Deletion of Endothelial TRPV4 Protects Heart From Pressure Overload-Induced Hypertrophy.

BACKGROUND: Left ventricular hypertrophy is a bipolar response, starting as an adaptive response to the hemodynamic challenge, but over time develops maladaptive pathology partly due to microvascular rarefaction and impaired coronary angiogenesis. Despite the profound influence on cardiac function, the mechanotransduction mechanisms that regulate coronary angiogenesis, leading to heart failure, are not well known. METHODS: We subjected endothelial-specific knockout mice of mechanically activated ion channel, TRPV4 (transient receptor potential cation channel subfamily V member 4; TRPV4ECKO) to pressure overload via transverse aortic constriction and examined cardiac function, cardiomyocyte hypertrophy, cardiac fibrosis, and apoptosis. Further, we measured microvascular density and underlying TRPV4 mechanotransduction mechanisms using human microvascular endothelial cells, extracellular matrix gels of varying stiffness, unbiased RNA sequencing, small interfering RNA, Western blot, quantitative-PCR, and confocal immunofluorescence techniques. RESULTS: We demonstrate that endothelial-specific deletion of TRPV4 preserved cardiac function, cardiomyocyte structure, and reduced cardiac fibrosis compared with TRPV4lox/lox mice, 28 days post-transverse aortic constriction. Interestingly, comprehensive RNA sequencing analysis revealed an upregulation of proangiogenic factors (VEGFα [vascular endothelial growth factor α], NOS3 [nitric oxide synthase 3], and FGF2 [fibroblast growth factor 2]) with concomitant increase in microvascular density in TRPV4ECKO hearts after transverse aortic constriction compared with TRPV4lox/lox. Further, an increased expression of VEGFR2 (vascular endothelial growth factor receptor 2) and activation of the YAP (yes-associated protein) pathway were observed in TRPV4ECKO hearts. Mechanistically, we found that downregulation of TRPV4 in endothelial cells induced matrix stiffness-dependent activation of YAP and VEGFR2 via the Rho/Rho kinase/large tumor suppressor kinase pathway. CONCLUSIONS: Our results suggest that endothelial TRPV4 acts as a mechanical break for coronary angiogenesis, and uncoupling endothelial TRPV4 mechanotransduction attenuates pathological cardiac hypertrophy by enhancing coronary angiogenesis.

TRPV4 Mechanotransduction in Fibrosis.

Fibrosis is an irreversible, debilitating condition marked by the excessive production of extracellular matrix and tissue scarring that eventually results in organ failure and disease. Differentiation of fibroblasts to hypersecretory myofibroblasts is the key event in fibrosis. Although both soluble and mechanical factors are implicated in fibroblast differentiation, much of the focus is on TGF-ß signaling, but to date, there are no specific drugs available for the treatment of fibrosis. In this review, we describe the role for TRPV4 mechanotransduction in cardiac and lung fibrosis, and we propose TRPV4 as an alternative therapeutic target for fibrosis.

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.

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.

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.

Extracellular Vesicles From Pathological Microenvironment Induce Endothelial Cell Transformation and Abnormal Angiogenesis via Modulation of TRPV4 Channels.

The soluble and mechanical microenvironment surrounding endothelial cells influences and instructs them to form new blood vessels. The cells in the pathological tumor microenvironment release extracellular vesicles (EVs) for paracrine signaling. EVs have been shown to induce angiogenesis by communicating with endothelial cells, but the underlying molecular mechanisms are not well known. We have recently shown that the mechanosensitive ion channel transient receptor vanilloid 4 (TRPV4) expression and activity is significantly reduced in tumor endothelial cells (TEC), and that activation of TRPV4 normalized the tumor vasculature and improved cancer therapy. However, whether and how the tumor microenvironment downregulates TRPV4 and transforms the normal endothelial cell phenotype remains unknown. To explore this, we exposed normal human endothelial cells (hNEC) to human lung tumor cell conditioned media (TCM) and measured phenotypic changes and angiogenesis. We found that treatment with TCM transformed hNEC to a TEC-like phenotype (hTEC) as evidenced by increased expression of tumor endothelial cell marker 8 (TEM8) and exhibition of abnormal angiogenesis on 2D-Matrigels compared to normal hNEC. Mechanistically, expression and activity of TRPV4 was decreased in hTEC. Further, when pre-treated with exosome inhibitor GW4869, TCM failed to induce hNEC transformation to hTEC. Finally, addition of purified EVs from TCM induced transformation of hNEC to hTEC as evidenced by abnormal angiogenesis in vitro. Taken together, our results suggest that the pathological (tumor) microenvironment transforms normal endothelial cells into a tumor endothelial cell-like phenotype through EVs via the downregulation of TRPV4.

Mechanosensitive TRPV4 channels stabilize VE-cadherin junctions to regulate tumor vascular integrity and metastasis.

The transient receptor potential vanilloid 4 (TRPV4) channel is a mechanosensor in endothelial cells (EC) that regulates cyclic strain-induced reorientation and flow-mediated nitric oxide production. We have recently demonstrated that TRPV4 expression is reduced in tumor EC and tumors grown in TRPV4KO mice exhibited enhanced growth and immature leaky vessels. However, the mechanism by which TRPV4 regulates tumor vascular integrity and metastasis is not known. Here, we demonstrate that VE-cadherin expression at the cell-cell contacts is significantly reduced in TRPV4-deficient tumor EC and TRPV4KO EC. In vivo angiogenesis assays with Matrigel of varying stiffness (700-900 Pa) revealed a significant stiffness-dependent reduction in VE-cadherin-positive vessels in Matrigel plugs from TRPV4KO mice compared with WT mice, despite an increase in vessel growth. Further, syngeneic Lewis Lung Carcinomatumor experiments demonstrated a significant decrease in VE-cadherin positive vessels in TRPV4KO tumors compared with WT. Functionally, enhanced tumor cell metastasis to the lung was observed in TRPV4KO mice. Our findings demonstrate that TRPV4 channels regulate tumor vessel integrity by maintaining VE-cadherin expression at cell-cell contacts and identifies TRPV4 as a novel target for metastasis.

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.

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.

TRPV4 channels regulate tumor angiogenesis via modulation of Rho/Rho kinase pathway.

Targeting angiogenesis is considered a promising therapy for cancer. Besides curtailing soluble factor mediated tumor angiogenesis, understanding the unexplored regulation of angiogenesis by mechanical cues may lead to the identification of novel therapeutic targets. We have recently shown that expression and activity of mechanosensitive ion channel transient receptor potential vanilloid 4 (TRPV4) is suppressed in tumor endothelial cells and restoring TRPV4 expression or activation induces vascular normalization and improves cancer therapy. However, the molecular mechanism(s) by which TRPV4 modulates angiogenesis are still in their infancy. To explore how TRPV4 regulates angiogenesis, we have employed TRPV4 null endothelial cells (TRPV4KO EC) and TRPV4KO mice. We found that absence of TRPV4 (TRPV4KO EC) resulted in a significant increase in proliferation, migration, and abnormal tube formation in vitro when compared to WT EC. Concomitantly, sprouting angiogenesis ex vivo and vascular growth in vivo was enhanced in TRPV4KO mice. Mechanistically, we observed that loss of TRPV4 leads to a significant increase in basal Rho activity in TRPV4KO EC that corresponded to their aberrant mechanosensitivity on varying stiffness ECM gels. Importantly, pharmacological inhibition of the Rho/Rho kinase pathway by Y-27632 normalized abnormal mechanosensitivity and angiogenesis exhibited by TRPV4KO EC in vitro. Finally, Y-27632 treatment increased pericyte coverage and in conjunction with Cisplatin, significantly reduced tumor growth in TRPV4KO mice. Taken together, these data suggest that TRPV4 regulates angiogenesis endogenously via modulation of EC mechanosensitivity through the Rho/Rho kinase pathway and can serve as a potential therapeutic target for cancer therapy.

TRPV4 channel activation selectively inhibits tumor endothelial cell proliferation.

Endothelial cell proliferation is a critical event during angiogenesis, regulated by both soluble factors and mechanical forces. Although the proliferation of tumor cells is studied extensively, little is known about the proliferation of tumor endothelial cells (TEC) and its contribution to tumor angiogenesis. We have recently shown that reduced expression of the mechanosensitive ion channel TRPV4 in TEC causes aberrant mechanosensitivity that result in abnormal angiogenesis. Here, we show that TEC display increased proliferation compared to normal endothelial cells (NEC). Further, we found that TEC exhibit high basal ERK1/2 phosphorylation and increased expression of proliferative genes important in the G1/S phase of the cell cycle. Importantly, pharmacological activation of TRPV4, with a small molecular activator GSK1016790A (GSK), significantly inhibited TEC proliferation, but had no effect on the proliferation of NEC or the tumor cells (epithelial) themselves. This reduction in TEC proliferation by TRPV4 activation was correlated with a decrease in high basal ERK1/2 phosphorylation. Finally, using a syngeneic tumor model revealed that TRPV4 activation, with GSK, significantly reduced endothelial cell proliferation in vivo. Our findings suggest that TRPV4 channels regulate tumor angiogenesis by selectively inhibiting tumor endothelial cell proliferation.

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.

Cysteinyl leukotrienes regulate endothelial cell inflammatory and proliferative signals through CysLT2 and CysLT1 receptors.

Cysteinyl leukotrienes (cys-LTs), LTC4, LTD4, LTE4 are potent inflammatory lipid mediators that act through two distinct G-protein-coupled receptors, CysLT1R and CysLT2R. Although cys-LTs are shown to induce vascular leakage and atherosclerosis, the molecular mechanism by which cys-LTs modulate endothelial function is not known. Here, we show that cys-LTs (LTC4 and LTD4) induce robust calcium influx in human umbilical vein endothelial cells (HUVECs) through CysLT2R, but not CysLT1R. Further, cys-LT treatment induced endothelial cell (EC) contraction leading to monolayer disruption via CysLT2R/Rho kinase dependent pathway. Furthermore, stimulation with cys-LTs potentiated TNFα-induced VCAM-1 expression and leukocyte recruitment to ECs through CysLT2R. In contrast, we found that both LTC4 and LTD4 stimulated EC proliferation through CysLT1R. Taken together, these results suggest that cys-LTs induce endothelial inflammation and proliferation via CysLT2R/Rho kinase and CysLT1R/Erk dependent pathways, respectively, which play critical role in the etiology of cardiovascular diseases such as atherosclerosis and myocardial infarction.

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.

Upregulation of thrombospondin-1 expression by leptin in vascular smooth muscle cells via JAK2- and MAPK-dependent pathways.

Hyperleptinemia, characteristic of diabetes and a hallmark feature of human obesity, contributes to the increased risk of atherosclerotic complications. However, molecular mechanisms mediating leptin-induced atherogenesis and gene expression in vascular cells remain incompletely understood. Accumulating evidence documents a critical role of a potent antiangiogenic and proatherogenic matricellular protein, thrombospondin-1 (TSP-1), in atherosclerosis. Although previous studies reported elevated TSP-1 levels in both diabetic and obese patients and rodent models, there is no direct information on TSP-1 expression in vascular cells in response to leptin. In the present study, we show that leptin upregulates TSP-1 expression in cultured human aortic smooth muscle cells (HASMC) in vitro, and this increase occurs at the level of transcription, revealed by mRNA stability and TSP-1 promoter-reporter assays. Utilizing specific pharmacological inhibitors and siRNA approaches, we demonstrate that upregulation of TSP-1 expression by leptin is mediated by JAK2/ERK/JNK-dependent mechanisms. Furthermore, we report that while ERK and JNK are required for both the constitutive and leptin-induced expression of TSP-1, JAK-2 appears to be specifically involved in leptin-mediated TSP-1 upregulation. Finally, we found that increased HASMC migration and proliferation in response to leptin is significantly inhibited by a TSP-1 blocking antibody, thereby revealing the physiological significance of leptin-TSP-1 crosstalk. Taken together, these findings demonstrate, for the first time, that leptin has a direct regulatory effect on TSP-1 expression in HASMCs, underscoring a novel role of TSP-1 in hyperleptinemia-induced atherosclerotic complications.

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.

Primary cilia regulates the directional migration and barrier integrity of endothelial cells through the modulation of hsp27 dependent actin cytoskeletal organization.

Cilia are mechanosensing organelles that communicate extracellular signals into intracellular responses. Altered functions of primary cilia play a key role in the development of various diseases including polycystic kidney disease. Here, we show that endothelial cells from the oak ridge polycystic kidney (Tg737(orpk/orpk) ) mouse, with impaired cilia assembly, exhibit a reduction in the actin stress fibers and focal adhesions compared to wild-type (WT). In contrast, endothelial cells from polycystin-1 deficient mice (pkd1(null/null) ), with impaired cilia function, display robust stress fibers, and focal adhesion assembly. We found that the Tg737(orpk/orpk) cells exhibit impaired directional migration and endothelial cell monolayer permeability compared to the WT and pkd1(null/null) cells. Finally, we found that the expression of heat shock protein 27 (hsp27) and the phosphorylation of focal adhesion kinase (FAK) are downregulated in the Tg737(orpk/orpk) cells and overexpression of hsp27 restored both FAK phosphorylation and cell migration. Taken together, these results demonstrate that disruption of the primary cilia structure or function compromises the endothelium through the suppression of hsp27 dependent actin organization and focal adhesion formation, which may contribute to the vascular dysfunction in ciliopathies.

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.

Leukotriene B4 is a physiologically relevant endogenous peroxisome proliferator-activated receptor-alpha agonist.

Peroxisome proliferator-activated receptors (PPARs) are nuclear transcription factors that play central roles in metabolism and inflammation. Although a variety of compounds have been shown to activate PPARs, identification of physiologically relevant ligands has proven difficult. In silico studies of lipid derivatives reported here identify specific 5-lipoxygenase products as candidate physiologically relevant PPAR-alpha activators. Subsequent studies show both in vitro and in a murine model of inflammation that 5-lipoxygenase stimulation induces PPAR-alpha signaling and that this results specifically from production of the inflammatory mediator and chemoattractant leukotriene B(4) (LTB(4)). Activation of PPAR-alpha is a direct effect of intracellularly generated LTB(4) binding to the nuclear receptor and not of secreted LTB(4) acting via its cell-surface receptors. Activation of PPAR-alpha reduces secretion of LTB(4) by stimulating degradation of this fatty acid derivative. We also show that the LTB(4) precursors leukotriene A(4) (LTA(4)) and 5-hydroperoxyeicosatetrenoic acid (5-HPETE) activate PPAR-alpha but have no significant endogenous effect independent of conversion to LTB(4). We conclude that LTB(4) is a physiologically relevant PPAR-alpha activator in cells of the immune system. This, together with previous findings, demonstrates that different types of lipids serve as endogenous PPAR-alpha ligands, with the relevant ligand varying between functionally different cell types. Our results also support the suggestion that regulation of inflammation may involve balancing proinflammatory effects of LTB(4), exerted through cell-surface receptors, and anti-inflammatory effects exerted through PPAR-alpha activation.

Curcumin is not a ligand for peroxisome proliferator-activated receptor-γ

Curcumin, a compound found in the spice turmeric, has been shown to possess a number of beneficial biological activities exerted through a variety of different mechanisms. Some curcumin effects have been reported to involve activation of the nuclear transcription factor peroxisome proliferator-activated receptor-γ (PPAR-γ), but the concept that curcumin might be a PPAR-γ ligand remains controversial. Results reported here demonstrate that, in contrast to the PPAR-γ ligands ciglitazone and rosiglitazone, curcumin is inactive in five different reporter or DNA-binding assays, does not displace [(3)H]rosiglitazone from the PPAR-γ ligand-binding site, and does not induce PPAR-γ-dependent differentiation of preadipocytes, while its ability to inhibit fibroblast-to-myofibroblast differentiation is not affected by any of four PPAR-γ antagonists. These multiple lines of evidence conclusively demonstrate that curcumin is not a PPAR-γ ligand and indicate the need for further investigation of the mechanisms through which the compound acts.