David X. Zhang

TRPV4-mediated endothelial Ca2+ influx and vasodilation in response to shear stress

The transient receptor potential vallinoid type 4 (TRPV4) channel has been implicated in the endothelial shear response and flow-mediated dilation, although the precise functions of this channel remain poorly understood. In the present study, we investigated the role of TRPV4 in shear stress-induced endothelial Ca(2+) entry and the potential link between this signaling response and relaxation of small resistance arteries. Using immunohistochemical analysis and RT-PCR, we detected strong expression of TRPV4 protein and mRNA in the endothelium in situ and endothelial cells freshly isolated from mouse small mesenteric arteries. The selective TRPV4 agonist GSK1016790A increased endothelial Ca(2+) and induced potent relaxation of small mesenteric arteries from wild-type (WT) but not TRPV4(-/-) mice. Luminal flow elicited endothelium-dependent relaxations that involved both nitric oxide and EDHFs. Both nitric oxide and EDHF components of flow-mediated relaxation were markedly reduced in TRPV4(-/-) mice compared with WT controls. Using a fura-2/Mn(2+) quenching assay, shear was observed to produce rapid Ca(2+) influx in endothelial cells, which was markedly inhibited by the TRPV4 channel blocker ruthenium red and TRPV4-specific short interfering RNA. Flow elicited a similar TRPV4-mediated Ca(2+) entry in HEK-293 cells transfected with TRPV4 channels but not in nontransfected cells. Collectively, these data indicate that TRPV4 may be a potential candidate of mechanosensitive channels in endothelial cells through which the shear stimulus is transduced into Ca(2+) signaling, leading to the release of endothelial relaxing factors and flow-mediated dilation of small resistance arteries.

H2O2 Is the Transferrable Factor Mediating Flow-Induced Dilation in Human Coronary Arterioles

Rationale: Endothelial derived hydrogen peroxide (H2O2) is a necessary component of the pathway regulating flow-mediated dilation (FMD) in human coronary arterioles (HCAs). However, H2O2 has never been shown to be the endothelium-dependent transferrable hyperpolarization factor (EDHF) in response to shear stress. Objective: We examined the hypothesis that H2O2 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 Ca2+-activated K+ (BKCa) 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 H2O2 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 H2O2. 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 H2O2, which acts as the transferrable EDHF activating BKCa channels on the smooth muscle cells.

Transient Receptor Potential Vanilloid Type 4–Deficient Mice Exhibit Impaired Endothelium-Dependent Relaxation Induced by Acetylcholine In Vitro and In Vivo

Agonist-induced Ca2+ entry is important for the synthesis and release of vasoactive factors in endothelial cells. The transient receptor potential vanilloid type 4 (TRPV4) channel, a Ca2+-permeant cation channel, is expressed in endothelial cells and involved in the regulation of vascular tone. Here we investigated the role of TRPV4 channels in acetylcholine-induced vasodilation in vitro and in vivo using the TRPV4 knockout mouse model. The expression of TRPV4 mRNA and protein was detected in both conduit and resistance arteries from wild-type mice. In small mesenteric arteries from wild-type mice, the TRPV4 activator 4α-phorbol-12,13-didecanoate increased endothelial [Ca2+]i in situ, which was reversed by the TRPV4 blocker ruthenium red. In wild-type animals, acetylcholine dilated small mesenteric arteries that involved both NO and endothelium-derived hyperpolarizing factors. In TRPV4-deficient mice, the NO component of the relaxation was attenuated and the endothelium-derived hyperpolarizing factor component was largely eliminated. Compared with their wild-type littermates, TRPV4-deficient mice demonstrated a blunted endothelial Ca2+ response to acetylcholine in mesenteric arteries and reduced NO release in carotid arteries. Acetylcholine (5 mg/kg, IV) decreased blood pressure by 37.0±6.2 mm Hg in wild-type animals but only 16.6±2.7 mm Hg in knockout mice. We conclude that acetylcholine-induced endothelium-dependent vasodilation is reduced both in vitro and in vivo in TRPV4 knockout mice. These findings may provide novel insight into mechanisms of Ca2+ entry evoked by chemical agonists in endothelial cells.

H2O2-Induced Dilation in Human Coronary Arterioles: Role of Protein Kinase G Dimerization and Large-Conductance Ca2+-Activated K+ Channel Activation

Rationale: Hydrogen peroxide (H2O2) serves as a key endothelium-derived hyperpolarizing factor mediating flow-induced dilation in human coronary arterioles (HCAs). The precise mechanisms by which H2O2 elicits smooth muscle hyperpolarization are not well understood. An important mode of action of H2O2 involves the oxidation of cysteine residues in its target proteins, including protein kinase G (PKG)-I&agr;, thereby modulating their activities. Objective: Here we hypothesize that H2O2 dilates HCAs through direct oxidation and activation of PKG-I&agr; leading to the opening of the large-conductance Ca2+-activated K+ (BKCa) channel and subsequent smooth muscle hyperpolarization. Methods and Results: Flow and H2O2 induced pressure gradient/concentration-dependent vasodilation in isolated endothelium-intact and -denuded HCAs, respectively. The dilation was largely abolished by iberiotoxin, a BKCa channel blocker. The PKG inhibitor Rp-8-Br-PET-cGMP also markedly inhibited flow- and H2O2-induced dilation, whereas the soluble guanylate cyclase inhibitor ODQ had no effect. Treatment of coronary smooth muscle cells (SMCs) with H2O2 elicited dose-dependent, reversible dimerization of PKG-I&agr;, 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 H2O2 into the bath solution significantly increased the probability of BKCa single-channel openings recorded from cell-attached patches, an effect that was blocked by the PKG-I&agr; inhibitor DT-2. H2O2 exhibited an attenuated stimulatory effect on BKCa channel open probability in inside-out membrane patches. Conclusions: H2O2 dilates HCAs through a novel mechanism involving protein dimerization and activation of PKG-I&agr; and subsequent opening of smooth muscle BKCa channels.

TRPV4 mediates myofibroblast differentiation and pulmonary fibrosis in mice

Idiopathic pulmonary fibrosis (IPF) is a fatal fibrotic lung disorder with no effective medical treatments available. The generation of myofibroblasts, which are critical for fibrogenesis, requires both a mechanical signal and activated TGF-β; however, it is not clear how fibroblasts sense and transmit the mechanical signal(s) that promote differentiation into myofibroblasts. As transient receptor potential vanilloid 4 (TRPV4) channels are activated in response to changes in plasma membrane stretch/matrix stiffness, we investigated whether TRPV4 contributes to generation of myofibroblasts and/or experimental lung fibrosis. We determined that TRPV4 activity is upregulated in lung fibroblasts derived from patients with IPF. Moreover, TRPV4-deficient mice were protected from fibrosis. Furthermore, genetic ablation or pharmacological inhibition of TRPV4 function abrogated myofibroblast differentiation, which was restored by TRPV4 reintroduction. TRPV4 channel activity was elevated when cells were plated on matrices of increasing stiffness or on fibrotic lung tissue, and matrix stiffness-dependent myofibroblast differentiation was reduced in response to TRVP4 inhibition. TRPV4 activity modulated TGF-β1-dependent actions in a SMAD-independent manner, enhanced actomyosin remodeling, and increased nuclear translocation of the α-SMA transcription coactivator (MRTF-A). Together, these data indicate that TRPV4 activity mediates pulmonary fibrogenesis and suggest that manipulation of TRPV4 channel activity has potential as a therapeutic approach for fibrotic 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.

Production and metabolism of ceramide in normal and ischemic-reperfused myocardium of rats.

Abstract Ceramide has been shown to be a key signaling molecule involved in the apoptotic effect of tumor necrosis factor α (TNF-α) and other cytokines. Given the importance of cytokines such as TNF-α in myocardial ischemia-reperfusion injury, we hypothesize that ceramide is increased during ischemia or reperfusion, and that the activity of enzymes responsible for its production or breakdown should be increased and/or decreased, respectively. Therefore, in the present study, we characterized the enzymatic activities responsible for ceramide production and metabolism in the myocardium of rats, and determined the contribution of these enzymes to altered ceramide levels during myocardial ischemia and reperfusion. The basal ceramide concentration in the myocardium of rats was 34.0 pmol/mg tissue. As determined by the conversion of 14C-sphingomyelin into ceramide and 14C-choline phosphate, both neutral (N-) and acidic (A-) SMase were detected in the myocardium, with a conversion rate of 0.09 ± 0.008 and 0.32 ± 0.05 nmol/min per mg protein, respectively. The activity of A-SMase (78 % of total cellular activity) was significantly higher in microsomes than in cytosol, while the activity of N-SMase was similar in both fractions. Ceramidase, a ceramide-metabolizing enzyme, was also detected in the myocardium of rats. It metabolized ceramide into sphingosine at a rate of 9.94 ± 0.42 pmol/min per mg protein. In anesthetized rats, 30 min of ischemia had no apparent effect on ceramide concentrations in the myocardium, while 30 min of ischemia followed by 3 h of reperfusion resulted in a significant increase in ceramide by 48 %. The activities of both N- and A-SMase were reduced by 44 % and 32 %, respectively, in the myocardium subjected to ischemia followed by reperfusion, but unaltered in the ischemic myocardium. It was also found that myocardial ischemia followed by reperfusion produced a marked inhibition of ceramidase (by 29 %). These results demonstrate that the myocardium of rats expresses N- and A-SMase and ceramidase, which contribute to the production and metabolism of ceramide, respectively. Tissue ceramide concentrations increased in reperfused myocardium. These increases in ceramide were not associated with enhanced SMase activity, but rather with reduced ceramidase activity.

Cyclic ADP-Ribose Contributes to Contraction and Ca2+ Release by M1 Muscarinic Receptor Activation in Coronary Arterial Smooth Muscle

The present study determined the role of cyclic ADP-ribose (cADPR) in mediating vasoconstriction and Ca2+ release in response to the activation of muscarinic receptors. Endothelium-denuded small bovine coronary arteries were microperfused under transmural pressure of 60 mm Hg. Both acetylcholine (ACh; 1 nmol/L to 1 µmol/L) and oxotremorine (OXO; 2.5–80 µmol/L) produced a concentration-dependent contraction. The vasoconstrictor responses to both ACh and OXO were significantly attenuated by nicotinamide (Nicot; an ADP-ribosyl cyclase inhibitor), 8-bromo-cADPR (8-Br-cADPR; a cADPR antagonist) or ryanodine (Ry; an Ry receptor antagonist). Intracellular Ca2+ ([Ca2+]i) was determined by fluorescence spectrometry using fura-2 as a fluorescence indicator. OXO produced a rapid increase in [Ca2+]i in freshly isolated single coronary arterial smooth muscle cells (CASMCs) bathed with Ca2+-free Hanks’ solution. This OXO-induced rise in [Ca2+]i was significantly reduced by pirenzepine (PIR; an M1 receptor-specific blocker), Nicot, 8-Br-cADPR or Ry. The effects of OXO on the activity of ADP-ribosyl cyclase (cADPR synthase) were examined in cultured CASMCs by measuring the rate of cyclic GDP- ribose (cGDPR) formation from β-nicotinamide guanine dinucleotide. It was found that OXO produced a concentration-dependent increase in the production of cGDPR. The stimulatory effect of OXO on ADP-ribosyl cyclase was inhibited by both PIR and Nicot. These results suggest that the cADPR signaling pathway participates in the contraction of small coronary arterial smooth muscle and Ca2+ release induced by activation of M1 muscarinic receptors.

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 Ca2+ concentration ([Ca2+]i) in endothelial cells. An essential component of this Ca2+ signal is the entry of Ca2+ from the extracellular space through plasma membrane Ca2+-permeable channels. Although the molecular identification of the potential Ca2+ 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 Ca2+-permeable cation channels, serve as an important mechanism of Ca2+ 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.