Endothelium-dependent Hyperpolarizing Factor Research Articles

Molecular mechanisms of endothelial dysfunction in coronary microcirculation dysfunction.

Coronary microvascular endothelial cells (CMECs) react to changes in coronary blood flow and myocardial metabolites and regulate coronary blood flow by balancing vasoconstrictors-such as endothelin-1-and the vessel dilators prostaglandin, nitric oxide, and endothelium-dependent hyperpolarizing factor. Coronary microvascular endothelial cell dysfunction is caused by several cardiovascular risk factors and chronic rheumatic diseases that impact CMEC blood flow regulation, resulting in coronary microcirculation dysfunction (CMD). The mechanisms of CMEC dysfunction are not fully understood. However, the following could be important mechanisms: the overexpression and activation of nicotinamide adenine dinucleotide phosphate oxidase (Nox), and mineralocorticoid receptors; the involvement of reactive oxygen species (ROS) caused by a decreased expression of sirtuins (SIRT3/SIRT1); forkhead box O3; and a decreased SKCA/IKCA expression in the endothelium-dependent hyperpolarizing factor electrical signal pathway. In addition, p66Shc is an adapter protein that promotes oxidative stress; although there are no studies on its involvement with cardiac microvessels, it is possible it plays an important role in CMD.

CP55940-induced vasorelaxation is endothelial-dependent and mediated by the CB1R through NOS, COX and EDHF pathways in porcine cerebral arteries.

Using swine as an experimental model, we chose to examine whether the cannabinoid receptors, e.g. CB1R and the CB2R, could affect vasomotor tone in isolated pial arteries. It was hypothesized that the CB1R would specifically mediate cerebral artery vasorelaxation in an endothelial-dependent manner. First-order pial arteries were isolated from female Landrace pigs (age = 2 months; N = 27) for wire and pressure myography. Arteries were pre-contracted with a thromboxane A2 analogue (U-46619) and vasorelaxation in response to the CB1R and CB2R receptor mixed agonist CP55940 was examined in the following conditions: 1) untreated; 2) inhibition of the CB1R (AM251); or 3) inhibition of the CB2R receptor (AM630). The data revealed that CP55940 elicits a CB1R-dependent relaxation in pial arteries. CB1R expression was confirmed using immunoblot and immunohistochemical analyses. Subsequently, the role of different endothelial-dependent pathways in the CB1R-mediated vasorelaxation was examined using: 1) denudation (removal of the endothelium); 2) inhibition of cyclooxygenase (COX; Naproxen); 3) inhibition of nitric oxide synthase (NOS; L-NAME); 4) combined inhibition of COX + NOS. The data revealed CB1R-mediated vasorelaxation was endothelial-dependent, with contributions from COX-derived prostaglandins, NO, and endothelium-dependent hyperpolarizing factor (EDHF). Pressurized arteries underwent myogenic curves (20-100 mmHg) under the following conditions: 1) untreated; 2) inhibition of the CB1R. The data revealed CB1R inhibition increased basal myogenic tone, but not myogenic reactivity. As the vascular responses were assessed in isolated pial arteries, this work reveals that the CB1R modulates cerebrovascular tone independently of changes in brain metabolism.

Pathophysiology and Outcomes of Endothelium Function in Coronary Microvascular Diseases: A Systematic Review of Randomized Controlled Trials and Multicenter Study.

Coronary macrovascular disease is a concept that has been well-studied within the literature and has long been the subject of debates surrounding coronary artery bypass grafting (CABG) vs. Percutaneous Coronary Intervention (PCI). ISCHEMIA trial reported no statistical difference in the primary clinical endpoint between initial invasive management and initial conservative management, while in the ORBITA trial PCI did not improve angina frequency score significantly more than placebo, albeit PCI resulted in more patient-reported freedom from angina than placebo. However, these results did not prove the superiority of the PCI against OMT, therefore do not indicate the benefit of PCI vs. the OMT. Please rephrase the sentence. We reviewed the role of different factors responsible for endothelial dysfunction from recent randomized clinical trials (RCTs) and multicentre studies. A detailed search strategy was performed using a dataset that has previously been published. Data of pooled analysis include research articles (human and animal models), CABG, and PCI randomized controlled trials (RCTs). Details of the search strategy and the methods used for data pooling have been published previously and registered with Open-Source Framework. The roles of nitric oxide (NO), endothelium-derived contracting factors (EDCFs), and vasodilator prostaglandins (e.g., prostacyclin), as well as endothelium-dependent hyperpolarization (EDH) factors, are crucial for the maintenance of vasomotor tone within the coronary vasculature. These homeostatic mechanisms are affected by sheer forces and other several factors that are currently being studied, such as vaping. The role of intracoronary testing is crucial when determining the effects of therapeutic medications with further studies on the horizon. The true impact of coronary microvascular dysfunction (CMD) is perhaps underappreciated, which supports the role of medical therapy in determining outcomes. Ongoing trials are underway to further investigate the role of therapeutic agents in secondary prevention.

Losartan metabolite EXP3179 is a unique blood pressure-lowering AT1R antagonist with direct, rapid endothelium-dependent vasoactive properties

Background and purposeLosartan is an anti-hypertensive angiotensin II (ANGII) type 1 receptor (AT1R) blocker (ARB) with many unexpected therapeutic properties, even in non-blood pressure (BP)-related diseases. Administered as a prodrug, losartan undergoes serial metabolism into EXP3179, a metabolite alleged to lack AT1R-blocking properties, and EXP3174, the dominant AT1R antagonist. Having observed that losartan can decrease vascular tone in mice with low AT1R expression and inhibit Marfan aortic widening at very high doses, we investigated whether EXP3179 may have unique, AT1R-independent effects on vascular tone and endothelial function. Experimental approachWe compared the AT1R blocking capabilities of EXP3179 and EXP3174 using AT1R-expressing cell lines. Their BP lowering and vasoactive properties were studied in normal, hypertensive and transgenic rodents, and ex vivo wire myography. Key resultsWe observed that both EXP3179 and EXP3174 can fully block (100%) AT1R signaling in vitro and significantly decrease BP in normotensive and spontaneously hypertensive rats. Only EXP3179 prevented PE-induced contraction by up to 65% (p < 0.01) in L-NAME and endothelium removal-sensitive fashion. Use of transgenic mice revealed that these effects involve the eNOS/caveolin-1 axis and the endothelium-dependent hyperpolarization factor (EDHF). Conclusion and implicationsWe provide direct structure-activity evidence that EXP3179 is a BP-lowering AT1R blocker with unique endothelial function-enhancing properties not shared with losartan or EXP3174. The major pharmacological effects of losartan in patients are therefore likely more complex than simple blockade of AT1R by EXP3174, which helps rationalize its therapeutic and prophylactic properties, especially at very high doses. Reports relying on EXP3179 as an AT1R-independent losartan analogue may require careful re-evaluation.

Endothelium in Coronary Macrovascular and Microvascular Diseases.

:The endothelium plays a pivotal role in the regulation of vascular tone by synthesizing and liberating endothelium-derived relaxing factors inclusive of vasodilator prostaglandins (eg, prostacyclin), nitric oxide (NO), and endothelium-dependent hyperpolarization factors in a distinct blood vessel size–dependent manner. Large conduit arteries are predominantly regulated by NO and small resistance arteries by endothelium-dependent hyperpolarization factors. Accumulating evidence over the past few decades has demonstrated that endothelial dysfunction and coronary vasomotion abnormalities play crucial roles in the pathogenesis of various cardiovascular diseases. Structural and functional alterations of the coronary microvasculature have been coined as coronary microvascular dysfunction (CMD), which is highly prevalent and associated with adverse clinical outcomes in many clinical settings. The major mechanisms of coronary vasomotion abnormalities include enhanced coronary vasoconstrictive reactivity at epicardial and microvascular levels, impaired endothelium-dependent and endothelium-independent coronary vasodilator capacities, and elevated coronary microvascular resistance caused by structural factors. Recent experimental and clinical research has highlighted CMD as the systemic small artery disease beyond the heart, emerging modulators of vascular functions, novel insights into the pathogenesis of cardiovascular diseases associated with CMD, and potential therapeutic interventions to CMD with major clinical implications. In this article, we will summarize the current knowledge on the endothelial modulation of vascular tone and the pathogenesis of coronary macrovascular and microvascular diseases from bench to bedside, with a special emphasis placed on the mechanisms and clinical implications of CMD.

Moderate Intensity Exercise Causes a Shift in the Relative Importance of the Endothelium‐Dependent Relaxing Factors in Mesenteric Arteries of Male UC Davis Type‐2 Diabetes Mellitus (UCD‐T2DM) Rats

Over the past decade obesity and type 2 diabetes (T2D) have reached epidemic levels worldwide. Cardiovascular disease (CVD) is one of the leading causes of mortality and morbidity in diabetic patients. Regular aerobic exercise is a well-characterized lifestyle intervention that improves endothelial dysfunction, insulin resistance, and CVD risk in T2D. However, there are still debates regarding the duration and intensity of exercise required to ameliorate the deleterious metabolic perturbations in patients with T2D. The objective of this study was to investigate the effects of moderate intensity exercise (MIE) on vascular reactivity of mesenteric artery (MA) of male UC Davis Type-2 Diabetes Mellitus Rat (UCD-T2DM) model. Specifically, we examined whether there were changes in the relative contributions of endothelium-derived relaxing factors in modulating vascular reactivity of MA from exercise-trained compared with control sedentary UCD-T2DM rats. Age-matched male control and UCD-T2DM rats underwent forced treadmill exercise (E) or remained sedentary (S) for 8 weeks. Endothelium-dependent vasorelaxation (EDV) to acetylcholine (ACh, 10-8 to 10-5 M) were measured in precontracted MA before and after indomethacin (10µM), a cyclooxygenase (COX) inhibitor; followed by addition of L-NAME (200µM), a nitric oxide synthase (NOS) inhibitor; followed by combination of apamin (1 µM) and TRAM-34 (1 µM) (SKCa and IKCa blockers, respectively). The diabetic exercise trained (DE) rats exhibited decreased visceral adiposity and plasma triglyceride levels compared with the diabetic sedentary (DS) group. However, MIE did not alter plasma glucose or insulin concentrations in either nondiabetic or diabetic UCD-T2DM Rats. Both sensitivity and maximal responses (Emax) to ACh were significantly reduced in MA from DS rats compared with those in control sedentary (CS). MIE improved the Emax to ACh only in MA of DE group. Inhibition of COX enhanced Emax to ACh in DS arteries suggesting the elevation of contractile COX metabolites in the DS group. The addition of L-NAME resulted in a reduction in ACh relaxation of arteries from both CS and DS rats, however the effect was more prominent in DS group, suggesting the contribution of NO was enhanced (or endothelium-dependent hyperpolarizing factor (EDHF)-type relaxation was diminished) in DS arteries. Remarkably, MIE reversed the loss of EDHF-type relaxation in UCD-T2DM Rats. Unlike in the DS group, inhibition of COX had no effect on ACh response in MA of the DE group, suggesting that MIE may cause a shift from contractile COX to NO and EDHF-type relaxation in UCD-T2DM Rats. In conclusion, we provide the first evidence for a positive impact of moderate exercise on mesenteric arterial function in male UCD-T2DM Rats, likely mediated by a contribution of EDHF-type (NO and prostanoid-independent) responses.

Reactive oxygen species in cardiovascular health and disease: special references to nitric oxide, hydrogen peroxide, and Rho-kinase.

The interaction between endothelial cells and vascular smooth muscle cells (VSMC) plays an important role in regulating cardiovascular homeostasis. Endothelial cells synthesize and release endothelium-derived relaxing factors (EDRFs), including vasodilator prostaglandins, nitric oxide (NO), and endothelium-dependent hyperpolarization (EDH) factors. Importantly, the contribution of EDRFs to endothelium-dependent vasodilatation markedly varies in a vessel size-dependent manner; NO mainly mediates vasodilatation of relatively large vessels, while EDH factors in small resistance vessels. We have previously identified that endothelium-derived hydrogen peroxide (H2O2) is an EDH factor especially in microcirculation. Several lines of evidence indicate the importance of the physiological balance between NO and H2O2/EDH factor. Rho-kinase was identified as the effectors of the small GTP-binding protein, RhoA. Both endothelial NO production and NO-mediated signaling in VSMC are targets and effectors of the RhoA/Rho-kinase pathway. In endothelial cells, the RhoA/Rho-kinase pathway negatively regulates NO production. On the contrary, the pathway enhances VSMC contraction with resultant occurrence of coronary artery spasm and promotes the development of oxidative stress and vascular remodeling. In this review, I will briefly summarize the current knowledge on the regulatory roles of endothelium-derived relaxing factors, with special references to NO and H2O2/EDH factor, in relation to Rho-kinase, in cardiovascular health and disease.

Nitric oxide and endothelium-dependent hyperpolarization mediated by hydrogen peroxide in health and disease.

The endothelium plays crucial roles in modulating vascular tone by synthesizing and releasing endothelium-derived relaxing factors (EDRFs), including vasodilator prostaglandins, nitric oxide (NO) and endothelium-dependent hyperpolarization (EDH) factors. Thus, endothelial dysfunction is the hallmark of atherosclerotic cardiovascular diseases. Importantly, the contribution of EDRFs to endothelium-dependent vasodilatation varies in a distinct vessel size-dependent manner; NO mainly mediates vasodilatation of relatively large, conduit vessels (eg epicardial coronary arteries), while EDH factors in small resistance vessels (eg coronary microvessels). Endothelium-derived hydrogen peroxide (H2 O2 ) is a physiological signalling molecule serving as one of the major EDH factors especially in microcirculations and has gained increasing attention in view of its emerging relevance for cardiovascular diseases. In the clinical settings, therapeutic approaches targeting NO (eg NO donors) or non-specific elimination of reactive oxygen species (eg antioxidant supplements) are disappointingly ineffective for the treatment of various cardiovascular diseases, in which endothelial dysfunction and coronary microvascular dysfunction are substantially involved. These lines of evidence indicate the potential importance of the physiological balance between NO and H2 O2 /EDH factor. Further characterization and better understanding of endothelium-dependent vasodilatations are important to develop novel therapeutic strategies in cardiovascular medicine. In this MiniReview, we will briefly summarize the current knowledge on the emerging regulatory roles of endothelium-dependent vasodilatations in the cardiovascular system, with a special reference to the two major EDRFs, NO and H2 O2 /EDH factor, in health and disease.

Gender Differences in Endothelial Function and Coronary Vasomotion Abnormalities

Introduction: Structural and functional abnormalities of coronary microvasculature, referred to as coronary microvascular dysfunction (CMD), have been implicated in a wide range of cardiovascular diseases and have gained growing attention in patients with chest pain with no obstructive coronary artery disease, especially in females. The central mechanisms of coronary vasomotion abnormalities encompass enhanced coronary vasoconstrictive reactivity (ie, coronary spasm), reduced endothelium-dependent and -independent coronary vasodilator capacities, and increased coronary microvascular resistance. The 2 major endothelium-derived relaxing factors, nitric oxide (NO) and endothelium-dependent hyperpolarization (EDH) factors, modulate vascular tone in a distinct vessel size–dependent manner; NO mainly mediates vasodilatation of relatively large, conduit vessels, while EDH factors in small resistance vessels. Endothelium-dependent hyperpolarization–mediated vasodilatation is more prominent in female resistance arteries, where estrogens exert beneficial effects on endothelium-dependent vasodilatation via multiple mechanisms. In the clinical settings, therapeutic approaches targeting NO are disappointing for the treatment of various cardiovascular diseases, where endothelial dysfunction and CMD are substantially involved. Significance: In this review, we will discuss the current knowledge on the pathophysiology and molecular mechanisms of endothelial function and coronary vasomotion abnormalities from bench to bedside, with a special reference to gender differences. Results: Recent experimental and clinical studies have demonstrated distinct gender differences in endothelial function and coronary vasomotion abnormalities with major clinical implications. Moreover, recent landmark clinical trials regarding the management of stable coronary artery disease have questioned the benefit of percutaneous coronary intervention, supporting the importance of the coronary microvascular physiology. Conclusion: Further characterization and a better understanding of the gender differences in basic vascular biology as well as those in cardiovascular diseases are indispensable to improve health care and patient outcomes in cardiovascular medicine.

Important Roles of Endothelium-Dependent Hyperpolarization in Coronary Microcirculation and Cardiac Diastolic Function in Mice.

Endothelium-dependent hyperpolarization (EDH) factor is one of endothelium-derived relaxing factors and plays important roles especially in microvessels. We have previously demonstrated that endothelium-derived hydrogen peroxide (H2O2) is an EDH factor produced by all types of nitric oxide synthases (NOSs), including endothelial NOS (eNOS), neuronal NOS (nNOS), and inducible NOS. Recent studies have suggested the association between coronary microvascular dysfunction and cardiac diastolic dysfunction. However, the role of EDH in this issue remains to be fully elucidated. We thus examined whether EDH plays an important role in coronary microcirculation and if so, whether endothelial dysfunction, especially impaired EDH, is involved in the pathogenesis of cardiac diastolic dysfunction in mice. Using a Langendorff-perfused heart experiment, we examined the increase in coronary flow in response to bradykinin in the presence of indomethacin and N-nitro-L-arginine (EDH condition) in wild-type, eNOS-knockout (KO), and nNOS/eNOS-double-KO mice. Compared with wild-type mice, EDH-mediated relaxations were increased in eNOS-KO mice but were significantly reduced in n/eNOS-KO mice. Catalase, a specific H2O2 scavenger, markedly inhibited EDH-mediated relaxations in all 3 genotypes, indicating compensatory roles of nNOS-derived H2O2 as an EDH factor in coronary microcirculation. Although both eNOS-KO and n/eNOS-KO mice exhibited similar extents of cardiac morphological changes, only n/eNOS-KO mice exhibited cardiac diastolic dysfunction. The expression of oxidized protein kinase G I-α (PKGIα) in the heart was significantly increased in eNOS-KO mice compared with n/eNOS-KO mice. These results indicate that EDH/H2O2 plays important roles in maintaining coronary microcirculation and cardiac diastolic function through oxidative PKGIα activation.

Chronic ethanol consumption enhances inducible endothelium-dependent hyperpolarizing factor-mediated relaxation in the rat artery

The inducible endothelium-dependent hyperpolarizing factor (iEDHF) pathway is activated as a compensatory response to adverse changes in the body. It causes vasorelaxation and maintains circulatory homeostasis in the organs. Small to moderate quantities of ethanol enhance vascular relaxation. However, its mechanism and the involvement of the iEDHF pathway in this process are unknown. Therefore, we studied iEDHF-mediated, acetylcholine-induced, endothelium-dependent relaxation in the superior mesenteric arteries (SMAs) of rats chronically fed ethanol. Rats were administered a standard diet (S-Control group), Lieber's control diet (L-Control group), or Lieber's ethanol diet (EtOH group). SMA relaxation was assessed by isometric tension measurements. Arachidonate 15-lipoxygenase (ALOX15) and soluble epoxide hydrolase (sEH) were determined by immunoblot. Acetylcholine-induced, endothelium-dependent relaxation was significantly greater in the EtOH than the control groups. These differences persisted after PGI2 and NO blockade. Thus, the increase in acetylcholine-induced relaxation was EDHF-mediated. In the EtOH group, however, it was prevented by iEDHF inhibitors. ALOX15 and sEH protein expression levels were higher in the EtOH than the L-Control group. The increase in acetylcholine-induced relaxation by chronic ethanol consumption was mediated by the iEDHF pathway. This mechanism may compensate for the blood pressure elevation induced by ethanol. This study suggests that iEDHF is induced during proper drinking and may help prevent the onset of cardiovascular conditions.

P4158Marked impairment of endothelium-dependent digital vasodilatations in patients with microvascular angina compared with those with vasospastic angina

Abstract Background/Introduction Nitric oxide (NO) and endothelium-dependent hyperpolarization (EDH) factor are the major endothelium-derived relaxing factors. NO plays an important role in conduit arteries, while the importance of EDH factor increases as the vessel size decrease in patients with microvascular angina (MVA) compared with those with vasospastic angina (VSA) remains to be fully elucidated. Purpose We evaluated the roles of NO and EDH factor in conduit (brachial) arteries and resistance (digital) arteries of the patients with MVA, VSA and comorbid MVA+VSA patients. Methods We enrolled 39 patients who underwent diagnostic cardiac catheterization and divided them into 3 groups based on acetylcholine (ACh) provocation test, index of microcirculation resistance (IMR), and coronary flow reserve (CFR); MVA (N=9, mean age 59.9±3.5 years), VSA (N=12, mean age 61.3±1.8 years), and comorbid MVA+VSA (N=18, mean age 64.0±2.2 years). Endothelium-dependent brachial and digital vasodilatations in response to intra-arterial infusion of bradykinin (BK, 25, 50, and 100 ng/min for 2 min) were simultaneously measured by ultrasonography and peripheral arterial tonometry, respectively. Measurements were repeated after oral administration of aspirin (486 mg) and intra-arterial infusion of NG-monomethyl-L-arginine (L-NMMA, 8μmol/min for 5 min) in order to inhibit the effects of vasodilator prostaglandins and NO, respectively. Finally, endothelium-independent brachial and digital vasodilatations in response to sublingual nitroglycerin (NTG, 0.3 mg) were measured in the same manner. Results In the brachial artery, dose-dependent vasodilatations to BK were comparable among the 3 groups, and L-NMMA equally attenuated the responses to BK (Figure 1). Endothelium-independent brachial vasodilatation in response to NTG was also comparable among the 3 groups. Surprisingly, dose-dependent digital vasodilatations to BK were almost absent in MVA patients compared with VSA or comorbid MVA+VSA group (Figure 2). Furthermore, the digital vasodilatations were unaffected by L-NMMA in VSA group, but were significantly reduced in comorbid MVA+VSA group (VSA, 16.8±15.1% vs. MVA+VSA, −0.23±6.2%, P&lt;0.05), suggesting reduced EDH and compensatory role of NO in the latter group. In contrast, endothelium-independent digital vasodilatation in response to NTG was comparable among the 3 groups. The main results of this study Conclusions These results provide the first evidence that endothelium-dependent digital vasodilatations (both NO and EDH factor) are markedly impaired in MVA patients compared with VSA or comorbid MVA+VSA patients, whereas the responses are comparable in the brachial artery among the 3 groups, suggesting the involvement of severe endothelial dysfunction in the pathogenesis of MVA.

Role of EDHF in Potentiation of ACh‐induced Relaxation of Aorta in Male UC Davis Type 2 Diabetes Mellitus (UCD‐T2DM) Rat: Sex Specific Responses

The UC Davis type 2 diabetes mellitus (UCD‐T2DM) Rat is a validated model of T2D that exhibits an etiology more similar to human disease than other rodent models. In spite of being a validated model, there are no published data on the vascular function in this model. We investigated whether vascular reactivity is altered, and if sex differences exist in aortic endothelial function in the UCD‐T2DM rats. We also examined the potential changes in the relative contributions of endothelium‐derived relaxing factors in modulating aortic reactivity of UCD‐T2DM.Endothelium‐dependent vasodilation (EDV) to acetylcholine (ACh, 10−8 to 10−5M) was measured in aortic rings pre‐contracted with phenylephrine (PE, 2μM) before and after pretreatment with indomethacin (Indo; 10 μM), a cyclooxygenase (COX) inhibitor, Indo plus ODQ (10 μM), an inhibitor of soluble guanylyl cyclase (sGC) and ultimately following incubation with Indo, ODQ and L‐NNA (100 μM), a nonselective NO synthase (NOS) inhibitor. Endothelium‐independent vasodilation to sodium nitroprusside (SNP, 10−9 to 10−5M) was assessed in endothelium‐denuded rings pre‐contracted with PE (2μM). Furthermore, constrictor response curves to PE (10−8 to 10−5 M) were also generated. The contribution of K+ to ACh‐induced relaxation was assessed in male animals by obtaining the concentration response curves to ACh before and after treatment with TRAM‐34 (1μM), an intermediate‐conductance Ca2+ activated K+ (IKCa) channel inhibitor; apamin (1 μM), a small‐conductance Ca2+ activated K+ (SKCa) channel inhibitor. Moreover, the expression of K+ channels were evaluated in aortic tissues.Diabetes significantly impaired relaxation responses to ACh and SNP in aortic rings from female UCD‐T2DM Rats, however, potentiated the relaxation in males. The responsiveness to PE was significantly enhanced in diabetic groups, regardless of sex. Blocking of COX, sGC and NOS completely abolished the relaxation response to ACh in all of experimental groups except for the diabetic male rats, indicating the presence of endothelium‐dependent hyperpolarizing factor (EDHF)‐type relaxation in this group. To further study the contribution of EDHF in diabetic males, ACh‐induced relaxation was measured before and after TRAM 34 or Apamin in male tissues. Incubation of male aortic rings with TRAM 34, but not Apamin, significantly blunted the relaxation responses to ACh only in diabetic group. Accordingly, the level of expression of IKca channels was significantly higher in diabetic males than that in controls.These data, for the first time, show that the vascular function in aortic rings of UCD‐T2DM is altered in both sexes. It also suggests that an increased contribution of EDHF, specifically IKca channel, plays a role in potentiated relaxation responses to ACh in male UCD‐T2DM rats.Support or Funding InformationSupported by NHLBIThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Important roles of endothelial caveolin-1 in endothelium-dependent hyperpolarization and ischemic angiogenesis in mice.

Although increased levels of reactive oxygen species (ROS) are involved in the pathogenesis of cardiovascular diseases, the importance of physiological ROS has also been emerging. We have previously demonstrated that endothelium-derived H2O2 is an endothelium-dependent hyperpolarization (EDH) factor and that loss of endothelial caveolin-1 reduces EDH/H2O2 in the microcirculation. Caveolin-1 (Cav-1) is a scaffolding/regulatory protein that interacts with diverse signaling pathways, including angiogenesis. However, it remains unclear whether endothelial Cav-1 plays a role in ischemic angiogenesis by modulating EDH/H2O2. In the present study, we thus addressed this issue in a mouse model of hindlimb ischemia using male endothelium-specific Cav-1 (eCav-1) knockout (KO) mice. In isometric tension experiments with femoral arteries from eCav-1-KO mice, reduced EDH-mediated relaxations to acetylcholine and desensitization of sodium nitroprusside-mediated endothelium-independent relaxations were noted ( n = 4~6). An ex vivo aortic ring assay also showed that the extent of microvessel sprouting was significantly reduced in eCav-1-KO mice compared with wild-type (WT) littermates ( n = 12 each). Blood flow recovery at 4 wk assessed with a laser speckle flowmeter after femoral artery ligation was significantly impaired in eCav-1-KO mice compared with WT littermates ( n = 10 each) and was associated with reduced capillary density and muscle fibrosis in the legs ( n = 6 each). Importantly, posttranslational protein modifications by reactive nitrogen species and ROS, as evaluated by thiol glutathione adducts and nitrotyrosine, respectively, were both increased in eCav-1-KO mice ( n = 6~7 each). These results indicate that endothelial Cav-1 plays an important role in EDH-mediated vasodilatation and ischemic angiogenesis through posttranslational protein modifications by nitrooxidative stress in mice in vivo. NEW & NOTEWORTHY Although increased levels of reactive oxygen species (ROS) are involved in the pathogenesis of cardiovascular diseases, the importance of physiological ROS has also been emerging. The present study provides a line of novel evidence that endothelial caveolin-1 plays important roles in endothelium-dependent hyperpolarization and ischemic angiogenesis in hindlimb ischemia in mice through posttranslational protein modifications by reactive nitrogen species and ROS in mice in vivo.

Total Flavone of Rhododendron Improves Cerebral Ischemia Injury by Activating Vascular TRPV4 to Induce Endothelium-Derived Hyperpolarizing Factor-Mediated Responses.

Background Total flavonoids of Rhododendron (TFR) is extracted from Rhododendron, a herbal medicine widely used in China. The main components are flavone compounds such as warfarin, rutin, quercetin, and hyperoside. We investigated the role of TRPV4 channel in the TFR induced endothelium-dependent hyperpolarizing factor- (EDHF-) mediated responses against ischemia/reperfusion injury (IR) in cerebral IR (CIR) rats. Methods The morphological changes of cerebral cortex, the relaxation of cerebral basal artery (CBA), and cell membrane potential recording were studied in CIR rats. The outward potassium current in smooth muscle cell was recorded by whole-cell patch clamp recording. The protein expression of TRPV4, SKca, and IKca was determined. Confocal laser was used to measure the Ca2+ fluorescence intensity. Results After treatment with TFR, the number of pyramidal cells in brain tissue increased and the number of empty or lightly stained cells decreased and these effects were eliminated by using HC-067047, Apamin, or TRAM-34. TFR induced and EDHF-mediated dilatation and hyperpolarization in CBA were also attenuated by using these inhibitors. The increased outward current density elicited by TFR in acutely isolated CBA smooth muscle cells was abolished by using TRAM-34 and Apamin. TFR upregulated the protein expression of TRPV4, SKca, and IKca that was also eliminated by these inhibitors. Laser scanning showed that the increased mean fluorescence intensity of Ca2+ by CIR was decreased by using TFR and that this effect was again eliminated by the above inhibitors. Conclusions We conclude that in the CBA of the CIR rats the protective effect of TFR on ischemic cerebrovascular injury may be related to the activation of the TRPV4 in both endothelium and smooth muscle by increasing its expression and activity. The activation of TRPV4 channel in the endothelium may be linked to the opening of endothelial IKca/SKca channels that induces EDHF-mediated relaxation and hyperpolarization in the smooth muscle cell. In addition, the activation of TRPV4 in the smooth muscle cell in CBA may be linked with the activation of BKCa channel through a TRPV4-dependent pathway, reduce Ca2+ concentration in the cell, and relaxes the vessel. These findings may form a new therapeutic target for protection of ischemic brain injury and facilitate the use of Chinese medicine in brain protection.

Differential participation of calcium-activated potassium channel in endothelium-dependent hyperpolarization-type relaxation in superior mesenteric arteries of spontaneously hypertensive rats.

The purpose of this study was to determine the relationship of KCa channels to endothelium-dependent hyperpolarizing factor (EDHF)-mediated relaxation induced by acetylcholine (ACh) in the superior mesenteric arteries of 7-month-old spontaneously hypertensive rats (SHR). Upon inhibition of nitric oxide synthase and cyclooxygenase, ACh-induced EDHF-mediated relaxation was found to be weaker in SHR than in age-matched Wistar Kyoto rats (WKY). These relaxations in both group were attenuated by combined treatment with small-conductance and intermediate-conductance Ca2+-activated K+ channels (SKCa and IKCa) inhibitors, with the exception of relaxation resistant to inhibition of these channels in SHR (vs. WKY). Treatment with large-conductance Ca2+-activated K+ channels (BKCa) inhibitor specifically attenuated relaxation in SHR, but not in WKY. Protein expression of IKCa and SKCa in the arteries did not differ between the 2 groups, whereas ratio of sloβ1 subunit to α subunit of BKCa was increased in SHR (vs. WKY). These results suggest that EDHF-mediated relaxations in superior mesenteric arteries are impaired in SHR, and utilize components of BKCa in addition to SKCa/IKCa channel activities, that the increased participation of BKCa may be attributable to alterations in α and sloβ1 subunit ratio, and that components unrelated to KCa activity may also contribute to the difference between SHR and WKY arteries.

Inhibitory Effect of Interferons on Contractive Activity of Bovine Mesenteric Lymphatic Vessels and Nodes.

We studied the effect of IFNα-2b and IFNβ-1a on phasic and tonic contractions of isolated bovine mesenteric lymphatic vessels and nodes. IFNα-2b and IFNβ-1a in concentrations of 250-1000 U/ml produced dose-dependent negative chronotropic and inotropic effects on spontaneous phasic contractions and tonus of lymphatic vessels and nodes. In de-endothelialized lymphatic vessels and nodes, IFNα-2b and IFNβ-1a in the same concentrations had less pronounced inhibitory effect on spontaneous contraction and tonus. L-NAME (100 μM) and charybdotoxin (0.1 μM with 0.5 μM apamine) significantly attenuated the inhibitory effect of IFNα-2b on phasic and tonic contractions of lymph nodes. L-NAME (100 μM) and indomethacin (10 μM) significantly reduced the IFNα-2b-induced inhibitory effect on phasic and tonic contractions of lymph node. These results indicate that IFNα-2b and IFNβ-1a have a pronounced inhibitory effect on the phasic and tonic contractions of bovine mesenteric lymphatic vessels and nodes. The responses are endothelium-dependent and are determined by production of NO and endothelium-dependent hyperpolarizing factor by endotheliocytes in lymphatic vessels and by production of NO and prostacyclin by endotheliocytes in the lymphatic nodes.

Endothelial Functions.

The endothelium plays important roles in modulating vascular tone by synthesizing and releasing a variety of endothelium-derived relaxing factors, including vasodilator prostaglandins, NO, and endothelium-dependent hyperpolarization factors, as well as endothelium-derived contracting factors. Endothelial dysfunction is mainly caused by reduced production or action of these relaxing mediators. Accumulating evidence has demonstrated that endothelial functions are essential to ensure proper maintenance of vascular homeostasis and that endothelial dysfunction is the hallmark of a wide range of cardiovascular diseases associated with pathological conditions toward vasoconstriction, thrombosis, and inflammatory state. In the clinical settings, evaluation of endothelial functions has gained increasing attention in view of its emerging relevance for cardiovascular disease. Recent experimental and clinical studies in the vascular biology field have demonstrated a close relationship between endothelial functions and cardiovascular disease and the highlighted emerging modulators of endothelial functions, new insight into cardiovascular disease associated with endothelial dysfunction, and potential therapeutic and diagnostic targets with major clinical implications. We herein will summarize the current knowledge on endothelial functions from bench to bedside with particular focus on recent publications in Arteriosclerosis, Thrombosis, and Vascular Biology.

Neuronal nitric oxide synthase-derived hydrogen peroxide effect in grafts used in human coronary bypass surgery.

Recently, H2O2 has been identified as the endothelium-dependent hyperpolarizing factor (EDHF), which mediates flow-induced dilation in human coronary arteries. Neuronal nitric oxide synthase (nNOS) is expressed in the cardiovascular system and, besides NO, generates H2O2 The role of nNOS-derived H2O2 in human vessels is so far unknown. The present study was aimed at investigating the relevance of nNOS/H2O2 signaling in the human internal mammary artery (IMA) and saphenous vein (SV), the major conduits used in coronary artery bypass grafting. In the IMA, but not in the SV, ACh (acetylcholine)-induced vasodilatation was decreased by selective nNOS inhibition with TRIM or Inhibitor 1, and by catalase, which specifically decomposes H2O2 Superoxide dismutase (SOD), which generates H2O2 from superoxide, decreased the vasodilator effect of ACh on SV. In the IMA, SOD diminished phenylephrine-induced contraction in endothelium-containing, but not in endothelium-denuded vessels. Importantly, while exogenous H2O2 produced vasodilatation in IMA, it constricted SV. ACh increased H2O2 production in both sets of vessels. In the IMA, the increase in H2O2 was inhibited by catalase and nNOS blockade. In SV, H2O2 production was abolished by catalase and reduced by nNOS inhibition. Immunofluorescence experiments showed the presence of nNOS in the vascular endothelium and smooth muscle cells of both the IMA and SV. Together, our results clearly show that H2O2 induced endothelium-dependent vascular relaxation in the IMA, whereas, in the SV, H2O2 was a vasoconstrictor. Thus, H2O2 produced in the coronary circulation may contribute to the susceptibility to accelerated atherosclerosis and progressive failure of the SV used as autogenous graft in coronary bypass surgery.

Gender Differences in Sepsis‐Induced Nitric Oxide and EDHF Signaling Pathways in Rat Aorta

Despite recent advances in managing sepsis, it still remains a major health concern in the US. Sepsis is characterized by systemic hypotension, pulmonary hypertension and refractoriness of the vasculature to contractile factors. Nitric Oxide (NO) and Endothelium‐Dependent Hyperpolarizing Factor (EDHF) are crucial in the regulation of vascular tone. The influence of gender on endothelial function in sepsis remains unknown. Therefore, this study was aimed to investigate the influence of gender on sepsis‐induced NO and EDHF signaling pathways in rat aorta. Results indicate that NO and EDHF play a major role in sepsis‐induced endothelial dysfunction in rat aorta in a experimental model of sepsis. Sepsis was induced in rats by cecal ligation and puncture (CLP). 18h after surgery septic rats showed increase in rectal temperature, neutrophilia, pulmonary edema and an increase in plasma nitrate/nitrite levels. However, mean arterial pressure was decreased. Arterial vasoreactivity was measured using an isometric tension protocol. Phenylephrine (PE) was more potent in contracting aortic rings from female than male rats, but the amplitude of contraction was identical in both the sexes. Sepsis significantly decreased the efficacy of PE in aortas from both the sexes. Acetylcholine (ACh) elicited concentration‐dependent relaxation in endothelium intact aortas from male and female rats. Sepsis had no significant effect on the endothelium‐dependent relaxation in the female aorta, but caused significant reduction in the maximal response to ACh in male aortas. There was no evidence of EDHF activity in aortic rings from either sex. Sodium nitroprusside (SNP) was more potent in dilating aortas from male than female rats. Sepsis had no significant effect on the potency or efficacy of SNP‐induced dilation in both genders. Real time PCR was used to measure mRNA expression levels of potassium channels important in the endothelium. In conclusion, the results of present study suggest that sepsis‐induced alteration in the reactivity of aorta to vasoconstrictors or vasodilators is dependent on the gender of the animal.