The isoflavone metabolites, O-desmethylangolensin and (S)-equol, relax isolated arteries ex vivo and decrease arterial blood pressure in vivo.

Aldosterone (Aldo), a mineralocorticoid hormone, modulates cardiovascular function by regulating the expression of intracellular Ca2+ handling proteins, among other effects. In rat resistance-sized mesenteric arteries (MA), Aldo treatment (10 nM, 24 h) upregulates both the L-type voltage-gated Ca2+ channel α1C subunit (CaV1.2) and the Sarco/Endoplasmic Reticulum Ca2+ ATPase (SERCA pump), thereby increasing Sarcoplasmic Reticulum (SR) Ca2+ load. Two SERCA isoforms, SERCA2a and SERCA2b, are expressed in rat MA, but their specific physiological contributions to distinct intracellular Ca2+ signals, remain unclear. In this study, we characterized the relative abundance and subcellular distribution of SERCA2a and SERCA2b in rat MA, their regulation by Aldo, and the impact of Aldo-induced SERCA remodeling on local Ca2+ signals relevant to vascular function, such as Ca2+ sparks and Ca2+ waves. Aldo-treated MA smooth muscle cells (MASMC) exhibited increased Ca2+ spark frequency and a higher incidence of spontaneous Ca2+ waves. Aldo augmented both protein and mRNA levels of SERCA2a and SERCA2b, effects that were blocked by the mineralocorticoid receptor (MR) antagonist RU28318. Under control conditions, SERCA2a was predominantly localized in the perinuclear region, whereas SERCA2b was distributed across both subplasmalemmal and perinuclear regions. Aldo treatment increased the expression of both isoforms in all analyzed subcellular compartments (subplasmalemmal, cytoplasmic, and perinuclear), with a pronounced redistribution towards the subplasmalemmal region of MASMC. This shift in SERCA subcellular distribution likely contributes to enhanced superficial Ca2+ buffering and the ignition of Ca2+ sparks and Ca2+ waves. Furthermore, Aldo increased mRNA levels of mitochondrial transcription factors A and B2 (TFAM and TFB2M), previously implicated in SERCA regulation in human aorta, suggesting a transcriptional mechanism whereby MR activation of the SERCA2 gene is associated with increased TFAM and TFB2M expression. Collectively, these findings demonstrate for the first time that Aldo increases the expression and promotes the subplasmalemmal localization of SERCA2a and SERCA2b in MASMC. This remodeling underscores their critical role in maintaining the superficial Ca2+ buffering system and SR Ca2+ load to prevent pathological elevations in the intracellular Ca2+ concentration. Our results highlight the SERCA pump as a potential therapeutic target in hypertension associated with hyperaldosteronism.

The inhibitory effect of exogenous nitric oxide (NO) on the open state of ATP-sensitive potassium channels (K ATP) and its underlying mechanism remain unclear. In this study, patch-clamp and molecular biology techniques are used to investigate this issue. In acutely isolated rat mesenteric artery smooth muscle cells and human embryonic kidney 293 cells (HEK293) expressing inwardly rectifying potassium channel 6.1 subunit/sulfonylurea receptor 2B subunit (Kir6.1/SUR2B), sodium nitroprusside (SNP) was found to significantly inhibit the activity of open K ATP channels. Detection using biotin-labeled glutathione ethyl ester (BioGEE) combined with Western blotting showed that Kir6.1 subunit glutathionylation level was significantly decreased after SNP treatment. These results indicate that exogenous NO directly inhibits the activity of open K ATP channels by nitrosylating key cysteine residues of the Kir6.1 subunit and competitively inhibiting glutathionylation at this site. This study provides new experimental evidence for the molecular mechanism of NO in vascular regulation.

β-Asarone-induced vasorelaxation in isolated rat mesenteric artery: An efficacy vs toxicity paradox of Acorus calamus.

Detection of active vasodilatory compounds from hawthorn: Screening and determination of mechanism of action.

Perivascular adipose tissue (PVAT), found outside blood vessels, enhances vasorelaxation when endothelium-mediated vasorelaxation fails, as observed in the mesenteric arteries of rats with metabolic syndrome (MetS) and hypertension; however, whether high blood pressure contributes to PVAT dysfunction remains unclear. We, therefore, aimed to investigate vascular function and PVAT modulation in the mesenteric arteries of rats with MetS but no hypertension. Mesenteric arteries were isolated from both lean and obese male Zucker fatty (ZF) and ZF diabetes mellitus (ZFDM) rats at 20 and 30 weeks of age. Vasorelaxation was examined in arteries with or without PVAT; mRNA levels in PVAT and arteries were examined using the organ bath method and quantitative reverse transcription polymerase chain reaction, respectively. Sodium nitroprusside-induced relaxations were lower in the obese versus lean ZF rats, and PVAT increased them to levels comparable to those in lean rats at 20 weeks. These enhancements disappeared after 30 weeks. The mRNA expression of apelin in PVAT and its receptor was upregulated in the arteries of obese rats, and its levels correlated positively with the enhancing effect of PVAT. In obese ZFDM rats, PVAT attenuated relaxation at both 20 and 30 weeks. Levels of chemerin mRNA expression in PVAT were higher in ZFDM versus ZF rats; however, treatment with chemerin or a chemerin receptor antagonist did not alter vasorelaxation in ZFDM rats. Apelin may contribute to compensatory PVAT modulation during vascular dysfunction in obese ZF rats. PVAT inhibits vasorelaxation by releasing a contractile factor other than chemerin in ZFDM rats. Thus, it may modulate vascular tone differently depending on MetS stage.

Abstract Arsenic trioxide (ATO) therapy is limited by its severe vascular toxicity, which manifests as endothelial injury through mechanisms that remain poorly defined. This study identifies the SUMOylation of the Transient Receptor Potential Melastatin 4 (TRPM4) channel as a pivotal pathological driver of this process. We demonstrate that the SUMOylation inhibitor ginkgolic acid (GA) confers protection by attenuating ATO‐induced TRPM4 overactivation, thereby rescuing endothelial cells from ionic dysregulation (Ca 2 ⁺, Na⁺), oxidative stress, membrane depolarization, and apoptosis. Critically, we identified lysine 618 (K618) as the key SUMOylation site on TRPM4. Expression of a SUMOylation‐deficient TRPM4 mutant (K618R) phenocopied the protective effects of GA, significantly mitigating ATO‐induced endothelial dysfunction. This confirms that the detrimental effects of TRPM4 are strictly dependent on its SUMOylation at this residue. The protective efficacy of GA was further validated ex vivo, where it restored endothelium‐dependent vasodilation in rat mesenteric arteries impaired by ATO. Complementing these findings, Raman microimaging provided unique, label‐free insights into the biochemical alterations induced by ATO and their reversal by GA at the single‐cell level. Our findings unveil TRPM4‐SUMOylation at K618 as a fundamental mechanism underlying ATO‐induced endothelial injury. These results position the inhibition of this specific post‐translational modification as a promising therapeutic strategy for managing the vascular toxicity associated with ATO chemotherapy.

Introduction: Increasing evidence shows that noble gas postconditioning by inhaled Xenon or Argon (Ar) after return of spontaneous circulation (ROSC) improves survival with favorable neurological function. When given during cardiopulmonary resuscitation (CPR), though, i.e. before ROSC, Ar increases cardiac output during chest compressions in a pig and a rat model of cardiac arrest. We believe it does so by decreasing pulmonary resistance as shown in isolated lungs and by decreasing systemic vascular resistance, subject of the present IACUC-approved study on isolated arteries. Methods: In a first set of experiments, we used pressurized (20 mmHg, no flow) 4 th generation mesenteric arteries isolated freshly from euthanized adult Sprague Dawley rats to measure diameter changes by confocal microscopy. Vessels were super-fused by oxygenated Krebs buffer balanced with 87.5% Ar; 7.5% O 2 ; 5% CO 2 or 87.5% N 2 ; 7.5% O 2 ; 5% CO 2 in a micro-vessel perfusion chamber. To test whether Ar promotes vasodilation through endothelial nitric oxide (NO), we then repeated these experiments in the presence of either the NO synthase inhibitor L-NG-Nitroarginine methyl ester (LN, 0.5 mM) or sodium deoxycholate (SDC, 0.2 mM), a detergent to inactivate endothelial cells and eliminate endothelium-dependent relaxation. All vessels were pre-constricted with 1 µM norepinephrine throughout the experiment and vasoconstricted by 25 mM KCl or vasodilated by 1 µM of the endothelium-independent vasodilator sodium nitroprusside (SNP) at the end. Diameters were normalized to baseline %, data are mean ± SEM. Statistics: ANOVA for repeated measures, p<.05 * vs control. Results: Argon increased the diameter by +6.7±1.4%* (n=15) from baseline; KCl decreased it by -5.5±2.3%* (Fig 1). LN (n=6) vs SDC (n=7) alone had no significant effects on diameter: -1.9±1.4% vs -0.4±0.8%, respectively, but abolished dilation by Argon completely: -3.1±1.7% vs -0.6±0.9%, respectively. All vessels still significantly dilated with SNP afterwards: +4.7±1.1%* vs +6.2±1.2%*, respectively. Conclusion: Argon’s mild but significant vasodilatory effect on peripheral vasculature appears mediated by endothelial NO release. Along with increased cardiac output by improved right-to-left heart flow due to pulmonary vasodilation, it may aid to improved peripheral perfusion during and after cardiac arrest. Future studies will address underlying signaling pathways and clinical applicability in more detail.

O304 is a mitochondrial uncoupler which extends C. elegans lifespan and induces vasorelaxation of rat mesenteric arteries.

Pharmacological characterization of vampire bat-derived CGRP at the human CGRP receptor in the Xenopus oocyte system.

Ertugliflozin induces vasodilation by inhibiting AMPK-mediated mitochondrial fission in vascular smooth muscle cells.

Previous studies have reported the involvement of argininosuccinate synthase (AsS) as a putative target for the cardiovascular effects of proline-rich oligopeptides. The present study investigated the mechanisms underlying the vasorelaxant effect of BPP-BrachyNH2 in rat small mesenteric arteries and applied in silico molecular dynamics studies to explore the interaction between AsS and BPP-BrachyNH2. Segments of male rat mesenteric arteries mounted in microvascular myographs were treated with 10μg/mL lipopolysaccharides (LPS), and then, BPP-BrachyNH2 was cumulatively added (10-9 - 3×10-5M) to noradrenalin-contracted (1-10μM) preparations. Molecular Dynamics calculations were performed between AsS enzyme (RCSB Protein Data Bank ID: 2NZ2) as the target and both BPP-BrachyNH2 and L-citrulline (PubChem CID 9750 code) as ligands. Immunohistochemistry showed expression of AsS and endothelial nitric oxide (NO) synthase (eNOS) in mesenteric arteries and of inducible NO synthase (iNOS) in segments exposed to LPS. The vasorelaxant effect of BPP-BrachyNH2 was abolished in the presence of 100μM L-NNA (L-NG-nitroarginine) and 3μM ODQ ([1H-[1,2,4]-oxadiazolo-[4,3-a] quinoxalin-1-one]), and attenuated in the presence of 10μM 1400W (N-(3-(aminomethyl)-benzyl)-acetamidine) and 1mM MDLA (α-methyl-D, L-aspartic acid), inhibitors of iNOS and AsS, respectively. The AsS_BPP-BrachyNH2 complex showed increased binding energy, inhibition constant, and the number of interactions with amino acids when compared with the AsS_L-citrulline complex. These results suggest that the positive interaction of BPP-BrachyNH2 with AsS leads to L-citrulline recycling and increases L-arginine bioavailability, thereby improving the mechanism for the vasorelaxant effect. Our findings open new perspectives for potential therapeutic applications of proline-rich oligopeptides in vascular dysfunction.

ObjectiveExposure to gestational diabetes mellitus appears to produce several effects on offspring, including increased rates of early-onset cardiovascular disease from childhood to early adulthood. In this study, we investigated the protection of human omentin-1 against endothelial dysfunction resulting from exposure to maternal diabetes in adult rat offspring.MethodsTwelve adult control mother offspring (CMO) were injected with rh-omentin or saline, and 12 adult diabetic mother offspring (DMO) were injected with rh-omentin or saline. The mesenteric artery rings of rh-omentin-injected DMO were incubated with Compound C (an AMPK inhibitor). The vascular reactivity of rat mesenteric artery rings was evaluated by treating with PE (10-9–10-5 M) and Ach (10-9–10-5 M). The mesenteric arterial endothelial cells (AECs) isolated from different groups were incubated with A769662 (an AMPK agonist) and/or transfected with siRNA against KLF2 (si-KLF2) to confirm the AMPK/KLF2 pathway involved in the protection of omentin-1 against endothelial dysfunction.ResultsInjection of rh-omentin alleviated PE-induced vasoconstriction and improved Ach-induced vasorelaxation in the mesenteric artery rings, inhibited phosphorylations of endoplasmic reticulum (ER) stress markers, prevented loss of phosphorylations of AMPK and endothelial nitric-oxide synthase (eNOS), increased nitric oxide production, reduced the level of reactive oxygen species, and promoted KLF2 expression in DMO. The AMPK inhibitor and KLF2 knockdown both eliminated these effects of omentin-1 on adult rat offspring exposed to maternal diabetes. KLF2 knockdown also weakened the effects of the AMPK agonist on adult rat offspring exposed to maternal diabetes.ConclusionThese findings point out that mentin-1 could protect adult rat offspring against endothelial dysfunction, including endothelium impairment, ER stress, and oxidative stress resulting from exposure to maternal diabetes through the AMPK/KLF2/eNOS pathway.

Ponatinib, a tyrosine kinase inhibitor (TKI) leads to hypertension; however, the mechanisms remain elusive. We aimed to investigate whether lysine acetyltransferase 7 (KAT7), a key regulator of cellular senescence that is closely associated with cardiovascular diseases, involves in ponatinib-induced hypertension. After administering ponatinib to Sprague-Dawley (SD) rats for 8 days, we measured blood pressure, vasodilation, and endothelial function using tail-cuff plethysmography, isometric myography, and the Total NO Assay kit, respectively. The results indicated that ponatinib increased blood pressure, impaired endothelium-dependent relaxation (EDR), and caused injury to endothelial cells in SD rats. Furthermore, PCR and Western blot experiments demonstrated an upregulation of KAT7 expression in rat mesenteric artery endothelial cells (MAECs) following ponatinib treatment. To further study the role of KAT7 in ponatinib-induced hypertension, we divided the SD rats into four groups: control, ponatinib, WM-3835 (a KAT7 inhibitor), and ponatinib plus WM-3835. Notably, WM-3835 administration significantly improved ponatinib-induced hypertension and EDR dysfunction in SD rats. Mechanistically, over-expression of KAT7 (OE-KAT7) in MAECs led to cellular senescence and inflammation, phenomena that were also observed in the mesenteric arteries of ponatinib-treated rats and in MAECs exposed to ponatinib. However, WM-3835 mitigated these detrimental effects in both in vivo and in vitro experiments. Additionally, both OE-KAT7 and ponatinib treatment induced H3K14 acetylation (H3K14ac), with OE-KAT7 also elevating the recruitment of the H3K14ac to the p21 promoter. Moreover, BAY 11-7085, a nuclear factor (NF)-κB inhibitor, potently alleviated the accumulation of IL-6 and IL-8, as well as endothelial cell senescence induced by ponatinib and KAT7 overexpression. Our data indicate that ponatinib-induced elevation of KAT7 led to endothelial cells senescence and inflammatory responses through H3K14 acetylation and NF-κB signaling pathway, subsequently caused vasotoxicity and hypertension.

Vasorelaxant effect of fennel seeds (Foeniculum vulgare Mill) extracts on rat mesenteric arteries: Assessment of phytochemical profiling and antioxidant potential.

Protein kinase C (PKC) reportedly plays a role in the pathogenesis of many vascular dysfunction-related conditions. In this study, we investigated whether PKCβ is associated with vascular contractile changes induced by angiotensin II (Ang II) exposure. Long-term (24 h) treatment of rat aortae and mesenteric arteries in Ang II-containing culture medium enhanced 5-hydroxytryptamine (5-HT)-induced vascular contraction in a dose-dependent manner, in association with enhanced phosphorylation of PKCβ S660. Increased contraction induced by Ang II treatment was also observed in endothelium-denuded aorta. Enhanced contraction induced by Ang II was markedly diminished by the knockout of the PKCβ gene or treatment with LY333531 and CGP53353 (PKCβ inhibitors). Cycloheximide (protein synthesis inhibitor) blocked the Ang II-induced enhanced contraction. Gene expression profiling and real-time PCR analyses showed marked upregulation of endothelin-1 (ET-1) expression in Ang II-treated aorta but was not observed in PKCβ-knockout aorta. Ang II increased the secretion of ET-1 peptide in endothelium-intact and -denuded aortae. Ang II-induced enhancement of vascular contraction was diminished by BQ-123 (ETAR blocker). In vivo treatment with Ang II (250 ng/kg/min) for 7 days increased phosphorylation of PKCβ S660 in rat vascular tissue and increased the in vitro contractile responses to 5-HT and in vivo systolic blood pressure, but these changes were largely absent in PKCβ-knockout experiments. These data suggest that long-term exposure to Ang II increases vascular smooth muscle contraction and blood pressure elevation, mediated by activation of PKCβ and subsequent biosynthesis of ET-1 in vascular smooth muscle cells.NEW & NOTEWORTHY We studied the role of PKCβ in Ang II-induced hypertension using a rat model. Our study showed that PKCβ plays a key role in Ang II-induced vascular hypercontractility. Our results also suggest that transcriptional activation-mediated ET-1 expression in vascular smooth muscle is responsible for this vascular change as a downstream pathway of PKCβ activation, which leads to blood pressure elevation. This Ang II-PKCβ-ET-1 mechanism may affect vascular homeostasis in hypertension.

Nitazoxanide protects against heart failure with preserved ejection and metabolic syndrome induced by high-fat diet (HFD) plus L-NAME "two-hit" in mice.

Aim: Adiponectin is an anti-diabetic and anti-atherogenic protein secreted primarily from adipose tissue. Adiponectin and modified LDL (mLDL) form a complex to modulate their biological activity. To elucidate the significance of the complex formation, we analyzed its effects on vascular tissue and developed and verified novel quantifying methods for adiponectin.Methods: To study the significance of the mLDL-adiponectin complex (MAC) formation, we used the wire-myography method on rat mesenteric artery. We developed a method to measure MAC by using LOX-1 as the capture protein and anti-adiponectin antibody for detection. We compared serum MAC levels between hemodialysis patients and control subjects.Results: Administering mLDL alone to rat mesenteric artery impaired endothelium-dependent vasorelaxation, whereas simultaneously administering adiponectin with mLDL protected rat mesenteric artery from the mLDL-induced impairment of vasorelaxation. This finding indicates MAC formation prevents endothelium from mLDL-induced dysfunction in tissue. Using our novel ELISA for MAC, we found that MAC was increasingly detectable depending on the doses of mLDL and adiponectinin vitro. In serum, hemodialysis patients showed a significantly higher ratio of MAC-high patients (higher than the median level of MAC) than did healthy controls. Furthermore, the MAC-high hemodialysis group had lower mLDL activity measured as LOX-1 ligand containing apoB.Conclusion: Using our ELISA, we detected MAC in human serum that protected blood vessels from the deleterious effects of oxidized LDL.

Modified latex glove model for simulating a range of supermicrosurgical vessels.

Understanding protein-protein interactions is crucial for unravelling subcellular protein distribution, contributing to our understanding of cellular organisation. Moreover, interaction studies can reveal insights into the mechanisms that cover protein trafficking within cells. Although various techniques such as Förster resonance energy transfer (FRET), co-immunoprecipitation, and fluorescence microscopy are commonly employed to detect protein interactions, their limitations have led to more advanced techniques such as the in situ proximity ligation assay (PLA) for spatial co-localisation analysis. The PLA technique, specifically employed in fixed cells and tissues, utilises species-specific secondary PLA probes linked to DNA oligonucleotides. When proteins are within 40 nm of each other, the DNA oligonucleotides on the probes interact, facilitating circular DNA formation through ligation. Rolling-circle amplification then produces DNA circles linked to the PLA probe. Fluorescently labelled oligonucleotides hybridise to the circles, generating detectable signals for precise co-localisation analysis. We employed PLA to examine the co-localisation of dynein with the Kv7.4 channel protein in isolated vascular smooth muscle cells from rat mesenteric arteries. This method enabled us to investigate whether Kv7.4 channels interact with dynein, thereby providing evidence of their retrograde transport by the microtubule network. Our findings illustrate that PLA is a valuable tool for studying potential novel protein interactions with dynein, and the quantifiable approach offers insights into whether these interactions are changed in disease.