduction: Erectile dysfunction (ED) may be an early marker for cardiovascular diseases, and it is often associated with an hypercontractility of the corpus cavernosum (CC) and pudendal artery (PA) because of increased reactive oxygen species (ROS). Leucine Rich Repeat Containing 8 (LRRC8) Volume Regulated Anion Channels (VRACs) regulates Nox1 and modulates the influx of extracellular superoxide anion (O2•-), contributing to the creation of an oxidized domain, enhancing calcium sensitivity of the smooth muscle which promotes contraction. Objective: We hypothesize that downregulation of NOX1 or LRRC8A would decrease the contractile responses to phenylephrine (PE) and electrical field stimulation (EFS) in the CC, PA and mesenteric resistance artery (MRA) of diabetic mice (dbdb−/−). Methods: The CC and PA of diabetic (dbdb−/−) mice were mounted in myographs. The contraction elicited by PE or EFS was performed in the presence of apocynin (non-selective NOX inhibitor) and GKT137831 (NOX1/4 inhibitor). In LRRC8A KO dbdb−/− mice, contractions to PE and EFS were obtained in the absence or in the presence of GKT137831.Results: In the CC, the maximal contraction to PE was increased in dbdb−/− animals, and in the presence of 3 mM of apocynin, the contractile response was markedly diminished in both control and dbdb−/−. Incubation with GKT137831 reduced the contraction elicited by PE only in the dbdb−/− group (68 ± 9% of the maximal contraction), whereas in the control group this reduction was not significant (82 ± 14% of the PE-maximal contraction). In the PA, the potency of PE was increased in the dbdb−/− group, and apocynin 1mM restored the contraction to normal levels. In the LRRC8A KO dbdb−/− mice, the contraction to PE in the MRA and PA was reduced by 30% compared to dbdb−/−. The response to EFS in CC was similar to dbdb−/−, however, GKT137831 reduced the contraction to EFS with lesser magnitude of inhibition in the LRRC8A KO dbdb−/− mice (35% of inhibition) compared to dbdb−/− mice (50% of inhibition), suggesting that the participation of O2•- in the contractile response was reduced by the lack of functional LRRC8A. Conclusion: LRRC8A modulates the contractile response by managing the influx of reactive oxygen species (ROS) in smooth muscle cells. Inhibition of LRRC8A may be a therapeutic target to prevent hypercontractility of arteries and CC in conditions where there are marked increases in ROS production, such as diabetes. NIH – R01 132948; Institute on Cardiovascular Disease Research, University of South Carolina. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Phenotyping adult mice with inducible depletion of sequestosome-1 (p62) in endothelial cells

Macroautophagy (autophagy) maintains intracellular homeostasis by targeting damaged / dysregulated organelles for their transport to and degradation by the lysosome. Autophagy regulated gene 3 ( Atg3) enables autophagosome maturation which is required for the process of autophagy to occur. We reported that inducible depletion of Atg3 specifically in endothelial cells (ECs) impairs arterial function in the absence of metabolic disruptions (Cho et al., Cardiovasc Res, 2023). Sequestosome-1 (p62/SQSTM1) tethers proteins into the autophagic pathway for subsequent lysosomal degradation. The contribution from EC p62 to arterial and metabolic function is unknown and we addressed this. Adult male and female C57BL/6 mice with tamoxifen inducible depletion of p62 specifically in ECs ( p62EC−/−) were compared to littermate controls ( p62EC+/+). p62/18S mRNA expression was lower (p<0.05) in ECs but not vascular smooth muscle cells (VSM) from p62EC−/− [n=6] vs. p62EC+/+ [n=9] mice, whereas Beclin-1/18S was similar in ECs and VSM from both groups. Neither body composition, glucose disposal nor insulin tolerance were impacted by EC specific p62 depletion. Vascular contractile responses to: (i) phenylephrine mediated α1-activation and (ii) potassium chloride evoked L-type Ca2+ channel opening; and relaxation responses to (iii) acetylcholine (endothelium-dependent) and (iv) sodium nitroprusside (endothelium-independent); were similar between groups in mesenteric resistance arteries (isometric myography) and middle cerebral arteries (isobaric myography). Further, intrinsic arterial stiffness was not different between p62EC−/− [n=6] and p62EC+/+ [n=9] mice i.e., neither the elastic modulus nor stress vs. strain curves generated using segments of thoracic aorta were altered. These findings indicate EC specific p62 depletion does not impact arterial function or the indexes of systemic metabolism we measured. Because p62 is upregulated during cellular stress, it is possible that a challenge (e.g., hypoxia, ischemia) might be required to unmask a protective role for intact EC p62 and studies are ongoing to test this. Supported by AHA23PRE1025910 (SM); Rural and Underserved Utah Training Experience T32 (CG), Pacific Islander Research Internship Program (LG), NIHR01HL149870-01A1 (SB) and NIHRO1HL141540 (JDS). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Alteration of Piezo1 signaling in type 2 diabetic mice: Focus on endothelium and BKca channel

Aim: Piezo1, a mechanosensitive ion channel, plays a significant role in vascular physiology and disease. This study aimed to elucidate the altered signaling elicited by Piezo1 activation in the arteries of type 2 diabetes. Methods: Ten-to 12-week-old male C57BL/6 (control) and type 2 diabetic mice (db−/db−) were used. The second-order mesenteric arteries (~150 mm) were used for isometric tension experiments. Western blot analysis and immunofluorescence staining were performed to observe protein expression. Results: Piezo1 was significantly decreased in mesenteric arteries of type 2 diabetic mice compared to control mice, as analyzed by western blot and immunofluorescence staining. Piezo1 agonist, Yoda1, concentration-dependently induced relaxation of mesenteric arteries in both groups. Interestingly, the relaxation response was significantly greater in control mice than in db−/db− mice. The removal of endothelium reduced relaxation responses induced by Yoda1, which was greater in control mice than db−/db− mice. Furthermore, the relaxation response was reduced by pre-treatment with various types of K+ channel blockers in endothelium-intact arteries in control mice. In endothelium-denuded arteries, pre-incubation with charybdotoxin, an Ca2+-activated K+ channel (BKCa channel) blocker, significantly attenuated Yoda1-induced relaxation in db−/db− mice, while there was no effect in control mice. Co-immunofluorescence staining showed more structural coupling between Piezo1 and BKCa channel was observed in db−/db− mice than in control mice. Conclusions: The present study is the first report on the distinct aspects of vascular response induced by Piezo1 activation in the mesenteric resistance arteries in type 2 diabetic mice. National Research Foundation of Korea (RS-2023-00272628). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Glyphosate, the active ingredient of RoundUp, promotes gut dysbiosis, vascular dysfunction and hypertension

Introduction: Glyphosate was developed and marketed as an herbicide by Monsanto, USA, in the 1970s. Since then, multiple studies have documented its adverse effects on human health, whereby the use of glyphosate is banned in Europe and its renewal is currently under discussion by European policy. However, glyphosate remains the most widely used herbicide in the United States. Glyphosate exposure can occur through consumption of genetically modified crops tolerant to glyphosate. Glyphosate targets 5-enolpyruvylshikimate-3-phosphate synthase, which is only present in plants and microbes. Therefore, glyphosate is considered safe for humans. However, humans are holobionts with symbiotic gut microbiota, which can be targeted by glyphosate to result in gut dysbiosis. Because our previous studies demonstrate that gut microbiota dysbiosis causes hypertension, we hypothesized that exposure to glyphosate aggravates gut dysbiosis-mediated hypertension. Methods: Two groups of adult (8–10 week old) genetically hypertensive Dahl salt-sensitive (S) male rats were surgically implanted with radiotelemetry transmitters to monitor blood pressure (BP) and administered with or without glyphosate (175 mg/kg body weight per day) in drinking water for 3 weeks (Glyphosate-treated group, n=8; control, n=6). Fecal 16S rRNA gene sequencing and Oxford Nanopore whole genome sequencing were performed for microbiota analyses. Vascular function was examined ex vivo in isolated mesenteric arteries using wire myography. Results: As early as 1 week of exposure to glyphosate was suffcient to significantly increase systolic (173 mmHg vs. 160 mmHg, p<0.0001) and mean arterial pressure (148 mmHg vs 141 mmHg, p<0.0002), but not diastolic BP of S rats. Diastolic BP (142 mmHg vs 135 mmHg, p<0.0001), systolic BP (195 mmHg vs 176 mmHg, p<0.0001), and mean arterial pressure (168 mmHg vs 155 mmHg, p<0.0001) were all significantly increased by week 3, indicating a temporally sustained detrimental effect of glyphosate on BP. The elevation in BP was accompanied by increased contractility of isolated mesenteric resistance arteries as determined by their increased vasocontractile response to phenylephrine (p=0.034), and decreased vasorelaxant response to acetylcholine (p=0.047). 16S metagenomics data revealed distinct rearrangement of gut microbiota composition in the glyphosate-treated group compared to control. Significant shifts in both α-diversity (Shannon entropy; p=0.049) and β-diversity (R=0.24, p<0.032) were observed in the glyphosate-treated group. Bacterial genera such as Candidatus, Erysipelatoclostridium, and Ruminicoccus were more abundant in the glyphosate group whereas Bifidobacterium that produces short-chain fatty acids, was depleted. Summary: Oral glyphosate exposure resulted in elevated BP, increased vascular contractility, and altered gut microbiota composition. Conclusion: This is the first proof-of-concept study to demonstrate that glyphosate is harmful for human health as it contributes to the escalation of hypertension. While more mechanistic studies are warranted, our study supports the reasoning that the unabated use of glyphosate could be a contributor to the rising incidence of hypertension in the western society. NHLBI to Bina Joe (R01HL1430820). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

A novel dual inhibitor of the angiotensin and endothelin systems for the treatment of hypertension

Hypertension, affecting nearly half of the US adult population, is a primary cause of morbidity and mortality worldwide. Despite the availability of several classes of antihypertensive medications, only 25% of patients have their blood pressure under control, highlighting the need for novel antihypertensive therapeutics with better effcacy. Among hypertensive cases, salt-sensitive and drug-resistant hypertension are particularly concerning, as they are unresponsive to a combined regimen of three or more classes of medications, including angiotensin II (Ang II) receptor blockers (ARBs), angiotensin-converting enzyme inhibitors (ACEis), and diuretics. Salt-sensitive, observed in 50% of the general and 73% of black hypertensive patients, disproportionately affects the latter, leading to significantly higher mortality. Salt-sensitive hypertension in humans, as well as in various experimental models of hypertension (e.g., Ang II-induced, Dahl-salt sensitive [DSS] rats, deoxycorticosterone acetate [DOCA]-salt hypertensive rats), is characterized by significantly elevated levels of plasma endothelin-1 (ET-1), oxidative stress, and kidney dysfunction. ET-1 is often considered a final common pathway in orchestrating cardiovascular dysfunction through a range of proinflammatory mediators such as Ang II, cytokines, hypoxia, thrombin, reactive oxygen species (ROS), oxidized LDL, and vasopressin, all of which stimulate ET-1 production. In this capacity, we report the discovery of a gut microbiota-derived metabolite, indole-3-acetic acid (IAA), that simultaneously blocks ET-1 receptors (ETAR and ETBR) and the ET-biosynthetic enzyme ECE-1. Our data demonstrate that IAA inhibits ET-1-induced constriction of resistance mesenteric arteries, ROS production in human aortic smooth muscle cells, activation of cultured endothelial cells, adhesion molecule expression, and leukocyte adhesion. IAA inhibited purified human ECE-1 and suppressed Ang-II-induced and hypoxia-elicited ET-1 production from endothelial cells in vitro. Administration of a once-daily, extended-release formulation of IAA inhibits blood pressure elevation and kidney dysfunction in both DSS and Ang II-induced hypertension. IAA treatment preserved endothelial nitric oxide (NO) production and maintained smooth muscle sensitivity to a NO donor, sodium nitroprusside. Furthermore, IAA inhibited the expression of ROS-associated proteins such as Nox2 and Nox4, reduced oxidative damages, and decreased the production of ET-1 in vivo. Overall, IAA is the first compound that, through novel dual targeting of both the angiotensin and endothelin systems, demonstrates profound antihypertensive action in both salt-sensitive and angiotensin II-induced hypertension. Thus, IAA has the potential to effectively treat hypertension that does not respond to conventional therapies. American Heart Association. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Melatonin alleviated splanchnic hyperdynamic circulation and portosystemic collaterals in cirrhotic rats

AbstractChronic liver damages may end up with cirrhosis and portal hypertension, featured by splanchnic hyperdynamic circulation, angiogenesis, and collaterals formation. Melatonin is used to improve sleep quality, which exerts anti‐inflammatory, anti‐angiogenesis, and vascular actions without significant side effects. However, the relevant impacts on aforementioned derangements are unclear. Liver cirrhosis was induced by bile duct ligation in Sprague‐Dawley rats. The rats received melatonin (40 mg/kg/day, i.p.) or vehicle for 28 days. Experiments were performed on the 28th day when cirrhosis developed. In cirrhotic rats, melatonin treatment significantly increased superior mesenteric artery resistance and reduced the blood flow. Melatonin enhanced the portosystemic collateral responsiveness to arginine vasopressin, reduced mesenteric vascular area, shunting degree, and down‐regulated mesenteric MMP‐2 protein expression. Melatonin improved the splanchnic hyperdynamic circulation, portosystemic collateral shunting, and mesenteric angiogenesis in cirrhotic rats. These beneficial effects make melatonin potentially feasible in clinical setting, but further investigation is required.

Testosterone modulates vasodilation in mesenteric arteries of hypertensive rats

AimsTo evaluate the effects of testosterone on endothelium-dependent vasodilation and oxidative stress in mesenteric resistance arteries. Main methodsSpontaneously hypertensive rats (SHR), aged 8 to 10 weeks, were divided into four groups: intact (SHAM), intact treated with testosterone (TTO; 3 mg/kg/day) via subcutaneous route (s.c.), intact treated with testosterone and anastrozole [aromatase enzyme inhibitor (TTO + ANA; 0.1 mg/kg/day, s.c.)] and intact treated with testosterone and finasteride [5 α-reductase enzyme inhibitor (TTO + FIN; 5 mg/kg/day, s.c.)] for four weeks. Concentration-response curves to acetylcholine (ACh, 0.1 nmol/L - 10 μmol/L) were obtained in mesenteric resistance arteries previously contracted with phenylephrine (PE, 3 μmol/L), before and after the use of selective inhibitors. Reactive oxygen species (ROS) levels were assessed in the vessels and the endothelium analyzed by scanning electron microscopy. Key findingsTTO group showed a lower participation of nitric oxide (NO), increased oxidative stress, and participation of prostanoids and endothelium-dependent hyperpolarization (EDH), possibly to maintain the vasodilator response. Lower participation of NO and prostanoids, combined to an increased participation of EDH, were observed in the TTO + ANA group, in addition to higher levels of ROS and altered endothelial morphology. The vasodilation to ACh was impaired in TTO + FIN, along increased participation of NO, reduction of prostanoids, and greater EDH-dependent vasodilation. SignificanceTestosterone contributes to endothelial vasodilation by enhancing EDH through an increased participation of epoxyeicosatrienoic acids. While the decrease in NO appears to involve the participation of dihydrotestosterone, 17 β-estradiol seems to stimulate the action of the NO pathway and prostanoids.

Histamine H2-receptor antagonism improves conduit artery endothelial function and reduces plasma aldosterone level without lowering arterial blood pressure in angiotensin II–hypertensive mice

Aldosterone through the mineralocorticoid receptor MR has detrimental effects on cardiovascular disease. It reduces the bioavailability of nitric oxide and impairs endothelium-dependent vasodilatation. In resistance arteries, aldosterone impairs the sensitivity of vascular smooth muscle cells to nitric oxide by promoting the local secretion of histamine which activates H2 receptors. The present experiments tested in vivo and ex vivo the hypothesis that systemic H2-receptor antagonism reduces arterial blood pressure and improves vasodilatation in angiotensin II–induced chronic hypertension. Hypertension was induced by intravenous infusion of angiotensin II (60 ng kg−1 min−1) in conscious, unrestrained mice infused concomitantly with the H2-receptor antagonist ranitidine (27.8 µg kg−1 min−1) or vehicle for 24 days. Heart rate and arterial blood pressure were recorded by indwelling arterial catheter. Resistance (mesenteric) and conductance (aortae) arteries were harvested for perfusion myography and isometric tension recordings by wire myography, respectively. Plasma was analyzed for aldosterone concentration. ANGII infusion resulted in elevated arterial blood pressure and while in vivo treatment with ranitidine reduced plasma aldosterone concentration, it did not reduce blood pressure. Ranitidine improved ex vivo endothelial function (acetylcholine 10−9 to 10−6 mol L−1) in mesenteric resistance arteries. This was abolished by ex vivo treatment with aldosterone (10−9 mol L−1, 1 h). In aortic segments, in vivo ranitidine treatment impaired relaxation. Activation of histamine H2 receptors promotes aldosterone secretion, does not affect arterial blood pressure, and protects endothelial function in conduit arteries but promotes endothelial dysfunction in resistance arteries during angiotensin II–mediated hypertension. Aldosterone contributes little to angiotensin II–induced hypertension in mice.

Nitric oxide, endothelium-derived hyperpolarizing factor, and smooth muscle-dependent mechanisms contribute to magnesium-dependent vascular relaxation in mouse arteries.

Magnesium (Mg2+ ) is a vasorelaxant. The underlying physiological mechanisms driving this vasorelaxation remain unclear. Studies were designed to test the hypothesis that multiple signaling pathways including nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) in endothelial cells as well as Ca2+ antagonization and TRPM7 channels in vascular smooth muscle cells mediate Mg2+ -dependent vessel relaxation. To uncover these mechanisms, force development was measured exvivo in aorta rings from mice using isometric wire myography. Concentration responses to Mg2+ were studied in intact and endothelium-denuded aortas. Key findings were confirmed in second-order mesenteric resistance arteries perfused exvivo using pressure myography. Effects of Mg2+ on NO formation were measured in Chinese Hamster Ovary (CHO) cells, isolated mesenteric vessels, and mouse urine. Mg2+ caused a significant concentration-dependent relaxation of aorta rings. This relaxation was attenuated significantly in endothelium-denuded aortas. The endothelium-dependent portion was inhibited by NO and cGMP blockade but not by cyclooxygenase inhibition. Mg2+ stimulated local NO formation in CHO cells and isolated mesenteric vessels without changing urinary NOx levels. High extracellular Mg2+ augmented acetylcholine-induced relaxation. SKCa and IKCa channel blockers apamin and TRAM34 inhibited Mg2+ -dependent relaxation. The endothelium-independent relaxation in aorta rings was inhibited by high extracellular Ca2+ . Combined blockade of NO, SKCa , and IKCa channels significantly reduced Mg2+ -dependent dilatation in mesenteric resistance vessels. In mouse conductance and resistance arteries Mg2+ -induced relaxation is contributed by endothelial NO formation, EDHF pathways, antagonism of Ca2+ in smooth muscle cells, and additional unidentified mechanisms.

Asymmetric Dimethylarginine Enables Depolarizing Spikes and Vasospasm in Mesenteric and Coronary Resistance Arteries.

Increased vasoreactivity due to reduced endothelial NO bioavailability is an underlying feature of cardiovascular disease, including hypertension. In small resistance arteries, declining NO enhances vascular smooth muscle (VSM) reactivity partly by enabling rapid depolarizing Ca2+-based spikes that underlie vasospasm. The endogenous NO synthase inhibitor asymmetric dimethylarginine (ADMA) is metabolized by DDAH1 (dimethylarginine dimethylaminohydrolase 1) and elevated in cardiovascular disease. We hypothesized ADMA might enable VSM spikes and vasospasm by reducing NO bioavailability, which is opposed by DDAH1 activity and L-arginine. Rat isolated small mesenteric arteries and myogenic rat-isolated intraseptal coronary arteries (RCA) were studied using myography, VSM intracellular recording, Ca2+ imaging, and DDAH1 immunolabeling. Exogenous ADMA was used to inhibit NO synthase and a selective DDAH1 inhibitor, NG-(2-methoxyethyl) arginine, to assess the functional impact of ADMA metabolism. ADMA enhanced rat-isolated small mesenteric arteries vasoreactivity to the α1-adrenoceptor agonist, phenylephrine by enabling T-type voltage-gated calcium channel-dependent depolarizing spikes. However, some endothelium-dependent NO-vasorelaxation remained, which was sensitive to DDAH1-inhibition with NG-(2-methoxyethyl) arginine. In myogenically active RCA, ADMA alone stimulated depolarizing Ca2+ spikes and marked vasoconstriction, while NO vasorelaxation was abolished. DDAH1 expression was greater in rat-isolated small mesenteric arteries endothelium compared with RCA, but low in VSM of both arteries. L-arginine prevented depolarizing spikes and protected NO-vasorelaxation in rat-isolated small mesenteric artery and RCA. ADMA increases VSM electrical excitability enhancing vasoreactivity. Endothelial DDAH1 reduces this effect, and low levels of DDAH1 in RCAs may render them susceptible to endothelial dysfunction contributing to vasospasm, changes opposed by L-arginine.

The Melatonin Derivative ITH13001 Prevents Hypertension and Cardiovascular Alterations in Angiotensin II-Infused Mice.

Inflammatory mechanisms and oxidative stress seem to contribute to the pathogenesis of hypertension. ITH13001 is a melatonin-phenyl-acrylate hybrid that moderately induces the antioxidant transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2) and has a potent oxidant scavenging effect compared with other derivatives of its family. Here we investigated the effect of ITH13001 on hypertension and the associated cardiovascular alterations. Angiotensin II (AngII)-infused mice were treated with ITH13001 (1 mg/kg per day, i.p.) for 2 weeks. The ITH13001 treatment prevented: 1) the development of hypertension, cardiac hypertrophy, and increased collagen and B-type natriuretic peptide (Bnp) expression in the heart; 2) the reduction of elasticity, incremental distensibility, fenestrae area, intraluminal diameter, and endothelial cell number in mesenteric resistance arteries (MRA); 3) the endothelial dysfunction in aorta and MRA; 4) the plasma and cardiovascular oxidative stress and the reduced aortic nitric oxide (NO) bioavailability; 5) the increased cardiac levels of the cytokines interleukin (IL)-1β, IL-6, and C-C motif chemokine ligand 2 (Ccl2), the T cell marker cluster of differentiation 3 (Cd3), the inflammasome NLRP3 (NOD-, LRR-, and pyrin domain-containing protein 3), the proinflammatory enzymes inducible nitric oxide synthase (iNOS) and COX-2, the toll-like receptor 4 (TLR4) adapter protein myeloid differentiation primary response 88 (MyD88), and the nuclear factor kappa B (NF-κB) subunit p65; 6) the greater aortic expression of the cytokines tumor necrosis factor alpha (Tnf-α), Ccl2 and IL-6, Cd3, iNOS, MyD88, and NLRP3. Although ITH13001 increased nuclear Nrf2 levels and heme oxygenase 1 (HO-1) expression in vascular smooth muscle cells, both cardiac and vascular Nrf2, Ho-1, and NADPH quinone dehydrogenase 1 (Nqo1) levels remained unmodified irrespective of AngII infusion. Summarizing, ITH13001 improved hypertension-associated cardiovascular alterations independently of Nrf2 pathway activation, likely due to its direct antioxidant and anti-inflammatory properties. Therefore, ITH13001 could be a useful therapeutic strategy in patients with resistant hypertension. SIGNIFICANCE STATEMENT: Despite the existing therapeutic arsenal, only half of the patients treated for hypertension have adequately controlled blood pressure; therefore, the search for new compounds to control this pathology and the associated damage to end-target organs (cerebral, cardiac, vascular, renal) is of particular interest. The present study demonstrates that a new melatonin derivative, ITH13001, prevents hypertension development and the associated cardiovascular alterations due to its antioxidant and anti-inflammatory properties, making this compound a potential candidate for treatment of resistant hypertensive patients.

Normotensive placental extracellular vesicles provide long-term protection against hypertension and cardiovascular disease

BackgroundWomen with normotensive pregnancy are at a reduced risk of developing cardiovascular disease postpartum compared to those who experience hypertensive conditions during pregnancy. However, the underlying mechanisms remain poorly understood. During normotensive pregnancy, vast numbers of placental extracellular vesicles are released into the maternal circulation, which protect endothelial cells from activation and alter maternal vascular tone. We hypothesized that placental extracellular vesicles play a mechanistic role in lowering risk of cardiovascular disease following normotensive pregnancy. ObjectiveThis study aimed to investigate the long-term effects of placental extracellular vesicles derived from normotensive term placentae on the cardiovascular system and explore the mechanisms underlying their biological effects. Study DesignSpontaneously hypertensive rats were injected with placental EVs from normotensive term pregnancies (2 mg /kg each time, n=8) or vehicle control (n=9) at 3 months of age. Blood pressure and cardiac function were regularly monitored from 3 months to 15 months of age. The response of mesenteric resistance arteries to vasoactive substances was investigated to evaluate vascular function. Cardiac remodelling, small artery remodelling, and renal function were investigated to comprehensively assess the impact of placental extracellular vesicles on cardiovascular and renal health. ResultsCompared to vehicle-treated control animals, rats treated with normotensive placental extracellular vesicles exhibited a significantly lower increase in blood pressure and improved cardiac function. Furthermore, the vasodilator response to the endothelium-dependent agonist acetylcholine was significantly enhanced in the normotensive placental extracellular vesicle-treated spontaneously hypertensive rats compared to the control. Moreover, treatment with placental extracellular vesicles reduced wall thickening of small renal vessels and attenuated renal fibrosis. ConclusionPlacental extracellular vesicles from normotensive term pregnancies have long-lasting protective effects reducing hypertension and mitigating cardiovascular damage in vivo.

Endothelin Receptor Blockade Improves Cerebral Blood Flow-Mediated Dilation in a Mouse Model of Alzheimer’s Disease

Introduction: Cerebral blood flow (CBF) is reduced in patients with Alzheimer’s disease (AD). Flow-mediated dilation (FMD), which plays a key role in the regulation of blood flow, is attenuated by endothelin-1. We hypothesized that endothelin receptor blockade may improve CBF in AD. Methods: We investigated cerebrovascular reactivity in a mouse model of AD (APP-PS1; 5-6-month-old male subjects). We assessed the in vivo response to normoxic hypercapnia and in vitro FMD in isolated cerebral and mesenteric resistance arteries before and after endothelin receptor blockade (bosentan). Results: Normoxic hypercapnia increased basilar trunk blood flow velocity (+12.3 ± 2.4%; p = 0.006, n = 6) in wild-type (WT) mice but reduced blood flow in APP-PS1 mice (−11.4 ± 1.2%; p < 0.0001, n = 8). Bosentan (50 mg/kg, acute intraperitoneal injection) restored cerebrovascular reactivity in APP-PS1 mice (+10.2 ± 2.2%; p < 0.0001, n = 8) but had no effect in WT. FMD was reduced in the posterior cerebral artery of APP-PS1 compared to WT and was normalized by bosentan (1 μmol/L, 30 min, or 50 mg/kg/day for 28 days). FMD was similar in the mesenteric artery of APPS-PS1 and WT. Conclusion: APP-PS1 mice exhibited cerebrovascular endothelial dysfunction. Acute and chronic blockade of endothelin receptors restored endothelial vasomotor function, suggesting a promising therapeutic approach to restoring cerebral vasoreactivity in AD.

Abstract 12477: ER Stress Chop Depletion in Endothelial Cells Protects From Renovascular Hypertension Pathogenesis

Introduction: Renovascular hypertension poses a significant risk for cardiovascular diseases, with endoplasmic reticulum (ER) stress playing a critical role in their development. However, the specific impact of ER stress-induced C/EBP homologous protein (CHOP) expression in endothelial cells on renovascular hypertension-induced cardiovascular complications remains unexplored. This study investigates the effects of disrupting ER stress CHOP in endothelial cells on microvascular dysfunction associated with renovascular hypertension. Hypothesis: We hypothesize that deleting ER stress CHOP in endothelial cells (EC) can mitigate renovascular hypertension-induced vascular endothelial dysfunction. Methods: Eight-week-old male and female mice (CHOP flx/flx and EC CHOP-/- ) were divided into eight groups: control groups undergoing a sham operation for 4 weeks and renovascular hypertension groups subjected to 2-kidney-1-clip (2K1C) surgery for 4 weeks. Body weight, blood pressure, running performance, cardiac hypertrophy and fibrosis, lung edema, inflammation, vascular endothelial function, and signaling were assessed. Results: Male and female CHOP flx/flx mice subjected to 2K1C for four weeks exhibited hypertension, cardiac hypertrophy and fibrosis, lung edema, impaired running performance, and endothelium-dependent vascular relaxation dysfunction. In contrast, male and female EC CHOP-/- mice subjected to 2K1C for four weeks were protected against the pathogenesis of renovascular hypertension. Furthermore, mesenteric resistance arteries from CHOP flx/flx mice displayed reduced endothelial nitric oxide synthase (eNOS) phosphorylation, while EC HOP-/- mice showed no such effect. Conclusions: These findings emphasize the significance of targeting ER stress CHOP in endothelial cells of male and female mice to protect against the development and pathogenesis of renovascular hypertension.

Myoendothelial feedback in mouse mesenteric resistance arteries is similar between the sexes, dependent on nitric oxide synthase, and independent of TPRV4.

Myoendothelial feedback (MEF), the endothelium-dependent vasodilation following sympathetic vasoconstriction (mediated by smooth muscle to endothelium gap junction communication), has been well studied in resistance arteries of males, but not females. We hypothesized that MEF responses would be similar between the sexes, but different in the relative contribution of the underlying nitric oxide and hyperpolarization mechanisms, given that these mechanisms differ between the sexes in agonist-induced endothelium-dependent dilation. We measured MEF responses (diameter changes) of male and female first- to second-order mouse mesenteric arteries to phenylephrine (10 µM) over 30 min using isolated pressure myography ± blinded inhibition of nitric oxide synthase (NOS) using Nω-nitro-l-arginine methyl ester (l-NAME; 0.1-1.0 mM), hyperpolarization using 35 mM KCl, or transient receptor potential vanilloid 4 (TRPV4) channels using GSK219 (0.1-1.0 µM) or RN-1734 (30 µM). MEF was similar [%dilation (means ± SE): males = 26.7 ± 2.0 and females = 26.1 ± 1.9 at 15 min] and significantly inhibited by l-NAME (1.0 mM) at 15 min [%dilation (means ± SE): males = 8.2 ± 3.3, P < 0.01; females = 6.8 ± 1.9, P < 0.001] and over time (P < 0.01) in both sexes. l-NAME (0.1 mM) + 35 mM KCl nearly eliminated MEF in both sexes (P < 0.001-0.0001). Activation of TRPV4 with GSK101 (0.1-10 µM) induced similar dilation between the sexes. Inhibition of TRPV4, which is reportedly involved in the hyperpolarization mechanism, did not inhibit MEF in either sex. Similar expression of eNOS was found between the sexes with Western blot. Thus, MEF is prominent and similar in murine first- and second-order mesenteric resistance arteries of both sexes, and reliant primarily on NOS and secondarily on hyperpolarization, but not TRPV4.NEW & NOTEWORTHY We found that female mesenteric resistance arteries have similar postconstriction dilatory responses (i.e., myoendothelial feedback) to a sympathetic neurotransmitter analog as male arteries. Both sexes use nitric oxide synthase (NOS) and hyperpolarization, but not TRPV4, in this response. Moreover, the key protein involved in this pathway (eNOS) is similarly expressed in these arteries between the sexes. These similarities are surprising given that agonist-induced endothelium-dependent dilatory mechanisms differ in these arteries between the sexes.

NTPDase1/CD39 Ectonucleotidase Is Necessary for Normal Arterial Diameter Adaptation to Flow.

NTPDase1/CD39, the major vascular ectonucleotidase, exerts thrombo-immunoregulatory function by controlling endothelial P2 receptor activation. Despite the well-described release of ATP from endothelial cells, few data are available regarding the potential role of CD39 as a regulator of arterial diameter. We thus investigated the contribution of CD39 in short-term diameter adaptation and long-term arterial remodeling in response to flow using Entpd1-/- male mice. Compared to wild-type littermates, endothelial-dependent relaxation was modified in Entpd1-/- mice. Specifically, the vasorelaxation in response to ATP was potentiated in both conductance (aorta) and small resistance (mesenteric and coronary) arteries. By contrast, the relaxing responses to acetylcholine were supra-normalized in thoracic aortas while decreased in resistance arteries from Entpd1-/- mice. Acute flow-mediated dilation, measured via pressure myography, was dramatically diminished and outward remodeling induced by in vivo chronic increased shear stress was altered in the mesenteric resistance arteries isolated from Entpd1-/- mice compared to wild-types. Finally, changes in vascular reactivity in Entpd1-/- mice were also evidenced by a decrease in the coronary output measured in isolated perfused hearts compared to the wild-type mice. Our results highlight a key regulatory role for purinergic signaling and CD39 in endothelium-dependent short- and long-term arterial diameter adaptation to increased flow.

Impaired vascular β-adrenergic relaxation in spontaneously hypertensive rats: The differences between conduit and resistance arteries

It was suggested that impaired β-adrenergic relaxation in spontaneously hypertensive rats (SHR) might contribute to their high blood pressure (BP). Our study was focused on isoprenaline-induced dilatation of conduit femoral or resistance mesenteric arteries and on isoprenaline-induced BP reduction in SHR and Wistar-Kyoto rats (WKY). We confirmed decreased β-adrenergic relaxation of SHR femoral arteries due to the absence of its endothelium-independent component, whereas endothelium-dependent component of β-adrenergic smooth muscle relaxation was similar in both strains. Conversely, isoprenaline-induced relaxation of resistance mesenteric arteries was similar in both strains and this was true for endothelium-dependent and endothelium-independent components. We observed moderately reduced sensitivity of SHR mesenteric arteries to salmeterol (β2-adrenergic agonist) and this strain difference disappeared after endothelium removal. However, there was no difference in mesenteric arteries relaxation by dobutamine (β1-adrenergic agonist) which was independent of endothelium. The increasing isoprenaline doses elicited similar BP decrease in both rat strains, although BP sensitivity to isoprenaline was slightly decreased in SHR. The blockade of cyclooxygenase (indomethacin) and NO synthase (L-NAME) further reduced BP sensitivity to isoprenaline in SHR. On the other hand, salmeterol elicited similar BP decrease in both strains and the blockade of cyclooxygenase and NO synthase increased BP sensitivity to salmeterol in SHR as compared to WKY. In conclusion, attenuated β-adrenergic vasodilatation of conduit arteries of SHR but similar β-adrenergic relaxation of resistance mesenteric arteries from WKY and SHR and their similar BP response to β-adrenergic agonists do not support major role of altered β-adrenergic vasodilatation for high BP in genetic hypertension.

Ezetimibe Induces Vasodilation in Rat Mesenteric Resistance Arteries through Inhibition of Extracellular Ca2+ Influx.

Ezetimibe is a lipid-lowering agent that selectively inhibits cholesterol absorption by binding to the Niemann-Pick C1-like 1 (NPC1L1) protein. Although it is well known that administration of ezetimibe in hypercholesterolemia patients reduces the risk of cardiovascular events through attenuation of atherosclerosis, studies on the direct effect of ezetimibe on vascular function are not sufficient. The aim of the present study was to investigate the vascular effects of ezetimibe in rat mesenteric arteries. In the present study, 12-week-old male Sprague Dawley rats were used. After the rats were sacrificed, the second branches of the mesenteric arteries were isolated and cut into 2-3 mm segments and mounted in a multi-wire myography system to measure isometric tension. Ezetimibe reduced vasoconstriction induced by U46619 (500 nM) in endothelium-intact and endothelium-denuded arteries. Ezetimibe-induced vasodilation was not affected by the endothelial nitric oxide synthase (eNOS) inhibitor Nω-Nitro-L-arginine (L-NNA, 300 μM) or the non-selective potassium channel blocker, tetraethylammonium (TEA, 10 mM). Moreover, ezetimibe also completely blocked the contraction induced by an increase in external calcium concentration. Ezetimibe significantly reduced vascular contraction induced by L-type Ca2+ channel activator (Bay K 8644, 30 nM). Treatment with ezetimibe decreased the phosphorylation level of 20 kDa myosin light chain (MLC20) in vascular smooth muscle cells. In the present study, we found that ezetimibe has a significant vasodilatory effect in rat mesenteric resistance arteries. These results suggest that ezetimibe may have beneficial cardiovascular effects beyond its cholesterol-lowering properties.