Sodium tanshinone IIA sulfonate protects vascular relaxation in ApoE-knockout mice by inhibiting the SYK-NLRP3 inflammasome-MMP2/9 pathway.

BACKGROUND: Hyperlipidemia damages vascular wall and serves as a foundation for diseases such as atherosclerosis, hypertension and stiffness. The NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome is implicated in vascular dysfunction associated with hyperlipidemia-induced vascular injury. Sodium tanshinone IIA sulfonate (STS), a well-established cardiovascular protective drug with recognized anti-inflammatory, antioxidant, and vasodilatory properties, is yet to be thoroughly investigated for its impact on vascular relaxant imbalance induced by hyperlipidemia. METHODS: In this study, we treated ApoE-knockout (ApoE-/-) mouse with STS and assessed the activation of the NLRP3 inflammasome, expression of MMP2/9, integrity of elastic fibers, and vascular constriction and relaxation. RESULTS: Our findings reveal that STS intervention effectively preserves elastic fibers, significantly restores aortic relaxation function in ApoE-/- mice, and reduces their excessive constriction. Furthermore, STS inhibits the phosphorylation of spleen tyrosine kinase (SYK), suppresses NLRP3 inflammasome activation, and reduces MMP2/9 expression. CONCLUSIONS: These results demonstrate that STS protects vascular relaxation against hyperlipidemia-induced damage through modulation of the SYK-NLRP3 inflammasome-MMP2/9 pathway. This research provides novel insights into the mechanisms underlying vascular relaxation impairment in a hyperlipidemic environment and uncovers a unique mechanism by which STS preserves vascular relaxation, offering valuable foundational research evidence for its clinical application in promoting vascular health.

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

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

The Effects of Novel Triazolopyrimidine Derivatives on H2S Production in Lung and Vascular Tonus in Aorta.

INTRODUCTION: Hydrogen sulfide (H2S), known as a third gasotransmitter, is a signaling molecule that plays a regulatory role in physiological and pathophysiological processes. Decreased H2S levels were reported in inflammatory respiratory diseases such as asthma, chronic obstructive pulmonary disease, and pulmonary hypertension. H2S donors or drugs that increase H2S have emerged as novel treatments for inflammatory respiratory diseases. We previously showed that resveratrol (RVT) causes vascular relaxation and antioxidant effects by inducing H2S production. In the current study, we synthesized a new molecule Cpd2, as an RVT analog. We examined the effect of Cpd2 and its precursor chalcone compound (Cpd1) on H2S formation under both healthy and oxidative stress conditions in the lung, as well as vascular relaxation in the aorta. METHODS: Cpd2 synthesized from Cpd1 with microwaved in basic conditions. H2S formation was measured by H2S biosensor in the mice lungs under both healthy and pyrogallol-induced oxidative stress conditions in the presence/absence of H2S synthesis inhibitor aminooxyacetic acid (AOAA). The effect of compounds on vascular tonus is investigated in mice aorta by DMT myograph. RESULTS: RVT and Cpd2 significantly increased l-cysteine (l-cys) induced-H2S formation in the lung homogenates of healthy mice, but Cpd1 did not. Superoxide anion generator pyrogallol caused a decrease in H2S levels in mice lungs and Cpd2 restored it. Inhibition of Cpd2-induced H2S formation by AOAA confirmed that Cpd2 increases endogenous H2S formation in both healthy and oxidative stress conditions. Furthermore, we found that both Cpd1 and Cpd2 (10-8-10-4 M) caused vascular relaxation in mice aorta. DISCUSSION AND CONCLUSION: We found that Cpd2, a newly synthesized RVT analog, is an H2S-inducing molecule and vasorelaxant similar to RVT. Since H2S has antioxidant and anti-inflammatory effects, Cpd2 has a potential for the treatment of respiratory diseases where oxidative stress and decreased H2S levels are present.

Independent endothelial functions of PIEZO1 and TRPV4 in hepatic portal vein and predominance of PIEZO1 in mechanical and osmotic stress.

BACKGROUND & AIMS: PIEZO1 and TRPV4 are mechanically and osmotically regulated calcium-permeable channels. The aim of this study was to determine the relevance and relationship of these channels in the contractile tone of the hepatic portal vein, which experiences mechanical and osmotic variations as it delivers blood to the liver from the intestines, gallbladder, pancreas and spleen. METHODS: Wall tension was measured in freshly dissected portal veins from adult male mice, which were genetically unmodified or modified for either a non-disruptive tag in native PIEZO1 or endothelial-specific PIEZO1 deletion. Pharmacological agents were used to activate or inhibit PIEZO1, TRPV4 and associated pathways, including Yoda1 and Yoda2 for PIEZO1 and GSK1016790A for TRPV4 agonism, respectively. RESULTS: PIEZO1 activation leads to nitric oxide synthase- and endothelium-dependent relaxation of the portal vein. TRPV4 activation causes contraction, which is also endothelium-dependent but independent of nitric oxide synthase. The TRPV4-mediated contraction is suppressed by inhibitors of phospholipase A2 and cyclooxygenases and mimicked by prostaglandin E2 , suggesting mediation by arachidonic acid metabolism. TRPV4 antagonism inhibits the effect of agonising TRPV4 but not PIEZO1. Increased wall stretch and hypo-osmolality inhibit TRPV4 responses while lacking effects on or amplifying PIEZO1 responses. CONCLUSIONS: The portal vein contains independently functioning PIEZO1 channels and TRPV4 channels in the endothelium, the pharmacological activation of which leads to opposing effects of vessel relaxation (PIEZO1) and contraction (TRPV4). In mechanical and osmotic strain, the PIEZO1 mechanism dominates. Modulators of these channels could present important new opportunities for manipulating liver perfusion and regeneration in disease and surgical procedures.

Endothelial PAR2 activation evokes resistance artery relaxation.

Protease-activated receptor-1 & -2 (PAR1 and PAR2) are expressed widely in cardiovascular tissues including endothelial and smooth muscle cells. PAR1 and PAR2 may regulate blood pressure via changes in vascular contraction or relaxation mediated by endothelial Ca2+ signaling, but the mechanisms are incompletely understood. By using single-cell Ca2+ imaging across hundreds of endothelial cells in intact blood vessels, we explored PAR-mediated regulation of blood vessel function using PAR1 and PAR2 activators. We show that PAR2 activation evoked multicellular Ca2+ waves that propagated across the endothelium. The PAR2-evoked Ca2+ waves were temporally distinct from those generated by muscarinic receptor activation. PAR2 activated distinct clusters of endothelial cells, and these cells were different from those activated by muscarinic receptor stimulation. These results indicate that distinct cell clusters facilitate spatial segregation of endothelial signal processing. We also demonstrate that PAR2 is a phospholipase C-coupled receptor that evokes Ca2+ release from the IP3 -sensitive store in endothelial cells. A physiological consequence of this PAR2 signaling system is endothelium-dependent relaxation. Conversely, PAR1 activation did not trigger endothelial cell Ca2+ signaling nor relax or contract mesenteric arteries. Neither did PAR1 activators alter the response to PAR2 or muscarinic receptor activation. Collectively, these results suggest that endothelial PAR2 but not PAR1 evokes mesenteric artery relaxation by evoking IP3 -mediated Ca2+ release from the internal store. Sensing mediated by PAR2 receptors is distributed to spatially separated clusters of endothelial cells.

Inhibitory Effects of Saururus chinensis Extract on Receptor for Advanced Glycation End-Products-Dependent Inflammation and Diabetes-Induced Dysregulation of Vasodilation.

Advanced glycation end-products (AGEs) and the receptor for AGEs (RAGE) are implicated in inflammatory reactions and vascular complications in diabetes. Signaling pathways downstream of RAGE are involved in NF-κB activation. In this study, we examined whether ethanol extracts of Saururus chinensis (Lour.) Baill. (SE) could affect RAGE signaling and vascular relaxation in streptozotocin (STZ)-induced diabetic rats. Treatment with SE inhibited AGEs-modified bovine serum albumin (AGEs-BSA)-elicited activation of NF-κB and could compete with AGEs-BSA binding to RAGE in a dose-dependent manner. Tumor necrosis factor-α (TNF-α) secretion induced by lipopolysaccharide (LPS)-a RAGE ligand-was also reduced by SE treatment in wild-type Ager+/+ mice as well as in cultured peritoneal macrophages from Ager+/+ mice but not in Ager-/- mice. SE administration significantly ameliorated diabetes-related dysregulation of acetylcholine-mediated vascular relaxation in STZ-induced diabetic rats. These results suggest that SE would inhibit RAGE signaling and would be useful for the improvement of vascular endothelial dysfunction in diabetes.

Novel Oxime Synthesized from a Natural Product of Senecio nutans SCh. Bip. (Asteraceae) Enhances Vascular Relaxation in Rats by an Endothelium-Independent Mechanism.

Senecio nutans Sch. Bip. and its constituents are reported to have antihypertensive effects. We isolated metabolite−1, a natural compound from S. nutans (4-hydroxy-3-(isopenten-2-yl)-acetophenone), and synthesized novel oxime − 1 (4-hydroxy-3-(isopenten-2-yl)-acetophenoxime) to evaluate their effect on vascular reactivity. Compounds were purified (metabolite−1) or synthetized (oxime−1) and characterized using IR and NMR spectroscopy and Heteronuclear Multiple Quantum Coherence (HMQC). Using pharmacological agents such as phenylephrine (PE) and KCl (enhancing contraction), acetylcholine (ACh), L-NAME (nitric oxide (NO) and endothelial function), Bay K8644-induced CaV1.2 channel (calcium channel modulator), and isolated aortic rings in an organ bath setup, the possible mechanisms of vascular action were determined. Pre-incubation of aortic rings with 10−5 M oxime−1 significantly (p < 0.001) decreased the contractile response to 30 mM KCl. EC50 to KCl significantly (p < 0.01) increased in the presence of oxime−1 (37.72 ± 2.10 mM) compared to that obtained under control conditions (22.37 ± 1.40 mM). Oxime−1 significantly reduced (p < 0.001) the contractile response to different concentrations of PE (10−7 to 10−5 M) by a mechanism that decreases Cav1.2-mediated Ca2+ influx from the extracellular space and reduces Ca2+ release from intracellular stores. At a submaximal concentration (10−5 M), oxime−1 caused a significant relaxation in rat aorta even without vascular endothelium or after pre-incubate the tissue with L-NAME. Oxime−1 decreases the contractile response to PE by blunting the release of Ca2+ from intracellular stores and blocking of Ca2+ influx by channels. Metabolite−1 reduces the contractile response to KCl, apparently by reducing the plasma membrane depolarization and Ca2+ influx from the extracellular space. These acetophenone derivates from S. nutans (metabolite−1 and oxime−1) cause vasorelaxation through pathways involving an increase of the endothelial NO generation or a higher bioavailability, further highlighting that structural modification of naturally occurring metabolites can enhance their intended pharmacological functions.

Vascular relaxing effect of Hydrocotyle umbellata L. is mediated by blocking of l-type Ca2+ channels.

ETHNOPHARMACOLOGICAL RELEVANCE: Hydrocotyle umbellata L. is a medicinal herb for the treatment of some health problems including hypertension, according to traditional medicine. Even so, its vascular effects and the pharmacological action mechanisms have not been analyzed. AIM OF THE STUDY: This experiment aimed to analyze the effects of hydroalcoholic extract of Hydrocotyle umbellata L. (HEHU) on isolated vessels and verify the interaction of hibalactone (chemical marker) against Cav1.2 channels using molecular docking. MATERIALS AND METHODS: Vascular reactivity experiments were performed using rat aortas with (E+) or without endothelium (E-) in an isolated organ bath. Computational molecular docking approaches were used to show the direct effect on L-type Ca2+ Channels. RESULTS: HEHU (0-560 µg/mL) induced relaxation of the pre-contracted arteries in a concentration-dependent manner. The maximum effect was higher in E+ (76.8 ± 4.1%) as compared to E- (47.3 ± 5.5%). Pre-treatment of E+ arteries with L-NAME or ODQ reduced the relaxation to similar level of E- arteries. The treatment of arteries with MDL-12,330 A, diclofenac, propranolol and atropine did not change the relaxation induced by HEHU. The contraction caused by internal Ca2+ release induced by caffeine was reduced after HEHU treatment. Moreover, the HEHU also impaired the contraction induced by Ca2+ influx stimulated with phenylephrine or high KCl. The docking study demonstrated the effectiveness of hibalactone in blocking the Cav1.2 channel. CONCLUSIONS: These findings show that HEHU induces vascular relaxation which is potentiated (but not dependent) by endothelial cells. Blocking of Ca2+ influx seems to be the main mechanism for the vascular effects of HEHU.

Perivascular adipose tissue-derived nitric oxide compensates endothelial dysfunction in aged pre-atherosclerotic apolipoprotein E-deficient rats.

BACKGROUND AND AIMS: Atherosclerosis is a major contributor to global mortality and is accompanied by vascular inflammation and endothelial dysfunction. Perivascular adipose tissue (PVAT) is an established regulator of vascular function with emerging implications in atherosclerosis. We investigated the modulation of aortic relaxation by PVAT in aged rats with apolipoprotein E deficiency (ApoE-/-) fed a high-fat diet as a model of early atherosclerosis. METHODS AND RESULTS: ApoE-/- rats (N = 7) and wild-type Sprague-Dawley controls (ApoE+/+, N = 8) received high-fat diet for 51 weeks. Hyperlipidemia was confirmed in ApoE-/- rats by elevated plasma cholesterol (p < 0.001) and triglyceride (p = 0.025) levels. Early atherosclerosis was supported by increased intima/media thickness ratio (p < 0.01) and ED1-positive macrophage influx in ApoE-/- aortic intima (p < 0.001). Inflammation in ApoE-/- PVAT was characteristic by an increased [18F]FDG uptake (p < 0.01), ED1-positive macrophage influx (p = 0.0003), mRNA expression levels of CD68 (p < 0.001) and IL-1ß (p < 0.01), and upregulated iNOS protein (p = 0.011). The mRNAs of MCP-1, IL-6 and adiponectin remained unchanged in PVAT. Aortic PVAT volume measured with micro-PET/CT was increased in ApoE-/- rats (p < 0.01). Maximal endothelium-dependent relaxation (EDR) to acetylcholine in ApoE-/- aortic rings without PVAT was severely impaired (p = 0.012) compared with controls, while ApoE-/- aortic rings with PVAT showed higher EDR than controls. All EDR responses were blocked by L-NMMA and the expression of eNOS mRNA was increased in ApoE-/- PVAT (p = 0.035). CONCLUSION: Using a rat ApoE-/- model of early atherosclerosis, we capture a novel mechanism by which inflammatory PVAT compensates severe endothelial dysfunction by contributing NO upon cholinergic stimulation.

Role of Phospholipases A2 in Vascular Relaxation and Sympatholytic Effects of Five Australian Brown Snake, Pseudonaja spp., Venoms in Rat Isolated Tissues.

Human envenoming by Australian brown snakes (Pseudonaja spp.) may result in potentially life-threatening hypotension and subsequent cardiovascular collapse. There have been relatively few studies of the cardiovascular and sympathetic effects of Pseudonaja spp. venoms. In this study, we have examined the effects of venom from five brown snake species-P. affinis, aspidorhyncha, inframacula, nuchalis, and textilis-on cardiac inotropic and chronotropic responses, vascular tone, and sympathetic nerve-induced vascular contractions in rat isolated tissues. The role of phospholipases A2 (PLA2s) in venom-induced effects was assessed with the sPLA2 inhibitor varespladib. In rat isolated left and right atria, there were no physiologically relevant effects of Pseudonaja venoms (0.1-30 µg/ml) on left atrial force of contraction (inotropy) or right atrial rate (chronotropy). In contrast, in isolated small mesenteric arteries precontracted with a thromboxane mimetic, each of the five brown snake venoms (at 30 µg/ml) caused marked vasorelaxation (-60 to -90% of contractile tone). Pretreatment with varespladib (1 µM) significantly inhibited the vasorelaxation caused by P. aspidorhyncha, P. nuchalis, and P. textilis venoms. Electrically induced sympathetic nerve-mediated contractions of mesenteric arteries were significantly attenuated by only P. textilis, and P. affinis venoms (30 µg/ml) and these sympatholytic effects were inhibited by varespladib (1 µM). Based on their inhibition with the sPLA2 inhibitor varespladib, we conclude that PLA2 toxins in P. aspidorhyncha, P. nuchalis, and P. textilis venoms are involved in brown snake venom-induced vasorelaxation and the sympatholytic effects of P. affinis, and P. textilis venoms. Our study supports the promising potential role of varespladib as an initial (pre-referral) and/or adjunct (in combination with antivenom) therapeutic agent for brown snake envenoming.

Impaired HSP70 Expression in the Aorta of Female Rats: A Novel Insight Into Sex-Specific Differences in Vascular Function.

Heat-shock protein 70 (HSP70) contributes to cellular calcium (Ca2+) handling mechanisms during receptor-mediated vascular contraction. Interestingly, previous studies have independently reported sex-related differences in HSP70 expression and Ca2+ dynamics. Still, it is unknown if sex, as a variable, plays a role in the impact that HSP70 has upon vascular contraction. To narrow this gap, we investigated if differences exist in the expression levels of HSP70 in the aorta, and if targeting this protein contributes to sex disparity in vascular responses. We report that, compared with male animals, female rats present a reduction in the basal levels of HSP70. More compelling, we found that the blockade of HSP70 has a greater impact on phenylephrine-induced phasic and tonic vascular contraction in female animals. In fact, it seems that the inhibition of HSP70 significantly affects vascular Ca2+ handling mechanisms in females, which could be associated with the fact that these animals have impaired HSP70 expression. Corroborating this idea, we uncovered that the higher sensitivity of female rats to HSP70 inhibition does not involve an increase in NO-dependent vasodilation nor a decrease in vascular oxidative stress. In summary, our findings reveal a novel mechanism associated with sex-specific differences in vascular responses to α-1 adrenergic stimulation, which might contribute to unraveling the network of intertwined pathways conferring female protection to (cardio)vascular diseases.

C-type natriuretic peptide-induced relaxation through cGMP-dependent protein kinase and SERCA activation is impaired in two kidney-one clip rat aorta.

AIMS: Hypertension underlies endothelial dysfunction, and activation of vasorelaxation signaling with low dependence on nitric oxide (NO) represents a good alternative for vascular modulation. C-type natriuretic peptide (CNP) causes relaxation by increasing cyclic guanosine 3',5'-monophosphate (cGMP) or Gi-protein activation through its natriuretic peptide receptor-B or -C, respectively. We have hypothesized that CNP could exerts its effects and could overcome endothelial dysfunction in two kidney-one clip (2K-1C) hypertensive rat aorta. Here, we investigate the intracellular signaling involved in CNP effects in hypertension. MATERIALS AND METHODS: The 2K-1C hypertension was induced in male Wistar rats (200 g). CNP-induced vascular relaxation and cGMP production were investigated in rat thoracic aortas. The natriuretic peptide receptor-B and -C localization was evaluated by immunofluorescence. Calcium mobilization was assessed in endothelial cells from rat aortas. KEY FINDINGS: CNP induced similar relaxation in normotensive and 2K-1C hypertensive rat aortas, which increased after endothelium removal. CNP-induced relaxation involved natriuretic peptide receptor-B and -C activation in 2K-1C rats. Nitric oxide synthase (NOS) and soluble guanylyl cyclase (sGC) counter-regulated CNP-particulate GC (pGC) activation in aortas. CNP reduced endothelial calcium and increased cGMP production, which was lower in 2K-1C. CNP-induced cGMP-dependent protein kinase (PKG) and sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) activation was impaired in 2K-1C rat aorta. SIGNIFICANCE: Our results indicated CNP triggered relaxation through its natriuretic peptide receptor-B and -C in 2K-1C rat aortas, and that CNP-induced relaxation overcomes endothelial dysfunction in hypertension. In addition, NOS and sGC activities counter-regulate CNP-pGC activation to induce vascular relaxation.

Endothelial dysfunction and body mass index: is there a role for plasma peroxynitrite?

BACKGROUND: Endothelial function is dependent on the balance between vasoconstrictive and vasodilatory substances. The endothelium ability to produce nitric oxide is one of the most crucial mechanisms in regulating vascular tone. An increase in inducible nitric oxide synthase contributes to endothelial dysfunction in overweight persons, while in underweight persons, oxidative stress contributes to the conversion of nitric oxide to peroxynitrite (measured as nitrotyrosine in vivo). The objective of this study was to elucidate the interaction of body composition and oxidative stress on vascular function and peroxynitrite. This was done through an experimental design with three weight groups (underweight normal weight and overweight), with four treatment arms in each. Plasma nitrotyrosine levels were measured 15-20 hours post lipopolysaccharide (LPS) treatment, as were aortic ring tension changes. Acetylcholine (ACh) and sodium nitroprusside (SNP) challenges were used to observe endothelial dependent and independent vascular relaxation after pre-constriction of aortic rings with phenylephrine. RESULTS: Nitrotyrosine levels in saline-treated rats were similar among the weight-groups. There was a significant increase in nitrotyrosine levels between saline-treated rats and those treated with the highest lipopolysaccharide doses in each of the weight groups. In response to ACh challenge, Rmax (percentage reduction in aortic tension) was lowest in overweight rats (112%). In response to SNP, there was an insignificantly lower Rmax in the underweight (106%) compared to the normal weight (112%). Overweight rats had a significant decrease in Rmax (83%) in response to SNP, signifying involvement of a more chronic process in tension reduction changes. A lower Rmax accompanied an increase in peroxynitrite after acetylcholine challenge in all weight-groups. CONCLUSIONS: Endothelial dysfunction, observed as an impairment in the ability to reduce tension, is associated with increased plasma peroxynitrite levels across the spectrum of body mass. In higher-BMI rats, an additional role is played by vascular smooth muscle in the causation of endothelial dysfunction.

Protective Effects of Grape Juice on Vascular Damage Induced by Chlorine Free Radical in Rats.

Grapes and their derivatives have antioxidant and cardioprotective properties. Therefore, we hypothesized that grape juice (GJ) could improve vascular oxidative damage caused by chlorine radicals (OCl-), which are excessively produced in vascular tissue during cardiovascular diseases (mainly diabetes and hypertension). The antioxidant capacity of GJ was analyzed by an electrochemical method, followed by administration in rats (100 or 300 mg/kg/d, via the oral) for seven days. Then, rats were sacrificed, and their aortas were isolated and subjected to isometric recordings or immuno-histochemical analyses with or without exposure to OCl- (5, 20, or 100 µM, 60 min). Concentration-effect curves for acetylcholine (ACh) and sodium nitroprusside (SNP) were derived to analyze endothelium-dependent or independent vasore-laxation. The GJ presented with high antioxidant capacity, and treatment with GJ did not alter vascular relaxation induced by ACh or SNP. After exposure to OCl-, endothelium-denuded arteries showed preserved relaxation with SNP, whereas endothelium-intact arteries showed reduced relaxation with ACh. OCl- at various concentrations induced significantly decreased relaxation of arteries (80.6±4.2%, 55.4±4.7%, and 28.1±5.9%, respectively) vs. control arteries (96.8±2.4%). However, treatment with GJ prevented loss in relaxation caused by 5 and 20 µM OCl- and improved relaxation after exposure to 100 µM OCl-. Exposure to OCl- induced increased nitrotyrosine immunostaining of endothelial cell layers, which was improved by GJ treatment. Altogether, vascular damage caused by OCl- was prevented by treatment with GJ, and GJ prevented nitrosative stress in these vessels.

Castanea sativa Mill. bark extract cardiovascular effects in a rat model of high-fat diet.

Ellagitannins may have a beneficial impact in cardiovascular diseases. The aim of the study was to evaluate the effect of high-fat diet (HFD) and the efficacy of Castanea sativa Mill. bark extract (ENC) on cardiac and vascular parameters. Rats were fed with regular diet, (RD, n = 15), HFD (n = 15), RD + ENC (20 mg/kg/day by gavage, n = 15), and HFD + ENC (same dose, n = 15) and the effects on body weight, biochemical serum parameters, and inflammatory cytokines determined. Cardiac functional parameters and aorta contractility were also assessed on isolated atria and aorta. Results showed that ENC reduced weight gain and serum lipids induced by HFD. In in vitro assays, HFD decreased the contraction force of left atrium, increased right atrium chronotropy, and decreased aorta K+ -induced contraction; ENC induced transient positive inotropic and negative chronotropic effects on isolated atria from RD and HFD rats and a spasmolytic effect on aorta. In ex vivo experiments, ENC reverted inotropic and chronotropic changes induced by HFD and enhanced Nifedipine effect more on aorta than on heart. In conclusion, ENC restores metabolic dysfunction and cardiac cholinergic muscarinic receptor function, and exerts spasmolytic effect on aorta in HFD rats, highlighting its potential as nutraceutical tool in obesity.

Defining the reducing system of the NO dioxygenase cytoglobin in vascular smooth muscle cells and its critical role in regulating cellular NO decay.

In smooth muscle, cytoglobin (Cygb) functions as a potent nitric oxide (NO) dioxygenase and regulates NO metabolism and vascular tone. Major questions remain regarding which cellular reducing systems regulate Cygb-mediated NO metabolism. To better define the Cygb-mediated NO dioxygenation process in vascular smooth muscle cells (SMCs), and the requisite reducing systems that regulate cellular NO decay, we assessed the intracellular concentrations of Cygb and its putative reducing systems and examined their roles in the process of NO decay. Cygb and the reducing systems, cytochrome b5 (B5)/cytochrome b5 reductase (B5R) and cytochrome P450 reductase (CPR) were measured in aortic SMCs. Intracellular Cygb concentration was estimated as 3.5 µM, while B5R, B5, and CPR were 0.88, 0.38, and 0.15 µM, respectively. NO decay in SMCs was measured following bolus addition of NO to air-equilibrated cells. siRNA-mediated knockdown experiments indicated that âˆ¼78% of NO metabolism in SMCs is Cygb-dependent. Of this, ∼87% was B5R- and B5-dependent. CPR knockdown resulted in a small decrease in the NO dioxygenation rate (VNO), while depletion of ascorbate had no effect. Kinetic analysis of VNO for the B5/B5R/Cygb system with variation of B5 or B5R concentrations from their SMC levels showed that VNO exhibits apparent Michaelis-Menten behavior for B5 and B5R. In contrast, linear variation was seen with change in Cygb concentration. Overall, B5/B5R was demonstrated to be the major reducing system supporting Cygb-mediated NO metabolism in SMCs with changes in cellular B5/B5R levels modulating the process of NO decay.

Potential role of hydrogen sulfide in diabetes-impaired angiogenesis and ischemic tissue repair.

Diabetes is one of the most prevalent metabolic disorders and is estimated to affect 400 million of 4.4% of population worldwide in the next 20 year. In diabetes, risk to develop vascular diseases is two-to four-fold increased. Ischemic tissue injury, such as refractory wounds and critical ischemic limb (CLI) are major ischemic vascular complications in diabetic patients where oxygen supplement is insufficient due to impaired angiogenesis/neovascularization. In spite of intensive studies, the underlying mechanisms of diabetes-impaired ischemic tissue injury remain incompletely understood. Hydrogen sulfide (H2S) has been considered as a third gasotransmitter regulating angiogenesis under physiological and ischemic conditions. Here, the underlying mechanisms of insufficient H2S-impaired angiogenesis and ischemic tissue repair in diabetes are discussed. We will primarily focuses on the signaling pathways of H2S in controlling endothelial function/biology, angiogenesis and ischemic tissue repair in diabetic animal models. We summarized that H2S plays an important role in maintaining endothelial function/biology and angiogenic property in diabetes. We demonstrated that exogenous H2S may be a theraputic agent for endothelial dysfunction and impaired ischemic tissue repair in diabetes.

PKR inhibitor imoxin prevents hypertension, endothelial dysfunction and cardiac and vascular remodelling in L-NAME-treated rats.

AIMS: Hypertension is one of the leading causes of cardiovascular mortality and morbidity. It is associated with severe cardiac and vascular dysfunction. Double-stranded RNA-dependent protein kinase (PKR), is a known inducer of inflammation and apoptosis. However, no research has been done to elucidate the role of the PKR in an experimental model of hypertension, and related cardiovascular complications. MAIN METHODS: L-NAME (NG-Nitro-L-arginine-methyl ester) was used to induce the hypertension. Imoxin treatment was given to Wistar rats for the four weeks along with the L-NAME, to investigate the influence on the hypertension. Changes in physiological parameter were assessed by recording non-invasive blood pressure. Expression of PKR and downstream markers for inflammation, fibrosis, and vascular damage in rat heart and aorta was determined by western blot and immunohistochemistry. Histological examination and fibrosis assessment were done by using assay kits. Vascular reactivity was determined by ex-vivo isometric tension studies on rat aortic rings. KEY FINDINGS: L-NAME-treated rats showed a significant increase in PKR expression followed by cardiac damage and vascular alterations compared to that of control animals. Results of western blot and immunohistochemistry indicate a significant increase in the inflammatory markers downstream to PKR. Endothelium-dependent vascular relaxation was significantly impaired in L-NAME administered rats. All effects of the L-NAME were attenuated by selective inhibition of PKR by imoxin. SIGNIFICANCE: Alterations in the heart and vasculature could be mediated in part by activation of the PKR pathway. Hence selective inhibition of PKR has therapeutic potential for combating hypertension and associated cardiovascular complications.

Progesterone promotes endothelial nitric oxide synthase expression through enhancing nuclear progesterone receptor-SP-1 formation.

Progesterone exerts antihypertensive actions partially by modulating endothelial nitric oxide synthase (eNOS) activity. Here, we aimed to investigate the effects and mechanisms of progesterone on eNOS expression. First, human umbilical vein endothelial cells (HUVECs) were exposed to progesterone and then the eNOS transcription factor specificity protein-1 (SP-1) and progesterone receptor (PRA/B) expression were assessed by Western blotting and qRT-PCR. The interaction between SP-1 and PRA/B was next determined through coimmunoprecipitation assay. The chromatin immunoprecipitation assay and luciferase assay were used to investigate the relationship of PRA/B, SP-1, and eNOS promoter. At last, rats were intraperitoneally injected with progesterone receptor antagonist RU-486, and then the expression of eNOS and vasodilation function in thoracic aorta and mesenteric artery were measured. The results showed that progesterone could increase eNOS expression in HUVECs. Further study showed that progesterone increased PRA-SP-1 complex formation and facilitated PRA/B and SP-1 binding to eNOS promoter. Mutating SP-1 or PR-binding motif on eNOS promoter abolished the effect of progesterone on eNOS gene transcription. We also observed that progesterone receptor antagonist RU-486 reduced eNOS expression and impaired vasodilation in rats. Those results suggest that progesterone modulates eNOS expression through promoting PRA-SP-1 complex formation, and progesterone antagonist attenuates eNOS expression, leading to the loss of vascular relaxation.NEW & NOTEWORTHY Progesterone directly upregulated endothelial nitric oxide synthase (eNOS) expression in human endothelial cells. Progesterone augmented eNOS promoter activity through a progesterone receptor A- and specificity protein-1-dependent manner. Antagonism of the progesterone receptor reduced eNOS expression and impaired vasodilation in rats.

Imipridone enhances vascular relaxation via FOXO1 pathway.

Cardiovascular complications are a side effect of cancer therapy, potentially through reduced blood vessel function. ONC201 (TIC10) is currently used in phase 2 clinical trials to treat high-grade gliomas. TIC10 is a phosphatidylinositol 3-kinase (PI3K)/AKT/extracellular signal-regulated kinase (ERK) inhibitor that induces apoptosis via upregulation of TNF-related apoptosis-inducing ligand, which via stimulation of FOXO and death receptor could increase eNOS upregulation. This has the potential to improve vascular function through increased NO bioavailability. Our aim was to investigate the role of TIC10 on vascular function to determine if it would affect the risk of CVD. Excised abdominal aorta from White New Zealand male rabbits were cut into rings. Vessels were incubated with TIC10 and AS1842856 (FOXO1 inhibitor) followed by cumulative doses of acetylcholine (Ach) to assess vessel function. Vessels were then processed for immunohistochemistry. Incubation of blood vessels with TIC10 resulted in enhanced vasodilatory capacity. Combination treatment with the FOXO1 inhibitor and TIC10 resulted in reduced vascular function compared to control. Immunohistochemical analysis indicated a 3-fold increase in death receptor 5 (DR5) expression in the TIC10-treated blood vessels but the addition of the FOXO1 inhibitor downregulated DR5 expression. The expression of DR4 receptor was not significantly increased in the presence of TIC10; however, addition of the FOXO1 inhibitor downregulated expression. TIC10 has the capacity to improve the function of healthy vessels when stimulated with the vasodilator Ach. This highlights its therapeutic potential not only in cancer treatment without cardiovascular side effects, but also as a possible drug to treat established CVD.