Receptors In Vascular Smooth Muscle Research Articles

Mineralocorticoid Receptors in Vascular Smooth Muscle: Blood Pressure and Beyond.

After half a century of evidence suggesting the existence of mineralocorticoid receptors (MR) in the vasculature, the advent of technology to specifically knockout the MR from smooth muscle cells (SMCs) in mice has elucidated contributions of SMC-MR to cardiovascular function and disease, independent of the kidney. This review summarizes the latest understanding of the molecular mechanisms by which SMC-MR contributes to (1) regulation of vasomotor function and blood pressure to contribute to systemic and pulmonary hypertension; (2) vascular remodeling in response to hypertension, vascular injury, obesity, and aging, and the impact on vascular calcification; and (3) cardiovascular pathologies including aortic aneurysm, heart valve dysfunction, and heart failure. Data are reviewed from in vitro studies using SMCs and in vivo findings from SMC-specific MR-knockout mice that implicate target genes and signaling pathways downstream of SMC-MR. By regulating expression of the L-type calcium channel subunit Cav1.2 and angiotensin II type-1 receptor, SMC-MR contributes to myogenic tone and vasoconstriction, thereby contributing to systemic blood pressure. MR activation also promotes SMC proliferation, migration, production and degradation of extracellular matrix, and osteogenic differentiation by regulating target genes including connective tissue growth factor, osteopontin, bone morphogenetic protein 2, galectin-3, and matrix metallopeptidase-2. By these mechanisms, SMC-MR promotes disease progression in models of aging-associated vascular stiffness, vascular calcification, mitral and aortic valve disease, pulmonary hypertension, and heart failure. While rarely tested, when sexes were compared, the mechanisms of SMC-MR-mediated disease were sexually dimorphic. These advances support targeting SMC-MR-mediated mechanisms to prevent and treat diverse cardiovascular disorders.

Comparison effect of high-intensity interval training and moderate intensity continuous training on vascular function in a mouse model of lower extremity peripheral artery disease

Introduction
 Lower extremity peripheral artery disease (PAD), due to narrowing of the arteries supplying the legs, represents a major cause of physical disability. Exercise training is an important part of PAD treatment, improving functional capacity. Recent research suggests that exercise training-induced endothelial function improvement is involved in the benefits of exercise in PAD (Lee et al., 2023); however, the underlying molecular mechanisms remain unknown. In addition, the potential vascular benefits of high intensity training in PAD remain unexplored. Therefore, the aim of the study was to compare the effects of moderate-intensity continuous training (MICT) and high-intensity interval training (HIIT) on endurance capacity, vascular endothelial function and expression of genes related to nitric oxide (NO) bioavailability in a mouse model of PAD.
 Methods
 PAD disease model was generated in male C57BL/6 mice by ligation of the right iliac artery. Mice with PAD were divided into 3 groups: sedentary control group (SED), MICT (40 min running at 70% of maximal aerobic speed) and HIIT (8 intervals of 2.5 minutes running at 90% of maximal aerobic speed combined with 2.5 minutes of running at 50% of maximal aerobic speed). Mice were exercised three times per week for 8 weeks. Maximal running time was determined by an incremental test until exhaustion. Vascular reactivity was assessed by measuring vasoconstrictor responses to phenylephrine as well as endothelium-dependent and -independent vasodilator responses to acetylcholine and DEA-NONOate, respectively, in isolated aortic rings. Gene expression of antioxidant enzymes (catalase and superoxide dismutase isoforms), prooxidant NADPH oxidase subunits (p22phox and p47phox), enzyme involved in NO production (endothelial NO synthase), vascular smooth muscle cell relaxation (soluble guanylate cyclase) and receptors involved in vascular tone regulation (a1 and b2 adrenergic receptors) was determined in aortic tissue by qPCR.
 Results
 Maximal running time was improved in both MICT and HIIT as compared to SED at the end of the study, but with no significant difference between the two exercise groups. Phenylephrine-induced vasoconstriction as well as vasodilator responses to acetylcholine and DEA-NONOate were significantly improved in MICT compared to SED while no significant differences were reported between HIIT and SED. There was no significant difference in gene expression among the three groups.
 Discussion/Conclusion
 MICT and HIIT were equally effective at improving endurance capacity in mice with PAD. However, only MICT improved vascular reactivity.
 References
 Lee, J., Zarezadehmehrizi, A., LaVoy, E., Markofski, M., & Park, Y. (2023). Exercise training improves brachial artery endothelial function, but does not alter inflammatory biomarkers in patients with peripheral artery disease: A systematic review and meta-analysis. Journal of Cardiovascular Translational Research. Advance online publication. https://doi.org/10.1007/s12265-023-10451-0

Recent Advances in Association Between Vitamin D Levels and Cardiovascular Disorders.

In this review, we discuss the evidence that vitamin D affects cardiovascular disease through interventional and observational studies and their corresponding association mechanisms. We also highlight the need for further research to definitively conclude clinical recommendations based on preliminary data and determine the extent to which vitamin D levels may impact the incidence and prognosis of major cardiovascular diseases in the future. Cardiovascular disease has long been recognized as the leading cause of morbidity and mortality worldwide, with many risk factors implicated in its pathogenesis. Vitamin D is a risk factor that, despite being known to be crucial for its role in maintaining bone health, also has several extra-skeletal effects due to vitamin D receptors in vascular smooth muscle and cardiomyocytes. Recent studies have documented a significant association between higher vitamin D levels and lower risk of each cardiovascular disease entity; 11 studies between serum vitamin D and heart failure, 7 studies between serum vitamin D and hypertension, 8 studies between serum vitamin D and coronary artery disease, and 5 studies between serum vitamin D and atrial fibrillation. More studies documenting a significant association between increased serum vitamin D and cardiovascular disease are in the context of heart failure compared to hypertension, coronary artery disease, and atrial fibrillation. Conversely, a significant association between increased serum vitamin D and a lower risk of atrial fibrillation is reported in fewer studies compared to the association of vitamin D with other cardiovascular disease entities. Although there is evidence documenting a clear significant association of vitamin D under each category, further research is still needed to definitively conclude the role of vitamin D in cardiovascular disease management.

Soluble CD93 lectin-like domain sequesters HMGB1 to ameliorate inflammatory diseases.

Rationale: CD93, a C-type lectin-like transmembrane glycoprotein, can be shed in a soluble form (sCD93) upon inflammatory stimuli. sCD93 effectively enhances apoptotic cell clearance and has been proposed as an inflammatory disease biomarker. The function of sCD93 involved directly in inflammation remains to be determined. Herein, we attempted to examine the hypothesis that sCD93 might sequester proinflammatory high-mobility group box 1 protein (HMGB1), exerting anti-inflammatory properties. Methods: Different forms of soluble recombinant human CD93 (rCD93) were prepared by a mammalian protein expression system. rCD93-HMGB1 interaction was assessed using co-immunoprecipitation and solid-phase binding assays. Effects of soluble rCD93 were evaluated in HMGB1-induced macrophage and vascular smooth muscle cells (VSMC) activation and receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis, CaCl2-induced and angiotensin II-infused abdominal aortic aneurysm (AAA) formation and ovariectomized-induced osteoporosis in mice. Results: Protein binding studies revealed that soluble rCD93, via the lectin-like domain (D1), can bind to HMGB1 and intercept HMGB1-receptor interaction. Soluble rCD93 containing D1 inhibited HMGB1-induced proinflammatory cytokine production and intracellular mitogen-activated protein kinase (MAPK)/nuclear factor (NF)-κB activation in macrophages and VSMCs, thereby attenuating CaCl2-induced and angiotensin II-infused AAA models. During osteoclastogenesis, RANKL stimulated HMGB1 secretion that promoted RANKL-induced osteoclastogenesis in return. Soluble rCD93 containing D1 impeded RANKL-induced osteoclastogenic marker gene expression and intracellular MAPK/NF-κB signaling, thereby mitigating ovariectomized-induced osteoporosis. Conclusion: These findings demonstrate the therapeutic potential of soluble recombinant CD93 containing D1 in inflammatory diseases. Our study highlights a novel anti-inflammatory mechanism, i.e., sequestration of HMGB1, through which sCD93 prevents HMGB1-receptor interaction on effector cells and alleviates inflammation.

MULTIVESSEL CORONARY VASOSPASM WITH ABORTED SUDDEN DEATH AND REFRACTORY ANGINA: EFFICACY OF ALPHA-BLOCKERS AND INDICATION OF IMPLANTABLE CARDIOVERTER-DEFIBRILLATOR

Vasospasm of the coronary arteries is an uncommon and underdiagnosed cause of angina pectoris. There is a myriad of clinical presentations with variable prognoses. The typical presentation in the scenario of chronic coronary syndrome includes angina at rest or with variable threshold, from late evening to early morning, including transient ischemia responsive to nitrates, detectable by 12-lead electrocardiogram or Holter. Concurrent multivessel vasospasm is rare and with worse prognosis, given the potential for acute ST-elevation myocardial infarction, ventricular arrhythmias and subsequent sudden cardiac death. The presence of refractory angina is a sign of persistent vasospasm, indicating severe manifestations. The complex physiopathology, including hypercontractility of the vascular smooth muscle, endothelial dysfunction and adrenergic receptor activation has not been fully elucidated, which makes it difficult to define a targeted therapy. We present a case of refractory angina after multivessel vasospasm with aborted sudden death, which responded to alpha-blocker and had implantable cardioverter-defibrillator implantation postponed.

Conditional deletion of the glucocorticoid receptor in vascular smooth muscle disrupts the circadian rhythm of vascular reactivity and reduces blood pressure

Glucocorticoids synchronise peripheral clocks with the master clock in the suprachiasmatic nucleus in response to the ambient light cycle. In humans and mice, arrhythmic glucocorticoids induce non‐dipping blood pressure and vascular dysfunction. The mechanisms of this are poorly understood. We hypothesize that arrhythmic activation of the glucocorticoid receptor attenuates the circadian clock signalling in the vasculature leading to changes in blood pressure. To test this hypothesis, we assessed vascular function and blood pressure rhythms in control mice and mice with smooth muscle specific deletion of the glucocorticoid receptor (SMGRKO).Renal and mesenteric arteries were isolated at 7am (ZT0; lights on) and 7pm (ZT12; lights off), mounted on a wire myograph and the responses to increasing doses of phenylephrine or sodium nitroprusside (SNP) assessed. In wild‐type mice, the sensitivity of the renal artery to phenylephrine‐mediated contraction was greater at ZT12 than at ZT0, by comparing the half maximal effective concentration of each group, (logEC50: 7.54±0.26 versus 7.29±0.17 respectively; p=0.04). The sensitivity to SNP‐mediated relaxation was also greater when measured at ZT12 compared to ZT0 (7.64±0.3 versus 7.09±0.5; p=0.03). Similar responses were also detected in the mesenteric arteries. In SMGRKO mice, the temporal differences in renal and mesenteric artery vascular reactivity were absent. We used radiotelemetry to assess the effect of this altered rhythm of vascular reactivity on blood pressure. SMGRKO had significantly lower systolic blood pressure throughout the 24h cycle compared to wild‐type littermates, both when lights were off (107.9 ± 22.13 versus 136.3 ± 17.17 mmHg respectively; p=0.015) and during the lights on period (99.6 ± 13.16 versus 118 ± 15.9 mmHg; p=0.014). Blood pressure rhythmicity was not altered in SMGRKO mice.These data show that the glucocorticoid receptor signalling in vascular smooth muscle contributes both to the diurnal variation of vascular reactivity and to blood pressure, but loss of the receptor does not affect blood pressure rhythm. Whether glucocorticoid receptor deletion in smooth muscle protects against non‐dipping blood pressure induced by glucocorticoids needs further investigation.

Differential Expression Profile of Sphingosine‐1‐Phosphate Receptors in Human Brain Vascular Smooth Muscle and Endothelial Cells Following Hypoxia Plus Glucose Deprivation

Sphingosine‐1‐phosphate receptors (S1PRs) and their signaling pathways play an important role in mediating vascular health and function. Upon ligand activation, S1PRs 1‐5 couple with diverse heterotrimeric G‐protein subunits (Gαi, Gαq/11, Gα12/13) initiating multimodal downstream signaling pathways which result in various physiological outcomes in the vasculature, including cell proliferation and migration, barrier integrity preservation or loss, contraction, and inflammation. Specifically, S1PR2 activation has been linked to endothelial activation, barrier integrity loss, and inflammation, whereas S1PR1 activation contributes to barrier integrity preservation, vasodilation, and anti‐inflammatory properties. Although the role of S1PRs during pathophysiological conditions such as acute ischemic stroke is under current investigation, S1PR expression in the brain vasculature following ischemic injury has not yet been investigated. Therefore, the present study characterized the expression profile of S1PRs 1‐5 in human brain microvascular endothelial cells (ECs) and brain vascular smooth muscle cells (VSMCs) following hypoxia plus glucose deprivation (HGD). Male ECs and VSMCs were exposed to either HGD (1% O2) or normoxia (21% O2) for 3h. Semi‐quantitative RT‐PCR was used to examine gene expression and concomitantly, protein levels were assessed with standard western blot and/or FIJI‐mediated ICC analysis. We hypothesized that ECs and VSMCs differentially express S1PRs 1‐5 both basally and during ischemia‐like conditions. At the mRNA level, S1PRs 1‐5 gene expression was observed in VSMCs with S1PR4 and 5 being just above the level of detection. S1PRs 1‐4 expression was detected in ECs with S1PR4 having the lowest expression. During normoxic conditions, we observed that S1PRs 1 and 3 mRNA levels were most prominently expressed in both the ECs and VSMCs. In ECs, HGD exposure did not alter the gene expression of S1PRs 1‐3. At the protein level, we observed that HGD increased S1PR1 levels, however S1PR2 and 3 protein levels were unaltered. In VSMCs, HGD increased both mRNA and protein levels of S1PR2 but did not alter S1PRs 1 and 3 mRNA expression and protein levels. In conclusion, acute exposure to HGD appears to differentially regulate S1PRs in VSMCs and ECs at the transcriptional and translational level, respectively. In VSMCs, HGD increased S1PR2 gene expression and protein levels, whereas in ECs, HGD increased S1PR1 protein levels. The differential expression in S1PRs both basally and following HGD exposure may suggest differential signaling mechanisms within the two brain vascular cell types. Furthermore, differential expression of S1PR1 and 2 highlight the potential beneficial and/or detrimental involvement of S1PRs in the pathophysiology of ischemic injury within the cerebrovasculature. Further investigation will be necessary to determine the signaling role of S1PRs and their impact on cerebrovascular cell function following acute ischemic injury in the brain.

Literatur Review : Peran Anti-Hipertensi dalam Penurunan Tekanan Darah

Hypertension is a silent killer because many sufferers do not show symptoms before reaching severe conditions and even complications to death. This review article aims to analyze the use of herbal ingredients in the treatment of hypertension. Quercetin and sterols act as anti-hypertension in the RAAS as ACEIs so that angiotensin 1 cannot be converted into angiotensin 2, which has various effects that can increase blood pressure. Flavonoids (beta-blockers) can reduce cardiac output, resulting in blood pressure. Allicin, Ginsenoside, and anthocyanin compounds exhibit vasodilating effects with an endo-intact mechanism involving nitric oxide (NO). Increased production and release of NO can cause relaxation resulting in vasodilation. And the endo-denuded effect on vascular smooth muscle. Angiotensin 2 binds to the AT1 receptor in vascular smooth muscle, and it causes vasoconstriction. This anti-hypertensive is found in Allium sativum, Moringa oleivera, Apium graveolens, Annona muricata, Andrographis paniculata, Alstonia scolaris, Panax ginseng, Phoenix sylvestris, Piper nigrum, and Hibiscus sabdariffa.

Histamine causes endothelium‐independent dilation of mouse bladder feeder arterioles

In addition to being highly vascularized, the urinary bladder vasculature has unique properties that help to maintain blood flow during voiding and filling. Unlike other vessels of similar size, however, bladder arterioles do not contract to increases in intraluminal pressure. Prolonged decreases in bladder blood flow are linked to many types of bladder dysfunction, including painful bladder syndrome and interstitial cystitis. Although the vasculature has an important role in bladder dysfunction, the mechanisms regulating vasculature diameter in the bladder are still widely unknown. We tested the hypothesis that histamine, an important pro-inflammatory compound released from mast cells in the bladder wall, also alters bladder arteriolar diameter. Using pressure myography with isolated bladder feed arterioles, concentration-response curves to histamine (1 nM – 10 µM) were performed in vessels constricted with the thromboxane mimetic U-46619 (10 nM). Histamine caused a concentration-dependent vasodilation that was inhibited by the histamine H2 receptor antagonist cimetidine (10 µM) but not the H1 receptor antagonist fexofenadine (10 µM). Histamine-dependent vasodilation was not inhibited by the NOS inhibitor L-NAME (200 µM), the inward rectifier K+ channel blocker BaCl2 (100 µM), or endothelial denudation, suggesting the dilation was endothelium-independent. Alternatively, increasing extracellular K+ (35 mM) or exposure to the KV channel blocker XE-991 (10 µM) attenuated dilations to histamine. These findings suggest the H2 receptors in bladder vascular smooth muscle, and not the endothelium, mediate histamine-induced dilation in bladder arterioles. Since H2 receptors are coupled to GaS, these findings also suggest that histamine dilation results from an increase in cAMP that may hyperpolarize smooth muscle cells via protein kinase A-mediated activation of smooth muscle KV channels.

Intramyometrial injection of vasopressin resulting in severe bradycardia during myomectomy.

To the Editor: Myomectomy is an important treatment option for removing a hysteromyoma, along with intramyometrial vasopressin to reduce intraoperative bleeding.[1,2] Sometimes, however, it results in life-threatening complications.[3] We report two rare cases of sudden severe bradycardia after intramyometrial injection of vasopressin during myomectomy. The two patients were diagnosed with uterine leiomyomas. The preoperative laboratory studies, chest radiography, electrocardiography, and blood pressure were normal in both patients. The first case was a 36-year-old woman who underwent laparoscopically assisted myomectomy under general anesthesia. After anesthesia induction, endotracheal intubation, and ensuring that mechanical ventilation was established, laparoscopic myomectomy was performed at 12 mmHg abdominal pressure in the Trendelenburg position. After trocar insertion, the abdominal cavity appeared smooth, and multiple uterine myomas were found covering the entire uterine wall. After negative aspiration, 10 mL of diluted vasopressin (0.6 U/mL) was injected into the myometrium using a 16-gauge needle. Approximately 2 min after the injection of the diluted vasopressin, sudden severe bradycardia was developed. The heart rate (HR) suddenly decreased from 68, to 47, to 25 beats/min. Both the operation and anesthesia were immediately discontinued, and the operative bed was adjusted to a supine position. The patient's HR returned to 55 beats/min and then, within 30 s, dropped to 30 beats/min, and then 25 beats/min. Intravenous atropine (0.5 mg) was administered immediately. Approximately 2 min later, the patient's HR gradually returned to baseline levels. After 10 min of observation, the operation continued and went smoothly. The patient recovered uneventfully and was discharged 4 days later. The second case was a 38-year-old woman who underwent abdominal myomectomy under combined spinal-epidural anesthesia. The operation commenced after anesthesia induction. Bleeding occurred during excision of the myoma, and 10 mL of diluted vasopressin (0.6 U/mL) was injected into the myometrium to relieve bleeding. Approximately 3 min after the injection, the patient's HR dropped suddenly from 70, to 50, to 25 beats/min. One minute later, her blood pressure was 70/40 mmHg. Intravenous atropine sulfate (0.5 mg) was administered. Severe bradycardia (30 beats/min) lasted for nearly 2 min. The patient was ventilated with 100% oxygen. Intravenous ephedrine (6 mg) was also administered immediately, resulting in the HR returning to 108 beats/min and the blood pressure to 125/80 mmHg 3 min later. The operation went smoothly. No more vasoactive drugs were administered. The patient recovered uneventfully and was discharged 6 days later. Vasopressin, an extremely potent vasoconstrictor, is widely used to control acute hemorrhage during myomectomy. The vasopressor effect of vasopressin is primarily mediated by the VIa receptors in vascular smooth muscle, resulting in increased vascular resistance and slowing of the HR. Our two patients showed that intramyometrial injection of vasopressin may result in severe bradycardia, regardless of whether general anesthesia or combined spinal–epidural anesthesia had been used or whether laparoscopically assisted myomectomy or abdominal myomectomy was performed. Anesthesiologists should be vigilant while injecting vasopressin during surgery. Severe bradycardia may be the first symptom. If left untreated, it may lead to life-threatening complications, including cardiac arrest.[4] Stopping the surgical procedure and ejecting carbon dioxide gas from the abdominal cavity to reduce abdominal pressure does not completely relieve this severe bradycardia. Early use of atropine alone may be an appropriate choice for alleviating the severe bradycardia associated with vasopressin and preventing it from progressing to cardiac arrest. We strongly recommend early cardiopulmonary resuscitation if atropine is not effective and the severe bradycardia deteriorates to <25 beats/min. Declaration of patient consent The authors certify that they have obtained all appropriate patient consent forms. In the form, the patients have given their consent for their images and other clinical information to be reported in the article. The patients understands that their names and initials will not be published and due efforts will be made to conceal the identity of the patients, although anonymity cannot be guaranteed. Conflicts of interest None.

Abstract 194: Effects of PEG-20k on Coronary Perfusion Pressure and Post-Resuscitation Myocardial and Cerebral Function in a Rat Model of Cardiac Arrest

Introduction: Epinephrine is the primary drug given during cardiopulmonary resuscitation (CPR), when given early, to reverse cardiac arrest (CA) by stimulation of α-adrenergic receptors in vascular smooth muscle. This increases coronary perfusion pressure (CPP) and increases rate of return of spontaneous circulation (ROSC). However, the use of epinephrine is not associated with a significant difference in long-term survival or favorable neurologic outcome when given late after arrest onset. In the present study, we compared the effects of aortic injection of polyethylene glycol-20k (PEG-20k) vs. epinephrine during CPR on CPP and postresuscitation myocardial and cerebral function in a rat model of CA. Hypothesis: Aortic injection of PEG-20k during CPR will increase CPP to the same extent as epinephrine without adversely affecting post-resuscitation myocardial and cerebral function. Methods: Twenty four male Sprague-Dawley rats weighing between 450-550 g were randomized into four groups: 1) PEG-20k 2) epinephrine 3) saline placebo 4) saline-intra-aorta (IA). Eight minutes of CPR was initiated after 6 minutes of untreated ventricular fibrillation. PEG-20k IA (10% est. total blood volume [1.8ml]), saline IA, saline IV or epinephrine IV (20ug/kg) was given after 4 minutes of CPR by continuous infusion over 3 minutes. CPP was recorded continuously and resuscitation was attempted with 4 Joule defibrillation. Myocardial function was measured at baseline, 2, 4, and 6 hours after ROSC by echocardiography and neurologic deficit scores (NDS) were recorded at 24, 48, and 72 hours after ROSC. Results: In both saline groups, CPP did not change. However, aortic injection of PEG-20k increased CPP significantly to the same extent as epinephrine (Fig 1). Post-resuscitation ejection fraction was significantly greater in PEG-20k compared to epinephrine (64 + 1 vs 45 + 3, p&lt;0.05) and NDS was significantly improved in PEG-20k compared to epinephrine (100 + 50 vs 450 + 50, p&lt;0.05).

Mechanisms of PAR-1 mediated kinase receptor transactivation: Smad linker region phosphorylation.

Protease activated receptors (PARs) transactivate both epidermal growth factor receptors (EGFR) and transforming growth factor (TGF)-β receptors (TGFBR1) in vascular smooth muscle leading to the increased expression of genes (CHST11 and CHSY1) which are rate limiting for the enzymes that mediate hyperelongation of glycosaminoglycan (GAG) chains on the lipid-binding proteoglycan, biglycan. This is an excellent model to investigate mechanisms of transactivation as the processes are biochemically distinct. EGFR transactivation is dependent on the classical matrix metalloprotease (MMP) based triple membrane bypass mechanism and TGFBR1 transactivation is dependent on Rho/ROCK signalling and integrins. We have shown that all kinase receptor signalling is targeted towards phosphorylation of the linker region of the transcription factor, Smad2. We investigated the mechanisms of thrombin mediated kinase receptor transactivation signalling using anti-phospho antibodies and Western blotting and gene expression by RT-PCR. Thrombin stimulation of phospho-Smad2 (Ser 245/250/255) and of phospho-Smad2(Thr220) via EGFR transactivation commences quickly and extends out to at least 4h whereas transactivation via TGFBR1 is delayed for 120min but also persists for at least 4h. Signalling of thrombin stimulated Smad linker region phosphorylation is approximately equally inhibited by the MMP inhibitor, GM6001 and the ROCK inhibitor, Y27632, and similarly expression of CHST11 and CHSY1 is approximately equally inhibited by GM6001 and Y27632. The data establishes Smad linker region phosphorylation as a central target of all transactivation signalling of GAG gene expression and thus an upstream kinase may be a target to prevent all transactivation signalling and its pathophysiological consequences.

α1-Adrenergic Receptors Function Within Hetero-Oligomeric Complexes With Atypical Chemokine Receptor 3 and Chemokine (C-X-C motif) Receptor 4 in Vascular Smooth Muscle Cells.

BackgroundRecently, we provided evidence that α1‐adrenergic receptors (ARs) in vascular smooth muscle are regulated by chemokine (C‐X‐C motif) receptor (CXCR) 4 and atypical chemokine receptor 3 (ACKR3). While we showed that CXCR4 controls α1‐ARs through formation of heteromeric receptor complexes in human vascular smooth muscle cells (hVSMCs), the molecular basis underlying cross‐talk between ACKR3 and α1‐ARs is unknown.Methods and ResultsWe show that ACKR3 agonists inhibit inositol trisphosphate production in hVSMCs on stimulation with phenylephrine. In proximity ligation assays and co‐immunoprecipitation experiments, we observed that recombinant and endogenous ACKR3 form heteromeric complexes with α1A/B/D‐AR. While small interfering RNA knockdown of ACKR3 in hVSMCs reduced α1B/D‐AR:ACKR3, CXCR4:ACKR3, and α1B/D‐AR:CXCR4 complexes, small interfering RNA knockdown of CXCR4 reduced α1B/D‐AR:ACKR3 heteromers. Phenylephrine‐induced inositol trisphosphate production from hVSMCs was abolished after ACKR3 and CXCR4 small interfering RNA knockdown. Peptide analogs of transmembrane domains 2/4/7 of ACKR3 showed differential effects on heteromerization between ACKR3, α1A/B/D‐AR, and CXCR4. While the transmembrane domain 2 peptide interfered with α1B/D‐AR:ACKR3 and CXCR4:ACKR3 heteromerization, it increased heteromerization between CXCR4 and α1A/B‐AR. The transmembrane domain 2 peptide inhibited ACKR3 but did not affect α1b‐AR in β‐arrestin recruitment assays. Furthermore, the transmembrane domain 2 peptide inhibited phenylephrine‐induced inositol trisphosphate production in hVSMCs and attenuated phenylephrine‐induced constriction of mesenteric arteries.Conclusionsα1‐ARs form hetero‐oligomeric complexes with the ACKR3:CXCR4 heteromer, which is required for α1B/D‐AR function, and activation of ACKR3 negatively regulates α1‐ARs. G protein–coupled receptor hetero‐oligomerization is a dynamic process, which depends on the relative abundance of available receptor partners. Endogenous α1‐ARs function within a network of hetero‐oligomeric receptor complexes.

Erratum: Uterine artery leptin receptors during the ovarian cycle and pregnancy regulate angiogenesis in ovine uterine artery endothelial cells.

Leptin regulates body weight, reproductive functions, blood pressure, endothelial function, and fetoplacental angiogenesis. Compared to the luteal phase, the follicular phase and pregnancy are physiological states of elevated estrogen, angiogenesis, and uterine blood flow (UBF). Little is known concerning regulation of uterine artery (UA) angiogenesis by leptin and its receptors. We hypothesized that (1) ex vivo expression of leptin receptors (LEPR) in UA endothelium (UAendo) and UA vascular smooth muscle (UAvsm) is elevated in pregnant versus nonpregnant (Luteal and Follicular) sheep; (2) in vitro leptin treatments differentially modulate mitogenesis in uterine artery endothelial cells from pregnant (P-UAECs) more than in nonpregnant (NP-UAECs) ewes; and (3) LEPR are upregulated in P-UAECs versus NP-UAECs in association with leptin activation of phospho-STAT3 signaling. Local UA adaptations were evaluated using a unilateral pregnant sheep model where prebreeding uterine horn isolation (nongravid) restricted gravidity to one horn. Immunolocalization revealed LEPR in UAendo and UAvsm from pregnant and nonpregnant sheep. Contrary to our hypothesis, western analysis revealed that follicular UAendo and UAvsm LEPR were greater than luteal, nongravid, gravid, and control pregnant. Compared to pregnant groups, LEPR were elevated in renal artery endothelium of follicular and luteal sheep. Leptin treatment significantly increased mitogenesis in follicular phase NP-UAECs and P-UAECs, but not luteal phase NP-UAECs. Although UAEC expression of LEPR was similar between groups, leptin treatment only activated phospho-STAT3 in follicular NP-UAECs and P-UAECs. Thus, leptin may play an angiogenic role particularly in preparation for the increased UBF during the periovulatory period and subsequently to meet the demands of the growing fetus.

Tannic Acid is a Tissue‐Specific Dual Modulator of Angiotensin Type 1 Receptor and Low‐Density Lipoprotein Receptor in Rat Aortic Vascular Smooth Muscle and Liver Epithelial Cells: An Insight into the “French Paradox”

“French Paradox,” suggests the protective effect of various polyphenols present in red wine against cardiovascular diseases. Although tannic acid (TA), one such hydrolyzable polyphenol, has diverse pharmacological effects, it's cardioprotective mechanism is not well understood. In this study, we have investigated the effects of tannic acid on angiotensin type 1 receptor (AT1R) and low‐density lipoprotein receptor (LDLR), two major players associated with cardiovascular disorders such as hypertension and hyperlipidemia. Under normal culture conditions, exposure of the primary rat aortic vascular smooth muscle cells (RASMCs) and rat liver epithelial (WB) cells to TA inhibited cell‐surface expression of AT1R an effect that is time and dose dependent without any significant change in receptor affinity to angiotensin II (AngII). The inhibitory effect of TA was reversible after providing a TA‐free media for 24 hours. The inhibition of AT1R was further correlated with a significant decrease in the AngII‐mediated release in intracellular calcium. The down‐regulation of AT1R protein is associated with an inhibition of AT1R mRNA expression. In these cells, TA phosphorylated p42/44 MAP Kinases. Nevertheless, pretreatment with a MEK‐specific inhibitor PD98059 prevented TA mediated down‐regulation of the AT1R suggesting a role for MAP Kinase. Moreover, AG1478, an EGFR inhibitor blocked TA mediated MAP kinase activation and down‐regulation of AT1R. Under similar conditions, we observed a significant increase in TA mediated LDLR expression in WB cells, which was not observed in RASMCs. Furthermore, proprotein convertase subtilisin/kexin type 9 (PCSK9), a crucial enzyme responsible for LDLR turn over, was decreased upon TA exposure in WB cells whereas increased in RASMCs. The inverse correlation between LDLR and PCSK9 expression suggests that TA induced LDLR expression is at least in part through preventing PCSK9‐mediated degradation of LDLR protein in these cells. Taken together, the present study show for the first time that TA mediated activation of MAP Kinase through EGFR is critical for the regulation of AT1R and LDLR in RASMCs and WB cells, which can simultaneously prevent hypertension and hyperlipidemia through distinctly targeting AT1R and LDLR, therefore, providing the mechanistic insight into the “French Paradox.”

Uterine artery leptin receptors during the ovarian cycle and pregnancy regulate angiogenesis in ovine uterine artery endothelial cells†.

Leptin regulates body weight, reproductive functions, blood pressure, endothelial function, and fetoplacental angiogenesis. Compared to the luteal phase, the follicular phase and pregnancy are physiological states of elevated estrogen, angiogenesis, and uterine blood flow (UBF). Little is known concerning regulation of uterine artery (UA) angiogenesis by leptin and its receptors. We hypothesized that (1) ex vivo expression of leptin receptors (LEPR) in UA endothelium (UAendo) and UA vascular smooth muscle (UAvsm) is elevated in pregnant versus nonpregnant (Luteal and Follicular) sheep; (2) in vitro leptin treatments differentially modulate mitogenesis in uterine artery endothelial cells from pregnant (P-UAECs) more than in nonpregnant (NP-UAECs) ewes; and (3) LEPR are upregulated in P-UAECs versus NP-UAECs in association with leptin activation of phospho-STAT3 signaling. Local UA adaptations were evaluated using a unilateral pregnant sheep model where prebreeding uterine horn isolation (nongravid) restricted gravidity to one horn. Immunolocalization revealed LEPR in UAendo and UAvsm from pregnant and nonpregnant sheep. Contrary to our hypothesis, western analysis revealed that follicular UAendo and UAvsm LEPR were greater than luteal, nongravid, gravid, and control pregnant. Compared to pregnant groups, LEPR were elevated in renal artery endothelium of follicular and luteal sheep. Leptin treatment significantly increased mitogenesis in follicular phase NP-UAECs and P-UAECs, but not luteal phase NP-UAECs. Although UAEC expression of LEPR was similar between groups, leptin treatment only activated phospho-STAT3 in follicular NP-UAECs and P-UAECs. Thus, leptin may play an angiogenic role particularly in preparation for the increased UBF during the periovulatory period and subsequently to meet the demands of the growing fetus.

Abstract 13832: GPR39 is a High Affinity Vascular Smooth Muscle Receptor for P450 Eicosanoids

Epoxyeicosatrienoic (EETs) and hydroxyeicosatetraenoic acids (HETEs) are endogenous vasoactive P450 eicosanoids that play important roles in cardiovascular physiology and disease, but their mechanisms of action are not fully understood. We developed a clickable photo-crosslinking probe (EET-P) based on EETs regio-isomer 14,15-EET, which allowed us to purify and enrich 14,15-EET binding proteins in mouse heart vascular smooth muscle cells (VSMCs). Proteomic analysis generated a list of proteins bound by EET-P that included the orphan G-protein coupled receptor 39 (GPR39). Consistent with the molecular weight of GPR39, sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) analysis revealed a prominent ~50 kD protein crosslinked to EET-P, and pretreatment with either 14,15-EET or its antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE) completely displaced EET-P binding. Heterologous expression of GPR39 but not a control plasmid in HEK293 cells conferred responsiveness to both 14,15-EET and 15-HETE, as assayed by extracellular-regulated kinase (ERK) activation. Immuno-histochemical analysis localized GPR39 expression in both mouse heart micro-vessels and primary cultured VSMCs. Live-cell imaging of VSMC intracellular calcium ([Ca 2+ ]i) revealed a dual and antagonistic regulatory mechanism in which 15-HETE induces and 14,15-EET suppresses VSMC calcium transients. Short hairpin RNA (shRNA) knockdown of GPR39 in VSMCs was sufficient to abolish effects of both 15-HETE and 14,15-EET on VSMC calcium. Our results identify GPR39 as a high-affinity, lipid-sensing GPCR antagonistically regulated by two endogenous P450 eicosanoids with opposing actions on VSMC calcium, and offer a unique window into multi-ligand GPCRs and their role in integrating the multitude of hormonal signals to regulate local tissue function and response to disease state.

Vasopressin V1A receptor mediates cell proliferation through GRK2-EGFR-ERK1/2 pathway in A7r5 cells

Abnormal proliferation and hypertrophy of vascular smooth muscle (VSMC), as the main structural component of the vasculature, is an important pathological mechanism of hypertension. Recently, increased levels of arginine vasopressin (AVP) and copeptin, the C-terminal fragment of provasopressin, have been shown to correlate with the development of preeclampsia. AVP targets on the Gq-coupled vasopressin V1A receptor and the Gs-coupled V2 receptor in VSMC and the kidneys to regulate vascular tone and water homeostasis. However, the role of the vasopressin receptor on VSM cell proliferation during vascular remodeling is unclear. Here, we studied the effects of AVP on the proliferation of the rat VSMC-derived A7r5 cells. AVP, in a time- and concentration-dependent manner, promoted A7r5 cell proliferation as indicated by the induction of proliferating cell nuclear antigen expression, methylthiazolyldiphenyl-tetrazolium reduction and incorporation of 5’-bromodeoxyuridine into cellular DNA. These effects, coupled with the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), were blocked by a V1A receptor antagonist SR45059 but not by a V2 receptor antagonist lixivaptan. Although acute activation of V1A receptor induced ERK1/2 phosphorylation via a protein kinase C-dependent pathway, this effect was not involved in cell proliferation. Cell proliferation and ERK1/2 phosphorylation in response to prolonged stimulation with AVP were abolished by inhibition of G protein-coupled receptor kinase 2 (GRK2) and epidermal growth factor receptor (EGFR) using specific inhibitors or small hairpin RNA knock-down. These results suggest that activation of V1A, but not V2 receptor, produces a cell proliferative signal in A7r5 cells via a GRK2/EGFR/ERK1/2–dependent mechanism.

Simultaneous Spectrophotometric Estimation of Candesartan Cilexetil and Hydrochlorothiazidein Tablet Dosage Form

A new simple derivative spectrophotometric method has been developed for the simultaneous determination of Candesartan Cilexetil and Hydrochlorothiazide in tablet dosage forms. Candesartan antagonizes the effect of angiotensin II (vasoconstriction and aldosterone secretion) by blocking the angiotensin II receptor in vascular smooth muscle and the adrenal gland and decrease the blood pressure whereas Hydrochlorothiazide increases chloride, sodium and water excretion by interfering with transport of sodium ions across renal tubular epithelium. And there by show the diuretic action. The first derivative method is based on the measurement of absorbance of one drug at the zero crossing point of another drug. Candesartan Cilexetil and Hydrochlorothiazide were determined at two different wavelengths 222.69 (zero crossing point of Hydrochlorothiazide) and 254.63 nm (zero crossing point of Candesartan Cilexetil) from the derivative spectra respectively. The methods hows linearity over the concentration range 0.5-50 and 0.1-50 µg/ml for Candesartan Cilexetil and Hydrochlorothiazide respectively in phosphate buffer. The proposed method was validated and can be used for routine analysis of combined tablet dosage forms containing Candesartan Cilexetil and Hydrochlorothiazide. Key words: Candesartan Cilexetil, Derivative spectroscopy, Hydrochlorothiazide, Simultaneous determination.

The ERK1/2 pathway participates in the upregulation of the expression of mesenteric artery α1 receptors by intravenous tail injections of mmLDL in mice

Minimally modified low density lipoprotein (mmLDL) is a risk factor for cardiovascular diseases. However, no studies examining the effect of mmLDL on vascular smooth muscle receptors have been released. The current study investigated the effect of mmLDL on the mesenteric artery α1 adrenoceptor and the molecular mechanisms. Mice were divided into the normal saline (NS), mmLDL, and mmLDL+U0126 groups. In the mmLDL+U0126 group, the animals were subjected to an intravenous tail injection of mmLDL and an intraperitoneal injection of U0126. Vascular tension caused by noradrenaline (NA) in mesenteric arteries was measured with a sensitive myograph system. The serum levels of oxLDL, TNF-α, and IL-1β were detected using enzyme-linked immunosorbent assays. The expressions of the α1 adrenoceptor, the α2 adrenoceptor, TNF-α, IL-1β, and pERK1/2 were detected using real-time polymerase chain reactions and Western blot analysis. Compared with the NS group, the mmLDL group exhibited a noticeably enhanced NA shrinkage dose–response curve and a significantly increased Emax value (P<0.01). Prazosin (α1 adrenoceptor antagonist) caused a noticeable right shift of the dose–response curve. U0126 inhibited the increases in the serum levels and vessel wall expression of IL-1β and TNF-α and enhanced the NA shrinkage dose–response curve caused by mmLDL, as observed by a significantly decreased Emax value (P<0.01). It inhibited the increased α1 adrenoceptor expression caused by mmLDL. The serum levels of IL-1β and TNF-α demonstrated a positive correlation with the NA-induced maximum shrinkage percentage. U0126 inhibited the mmLDL-induced increase in the pERK1/2 protein level in the vessel wall. In conclusion, mmLDL increased the serum levels of IL-1β and TNF-α in vivo by activating the ERK1/2 pathway, which resulted in α1 receptor-mediated vasoconstriction and an increase in the expression of α1 adrenoceptor. The results of this study may provide new ideas for the prevention and cure of cardiovascular diseases in the future.