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1.
Nat Commun ; 14(1): 3733, 2023 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-37353478

RESUMO

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.


Assuntos
Canais de Cátion TRPV , Proteína rhoA de Ligação ao GTP , Humanos , Repetição de Anquirina , Canais de Cátion TRPV/química , Proteína rhoA de Ligação ao GTP/química
2.
Biomolecules ; 12(6)2022 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-35740948

RESUMO

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).


Assuntos
Hipertensão , NADPH Oxidases , Células Endoteliais , Humanos , Peróxido de Hidrogênio , NADPH Oxidases/antagonistas & inibidores , Espécies Reativas de Oxigênio
3.
Basic Res Cardiol ; 117(1): 24, 2022 04 25.
Artigo em Inglês | MEDLINE | ID: mdl-35469044

RESUMO

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.


Assuntos
Doença da Artéria Coronariana , Vasodilatação , Arteríolas/metabolismo , Doença da Artéria Coronariana/metabolismo , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Humanos , NADPH Oxidase 4/metabolismo , Fosforilação , Canais de Cátion TRPV , Vasodilatação/fisiologia
6.
Front Cell Dev Biol ; 9: 687598, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34222255

RESUMO

Small GTPase Rap1 plays a prominent role in endothelial cell (EC) homeostasis by promoting NO release. Endothelial deletion of the two highly homologous Rap1 isoforms, Rap1A and Rap1B, leads to endothelial dysfunction ex vivo and hypertension in vivo. Mechanistically, we showed that Rap1B promotes NO release in response to shear flow by promoting mechanosensing complex formation involving VEGFR2 and Akt activation. However, the specific contribution of the Rap1A isoform to NO release and the underlying molecular mechanisms through which the two Rap1 isoforms control endothelial function are unknown. Here, we demonstrate that endothelial dysfunction resulting from knockout of both Rap1A and Rap1B isoforms is ameliorated by exogenous L-Arg administration to rescue NO-dependent vasorelaxation and blood pressure. We confirmed that Rap1B is rapidly activated in response to agonists that trigger eNOS activation, and its deletion in ECs attenuates eNOS activation, as detected by decreased Ser1177 phosphorylation. Somewhat surprising was the finding that EC deletion of Rap1A does not lead to impaired agonist-induced vasorelaxation ex vivo. Mechanistically, the deletion of Rap1A led to elevated eNOS phosphorylation both at the inhibitory, T495, and the activating Ser1177 residues. These findings indicate that the two Rap1 isoforms act via distinct signaling pathways: while Rap1B directly positively regulates eNOS activation, Rap1A prevents negative regulation of eNOS. Notably, the combined deficiency of Rap1A and Rap1B has a severe effect on eNOS activity and NO release with an in vivo impact on endothelial function and vascular homeostasis.

7.
Arterioscler Thromb Vasc Biol ; 41(2): 638-650, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33267664

RESUMO

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.


Assuntos
Aorta/metabolismo , Doenças da Aorta/prevenção & controle , Aterosclerose/prevenção & controle , Células Endoteliais/metabolismo , Mediadores da Inflamação/metabolismo , Inflamação/prevenção & controle , Proteínas rap de Ligação ao GTP/metabolismo , Animais , Aorta/patologia , Doenças da Aorta/genética , Doenças da Aorta/metabolismo , Doenças da Aorta/patologia , Aterosclerose/genética , Aterosclerose/metabolismo , Aterosclerose/patologia , Células Cultivadas , Citocinas/metabolismo , Dieta Hiperlipídica , Modelos Animais de Doenças , Progressão da Doença , Células Endoteliais/patologia , Feminino , Humanos , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Leucócitos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , NF-kappa B/metabolismo , Óxido Nítrico/metabolismo , Placa Aterosclerótica , Transdução de Sinais , Proteínas rap de Ligação ao GTP/genética , Proteínas rap1 de Ligação ao GTP/genética , Proteínas rap1 de Ligação ao GTP/metabolismo
8.
Br J Pharmacol ; 178(3): 709-725, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33184836

RESUMO

BACKGROUND AND PURPOSE: The TRPV cation channels have emerged as important regulators of vascular tone. TRPV1 channels and endothelin-1 are independently associated with the pathophysiology of coronary vasospasm, but the relationship between their vasomotor functions remains unclear. We characterized the vasomotor function of TRPV1 channels in human arterioles and investigated regulation of their vasomotor function by endothelin-1. EXPERIMENTAL APPROACH: Human arterioles (mainly from adipose tissue) were threaded on two metal wires, equilibrated in a physiological buffer at 37°C and exposed to increasing concentrations of capsaicin, with or without SB366791 (TRPV1-selective inhibitor) or GF109203X (PKC-selective inhibitor). Some arterioles were pre-constricted with endothelin-1 or phenylephrine or high potassium buffer. TRPV1 mRNA and protein expression in human arteries were also assessed. KEY RESULTS: TRPV1 transcripts and proteins were detected in human resistance arteries. Capsaicin (1 µM) induced concentration-dependent constriction of endothelium-intact and endothelium-denuded human adipose arterioles (HAA), which was significantly inhibited by SB366791. Pre-constriction of HAA with endothelin-1, but not high potassium buffer or phenylephrine, significantly potentiated capsaicin (0.1 µM)-induced constriction. GF109203X significantly inhibited potentiation of capsaicin-induced constriction by endothelin-1. CONCLUSION AND IMPLICATIONS: TRPV1 channels are expressed in the human vasculature and affect vascular tone of human arterioles on activation. Their vasomotor function is modulated by endothelin-1, mediated in part by PKC. These findings reveal a novel interplay between endothelin-1 signalling and TRPV1 channels in human VSMC, adding to our understanding of the ion channel mechanisms that regulate human arteriolar tone and may also contribute to the pathophysiology of coronary vasospasm.


Assuntos
Endotelina-1 , Vasoconstrição , Tecido Adiposo , Arteríolas , Capsaicina/farmacologia , Humanos , Proteína Quinase C , Canais de Cátion TRPV
10.
J Cell Mol Med ; 23(2): 761-774, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30450767

RESUMO

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.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Ciclo Celular/genética , Epiderme/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Matriz Extracelular/efeitos dos fármacos , Canais de Cátion TRPV/genética , Transativadores/genética , Actinas/genética , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Bleomicina/administração & dosagem , Caderinas/genética , Caderinas/metabolismo , Adesão Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Movimento Celular/efeitos dos fármacos , Epiderme/metabolismo , Epiderme/patologia , Transição Epitelial-Mesenquimal/genética , Matriz Extracelular/metabolismo , Fibrose/induzido quimicamente , Regulação da Expressão Gênica , Humanos , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Mecanotransdução Celular , Camundongos , Cultura Primária de Células , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína Smad2/genética , Proteína Smad2/metabolismo , Proteína Smad3/genética , Proteína Smad3/metabolismo , Canais de Cátion TRPV/antagonistas & inibidores , Canais de Cátion TRPV/deficiência , Transativadores/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Proteínas de Sinalização YAP
11.
FASEB J ; 33(2): 2636-2645, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30303741

RESUMO

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.


Assuntos
Cálcio/metabolismo , Hipertensão/fisiopatologia , Músculo Liso Vascular/fisiologia , Receptor de Endotelina A/metabolismo , Artéria Renal/fisiologia , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src/metabolismo , Resistência Vascular , Animais , Células Cultivadas , Endotelina-1/metabolismo , Hipertensão/metabolismo , Masculino , Músculo Liso Vascular/citologia , Ratos , Ratos Endogâmicos Dahl , Artéria Renal/citologia
12.
J Biol Chem ; 293(14): 5307-5322, 2018 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-29462784

RESUMO

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.


Assuntos
Canais de Cátion TRPV/metabolismo , Canais de Cátion TRPV/fisiologia , Ácido Araquidônico/metabolismo , Canais de Cálcio/metabolismo , Células Cultivadas , Vasos Coronários/metabolismo , Cristalografia por Raios X , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Células Endoteliais/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Fosforilação , Transdução de Sinais
13.
Microcirculation ; 25(1)2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29161755

RESUMO

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.


Assuntos
Vasos Coronários/química , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Vasodilatação/efeitos dos fármacos , Arteríolas/fisiologia , Doenças Cardiovasculares/etiologia , Estudos de Casos e Controles , AMP Cíclico/fisiologia , Humanos , Subunidades Proteicas , RNA Mensageiro/análise
14.
PLoS One ; 12(5): e0176796, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28472069

RESUMO

The transient receptor potential vallinoid type 4 (TRPV4) is a calcium entry channel known to modulate vascular function by mediating endothelium-dependent vasodilation. The present study investigated if isolated cerebral arterial myocytes of the Fawn Hooded hypertensive (FHH) rat, known to display exaggerated KCa channel current activity and impaired myogenic tone, express TRPV4 channels at the transcript and protein level and exhibit TRPV4-like single-channel cationic current activity. Reverse transcription polymerase chain reaction (RT-PCR), Western blot, and immunostaining analysis detected the expression of mRNA transcript and translated protein of TRPV4 channel in FHH rat cerebral arterial myocytes. Patch clamp recording of single-channel current activity identified the presence of a single-channel cationic current with unitary conductance of ~85 pS and ~96 pS at hyperpolarizing and depolarizing potentials, respectively, that was inhibited by the TRPV4 channel antagonist RN 1734 or HC 067074 and activated by the potent TRPV4 channel agonist GSK1016790A. Application of negative pressure via the interior of the patch pipette increased the NPo of the TRPV4-like single-channel cationic current recorded in cell-attached patches at a patch potential of 60 mV that was inhibited by prior application of the TRPV4 channel antagonist RN 1734 or HC 067047. Treatment with the TRPV4 channel agonist GSK1016790A caused concentration-dependent increase in the NPo of KCa single-channel current recorded in cell-attached patches of cerebral arterial myocytes at a patch potential of 40 mV, which was not influenced by pretreatment with the voltage-gated L-type Ca2+ channel blocker nifedipine or the T-type Ca2+ channel blocker Ni2+. These findings demonstrate that FHH rat cerebral arterial myocytes express mRNA transcript and translated protein for TRPV4 channel and display TRPV4-like single-channel cationic current activity that was stretch-sensitive and activation of which increased the open state probability of KCa single-channel current in these arterial myocytes.


Assuntos
Encéfalo/irrigação sanguínea , Circulação Cerebrovascular , Hipertensão/fisiopatologia , Músculo Liso Vascular/fisiopatologia , Canais de Cátion TRPV/fisiologia , Animais , Masculino , Ratos
15.
Am J Physiol Cell Physiol ; 312(5): C562-C572, 2017 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-28249987

RESUMO

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.


Assuntos
Matriz Extracelular/fisiologia , Mecanotransdução Celular/fisiologia , Miofibroblastos/citologia , Miofibroblastos/fisiologia , Canais de Cátion TRPV/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , Módulo de Elasticidade/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Humanos , Ativação do Canal Iônico/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pele/citologia , Especificidade da Espécie
17.
Circ Res ; 120(4): 658-669, 2017 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-27872049

RESUMO

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.


Assuntos
Arteríolas/fisiologia , Doença da Artéria Coronariana/fisiopatologia , Peróxido de Hidrogênio/farmacologia , Canal de Potássio Kv1.5/fisiologia , Vasodilatação/fisiologia , Adulto , Idoso , Arteríolas/efeitos dos fármacos , Arteríolas/patologia , Células Cultivadas , Doença da Artéria Coronariana/patologia , Vasos Coronários/efeitos dos fármacos , Vasos Coronários/patologia , Vasos Coronários/fisiologia , Feminino , Células HEK293 , Humanos , Canal de Potássio Kv1.5/antagonistas & inibidores , Masculino , Pessoa de Meia-Idade , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/fisiologia , Vasodilatação/efeitos dos fármacos
18.
Artigo em Inglês | MEDLINE | ID: mdl-27174801

RESUMO

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.


Assuntos
Astrócitos/metabolismo , Sistema Enzimático do Citocromo P-450/biossíntese , Ácidos Hidroxieicosatetraenoicos/biossíntese , Receptores de Glutamato Metabotrópico/metabolismo , Animais , Ácido Araquidônico/administração & dosagem , Ácido Araquidônico/metabolismo , Astrócitos/efeitos dos fármacos , Astrócitos/enzimologia , Encéfalo/metabolismo , Circulação Cerebrovascular/genética , Sistema Enzimático do Citocromo P-450/genética , Regulação Enzimológica da Expressão Gênica , Ácidos Hidroxieicosatetraenoicos/metabolismo , Músculo Liso Vascular/metabolismo , Ratos , Receptores de Glutamato Metabotrópico/genética
19.
Oncotarget ; 7(25): 37622-37635, 2016 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-27191895

RESUMO

TRPV4 ion channel mediates vascular mechanosensitivity and vasodilation. Here, we sought to explore whether non-mechanical activation of TRPV4 could limit vascular inflammation and atherosclerosis. We found that GSK1016790A, a potent and specific small-molecule agonist of TRPV4, induces the phosphorylation and activation of eNOS partially through the AMPK pathway. Moreover, GSK1016790A inhibited TNF-α-induced monocyte adhesion to human endothelial cells. Mice given GSK1016790A showed increased phosphorylation of eNOS and AMPK in the aorta and decreased leukocyte adhesion to TNF-α-inflamed endothelium. Importantly, oral administration of GSK1016790A reduced atherosclerotic plaque formation in ApoE deficient mice fed a Western-type diet. Together, the present study suggests that pharmacological activation of TRPV4 may serve as a potential therapeutic approach to treat atherosclerosis.


Assuntos
Aterosclerose/tratamento farmacológico , Monócitos/efeitos dos fármacos , Canais de Cátion TRPV/agonistas , Animais , Aterosclerose/metabolismo , Aterosclerose/patologia , Adesão Celular/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Células Endoteliais da Veia Umbilical Humana , Humanos , Leucina/análogos & derivados , Leucina/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/patologia , Sulfonamidas/farmacologia , Canais de Cátion TRPV/genética , Células U937
20.
Circ Heart Fail ; 9(1): e002424, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26763290

RESUMO

BACKGROUND: Diabetic heart disease is associated with tetrahydrobiopterin oxidation and high arginase activity, leading to endothelial nitric oxide synthase dysfunction. Sepiapterin (SEP) is a tetrahydrobiopterin precursor, and L-citrulline (L-Cit) is converted to endothelial nitric oxide synthase substrate, L-arginine. Whether SEP and L-Cit are effective at reducing diabetic heart disease is not known. The present study examined the effects of SEP and L-Cit on diabetic cardiomyopathy and ischemia/reperfusion injury in obese type 2 diabetic mice. METHODS AND RESULTS: Db/db and C57BLKS/J mice at 6 to 8 weeks of age received vehicle, SEP, or L-Cit orally alone or in combination for 8 weeks. Cardiac function was evaluated with echocardiography. Db/db mice displayed hyperglycemia, obesity, and normal blood pressure and cardiac function compared with C57BLKS/J mice at 6 to 8 weeks of age. After vehicle treatment for 8 weeks, db/db mice had reduced ejection fraction, mitral E/A ratio, endothelium-dependent relaxation of coronary arteries, tetrahydrobiopterin concentrations, ratio of endothelial nitric oxide synthase dimers/monomers, and nitric oxide levels compared with vehicle-treated C57BLKS/J mice. These detrimental effects of diabetes mellitus were abrogated by co-administration of SEP and L-Cit. Myocardial infarct size was increased, and coronary flow rate and ± dP/dt were decreased during reperfusion in vehicle-treated db/db mice subjected to ischemia/reperfusion injury compared with control mice. Co-administration of SEP and L-Cit decreased infarct size and improved coronary flow rate and cardiac function in both C57BLKS/J and db/db mice. CONCLUSIONS: Co-administration of SEP and L-Cit limits diabetic cardiomyopathy and ischemia/reperfusion injury in db/db mice through a tetrahydrobiopterin/endothelial nitric oxide synthase/nitric oxide pathway.


Assuntos
Cardiotônicos/administração & dosagem , Citrulina/administração & dosagem , Diabetes Mellitus Tipo 2/tratamento farmacológico , Cardiomiopatias Diabéticas/prevenção & controle , Infarto do Miocárdio/prevenção & controle , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Obesidade/complicações , Pterinas/administração & dosagem , Fatores Etários , Animais , Biopterinas/análogos & derivados , Biopterinas/metabolismo , Células Cultivadas , Circulação Coronária/efeitos dos fármacos , Vasos Coronários/efeitos dos fármacos , Vasos Coronários/metabolismo , Vasos Coronários/fisiopatologia , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/metabolismo , Cardiomiopatias Diabéticas/etiologia , Cardiomiopatias Diabéticas/metabolismo , Cardiomiopatias Diabéticas/patologia , Cardiomiopatias Diabéticas/fisiopatologia , Modelos Animais de Doenças , Esquema de Medicação , Quimioterapia Combinada , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Preparação de Coração Isolado , Camundongos Endogâmicos C57BL , Camundongos Obesos , Infarto do Miocárdio/etiologia , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Traumatismo por Reperfusão Miocárdica/etiologia , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Miocárdio/metabolismo , Miocárdio/patologia , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Fosforilação , Multimerização Proteica , Fatores de Tempo , Vasodilatação/efeitos dos fármacos , Função Ventricular Esquerda/efeitos dos fármacos
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