Prostaglandin E2 attenuates lung fibroblast differentiation via inactivation of yes-associated protein signaling.

Prostaglandin E2 (PGE2 ) has been implicated in counteracting fibroblast differentiation by TGFß1 during pulmonary fibrosis. However, the precise mechanism is not well understood. We show here that PGE2 via EP2 R and EP4 R inhibits the expression of mechanosensory molecules Lysyl Oxidase Like 2 (LOXL2), myocardin-related transcription factor A (MRTF-A), ECM proteins, plasminogen activation inhibitor 1 (PAI-1), fibronectin (FN), α-smooth muscle actin (α-SMA), and redox sensor (nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (NOX4)) required for TGFß1-mediated fibroblast differentiation. We further demonstrate that PGE2 inhibits fibrotic signaling via Yes-associated protein (YAP) but does so independently from its actions on SMAD phosphorylation and conserved cylindromatosis (CYLD; deubiquitinase) expression. Mechanistically, PGE2 phosphorylates/inactivates YAP downstream of EP2 R/Gαs and restrains its translocation to the nucleus, thus inhibiting its interaction with TEA domain family members (TEADs) and transcription of fibrotic genes. Importantly, pharmacological or siRNA-mediated inhibition of YAP significantly downregulates TGFß1-mediated fibrotic gene expression and myofibroblast formation. Notably, YAP expression is upregulated in the lungs of D. farinae-treated wild type (WT) mice relative to saline-treated WT mice. Our results unravel a unique role for PGE2 -YAP interactions in fibroblast differentiation, and that PGE2 /YAP inhibition can be used as a novel therapeutic target in the treatment of pathological conditions associated with myofibroblasts like asthma.

Epigenetic histone modification by butyrate downregulates KIT and attenuates mast cell function.

Short-chain fatty acid butyrate is produced from the bacterial fermentation of indigestible fiber in the intestinal lumen, and it has been shown to attenuate lung inflammation in murine asthma models. Mast cells (MCs) are initiators of inflammatory response to allergens, and they play an important role in asthma. MC survival and proliferation is regulated by its growth factor stem cell factor (SCF), which acts through the receptor, KIT. It has previously been shown that butyrate attenuates the activation of MCs by allergen stimulation. However, how butyrate mechanistically influences SCF signalling to impact MC function remains unknown. Here, we report that butyrate treatment triggered the modification of MC histones via butyrylation and acetylation, and inhibition of histone deacetylase (HDAC) activity. Further, butyrate treatment caused downregulation of SCF receptor KIT and associated phosphorylation, leading to significant attenuation of SCF-mediated MC proliferation, and pro-inflammatory cytokine secretion. Mechanistically, butyrate inhibited MC function by suppressing KIT and downstream p38 and Erk phosphorylation, and it mediated these effects via modification of histones, acting as an HDAC inhibitor and not via its traditional GPR41 (FFAR3) or GPR43 (FFAR2) butyrate receptors. In agreement, the pharmacological inhibition of Class I HDAC (HDAC1/3) mirrored butyrate's effects, suggesting that butyrate impacts MC function by HDAC1/3 inhibition. Taken together, butyrate epigenetically modifies histones and downregulates the SCF/KIT/p38/Erk signalling axis, leading to the attenuation of MC function, validating its ability to suppress MC-mediated inflammation. Therefore, butyrate supplementations could offer a potential treatment strategy for allergy and asthma via epigenetic alterations in MCs.

Protein Kinase C α and ß compensate for each other to promote stem cell factor-mediated KIT phosphorylation, mast cell viability and proliferation.

Mast cells (MCs) develop from hematopoietic progenitors and differentiate into mature MCs that reside within connective or mucosal tissues. Though the number of MCs in tissues usually remains constant, inflammation and asthma disturb this homeostasis, leading to proliferation of MCs. Understanding the signaling events behind this proliferative response could lead to the development of novel strategies for better management of allergic diseases. MC survival, proliferation, differentiation, and migration are all maintained by a MC growth factor, stem cell factor (SCF) via its receptor, KIT. Here, we explored how protein kinase C (PKC) redundancy influences MC proliferation in bone marrow-derived MC (BMMC). We found that SCF activates PKCα and PKCß isoforms, which in turn modulates KIT phosphorylation and internalization. Further, PKCα and PKCß activate p38 mitogen activated protein kinase (MAPK), and this axis subsequently regulates SCF-induced MC cell proliferation. To ascertain the individual roles of PKCα and PKCß, we knocked down either PKCα or PKCß or both via short hairpin RNA (shRNA) and analyzed KIT phosphorylation, p38 MAPK phosphorylation, and MC viability and proliferation. To our surprise, downregulation of neither PKCα nor PKCß affected MC viability and proliferation. In contrast, blocking both PKCα and PKCß significantly attenuated SCF-induced cell viability and proliferation, suggesting that PKCα and PKCß compensate for each other downstream of SCF signaling to enhance MC viability and proliferation. Our results not only suggest that PKC classical isoforms are novel therapeutic targets for SCF/MC-mediated inflammatory and allergic diseases, but they also emphasize the importance of inhibiting both PKCα and ß isoforms simultaneously to prevent MC proliferation.

Cysteinyl leukotriene 2 receptor promotes endothelial permeability, tumor angiogenesis, and metastasis.

Cysteinyl leukotrienes (cys-LTs) are proinflammatory mediators that enhance vascular permeability through distinct receptors (CysLTRs). We found that CysLT2R regulates angiogenesis in isolated mouse endothelial cells (ECs) and in Matrigel implants in WT mice and enhances EC contraction and permeability via the Rho-dependent myosin light chain 2 and vascular endothelial (VE)-cadherin axis. Since solid tumors utilize aberrant angiogenesis for their growth and metastasis and their vessels exhibit vascular hyperpermeability, we hypothesized that CysLT2R, via its actions on the endothelium, might regulate tumor growth. Both tumor growth and metastases of adoptively transferred syngeneic Lewis lung carcinoma (LLC) cells are significantly reduced in CysLT2R-null mice (Cysltr2-/-) compared with WT and CysLT1R-null mice (Cysltr1-/-). In WT recipients of LLC cells, CysLT2R expression is significantly increased in the tumor vasculature, compared with CysLT1R. Further, the tumor vasculature in Cysltr2-/- recipients exhibited significantly improved integrity, as revealed by increased pericyte coverage and decreased leakage of i.v.-administered Texas Red-conjugated dextran. Administration of a selective CysLT2R antagonist significantly reduced LLC tumor volume, vessel density, dextran leakage, and metastases in WT mice, highlighting CysLT2R as a VEGF-independent regulator of the vasculature promoting risk of metastasis. Thus, both genetic and pharmacological findings establish CysLT2R as a gateway for angiogenesis and EC dysregulation in vitro and ex vivo and in an in vivo model with a mouse tumor. Our data suggest CysLT2R as a possible target for intervention.

Transient receptor potential vanilloid 4 channel deletion regulates pathological but not developmental retinal angiogenesis.

Transient receptor potential vanilloid 4 (TRPV4) channels are mechanosensitive ion channels that regulate systemic endothelial cell (EC) functions such as vasodilation, permeability, and angiogenesis. TRPV4 is expressed in retinal ganglion cells, Müller glia, pigment epithelium, microvascular ECs, and modulates cell volume regulation, calcium homeostasis, and survival. TRPV4-mediated physiological or pathological retinal angiogenesis remains poorly understood. Here, we demonstrate that TRPV4 is expressed, functional, and mechanosensitive in retinal ECs. The genetic deletion of TRPV4 did not affect postnatal developmental angiogenesis but increased pathological neovascularization in response to oxygen-induced retinopathy (OIR). Retinal vessels from TRPV4 knockout mice subjected to OIR exhibited neovascular tufts that projected into the vitreous humor and displayed reduced pericyte coverage compared with wild-type mice. These results suggest that TRPV4 is a regulator of retinal angiogenesis, its deletion augments pathological retinal angiogenesis, and that TRPV4 could be a novel target for the development of therapies against neovascular ocular diseases.

Endothelial TRPV4 channels prevent tumor growth and metastasis via modulation of tumor angiogenesis and vascular integrity.

Transient receptor potential vanilloid 4 (TRPV4) is a ubiquitously expressed polymodally activated ion channel. TRPV4 has been implicated in tumor progression; however, the cell-specific role of TRPV4 in tumor growth, angiogenesis, and metastasis is unknown. Here, we generated endothelial-specific TRPV4 knockout (TRPV4ECKO) mice by crossing TRPV4lox/lox mice with Tie2-Cre mice. Tumor growth and metastasis were significantly increased in a syngeneic Lewis lung carcinoma tumor model of TRPV4ECKO mice compared to TRPV4lox/lox mice. Multiphoton microscopy, dextran leakage, and immunohistochemical analysis revealed increased tumor angiogenesis and metastasis that were correlated with aberrant leaky vessels (increased width and reduced pericyte and VE-cadherin coverage). Mechanistically, increases in VEGFR2, p-ERK, and MMP-9 expression and DQ gelatinase activity were observed in the TRPV4ECKO mouse tumors. Our results demonstrated that endothelial TRPV4 is a critical modulator of vascular integrity and tumor angiogenesis and that deletion of TRPV4 promotes tumor angiogenesis, growth, and metastasis.

TRPV4 deletion protects heart from myocardial infarction-induced adverse remodeling via modulation of cardiac fibroblast differentiation.

Cardiac fibrosis caused by adverse cardiac remodeling following myocardial infarction can eventually lead to heart failure. Although the role of soluble factors such as TGF-ß is well studied in cardiac fibrosis following myocardial injury, the physiological role of mechanotransduction is not fully understood. Here, we investigated the molecular mechanism and functional role of TRPV4 mechanotransduction in cardiac fibrosis. TRPV4KO mice, 8 weeks following myocardial infarction (MI), exhibited preserved cardiac function compared to WT mice. Histological analysis demonstrated reduced cardiac fibrosis in TRPV4KO mice. We found that WT CF exhibited hypotonicity-induced calcium influx and extracellular matrix (ECM)-stiffness-dependent differentiation in response to TGF-ß1. In contrast, TRPV4KO CF did not display hypotonicity-induced calcium influx and failed to differentiate on high-stiffness ECM gels even in the presence of saturating amounts of TGF-ß1. Mechanistically, TRPV4 mediated cardiac fibrotic gene promoter activity and fibroblast differentiation through the activation of the Rho/Rho kinase pathway and the mechanosensitive transcription factor MRTF-A. Our findings suggest that genetic deletion of TRPV4 channels protects heart from adverse cardiac remodeling following MI by modulating Rho/MRTF-A pathway-mediated cardiac fibroblast differentiation and cardiac fibrosis.

The role of TRPV4 channels in ocular function and pathologies.

Transient potential receptor vanilloid 4 (TRPV4) is an ion channel responsible for sensing osmotic and mechanical signals, which in turn regulates calcium signaling across cell membranes. TRPV4 is widely expressed throughout the body, and plays an important role in normal physiological function, as well as different pathologies, however, its role in the eye is not well known. In the eye, TRPV4 is expressed in various tissues, such as the retina, corneal epithelium, ciliary body, and the lens. In this review, we provide an overview on TRPV4 structure, activation, mutations, and summarize the current knowledge of TRPV4 function and signaling mechanisms in various locations throughout the eye, as well as its role in ocular diseases, such as glaucoma and diabetic retinopathy. Based on the available data, we highlight the therapeutic potential of TRPV4 as well as the shortcomings of current research. Finally, we provide future perspectives on the implications of targeting TRPV4 to treat various ocular pathologies.

Novel noncanonical regulation of soluble VEGF/VEGFR2 signaling by mechanosensitive ion channel TRPV4.

VEGF signaling via VEGF receptor-2 (VEGFR2) is a major regulator of endothelial cell (EC) functions, including angiogenesis. Although most studies of angiogenesis focus on soluble VEGF signaling, mechanical signaling also plays a critical role. Here, we examined the consequence of disruption of mechanical signaling on soluble signaling pathways. Specifically, we observed that small interfering RNA (siRNA) knockdown of a mechanosensitive ion channel, transient receptor potential vanilloid 4 (TRPV4), significantly reduced perinuclear (Golgi) VEGFR2 in human ECs with a concomitant increase in phosphorylation at Y1175 and membrane translocation. TRPV4 knockout (KO) ECs exhibited increased plasma membrane localization of phospho-VEGFR2 compared with normal ECs. The knockdown also increased phospho-VEGFR2 in whole cell lysates and membrane fractions compared with control siRNA-treated cells. siRNA knockdown of TRPV4 enhanced nuclear localization of mechanosensitive transcription factors, yes-associated protein/transcriptional coactivator with PDZ-binding motif via rho kinase, which were shown to increase VEGFR2 trafficking to the plasma membrane. Furthermore, TRPV4 deletion/knockdown enhanced VEGF-mediated migration in vitro and increased expression of VEGFR2 in vivo in the vasculature of TRPV4 KO tumors compared with wild-type tumors. Our results thus show that TRPV4 channels regulate VEGFR2 trafficking and activation to identify novel cross-talk between mechanical (TRPV4) and soluble (VEGF) signaling that controls EC migration and angiogenesis.-Kanugula, A. K., Adapala, R. K., Midha, P., Cappelli, H. C., Meszaros, J. G., Paruchuri, S., Chilian, W. M., Thodeti, C. K., Novel noncanonical regulation of soluble VEGF/VEGFR2 signaling by mechanosensitive ion channel TRPV4.

Mechanosensitive transient receptor potential vanilloid 4 regulates Dermatophagoides farinae-induced airway remodeling via 2 distinct pathways modulating matrix synthesis and degradation.

Contributions of mechanical signals to airway remodeling during asthma are poorly understood. Transient receptor potential vanilloid 4 (TRPV4), a mechanosensitive ion channel, has been implicated in cardiac and pulmonary fibrosis; however, its role in asthma remains elusive. Employing a Dermatophagoides farinae-induced asthma model, we report here that TRPV4-knockout mice were protected from D. farinae-induced airway remodeling. Furthermore, lung fibroblasts that were isolated from TRPV4-knockout mice showed diminished differentiation potential compared with wild-type mice. Fibroblasts from asthmatic lung exhibited increased TRPV4 activity and enhanced differentiation potential compared with normal human lung fibroblasts. Of interest, TGF-ß1 treatment enhanced TRPV4 activation in a PI3K-dependent manner in normal human lung fibroblasts in vitro Mechanistically, TRPV4 modulated matrix remodeling in the lung via 2 distinct but dependent pathways: one enhances matrix deposition by fibrotic gene activation, whereas the other slows down matrix degradation by increased plasminogen activator inhibitor 1. Of importance, both pathways are regulated by Rho/myocardin-related transcription factor-A and contribute to fibroblast differentiation and matrix remodeling in the lung. Thus, our results support a unique role for TRPV4 in D. farinae-induced airway remodeling and warrant further studies in humans for it to be used as a novel therapeutic target in the treatment of asthma.-Gombedza, F., Kondeti, V., Al-Azzam, N., Koppes, S., Duah, E., Patil, P., Hexter, M., Phillips, D., Thodeti, C. K., Paruchuri, S. Mechanosensitive transient receptor potential vanilloid 4 regulates Dermatophagoides farinae-induced airway remodeling via 2 distinct pathways modulating matrix synthesis and degradation.

Leukotriene D4 and prostaglandin E2 signals synergize and potentiate vascular inflammation in a mast cell-dependent manner through cysteinyl leukotriene receptor 1 and E-prostanoid receptor 3.

BACKGROUND: Although arachidonic acid metabolites, cysteinyl leukotrienes (cys-LTs; leukotriene [LT] C4, LTD4, and LTE4), and prostaglandin (PG) E2 are generated at the site of inflammation, it is not known whether crosstalk exists between these 2 classes of inflammatory mediators. OBJECTIVE: We sought to determine the role of LTD4-PGE2 crosstalk in inducing vascular inflammation in vivo, identify effector cells, and ascertain specific receptors and pathways involved in vitro. METHODS: Vascular (ear) inflammation was assessed by injecting agonists into mouse ears, followed by measuring ear thickness and histology, calcium influx with Fura-2, phosphorylation and expression of signaling molecules by means of immunoblotting, PGD2 and macrophage inflammatory protein 1ß generation by using ELISA, and expression of transcripts by using RT-PCR. Candidate receptors and signaling molecules were identified by using antagonists and inhibitors and confirmed by using small interfering RNA. RESULTS: LTD4 plus PGE2 potentiated vascular permeability and edema, gearing the system toward proinflammation in wild-type mice but not in Kit(W-sh) mice. Furthermore, LTD4 plus PGE2, through cysteinyl leukotriene receptor 1 (CysLT1R) and E-prostanoid receptor (EP) 3, enhanced extracellular signal-regulated kinase (Erk) and c-fos phosphorylation, inflammatory gene expression, macrophage inflammatory protein 1ß secretion, COX-2 upregulation, and PGD2 generation in mast cells. Additionally, we uncovered that this synergism is mediated through Gi, protein kinase G, and Erk signaling. LTD4 plus PGE2-potentiated effects are partially sensitive to CysLT1R or EP3 antagonists but completely abolished by simultaneous treatment both in vitro and in vivo. CONCLUSIONS: Our results unravel a unique LTD4-PGE2 interaction affecting mast cells through CysLT1R and EP3 involving Gi, protein kinase G, and Erk and contributing to vascular inflammation in vivo. Furthermore, current results also suggest an advantage of targeting both CysLT1R and EP3 in attenuating inflammation.

Differential regulation of TRPV1 channels by H2O2: implications for diabetic microvascular dysfunction.

We demonstrated previously that TRPV1-dependent coupling of coronary blood flow (CBF) to metabolism is disrupted in diabetes. A critical amount of H2O2 contributes to CBF regulation; however, excessive H2O2 impairs responses. We sought to determine the extent to which differential regulation of TRPV1 by H2O2 modulates CBF and vascular reactivity in diabetes. We used contrast echocardiography to study TRPV1 knockout (V1KO), db/db diabetic, and wild type C57BKS/J (WT) mice. H2O2 dose-dependently increased CBF in WT mice, a response blocked by the TRPV1 antagonist SB366791. H2O2-induced vasodilation was significantly inhibited in db/db and V1KO mice. H2O2 caused robust SB366791-sensitive dilation in WT coronary microvessels; however, this response was attenuated in vessels from db/db and V1KO mice, suggesting H2O2-induced vasodilation occurs, in part, via TRPV1. Acute H2O2 exposure potentiated capsaicin-induced CBF responses and capsaicin-mediated vasodilation in WT mice, whereas prolonged luminal H2O2 exposure blunted capsaicin-induced vasodilation. Electrophysiology studies re-confirms acute H2O2 exposure activated TRPV1 in HEK293A and bovine aortic endothelial cells while establishing that H2O2 potentiate capsaicin-activated TRPV1 currents, whereas prolonged H2O2 exposure attenuated TRPV1 currents. Verification of H2O2-mediated activation of intrinsic TRPV1 specific currents were found in isolated mouse coronary endothelial cells from WT mice and decreased in endothelial cells from V1KO mice. These data suggest prolonged H2O2 exposure impairs TRPV1-dependent coronary vascular signaling. This may contribute to microvascular dysfunction and tissue perfusion deficits characteristic of diabetes.

Modulation of mast cell proliferative and inflammatory responses by leukotriene d4 and stem cell factor signaling interactions.

Mast cells (MCs) are important effector cells in asthma and pulmonary inflammation, and their proliferation and maturation is maintained by stem cell factor (SCF) via its receptor, c-Kit. Cysteinyl leukotrienes (cys-LTs) are potent inflammatory mediators that signal through CysLT1 R and CysLT2 R located on the MC surface, and they enhance MC inflammatory responses. However, it is not known if SCF and cys-LTs cross-talk and influence MC hyperplasia and activation in inflammation. Here, we report the concerted effort of the growth factor SCF and the inflammatory mediator LTD4 in MC activation. Stimulation of MCs by LTD4 in the presence of SCF enhances c-Kit-mediated proliferative responses. Similarly, SCF synergistically enhances LTD4 -induced calcium, c-fos expression and phosphorylation, as well as MIP1ß generation in MCs. These findings suggest that integration of SCF and LTD4 signals may contribute to MC hyperplasia and hyper-reactivity during airway hyper-response and inflammation.

Absence of type VI collagen paradoxically improves cardiac function, structure, and remodeling after myocardial infarction.

RATIONALE: We previously reported that type VI collagen deposition increases in the infarcted myocardium in vivo. To date, a specific role for this nonfibrillar collagen has not been explored in the setting of myocardial infarction (MI). OBJECTIVE: To determine whether deletion of type VI collagen in an in vivo model of post-MI wound healing would alter cardiac function and remodeling in the days to weeks after injury. METHODS AND RESULTS: Wild-type and Col6a1(-/-) mice were subjected to MI, followed by serial echocardiographic and histological assessments. At 8 weeks after MI, infarct size was significantly reduced, ejection fraction was significantly preserved (43.9% ± 3.3% versus 29.1% ± 4.3% for wild-type), and left ventricular chamber dilation was attenuated in the Col6a1(-/-) MI group (25.8% ± 7.9% increase versus 62.6% ± 16.5% for wild-type). The improvement in cardiac remodeling was evident as early as 10 days after MI in the Col6a1(-/-) mice. Myocyte apoptosis within the infarcted zones was initially greater in the Col6a1(-/-) group 3 days after MI, but by day 14 this was significantly reduced. Collagen deposition also was reduced in the infarcted and remote areas of the Col6a1(-/-) hearts. The reductions in chronic myocyte apoptosis and fibrosis are critical events leading to improved long-term remodeling and functional outcomes. CONCLUSIONS: These unexpected results demonstrate for the first time that deletion of type VI collagen in this knockout model plays a critical protective role after MI by limiting infarct size, chronic apoptosis, aberrant remodeling, and fibrosis, leading to preservation of cardiac function.

Hyperglycemia enhances function and differentiation of adult rat cardiac fibroblasts.

Diabetes is an independent risk factor for cardiovascular disease that can eventually cause cardiomyopathy and heart failure. Cardiac fibroblasts (CF) are the critical mediators of physiological and pathological cardiac remodeling; however, the effects of hyperglycemia on cardiac fibroblast function and differentiation is not well known. Here, we performed a comprehensive investigation on the effects of hyperglycemia on cardiac fibroblasts and show that hyperglycemia enhances cardiac fibroblast function and differentiation. We found that high glucose treatment increased collagen I, III, and VI gene expression in rat adult cardiac fibroblasts. Interestingly, hyperglycemia increased CF migration and proliferation that is augmented by collagen I and III. Surprisingly, we found that short term hyperglycemia transiently inhibited ERK1/2 activation but increased AKT phosphorylation. Finally, high glucose treatment increased spontaneous differentiation of cardiac fibroblasts to myofibroblasts with increasing passage compared with low glucose. Taken together, these findings suggest that hyperglycemia induces cardiac fibrosis by modulating collagen expression, migration, proliferation, and differentiation of cardiac fibroblasts.

TRPV4 channels mediate cardiac fibroblast differentiation by integrating mechanical and soluble signals.

The phenotypic switch underlying the differentiation of cardiac fibroblasts into hypersecretory myofibroblasts is critical for cardiac remodeling following myocardial infarction. Myofibroblasts facilitate wound repair in the myocardium by secreting and organizing extracellular matrix (ECM) during the wound healing process. However, the molecular mechanisms involved in myofibroblast differentiation are not well known. TGF-ß has been shown to promote differentiation and this, combined with the robust mechanical environment in the heart, lead us to hypothesize that the mechanotransduction and TGF-ß signaling pathways play active roles in the differentiation of cardiac fibroblasts to myofibroblasts. Here, we show that the mechanosensitve ion channel TRPV4 is required for TGF-ß1-induced differentiation of cardiac fibroblasts into myofibroblasts. We found that the TRPV4-specific antagonist AB159908 and siRNA knockdown of TRPV4 significantly inhibited TGFß1-induced differentiation as measured by incorporation of α-SMA into stress fibers. Further, we found that TGF-ß1-induced myofibroblast differentiation was dependent on ECM stiffness, a response that was attenuated by TRPV4 blockade. Finally, TGF-ß1 treated fibroblasts exhibited enhanced TRPV4 expression and TRPV4-mediated calcium influx compared to untreated controls. Taken together these results suggest for the first time that the mechanosensitive ion channel, TRPV4, regulates cardiac fibroblast differentiation to myofibroblasts by integrating signals from TGF-ß1 and mechanical factors.

Angiotensin II induces endothelial dysfunction and vascular remodeling by downregulating TRPV4 channels.

Angiotensin II (Ang II) is a potent vasoconstrictor of vascular smooth muscle cells (VSMC) and is implicated in hypertension, but it's role in the regulation of endothelial function is not well known. We and others have previously shown that mechanically activated ion channel, Transient Receptor Potential Vanilloid 4 (TRPV4) mediates flow- and/or receptor-dependent vasodilation via nitric oxide (NO) production in endothelial cells. Ang II was demonstrated to crosstalk with TRPV4 via angiotensin 1 receptor (AT1R) and ß-arrestin signaling in epithelial and immortalized cells, however, the role of this crosstalk in endothelial cell function is not fully explored. Ang II treatment significantly downregulated TRPV4 protein expression and TRPV4-mediated Ca2+ influx in human EC without altering TRPV4 mRNA levels. Further, TRPV4-induced eNOS phosphorylation and NO production were significantly reduced in Ang II-treated human EC. Importantly, Ang II infusion in mice revealed that, TRPV4/p-eNOS expression and colocalization was reduced in endothelium in vivo. Finally, Ang II infusion induced vascular remodeling as evidenced by decreased lumen to wall ratio in resistant mesenteric arteries. These findings suggest that Ang II induces endothelial dysfunction and vascular remodeling via downregulation of TRPV4/eNOS pathway and may contribute to hypertension, independent of or in addition to its effect on vascular smooth muscle contraction.