NADPH oxidase 4 contributes to TRPV4-mediated endothelium-dependent vasodilation in human arterioles by regulating protein phosphorylation of TRPV4 channels.

Impaired endothelium-dependent vasodilation has been suggested to be a key component of coronary microvascular dysfunction (CMD). A better understanding of endothelial pathways involved in vasodilation in human arterioles may provide new insight into the mechanisms of CMD. The goal of this study is to investigate the role of TRPV4, NOX4, and their interaction in human arterioles and examine the underlying mechanisms. Arterioles were freshly isolated from adipose and heart tissues obtained from 71 patients without coronary artery disease, and vascular reactivity was studied by videomicroscopy. In human adipose arterioles (HAA), ACh-induced dilation was significantly reduced by TRPV4 inhibitor HC067047 and by NOX 1/4 inhibitor GKT137831, but GKT137831 did not further affect the dilation in the presence of TRPV4 inhibitors. GKT137831 also inhibited TRPV4 agonist GSK1016790A-induced dilation in HAA and human coronary arterioles (HCA). NOX4 transcripts and proteins were detected in endothelial cells of HAA and HCA. Using fura-2 imaging, GKT137831 significantly reduced GSK1016790A-induced Ca2+ influx in the primary culture of endothelial cells and TRPV4-WT-overexpressing human coronary artery endothelial cells (HCAEC). However, GKT137831 did not affect TRPV4-mediated Ca2+ influx in non-phosphorylatable TRPV4-S823A/S824A-overexpressing HCAEC. In addition, treatment of HCAEC with GKT137831 decreased the phosphorylation level of Ser824 in TRPV4. Finally, proximity ligation assay (PLA) revealed co-localization of NOX4 and TRPV4 proteins. In conclusion, both TRPV4 and NOX4 contribute to ACh-induced dilation in human arterioles from patients without coronary artery disease. NOX4 increases TRPV4 phosphorylation in endothelial cells, which in turn enhances TRPV4-mediated Ca2+ entry and subsequent endothelium-dependent dilation in human arterioles.

Endothelial Rap1 (Ras-Association Proximate 1) Restricts Inflammatory Signaling to Protect From the Progression of Atherosclerosis.

OBJECTIVE: Small GTPase Rap1 (Ras-association proximate 1) is a novel, positive regulator of NO release and endothelial function with a potentially key role in mechanosensing of atheroprotective, laminar flow. Our objective was to delineate the role of Rap1 in the progression of atherosclerosis and its specific functions in the presence and absence of laminar flow, to better define its role in endothelial mechanisms contributing to plaque formation and atherogenesis. Approach and Results: In a mouse atherosclerosis model, endothelial Rap1B deletion exacerbates atherosclerotic plaque formation. In the thoracic aorta, where laminar shear stress-induced NO is otherwise atheroprotective, plaque area is increased in Athero-Rap1BiΔEC (atherogenic endothelial cell-specific, tamoxifen-inducible Rap1A+Rap1B knockout) mice. Endothelial Rap1 deficiency also leads to increased plaque size, leukocyte accumulation, and increased CAM (cell adhesion molecule) expression in atheroprone areas, whereas vascular permeability is unchanged. In endothelial cells, in the absence of protective laminar flow, Rap1 deficiency leads to an increased proinflammatory TNF-α (tumor necrosis factor alpha) signaling and increased NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) activation and elevated inflammatory receptor expression. Interestingly, this increased signaling to NF-κB activation is corrected by AKTVIII-an inhibitor of Akt (protein kinase B) translocation to the membrane. Together, these data implicate Rap1 in restricting Akt-dependent signaling, preventing excessive cytokine receptor signaling and proinflammatory NF-κB activation. CONCLUSIONS: Via 2 distinct mechanisms, endothelial Rap1 protects from the atherosclerosis progression in the presence and absence of laminar flow; Rap1-stimulated NO release predominates in laminar flow, and restriction of proinflammatory signaling predominates in the absence of laminar flow. Our studies provide novel insights into the mechanisms underlying endothelial homeostasis and reveal the importance of Rap1 signaling in cardiovascular disease.

Endothelin receptor A and p66Shc regulate spontaneous Ca2+ oscillations in smooth muscle cells controlling renal arterial spontaneous motion.

Adaptor protein p66Shc is overexpressed in smooth muscle cells of renal resistance vessels of hypertensive salt-sensitive rats and is involved in the regulation of renal vascular tone. We applied 2-photon laser scanning fluorescence microscopy to analyze spontaneous dynamic fluctuations in intracellular calcium concentrations ([Ca2+]i) in smooth muscle cells embedded in the walls of freshly isolated renal resistance arteries. The amplitude, number of events, and frequency of spontaneous [Ca2+]i oscillations triggered by endogenously released endothelin-1 were recorded in smooth muscle cells of the renal arteries. Endothelin receptor A antagonist BQ123 dramatically reduced the amplitude and frequency of spontaneous Ca2+ events, producing marked inhibition of renal vessels spontaneous motion. Spontaneous Ca2+ fluctuations in smooth muscle cells of p66Shc knockout (p66ShcKO) rats had significantly higher amplitude than in control rats. The frequency of spontaneous [Ca2+]i oscillations did not change in p66ShcKO rats, suggesting that p66Shc expression did not affect endothelin-1 release from resident endothelial cells. Acute application of endothelin-1 revealed significantly elevated production of the total [Ca2+]i in p66ShcKO rats. Spontaneous cytosolic Ca2+ oscillations in smooth muscle cells of renal vessels mediate their spontaneous motion via the endothelin-1/endothelin receptor A pathway. p66Shc decreases the amplitude of individual changes in [Ca2+]i, which mitigates the spontaneous motion of renal vessels.-Palygin, O., Miller, B. S., Nishijima, Y., Zhang, D. X., Staruschenko, A., Sorokin, A. Endothelin receptor A and p66Shc regulate spontaneous Ca2+ oscillations in smooth muscle cells controlling renal arterial spontaneous motion.

TRPV4 regulates matrix stiffness and TGFß1-induced epithelial-mesenchymal transition.

Substrate stiffness (or rigidity) of the extracellular matrix has important functions in numerous pathophysiological processes including fibrosis. Emerging data support a role for both a mechanical signal, for example, matrix stiffness, and a biochemical signal, for example, transforming growth factor ß1 (TGFß1), in epithelial-mesenchymal transition (EMT), a process critically involved in fibrosis. Here, we report evidence showing that transient receptor potential vanilloid 4 (TRPV4), a mechanosensitive channel, is the likely mediator of EMT in response to both TGFß1 and matrix stiffness. Specifically, we found that: (a) genetic ablation or pharmacological inhibition of TRPV4 blocked matrix stiffness and TGFß1-induced EMT in normal mouse primary epidermal keratinocytes (NMEKs) as determined by changes in morphology, adhesion, migration and alterations of expression of EMT markers including E-cadherin, N-cadherin (NCAD) and α-smooth muscle actin (α-SMA), and (b) TRPV4 deficiency prevented matrix stiffness-induced EMT in NMEKs over a pathophysiological range. Intriguingly, TRPV4 deletion in mice suppressed expression of mesenchymal markers, NCAD and α-SMA, in a bleomycin-induced murine skin fibrosis model. Mechanistically, we found that: (a) TRPV4 was essential for the nuclear translocation of YAP/TAZ (yes-associated protein/transcriptional coactivator with PDZ-binding motif) in response to matrix stiffness and TGFß1, (b) TRPV4 deletion inhibited both matrix stiffness- and TGFß1-induced expression of YAP/TAZ proteins and (c) TRPV4 deletion abrogated both matrix stiffness- and TGFß1-induced activation of AKT, but not Smad2/3, suggesting a mechanism by which TRPV4 activity regulates EMT in NMEKs. Altogether, these data identify a novel role for TRPV4 in regulating EMT.

Transient receptor potential vanilloid 4 (TRPV4) activation by arachidonic acid requires protein kinase A-mediated phosphorylation.

Transient receptor potential vanilloid 4 (TRPV4) is a Ca2+-permeable channel of the transient receptor potential (TRP) superfamily activated by diverse stimuli, including warm temperature, mechanical forces, and lipid mediators such as arachidonic acid (AA) and its metabolites. This activation is tightly regulated by protein phosphorylation carried out by various serine/threonine or tyrosine kinases. It remains poorly understood how phosphorylation differentially regulates TRPV4 activation in response to different stimuli. We investigated how TRPV4 activation by AA, an important signaling process in the dilation of coronary arterioles, is affected by protein kinase A (PKA)-mediated phosphorylation at Ser-824. Wildtype and mutant TRPV4 channels were expressed in human coronary artery endothelial cells (HCAECs). AA-induced TRPV4 activation was blunted in the S824A mutant but was enhanced in the phosphomimetic S824E mutant, whereas the channel activation by the synthetic agonist GSK1016790A was not affected. The low level of basal phosphorylation at Ser-824 was robustly increased by the redox signaling molecule hydrogen peroxide (H2O2). The H2O2-induced phosphorylation was accompanied by an enhanced channel activation by AA, and this enhanced response was largely abolished by PKA inhibition or S824A mutation. We further identified a potential structural context dependence of Ser-824 phosphorylation-mediated TRPV4 regulation involving an interplay between AA binding and the possible phosphorylation-induced rearrangements of the C-terminal helix bearing Ser-824. These results provide insight into how phosphorylation specifically regulates TRPV4 activation. Redox-mediated TRPV4 phosphorylation may contribute to pathologies associated with enhanced TRPV4 activity in endothelial and other systems.

Contribution of KV1.5 Channel to Hydrogen Peroxide-Induced Human Arteriolar Dilation and Its Modulation by Coronary Artery Disease.

RATIONALE: Hydrogen peroxide (H2O2) regulates vascular tone in the human microcirculation under physiological and pathophysiological conditions. It dilates arterioles by activating large-conductance Ca2+-activated K+ channels in subjects with coronary artery disease (CAD), but its mechanisms of action in subjects without CAD (non-CAD) when compared with those with CAD remain unknown. OBJECTIVE: We hypothesize that H2O2-elicited dilation involves different K+ channels in non-CAD versus CAD, resulting in an altered capacity for vasodilation during disease. METHODS AND RESULTS: H2O2 induced endothelium-independent vasodilation in non-CAD adipose arterioles, which was reduced by paxilline, a large-conductance Ca2+-activated K+ channel blocker, and by 4-aminopyridine, a voltage-gated K+ (KV) channel blocker. Assays of mRNA transcripts, protein expression, and subcellular localization revealed that KV1.5 is the major KV1 channel expressed in vascular smooth muscle cells and is abundantly localized on the plasma membrane. The selective KV1.5 blocker diphenylphosphine oxide-1 and the KV1.3/1.5 blocker 5-(4-phenylbutoxy)psoralen reduced H2O2-elicited dilation to a similar extent as 4-aminopyridine, but the selective KV1.3 blocker phenoxyalkoxypsoralen-1 was without effect. In arterioles from CAD subjects, H2O2-induced dilation was significantly reduced, and this dilation was inhibited by paxilline but not by 4-aminopyridine, diphenylphosphine oxide-1, or 5-(4-phenylbutoxy)psoralen. KV1.5 cell membrane localization and diphenylphosphine oxide-1-sensitive K+ currents were markedly reduced in isolated vascular smooth muscle cells from CAD arterioles, although mRNA or total cellular protein expression was largely unchanged. CONCLUSIONS: In human arterioles, H2O2-induced dilation is impaired in CAD, which is associated with a transition from a combined large-conductance Ca2+-activated K+- and KV (KV1.5)-mediated vasodilation toward a large-conductance Ca2+-activated K+-predominant mechanism of dilation. Loss of KV1.5 vasomotor function may play an important role in microvascular dysfunction in CAD or other vascular diseases.

Shaker-related voltage-gated K+ channel expression and vasomotor function in human coronary resistance arteries.

OBJECTIVES: KV channels are important regulators of vascular tone, but the identity of specific KV channels involved and their regulation in disease remain less well understood. We determined the expression of KV 1 channel subunits and their role in cAMP-mediated dilation in coronary resistance arteries from subjects with and without CAD. METHODS: HCAs from patients with and without CAD were assessed for mRNA and protein expression of KV 1 channel subunits with molecular techniques and for vasodilator response with isolated arterial myography. RESULTS: Assays of mRNA transcripts, membrane protein expression, and vascular cell-specific localization revealed abundant expression of KV 1.5 in vascular smooth muscle cells of non-CAD HCAs. Isoproterenol and forskolin, two distinct cAMP-mediated vasodilators, induced potent dilation of non-CAD arterioles, which was inhibited by both the general KV blocker 4-AP and the selective KV 1.5 blocker DPO-1. The cAMP-mediated dilation was reduced in CAD and was accompanied by a loss of or reduced contribution of 4-AP-sensitive KV channels. CONCLUSIONS: KV 1.5, as a major 4-AP-sensitive KV 1 channel expressed in coronary VSMCs, mediates cAMP-mediated dilation in non-CAD arterioles. The cAMP-mediated dilation is reduced in CAD coronary arterioles, which is associated with impaired 4-AP-sensitive KV channel function.

TRPV4 ion channel is a novel regulator of dermal myofibroblast differentiation.

Scleroderma is a multisystem fibroproliferative disease with no effective medical treatment. Myofibroblasts are critical to the fibrogenic tissue repair process in the skin and many internal organs. Emerging data support a role for both matrix stiffness, and transforming growth factor ß1 (TGFß1), in myofibroblast differentiation. Transient receptor potential vanilloid 4 (TRPV4) is a mechanosensitive ion channel activated by both mechanical and biochemical stimuli. The objective of this study was to determine the role of TRPV4 in TGFß1- and matrix stiffness-induced differentiation of dermal fibroblasts. We found that TRPV4 channels are expressed and functional in both human (HDF) and mouse (MDF) dermal fibroblasts. TRPV4 activity (agonist-induced Ca2+ influx) was induced by both matrix stiffness and TGFß1 in dermal fibroblasts. TGFß1 induced expression of TRPV4 proteins in a dose-dependent manner. Genetic ablation or pharmacological antagonism of TRPV4 channel abrogated Ca2+ influx and both TGFß1-induced and matrix stiffness-induced myofibroblast differentiation as assessed by 1) α-smooth muscle actin expression/incorporation into stress fibers, 2) generation of polymerized actin, and 3) expression of collagen-1. We found that TRPV4 inhibition abrogated TGFß1-induced activation of AKT but not of Smad2/3, suggesting that the mechanism by which profibrotic TGFß1 signaling in dermal fibroblasts is modified by TRPV4 may be through non-Smad pathways. Altogether, these data identify a novel reciprocal functional link between TRPV4 activation and TGFß1 signals regulating dermal myofibroblast differentiation. These findings suggest that therapeutic inhibition of TRPV4 activity may provide a targeted approach to the treatment of scleroderma.

Rap1 promotes endothelial mechanosensing complex formation, NO release and normal endothelial function.

Decreased nitric oxide (NO) bioavailability underlies a number of cardiovascular pathologies, including hypertension. The shear stress exerted by flowing blood is the main determinant of NO release. Rap1 promotes integrin- and cadherin-mediated signaling. Here, we show that Rap1 is a critical regulator of NO production and endothelial function. Rap1 deficiency in murine endothelium attenuates NO production and diminishes NO-dependent vasodilation, leading to endothelial dysfunction and hypertension, without deleterious effects on vessel integrity. Mechanistically, Rap1 is activated by shear stress, promotes the formation of the endothelial mechanosensing complex-comprised of PECAM-1, VE-cadherin and VEGFR2- and downstream signaling to NO production. Our study establishes a novel paradigm for Rap1 as a regulator of mechanotransduction.

Expression of CYP 4A ω-hydroxylase and formation of 20-hydroxyeicosatetreanoic acid (20-HETE) in cultured rat brain astrocytes.

Astrocytes secrete vasodilator and vasoconstrictor factors via end feet processes, altering blood flow to meet neuronal metabolic demand. Compared to what is known about the ability of astrocytes to release factors that dilate local cerebral vasculature, very little is known regarding the source and identity of astrocyte derived constricting factors. The present study investigated if astrocytes express CYP 4A ω-hydroxylase and metabolize arachidonic acid (AA) to 20-hydroxyeicotetraenoic acid (20-HETE) that regulates KCa channel activity in astrocytes and cerebral arterial myocyte contractility. Here we report that cultured astrocytes express CYP 4A2/3 ω-hydroxylase mRNA and CYP 4A protein and produce 20-HETE and the CYP epoxygenase metabolites epoxyeicosatrienoic acids (EETs) when incubated with AA. The production of 20-HETE and EETs was enhanced following stimulation of metabotropic glutamate receptors (mGluR) on the astrocytes. Exogenous application of 20-HETE attenuated, whereas inhibition of 20-HETE production with HET-0016 increased the open state probabilities (NPo) of 71pS and 161pS KCa single-channel currents recorded from astrocytes. Exposure of isolated cerebral arterial myocytes to conditioned media from cultured astrocytes caused shortening of the length of freshly isolated cerebral arterial myocytes that was not evident following inhibition of astrocyte 20-HETE synthesis and action. These findings suggest that astrocytes not only release vasodilator EETs in response to mGluR stimulation but also synthetize and release the cerebral arterial myocyte constrictor 20-HETE that also functions as an endogenous inhibitor of the activity of two types of KCa channel currents found in astrocytes.

Rap1b in smooth muscle and endothelium is required for maintenance of vascular tone and normal blood pressure.

OBJECTIVE: Small GTPase Ras-related protein 1 (Rap1b) controls several basic cellular phenomena, and its deletion in mice leads to several cardiovascular defects, including impaired adhesion of blood cells and defective angiogenesis. We found that Rap1b(-/-) mice develop cardiac hypertrophy and hypertension. Therefore, we examined the function of Rap1b in regulation of blood pressure. APPROACH AND RESULTS: Rap1b(-/-) mice developed cardiac hypertrophy and elevated blood pressure, but maintained a normal heart rate. Correcting elevated blood pressure with losartan, an angiotensin II type 1 receptor antagonist, alleviated cardiac hypertrophy in Rap1b(-/-) mice, suggesting a possibility that cardiac hypertrophy develops secondary to hypertension. The indices of renal function and plasma renin activity were normal in Rap1b(-/-) mice. Ex vivo, we examined whether the effect of Rap1b deletion on smooth muscle-mediated vessel contraction and endothelium-dependent vessel dilation, 2 major mechanisms controlling basal vascular tone, was the basis for the hypertension. We found increased contractility on stimulation with a thromboxane analog or angiotensin II or phenylephrine along with increased inhibitory phosphorylation of myosin phosphatase under basal conditions consistent with elevated basal tone and the observed hypertension. Cyclic adenosine monophosphate-dependent relaxation in response to Rap1 activator, Epac, was decreased in vessels from Rap1b(-/-) mice. Defective endothelial release of dilatory nitric oxide in response to elevated blood flow leads to hypertension. We found that nitric oxide-dependent vasodilation was significantly inhibited in Rap1b-deficient vessels. CONCLUSIONS: This is the first report to indicate that Rap1b in both smooth muscle and endothelium plays a key role in maintaining blood pressure by controlling normal vascular tone.

H2O2-induced dilation in human coronary arterioles: role of protein kinase G dimerization and large-conductance Ca2+-activated K+ channel activation.

RATIONALE: Hydrogen peroxide (H(2)O(2)) serves as a key endothelium-derived hyperpolarizing factor mediating flow-induced dilation in human coronary arterioles (HCAs). The precise mechanisms by which H(2)O(2) elicits smooth muscle hyperpolarization are not well understood. An important mode of action of H(2)O(2) involves the oxidation of cysteine residues in its target proteins, including protein kinase G (PKG)-Iα, thereby modulating their activities. OBJECTIVE: Here we hypothesize that H(2)O(2) dilates HCAs through direct oxidation and activation of PKG-Iα leading to the opening of the large-conductance Ca(2+)-activated K(+) (BK(Ca)) channel and subsequent smooth muscle hyperpolarization. METHODS AND RESULTS: Flow and H(2)O(2) induced pressure gradient/concentration-dependent vasodilation in isolated endothelium-intact and -denuded HCAs, respectively. The dilation was largely abolished by iberiotoxin, a BK(Ca) channel blocker. The PKG inhibitor Rp-8-Br-PET-cGMP also markedly inhibited flow- and H(2)O(2)-induced dilation, whereas the soluble guanylate cyclase inhibitor ODQ had no effect. Treatment of coronary smooth muscle cells (SMCs) with H(2)O(2) elicited dose-dependent, reversible dimerization of PKG-Iα, and induced its translocation to the plasma membrane. Patch-clamp analysis identified a paxilline-sensitive single-channel K(+) current with a unitary conductance of 246-pS in freshly isolated coronary SMCs. Addition of H(2)O(2) into the bath solution significantly increased the probability of BK(Ca) single-channel openings recorded from cell-attached patches, an effect that was blocked by the PKG-Iα inhibitor DT-2. H(2)O(2) exhibited an attenuated stimulatory effect on BK(Ca) channel open probability in inside-out membrane patches. CONCLUSIONS: H(2)O(2) dilates HCAs through a novel mechanism involving protein dimerization and activation of PKG-Iα and subsequent opening of smooth muscle BK(Ca) channels.

H2O2 is the transferrable factor mediating flow-induced dilation in human coronary arterioles.

RATIONALE: Endothelial derived hydrogen peroxide (H(2)O(2)) is a necessary component of the pathway regulating flow-mediated dilation (FMD) in human coronary arterioles (HCAs). However, H(2)O(2) has never been shown to be the endothelium-dependent transferrable hyperpolarization factor (EDHF) in response to shear stress. OBJECTIVE: We examined the hypothesis that H(2)O(2) serves as the EDHF in HCAs to shear stress. METHODS AND RESULTS: Two HCAs were cannulated in series (a donor intact vessel upstream and endothelium-denuded detector vessel downstream). Diameter changes to flow were examined in the absence and presence of polyethylene glycol catalase (PEG-CAT). The open state probability of large conductance Ca(2+)-activated K(+) (BK(Ca)) channels in smooth muscle cells downstream from the perfusate from an endothelium-intact arteriole was examined by patch clamping. In some experiments, a cyanogen bromide-activated resin column bound with CAT was used to remove H(2)O(2) from the donor vessel. When flow proceeds from donor to detector, both vessels dilate (donor:68±7%; detector: 45±11%). With flow in the opposite direction, only the donor vessel dilates. PEG-CAT contacting only the detector vessel blocked FMD in that vessel (6±4%) but not in donor vessel (61±13%). Paxilline inhibited dilation of endothelium-denuded HCAs to H(2)O(2). Effluent from donor vessels elicited K(+) channel opening in an iberiotoxin- or PEG-CAT-sensitive fashion in cell-attached patches but had little effect on channel opening on inside-out patches. Vasodilation of detector vessels was diminished when exposed to effluent from CAT-column. CONCLUSIONS: Flow induced endothelial production of H(2)O(2), which acts as the transferrable EDHF activating BK(Ca) channels on the smooth muscle cells.

Structure of human TRPV4 in complex with GTPase RhoA.

Transient receptor potential (TRP) channel TRPV4 is a polymodal cellular sensor that responds to moderate heat, cell swelling, shear stress, and small-molecule ligands. It is involved in thermogenesis, regulation of vascular tone, bone homeostasis, renal and pulmonary functions. TRPV4 is implicated in neuromuscular and skeletal disorders, pulmonary edema, and cancers, and represents an important drug target. The cytoskeletal remodeling GTPase RhoA has been shown to suppress TRPV4 activity. Here, we present a structure of the human TRPV4-RhoA complex that shows RhoA interaction with the membrane-facing surface of the TRPV4 ankyrin repeat domains. The contact interface reveals residues that are mutated in neuropathies, providing an insight into the disease pathogenesis. We also identify the binding sites of the TRPV4 agonist 4α-PDD and the inhibitor HC-067047 at the base of the S1-S4 bundle, and show that agonist binding leads to pore opening, while channel inhibition involves a π-to-α transition in the pore-forming helix S6. Our structures elucidate the interaction interface between hTRPV4 and RhoA, as well as residues at this interface that are involved in TRPV4 disease-causing mutations. They shed light on TRPV4 activation and inhibition and provide a template for the design of future therapeutics for treatment of TRPV4-related diseases.

Activation of endothelial TRPV4 channels mediates flow-induced dilation in human coronary arterioles: role of Ca2+ entry and mitochondrial ROS signaling.

In human coronary arterioles (HCAs) from patients with coronary artery disease, flow-induced dilation is mediated by a unique mechanism involving the release of H(2)O(2) from the mitochondria of endothelial cells (ECs). How flow activates ECs to elicit the mitochondrial release of H(2)O(2) remains unclear. Here, we examined the role of the transient receptor potential vanilloid type 4 (TRPV4) channel, a mechanosensitive Ca(2+)-permeable cation channel, in mediating ROS formation and flow-induced dilation in HCAs. Using RT-PCR, Western blot analysis, and immunohistochemical analysis, we detected the mRNA and protein expression of TRPV4 channels in ECs of HCAs and cultured human coronary artery ECs (HCAECs). In HCAECs, 4α-phorbol-12,13-didecanoate (4α-PDD), a selective TRPV4 agonist, markedly increased (via Ca(2+) influx) intracellular Ca(2+) concentration. In isolated HCAs, activation of TRPV4 channels by 4α-PDD resulted in a potent concentration-dependent dilation, and the dilation was inhibited by removal of the endothelium and by catalase, a H(2)O(2)-metabolizing enzyme. Fluorescence ROS assays showed that 4α-PDD increased the production of mitochondrial superoxide in HCAECs. 4α-PDD also enhanced the production of H(2)O(2) and superoxide in HCAs. Finally, we found that flow-induced dilation of HCAs was markedly inhibited by different TRPV4 antagonists and TRPV4-specific small interfering RNA. In conclusion, the endothelial TRPV4 channel is critically involved in flow-mediated dilation of HCAs. TRPV4-mediated Ca(2+) entry may be an important signaling event leading to the flow-induced release of mitochondrial ROS in HCAs. Elucidation of this novel TRPV4-ROS pathway may improve our understanding of the pathogenesis of coronary artery disease and/or other cardiovascular disorders.

Inhibiting NADPH Oxidases to Target Vascular and Other Pathologies: An Update on Recent Experimental and Clinical Studies.

Reactive oxygen species (ROS) can be beneficial or harmful in health and disease. While low levels of ROS serve as signaling molecules to regulate vascular tone and the growth and proliferation of endothelial cells, elevated levels of ROS contribute to numerous pathologies, such as endothelial dysfunctions, colon cancer, and fibrosis. ROS and their cellular sources have been extensively studied as potential targets for clinical intervention. Whereas various ROS sources are important for different pathologies, four NADPH oxidases (NOX1, NOX2, NOX4, and NOX5) play a prominent role in homeostasis and disease. NOX1-generated ROS have been implicated in hypertension, suggesting that inhibition of NOX1 may be a promising therapeutic approach. NOX2 and NOX4 oxidases are of specific interest due to their role in producing extra- and intracellular hydrogen peroxide (H2O2). NOX4-released hydrogen peroxide activates NOX2, which in turn stimulates the release of mitochondrial ROS resulting in ROS-induced ROS release (RIRR) signaling. Increased ROS production from NOX5 contributes to atherosclerosis. This review aims to summarize recent findings on NOX enzymes and clinical trials inhibiting NADPH oxidases to target pathologies including diabetes, idiopathic pulmonary fibrosis (IPF), and primary biliary cholangitis (PBC).

PKCα mediates acetylcholine-induced activation of TRPV4-dependent calcium influx in endothelial cells.

Transient receptor potential vanilloid channel 4 (TRPV4) is a polymodally activated nonselective cationic channel implicated in the regulation of vasodilation and hypertension. We and others have recently shown that cyclic stretch and shear stress activate TRPV4-mediated calcium influx in endothelial cells (EC). In addition to the mechanical forces, acetylcholine (ACh) was shown to activate TRPV4-mediated calcium influx in endothelial cells, which is important for nitric oxide-dependent vasodilation. However, the molecular mechanism through which ACh activates TRPV4 is not known. Here, we show that ACh-induced calcium influx and endothelial nitric oxide synthase (eNOS) phosphorylation but not calcium release from intracellular stores is inhibited by a specific TRPV4 antagonist, AB-159908. Importantly, activation of store-operated calcium influx was not altered in the TRPV4 null EC, suggesting that TRPV4-dependent calcium influx is mediated through a receptor-operated pathway. Furthermore, we found that ACh treatment activated protein kinase C (PKC) α, and inhibition of PKCα activity by the specific inhibitor Go-6976, or expression of a kinase-dead mutant of PKCα but not PKCε or downregulation of PKCα expression by chronic 12-O-tetradecanoylphorbol-13-acetate treatment, completely abolished ACh-induced calcium influx. Finally, we found that ACh-induced vasodilation was inhibited by the PKCα inhibitor Go-6976 in small mesenteric arteries from wild-type mice, but not in TRPV4 null mice. Taken together, these findings demonstrate, for the first time, that a specific isoform of PKC, PKCα, mediates agonist-induced receptor-mediated TRPV4 activation in endothelial cells.

Transient receptor potential channel activation and endothelium-dependent dilation in the systemic circulation.

The endothelium plays a crucial role in the regulation of vascular tone by releasing a number of vasodilator mediators, including nitric oxide, prostacyclin, and endothelium-derived hyperpolarizing factor(s). The production of these mediators is typically initiated by an increase in intracellular Ca(2+) concentration ([Ca(2+)]i) in endothelial cells. An essential component of this Ca(2+) signal is the entry of Ca(2+) from the extracellular space through plasma membrane Ca(2+)-permeable channels. Although the molecular identification of the potential Ca(2+) entry channel(s) responsible for the release of endothelial relaxing factors is still evolving, accumulating evidence indicates that the transient receptor potential (TRP) channels, a superfamily of Ca(2+)-permeable cation channels, serve as an important mechanism of Ca(2+) entry in endothelial cells and other nonexcitable cells. The activation of these channels has been implicated in diverse endothelial functions ranging from control of vascular tone and regulation of vascular permeability to angiogenesis and vascular remodeling. This review summarizes recent evidence concerning TRP channels and endothelium-dependent dilation in several systemic vascular beds. In particular, we highlight the emerging roles of several TRP channels from the canonical and vanilloid subfamilies, including TRPV4, TRPC4, and TRPC6, in vasodilatory responses to shear stress and receptor agonists and discuss potential signaling mechanisms linking the TRP channel activation and the initiation of endothelium-derived hyperpolarizing factor-mediated responses in endothelial cells.

Corrigendum: Distinct Signaling Functions of Rap1 Isoforms in NO Release From Endothelium.

[This corrects the article DOI: 10.3389/fcell.2021.687598.].