Artery Rings Research Articles

Antihypertensive effects of allicin on spontaneously hypertensive rats via vasorelaxation and hydrogen sulfide mechanisms

BackgroundAllicin, the principle active constituent in garlic, has been reported to have antihypertensive effects on drug-induced hypertension or renal hypertension in rats, but reports on spontaneously hypertensive rats (SHRs) are rare. Allicin is comprised of a variety of sulfur-containing compounds, and hydrogen sulfide (H2S) has been shown to have specific vasomotor effects. We therefore hypothesize that allicin may exert a vasorelaxant activity by inducing H2S production, and this eventually result in a reduction in blood pressure in SHRs. MethodsThe in vivo antihypertensive effect of allicin was assessed using a tail-cuff method on SHRs. The in vitro vasorelaxant effect and in-depth mechanisms of allicin were explored on rat mesenteric arterial rings (RMARs) isolated from SD rats. ResultsIn the in vivo study, administration of allicin (7 mg/kg and 14 mg/kg, 4 weeks, i.g) dramatically decreased the blood pressure in SHRs, which was also shown to be attenuated by H2S synthase inhibitor (PAG, 32 mg/kg, i.g). In in vitro studies, allicin (2.50–15.77 mM) produced a concentration-dependent vasorelaxation on RMARs, which was obviously reduced by preincubation with PAG. The removal of endothelium led to a decline in allicin’s vasorelaxation, which was almost completely mitigated when treatment was followed with PAG. Inhibitors of nitric oxide (NO) and prostaglandin (PGI2) pathways separately suppressed the vasorelaxation induced by allicin to a certain degree. When the RMARs incubated with PAG were treated with or without the above inhibitors in separate groups, the relaxations caused by allicin were almost identical under both these conditions. Moreover, allicin treatment increased cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) levels (downstream products of NO and PGI2 pathways), which was decreased by PAG. Additionally, allicin increased the acetylcholine-induced endothelium-derived hyperpolarizing factor (EDHF) -mediated relaxation, which was unaffected by PAG. ConclusionAllicin exhibits a potent antihypertensive effect through vasodilatory properties and H2S mechanisms. Moreover, the vasodilation of allicin is partially dependent on endothelium. The endothelium-dependent vasodilation of allicin is mediated by the NO-sGC-cGMP, PGI2-AC-cAMP and EDHF pathways, of which H2S participates in the first two but not the third one. The endothelium independent vasodilation can be predominantly attributed to H2S production.

Validation of canine uterine and testicular arteries for the functional characterisation of receptor-mediated contraction as a replacement for laboratory animal tissues in teaching.

Teaching practicals for receptor physiology/pharmacology in medical and veterinary schools have involved the use of in vitro experiments using tissues from laboratory animals, which have been killed for isolated vascular strip or ring preparations. However, the use of scavenged tissues has been advocated to reduce animal use. Utilising discarded tissues from routine surgical procedures, such as canine neutering, has not previously been investigated. Canine testicular and uterine tissues (discarded tissues) were obtained from routine neutering procedures performed by the veterinary team at a local animal neutering clinic for stray dogs. Rings of uterine and testicular artery were dissected and mounted on a Mulvany-Halpern wire myograph in order to characterize the adrenergic and serotonergic receptors mediating vasoconstriction. Cumulative contractile concentration-response curves were constructed for the alpha adrenoceptor agonists epinephrine (α1 and α2 receptors), phenylephrine (α1 selective) and UK14304 (α2 selective). Pre-treatment with the α1-selective antagonist, prazosin, was also investigated. The response to serotonin (5-HT) receptor agonists were also investigated, including 5-HT (acting at both 5-HT1 and 5-HT2 receptors), 5-carboxamidotryptamine (5-CT; 5-HT1 selective) and α-methyl 5-HT (5-HT2 selective). A contractile response was observed in both canine uterine and testicular arteries to epinephrine and phenylephrine, and prazosin caused a dose-dependent parallel rightward shift in the phenylephrine dose-response curve (pA2 values of 7.97 and 8.39, respectively). UK14304 caused a contractile response in canine testicular arteries but very little appreciable contractile response in uterine arteries. The maximum responses produced by the uterine arteries to 5-HT was significantly lower than those of the testicular arteries. In the testicular artery, the 5-HT2 receptor selective agonist, α-methyl 5-HT, produced a similar contractile response to 5-HT but the administration of 5-CT failed to produce a response in either the testicular or uterine artery segments. These results validate the use of discarded tissue from routine canine neutering procedures as a useful source of vascular tissue for pharmacological teaching, for characterizing alpha and 5-HT receptor contractile responses.

SUN-559 Involvement of Sarco/Endoplasmic Reticulum Ca- ATPase (SERCA) in Membrane Progesterone Receptor Alpha (PAQR7)-Mediated Progesterone Induction of Vascular Smooth Muscle Relaxation

Progesterone (P4) exerts multiple beneficial effects on the human cardiovascular system through its actions on vascular endothelial cells and also by acting directly on vascular smooth muscle cells (VSMCs). Membrane progesterone receptor alpha (mPRα) has been shown to mediate the rapid P4-induction of human VSMC relaxation through activation of MAPK, Akt/Pi3k and RhoA/ROCK signaling pathways and the resulting reduction of calcium influx through calcium channels. In this study, we demonstrate that treatment of cultured human VSMCs with P4 for 1-2 hours increases both the mRNA and protein expression of sarco/endoplasmic reticulum Ca- ATPase (SERCA), the major transporter of calcium from the cytosol into the sarcoplasmic reticulum (SR) during muscle relaxation. Knockdown of mPRα with siRNA completely blocked this stimulatory effect of P4 as well as that of OD 02-0, a mPR selective agonist, on SERCA protein expression. In contrast, expression levels of phospholamban (PLB), a SR protein that reversibly inhibits SERCA were downregulated by this P4 treatment, and mRNA expression of a channel that releases calcium from the SR, inositol trisphosphate receptor (IP3R), was unaltered after treatment with P4. Moreover, treatments with P4 and OD 02-0, but not with R5020, a nuclear PR agonist, increased PLB phosphorylation, which would result in disinhibition of SERCA function. P4 and OD 02-0 significantly increased calcium levels in the SR detected with Fluo-5N, a specific SR calcium indicator, and caused VSMC relaxation. These effects were blocked by cyclopiazonic acid (CPA, a SERCA inhibitor), suggesting that SERCA plays a critical role in P4 induction of VSMC relaxation. Similarly, the effects of P4 and OD 02-0 on relaxation of umbilical artery rings measured with a myograph were significantly attenuated by CPA, which confirms the critical role of SERCA in the rapid action of P4 and 02-0 on vascular muscle relaxation. P4 has previously been shown to activate MAPK and Akt signaling pathways to induce VSMC relaxation. The P4- and OD 02-0-induced increases in calcium in the SR were blocked by MAPK and Akt/Pi3k signaling inhibitors, AZD6244 and wortmannin. Taken together, these results suggest that the direct, rapid effects of P4 on relaxation of VSMCs through mPRα involves regulation of the expression and function of the SR proteins SERCA and PLB through MAPK and Akt signaling pathways.

Antihypertensive activity of a new c-Jun N-terminal kinase inhibitor in spontaneously hypertensive rats.

c-Jun N-terminal kinases (JNKs) are involved in the myocardial and aortic remodeling, increased arterial tone, and arterial blood pressure elevation associated with hypertension. The aim of the present study was to investigate the antihypertensive effect of a new JNK inhibitor, 1H-indeno[1,2-b]quinoxalin-11-one oxime sodium salt (IQ-1S), on spontaneously hypertensive rats (SHRs). Experiments were performed using normotensive Wistar-Kyoto (WKY) rats and SHRs. Experimental groups of SHRs received IQ-1S intragastrically for 6 weeks in daily doses of 5 and 50 mg/kg; experimental groups of WKY rats received 50 mg/kg IQ-1S according to the same regimen. The IQ-1S administration regimen induced decreases in systolic blood pressure, mean arterial blood pressure, total peripheral resistance, blood viscosity, hematocrit, myocardial cell cross-sectional area, and aortic wall thickness in SHRs vs untreated SHRs. There were no significant differences in systolic blood pressure values between the control and experimental groups of WKY rats during the treatment period. A concentration-dependent decrease in the tone of carotid arterial rings isolated from SHRs was observed after JNK inhibitor application in vitro. Application of the JNK inhibitor diminished endothelin-1 secretion by human umbilical vein endothelial cells in vitro. The main mechanisms of the antihypertensive effect of IQ-1S included the attenuation of blood viscosity due to decreased hematocrit, a vasodilatory effect on arterial smooth muscle cells, and a decrease in endothelin-1 production by endothelial cells.

Biochanin-A elicits relaxation in coronary artery of goat through different mechanisms

Flavonoids have shown beneficial effects in various disease conditions as reported by various previous studies. Biochanin-A is a flavonoid present in various plants in nature. Present investigation was done to assess the vasorelaxant potential of biochanin-A on isolated coronary artery of goat and its possible mechanism of action. Vascular reactivity experiments were done on circumflex coronary artery of goats using the tension experiments. Goat coronary arterial rings were relaxed with biochanin-A in concentration (0.1–100 μM)-dependent manner. Endothelium had no effect on biochanin-A-induced relaxation. Maximum relaxation induced by biochanin-A was 116.54 ± 12.21% in endothelium-intact artery and it was not significantly different with maximal relaxation (108.22 ± 1.88%) of endothelium-denuded vessel. L-NAME (100 μM) did not show any effect on biochanin-A-induced relaxation. TEA (BKCa channel blocker), and BaCl2 (KIR blocker) had no effect on biochanin-A-induced relaxation. However, biochanin-A-induced maximal relaxation (71.72 ± 4.50%) was reduced significantly (P < .001) in the presence of 4-aminopyridine (KV channel blocker, 3 mM) in comparison with control (114.07 ± 4.33%). Glibenclaminde (KATP channel blocker), H89 (PKA inhibitor), ICI182780 (estrogen receptor antagonist) showed partial attenuation in the biochanin-A-induced relaxation. ODQ (sGC blocker) and HC067047 (TRPV4 channel blocker) had no effect on biochanin-A-induced relaxation. In K+-depolarized endothelium-denuded arterial rings, biochanin-A (30 μM) significantly (P < .05; P < .001) decreased CaCl2-induced contractions (0.02 ± 0.01 g vs. control 0.73 ± 0.30 g). Biochanin-A did not influence the fasudil (rho kinase inhibitor) and SNP (NO-donor)-induced relaxation in this vessel. Biochanin-A showed relaxation in goat coronary artery in endothelium-independent pathways and showed the partial involvement of KATP, protein kinase A and estrogen receptors and full involvement of Cav1.2 channels.

Structure and Function of Porcine Arteries Are Preserved for up to 6 Days Using the HypoRP Cold-storage Solution.

Maintaining functional vessels during preservation of vascularized composite allografts (VCAs) remains a major challenge. The University of Wisconsin (UW) solution has demonstrated significant short-term benefits (4-6 h). Here we determined whether the new hypothermic resuscitation and preservation solution HypoRP improves both structure, survival, and function of pig arteries during storage for up to 6 days. Using porcine swine mesenteric arteries, the effects of up to 6-day incubation in a saline (PBS), UW, or HypoRP solution on the structure, cell viability, metabolism, and function were determined. After incubation at 4°C, for up to 6 days, the structures of the arteries were significantly disrupted, especially the tunica media, following incubation in PBS, in contrast with incubation in the HypoRP solution and to a lesser extent, in UW solution. Those disruptions were associated with increased active caspase 3 indicative of apoptosis. Additionally, while incubation in PBS led to a significant decrease in the metabolic activity, UW and HypoRP solutions allowed a stable to increased metabolic activity following 6 days of cold storage. Functional responsiveness to phenylephrine (PE) and sodium nitroprusside (SNP) decreased over time for artery rings stored in PBS and UW solution but not for those stored in HypoRP solution. Moreover, artery rings cold-stored in HypoRP solution were more sensitive to ATP. The HypoRP solution improved long-term cold storage of porcine arteries by limiting structural alterations, including the collagen matrix, reducing apoptosis, and maintaining artery contraction-relaxation functions for up to 6 days.

Reversal of Hypoxic Pulmonary Hypertension by Hypoxia-Inducible Overexpression of Angiotensin-(1-7) in Pulmonary Endothelial Cells

Hypoxia-induced pulmonary vascular constriction and structure remodeling are the main causes of hypoxic pulmonary hypertension. In the present study, an adeno-associated virus vector, containing Tie2 promoter and hypoxia response elements, was designed and named HTSFcAng(1-7). Its targeting, hypoxic inducibility, and vascular relaxation were examined in vitro, and its therapeutic effects on hypobaric hypoxia-induced pulmonary hypertension were examined in rats. Transfection of HTSFcAng(1-7) specifically increased the expression of angiotensin-(1-7) in endothelial cells in normoxia. Hypoxia increased the expression of angiotensin-(1-7) in HTSFcAng(1-7)-transfected endothelial cells. The condition medium from HTSFcAng(1-7)-transfected endothelial cells inhibited the hypoxia-induced proliferation of pulmonary artery smooth muscle cells, relaxed the pulmonary artery rings, totally inhibited hypoxia-induced early contraction, enhanced maximum relaxation, and reversed phase II constriction to sustained relaxation. In hypoxic pulmonary hypertension rats, treatment with HTSFcAng(1-7) by nasal drip adeno-associated virus significantly reversed hypoxia-induced hemodynamic changes and pulmonary artery-wall remodeling, accompanied by the concomitant overexpression of angiotensin-(1-7), mainly in the endothelial cells in the lung. Therefore, hypoxia-inducible overexpression of angiotensin-(1-7) in pulmonary endothelial cells may be a potential strategy for the gene therapy of hypoxic pulmonary hypertension.

Synchronous detection of vascular tension and nitric oxide release in pulmonary artery: A combined application of confocal wire myograph with confocal laser scanning microscopy.

To detect the vascular tension and nitric oxide (NO) release synchronously in mice pulmonary artery, we perform two experiments and present a novel application of confocal wire myograph coupled with the confocal laser scanning microscopy. In the first experiment, viable endothelium-intact mouse pulmonary artery (outer diameter 100-300 μM) rings underwent a one-hour preincubation with a NO-specific fluorescent dye, 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate Calbiochem (2.5 μM), and then precontracted with phenylephrine (Phen, 10-6 M), and subsequently dilated in acetylcholine (ACh, 10-6 M - 10-4 M). The endothelium-dependent vasorelaxation and NO generation in pulmonary artery rings were simultaneously recorded. In the second experiment, after 30-min incubation with the former NO fluorescent dye, the qualified pulmonary artery rings were co-incubated for another 30 min with a nitric oxide synthase inhibitor, 10-4 M Nω-nitro-L-arginine-methyl-ester (L-NAME), and then pretreated with Phen (10-6 M) followed by ACh (10-5 M). The Ach-induced vasodilation and NO release were recorded simultaneously. ACh (10-6 M - 10-4 M) promoted pulmonary artery relaxation and intracellular NO release in a dose-dependent manner. Additionally, L-NAME (10-4 M) significantly attenuated the vasodilatation and the intracellular NO release. This combined application visually confirms that the synchronous changes in Ach induced vasodilation and NO release, which provides a new method for cardiovascular research.

Introducing a New Classification System for the Cerebral Arterial Circle of Willis

The cerebral arterial circle of Willis (CoW) is an arterial ring with an extensive range of variation and implications in cerebrovascular disease. Variations of the CoW are implicated in varied pathologies including aneurysms. This study analyzed the variations of the CoW in 177 cadavers. Each vessel in the CoW was identified as normal, hyperplastic, hypoplastic, aplastic, duplicated, triplicated, or plexiform. There were 73 total variation patterns identified. These variations were used to inform the development of a novel classification system that categorizes a CoW into one of 20 morphological types. The categorization is streamlined by assessing the anterior and posterior CoW vasculature independently. The anterior (A) variations are paired with their corresponding posterior (P) variations for a more complete description of the entire CoW variation. An unremarkable CoW is referenced as “A0P0.” The most common variant type is “A0P1” (16.95%) which has a normal anterior and a hypoplastic P1/hyperplastic P2/hyperplastic PCoA. This is followed by “A0P2” (12.99%) which has a normal anterior and a hypoplastic/aplastic PCoA. The circle is incomplete in 9.03% and bilaterally asymmetric in 49.16%. The proposed classification system will help improve the way CoW variations are classified and improve the understanding of how CoW variations are related to cerebrovascular pathology.Support or Funding InformationFunding: WV Research Challenge Fund [HEPC.dsr.17.06]

Hypoxic Pulmonary Vasoconstriction in Isolated Mouse Lungs

BackgroundHypoxic pulmonary vasoconstriction (HPV) is a physiological mechanism of lung to ensure maximal gas exchange and blood oxygenation. Persistent and global alveolar hypoxia leads to sustained vasoconstriction and vascular remodeling causing pulmonary hypertension. It has been demonstrated that acute HPV is initiated by an increase in cytosolic [Ca2+] ([Ca2+]cyt) in pulmonary arterial smooth muscle cells (PASMC) and modulated by factors released from endothelium. Acute HPV is well reserved in isolated perfused/ventilated lungs, whereas HPV in isolated pulmonary arterial rings shows different pattern in comparison to HPV in intact animals and humans. In this study, we aimed to use isolated perfused/ventilated mouse lungs to further study potential mechanisms involved in development of HPV.MethodsC57BL/6 mice (male, 8–10 weeks old) were anesthetized by pentobarbital sodium (120 mg/kg). After tracheostomy, isolated lungs were immediately ventilated with normoxic gas (21% O2/5% CO2) using a rodent ventilator (80 breaths/min with a tidal volume of 250 μl). The mice were then placed in an isolated lung system chamber with a heated water jacket at 37°C. After tracheal intubation, the chest was opened and a catheter was inserted into the main PA via the right ventricle to measure PAP. The lung vasculature was consistently superfused with physiological salt solution (PSS) via a pump while lung airway and alveoli were ventilated with normoxic or hypoxic gas. Before experimentation, the isolated lung was first superfused with the 40 mM K+‐containing PSS (40K) to stabilize the basal PAP. The lung was then repetitively challenged by ventilation of hypoxic gas (1% O2 in N2, for 4 min) to induce an increase in PAP (due to HPV). Pharmacological effects of various inhibitors on HPV were examined by superfusion of PSS containing the inhibitors while the lung was ventilated with hypoxic gas.ResultsOur results show that HPV was dependent on Ca2+ influx through a) nifedipine‐sensitive L‐type voltage‐dependent Ca2+ channels; b) SKF‐sensitive non‐selective cation channels; and c) TRP channels like TRPC6, TRPV1 and TRPP. HPV in isolated perfused lung was also significantly inhibited by blocking Ca2+‐sensing receptors (CaSR) and Notch receptors and by extracellular Mg2+ and Mn2+. Interestingly, blockade of Ca2+‐activated Cl− channels and inhibition of muscarinic receptors and endothelin receptors had little effect on HPV. Furthermore, inhibition of nitric oxide synthase (eNOS) potentiated HPV.ConclusionAn increase in [Ca2+]cyt due to Ca2+ influx through various Ca2+‐permeable cation channels in PASMC influx is required for the initiation of acute HPV. Both voltage‐gated Ca2+ channels (e.g., CaV1.2) and receptor‐operated Ca2+ channels (TRPC6/V1/P) are involved in mediating Ca2+ influx to induce HPV. G protein‐coupled receptor/CaSR‐mediated synthesis of intracellular signaling molecules (e.g., diacylglycerol and inositol 1, 4, 5‐trisphosphate) and Notch‐associated cell‐to‐cell interaction also play important roles in HPV. These data indicate that multiple mechanisms are involved in the initiation and regulation of HPV.Support or Funding InformationNIH NIHLBI HL135807

Signaling Pathways Involved in Enhanced 5‐HT‐Induced Contraction of Pulmonary Artery Segments From Guinea‐Pigs With Hyperthyroidism

We have previously reported that hyperthyroidism in rats resulted in enhanced 5‐HT‐induced contractions of pulmonary artery segments by a mechanism involving activation of calcium‐activated chloride channels. We have investigated other possible pathways that might be involved in this response in this study. Pulmonary artery ring segments were set up in Krebs’ solution for isometric tension recording. 5‐HT‐induced contraction was significantly enhanced in main pulmonary artery segments from guinea‐pigs with hyperthyroidism compared with a similar preparation from control guinea pigs. Nifedipine (10−6M) and tempol (3×10−3M) significantly reduced 5‐HT‐induced contraction in artery segments from thyroxine‐treated while having no effect in artery segments from control guinea pigs. Y‐27632 (10−5M) and SB 202190 (10−5M) but not PD 98059 (10−5M) significantly reduced 5‐HT‐induced contractions in artery segments from control and thyroxine‐treated guinea pigs. However, the reduction was greater in artery segments from thyroxine‐treated guinea pigs. Genistein (10−5M) significantly reduced 5‐HT‐induced contractions in artery segments from control or thyroxine‐treated guinea pigs. Genistein was equipotent in both cases. These results would suggest that hyperthyroidism‐induced increased 5‐HT‐induced contraction of the guinea pig pulmonary artery is mediated by multiple signaling pathways including increased influx of extracellular calcium through L‐type dihydropyridine channels, tyrosine kinase pathway, Rho‐kinase pathway of calcium sensitization, increased generation of reactive oxygen species and p38 MAPK pathway.

Single Endothelial Cell mRNA Sequencing Better Captures the Severity of Coronary Artery Disease Than Targeted Total Arterial Markers of Inflammation and Senescence

Endothelial dysfunction is the initial step towards atherosclerotic plaque development and coronary artery disease (CAD). Senescent endothelial cells (SnEC) have been linked to the atherosclerotic burden. In particular, the senescent‐associated secretory phenotype protein angiopoietin‐like 2 (ANGPTL2) was shown to be elevated in the plasma of CAD patients and related to the senescent cellular load in human internal mammary artery (IMA) segments discarded during coronary artery bypass grafting (CABG) surgery. We tested the hypothesis that the accumulation of vascular SnEC causes endothelial dysfunction and precedes the appearance of atherosclerotic lesions.Discarded atheroma‐free IMA segments from 12 patients (11 men and 1 woman, 69±3 years) were collected during consecutive elective CABG surgeries. Arterial rings of IMA segments were mounted in a wire myograph to record isometric changes in tension: arteries were pre‐contracted with U46619, a synthetic analogue of PGH2, and endothelium‐dependent relaxations to increasing concentrations of acetylcholine (ACh) were recorded. The maximal relaxation (Emax) and the concentration of ACh inducing 50% of relaxation (EC50) were calculated. Afterwards total mRNA was extracted from the IMA segments. Vascular gene expression of ANGPTL2 and p21 (senescence markers), and CD68 and PAI‐1 (inflammatory markers) were assessed by RT‐qPCR. In parallel, single‐cell RNA sequencing was performed in IMA segments from two age‐matched male patients obtained either during elective (stable CAD) or emergency (unstable CAD, with numerous risk factors) procedures, and differential gene expression was specifically analyzed in vascular EC.Endothelium‐dependent relaxations were characterized by Emax (56±7 %, [18–100%], n=12) and pD2 (‐log EC50: 6.9±0.1 [5.8–7.3], n=12). Patients were divided into 2 groups according to their Emax (&lt; or &gt; to 50%) to define low and high endothelial function; vascular gene expression of the 4 senescence and inflammatory markers were similar between the two groups, demonstrating that global arterial wall senescence and inflammation did not distinguish severity of endothelial dysfunction. We then used single‐cell mRNA sequencing and analysed the unbiased differential gene expression specifically in vascular EC. From the total IMA cellular counts, EC represented ~2%. Nine genes were identified that were differentially overexpressed in EC from unstable CAD patient that have been associated with senolytic drug targets and cardiovascular diseases (Table ), potentially attesting to the severity of endothelial dysfunction and CAD.In conclusion, unlike total arterial wall mRNA quantification, unbiased single‐cell mRNA sequencing identified differentially upregulated endothelial pathways that may contribute to the severity of the CAD by inducing precocious endothelial dysfunction and senescence. Top genes differentially expressed in endothelial cells between a stable and unstable CAD patient. Percentages represent the percent of endothelial cells expressing the gene in each patient dataset. P‐values are Bonferroni corrected. Stable CAD patient Unstable CAD patient p‐value Gene Associated CVD disorder or drug target 0.0% 41.7% 0.00061 TGFB2 Atrial fibrillation 0.0% 41.7% 0.00061 SQLE Statins 2.9% 58.3% 0.00090 RPPH1 Quercetin (senolytic drug) 7.1% 66.7% 0.00339 ARL14EP Systolic blood pressure 25.7% 100.0% 0.00914 RPL17 Myocardial ischemia 17.1% 83.3% 0.01556 IGFBP7 Ruptured aneurysm 0.0% 33.3% 0.01699 TMEM8B Heart valve disorders 0.0% 33.3% 0.01699 ZNF675 Congestive heart failure 8.6% 66.7% 0.04848 SELL Vaso‐occlusive crisis

Role of protein kinase D1 in pulmonary hypertension in rats

Protein kinase D (PKD) is known to be implicated in a variety of physiological functions and pathological conditions including cardiovascular diseases, such as cardiac hypertrophy and ischemia reperfusion injury. PKD has been thought to be a new target of the diseases for treatments. Previously, we showed that PKD played a role in pulmonary arterial contraction through MYPT1 phosphorylation and actin polymerization and that inhibition of PKD activity decreased right cardiac ventricular pressure in monocrotaline‐induced pulmonary hypertension (PH) model,. In this study, we further investigated a precise role of PKD in PH and in human pulmonary smooth muscle cells (HPSMCs).Material and methodsEight‐week‐old male rats were given either 60 mg/kg MCT or saline 4 weeks before experiments. Isometric tension was measured using pulmonary arterial ring samples to examine the effect of PKD inhibitor, CRT0066101 on norepinephrine (NE)‐induced contraction and phosphorylation of PKD, myosin phosphatase target subunit 1 (MYPT1), regulatory myosin light chain (RLC) and other PKD substrate proteins. Expression of PKD and phosphorylated‐PKD in rats lungs were also examined with immunohistochemistry using anti‐phospho‐PKD antibodies. We also investigated precise roles of PKD isoforms using HPSMCs with RNA interference.ResultsIn pulmonary arterial rings, basal expression of PKD1 and phosphorylated PKD were increased in pulmonary arteries in PH rats. NE increased phosphorylation of PKD, MYPT1 and RLC in ring samples during contraction, which were attenuated by pretreatment of CRT0066101. In immunohistochemistry, increased expression of PKD1 was observed in pulmonary arteries in PH rats (Fig 1). In HASMCs, increased phosphorylation of MYPT1 and RLC with NE were significantly inhibited by PKD1 but not PKD2, 3 knock down.ConclusionsPKD1 may play role in contraction of pulmonary arteries and at least partially via MYPT1 phosphorylation. Increased PKD1 could be involved in PH development in rats.Support or Funding InformationGrant‐in‐Aid for Scientific Research (C) 17K11058Immunohistochemistry with anti‐PKD antibody in lung tissue from PH rats.Figure 1

L‐Arginase induces Vascular Dysfunction in Old Spontaneously Hypertensive Rats

BackgroundAging is a major non‐modifiable risk factor for hypertension. Structural, functional and mechanical changes occur with aging. These changes are similar to those seen in hypertension in the vasculature. Also, aging increases the vascular dysfunction that occurs in hypertension. L‐arginase is a ureohydrolase enzyme that converts L‐arginine to L‐ornithine and urea. Arginase action reduces substrate (L‐arginine) availability for the formation of nitric oxide (NO). This reduces the level of NO and leads to reduced vasodilation and ultimately, vascular dysfunction.HypothesisThis study tests the hypothesis that the age dependent vascular dysfunction in SHRs is mediated by arginase.MethodsYoung (12–14 weeks) and old (11–12 months) male Wistar and spontaneously hypertensive rats (SHR) were used in this study. Mean arterial pressure (MAP) was measured in the rats. They were then euthanized and 5ml of blood was drawn from the heart to obtain serum for the assay of arginase activity. Mesenteric resistance arteries (MRAs) and thoracic aortae were excised and placed in ice‐cold physiological salt solution (PSS). Arterial segments were either snap‐frozen in liquid nitrogen and stored for immunoblotting studies or cut into 2mm rings for reactivity studies. Cumulative concentration‐response curves to phenylephrine (PE; 10‐9‐3×10‐5M), acetylcholine (Ach; 10‐9 – 3×10‐5M) and sodium nitroprusside (SNP; 10‐12 – 3×10‐5 M) were performed in the absence or presence (30‐minute exposure) of various agents: L‐arginase, 0.05U/ML (MRA) or 0.5U/ML (aorta); Rho kinase inhibitor, Y27632 − 10‐5mmol/L; or 0.5U/ML L‐arginase + 10‐5mmol/L Y27632 (aorta). All chemicals were obtained from Sigma‐Aldrich MO, USA. Data are expressed as mean + S.E.M. of 6 rats per group. Statistical differences were calculated using Student’s t‐test and two‐way ANOVA with repeated measures followed by Bonferroni post hoc test. Significance was set at p&lt;0.05.ResultsMAP increased during aging in the SHRs (132.2±10.2 vs 166.2±1.9mmHg p&lt;0.05.) but not in the Wistar rats (104.4±3.0 vs 92.0±1.5mmHg). Arginase impaired the endothelium‐dependent relaxation responses of thoracic aortic and MRA arterial rings to Ach in the old Wistars (Emax aorta: 68.66248±1.96% vs 56.46116±3.42%) and SHRs (Emax aorta: 29.41753±2.19% vs 7.943045±1.86%). Arginase also impaired endothelium‐independent relaxation response to SNP in the old SHRs only (Emax aorta: 88.62396±4.10% vs 31.45081±10.61%). Correspondingly, arginase potentiated contractile responses of thoracic aortic and MRA arterial rings to PE in the old SHRs (Emax MRA: 25.24835±1.93mN vs 36.43248±2.99mN) which was attenuated by the Rho Kinase inhibitor (Y26732) in the presence or absence of arginase. We also observed no differences in the serum arginase activity in the four groups of rats. On the contrary, arginase activity in the thoracic aortae of young Wistar rats was reduced compared to the other groups.ConclusionsThe results of this study suggest that arginase impairs both endothelium‐dependent and –independent vasorelaxation responses, through the NO signaling pathway.Support or Funding InformationNIH

Pharmacokinetics and Antihypertensive effect of linalool is improved after incorporation in β‐cyclodextrin as a drug delivery system

Linalool (LIN) is a monoterpene alcohol present in some aromatic medicinal plants with biological activities that can impact cardiovascular disease. Previous studies from our research group have shown that LIN has antihypertensive activity, but as an essential oil it has some administration restrictions. The study aimed to investigate the pharmacokinetics (PK) and cardiovascular effects of LIN complexed with beta‐cyclodextrin (LIN/β‐CD) as a potential antihypertensive drug. All the experiments were conducted in accordance with FASEB statement of principles for the use of animals in research and education and approved by the ethics committee under the protocol CEUA‐ICS/UFBA n° 085/2015. For PK studies, LIN and LIN/β‐CD were administered to male normotensive wistar rats orally and LIN plasma concentrations were validated by HPLC‐UV. The individual and mean PK parameters of elimination rate, absorption rate, clearance, volume of distribution, half‐life, mean residence time, absolute bioavailability and relative bioavailability were estimated for LIN free or complexed form. For the cardiovascular experiments, spontaneously hypertensive rats (SHR) and wistar were used. Rats were randomly divided into 3 groups, each treated daily for 60 days, in the following manner: group 1 (vehicle solution); group 2 (LIN; 50 mg/kg/day); group 3 group (LIN/β‐CD; 50 mg/kg/day). Blood pressure, daily body weight and vascular reactivity were studied. The results of PK parameters showed that bioavailability of the LIN/β‐CD was approximately 20‐fold higher compared to LIN, after oral administration. Population modeling studies demonstrated that LIN/β‐CD had less tissues distribution compared to non‐complexed form, consequently remaining in the bloodstream or more irrigated organ and tissues. In addition, LIN/β‐CD, but not LIN, was able to induce acute hypotensive effect after oral administration. Furthermore, during the 60 days of chronic treatment, SHR treated with vehicle (192 ± 2.5 mmHg, p&lt;0.05; 40th day) demonstrated progressive increase in mean arterial pressure compared to wistar normotensive rats (103 ± 5 mmHg, 40th day). However, the blood pressure of the SHR treated by LIN/β‐CD was significantly reduced during the treatment, presenting similar levels to the normotensive healthy animals (113 ± 5 mmHg; 40th day). This antihypertensive effect induced by complexed form was not observed with linalool free (167 ± 8 mmHg 40th day). Vascular reactivity assays using superior mesenteric artery rings demonstrated that LIN/β‐CD or LIN ameliorated the vascular function of SHR, increasing vasodilator responsiveness and reducing sensitivity to the sympathetic agonist. Taking together, our results suggested that incorporation of linalool into β‐cyclodextrin improved the bioavailability and antihypertensive effect of this monoterpene, making it a possible new pharmaceutical formulation for the treatment of hypertension in the future.Support or Funding InformationFIOCRUZ, National Council for Scientific and Technological Development (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior ‐ Brasil (CAPES).

The vascular dilatation induced by Hydroxysafflor yellow A (HSYA) on rat mesenteric artery through TRPV4-dependent calcium influx in endothelial cells

Ethnopharmacological relevanceHydroxysafflor yellow A (HSYA) is the principal constituent of the flowers of Carthamus tinctorius L., a traditional Chinese herbal medicine, which has been used for the treatment of cerebrovascular and cardiovascular diseases due to its property of promoting blood circulation and removing blood stasis. It is dominated in the water extract of Carthamus tinctorius L., which has been used in the clinical treatment for cardiovascular diseases. HSYA exerts a variety of pharmacological efficacy upon the vascular system. However, the underlying mechanisms remain unclear. Aim of the studyTo investigate the vascular dilatation effect of HSYA on rat mesenteric artery (MA) and its potential mechanism. Materials and methodsAdult male Wistar rats were applied to the study. Tension studies were conducted to determine the dilatation activity of HSYA against pre-contracted mesenteric arterial (MA) rings by U 46619 and Phenylephrine (PE). The vascular activities were measured with or without incubation with some selective inhibitors, including L-N(ω)-nitro-L-arginine methyl ester (L-NAME, a nitro oxide synthase inhibitor), HC-067047 (a selective TRPV4 antagonist), BaCl2 (a Kir channel blocker), and Indomethacin (Indo, a nonselective cyclooxygenase inhibitor), respectively. Immunocytochemistry, Calcium Imaging, NO Production detection, and Western Blot were also employed to further study the underlying mechanism. ResultsHSYA reversed the constriction of MAs induced by U 46619 in a manner of concentration dependency, and the dilatation capability was reversed by L-NAME. This effect was significantly dependent on the intactness of MA endothelium, accompanying an increment of NO production in mesenteric arterial endothelium cells. The increment of NO production was reversed by inhibiting the PKA. Also, the expression of p-eNOS was activated by HSYA shown in Western Blot assays. The cells imaging revealed a significant increase and drop of the influx of Ca2+ before and after treatment with HC-067047. ConclusionsThese findings suggest that HSYA exerts vessel dilation effect on MAs via a TRPV4-dependent influx of Ca2+ in endothelium cells, PKA-dependent eNOS phosphorylation and NO production mechanism. The present study indicates that HSYA has the potential to be a future candidate for the treatment of hypertension.

Dilatation Reserve of Pulmonary Arteries at Stages of the Chronic Obstructive Pulmonary Disease Model.

To assess the state of pulmonary vascular mediator systems during the stepwise formation of the chronic obstructive pulmonary disease (COPD) model. The COPD model was induced in rats by nitrogen dioxide (NO2) inhalation for 60 days. At different stages of COPD (15, 30, and 60 days), the effect of reagents-vasodilators (β-adrenoceptor agonist isoproterenol, nitric oxide donor nitrosorbide, acetylcholine, activator of C-fibers capsaicin, corticosteroid beclometasone) on the isolated pulmonary arteries (diameter <0.5 mm) was studied. Vascular reactivity was assessed by determining isometric contraction (tension in milligrams) of arterial rings by using an electromechanical transducer. All vasodilators dose-dependently decreased the vascular tone of pulmonary arteries isolated from intact rats. After 15 days of NO2 exposure, dilatation effect of low doses of vasodilators did not differ from that of intact specimens. The functional state of the adrenergic system deteriorated faster than that of the nonadrenergic noncholinergic system as reflected by the weakening of the isoproterenol relaxation effect. On prolonged NO2 exposure, pulmonary arteries responded to the impact of all vasodilators with smaller relaxation. Dose dependence of the dilatation reaction disappeared for isoproterenol, capsaicin, beclometasone, and was less expressed for nitrosorbide and acetylcholine after 60 days of exposure. In the course of COPD model generation, the functioning of almost all neurotransmitter systems of pulmonary artery wall was negatively affected. This led to a decrease in the influence of vasodilators on pulmonary artery vascular tone and could facilitate the development of pulmonary hypertension, which is typical of COPD.

Methylglyoxal-induced miR-223 suppresses rat vascular KATP channel activity by downregulating Kir6.1 mRNA in carbonyl stress

The vascular ATP-sensitive K+ (KATP) channel composed of Kir6.1 and SUR2B subunits regulates cellular activity by coupling intermediary metabolism to membrane excitability. Our previous studies have shown that both Kir6.1 and SUB2B are post-transcriptionally downregulated by methylglyoxal (MGO) which is a reactive carbonyl specie and can cause disruption of vascular tone regulation under diabetic conditions. We have shown that the SUB2B downregulation is mediated by the microRNA (miR) miR-9a, while the mechanism underlying Kir6.1 inhibition is still unclear. Studying the microRNA databases, we found that miR-223 has sequence similarities to the 3′ untranslated sequence (3’UTR) of Kir6.1 mRNA suggesting their potential interactions. Therefore, we explored the role of miR-233 in KATP channel regulation by up/down-regulation of miR-223 in smooth muscle cells (SMCs) and mesenteric arterials. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analysis showed augmentation of miR-223 expression in the cultured SMCs after 300 μM MGO exposure by 5–6 folds. miR-223 overexpression down-regulated Kir6.1 mRNA levels by ~2.6 times while miR-223 knockdown diminished the effect of 300 μM MGO by ~50% in the SMCs. Luciferase assay and mutagenesis studies showed that the effect of miR-223 was abolished when the potential interaction site in the 3’ UTR was mutated. Studies with Western blot, patch clamp, and perfused mesenteric arterial rings showed that transfection of miR-223 downregulated KATP protein expression, inhibited KATP channel activity and enhanced vasoconstriction. These results therefore suggest that miR-223 is induced by MGO exposure, which subsequently downregulates the Kir6.1 mRNA, suppresses KATP channel function, and impairs functional regulation of vascular tones. BackgroundMethylglyoxal causes transcriptional inhibition of the vascular KATP channel. ResultsExogenous miR-223 down-regulated Kir6.1. miR-223 knockdown alleviated the effect of MGO. ConclusionVascular KATP channel is important for miR-223 targeting. SignificanceRegulation of the miR-223 level may be a novel strategy for clinical treatment of diabetes.

Distinct hemodynamic responses to (pyr)apelin-13 in large animal models.

This study tested the hypothesis that (pyr)apelin-13 dose-dependently augments myocardial contractility and coronary blood flow, irrespective of changes in systemic hemodynamics. Acute effects of intravenous (pyr)apelin-13 administration (10 to 1,000 nM) on blood pressure, heart rate, left ventricular pressure and volume, and coronary parameters were measured in dogs and pigs. Administration of (pyr)apelin-13 did not influence blood pressure (P = 0.59), dP/dtmax (P = 0.26), or dP/dtmin (P = 0.85) in dogs. However, heart rate dose-dependently increased > 70% (P < 0.01), which was accompanied by a significant increase in coronary blood flow (P < 0.05) and reductions in left ventricular end-diastolic volume and stroke volume (P < 0.001). In contrast, (pyr)apelin-13 did not significantly affect hemodynamics, coronary blood flow, or indexes of contractile function in pigs. Furthermore, swine studies found no effect of intracoronary (pyr)apelin-13 administration on coronary blood flow (P = 0.83) or vasorelaxation in isolated, endothelium-intact (P = 0.89) or denuded (P = 0.38) coronary artery rings. Examination of all data across (pyr)apelin-13 concentrations revealed an exponential increase in cardiac output as peripheral resistance decreased across pigs and dogs (P < 0.001; R2 = 0.78). Assessment of the Frank-Starling relationship demonstrated a significant linear relationship between left ventricular end-diastolic volume and stroke volume across species (P < 0.001; R2 = 0.70). Taken together, these findings demonstrate that (pyr)apelin-13 does not directly influence myocardial contractility or coronary blood flow in either dogs or pigs.NEW & NOTEWORTHY Our findings provide much needed insight regarding the pharmacological cardiac and coronary effects of (pyr)apelin-13 in larger animal preparations. In particular, data highlight distinct hemodynamic responses of apelin across species, which are independent of any direct effect on myocardial contractility or perfusion.

Empagliflozin improved systolic blood pressure, endothelial dysfunction and heart remodeling in the metabolic syndrome ZSF1 rat

BackgroundEmpagliflozin (empa), a selective sodium–glucose cotransporter (SGLT)2 inhibitor, reduced cardiovascular mortality and hospitalization for heart failure in patients with type 2 diabetes at high cardiovascular risk independent of glycemic control. The cardiovascular protective effect of empa was evaluated in an experimental model of metabolic syndrome, the obese ZSF1 rat, and its’ lean control.MethodsLean and obese ZSF1 rats were either non-treated or treated with empa (30 mg/kg/day) for 6 weeks. Vascular reactivity was assessed using mesenteric artery rings, systolic blood pressure by tail-cuff sphygmomanometry, heart function and structural changes by echocardiography, and protein expression levels by Western blot analysis.ResultsEmpa treatment reduced blood glucose levels from 275 to 196 mg/dl in obese ZSF1 rats whereas normoglycemia (134 mg/dl) was present in control lean ZSF1 rats and was unaffected by empa. Obese ZSF1 rats showed increased systolic blood pressure, and blunted endothelium-dependent relaxations associated with the appearance of endothelium-dependent contractile responses (EDCFs) compared to control lean rats. These effects were prevented by the empa treatment. Obese ZSF1 rats showed increased weight of the heart and of the left ventricle volume without the presence of diastolic or systolic dysfunction, which were improved by the empa treatment. An increased expression level of senescence markers (p53, p21, p16), tissue factor, VCAM-1, SGLT1 and SGLT2 and a down-regulation of eNOS were observed in the aortic inner curvature compared to the outer one in the control lean rats, which were prevented by the empa treatment. In the obese ZSF1 rats, no such effects were observed. The empa treatment reduced the increased body weight and weight of lungs, spleen, liver and perirenal fat, hyperglycemia and the increased levels of total cholesterol and triglycerides in obese ZSF1 rats, and increased blood ketone levels and urinary glucose excretion in control lean and obese ZSF1 rats.ConclusionEmpa reduced glucose levels by 28% and improved both endothelial function and cardiac remodeling in the obese ZSF1 rat. Empa also reduced the increased expression level of senescence, and atherothrombotic markers at arterial sites at risk in the control lean, but not obese, ZSF1 rat.