Channels In Endothelial Cells Research Articles

TMAO Impairs Mouse Aortic Vasodilation by Inhibiting TRPV4 Channels in Endothelial Cells.

Trimethylamine oxide (TMAO) is an intestinal flora metabolite associated with risk of cardiovascular diseases. Transient receptor potential vanilloid 4 (TRPV4) is a Ca2+-permeable ion channel that is essential for vasodilation and endothelial function. Currently, there are few studies on the effect of TMAO on TRPV4 channels. In the present study, Ca2+ imaging of vascular tissue showed that TMAO inhibited TRPV4-mediated Ca2+ influx into aortic endothelial cells in a dose-dependent manner. Furthermore, a whole-cell patch clamp assay showed that TMAO blocked TRPV4-mediated cation currents. Notably, results of aortic vascular tension measurement showed that TMAO impaired endothelium-dependent vasodilation in mouse aortic vessels through the TRPV4-NO pathway. Our results indicated that TMAO inhibited Ca2+ entry in endothelial cells and impaired vasodilation through the TRPV4-NO pathway in mice. These results provide scientific evidence for novel pathogenic mechanisms underlying the role of TMAO in cardiovascular disease.

Vasodilators activate the anion channel TMEM16A in endothelial cells to reduce blood pressure.

Systemic blood pressure is acutely controlled by total peripheral resistance as determined by the diameter of small arteries and arterioles, the contractility of which is regulated by endothelial cells lining the lumen of blood vessels. We investigated the physiological functions of the chloride (Cl-) channel TMEM16A in endothelial cells. TMEM16A channels generated calcium (Ca2+)-activated Cl- currents in endothelial cells from control (TMEM16Afl/fl) mice that were absent in those from mice with tamoxifen-inducible, endothelial cell-specific knockout of TMEM16A (TMEM16A ecKO). TMEM16A currents in endothelial cells were activated by the muscarinic receptor agonist acetylcholine and an agonist of the Ca2+ channel TRPV4, which localized in nanoscale proximity with TMEM16A as assessed by single-molecule localization imaging of endothelial cells. Acetylcholine stimulated TMEM16A currents by activating Ca2+ influx through surface TRPV4 channels without altering the nanoscale properties of TMEM16A and TRPV4 surface clusters or their colocalization. In pressurized arteries, activation of TMEM16A channels in endothelial cells induced by acetylcholine; TRPV4 channel stimulation; or intraluminal ATP, another vasodilator, produced hyperpolarization and dilation. Furthermore, deficiency of TMEM16A channels in endothelial cells resulted in increased systemic blood pressure in conscious mice. These data indicate that vasodilators stimulate TRPV4 channels, leading to Ca2+-dependent activation of nearby TMEM16A channels in endothelial cells to produce arterial hyperpolarization, vasodilation, and reduced blood pressure. Thus, TMEM16A is an anion channel in endothelial cells that regulates arterial contractility and blood pressure.

Vasodilators mobilize SK3 channels in endothelial cells to produce arterial relaxation

Endothelial cells (ECs) line the lumen of all blood vessels and regulate functions, including contractility. Physiological stimuli, such as acetylcholine (ACh) and intravascular flow, activate transient receptor potential vanilloid 4 (TRPV4) channels, which stimulate small (SK3)- and intermediate (IK)-conductance Ca2+-activated potassium channels in ECs to produce vasodilation. Whether physiological vasodilators also modulate the surface abundance of these ion channels in ECs to elicit functional responses is unclear. Here, we show that ACh and intravascular flow stimulate rapid anterograde trafficking of an intracellular pool of SK3 channels in ECs of resistance-size arteries, which increases surface SK3 protein more than two-fold. In contrast, ACh and flow do not alter the surface abundance of IK or TRPV4 channels. ACh triggers SK3 channel trafficking by activating TRPV4-mediated Ca2+ influx, which stimulates Rab11A, a Rab GTPase associated with recycling endosomes. Superresolution microscopy data demonstrate that SK3 trafficking specifically increases the size of surface SK3 clusters which overlap with TRPV4 clusters. We also show that Rab11A-dependent trafficking of SK3 channels is an essential contributor to vasodilator-induced SK current activation in ECs and vasorelaxation. In summary, our data demonstrate that vasodilators activate Rab11A, which rapidly delivers an intracellular pool of SK3 channels to the vicinity of surface TRPV4 channels in ECs. This trafficking mechanism increases surface SK3 cluster size, elevates SK3 current density, and produces vasodilation. These data also demonstrate that SK3 and IK channels are differentially regulated by trafficking-dependent and -independent signaling mechanisms in endothelial cells.

Vasoprotective effects of NOX4 are mediated via polymerase and transient receptor potential melastatin 2 cation channels in endothelial cells.

NOX4 activation has been implicated to have vasoprotective and blood pressure (BP)-lowering effects. Molecular mechanisms underlying this are unclear, but NOX4-induced regulation of the redox-sensitive Ca2+ channel TRPM2 and effects on endothelial nitric oxide synthase (eNOS)-nitric oxide signalling may be important. Wild-type and LinA3, renin-expressing hypertensive mice, were crossed with NOX4 knockout mice. Vascular function was measured by myography. Generation of superoxide (O2-) and hydrogen peroxide (H2O2) were assessed by lucigenin and amplex red, respectively, and Ca2+ influx by Cal-520 fluorescence in rat aortic endothelial cells (RAEC). BP was increased in NOX4KO, LinA3 and LinA3/NOX4KO mice. This was associated with endothelial dysfunction and vascular remodelling, with exaggerated effects in NOX4KO groups. The TRPM2 activator, ADPR, improved vascular relaxation in LinA3/NOX4KO mice, an effect recapitulated by H2O2. Inhibition of PARP and TRPM2 with olaparib and 2-APB, respectively, recapitulated endothelial dysfunction in NOX4KO. In endothelial cells, Ang II increased H2O2 generation and Ca2+ influx, effects reduced by TRPM2 siRNA, TRPM2 inhibitors (8-br-cADPR, 2-APB), olaparib and GKT137831 (NOX4 inhibitor). Ang II-induced eNOS activation was blocked by NOX4 and TRPM2 siRNA, GKT137831, PEG-catalase and 8-br-cADPR. Our findings indicate that NOX4-induced H2O2 production activates PARP/TRPM2, Ca2+ influx, eNOS activation and nitric oxide release in endothelial cells. NOX4 deficiency impairs Ca2+ homeostasis leading to endothelial dysfunction, an effect exacerbated in hypertension. We define a novel pathway linking endothelial NOX4/H2O2 to eNOS/nitric oxide through PARP/TRPM2/Ca2+. This vasoprotective pathway is perturbed when NOX4 is downregulated and may have significance in conditions associated with endothelial dysfunction, including hypertension.

Adenosine signaling activates ATP-sensitive K + channels in endothelial cells and pericytes in CNS capillaries

The dense network of capillaries composed of capillary endothelial cells (cECs) and pericytes lies in close proximity to all neurons, ideally positioning it to sense neuron- and glial-derived compounds that enhance regional and global cerebral perfusion. The membrane potential (VM) of vascular cells serves as the physiological bridge that translates brain activity into vascular function. In other beds, the ATP-sensitive K+ (KATP) channel regulates VM in vascular smooth muscle, which is absent in the capillary network. Here, with transgenic mice that expressed a dominant-negative mutant of the pore-forming Kir6.1 subunit specifically in brain cECs or pericytes, we demonstrated that KATP channels were present in both cell types and robustly controlled VM. We further showed that the signaling nucleotide adenosine acted through A2A receptors and the Gαs/cAMP/PKA pathway to activate capillary KATP channels. Moreover, KATP channel stimulation in vivo increased cerebral blood flow (CBF), an effect that was blunted by expression of the dominant-negative Kir6.1 mutant in either capillary cell type. These findings establish an important role for KATP channels in cECs and pericytes in the regulation of CBF.

Beneficial effect of capsaicin via TRPV4/EDH signals on mesenteric arterioles of normal and colitis mice

IntroductionAlthough capsaicin has long been used as food additive and medication worldwide, its actions on gastrointestinal tract as its most delivery pathway have not been well addressed. ObjectivesIn the present study, we aimed to study GI actions of capsaicin on mesenteric arterioles in normal and colitis mice and to elucidate the underlying mechanisms. MethodsVasorelaxation of human submucosal arterioles and the mesenteric arterioles from wide-type (WT) mice, TRPV1−/− and TRPV4−/− (KO) mice were measured. The expression and function of TRPV channels in endothelial cells were examined by q-PCR, immunostaining, Ca2+ imaging and membrane potential measurements. ResultsCapsaicin dose-dependently induced vasorelaxation of human submucosal arterioles and mouse mesenteric arterioles in vitro and in vivo through endothelium-dependent hyperpolarization (EDH), nitric oxide (NO), and prostacyclin (PGI2). Using TRPV1 and TRPV4 KO mice, we found that capsaicin-induced vasorelaxation was predominately through TRPV4/EDH, but marginally through TRPV1/NO/PGI2. Capsaicin induced hyperpolarization through activation of endothelial TRPV4 channels and intermediate-conductance of Ca2+-activated K+ channels to finally stimulate vasorelaxation. Importantly, capsaicin exerted anti-colitis action by rescuing the impaired ACh-induced vasorelaxation in WT colitis mice but not in TRPV4 KO colitis mice. ConclusionsCapsaicin increases intestinal mucosal blood perfusion to potentially prevent/treat colitis through a novel TRPV4/EDH-dependent vasorelaxation of submucosal arterioles in health and colitis. This study further supports our previous notion that TRPV4/EDH in mesenteric circulation plays a critical role in the pathogenesis of colitis.

Role of TRP ion channels in cerebral circulation and neurovascular communication

The dynamic regulation of blood flow is essential for meeting the high metabolic demands of the brain and maintaining brain function. Cerebral blood flow is regulated primarily by 1) the intrinsic mechanisms that determine vascular contractility and 2) signals from neurons and astrocytes that alter vascular contractility. Stimuli from neurons and astrocytes can also initiate a signaling cascade in the brain capillary endothelium to increase regional blood flow. Recent studies provide evidence that TRP channels in endothelial cells, smooth muscle cells, neurons, astrocytes, and perivascular nerves control cerebrovascular contractility and cerebral blood flow. TRP channels exert their functional effects either through cell membrane depolarization or by serving as a Ca2+ influx pathway. Endothelial cells and astrocytes also maintain the integrity of the blood-brain barrier. Both endothelial cells and astrocytes express TRP channels, and an increase in endothelial TRP channel activity has been linked with a disrupted endothelial barrier function. Therefore, TRP channels can play a potentially important role in regulating blood-brain barrier integrity. Here, we review the regulation of cerebrovascular contractility by TRP channels under healthy and disease conditions and their potential roles in maintaining blood-brain barrier function.

Cytoprotective effects of the flavonoid quercetin by activating mitochondrial BKCa channels in endothelial cells.

Mitochondrial potassium channels have been implicated in cytoprotective mechanisms. Activation of the mitochondrial large-conductance Ca2+-regulated potassium (mitoBKCa) channel is important for protecting brain tissue against stroke damage as well as heart tissue against ischemia damage. In this paper, we examine the effect of the natural flavonoid quercetin as an activator of the mitoBKCa channel. Quercetin has a beneficial effect on many processes in the human body and interacts with many receptors and signaling pathways. We found that quercetin acts on mitochondria as a mitoBKCa channel opener. The activation observed with the patch-clamp technique was potent and increased the channel open probability from approximately 0.35 to 0.95 at + 40 mV in the micromolar concentration range. Moreover, quercetin at a concentration of 10 µM protected cells by reducing damage from treatment factors (tumor necrosis factor α and cycloheximide) by 40%, enhancing cellular migration and depolarizing the mitochondrial membrane. Moreover, the presence of quercetin increased the gene expression and protein level of the mitoBKCa β3 regulatory subunit. The observed cytoprotective effects suggested the involvement of BKCa channel activation. Additionally, the newly discovered mitoBKCa activator quercetin elucidates a new mitochondrial pathway that is beneficial for vascular endothelial cells.

Homocysteine Causes Endothelial Dysfunction via Inflammatory Factor-Mediated Activation of Epithelial Sodium Channel (ENaC).

BackgroundHyperhomocysteinemia (HHcy) causes cardiovascular diseases via regulating inflammatory responses. We investigated whether and how the epithelial sodium channel (ENaC), a recently identified ion channel in endothelial cells, plays a role in HHcy-induced endothelial dysfunction.MethodsCell-attached patch-clamp recording in acute split-open aortic endothelial cells, western blot, confocal imaging, and wire myograph combined with pharmacological approaches were used to determine whether HHcy-mediated inflammatory signaling leads to endothelial dysfunction via stimulating ENaC.ResultsThe data showed that 4 weeks after L-methionine diet the levels of plasma Hcy were significantly increased and the ENaC was dramatically activated in mouse aortic endothelial cells. Administration of benzamil, a specific ENaC blocker, ameliorated L-methionine diet-induced impairment of endothelium-dependent relaxation (EDR) and reversed Hcy-induced increase in ENaC activity. Pharmacological inhibition of NADPH oxidase, reactive oxygen species (ROS), cyclooxygenase-2 (COX-2)/thromboxane B2 (TXB2), or serum/glucocorticoid regulated kinase 1 (SGK1) effectively attenuated both the Hcy-induced activation of endothelial ENaC and impairment of EDR. Our in vitro data showed that both NADPH oxidase inhibitor and an ROS scavenger reversed Hcy-induced increase in COX-2 expression in human umbilical vein endothelial cells (HUVECs). Moreover, Hcy-induced increase in expression levels of SGK-1, phosphorylated-SGK-1, and phosphorylated neural precursor cell-expressed developmentally downregulated protein 4-2 (p-Nedd4-2) in HUVECs were significantly blunted by a COX-2 inhibitor.ConclusionWe show that Hcy activates endothelial ENaC and subsequently impairs EDR of mouse aorta, via ROS/COX-2-dependent activation of SGK-1/Nedd4-2 signaling. Our study provides a rational that blockade of the endothelial ENaC could be potential method to prevent and/or to treat Hcy-induced cardiovascular disease.

TRPV4 regulates matrix stiffness‐dependent activation of YAP/VEGFR2 signaling via Rho/Rho kinase/LATS1/2 pathway

TRPV4 is a mechanosensitive ion channel in endothelial cells and shown to regulate physiological and pathological angiogenesis. We have recently shown that TRPV4 deletion and/or down regulation activates vascular endothelial growth factor receptor-2 (VEGFR2) via yes-associated protein (YAP) in human EC. However, the molecular mechanisms of TRPV4-dependant YAP/VEGFR2 activation are unknown. We have employed human microvascular endothelial cells (HMEC-1; EC) on ECM gels of varying stiffness that mimic normal and failing hearts (8 and 50 kPa) and examined nuclear translocation of YAP and disappearance of perinuclear VEGFR2, after siRNA knockdown of TRPV4. Next, we investigated the role of upstream and downstream regulators of YAP translocation by focusing on LATS-1, CYR61 and Rho/Rho Kinase, using phospho-specific antibodies (pYAP and pLATS) and pharmacological inhibitor of Rho kinase (Y-27632). siRNA knockdown of TRPV4 significantly increased nuclear translocation of YAP with concomitant disappearance of perinuclear-VEGFR2 in matrix stiffness-dependent manner. Further, we found increased Rho-GTP and decreased LATS-1 phosphorylation in TRPV4 knockdown EC compared to control siRNA treated EC. Furthermore, pharmacological inhibition of Rho kinase with Y-27632, increased phosphorylation of LATS1/2 kinase, inhibited nuclear translocation of YAP and VEGFR2 redistribution. Consistent with above results, YAP target genes CYR61 and CTGF mRNA expression levels were increased in a matrix stiffness-dependent manner in TRPV4 knockdown EC than the control siRNA treated EC. Our results suggest that TRPV4 regulates YAP/VEGFR2 activation in a matrix stiffness dependent manner during angiogenesis via Rho/Rho kinase/LATS1/2 pathway.

Quantitative Examination of Changes in Fluidity and Cholesterol Content in Bovine Aortic Endothelial Cells using Phasor Analysis of Laurdan Fluorescence Lifetime

Cholesterol is important for maintaining cell membrane integrity and is known to have an effect on the function of ion channels in endothelial cells. Here, we use the properties of Laurdan to investigate how various cellular cholesterol manipulations, altering membrane fluidity and adding a cholesterol analog affect physical properties of bovine aortic endothelial cells (BAEC). Laurdan, a solvatochromic dye, has a spectral red shift in its emission when in a more liquid disordered (Ld) phase compared to a liquid ordered phase (LO). BAECs labeled with Laurdan were treated with methyl-ß-cyclodextrin (MßCD) to remove cholesterol, MßCD saturated with cholesterol to enrich cholesterol content, the cholesterol analog epicholesterol, or fluidizing alcohols benzyl alcohol (BA) or dodecanol and compared to BAECs treated with PBS+Ca2+ as a control. Two-photon excitation was used to excite Laurdan and lifetime measurements were compared at two emission channels (‘blue channel’ and ‘green channel’) in order to differentiate between changes in membrane fluidity and cholesterol content using two methods: traditional phasor analysis and a multiparametric distance analysis using phasor data. Treatment with MßCD, BA and dodecanol resulted in a shift to shorter lifetimes in the blue channel, indicative a more polar (Ld) environment, while enriching cells with cholesterol shifted phasors to longer lifetimes. In the green channel, the phasors of cholesterol enriched cells were shifted in the direction that corresponds to an increase in dipolar relaxation. Only treatment with a higher concentration of MßCD caused a shift in the opposite direction. Using Laurdan and phasor analysis to distinguish changes in fluidity from changes in cholesterol gives us the ability to manipulate these parameters to assist in determining the mechanisms by which cholesterol can impair cell signaling processes.

Abstract 15033: Chronic Inhibition of Mitochondrial Reactive Oxygen Species Protects Against Endothelial SK Channel Dysfunction in Diabetes

Introduction: Oxidative stress plays an important role in the development of pathological condition in diabetes. Persistent oxidative stress during diabetes may result in impaired endothelium-independent vasodilation, by reducing small-conductance Ca 2+ -activated K + (SK) channel activity. The mitochondria are main sources of reactive oxygen species (ROS) in diabetes, and mitochondria-targeted antioxidant mito-TEMPO (MT) prevents ROS generation has been found in a variety of disorders. However, few studies are available on the dysfunction of vasodilation and endothelial SK channels in diabetes after a chronic use of MT. Purpose: We investigated the chronic administration of MT on vasodilation in coronary arteries, and endothelial SK channel activity with or without diabetes. Methods: MT (1mg/kg) was applied to diabetic and health mice (n = 6/group) for 1 month. In-vitro relaxation response of pre-contracted arteries was examined in the presence or absence of the vasodilatory agents. Isolated endothelium was studied using whole-cell patch clamp recording SK channels currents. Results: Coronary arteries dilation was decreased in diabetic mice compared with that in health ( p <0.05). After one month treatment with MT, coronary arteries dilation was significantly improved when compared with that in non-treatment diabetic mice ( p <0.05). Interestingly, coronary arteries dilation was no significantly changed in health mice whether to treat with MT or not. Similar changes were seen in patch clamp recording, impaired currents of SK channels in endothelial cells of diabetic mice were significantly improved after chronic treatment with MT ( p <0.05, Fig) and currents of SK channels were no changed in health mice whether to treat with MT or not. Conclusions: MT appears to improve coronary endothelial function/dilation by protection of SK channel activity in diabetes, and administration of MT may be a novel strategy to treat vascular complications in diabetes.

Stimulation of Epithelial Sodium Channels in Endothelial Cells by Bone Morphogenetic Protein-4 Contributes to Salt-Sensitive Hypertension in Rats.

Previous studies have shown that high salt induces artery stiffness by causing endothelial dysfunction via increased sodium influx. We used our unique split-open artery technique combined with protein biochemistry and in vitro measurement of vascular tone to test a hypothesis that bone morphogenetic protein 4 (BMP4) mediates high salt-induced loss of vascular relaxation by stimulating the epithelial sodium channel (ENaC) in endothelial cells. The data show that high salt intake increased BMP4 both in endothelial cells and in the serum and that exogenous BMP4 stimulated ENaC in endothelial cells. The data also show that the stimulation is mediated by p38 mitogen-activated protein kinases (p38 MAPK) and serum and glucocorticoid-regulated kinase 1 (Sgk1)/neural precursor cell expressed developmentally downregulated gene 4-2 (Nedd4-2) (Sgk1/Nedd4-2). Furthermore, BMP4 decreased mesenteric artery relaxation in a benzamil-sensitive manner. These results suggest that high salt intake stimulates endothelial cells to express and release BMP4 and that the released BMP4 reduces artery relaxation by stimulating ENaC in endothelial cells. Therefore, stimulation of ENaC in endothelial cells by BMP4 may serve as another pathway to participate in the complex mechanism of salt-sensitive (SS) hypertension.

Analyzing the shear-induced sensitization of mechanosensitive ion channel Piezo-1 in human aortic endothelial cells.

Mechanosensitive ion channels mediate endothelial responses to blood flow and orchestrate their physiological function in response to hemodynamic forces. In this study, we utilized microfluidic technologies to study the shear-induced sensitization of endothelial Piezo-1 to its selective agonist, Yoda-1. We demonstrated that shear stress-induced sensitization is brief and can be impaired when exposing aortic endothelial cells to low and proatherogenic levels of shear stress. Our results suggest that shear stress-induced sensitization of Piezo-1 to Yoda-1 is independent of cell-cell adhesion and is mediated by the PI3K-AKT signaling pathway. We also found that shear stress increases the membrane density of Piezo-1 channels in endothelial cells. To further confirm our findings, we performed experiments using a carotid artery ligation mouse model and demonstrated that transient changes in blood-flow pattern, resulting from a high-degree ligation of the mouse carotid artery alters the distribution of Piezo-1 channels across the endothelial layer. These results suggest that shear stress influences the function of Piezo-1 channels via changes in membrane density, providing a new model of shear-stress sensitivity for Piezo-1 ion channel.

Endothelial Ca2+ signaling-dependent vasodilation through transient receptor potential channels.

Ca2+ signaling of endothelial cells plays a critical role in controlling blood flow and pressure in small arteries and arterioles. As the impairment of endothelial function is closely associated with cardiovascular diseases (e.g., atherosclerosis, stroke, and hypertension), endothelial Ca2+ signaling mechanisms have received substantial attention. Increases in endothelial intracellular Ca2+ concentrations promote the synthesis and release of endothelial-derived hyperpolarizing factors (EDHFs, e.g., nitric oxide, prostacyclin, or K+ efflux) or directly result in endothelial-dependent hyperpolarization (EDH). These physiological alterations modulate vascular contractility and cause marked vasodilation in resistance arteries. Transient receptor potential (TRP) channels are nonselective cation channels that are present in the endothelium, vascular smooth muscle cells, or perivascular/sensory nerves. TRP channels are activated by diverse stimuli and are considered key biological apparatuses for the Ca2+ influx-dependent regulation of vasomotor reactivity in resistance arteries. Ca2+-permeable TRP channels, which are primarily found at spatially restricted microdomains in endothelial cells (e.g., myoendothelial projections), have a large unitary or binary conductance and contribute to EDHFs or EDH-induced vasodilation in concert with the activation of intermediate/small conductance Ca2+-sensitive K+ channels. It is likely that endothelial TRP channel dysfunction is related to the dysregulation of endothelial Ca2+ signaling and in turn gives rise to vascular-related diseases such as hypertension. Thus, investigations on the role of Ca2+ dynamics via TRP channels in endothelial cells are required to further comprehend how vascular tone or perfusion pressure are regulated in normal and pathophysiological conditions.

Correction: Intravascular flow stimulates PKD2 (polycystin-2) channels in endothelial cells to reduce blood pressure.

PKD2 (polycystin-2, TRPP1), a TRP polycystin channel, is expressed in endothelial cells (ECs), but its physiological functions in this cell type are unclear. Here, we generated inducible, EC-specific Pkd2 knockout mice to examine vascular functions of PKD2. Data show that a broad range of intravascular flow rates stimulate EC PKD2 channels, producing vasodilation. Flow-mediated PKD2 channel activation leads to calcium influx that activates SK/IK channels and eNOS serine 1176 phosphorylation in ECs. These signaling mechanisms produce arterial hyperpolarization and vasodilation. In contrast, EC PKD2 channels do not contribute to acetylcholine-induced vasodilation, suggesting stimulus-specific function. EC-specific PKD2 knockout elevated blood pressure in mice without altering cardiac function or kidney anatomy. These data demonstrate that flow stimulates PKD2 channels in ECs, leading to SK/IK channel and eNOS activation, hyperpolarization, vasodilation and a reduction in systemic blood pressure. Thus, PKD2 channels are a major component of functional flow sensing in the vasculature.

Intravascular flow stimulates PKD2 (polycystin-2) channels in endothelial cells to reduce blood pressure.

PKD2 (polycystin-2, TRPP1), a TRP polycystin channel, is expressed in endothelial cells (ECs), but its physiological functions in this cell type are unclear. Here, we generated inducible, EC-specific Pkd2 knockout mice to examine vascular functions of PKD2. Data show that a broad range of intravascular flow rates stimulate EC PKD2 channels, producing vasodilation. Flow-mediated PKD2 channel activation leads to calcium influx that activates SK/IK channels and eNOS serine 1176 phosphorylation in ECs. These signaling mechanisms produce arterial hyperpolarization and vasodilation. In contrast, EC PKD2 channels do not contribute to acetylcholine-induced vasodilation, suggesting stimulus-specific function. EC-specific PKD2 knockout elevated blood pressure in mice without altering cardiac function or kidney anatomy. These data demonstrate that flow stimulates PKD2 channels in ECs, leading to SK/IK channel and eNOS activation, hyperpolarization, vasodilation and a reduction in systemic blood pressure. Thus, PKD2 channels are a major component of functional flow sensing in the vasculature.

Metformin Attenuates High Glucose‐Induced Coronary Vascular Endothelial Hyper Permeability Via Inhibition of Orai‐1 Mediated Store‐Operated Calcium Entry

Increase in vascular endothelial permeability is an earliest pathological hallmark in diabetes mellitus, which progressively leads to cardiovascular disease. Stromal‐interaction molecule 1 (STIM1), upon sensing the depletion of calcium (Ca2+) from the endoplasmic reticulum (ER) store, organizes as a puncta that triggers store‐operated Ca2+ entry (SOCE) via plasmalemmal Ca2+‐selective Orai1 channels in endothelial cells. Thus, signals to disrupt endothelial cell‐cell junctions’ integrity and increase in endothelial permeability. Recent studies show that the anti‐diabetic drug metformin confers vascular benefits beyond glycaemia control and reduced the high risk of cardiovascular events in pre‐diabetes patients. However, the precise pharmacological role of metformin and the mechanism to regulate SOCE‐induced endothelial hyper permeability response in coronary vasculature remain enigmatic in the pathogenesis of hyperglycemia. Here, we demonstrate a previously undetermined role of metformin in inhibiting the Orai1 mediated induction of SOCE and thereby preventing the disintegration of vascular endothelial (VE) cadherin in high‐glucose exposed coronary vascular endothelial cells (ECs). Our data showed that exposing the human coronary endothelial cell monolayer to high glucose media (25mM) for 48 hours increased the expression of Orai1, protracted SOCE and the increased in endothelial permeability. We found that SOCE mediated by Orai1 activated the Pyk2 in ECs. Intriguingly, we observed the tyrosine phosphorylation of vascular endothelial‐protein tyrosine phosphatase (VE‐PTP) at tyrosine (Y) 1981 residue downstream of the Pyk2 activation in high glucose milieu. Our results showed that metformin prevented the SOCE by decreasing the expression of Orai1 and thereby abrogated the Pyk2 mediated phosphorylation of VE‐PTP in high glucose condition. Thus, led to the stabilization and maintenance of VE‐cadherin at interendothelial junctions and strengthening the barrier function. Our data identify heretofore unprecedented signaling mechanism by which metformin via inhibiting SOCE, maintains VE‐cadherin integrity and henceforth decreased high glucose induced hyper permeability response in human coronary endothelial cells.Support or Funding InformationThis work is supported by the Department of Pharmaceutical Sciences, Chicago State University College of Pharmacy (CSU‐COP) research funding to Mohammad Tauseef.

“Spatial localization determines preferential calcium signal‐target coupling in microvascular endothelium”

Endothelial cell layer is an important regulator of vascular resistance. Localized, spatially restricted Ca2+ signals in endothelial cells promote vasodilation in systemic and pulmonary arteries, thereby lowering vascular resistance. Ca2+ signals are known to activate two main vasodilatory targets in endothelial cells: intermediate and small conductance Ca2+‐activated K+ (IK and SK, respectively) channels and endothelial nitric oxide synthase (eNOS). The goal of this study was to determine the specific mechanisms underlying preferential Ca2+ signal to vasodilatory target coupling. Based on the literature, we used resistance mesenteric arteries (MA) as a prototype for Ca2+ signal‐IK/SK channel coupling, and resistance pulmonary arteries (PA) as a prototype for Ca2+ signal‐eNOS coupling. Unitary Ca2+ influx signals through endothelial transient receptor potential vanilloid 4 (TRPV4) channels, termed TRPV4 sparklets, dilate both mesenteric and pulmonary arteries, thereby lowering resting systemic and pulmonary arterial pressures. Therefore, the study focused on the coupling of endothelial TRPV4 channels with their vasodilator targets. Whole‐cell patch clamp studies in freshly isolated endothelial cells showed that TRPV4 channel activator GSK1016790A (GSK101, 10 nM) induced IK/SK currents in MA endothelial cells, but not in PA endothelial cells. However, direct IK/SK channel activator NS309 (1 mM) or 3 mM intracellular Ca2+ was able to elicit similar IK/SK currents in both MA and PA endothelial cells, confirming the presence of IK/SK channels in endothelial cells from PA and supporting a lack of TRPV4‐IK/SK channel coupling in PAs. NO measurements with fluorescent indicator DAF‐FM showed increased endothelial NO levels in response to GSK101 in PAs but not in MAs, supporting a lack of TRPV4 sparklet‐eNOS coupling in MAs. Because of the localized nature of TRPV4 sparklets, we hypothesized that spatial co‐localization determines Ca2+ signal‐vasodilator target coupling. While TRPV4 channels were localized at endothelial projections to smooth muscle or myoendothelial projections (MEPs) in both MAs and PAs, IK/SK channels were seen at MEPs only in MAs, supporting the importance of spatial co‐localization for TRPV4‐IK/SK channel coupling. Interestingly, eNOS was localized at MEPs in both MAs and PAs, raising the question why TRPV4 sparklets cannot activate eNOS in MAs despite the spatial proximity. Therefore, we postulated that hemoglobin α (Hbα), which is known to bind eNOS and scavenge NO, silences TRPV4 channel‐eNOS coupling in MAs. Hbα immunostaining was seen mostly at MEPs in MAs, but was absent in PA endothelium. Moreover, Hbα existed in nanometer proximity with eNOS in MAs. Finally, a synthetic peptide that disrupts eNOS‐Hbα interaction uncovered the TRPV4‐eNOS coupling in MAs. Our results suggest that spatial co‐localization and the presence of Hbα determine Ca2+ signal‐vasodilator target coupling in endothelial cells. Understanding the differential coupling mechanisms may lead to novel therapeutic approaches that can selectively dilate pulmonary versus systemic vasculature.Support or Funding InformationHL121484‐01, HL138496

Epithelial sodium channels in endothelial cells mediate diet-induced endothelium stiffness and impaired vascular relaxation in obese female mice

ObjectiveMineralocorticoid receptor activation of the epithelial sodium channel in endothelial cells (ECs) (EnNaC) is accompanied by aldosterone induced endothelial stiffening and impaired nitric oxide (NO)-mediated arterial relaxation. Recent data support enhanced activity of the alpha subunit of EnNaC (αEnNaC) mediates this aldosterone induced endothelial stiffening and associated endothelial NO synthase (eNOS) activation. There is mounting evidence that diet induced obesity diminishes expression and activation of AMP-activated protein kinase α (AMPKα), sirtuin 1 (Sirt1), which would be expected to lead to impaired downstream eNOS activation. Thereby, we posited that enhanced EnNaC activation contributes to diet induced obesity related increases in stiffness of the endothelium and diminished NO mediated vascular relaxation by increasing oxidative stress and related inhibition of AMPKα, Sirt1, and associated eNOS inactivation. Materials/MethodsSixteen to twenty week-old αEnNaC knockout (αEnNaC−/−) and wild type littermate (EnNaC+/+) female mice were fed a mouse chow or an obesogenic western diet (WD) containing excess fat (46%) and fructose (17.5%) for 16 weeks. Sodium currents of ECs, endothelial stiffness and NO mediated aortic relaxation were examined along with indices of aortic oxidative stress, vascular remodeling and fibrosis. ResultsEnhanced EnNaC activation-mediated WD-induced increases in sodium currents in isolated lung ECs, increased endothelial stiffness and impaired aortic endothelium-dependent relaxation to acetylcholine (10−9–10−4 mol/L). These abnormalities occurred in conjunction with WD-mediated aortic tissue oxidative stress, inflammation, and decreased activation of AMPKα, Sirt1, and downstream eNOS were substantially mitigated in αEnNaC−/− mice. Importantly, αEnNaC−/− prevented WD induced increases in endothelial stiffness and related impairment of endothelium-dependent relaxation as well as aortic fibrosis and remodeling. However, EnNaC signaling was not involved in diet-induced abnormal expression of adipokines and CYP11b2 in abdominal aortic perivascular adipose tissue. ConclusionThese data suggest that endothelial specific EnNaC activation mediates WD-induced endothelial stiffness, impaired eNOS activation, aortic fibrosis and remodeling through increased aortic oxidative stress and increased inflammation related to a reduction of AMPKα and Sirt 1 mediated eNOS phosphorylation/activation and NO production.