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Background Thromboxane A2 (TXA2 ) participates in many pathophysiological processes of coronary artery disease. However, its mechanism of TXA2 -induced contraction in the coronary artery remains to be clarified. Methods Multi Myograph System was used to measure the isometric tension of the mouse coronary arteries and identify the effect and pathway of TXA2 analogues U46619. Confocal laser scanning microscopy was used to measure the intracellular calcium concentration ([Ca2+ ]i ) in mouse coronary artery smooth muscle cells. Results Results from the experiment had shown that contraction in coronary artery was generated by U46619 in a concentration- dependent manner, which was completely abolished by a specific TXA2 receptor blocker, GR32191. PI-PLC inhibitors U73122 and D609 and Rho-Kinase inhibitor Y-27632 can block the U46619 elicited coronary artery contraction in a dose-dependent manner. Then, the vasoconstriction response to U46619 was obviously inhibited by two pan-PKC inhibitors chelerythrine or Gӧ6983, and a selective PKCδ inhibitor rottlerin, but wasn't blocked by a selective PKCζ inhibitor PKC-PS or a selective PKCβ inhibitor hispidin. Meanwhile, the PKC activator PDBu-induced vasoconstriction was significantly inhibited by 1 μM nifedipine,then mostly inhibited by 100 μM 2-APB and 10 μM Y27632. We further found that the response to U46619 was inhibited respectively by three calcium channel blockers nifedipine, SKF96356 or 2-APB in a concentration-dependent manner. Although Store-operated Ca2+ (SOC) channels generated the increase of [Ca2+ ]i in mouse coronary artery smooth muscle cells, SOC channels didn't contribute to the vasoconstriction in mouse coronary arteries. Caffeine-induced sarcoplasmic reticulum (SR) Ca2+ release could obviously induce coronal vasoconstriction. In addition, NPPB, a cell membrane Ca2+ activated C1- channel blocker, could obviously inhibit the U46619-induced vasoconstriction. Conclusions The U46619-induced mouse coronary artery contraction was involved in the increase in [Ca2+ ]i mediated by Cav1.2, TRPC channels and SR release through the activation of G-protein-coupled TP receptors and the kinases signaling pathway in TP downstream proteins, while SOC channels didn't participate in the vasoconstriction.

It is well-established that long-term exposure of the vasculature to metabolic disturbances leads to abnormal vascular tone, while the physiological regulation of vascular tone upon acute metabolic challenge remains unknown. Here, we found that acute glucose challenge induced transient increases in blood pressure and vascular constriction in humans and mice. Ex vivo study in isolated thoracic aortas from mice showed that glucose-induced vascular constriction is dependent on glucose oxidation in vascular smooth muscle cells. Specifically, mitochondrial membrane potential (ΔΨm), an essential component in glucose oxidation, was increased along with glucose influx and positively regulated vascular smooth muscle tone. Mechanistically, mitochondrial hyperpolarization inhibited the activity of myosin light chain phosphatase (MLCP) in a Ca2+-independent manner through activation of Rho-associated kinase, leading to cell contraction. However, ΔΨm regulated smooth muscle tone independently of the small G protein RhoA, a major regulator of Rho-associated kinase signaling. Furthermore, myosin phosphatase target subunit 1 (MYPT1) was found to be a key molecule in mediating MLCP activity regulated by ΔΨm. ΔΨm positively phosphorylated MYPT1, and either knockdown or knockout of MYPT1 abolished the effects of glucose in stimulating smooth muscle contraction. In addition, smooth muscle-specific Mypt1 knockout mice displayed blunted response to glucose challenge in blood pressure and vascular constriction and impaired clearance rate of circulating metabolites. These results suggested that glucose influx stimulates vascular smooth muscle contraction via mitochondrial hyperpolarization-inactivated myosin phosphatase, which represents a novel mechanism underlying vascular constriction and circulating metabolite clearance.

Introduction: Endothelial dysfunction is an imbalance in bioavailability of endothelium-derived relaxing (Nitric Oxide) and contracting factors (Endothelin). Studies have shown that NO has antiatherogenic property as it inhibits vascular smooth muscle contraction and proliferation, platelet aggregation and leukocyte adhesion. On the other hand, Endothelin-1 causes proliferation and vasoconstriction of myocytes resulting in elevated vascular tone thereby contributing to endothelial dysfunction. This dysfunction is a well-established response to cardiovascular risk factors and precedes the development of atherosclerosis. As a result, status of an endothelial dysfunction may reflect the propensity to develop atherosclerotic disease and may serve as a marker of an unfavourable cardiovascular prognosis. Materials and Methods: 50 cases of angiographically proven ischemic heart disease and 50 age and sex matched patients with no evidence of ischaemic heart disease on angiography were selected from G.B Pant Hospital, New Delhi, India. Serum levels of Endothelin were estimated by sandwich ELISA and nitric oxide levels by modified Griess reaction. Results: Serum levels of Endothelin were raised while nitric oxide levels were significantly reduced (p=0.049) in the cases as compared to controls. The mean plasma nitric oxide level in the study group (cases) was 23.51±13.48 µM and in the control group was 29.35±15.73 µM. The difference between the two was significant (p=0.049*). The mean plasma endothelin level in the study group was 3.04±1.37fmol/ml and in the control group was 2.95±1.70 fmol/ml. The difference between the two was not significant (p = 0.764). Conclusion: Higher Endothelin-1 and lower NO levels in cases suggest their role in causing endothelial dysfunction in ischemic heart disease patients. However, larger sample size is required to assess the role of Endothelin as a biomarker of endothelial dysfunction in patients of ischemic heart disease.

To investigate the biological role of protein phosphorylation in human nonfunctional pituitary neuroendocrine tumors (NF-PitNETs), proteins extracted from NF-PitNET and control tissues were analyzed with tandem mass tag (TMT)-based quantitative proteomics coupled with TiO2 enrichment of phosphopeptides. A total of 595 differentially phosphorylated proteins (DPPs) with 1412 phosphosites were identified in NF-PitNETs compared to controls (p &lt, 0.05). KEGG pathway network analysis of 595 DPPs identified nine statistically significant signaling pathways, including the spliceosome pathway, the RNA transport pathway, proteoglycans in cancer, SNARE interactions in vesicular transport, platelet activation, bacterial invasion of epithelial cells, tight junctions, vascular smooth muscle contraction, and protein processing in the endoplasmic reticulum. GO analysis revealed that these DPPs were involved in multiple cellular components (CCs), biological processes (BPs), and molecule functions (MFs). The kinase analysis of 595 DPPs identified seven kinases, including GRP78, WSTF, PKN2, PRP4, LOK, NEK1, and AMPKA1, and the substrate of these kinases could provide new ideas for seeking drug targets for NF-PitNETs. The randomly selected DPP calnexin was further confirmed with immunoprecipitation (IP) and Western blot (WB). These findings provide the first DPP profiling, phosphorylation-mediated molecular network alterations, and the key kinase profiling in NF-PitNET pathogenesis, which are a precious resource for understanding the biological roles of protein phosphorylation in NF-PitNET pathogenesis and discovering effective phosphoprotein biomarkers and therapeutic targets and drugs for the management of NF-PitNETs.

Hypertension is a major cause of cardiovascular morbidity and mortality, despite the availability of antihypertensive drugs with different targets and mechanisms of action. There is an unmet need for antihypertensive drugs with novel mechanisms of action to better control hypertension and reduce cardiovascular morbidity and mortality. Here, we provide evidence that pharmacological inhibition of TMEM16A (transmembrane member 16A or anoctamin-1), a Ca 2+ -activated Cl - channel expressed in vascular smooth muscle cells, reduces in vitro vascular smooth muscle contraction and decreases blood pressure in spontaneously hypertensive rats (SHR). We recently identified by high-throughput screening and subsequent medicinal chemistry, small molecule TMEM16A inhibitor TM inh -23 (2-bromodifluoroacetylamino-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-carboxylic acid o-tolylamide) that inhibits TMEM16A current fully, with IC 50 ~ 30 nM. TM inh -23 pretreatment blocked maximum in vitro vascular smooth muscle contractions induced by a thromboxane mimetic (U46619) in rat mesenteric arteries by 90%. Intraperitoneal (ip) administration of TM inh -23 to rodents at 10 mg/kg produced sustained serum concentrations of >10 μM for >4 hours. BP measurements by tail-cuff and telemetry showed a maximum ~45 mmHg reduction in SBP in spontaneously hypertensive rats (SHR) after a single dose (10 mg/kg, ip) TM inh -23 administration compared to vehicle treatment, with BP gradually returning to baseline values within 6-8 hours after TM inh -23 treatment. Minimal effect on BP (less than 10 mmHg decrease in SBP) was seen in wild-type rats and mice with TM inh -23 treatment (10 mg/kg, ip). Chronic 5-day treatment of SHR with TM inh -23 (10 mg/kg, ip, twice daily) caused sustained decreases (~20-25 mmHg) in daily average SBP, DBP and MAP during the treatment period. TM inh -23 action was reversible, with BP returning to baseline (~170/115 mmHg) by 3 days after discontinuation of treatment. These studies provide validation for TMEM16A as a target for antihypertensive therapy, and demonstrate the proof-of-concept for efficacy of TM inh -23 as an antihypertensive with a novel mechanism of action.

The selection of raw milk with high levels of functional components that have health-promoting activities is very important for the exploitation and production of functional milk, but the differences in the functional components of whey from buffalo raised at different altitudes have not been thoroughly investigated. Here, we detected the effects of altitudes on the functional components in whey from dairy buffalo farms situated at low altitude (LA), medium altitude (MA), and high altitude (HA) sites with data-independent acquisition proteomic approaches. In 33 samples, 9331 peptides corresponding to 1008 high-confidence proteins were detected. HA-whey had a lower level of angiotensinogen than that of the LA- and MA-whey, and conversely contained higher levels of immune-enhancing components than for the latter two groups. Differential proteins were involved in vascular smooth muscle contraction, complement and coagulation cascades, and the secretion, production and regulation pathways in immune components. LA-whey showed higher levels of lymphocyte antigen and selenoprotein F than that of the HA-whey. Owing to the biological functions of their most abundant components, HA- and LA-whey are suitable for the processing of functional milk for lowering blood pressure, and the production of immune milk, respectively.

Class II phosphatidylinositol 3-kinases (PI3K), PI3K-C2α and PI3K-C2β, are involved in cellular processes including endocytosis, cilia formation and autophagy. However, the role of PI3K-C2α and PI3K-C2β at the organismal level is not well understood. We found that double knockout (KO) mice with both smooth muscle-specific KO of PI3K-C2α and global PI3K-C2β KO, but not single KO mice of either PI3K-C2α or PI3K-C2β, exhibited reductions in arterial blood pressure and substantial attenuation of contractile responses of isolated aortic rings. In wild-type vascular smooth muscle cells, double knockdown of PI3K-C2α and PI3K-C2β but not single knockdown of either PI3K markedly inhibited contraction with reduced phosphorylation of 20-kDa myosin light chain and MYPT1 and Rho activation, but without inhibition of the intracellular Ca2+ mobilization. These data indicate that PI3K-C2α and PI3K-C2β play the redundant but essential role for vascular smooth muscle contraction and blood pressure regulation mainly through their involvement in Rho activation.

Background: A large percentage of maternal mortality is related to hypertensive disorders of pregnancy. The etiology of preeclampsia is uncertain with imbalance between vasoconstrictor and vasodilators leading to vasospasm and endothelial dysfunction. Magnesium and Calcium plays a role in vascular smooth muscle contraction and thus regulating blood pressure. Aim: We compared the serum level of Calcium and Magnesium of preeclamptic women with normotensive women at SRMS-IMS, Bareilly. Method: 30 preeclamptic patient between 18-35 years with BP>140/90mmHg and gestation age >30 weeks were selected. Blood sample were taken for serum calcium and magnesium level and the results were compared with 20 normotensives pregnant. Result: Mean serum Calcium and Magnesium level in preeclamptic woman was 9.00± 0.47 and 1.90±0.31 mg/dl, respectively. Mean serum Calcium and Magnesium in normal pregnant woman was 9.12±0.37 and 1.92±0.23mg/dl, respectively. There was statistically no significant difference with p value of 0.34 and 1.00, respectively. Conclusion: Serum Calcium and Magnesiumlevelwere observed to be same in preeclamptic and normotensive pregnant women.

To investigate the effects of Zhizi Chuanxiong Capsule (ZCC, 栀子川芎胶囊) on abnormal DNA methylation in a rabbit model of atherosclerosis (AS). After 1 week of adaptive feeding, 48 New Zealand white rabbits were randomly divided into 4 groups: a control group (n=12) fed with normal diet for 22 weeks; a model group (n=12) fed with high fat diet for 14 weeks followed by 8 weeks of normal diet feeding; a low-dose ZCC group (n=12) fed with high fat diet and low-dose ZCC for 14 weeks, followed by 8 weeks of normal diet and low-dose drug; a high-dose ZCC group (n=12) fed with high fat diet and high-dose drug for 14 weeks, followed by 8 weeks of normal diet and high-dose drug. After 22 weeks of feeding, blood samples were taken from the rabbit ear vein, and the genomic DNA was extracted for methylation immunoprecipitation sequencing (Medip-seq). The aorta tissues were collected for hematoxylin-eosin (HE) staining. Eight rabbits died during the feeding process. HE staining showed that the size of the lipid deposition on vessel wall and atherosclerotic plaque formation were reduced in both low- and high-dose group. The Medip-seq results showed that there were 146 abnormally methylated genes (including both hypermethylated gene and hypomethylated genes) in the model group, compared with the control group. Gene Ontology (GO) and Pathway analysis showed that these abnormally methylated genes were found to be involved in multiple AS-related functions and pathways, such as protein kinase C activity, cholesterol transport, mitogen-activated protein kinase (MAPK) signaling pathway, peroxisome proliferater-activated receptor signaling pathway, vascular smooth muscle contraction, inflammation and so on. The abnormal methylated genes in AS model group were altered in both low- and high-dose groups: low-dose ZCC could change 72 of the 146 abnormally methylated genes, high-dose ZCC could change 71. Through GO and Pathway analysis, these altered methylated genes were involved in protein kinase C activity, inflammatory pathway, MAPK signaling pathway, vascular endothelial growth factor signaling pathway, etc. ZCC could treat AS through regulating the abnormal hypermethylated and hypomethylated genes in AS rabbit model.

Systems toxicology approaches have been used as important tools in the drug discovery and medicine quality control processes. The aim of this study was to assess the pharmacokinetic and toxicity properties of cephalosporins with an aminothiazoyl ring at the C-7 position (CATRs). Cardiac toxicity of the compounds was assessed in zebrafish embryos, and it was determined that CATRs disturbed the formation and development of the heart in a dose-dependent manner. Differentially expressed genes (DEGs) related to the heart were also identified by transcriptome analysis, and co-DEGs were obtained in the protein-protein interaction (PPI) network. Several Gene Ontology (GO) terms and pathways that were enriched by DEGs were identified, and the most significantly enriched pathways were adrenergic signaling in cardiomyocytes, cardiac muscle contraction, and vascular smooth muscle contraction. Combined molecular docking results elucidated that cardiac toxicity mainly depends on the mother nucleus structure 7-aminocephalosporanic acid (7-ACA). The predicted absorption, distribution, metabolism and excretion (ADME) profile suggests that there is a modification at the C-3 side chain of 7-ACA that could change the compound distribution in vivo. The 7-ACA mother nucleus is responsible for the CATRs induced cardiac toxicity, and the three DEGs (nppa, adra2c, and tnni1c) may potentially be utilized as novel biomarkers for CATRs. Our results show that zebrafish embryos may be used to reveal the pathways of cardiac toxicity and they play a vital role in drug safety assessments.

Scutellaria baicalensis Georgi is an extensively used medicinal herb for the treatment of hypertension in traditional Chinese medicine. Baicalin, is an important flavonoid in Scutellaria baicalensis Georgi extracts, which exhibits therapeutic effects on anti-hypertension, but its underlying mechanisms remain to be further explored. Therefore, we investigated the effects and molecular mechanisms of Baicalin on anti-hypertension. In vivo studies revealed that Baicalin treatment significantly attenuated the elevation in blood pressure, the pulse propagation and thickening of the abdominal aortic wall in C57BL/6 mice infused with Angiotensin II (Ang II). Moreover, RNA-sequencing and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses identified 537 differentially expressed transcripts and multiple enriched signaling pathways (including vascular smooth muscle contraction and calcium signaling pathway). Consistently, we found that Baicalin pretreatment significantly alleviated the Ang II induced constriction of abdominal aortic ring, while promoted NE pre-contracted vasodilation of abdominal aortic ring at least partly dependent on L-type calcium channel. In addition, Ang II stimulation significantly increased cell viability and PCNA expression, while were attenuated after Baicalin treatment. Moreover, Baicalin pretreatment attenuated Ang II-induced intracellular Ca2+ release, Angiotensin II type 1 receptor (AT1R) expression and activation of MLCK/p-MLC pathway in vascular smooth muscle cells (VSMCs). The present work further addressed the pharmacological and mechanistic insights on anti-hypertension of Baicalin, which may help better understand the therapeutic effect of Scutellaria baicalensis Georgi on anti-hypertension.

Background Hypertension is one of the primary modifiable risk factors for cardiovascular disease. Adequate vitamin D (vit D) levels have been shown to reduce vascular smooth muscle contraction and to increase arterial compliance, which may be beneficial in hypertension. Further, coenzyme Q10 (COQ10) through its action to lower oxidative stress has been reported to have beneficial effects on hypertension and heart failure. This study examined the possible cardiac and renal protective effects of vit D and COQ10 both separately and in combination with an angiotensin II receptor blocker, valsartan (vals) in l -NAME hypertensive rats. Materials and Methods Hypertension was induced in rats by l -NAME administration. Following induction of hypertension, the rats were assigned into the following 6 subgroups: an l -NAME alone group and treated groups receiving the following drugs intraperitoneally for 6 weeks; vals, vit D, COQ10 and combination of vals with either vit D or COQ10. A group of normotensive rats were used as negative controls. At the end of the treatment period, blood pressure, serum creatinine, blood urea nitrogen, lipids and serum, cardiac and renal parameters of oxidative stress were measured. Results Compared to the l -NAME only group, all treatments lowered systolic, diastolic, mean arterial pressure, total cholesterol, low-density lipoprotein cholesterol, and creatinine levels as well as TNF-α and malondialdehyde. Further, the agents increased serum, cardiac and renal total antioxidant capacity. Interestingly, the combination of agents had further effects on all the parameters compared to treatment with each single agent. Conclusions The study suggests that the additive protective effects of vit D and COQ10 when used alone or concurrent with vals treatment in hypertensive rats may be due to their effects as antioxidants, anticytokines and blood pressure conservers.

Objective To examine the effects of accumulated heat in GI tract (AHGIT) on lung tissue protein expression in pneumonic mice. Methods Nebulized lipopolysaccharides (LPS) were administered to induce a pneumonic mouse model (M1), and a high-calorie/protein diet combined with nebulized LPS was used to induce AHGIT pneumonia (M2). Isobaric tag for relative and absolute quantitation (iTRAQ) proteomics was applied to study lung protein expression, followed by bioinformatics analysis. Results M1 mice developed alveolar damage with prominent septum thickening, vascular dilation, hyperaemia and infiltration of large amounts of inflammatory cells. M2 mice developed more severe pathological responses. A total of 2626 proteins were reliably identified in the lung tissue. Compared with normal mice, the M1 mice had 344 differentially expressed proteins in their lungs, which are involved in the following biological processes: response to organic substance, response to cytokine, response to external stimulus, defense response and immune system process. They are also involved in the following Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways: ECM-receptor interaction, leukocyte transendothelial migration, Fc gamma R-mediated phagocytosis, complement and coagulation cascades, and antigen processing and presentation. Compared with the M1 group, the M2 mice had 164 differentially expressed proteins in their lungs, including 14 upregulated and 150 downregulated proteins. These proteins are involved in the following biological processes: small molecule metabolism, ribose phosphate metabolic process, cell adhesion and biological adhesion. The relevant KEGG pathways included oxidative phosphorylation, Citrate cycle (TCA cycle), complement and coagulation cascades, and vascular smooth muscle contraction. Conclusions AHGIT aggravated the lung inflammatory damage in the mice with LPS-induced pneumonia. It may affect the mouse substance/energy metabolism, and therefore the immune function, to aggravate the LPS-induced inflammatory damage.

The pathological process and mechanism of myocardial ischemia (MI) is very complicated, and remains unclear. An integrated proteomic-metabolomics analysis was applied to comprehensively understand the pathological changes and mechanism of MI. Male Sprague-Dawley rats were randomly divided into a mock surgery (MS) group and an MI group. The MI model was made by ligating the left anterior descending coronary artery, twenty-four hours after which, echocardiography was employed to assess left ventricular (LV) function variables. Blood samples and left ventricular tissues were collected for ELISA, metabolomics and proteomics analysis. The results showed that LV function, including ejection fraction (EF) and fractional shortening (FS), was significantly reduced and the level of cTnT in the serum increased after MI. iTRAQ proteomics showed that a total of 169 proteins were altered including 52 and 117 proteins with increased and decreased expression, respectively, which were mainly involved in the following activities: complement and coagulation cascades, tight junction, regulation of actin cytoskeleton, MAPK signaling pathway, endocytosis, NOD-like receptor signaling pathway, as well as phagosome coupled with vitamin digestion and absorption. Altered metabolomic profiling of this transition was mostly enriched in pathways including ABC transporters, glycerophospholipid metabolism, protein digestion and absorption and aminoacyl-tRNA biosynthesis. The integrated metabolomics and proteomics analysis indicated that myocardial injury after MI is closely related to several metabolic pathways, especially energy metabolism, amino acid metabolism, vascular smooth muscle contraction, gap junction and neuroactive ligand-receptor interaction. These findings may contribute to understanding the mechanism of MI and have implication for new therapeutic targets.

Pulmonary hypertension (PH) is a serious disorder with high morbidity and mortality rate. We analyzed the right-ventricular systolic pressure (RVSP), right-ventricular hypertrophy (RVH), lung histology, and transcriptomes of six-week-old male rats with PH induced by (1) hypoxia (HO), (2) administration of monocrotaline (CM), or (3) administration of monocrotaline and exposure to hypoxia (HM). The results in PH rats were compared to those in control rats (CO). After four weeks exposure, increased RVSP and RVH, pulmonary arterial wall thickening, and alteration of the lung transcriptome were observed in all PH groups. The HM group exhibited the largest alterations, as well as neointimal lesions and obliteration of the lumen in small arteries. We found that PH increased the expression of caveolin1, matrix metallopeptidase 2, and numerous inflammatory and cell proliferation genes. The cell cycle, vascular smooth muscle contraction, and oxidative phosphorylation pathways, as well as their interplay, were largely perturbed. Our results also suggest that the upregulated Rhoa (Ras homolog family member A) mediates its action through expression coordination with several ATPases. The upregulation of antioxidant genes and the extensive mitochondrial damage observed, especially in the HM group, indicate metabolic shift toward aerobic glycolysis.

Mastoparan-7 activates guanine nucleotide-binding proteins (G-proteins) and stimulates both apoptosis and increases in cytoplasmic calcium concentration and may induce smooth muscle contraction. The primary aim of the present study was to evaluate the modulatory effect of laser stimulation on vascular smooth muscle contraction induced by direct stimulation of G-protein with mastoparan-7. Experiments were performed on isolated and perfused tail arteries of Wistar rats. Contraction force in the model was measured by increased levels of perfusion pressure with a constant flow. Irradiation treatment was applied directly to the blood vessels. The laser was applied in increasing doses of 10 mW (E=1.8 J), 30 mW (E=5.5 J) and 110 mW (E=19.8 J). Time of exposure was 3 min for each irradiation. In the laser-stimulated arteries, a significant and dose-dependent decrease was observed. The half maximal effective concentration values were 4.43±2.2x10(-8), 2.4±0.56x10(-7), 3.2±0.72x10(-7) and 7.7±0.3x10(-7) M/l in the control and 10, 30 and 110 mW laser irradiation groups, respectively. Significant (P

Background Compromised microvasculature resulting from disrupted bronchial arterial circulation appears to trigger chronic lung allograft dysfunction. Maintaining the microvasculature throughout the transplant process could improve the long-term health of transplanted lungs. We recently developed a bronchial-arterial-circulation-sparing (BACS) lung preservation approach and tested whether this approach would decrease microvascular damage and improve allograft function. Methods The lungs of Lewis rats were procured using either the BACS approach, where the bronchial and pulmonary arteries were synchronously perfused; a conventional approach, where only the pulmonary artery was perfused; or a conventional approach with a prostaglandin flush. After 4 hours of cold ischemia, physiologic function and vascular tone of the grafts were evaluated during ex vivo lung perfusion (EVLP), and microvasculature damage was assessed using 2-photon microscopy analysis. Lung function was compared after transplant among the groups. Results After 4 hours of cold ischemia, the BACS group exhibited significantly higher adenosine triphosphate levels and lower expression of phosphorylated myosin light chain, which is essential for vascular smooth muscle contraction. On EVLP, the BACS and prostaglandin groups showed lower pulmonary vascular resistance and less arterial stiffness. BACS attenuated microvasculature damage in the lung grafts when compared with conventional preservation. After transplantation, the lungs preserved with the BACS approach exhibited significantly better graft function and lower expression of phosphorylated myosin light chain. Conclusions Our data suggest that BACS lung preservation protects the dual circulation inherent to the lungs, facilitating robust microvasculature in lung grafts after transplantation, leading to better posttransplant outcomes.

Smooth muscle (SM) contraction is one of the important physiological functions of the human body, and SM abnormal contraction will induce many diseases. The phosphorylated regulatory light chains (p-RLC) play a decisive role in SM contraction, and dephosphorylation of p-RLC is an effective way to relax SM. Our previous study showed that the novel benzylisoquinoline alkaloid, neoliensinine (Neo), could relax microvascular SM contracted by KCl hyperpolarization. In this study, mesenteric capillaries isolated from 45 mice were divided into normal tension group (Control), 124 mM KCl induced contraction model group (Model), and KCl and Neo-treatment group (Drug). The dephosphorylation levels of RLC in the three groups were measured. Compared with the model group, the phosphorylation of RLC in the drug group was decreased dramatically as expected, suggesting that the relaxation effect of Neo was caused by downregulating p-RLC of microvessel SM. In order to fully understand its fundamental mechanism, our research focused on the identification of target proteins in mice with KCl-induced contractile mesenteric capillary. Isobaric tags for relative and absolute quantification (ITRAQ) tagging was carried out by nanospray liquid chromatography–tandem mass spectrometry. The results allowed the upregulation of 164 differential abundance proteins (DAPs) among the 3,474 protein abundance disturbances identified from the model/control samples. Further comparison showed that there were 16 DAP convergences associated with vascular SM contraction between the drug/model and the drug/control samples. Among them, two proteins with known function, PLCβ and RhoGEF12, were selected as target proteins of the relaxation effect of Neo. The two selective target DAPs were verified by Western blot at protein level. The results suggested that changes of the two proteins were consistent with that of the iTRAQ results. Our present work reveals that Neo relaxes vascular smooth muscle via inhibition of RLC phosphorylation, and PLCβ and RhoGEF12 may be potential biomarkers for evaluating the effects mediated by Neo.

Angina pectoris can be used as an early warning for coronary artery disease. Vasodilation is an important mechanism of angina pectoris. Traditional Chinese medicine - Compound Danshen Dripping Pill (CDDP) is widely used to improve the symptoms of cardiovascular diseases (CVDs). To investigate the influence of vasodilation effect and underlying mechanisms of CDDP, we determined the vasodilation effect of thoracic aorta ring on rat induced by norepinephrine (NE). Then targets-fishing method was used to predict the potential mechanism of CDDP on vasodilation, based on the structures of the main components. Then, iTRAQ-based quantitative proteomics analysis was used for verification of the candidate target proteins and pathways to illustrate the underlying mechanisms. Furthermore, the differentially expressed proteins in the enriched pathways were validated by western blotting. In this study, we found that CDDP could significantly inhibit NE induced aortic contraction tension, and the mechanism may be related to platelet activation, cGMP - PKG signaling pathway and vascular smooth muscle contraction. The method provides a new way to uncover the vasodilation mechanism of CDDP, as well as other multi-component herbal medicines.

Preterm birth (PTB), the leading cause of neonatal morbidity and mortality, urgently requires novel therapeutic agents. Spontaneous PTB, resulting from preterm labor, is commonly caused by intrauterine infection/ inflammation. Statins are well‐established, cholesterol‐lowering drugs that can reduce inflammation and inhibit vascular smooth muscle contraction. We show that simvastatin reduced the incidence of PTB in a validated intrauterine LPS‐induced PTB mouse model, decreased uterine proinflammatory mRNA concentrations (IL‐6, Cxcl1, and Ccl2), and reduced serum IL‐6 concentration. In human myometrial cells, simvastatin reduced proinflammatory mediator mRNA and protein expression (IL‐6 and IL‐8) and increased anti‐inflammatory cytokine mRNA expression (IL‐10 and IL‐13). Critically, simvastatin inhibited myometrial cell contraction, basally and during inflammation, and reduced phosphorylated myosin light chain concentration. Supplementation with mevalonate and geranylgeranyl pyrophosphate, but not farnesyl pyrophosphate, abolished these anticontractile effects, indicating that the Rho/Rho‐associated protein kinase pathway is critically involved. Thus, simvastatin reduces PTB incidence in mice, inhibits myometrial contractions, and exhibits key anti‐inflammatory effects, providing a rationale for investigation into the repurposing of statins to treat preterm labor in women.—Boyle, A. K., Rinaldi, S. F., Rossi, A. G., Saunders, P. T. K., Norman, J. E. Repurposing simvastatin as a therapy for preterm labor: evidence from preclinical models. FASEB J. 33, 2743–2758 (2019). www.fasebj.org

OBJECTIVES: To investigate the effects of plant sterols intake on systematic and tissue specific lipid metabolism in C57BL/6J mice. METHODS: Male C57BL/6J mice were randomly divided into control diet group (CS) and plant sterol group (PS, 2% plant sterols). After 28 weeks of continuous feeding, the serum of the mice were collected for biochemical and mass spectrometry tests. Serum levels of total cholesterol (TC), triglyceride (TG) and free sterols were determined. The livers and lungs were collected for free sterol quantification and RNA-seq analysis. RESULTS: Compared with the CS group, 2% plant sterols intake significantly reduced the levels of TC in the serum of mice (P

Lysophosphatidic acid (LPA) has been recognized recently as an endothelium-dependent vasodilator, but several lines of evidence indicate that it may also stimulate vascular smooth muscle cells (VSMCs), thereby contributing to vasoregulation and remodeling. In the present study, mRNA expression of all 6 LPA receptor genes was detected in murine aortic VSMCs, with the highest levels of LPA1, LPA2, LPA4, and LPA6 In endothelium-denuded thoracic aorta (TA) and abdominal aorta (AA) segments, 1-oleoyl-LPA and the LPA1-3 agonist VPC31143 induced dose-dependent vasoconstriction. VPC31143-induced AA contraction was sensitive to pertussis toxin (PTX), the LPA1&3 antagonist Ki16425, and genetic deletion of LPA1 but not that of LPA2 or inhibition of LPA3, by diacylglycerol pyrophosphate. Surprisingly, vasoconstriction was also diminished in vessels lacking cyclooxygenase-1 [COX1 knockout (KO)] or the thromboxane prostanoid (TP) receptor (TP KO). VPC31143 increased thromboxane A2 (TXA2) release from TA of wild-type, TP-KO, and LPA2-KO mice but not from LPA1-KO or COX1-KO mice, and PTX blocked this effect. Our findings indicate that LPA causes vasoconstriction in VSMCs, mediated by LPA1-, Gi-, and COX1-dependent autocrine/paracrine TXA2 release and consequent TP activation. We propose that this new-found interaction between the LPA/LPA1 and TXA2/TP pathways plays significant roles in vasoregulation, hemostasis, thrombosis, and vascular remodeling.-Dancs, P. T., Ruisanchez, E., Balogh, A., Panta, C. R., Miklos, Z., Nusing, R. M., Aoki, J., Chun, J., Offermanns, S., Tigyi, G., Benyo, Z. LPA1 receptor-mediated thromboxane A2 release is responsible for lysophosphatidic acid-induced vascular smooth muscle contraction.

The pharmacology of cannabidiol, the non-psychoactive major component of Cannabis sativa, is of growing interest as it becomes more widely prescribed. This study aimed to examine the effects of cannabidiol on a wide range of contractile agents in rat small resistance arteries, in comparison with large arteries, and to explore its mechanism of action. The vascular actions of cannabidiol were also contrasted with effects on the contractions of bronchial, urogenital, cardiac and skeletal muscles. Isolated small or large arteries were incubated with cannabidiol (0.3-3 μM) or vehicle and concentration-contraction response curves were completed to various agents, including endothelin-1, arginine vasopressin, methoxamine, 5-HT, α-methyl 5-HT and U46619. In small arteries, the effects of cannabidiol were tested in the presence of antagonists of CB1 or CB2 receptors, calcitonin gene-related peptide (CGRP), nitric oxide synthase, cyclooxygenase, PPARγ or a combination. The role of L-type voltage-operated calcium channels was also assessed. Cannabidiol 1-3 μM significantly inhibited the contraction of small resistance arteries to all tested agents through a combination of mechanisms that include CGRP and L-type calcium channels. However, large arteries were insensitive to cannabidiol. Cannabidiol (10-100 μM) was largely without effect in bronchi, atria and hemidiaphragm, but 100 μM attenuated maximum contractions in vasa deferentia. Cannabidiol's effects in the clinical range (1-3 μM) appear to be specific to small resistance arteries. This high sensitivity of the resistance arterial circulation to cannabidiol may offer a therapeutic opportunity in peripheral vascular disease that excludes off-target sites such as the heart and non-vascular smooth muscle.

Fluorine, an environmental toxicant in our daily life, has been reported to have adverse effects on nervous system. Previous studies demonstrated that fluorine exposure could induce brain injury in fish and human. However, the possible mechanism remains unclear. In the present study, we aimed to reveal the mechanism of fluorine exposure on brain injury of common carp through transcriptome analysis. In the fluorine-exposed carp, 444 brain genes were up-regulated, whereas 742 genes were down-regulated. DNA-templated (regulation of transcription) and multicellular organism development in the GO function annotation accounted for the most biological processes. Nucleus and membrane accounted for the most cellular components and DNA binding and metal ion binding accounted for the most molecular function. Meanwhile, 196 metabolic pathways were identified in Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway significant enrichment analysis, including long-term depression, Cushing syndrome, nuclear receptors, vascular smooth muscle contraction, Ion channels, and other pathways. Furthermore, we found that the up-regulated and down-regulated trends were similar between the quantitative real-time-PCR and RNA-Seq results, which indicate the transcriptome sequencing data is reliable. In conclusion, our data may provide insights into the mechanisms underlying brain injury induced by fluorine exposure.

The fermentation of mare's milk into koumiss produces many beneficial functional compounds depending on the metabolism of the initial microbial flora. In this study, metabolites found in mare's milk and resulting koumiss were identified. Major metabolic pathways in the fermentation were also identified using an UPLC-Q-TOF-MS-based metabolomics method. In total, 354 metabolites were identified: 61 were up-regulated and 105 were down-regulated. Metabolic pathway analyses revealed that c-5-branched dibasic acid metabolism, valine, leucine and isoleucine degradation, arginine and proline metabolism, valine, leucine and isoleucine biosynthesis, vascular smooth muscle contraction, aminoacyl-tRNA biosynthesis and ß-alanine metabolism showed significant increases. A hierarchical cluster analysis of metabolites indicated a clear grouping pattern in which the relative concentrations of p-pyruvate, 20-HETE, 4-aminobutanoate, uracil, acetoacetate, and γ-linolenic acid differed significantly between milk and koumiss. This study provides reference values for metabolic isolates and bioactive compounds purification in mare's milk and koumiss.

Background Vitex pubescens has been used traditionally in hypertension treatment but not yet scientifically assessed. The objective of the study is to investigate the antihypertensive and vasorelaxant activities of V. pubescens, study its underlying pharmacological mechanisms, and identify the relevant vasoactive compounds. Methods Successive extractions of V. pubescens leaf were carried out to produce petroleum ether (VPPE), chloroform (VPCE), methanol (VPME), and water (VPWE) extracts. Spontaneously hypertensive rats (SHRs) received a daily oral administration of the extracts (500 mg/kg/day; n = 6) or verapamil (15 mg/kg/day; n = 6) for 2 weeks, while the systolic and diastolic blood pressures were measured using non-invasive tail-cuff method. Vasorelaxation assays of the extracts were later conducted using phenylephrine (PE, 1 μM) pre-contracted aortic ring preparation. Mechanisms of vasorelaxation by the most potent fraction were studied using vasorelaxation assays with selected blockers/inhibitors. GC-MS was conducted to determine the active compounds. Results VPPE elicited the most significant diminution in systolic and diastolic blood pressure of treated SHRs and produced the most significant vasorelaxation in the aortic rings. Vasorelaxant effects of F2-VPPE were significantly reduced in endothelium-denuded aortic rings by glibenclamide (1 μM), whereas calcium chloride and PE-induced contractions were significantly suppressed. Endothelium removal of the aortic rings or incubation with indomethacin (10 μM), atropine (1 μM), methylene blue (10 μM), propranolol (1μM) and L-NAME (10 μM) did not significantly alter F2-VPPE-induced vasorelaxation. Seven compounds were identified using GC-MS, including spathulenol. Conclusion F2-VPPE exerted its endothelium-independent vasorelaxation by inhibition of vascular smooth muscle contraction induced by extracellular Ca+2 influx through trans-membrane Ca+2 channels and/or Ca+2 release from intracellular stores, and by activation of KATP channels. The vasorelaxation effects of V. pubescens could be mediated by the compound, spathulenol., Vitex pubescens; Antihypertensive; Vasorelaxation; Aortic ring; GC-MS; Spathulenol; Pharmaceutical chemistry; Plant biology; Pharmaceutical science; Cardiology; Pharmacology

Breed improvement is an important genetic process affecting meat quality. Compared with Kazakh cattle, Xinjiang brown cattle have significantly improved carcass quality and meat quality. To elucidate the molecular mechanisms underlying the improvements in Xinjiang brown cattle and the differences in beef quality between the two breeds, we used RNA-Seq to study differentially expressed genes and miRNAs and regulatory pathways related to adipogenesis, myogenesis and fibrogenesis in the longissimus dorsi muscles of Xinjiang brown cattle and Kazakh cattle. The results showed that 1669 genes were differentially expressed in the longissimus dorsi muscle tissues of Xinjiang brown cattle and Kazakh cattle; 879 genes were upregulated and 790 genes were downregulated in Xinjiang brown cattle compared to Kazakh cattle. These genes were mainly involved in PPAR signaling, unsaturated fatty acid biosynthesis and vascular smooth muscle contraction. Additionally, 346 differentially expressed miRNAs were identified, of which 265 miRNAs were downregulated and 81 miRNAs were upregulated in Xinjiang brown cattle compared to Kazakh cattle. Association analysis of the differentially expressed genes and miRNAs revealed that 86 differentially expressed miRNAs related to adipogenesis were associated with 31 differentially expressed genes, 76 differentially expressed miRNAs associated with myogenesis were associated with 28 differentially expressed genes, and 54 differentially expressed miRNAs associated with fibrogenesis were associated with 19 differentially expressed genes. miRNA-target gene networks were also constructed. Finally, the expression levels of 19 genes and miRNAs were verified by qRT-PCR. Some differentially expressed genes, including FABP4, ACTA2 and ACTG2, were shown to play an important role in beef meat quality. This is the first study to perform transcriptomic analysis of muscle tissues from Xinjiang brown and Kazakh cattle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K(+) solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K(+) solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K(+) solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

Background Cerebrovascular dysfunction plays a critical role in the pathogenesis of Alzheimer’s disease (AD): the most common cause of dementia in the elderly. The involvement of neurovasculature disorders in the progression of AD is now increasingly appreciated, but whether they represent initial factors or late-stage pathological changes during the disease is unclear. Using senescence-accelerated OXYS rats, which simulate key characteristics of sporadic AD, we evaluated contributions of cerebrovascular alterations to the disease development. At preclinical, early, and advanced stages of AD-like pathology, in the hippocampus of OXYS and Wistar (control) rats, we evaluated (i) the blood vessel state by histological and electron-microscopic analyses; (ii) differences in gene expression according to RNA sequencing (RNA-Seq) to identify the metabolic processes and pathways associated with blood vessel function; (iii) the amount of vascular endothelial growth factor (VEGF) by western blot and immunohistochemical analysis. Results We observed a loss of hippocampal blood vessel density and ultrastructural changes of those blood vessels in OXYS rats at the early stage of AD-like pathology. There were significant alterations in the vessels and downregulation of VEGF with an increased amount of amyloid β1–42 there at the advanced stage of the disease. According to RNA-Seq data analysis, major alterations in cerebrovascular processes of OXYS rats were associated with blood vessel development, circulatory system processes, the VEGF signaling pathway, and vascular smooth muscle contraction. At preclinical and early stages of the AD-like pathology, these processes were upregulated and then downregulated with age. At the advanced stage in OXYS rats, differentially expressed genes (DEGs) were associated with downregulation of cerebrovascular function as compared to Wistar rats. Among the 46 DEGs at the preclinical stage of the disease, 28 DEGs at the early stage, and among 85 DEGs at the advanced stage, using functional analysis and gene network construction, we identified genes (Nos1, P2rx4, Pla2g6, and Bdkrb2) probably playing a significant role in the development of cerebrovascular dysfunction in OXYS rats. Conclusions Changes in expression of the genes functionally associated with cerebrovascular processes already in the early period of life may contribute to the development of AD-like pathology in OXYS rats. Electronic supplementary material The online version of this article (10.1186/s12864-018-4480-9) contains supplementary material, which is available to authorized users.

The L-type calcium channel (LCC) plays a regulatory role in various physical and pathological processes. In the vasculature, LCCs mediate agonist-induced vascular smooth muscle contraction. However, whether LCC-mediated vessel responses to certain vasoconstrictors vary among species remains unclear. The coronary arteries were dissected from the hearts of rats and mice. Coronary arterial ring contraction was measured using the Multi Myograph system. High K+ (60 mM)-induced coronary artery contractions were stronger in rats than in mice, whereas CaCl2-induced contraction curves did not differ significantly between the two groups. Endothelin-1, U46619 (thromboxane A2 receptor agonist), and 5-hydroxytryptamine (5-HT) induced concentration-dependent vasoconstriction of coronary arterial rings in rats and mice. The vessel rings of mice were more sensitive to ET-1 and U46619 and less sensitive to 5-HT than those of rats. The LCC blocker nifedipine significantly inhibited coronary artery contractions induced by ET-1, U46619, and 5-HT. The inhibitory effect of 1 μM nifedipine on ET-1- and 5-HT-induced coronary artery contractions was stronger in mice than in rats, whereas its effect on U46619-induced vessel contractions was weaker in mice than in rats. The 5-HT2A receptor and LCC mRNA levels were higher in the coronary arteries of rats than in those of mice, whereas the expressions of the ETA and TXA2 receptors and Orai1 mRNA levels were comparable between the two groups. LCC plays an important role in coronary arterial contraction. Rats and mice show different responses to vasoconstrictors and LCC blockers, suggesting that the coronary arteries of rats and mice have different biological characteristics.

Depolarization of the plasma membrane is a key mechanism of activation of contraction of vascular smooth muscle. This is commonly achieved in isolated, de-endothelialized vascular smooth muscle strips by increasing extracellular [K(+)] (replacing Na(+) by K(+)) and leads to a rapid phasic contraction followed by a sustained tonic contraction. The initial phasic contractile response is due to opening of voltage-gated Ca(2+) channels and entry of extracellular Ca(2+), which binds to calmodulin, leading to activation of myosin light chain kinase, phosphorylation of the regulatory light chains of myosin II at Ser19 and cross-bridge cycling. The subsequent tonic contractile response involves, in addition to myosin light chain kinase activation, Ca(2+)-induced Ca(2+) sensitization whereby Ca(2+) entry activates the RhoA/Rho-associated kinase pathway leading to phosphorylation of MYPT1 (the myosin targeting subunit of myosin light chain phosphatase) and inhibition of the phosphatase. Investigations into the mechanism of activation of RhoA by Ca(2+) have implicated a genistein-sensitive tyrosine kinase, and recent evidence indicates this to be the Ca(2+)-dependent tyrosine kinase, Pyk2.

金沢大学医薬保健研究域医学系, Class II phosphoinositide 3-kinases (PI3Ks), PI3K-C2α and PI3K-C2β, are highly homologous and distinct from class I and class III PI3Ks in catalytic products and domain structures. In contrast to class I and class III PI3Ks, physiological roles of PI3K-C2α and PI3K-C2β are not fully understood. Because we previously demonstrated that PI3K-C2α is involved in vascular smooth muscle contraction, we studied the phenotypes of smooth muscle-specific knockout (KO) mice of PI3K-C2α and PI3K-C2β. The pup numbers born from single PI3K-C2α-KO and single PI3K-C2β-KO mothers were similar to those of control mothers, but those from double KO (DKO) mothers were smaller compared with control mice. However, the number of intrauterine fetuses in pregnant DKO mothers was similar to that in control mice. Both spontaneous and oxytocin-induced contraction of isolated uterine smooth muscle (USM) strips was diminished in DKO mice but not in either of the single KO mice, compared with control mice. Furthermore, contraction of USM of DKO mice was less sensitive to a Rho kinase inhibitor. Mechanistically, the extent of oxytocin-induced myosin light chain phosphorylation was greatly reduced in USM from DKO mice compared with control mice. The oxytocin-induced rise in the intracellular Ca2+ concentration in USM was similar in DKO and control mice. However, Rho activation in the intracellular compartment was substantially attenuated in DKO mice compared with control mice, as evaluated by fluorescence resonance energy transfer imaging technique. These data indicate that both PI3K-C2α and PI3K-C2β are required for normal USM contraction and parturition mainly through their involvement in Rho activation., Embargo Period 12 months

Vascular smooth muscle (VSM) plays an important role in the regulation of vascular function. Identifying the mechanisms of VSM contraction has been a major research goal in order to determine the causes of vascular dysfunction and exaggerated vasoconstriction in vascular disease. Major discoveries over several decades have helped to better understand the mechanisms of VSM contraction. Ca(2+) has been established as a major regulator of VSM contraction, and its sources, cytosolic levels, homeostatic mechanisms and subcellular distribution have been defined. Biochemical studies have also suggested that stimulation of Gq protein-coupled membrane receptors activates phospholipase C and promotes the hydrolysis of membrane phospholipids into inositol 1,4,5-trisphosphate (IP(3)) and diacylglycerol (DAG). IP(3) stimulates initial Ca(2+) release from the sarcoplasmic reticulum, and is buttressed by Ca(2+) influx through voltage-dependent, receptor-operated, transient receptor potential and store-operated channels. In order to prevent large increases in cytosolic Ca(2+) concentration ([Ca(2+)](c)), Ca(2+) removal mechanisms promote Ca(2+) extrusion via the plasmalemmal Ca(2+) pump and Na(+)/Ca(2+) exchanger, and Ca(2+) uptake by the sarcoplasmic reticulum and mitochondria, and the coordinated activities of these Ca(2+) handling mechanisms help to create subplasmalemmal Ca(2+) domains. Threshold increases in [Ca(2+)](c) form a Ca(2+)-calmodulin complex, which activates myosin light chain (MLC) kinase, and causes MLC phosphorylation, actin–myosin interaction, and VSM contraction. Dissociations in the relationships between [Ca(2+)](c), MLC phosphorylation, and force have suggested additional Ca(2+) sensitization mechanisms. DAG activates protein kinase C (PKC) isoforms, which directly or indirectly via mitogen-activated protein kinase phosphorylate the actin-binding proteins calponin and caldesmon and thereby enhance the myofilaments force sensitivity to Ca(2+). PKC-mediated phosphorylation of PKC-potentiated phosphatase inhibitor protein-17 (CPI-17), and RhoA-mediated activation of Rho-kinase (ROCK) inhibit MLC phosphatase and in turn increase MLC phosphorylation and VSM contraction. Abnormalities in the Ca(2+) handling mechanisms and PKC and ROCK activity have been associated with vascular dysfunction in multiple vascular disorders. Modulators of [Ca(2+)](c), PKC and ROCK activity could be useful in mitigating the increased vasoconstriction associated with vascular disease.

Farrerol, a typical natural flavanone and major active component in Rhododendron dauricum var. ciliatum, has been shown to possess vasoactive ability in vitro. The aim of this study was to investigate its effect on aorta gene expression in spontaneously hypertensive rats. Twelve-week-old male normotensive Wistar Kyoto rats and spontaneously hypertensive rats were treated with orally administered farrerol (50 mg/kg body weight) for 8 wk before they were sacrificed. We found that aorta samples showed 444 upregulated genes in control spontaneously hypertensive rats compared with the control Wistar Kyoto rats. Administration of farrerol in spontaneously hypertensive rats increased the expression of 2329 genes in the aorta compared with the control spontaneously hypertensive rats. Gene expression profiles performed on the aorta revealed that farrerol induced changes in vascular smooth muscle contraction, mitogen-activated protein kinase signaling pathway, regulation of actin cytoskeleton, vascular endothelial growth factor signaling pathway, calcium signaling pathway, and renin angiotensin system. Furthermore, 10 genes involved in the pathway of vascular smooth muscle contraction were verified using real-time polymerase chain reaction technique, and several novel potential target genes for the farrerol treatment of hypertension were identified. The findings of this study lend support to the potential use of farrerol as a novel therapeutic and antihypertensive candidate drug to prevent the development of hypertension.

There is accumulating evidence that makes the link between the circadian variation in blood pressure and circadian variations in vascular contraction. The importance of vascular endothelium-derived redox-active and redox-derived species in the signalling pathways involved in controlling vascular smooth muscle contraction are well known, and when linked to the circadian variations in the processes involved in generating these species, suggests a cellular mechanism for the circadian variations in blood pressure that links directly to the peripheral circadian clock. Relaxation of vascular smooth muscle cells involves endothelial-derived relaxing factor (EDRF) which is nitric oxide (NO) produced by endothelial NO synthase (eNOS), and endothelial-derived hyperpolarising factor (EDHF) which includes hydrogen peroxide (H2O2) produced by NADPH oxidase (Nox). Both of these enzymes appear to be under the direct control of the circadian clock mechanism in the endothelial cells, and disruption to the clock results in endothelial and vascular dysfunction. In this review, we focus on EDRF and EDHF and summarise the recent findings on the influence of the peripheral circadian clock mechanism on processes involved in generating the redox species involved and how this influences vascular contractility, which may account for some of the circadian variations in blood pressure and peripheral resistance. Moreover, the direct link between the peripheral circadian clock and redox-signalling pathways in the vasculature, has a bearing on vascular endothelial dysfunction in disease and aging, which are both known to lead to dysfunction of the circadian clock.

Vascular smooth muscle contraction is suppressed by feedback dilation mediated by the endothelium. In skeletal muscle arterioles, this feedback can be activated by Ca2+ signals passing from smooth muscle through gap junctions to endothelial cells, which protrude through holes in the internal elastic lamina to make contact with vascular smooth muscle cells. Although hypothetically either Ca2+ ions or inositol trisphosphate (IP3) may provide the intercellular signal, it is generally thought that IP3 diffusion is responsible. Here, we provide evidence that Ca2+ entry through L-type voltage-dependent Ca2+ channels (VDCCs) in vascular smooth muscle can pass to the endothelium through positions aligned with holes in the internal elastic lamina in amounts sufficient to activate endothelial cell Ca2+ signalling. In endothelial cells in which IP3 receptors (IP3Rs) were blocked, VDCC-driven Ca2+ events were transient and localized to the endothelium that protrudes through the internal elastic lamina to contact vascular smooth muscle cells. In endothelial cells in which IP3Rs were not blocked, VDCC-driven Ca2+ events in endothelial cells were amplified to form propagating waves. These waves activated voltage-insensitive, intermediate-conductance, Ca2+-activated K+ (IKCa) channels, thereby providing feedback that effectively suppressed vasoconstriction and enabled cycles of constriction and dilation called vasomotion. Thus, agonists that stimulate vascular smooth muscle depolarization provide Ca2+ to endothelial cells to activate a feedback circuit that protects tissue blood flow.

Enantioselectivity in the aquatic toxicity of chiral pesticides has been widely investigated, while the molecular mechanisms remain unclear. Thus far, few studies has focused on genomic expression related to selective toxicity in chiral pesticide, nor on epigenetic changes, such as DNA methylation. Here, we used fipronil, a broad-spectrum insecticide, as a model chemical to probe its enantioselective toxicity in embryo development. Our results showed that S-(+)-fipronil caused severer developmental toxicity in embryos. The MeDIP-Seq analysis demonstrated that S-(+)-fipronil dysregulated a higher level of genomic DNA methylation than R-(−)-fipronil. Gene Ontology analysis revealed that S-(+)-fipronil caused more differentially methylated genes that are involved in developmental processes. Compared with R-(−)-fipronil, S-(+)-fipronil significantly disrupted 7 signaling pathways (i.e., mitogen-activated protein kinases, tight junctions, focal adhesion, transforming growth factor-β, vascular smooth muscle contraction, and the hedgehog and Wnt signaling pathways) by hyper-methylation of developmentally related genes, which further induced the downregulation of those genes. Together, these data suggest that differences in DNA methylation may partly explain the enantioselectivity of fipronil to zebrafish embryos. The application of epigenetics to investigate the enantioselective toxicity mechanism of chiral chemicals would provide a further understanding of their stereoselectivity biological effects.

We previously reported that eicosapentaenoic acid (EPA), an omega-3 polyunsaturated fatty acid (n-3 PUFA), effectively inhibits sphingosylphosphorylcholine (SPC)-induced Ca2+-sensitization of vascular smooth muscle (VSM) contraction which is a major cause of cardiovascular and cerebrovascular vasospasm, and EPA is utilized clinically to prevent cerebrovascular vasospasm. In this study, we clearly demonstrate that docosapentaenoic acid (DPA), which exists in two forms as omega-3 (n-3) and omega-6 (n-6) PUFA, strongly inhibits SPC-induced contraction in VSM tissue and human coronary artery smooth muscle cells (CASMCs), with little effect on Ca2+-dependent contraction. Furthermore, n-3 and n-6 DPA inhibited the activation and translocation of Rho-kinase from cytosol to cell membrane. Additionally, SPC-induced phosphorylation of myosin light chain (MLC) was inhibited in n-3 and n-6 DPA pretreated smooth muscleVSM cells and tissues. In summary, we provide direct evidence that n-3 and n-6 DPA effectively equally inhibits SPC-induced contraction by inhibiting Rho-kinase activation and translocation to the cell membrane.

The article discusses the data on the functioning of Na + ,K + ,2Cl – cotransport – the carrier providing electroneutral symport of sodium, potassium and chloride, as well as molecular mechanisms of the regulation and physiological significance of this carrier. We analyzed the novel data on involvement of ubiquitous isoform of Na + ,K + ,2Cl – cotransporter (NKCC1) in regulation of vascular smooth muscle contraction, and role of this carrier in the regulation of cell volume and intracellular chloride concentration.

This review summarizes the data on the functioning of carriers providing electroneutral symport of sodium, potassium, and chloride (Na(+),K(+),2Cl(-) cotransport), potassium and chloride (K(+),Cl(-) cotransport), and sodium and chloride (K(+),Cl(-) cotransport) as well as molecular mechanisms of the regulation of these carriers and their physiological significance. We emphasized the involvement of chloride-coupled carriers in the regulation of cell volume and intracellular chloride concentration and novel data on the role of ubiquitous isoform of Na(+),K(+),2Cl(-) cotransporter NKCC1 in regulation of vascular smooth muscle contraction and activity of GABA(A) receptors. Finally, we analyzed the data on activation of NKCC1 in patients with essential hypertension and its role in the long-term maintenance of elevated systemic blood pressure and myogenic response in microcirculatory beds.

Background and Purpose Calcium-activated chloride channels (CaCCs) are key depolarizing mechanisms that have an important role in vascular smooth muscle contraction. Here, we investigated whether these channels are regulated by phosphatidylinositol (4,5) bisphosphate [P(4,5)P2], a known regulator of various ion channels. Experimental Approach Calcium-activated Cl− currents (IClCa) were recorded by patch clamp electrophysiology of rat isolated pulmonary artery smooth muscle cells. TMEM16A protein-phosphoinositide interaction was studied by co-immunoprecipitation and phosphoinositide binding arrays on protein lysates from whole pulmonary arteries and HEK293 cells overexpressing TMEM16A, the molecular correlate. Key Results PI(4,5)P2 and other phospholipids were shown to bind directly to TMEM16A isolated from whole pulmonary artery (PA) and TMEM16A-eGFP expressed in HEK293 cells. Agents that reduced PI(4,5)P2 levels through different routes [PLC activation, PI4K inhibition, PI(4,5)P2 scavenging and absorption] all increased IClCa evoked by solutions containing clamped-free [Ca2+], whereas enrichment of activating solutions with PI(4,5)P2 inhibited IClca in PA smooth muscle cells with approximately 50% reduction at 1 μM. Conclusions and Implications These data are the first to show a negative regulation of TMEM16A-encoded CaCCs by PI(4,5)P2 and propose that control of PI(4,5)P2 levels is a key determinant of arterial physiology.

The purpose of this study was to determine the effects of resveratrol (RSV) and the molecular mechanisms by which it regulates vascular smooth muscle contraction and blood pressure in mice. In cultured human vascular smooth muscle cells (VSMCs), we found that the activation of AMP-activated protein kinase (AMPK) by RSV inhibited angiotensin II (AngII)-induced phosphorylation of myosin phosphatase-targeting subunit 1 (MYPT1) and myosin light chain (MLC). Inversely, AMPK inhibition with RNA interference and compound C, an AMPK inhibitor, abolished the inhibitory effect of RSV on AngII-induced MYPT1 and MLC phosphorylation. Thiazovivin, a Rho-associated kinase (ROCK) inhibitor, reversed AngII-induced MYPT1 and MLC phosphorylation, suggesting that ROCK functions as an upstream kinase for MYPT1/MLC. RSV reversed AngII-induced Ras homolog gene family member A (RhoA) and ROCK activity, whereas AMPK inhibition via pharmacological or genetic means abolished this effect. In addition, gene silencing of p190-guanosine triphosphatase-activating protein blocked the effects of RSV-induced AMPK activation on MLC, MYPT1 and RhoA in VSMCs. Ex vivo analyses demonstrated that AngII-induced aorta contractions were dramatically inhibited by RSV, and this effect was abolished by AMPK inhibition. Finally, daily chronic administration of RSVl alleviated hypertension in the experimental model of AngII-induced hypertensive mice, and these effects were accompanied by the activation of AMPK, significantly decreased RhoA activity and phosphorylation levels of MYPT1 and MLC in AngII-treated murine aortic VSMCs. More importantly, administration of compound C significantly abolished the effects of RSV. In conclusion, AMPK suppression of the p190-GAP-dependent RhoA/ROCK/MYPT1/MLC pathway contributes to the hypotensive effect of RSV in AngII-treated mice.

Myosin regulatory light chain (LC20) phosphorylation plays an important role in vascular smooth muscle contraction and cell migration. Ca2+/calmodulin-dependent myosin light chain kinase (MLCK) phosphorylates LC20 (its only known substrate) exclusively at S19. Rho-associated kinase (ROCK) and zipper-interacting protein kinase (ZIPK) have been implicated in the regulation of LC20 phosphorylation via direct phosphorylation of LC20 at T18 and S19 and indirectly via phosphorylation of MYPT1 (the myosin targeting subunit of myosin light chain phosphatase, MLCP) and Par-4 (prostate-apoptosis response-4). Phosphorylation of MYPT1 at T696 and T853 inhibits MLCP activity whereas phosphorylation of Par-4 at T163 disrupts its interaction with MYPT1, exposing the sites of phosphorylation in MYPT1 and leading to MLCP inhibition. To evaluate the roles of MLCK, ROCK and ZIPK in these phosphorylation events, we investigated the time courses of phosphorylation of LC20, MYPT1 and Par-4 in serum-stimulated human vascular smooth muscle cells (from coronary and umbilical arteries), and examined the effects of siRNA-mediated MLCK, ROCK and ZIPK knockdown and pharmacological inhibition on these phosphorylation events. Serum stimulation induced rapid phosphorylation of LC20 at T18 and S19, MYPT1 at T696 and T853, and Par-4 at T163, peaking within 30-120 s. MLCK knockdown or inhibition, or Ca2+ chelation with EGTA, had no effect on serum-induced LC20 phosphorylation. ROCK knockdown decreased the levels of phosphorylation of LC20 at T18 and S19, of MYPT1 at T696 and T853, and of Par-4 at T163, whereas ZIPK knockdown decreased LC20 diphosphorylation, but increased phosphorylation of MYPT1 at T696 and T853 and of Par-4 at T163. ROCK inhibition with GSK429286A reduced serum-induced phosphorylation of LC20 at T18 and S19, MYPT1 at T853 and Par-4 at T163, while ZIPK inhibition by HS38 reduced only LC20 diphosphorylation. We also demonstrated that serum stimulation induced phosphorylation (activation) of ZIPK, which was inhibited by ROCK and ZIPK down-regulation and inhibition. Finally, basal phosphorylation of LC20 in the absence of serum stimulation was unaffected by MLCK, ROCK or ZIPK knockdown or inhibition. We conclude that: (i) serum stimulation of cultured human arterial smooth muscle cells results in rapid phosphorylation of LC20, MYPT1, Par-4 and ZIPK, in contrast to the slower phosphorylation of kinases and other proteins involved in other signaling pathways (Akt, ERK1/2, p38 MAPK and HSP27), (ii) ROCK and ZIPK, but not MLCK, are involved in serum-induced phosphorylation of LC20, (iii) ROCK, but not ZIPK, directly phosphorylates MYPT1 at T853 and Par-4 at T163 in response to serum stimulation, (iv) ZIPK phosphorylation is enhanced by serum stimulation and involves phosphorylation by ROCK and autophosphorylation, and (v) basal phosphorylation of LC20 under serum-free conditions is not attributable to MLCK, ROCK or ZIPK.

Microbial transformation stands out among the many possible semi-synthetic strategies employed to increase the variety of chemical structures that can be applied in the search for novel bioactive compounds. In this paper we obtained ent -pimaradienoic acid ( 1 , PA, ent- pimara-8(14),15-dien-19-oic acid) derivatives by fungal biotransformation using Aspergillus niger strains. To assess the ability of such compounds to inhibit vascular smooth muscle contraction, we also investigated their spasmolytic effect, along with another five PA derivatives previously obtained in our laboratory, on aortic rings isolated from male Wistar rats. The microbial transformation experiments were conducted at 30 °C using submerged shaken liquid culture (120 rpm) for 10 days. One known compound, 7α-hydroxy ent -pimara-8(14),15-dien-19-oic acid ( 2 ), and three new derivatives, 1β-hydroxy ent -pimara-6,8(14),15-trien-19-oic acid ( 3 ), 1α,6β,14β-trihydroxy ent -pimara-7,15-dien-19-oic acid ( 4 ), and 1α,6β,7α,11α-tetrahydroxy ent -pimara-8(14),15-dien-19-oic acid ( 5 ), were isolated and identified on the basis of spectroscopic analyses and computational studies. The compounds obtained through biotransformation ( 2 – 5 ) did not display a significant antispasmodic activity (values ranging from 0% to 16.8% of inhibition); however the previously obtained diterpene, methyl 7α-hydroxy ent -pimara-8(14),15-dien-19-oate ( 8 ), showed to be very effective (82.5% of inhibition). In addition, our biological results highlight the importance to study the antispasmodic potential of a large number of novel diterpenes, to conduct further structure–activity relationship investigations.

Potassium channels importantly contribute to the regulation of vascular smooth muscle (VSM) contraction and growth. They are the dominant ion conductance of the VSM cell membrane and importantly determine and regulate membrane potential. Membrane potential, in turn, regulates the open-state probability of voltage-gated Ca2+ channels (VGCC), Ca2+ influx through VGCC, intracellular Ca2+ and VSM contraction. Membrane potential also affects release of Ca2+ from internal stores and the Ca2+ sensitivity of the contractile machinery such that K+ channels participate in all aspects of regulation of VSM contraction. Potassium channels also regulate proliferation of VSM cells through membrane potential-dependent and membrane potential-independent mechanisms. Vascular smooth muscle cells express multiple isoforms of at least five classes of K+ channels contribute to the regulation of contraction and cell proliferation (growth). This review will examine the structure, expression and function of large-conductance, Ca2+-activated K+ (BKCa) channels, intermediate-conductance Ca2+-activated K+ (KCa3.1) channels, multiple isoforms of voltage-gated K+ (KV) channels, ATP-sensitive K+ (KATP) channels, and inward-rectifier K+ (KIR) channels in both contractile and proliferating VSM cells.

Vascular smooth muscle contraction is an important physiological process contributing to cardiovascular homeostasis. The principal determinant of smooth muscle contraction is the intracellular free Ca2+ concentration, and phosphorylation of myosin light chain (MLC) by activated myosin light chain kinase (MLCK) in response to increased Ca2+ is the main pathway by which vasoconstrictor stimuli induce crossbridge cycling of myosin and actin filaments. A secondary pathway for vascular smooth muscle contraction that is not directly dependent on Ca2+ concentration, but rather mediating Ca2+ sensitization, is the RhoA/Rho kinase pathway. In response to contractile stimuli, the small GTPase RhoA activates its downstream effector Rho kinase which, in turn, promotes contraction via myosin light chain phosphatase (MLCP) inhibition. RhoA/Rho kinase-mediated MLCP inhibition occurs mainly by phosphorylation and inhibition of MYPT1, the regulatory subunit of MLCP, or by CPI-17-mediated inhibition of the catalytic subunit of MLCP. In this review, we describe the molecular mechanisms underlying the pivotal role exerted by Rho kinase on vascular smooth muscle contraction and discuss the main regulatory pathways for its activity.