Endothelium-independent Relaxation Research Articles

Characterization and quantification of intact glucosinolates in Catozza rapeseeds: A promising food matrix for nutraceuticals development as a source of hydrogen sulfide

Glucosinolates (GSLs), anionic sulfur-containing secondary metabolites of Brassicales order, have shown beneficial effects for the prevention of cardiovascular diseases. This study has focused on HPLC-HESI-MS/MS analysis of intact GSLs in a poorly studied food matrix, Catozza rapeseeds (Brassica rapa L. var. rapa DC.), providing the first qualitative profile of this matrix by the identification of fifteen GSLs and the quantification of thenine most abundant compounds. After the evaluation of different powder granulometries, the best myrosinase inactivation treatment condition was achieved with a 30-minute heating treatment at 80 °C, resulting in the highest GSLs content. Two extraction methods were performed on the rapeseed meal to obtain hydroalcoholic Catozza rapeseed extracts (HCRE) and aqueous Catozza rapeseed extracts (ACRE) showing a concentration-dependent and endothelium-independent relaxing effect in mice aorta by releasing hydrogen sulfide (H2S). This study represents the starting point for further investigation of Catozza rapeseed use for GSLs-based formulations for nutraceuticals purpose.

HTK vs. HTK-N for Coronary Endothelial Protection during Hypothermic, Oxygenated Perfusion of Hearts Donated after Circulatory Death.

Protection of the coronary arteries during donor heart maintenance is pivotal to improve results and prevent the development of coronary allograft vasculopathy. The effect of hypothermic, oxygenated perfusion (HOP) with the traditional HTK and the novel HTK-N solution on the coronary microvasculature of donation-after-circulatory-death (DCD) hearts is known. However, the effect on the coronary macrovasculature is unknown. Thus, we maintained porcine DCD hearts by HOP with HTK or HTK-N for 4 h, followed by transplantation-equivalent reperfusion with blood for 2 h. Then, we removed the left anterior descending coronary artery (LAD) and compared the endothelial-dependent and -independent vasomotor function of both groups using bradykinin and sodium-nitroprusside (SNP). We also determined the transcriptome of LAD samples using microarrays. The endothelial-dependent relaxation was significantly better after HOP with HTK-N. The endothelial-independent relaxation was comparable between both groups. In total, 257 genes were expressed higher, and 668 genes were expressed lower in the HTK-N group. Upregulated genes/pathways were involved in endothelial and vascular smooth muscle cell preservation and heart development. Downregulated genes were related to ischemia/reperfusion injury, oxidative stress, mitochondrion organization, and immune reaction. The novel HTK-N solution preserves the endothelial function of DCD heart coronary arteries more effectively than traditional HTK.

(247) A Rat Model for Bipolar Androgen Therapy is Evaluated for Its Effects on Erectile Dysfunction after Castration

Abstract Introduction Prostate cancer (PCa), an androgen-dependent tumor and one of the most prevalent cancers in males, has traditionally been treated with androgen suppression. In recent years, bipolar androgen treatment (BAT) has emerged as a potential therapeutic approach for men with metastatic castrate-resistant PCa. The control of testosterone (T) between castration and supraphysiological levels inhibits the proliferation of cancer cells and slows the progression of cancer. Furthermore, while there has been prior research between T and erectile function, there is no evidence linking BAT and erectile dysfunction (ED). Objective The purpose of the current investigation was to determine if BAT therapy was effective in treating ED in a castration-prone rat model. Methods Sprague-Dawley rats (n=30) were randomly divided into four groups: 1) Control, 2) Control rats-received BAT treatment, 3) castrated Rats, and 4) castrated rats-received BAT treatment. The rats were castrated by making bilateral scrotal incisions. A subcutaneous injection of testosterone propionate (250 mg/kg/week, intramuscular) was administered four weeks following castration. Four weeks after the initial dose, the second dose of the medication was given in the same manner. By stimulating the cavernous nerve, the intracavernosal pressure (ICP) to mean arterial pressure (MAP) ratio and total ICP were determined. Corpus cavernosum (CC) strips were used as in vitro preparations for evaluating the relaxant and contractile responses. Nitric oxide synthase (nNOS) and endothelial NOS (eNOS) protein expression was analyzed using Western blotting. Using Masson trichrome staining, the location of the smooth muscle in relation to the collagen was identified. Results Castrated rats' prostate weight was reduced when compared to uncastrated rats (P< 0.001). Compared to controls and castrated rats, BAT marginally boosted prostate weight reduction. The ICP/MAP ratio and total ICP for 7.5 V were lowest in castrated rats (P< 0.001 vs. controls). BAT enhanced ICP/MAP and total ICP responses (P<0.01 vs. castrated rats). In vitro, endothelium-dependent acetylcholine-induced relaxation at 1 nM (P 0.05 vs controls), endothelium-independent sodium nitroprusside-induced relaxation at 1 nM (P 0.01 vs controls), electrical field stimulation-induced relaxant response at 20 Hz (P<0.05 vs. controls) and sildenafil-induced relaxation at 10 M (P 0.05 vs controls) were all reduced in the CC strips from the castrated group. Additionally, the castrated group showed increased neurogenic contractile response at 40 Hz and the contractile response to phenylephrine at 1 mM (P 0.001 vs. controls). The variations in the relaxation and contractile responses were returned by BAT. The smooth muscle cell-to-collagen ratio and nNOS and eNOS protein expression were both markedly reduced in castrated rats, though BAT reversed these modifications. Conclusions The results of the study show that BAT improves neurogenic relaxation, endothelium relaxation, sildenafil-induced relaxing of the CC, and changes in penile morphology after castration. BAT might be a cutting-edge therapeutic approach for patients with castration-resistant prostate cancer who have an ED route. Disclosure No.

(370) Overview of Nonclinical Studies of LIB-01, a Novel Promising Oral Drug Under Development for Treatment of Erectile Dysfunction

Abstract Introduction LIB-01 is a novel molecule being developed as an oral drug for the treatment of erectile dysfunction (ED). It is a semi-synthetic analog, developed based on research on active compounds identified in root bark that had been used for treatment of male sexual disability since time immemorial. Objective In preparation for clinical development, a number of nonclinical studies on erectile function were conducted in normal and diabetic rats with ED. Studies to investigate the mechanism supporting the pro-erectile effect of LIB-01 were also conducted. A summary of the results from the nonclinical research program is presented. Methods Erectile response elicited by electrical stimulation of the cavernous nerve (ES CN) in anaesthetized rats was assessed. Intracavernous and arterial blood pressures were monitored according to a standardized experimental procedure. Mechanism of action studies were conducted with rat cavernosal tissue samples using immunohistochemistry and in ex vivo/organ chamber experiments. The isometric tension studies were conducted on cavernosal strips from LIB-01 treated and untreated Wistar rats. Cavernosal strips from untreated rats were incubated with either LIB-01 or DMSO (control) and then pre-contracted by phenylephrine (PHE). Concentration- or frequency response curves were performed on PHE pre-contracted strips in 3 consecutive steps: (i) cumulative addition of increasing acetylcholine (ACh) concentrations, (ii) electrical field stimulation (EFS) with increasing frequencies, and (iii), cumulative addition of increasing sodium nitroprusside (SNP) concentrations. Results A statistically significant pro-erectile facilitatory effect of LIB-01 has been repeatedly evidenced in all the studies conducted in anaesthetized control Wistar rats, by oral or subcutaneous administration of LIB-01 at 15 mg/kg/d, with an effect in the similar range as iv sildenafil 0.3mg/kg, when compared. The pro-erectile effect of LIB-01 was not acute, instead, the effect gradually increased and remained for at least 7 days post treatment. In addition, LIB-01 significantly improved erectile responses in anaesthetized diabetic Goto-Kakizaki ED rats compared to vehicle-treated rats, demonstrating the pro-erectile effect of LIB-01 in a robust validated pathophysiological rat model of type 2 diabetes. Immunohistochemistry experiments on cavernosal tissue from LIB-01 treated normal rats showed that neither CD31 nor nNOS expression was altered, thus likely ruling out any effect of LIB-01 on cavernosal micro-vascularization or cavernosal nNOS. Furthermore, only at a high concentration (10-5 M) was there a slight increase in the endothelium dependent relaxations induced by ACh compared to vehicle while LIB-01 did not exert any effect neither on endothelium-independent relaxations induced by SNP nor on nitrergic relaxation induced by EFS of untreated cavernosal strips exposed ex-vivo to different concentrations of LIB-01. Conclusions LIB-01 is a novel molecule which displays a significant pro-erectile effect in anesthetized normal rats as well as in diabetic rats with ED. The pro-erectile effect of LIB-01 is not acute, instead, the effect gradually increases and remains for at least 7 days post-treatment. Results from several mechanistic studies lead to postulate that the mechanism of LIB-01 does not recruit the NO-cGMP pathway thereby differing from phosphodiesterase type 5 inhibitors. A first-in-human clinical trial with LIB-01 is planned to start in 2023. Disclosure Yes, this is sponsored by industry/sponsor: Dicot AB. Clarification: Industry initiated, executed and funded study. Any of the authors act as a consultant, employee or shareholder of an industry for: Dicot AB.

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

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. To uncover these mechanisms, force development was measured exvivo 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 exvivo using pressure myography. Effects of Mg2+ on NO formation were measured in Chinese Hamster Ovary (CHO) cells, isolated mesenteric vessels, and mouse urine. 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. 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.

LCZ696, an Angiotensin Receptor-Neprilysin Inhibitor, Ameliorates Endothelial Dysfunction in Diabetic C57BL/6 Mice.

LCZ696 (sacubitril/valsartan) exerts cardioprotective effects. Recent studies have suggested that it improves the endothelial function; however, the underlying mechanisms have not been thoroughly investigated. We investigated whether LCZ696 ameliorates diabetes-induced endothelial dysfunction. Diabetes was induced using streptozotocin in 8-week-old male C57BL/6 mice. Diabetic mice were randomly assigned to receive LCZ696 (100 mg/kg/day), valsartan (50 mg/kg/day), or a vehicle for three weeks. The endothelium-dependent and endothelium-independent vascular responses of the aortic segments were determined based on the response to acetylcholine and sodium nitroprusside, respectively. Human umbilical vein endothelial cells (HUVEC) and aortic segments obtained from C57BL/6 mice were used to perform in vitro and ex vivo experiments, respectively. LCZ696 and valsartan reduced the blood pressure in diabetic mice (P＜0.05). The administration of LCZ696 (P＜0.001) and valsartan (P＜0.01) ameliorated endothelium-dependent vascular relaxation, but not endothelium-independent vascular relaxation, under diabetic conditions. LCZ696, but not valsartan, increased eNOSSer1177 (P=0.06) and Akt (P＜0.05) phosphorylation in the aorta. In HUVEC, methylglyoxal (MGO), a major precursor of advanced glycation end products, decreased eNOSSer1177 phosphorylation (P＜0.05) and increased eNOSThr495 phosphorylation (P＜0.001). However, atrial natriuretic peptide (ANP) reversed these effects. ANP also ameliorated the MGO-induced impairment of endothelium-dependent vascular relaxation in the aortic segments (P＜0.05), although L-NAME completely blocked this effect (P＜0.001). LCZ696 ameliorated diabetes-induced endothelial dysfunction by increasing the bioavailability of ANP. Our findings suggest that LCZ696 has a vascular protective effect in a diabetic model and highlight that it may be more effective than valsartan.

Evidence for the involvement of TRPV2 channels in the modulation of vascular tone in the mouse aorta

AimsTransient receptor potential vanilloid 2 (TRPV2) channels are expressed in both smooth muscle and endothelial cells and participate in vascular mechanotransduction and sensing of high temperatures and lipids. Nevertheless, the impact of TRPV2 channel activation by agonists on the coordinated and cell-type specific modulation of vasoreactivity is unknown. Main methodsAorta from 2- to 4-months-old male Oncins France 1 mice was dissected and mounted in tissue baths for isometric tension measurements. TRPV2 channel expression was assessed by immunofluorescence and western blot in mice aortas and in cultured A7r5 rat aortic smooth muscle cells. Key findingsTRPV2 channels were expressed in all three mouse aorta layers. Activation of TRPV2 channels with probenecid evoked endothelium-dependent relaxations through a mechanism that involved activation of smooth muscle Kir and Kv channels. In addition, TRPV2 channel inhibition with tranilast increased endothelium-independent relaxations to probenecid and this effect was abrogated by the KATP channel blocker glibenclamide, revealing that smooth muscle TRPV2 channels induce negative feedback on probenecid relaxations mediated via KATP channel inhibition. Exposure to the NO donor sodium nitroprusside increased TRPV2 channel translocation to the plasma membrane in cultured smooth muscle cells and enhanced negative feedback on probenecid relaxations. SignificanceIn conclusion, we present the first evidence that TRPV2 channels may modulate vascular tone through a balance of opposed inputs from the endothelium and the smooth muscle leading to net vasodilation. The fact that TRPV2 channel-induced activity can be amplified by NO emphasizes the pathophysiological relevance of these findings.

Cardiovascular phenotype of the duchenne muscular dystrophy carrier female rat

Abstract Background Duchenne muscular dystrophy (DMD) is a lethal, severely progressive muscle-wasting disease. It is an X-linked recessive disease caused by mutations in the DMD gene encoding dystrophin protein and consequently the absence of dystrophin protein synthesis. Besides the skeletal muscle abnormalities, DMD patients are often suffering cardiovascular symptoms include cardiac fibrosis, dilated cardiomyopathy, arrhythmia, and congestive heart failure. Heterozygous female DMD carriers rarely acquire skeletal muscle symptoms, however the cardiac abnormalities are often underrepresented. There are few studies to date characterizing cardiovascular abnormalities in female DMD carriers and the mechanisms underlying the cardiac manifestations are still not fully known. Aims and Methods: Therefore, the aim of this study was to investigate the cardiovascular phenotype of 9 months old female DMD carrier rats and compare with age-matched wildtype rats. Objectives included assessment of cardiac function via transthoracic echocardiography, vascular function using wire myography, cardiac fibrosis and dystrophin via histology, mRNA expression of inflammatory markers via RT-qPCR, measurements of ACE1 and ACE2 activity, as well as expression of dystrophin, SERCA, sarcolipin, BAG3, and Connexin43 (Cx43) using western blotting. The cardiac function, vascular endothelial function, ACE1 and ACE2 activity, and collagen deposition in cardiac tissue were similar between the female DMD carrier and wildtype rats. The echocardiography data revealed thinning of the left-ventricular posterior wall indicating trend towards cardiac atrophy and dysfunction (p<0.05). Wire myography data showed trends of impaired endothelium-independent relaxation and hypercontractility, suggesting the potential vascular abnormalities. Histological analysis showed thickening of intima-media layer in coronary arteries, addition to reduced dystrophin staining, further affirming dystrophin loss impairs smooth muscle cell function (p<0.05, respectively). Protein expression of dystrophin, SERCA2, and BAG3 was decreased in female DMD carrier rats in the myocardium. We also found decreased levels of Cx43 at the intercalated discs, with a concomitant increase in the phosphorylation of Serine368 on Cx43 that very often precedes ubiquitination and degradation of Cx43 channels. In agreement, higher levels of the ubiquitin ligase Nedd4 were observed in hearts of female DMD carrier rats, implicating Cx43 degradation in cardiac dysfunction observed in these animals. Conclusions These findings indicate that female DMD carrier rats do not develop severe cardiac systolic dysfunction nor dilated cardiomyopathy at the age of 9 months unlike male DMD rats as previously described(1). However, the female DMD carrier rats begin to manifest tendencies towards cardiac and vascular dysfunction in association with a changes of calcium handling protein and Cx 43 expression in the heart.

Relaxation Effect of Schisandrol A on Isolated Thoracic Aorta and Its Mechanism in Rats

Background Schisandra chinensis (S. chinensis) is a drug commonly used in the clinical treatment of cardiovascular diseases in Traditional Chinese Medicine. However, the specific components and mechanisms of its action are still unclear. We screened six kinds of lignans from S. chinensis with high content and found that schisandrol A and schisantherin A had a strong vasorelaxant effect. The purpose of this study was to investigate the relaxation and underlying mechanism of schisandrol A in the isolated thoracic aorta of rats. Materials and Methods Isolated rat endothelium-intact and endothelium-removed thoracic aorta strips were pre-constricted with phenylephrine (PE), and the relaxation of schisandrol A on the strips was observed. Then, the mechanism was explored by pre-incubating the strips with nitric oxide synthetase inhibitor Nɷ-nitro-l-arginine methyl ester (L-NAME), cyclooxygenase inhibitor (indomethacin), potassium channel blockers 4-aminopyridine (4-AP), barium chloride (BaCl2), tetraethylamine (TEA), and glibenclamide, respectively, and changing the calcium concentration in the bath. In addition, expressions of endothelial nitric oxide synthetase (eNOS) mRNA and protein in rat thoracic aorta were detected. Results Schisandrol A induced both endothelium-dependent and endothelium-independent relaxation of isolated thoracic aorta strips of rats, and the mechanism might be related to promoting the synthesis of NO, inhibiting Ca2+ release from the sarcoplasmic reticulum, and blocking the Ca2+ channels. Conclusion These discoveries may provide a theoretical basis for the traditional application of S. chinensis to treat cardiovascular disease.

Varied effects of tobacco smoke and e-cigarette vapor suggest that nicotine does not affect endothelium-dependent relaxation and nitric oxide signaling

Chronic smoking causes dysfunction of vascular endothelial cells, evident as a reduction of flow-mediated dilation in smokers, but the role of nicotine is still controversial. Given the increasing use of e-cigarettes and other nicotine products, it appears essential to clarify this issue. We studied extracts from cigarette smoke (CSE) and vapor from e-cigarettes (EVE) and heated tobacco (HTE) for their effects on vascular relaxation, endothelial nitric oxide signaling, and the activity of soluble guanylyl cyclase. The average nicotine concentrations of CSE, EVE, and HTE were 164, 800, and 85 µM, respectively. At a dilution of 1:3, CSE almost entirely inhibited the relaxation of rat aortas and porcine coronary arteries to acetylcholine and bradykinin, respectively, while undiluted EVE, with a 15-fold higher nicotine concentration, had no significant effect. With about 50% inhibition at 1:2 dilution, the effect of HTE was between CSE and EVE. Neither extract affected endothelium-independent relaxation to an NO donor. At the dilutions tested, CSE was not toxic to cultured endothelial cells but, in contrast to EVE, impaired NO signaling and inhibited NO stimulation of soluble guanylyl cyclase. Our results demonstrate that nicotine does not mediate the impaired endothelium-dependent vascular relaxation caused by smoking.

GYY4137, a hydrogen sulfide donor, protects against endothelial dysfunction in porcine coronary arteries exposed to myeloperoxidase and hypochlorous acid

Background and aimsMyeloperoxidase (MPO) and its principal reaction product hypochlorous acid (HOCl) are part of the innate immune response but are also associated with endothelial dysfunction, thought to involve a reduction in nitric oxide (NO) bioavailability. We aimed to investigate the effect of MPO and HOCl on vasorelaxation of coronary arteries and to assess directly the involvement of NO. In addition, we hypothesised that the slow release hydrogen sulfide (H2S) donor GYY4137 would salvage coronary artery endothelial function in the presence of MPO and HOCl. Methods and resultsContractility of porcine coronary artery segments was measured using isometric tension recording. Incubation with MPO (50 ng/ml) plus hydrogen peroxide (H2O2) (30 μM; substrate for MPO) impaired endothelium-dependent vasorelaxation to bradykinin in coronary arteries. HOCl (10–500 μM) also impaired endothelium-dependent relaxations. There was no effect of MPO plus H2O2, or HOCl, on endothelium-independent relaxations to 5′-N-ethylcarboxamidoadenosine and sodium nitroprusside. L-NAME (300 μM), a NO synthase inhibitor, attenuated bradykinin relaxations, leaving L-NAME-resistant relaxations to bradykinin mediated by endothelium-dependent hyperpolarization. In the presence of L-NAME, MPO plus H2O2 largely failed to impair endothelium-dependent relaxations to bradykinin. Similarly, HOCl failed to inhibit endothelium-dependent relaxations to bradykinin in the presence of L-NAME. GYY4137 (1–100 μM) protected endothelium-dependent relaxations to bradykinin from dysfunction caused by MPO plus H2O2, and HOCl, with no effect alone on bradykinin relaxation responses. The specific MPO inhibitor aminobenzoic acid hydrazide (ABAH) (1 and 10 μM) also protected against MPO plus H2O2-induced endothelial dysfunction (at 10 μM ABAH), but was less potent than GYY4137. ConclusionsMPO plus H2O2, and HOCl, impair coronary artery endothelium-dependent vasorelaxation via inhibition of NO. GYY4137 protects against endothelial dysfunction in arteries exposed to MPO plus H2O2, and HOCl. H2S donors such as GYY4137 are possible therapeutic options to control excessive MPO activity in cardiovascular diseases.

Sex-Specific Protection of Endothelial Function after Vascular Ischemia/Reperfusion Injury by the Senomorphic Agent Ruxolitinib.

Ischemia/reperfusion (I/R)-induced endothelial dysfunction occurs in various cardiovascular disorders. I/R injury is partially driven by the release of cytokines. Known for its use in senotherapy, the JAK inhibitor ruxolitinib is able to block the release of cytokines. We investigated the effect of ruxolitinib on the cytokine release and endothelial-dependent vasorelaxation in an in vitro model of I/R. Aortic segments of C57BL/6J mice (N = 12/group) were divided into three groups: control, in vitro I/R (I/R group), and in vitro I/R with ruxolitinib during ischemic incubation (I/R+Ruxo group). We determined cytokine expression. In organ bath chambers, we investigated the maximal endothelial-dependent relaxation to acetylcholine (RmaxACh) and maximal endothelial-independent relaxation to sodium-nitroprusside (RmaxSNP). RmaxACh was decreased in I/R compared to the control (83.6 ± 2.4 vs. 48.6 ± 3.4%; p < 0.05) and I/R+Ruxo (74.4 ± 2.6 vs. 48.6 ± 3.4%; p < 0.05). RmaxSNP was comparable between all groups. IL-10 was detectable only in I/R+Ruxo. CXCL5, CCL2, CCL3, CCL8, CCL11, ICAM-1, IL-1α, IL-7, TNF-α, and G-CSF were decreased or not detectable in I/R+Ruxo. In I/R+Ruxo, ICAM-1 was reduced in rings only from male mice. Treatment of the aorta from mice during in vitro ischemia with the senomorphic agent ruxolitinib reduces cytokine release and protects the endothelium from I/R-mediated dysfunction.

Rutin ameliorates nitrergic and endothelial dysfunction on vessels and corpora cavernosa of diabetic animals

Endothelial dysfunction is an early complication of diabetes and it is related to both micro- and macroangiopathies. In addition, >70% of diabetic patients develop autonomic neuropathies. Increased oxidative stress has a major role in the development of both nitrergic and endothelial dysfunction. The aim of this work is to evaluate whether rutin, a potent antioxidant, could ameliorate nitrergic and/or endothelial dysfunction in diabetic animals. Primary and secondary treatment protocols with rutin were investigated on rat aortic rings and the mesenteric arteriolar bed, and on rabbit aortic rings and corpora cavernosa (RbCC) from both euglycemic and alloxan-diabetic animals. Acetylcholine endothelium-dependent and sodium nitroprusside endothelium-independent relaxations were compared in tissues from euglycemic or diabetic animals. Electrical field stimulation (EFS)-induced relaxation was performed only in the RbCC. Endothelial-dependent relaxations were blunted by 40% in vessels and neuronal relaxation was blunted by 50% in RbCC taken from diabetic animals when compared to euglycemic animals. Pre-treatment with rutin restored both neuronal and endothelial dependent relaxations in diabetic animals towards the values achieved in control euglycemic tissues. Rutin was able to ameliorate both endothelial dysfunction and nitrergic neuropathy in animal experimental models. Rutin could be a lead compound in the primary or secondary preventive ancillary treatment of endothelial and nitrergic dysfunction in the course of diabetes.

(224) Icilin, a TRPM8 Agonist, Induces Relaxation and Reduces Sensitivity to Phenylephrine in Pudendal Artery from Control and Diabetic Mice

Abstract Introduction Diabetes mellitus (DM) is a chronic metabolic disease with epidemiological relevance worldwide. One of the most common and most underestimated complications of diabetes is erectile dysfunction (ED). It is estimated that approximately 35-90% of diabetic men present ED as an early complication. Previous studies have shown that TRPM8 activation triggers relaxation of pudendal artery with increased sensitivity in the hypertensive rats. However, despite the correlation between ED and diabetes, research on the role of TRPM8 channels in the vasculature responsible for sexual function in diabetic condition still needs to be elucidated. Objective In this way, the aim of this study was to investigate TRPM8 channels in arteries vital to erection of the penis. We hypothesized that TRPM8 activation would induce relaxation and reduce sensitivity to alpha-adrenergic stimulation in pudendal arteries from diabetic mice. Methods Male control and db/db mice were euthanized, and the internal pudendal arteries were mounted on DMT wire myographs for the measurement of isometric force. Results Cumulative administration of icilin (10-8 to 10-4M), a TRPM8 agonist, induced endothelium-dependent and endothelium-independent relaxation in pre-contracted rings with phenylephrine (Phe,1μM). Relaxant effect induced by icilin was similar between pudendal arteries from db/db (Emax: 56.31 +/− 1.90%, n=5) and control (Emax:46.57 +/− 4.45%, n=5) mice. Diabetes increased the contractility of the pudendal artery in response to phenylephrine in concentration of 3x10-6M (99.40 +/− 4.65%, n=4) compared to control (85.44 +/− 2.36%, n=4). Interestingly, pre-incubation with icilin (10-4M) decreased the potency of the contraction induced by phenylephrine (10-9 to 3x10-5M) compared to untreated rings in both diabetic (pD2: 6.39 +/− 0.07 vs 6.67 +/− 0.05, n=4) and control (pD2: 6.43 +/− 0.09 vs 6.82 +/− 0.05, n=4) animals. In the pudendal artery, diabetes decreased the sensitivity to acetylcholine compared to control (pD2: 7.17 +/− 0.06 vs 7.42 +/− 0.03, n=4). However, the relaxation induced by acetylcholine (10-9 to 3x10-5M) was not changed by pre-incubation with icilin. Conclusions Icilin is able to promote relaxation with the same magnitude in pudendal arteries from diabetic and control mice. Increased responses to phenylephrine in pudendal artery may contribute to the development of ED. In addition, pre-incubation with icilin reduced sensitivity to phenylephrine, suggesting that the icilin could be a helpful treatment for ED. Disclosure No

Helicobacter pylori infection selectively attenuates endothelial function in male mice via exosomes-mediated ROS production.

Substantial sex differences exist in atherosclerosis. Excessive reactive oxygen species (ROS) formation could lead to endothelial dysfunction which is critical to atherosclerosis development and progression. Helicobacter pylori (H. pylori) infection has been shown to attenuate endothelial function via exosomes-mediated ROS formation. We have demonstrated that H. pylori infection selectively increases atherosclerosis risk in males with unknown mechanism(s). The present study was to test the hypothesis that H. pylori infection impaired endothelial function selectively in male mice through exosome-mediated ROS formation. Age-matched male and female C57BL/6 mice were infected with CagA+ H. pylori to investigate sex differences in H. pylori infection-induced endothelial dysfunction. H. pylori infection attenuated acetylcholine (ACh)-induced endothelium-dependent aortic relaxation without changing nitroglycerine-induced endothelium-independent relaxation in male but not female mice, associated with increased ROS formation in aorta compared with controls, which could be reversed by N-acetylcysteine treatment. Treatment of cultured mouse brain microvascular endothelial cells with exosomes from H. pylori infected male, not female, mice significantly increased intracellular ROS production and impaired endothelial function with decreased migration, tube formation, and proliferation, which could be prevented with N-acetylcysteine treatment. H. pylori infection selectively impairs endothelial function in male mice due to exosome-mediated ROS formation.

The Beneficial Effect of Swimming Training Associated with Quercetin Administration on the Endothelial Nitric Oxide-Dependent Relaxation in the Aorta of Rats with Experimentally Induced Type 1 Diabetes Mellitus.

Type 1 diabetes mellitus is related to the vascular oxidative and nitrosative stress, the trigger for atherosclerosis and cardiovascular complications. The effects of moderate swimming training associated with quercetin oral administration were evaluated in aorta of rats with experimentally induced type 1 diabetes mellitus (T1DM), by analysing the nitric oxide-endothelial dependent relaxation (NO-EDR). T1DM rats received daily quercetin 30 mg/kg and followed the protocol of 5-weeks swimming exercise (30 min/day; 5 days/week). Aorta relaxation to acetylcholine (Ach) and sodium nitroprusside (SNP) were measured at the end of the experiment. Ach-induced endothelial dependent relaxation was significantly decreased in phenylephrine (PE) pre-contracted aorta of diabetic rats. Swimming exercise with quercetin administration preserved Ach-induced EDR but did not have any impact on SNP-induced endothelium-independent relaxation in the diabetic aorta. These findings suggest that quercetin administration associated with moderate swimming exercise could improve the endothelial NO-dependent relaxation in the aorta of rats with experimentally induced type 1 diabetes mellitus, showing that this therapeutical combination may improve and even prevent the vascular complications that occur in diabetic patients.

Indoxyl Sulphate Retention Is Associated with Microvascular Endothelial Dysfunction after Kidney Transplantation.

Kidney transplantation (KTx) is the preferred form of renal replacement therapy in chronic kidney disease (CKD) patients, owing to increased quality of life and reduced mortality when compared to chronic dialysis. Risk of cardiovascular disease is reduced after KTx; however, it is still a leading cause of death in this patient population. Thus, we aimed to investigate whether functional properties of the vasculature differed two years post-KTx (postKTx) compared to baseline (time of KTx). Using the EndoPAT device in 27 CKD patients undergoing living-donor KTx, we found that vessel stiffness significantly improved while endothelial function worsened postKTx vs. baseline. Furthermore, baseline serum indoxyl sulphate (IS), but not p-cresyl sulphate, was independently negatively associated with reactive hyperemia index, a marker of endothelial function, and independently positively associated with P-selectin postKTx. Finally, to better understand the functional effects of IS in vessels, we incubated human resistance arteries with IS overnight and performed wire myography experiments ex vivo. IS-incubated arteries showed reduced bradykinin-mediated endothelium-dependent relaxation compared to controls via reduced nitric oxide (NO) contribution. Endothelium-independent relaxation in response to NO donor sodium nitroprusside was similar between IS and control groups. Together, our data suggest that IS promotes worsened endothelial dysfunction postKTx, which may contribute to the sustained CVD risk.

Red blood cells from endothelial nitric oxide synthase-deficient mice induce vascular dysfunction involving oxidative stress and endothelial arginase I

Background & aimsNitric oxide bioactivity (NO) from endothelial NO synthase (eNOS) importantly contributes to the maintenance of vascular homeostasis, and reduced eNOS activity has been associated with cardiovascular disease. Emerging evidence suggests interaction(s) between red blood cells (RBCs) and the endothelium in vascular control; however, the specific role of RBC eNOS is less clear. We aimed to investigate the hypothesis that a lack of RBC eNOS induces endothelial dysfunction. Methods & ResultsRBCs from global eNOS knockout (KO) and wildtype (WT) mice were co-incubated ex vivo overnight with healthy mouse aortic rings, followed by functional and mechanistic analyses of endothelium-dependent and independent relaxations. RBCs from eNOS KO mice induced endothelial dysfunction and vascular oxidative stress, whereas WT RBC did not. No differences were observed for endothelium-independent relaxations. This eNOS KO RBC-induced endothelial dysfunctional phenotype was prevented by concomitant co-incubation with reactive oxygen species scavenger (TEMPOL), arginase inhibitor (nor-NOHA), NO donor (detaNONOate) and NADPH oxidase 4 (NOX4) inhibitor. Moreover, vessels from endothelial cell-specific arginase 1 KO mice were resistant to eNOS KO-RBC-induced endothelial dysfunction. Finally, in mice aortae co-incubated with RBCs from women with preeclampsia, we observed a significant reduction in endothelial function compared to when using RBCs from healthy pregnant women or from women with uncomplicated gestational hypertension. ConclusionsRBCs from mice lacking eNOS, and patients with preeclampsia, induce endothelial dysfunction in adjacent blood vessels. Thus, RBC-derived NO bioactivity acts to prevent induction of vascular oxidative stress occurring via RBC NOX4-derived ROS in a vascular arginase-dependent manner. Our data highlight the intrinsic protective role of RBC-derived NO bioactivity in preventing the damaging potential of RBCs. This provides novel insight into the functional relationship between RBCs and the vasculature in health and cardiovascular disease, including preeclampsia.

Comparison of arterial storage conditions for delayed arterial ring testing

ObjectiveArterial ring testing is the gold standard for measuring arterial function. Increased arterial tone through arterial contraction and impaired endothelial relaxation (endothelial dysfunction) are key metrics of impaired arterial health in peripheral arterial disease (PAD). To allow for comparative testing of arteries during standard laboratory hours, storage buffers and conditions have been used to extend the functional life of arteries. Various storage conditions have been compared, but there has not been a robust comparison or validation in human arteries. The objective of this work is to optimize storage of arterial segments for endothelial cell (EC) testing in a murine model and to test EC function in human PAD arteries. We hypothesized that certain storage conditions would be superior to others. MethodsHealthy murine aortas were harvested from 10- to 14-week-old C57/Bl6J male and female mice and compared under different storage protocols (24 hours) to immediate arterial testing. The storage conditions tested were: Opti-MEM (37°C or 4°C), Krebs-HEPES with 1.8 mmol/L or 2.5 mmol/L calcium (4°C), or Wisconsin (WI) solution at 4°C. Vascular function was evaluated by isometric force testing. Endothelium-dependent and -independent relaxation were measured after precontraction with addition of methacholine or sodium nitroprusside, respectively. Arterial contraction was stimulated with potassium chloride or phenylephrine. Analysis of variance was used to determine significance compared with immediate testing with P < .05. Under institutional review board approval, 28 PAD arteries were collected at amputation and underwent vascular function testing as described. Disturbed flow conditions were determined by indirect (upstream occlusion) flow to the harvested tibial arteries. Stable flow arteries had in-line flow. Arterial calcification was quantified manually as present or not present. ResultsWe found that 4°C WI and 37°C Opti-MEM best preserved endothelium-dependent relaxation and performed similarly to immediately testing aortas (termed fresh for freshly tested) (P > .95). Other storage conditions were inferior to freshly tested aortas (P < .05). Vascular smooth muscle function was tested by endothelial-independent relaxation and contractility. All storage conditions preserved endothelial-independent relaxation and contractility similar to freshly tested arteries. However, 4°C WI and 37°C Opti-MEM storage conditions most closely approximated the maximum force of contraction of freshly tested arteries in response to potassium chloride (P > .39). For human arterial testing, 28 tibial arteries were tested for relaxation and contraction with 16 arteries with peripheral artery occlusive disease (PAD with disturbed flow) and 12 without peripheral artery occlusive disease (PAD with stable flow), of which 14 were calcified and 14 were noncalcified. Endothelial-dependent relaxation data was measurable in 9 arteries and arterial contraction data was measurable in 14 arteries. When comparing flow conditions, arteries exposed to disturbed flow (n = 4) had significantly less relaxation (2% vs 59%; P = .03) compared with stable flow conditions (n = 5). In contrast, presence the (n = 6) or absence of calcification (n = 3) did not impact arterial relaxation. Arterial contraction was not different between groups in either comparison by flow (n = 9 disturbed; n = 5 stable) or calcification (n = 6 present; n = 8 absent). ConclusionsIn healthy murine aortas, arterial storage for 24 hours in 4°C WI or 37°C Opti-MEM both preserved endothelium-dependent relaxation and maximum force of contraction. In human PAD arteries stored in 4° WI, flow conditions before arterial harvest, but not arterial calcification, led to differences in arterial relaxation in human PAD arteries. Arterial contractility was more robust (11/28 arteries) compared with arterial relaxation (7/28 arteries), but was not significantly different under flow or calcification parameters. This work defines ideal storage conditions for arterial ring testing and identifies that EC dysfunction from disturbed flow may persist in delayed ex vivo arterial testing.

Red blood cells induce endothelial dysfunction in patients with ST-elevation myocardial infarction and elevated C-reactive protein

Abstract Background The important role of inflammation in atherosclerotic plaque progression and instability leading to myocardial infarction has been widely demonstrated. C-reactive protein (CRP) has been shown to be of predictive value in atherosclerotic cardiovascular disease. The red blood cell (RBC) is an important regulator of cardiovascular function through nitric oxide bioactivity and oxidative stress in ischemic heart disease. Also, arginase-1 has been shown to greatly influence nitric oxide bioactivity in RBCs and to cause endothelial dysfunction. However, the mechanisms by which RBCs regulate vascular function in patients with myocardial infarction and its relation to inflammation and arginase-1 remain unknown. Objective The study aimed to investigate the effect of RBCs on endothelial function in patients with ST-elevation myocardial infarction (STEMI) and its possible association with systemic inflammation and arginase-1. Material and methods Blood samples were collected from patients with STEMI within 36 h after admission and from age-matched healthy controls. RBCs were incubated with isolated rat aortic segments for 18 h after which the aortas were assessed for endothelium-dependent and endothelium-independent relaxations using wire myographs and application of acetylcholine and nitroprusside, respectively. The vascular response was evaluated in relation to the level of inflammation defined as CRP &amp;lt;2 and ≥2 mg/L at admission. The levels of 4-hydroxynonenal (4-HNE; a marker of oxidative stress formed by lipid peroxidation) and the expression of arginase-1 were visualized in rat aortas following incubation with RBCs by immunohistochemistry. All handling and procedures regarding human subjects and sampling were performed according to the Declaration of Helsinki. All animal experiments and procedures were performed according to the guidelines by the U.S National Institutes of Health (NIH publication no 85–23, revised 1996). Results RBCs from patients with STEMI and elevated CRP (≥2 mg/L, mean of 9.6 mg/L) induced significant impairment of endothelium-dependent relaxation compared to RBCs from patients with STEMI and low CRP (&amp;lt;2 mg/L, mean of 1.0 mg/L) and to the healthy controls (Fig. 1). Endothelium-independent relaxations did not differ between the groups. Immunohistochemical staining of the aorta revealed that incubation with RBCs from patients with STEMI and high CRP increased the expression of 4-HNE and arginase-1 compared to incubation of RBCs from healthy controls and patients with STEMI and low CRP (Fig. 2). Conclusion RBCs from patients with STEMI and elevated CRP induce endothelial dysfunction and increase the expression of 4-HNE and arginase-1, indicating that inflammation is involved in the mechanism by which RBCs induce endothelial dysfunction in STEMI. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Swedish Heart and Lung foundation