RESUMEN
Mechanosensitive cation channels and Ca2+ influx through these channels play an important role in the regulation of endothelial cell functions. Transient receptor potential canonical channel 6 (TRPC6) is a diacylglycerol-sensitive nonselective cation channel that forms receptor-operated Ca2+ channels in a variety of cell types. Piezo1 is a mechanosensitive cation channel activated by membrane stretch and shear stress in lung endothelial cells. In this study, we report that TRPC6 and Piezo1 channels both contribute to membrane stretch-mediated cation currents and Ca2+ influx or increase in cytosolic-free Ca2+ concentration ([Ca2+]cyt) in human pulmonary arterial endothelial cells (PAECs). The membrane stretch-mediated cation currents and increase in [Ca2+]cyt in human PAECs were significantly decreased by GsMTX4, a blocker of Piezo1 channels, and by BI-749327, a selective blocker of TRPC6 channels. Extracellular application of 1-oleoyl-2-acetyl-sn-glycerol (OAG), a membrane permeable analog of diacylglycerol, rapidly induced whole cell cation currents and increased [Ca2+]cyt in human PAECs and human embryonic kidney (HEK)-cells transiently transfected with the human TRPC6 gene. Furthermore, membrane stretch with hypo-osmotic or hypotonic solution enhances the cation currents in TRPC6-transfected HEK cells. In HEK cells transfected with the Piezo1 gene, however, OAG had little effect on the cation currents, but membrane stretch significantly enhanced the cation currents. These data indicate that, while both TRPC6 and Piezo1 are involved in generating mechanosensitive cation currents and increases in [Ca2+]cyt in human PAECs undergoing mechanical stimulation, only TRPC6 (but not Piezo1) is sensitive to the second messenger diacylglycerol. Selective blockers of these channels may help develop novel therapies for mechanotransduction-associated pulmonary vascular remodeling in patients with pulmonary arterial hypertension.
Asunto(s)
Células Endoteliales , Canales Iónicos , Mecanorreceptores , Canal Catiónico TRPC6 , Calcio/metabolismo , Cationes/metabolismo , Diglicéridos/metabolismo , Diglicéridos/farmacología , Células Endoteliales/metabolismo , Humanos , Soluciones Hipotónicas/metabolismo , Soluciones Hipotónicas/farmacología , Canales Iónicos/genética , Canales Iónicos/metabolismo , Mecanorreceptores/metabolismo , Mecanotransducción Celular/genética , Mecanotransducción Celular/fisiología , Arteria Pulmonar/citología , Arteria Pulmonar/metabolismo , Canal Catiónico TRPC6/genética , Canal Catiónico TRPC6/metabolismoRESUMEN
Concentric pulmonary vascular wall thickening due partially to increased pulmonary artery (PA) smooth muscle cell (PASMC) proliferation contributes to elevating pulmonary vascular resistance (PVR) in patients with pulmonary hypertension (PH). Although pulmonary vasoconstriction may be an early contributor to increasing PVR, the transition of contractile PASMCs to proliferative PASMCs may play an important role in the development and progression of pulmonary vascular remodeling in PH. A rise in cytosolic Ca2+ concentration ([Ca2+]cyt) is a trigger for PASMC contraction and proliferation. Here, we report that upregulation of Piezo1, a mechanosensitive cation channel, is involved in the contractile-to-proliferative phenotypic transition of PASMCs and potential development of pulmonary vascular remodeling. By comparing freshly isolated PA (contractile PASMCs) and primary cultured PASMCs (from the same rat) in a growth medium (proliferative PASMCs), we found that Piezo1, Notch2/3, and CaSR protein levels were significantly higher in proliferative PASMCs than in contractile PASMCs. Upregulated Piezo1 was associated with an increase in expression of PCNA, a marker for cell proliferation, whereas downregulation (with siRNA) or inhibition (with GsMTx4) of Piezo1 attenuated PASMC proliferation. Furthermore, Piezo1 in the remodeled PA from rats with experimental PH was upregulated compared with PA from control rats. These data indicate that PASMC contractile-to-proliferative phenotypic transition is associated with the transition or adaptation of membrane channels and receptors. Upregulated Piezo1 may play a critical role in PASMC phenotypic transition and PASMC proliferation. Upregulation of Piezo1 in proliferative PASMCs may likely be required to provide sufficient Ca2+ to assure nuclear/cell division and PASMC proliferation, contributing to the development and progression of pulmonary vascular remodeling in PH.
Asunto(s)
Hipertensión Pulmonar , Proteínas de la Membrana/metabolismo , Arteria Pulmonar , Animales , Señalización del Calcio/fisiología , Proliferación Celular , Células Cultivadas , Humanos , Hipertensión Pulmonar/metabolismo , Canales Iónicos/genética , Canales Iónicos/metabolismo , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Arteria Pulmonar/metabolismo , Ratas , Remodelación VascularRESUMEN
Idiopathic pulmonary arterial hypertension (PAH) is a fatal and progressive disease. Sustained vasoconstriction due to pulmonary arterial smooth muscle cell (PASMC) contraction and concentric arterial remodeling due partially to PASMC proliferation are the major causes for increased pulmonary vascular resistance and increased pulmonary arterial pressure in patients with precapillary pulmonary hypertension (PH) including PAH and PH due to respiratory diseases or hypoxemia. We and others observed upregulation of TRPC6 channels in PASMCs from patients with PAH. A rise in cytosolic Ca2+ concentration ([Ca2+]cyt) in PASMC triggers PASMC contraction and vasoconstriction, while Ca2+-dependent activation of PI3K/AKT/mTOR pathway is a pivotal signaling cascade for cell proliferation and gene expression. Despite evidence supporting a pathological role of TRPC6, no selective and orally bioavailable TRPC6 antagonist has yet been developed and tested for treatment of PAH or PH. In this study, we sought to investigate whether block of receptor-operated Ca2+ channels using a nonselective blocker of cation channels, 2-aminoethyl diphenylborinate (2-APB, administered intraperitoneally) and a selective blocker of TRPC6, BI-749327 (administered orally) can reverse established PH in mice. The results from the study show that intrapulmonary application of 2-APB (40 µM) or BI-749327 (3-10 µM) significantly and reversibly inhibited acute alveolar hypoxia-induced pulmonary vasoconstriction. Intraperitoneal injection of 2-APB (1 mg/kg per day) significantly attenuated the development of PH and partially reversed established PH in mice. Oral gavage of BI-749327 (30 mg/kg, every day, for 2 wk) reversed established PH by â¼50% via regression of pulmonary vascular remodeling. Furthermore, 2-APB and BI-749327 both significantly inhibited PDGF- and serum-mediated phosphorylation of AKT and mTOR in PASMC. In summary, the receptor-operated and mechanosensitive TRPC6 channel is a good target for developing novel treatment for PAH/PH. BI-749327, a selective TRPC6 blocker, is potentially a novel and effective drug for treating PAH and PH due to respiratory diseases or hypoxemia.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Hipertensión Pulmonar/patología , Músculo Liso Vascular/patología , Arteria Pulmonar/patología , Canal Catiónico TRPC6/metabolismo , Vasoconstricción , Animales , Compuestos de Boro/farmacología , Señalización del Calcio , Células Cultivadas , Humanos , Hipertensión Pulmonar/tratamiento farmacológico , Hipertensión Pulmonar/metabolismo , Ratones , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Arteria Pulmonar/efectos de los fármacos , Arteria Pulmonar/metabolismo , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Canal Catiónico TRPC6/antagonistas & inhibidores , Canal Catiónico TRPC6/genéticaRESUMEN
Idiopathic pulmonary arterial hypertension (IPAH) is a rare disease, with an estimated 500-1000 new cases diagnosed every year. A portion of these cases may be caused by mutations in the BMPR2 gene, suggesting a possible genetic component in the development of the disease. Here, we report two human induced pluripotent stem cell (iPSC) lines generated from IPAH patients. Both cell lines provide valuable insight into the molecular and cellular mechanisms of IPAH and can be used to further understand the disease.
Asunto(s)
Hipertensión Pulmonar , Células Madre Pluripotentes Inducidas , Humanos , Hipertensión Pulmonar/genética , MutaciónRESUMEN
Cancers and cardiovascular diseases are the top two causes of death in the United States. Over the past decades, novel therapies have slowed the cancer mortality rate, yet cardiac failures have risen due to the toxicity of cancer treatments. The mechanisms behind this relationship are poorly understood and it is crucial that we properly treat patients at risk of developing cardiac failure in response to cancer treatments. Currently, we rely on early-stage biomarkers of inflammation and angiogenesis to detect cardiotoxicity before it becomes irreversible. Identification of such biomarkers allows healthcare professionals to decrease the adverse effects of cancer therapies. Angiogenesis and inflammation have a systemic influence on the heart and vasculature following cancer therapy. In the field of cardio-oncology, there has been a recent emphasis on gender and racial disparities in cardiotoxicity and the impact of these disparities on disease outcomes, but there is a scarcity of data on how cardiotoxicity varies across diverse populations. Here, we will discuss how current markers of angiogenesis and inflammation induced by cancer therapy are related to disparities in cardiovascular health.
Asunto(s)
Antineoplásicos , Insuficiencia Cardíaca , Neoplasias , Humanos , Cardiotoxicidad/etiología , Cardiotoxicidad/tratamiento farmacológico , Antineoplásicos/uso terapéutico , Neoplasias/terapia , Insuficiencia Cardíaca/tratamiento farmacológico , Inflamación/tratamiento farmacológico , BiomarcadoresRESUMEN
INTRODUCTION: Piezo1 is an important mechanosensitive channel implicated in vascular remodeling. However, the role of Piezo1 in different types of vascular cells during the development of pulmonary hypertension (PH) induced by high shear stress is largely unknown. MATERIALS AND METHODS: We used a rat PH model established by left pulmonary artery ligation (LPAL, for 2-5 weeks), which mimics the high flow and hemodynamic stress, to study Piezo1 contribution to pulmonary vascular remodeling. RESULTS: Right ventricular systolic pressure (RVSP), a surrogate measure for pulmonary arterial systolic pressure, and right ventricular wall thickness, a measure for right ventricular hypertrophy, were significantly increased in LPAL rats compared with Sham-control (SHAM) rats. Rats in LPAL-5w groups developed remarkable pulmonary vascular remodeling, while phenylephrine-induced contraction and acetylcholine-induced relaxation were both significantly inhibited in these rats. Upregulation of Piezo1, in association with increase in cytosolic Ca2+ concentration ([Ca2+]cyt), was observed in pulmonary arterial smooth muscle cells (PASMCs) from LPAL-2w and LPAL-5w rats in comparison to the SHAM controls. Piezo1 upregulation in PASMCs from LPAL rats was directly related to Yes-associated protein (YAP)/ TEA domain transcription factor 4 (TEAD4). Piezo1 expression was also upregulated in the whole-lung tissue of LPAL rats. The endothelial upregulation of Piezo1 was related to transcriptional regulation by RELA (p65) and lung inflammation. CONCLUSION: The upregulation of Piezo1 in both PASMCs and ECs coordinates with each other via different cell signaling pathways to cause pulmonary vascular remodeling in LPAL-PH rats, providing novel insights into the cell-type specific pathogenic roles of Piezo1 in shear stress-associated experimental PH.
Asunto(s)
Hipertensión Pulmonar , Proteínas de la Membrana , Animales , Ratas , Acetilcolina/metabolismo , Proliferación Celular , Hipertensión Pulmonar/etiología , Proteínas de la Membrana/metabolismo , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Fenilefrina/metabolismo , Arteria Pulmonar/patología , Factor de Transcripción 4/metabolismo , Regulación hacia Arriba , Remodelación Vascular , Proteínas Señalizadoras YAPRESUMEN
Pulmonary arterial hypertension is a progressive and fatal disease and rodents with experimental pulmonary hypertension (PH) are often used to study pathogenic mechanisms, identify therapeutic targets, and develop novel drugs for treatment. Here we describe a hands-on set of experimental approaches including ex vivo lung angiography and histology and in vivo right heart catheterization (RHC) to phenotypically characterize pulmonary hemodynamics and lung vascular structure in normal mice and mice with experimental PH. We utilized Microfil polymer as contrast in our ex vivo lung angiogram to quantitatively examine pulmonary vascular remodeling in mice with experimental PH, and lung histology to estimate pulmonary artery wall thickness. The peripheral lung vascular images were selected to determine the total length of lung vascular branches, the number of branches and the number of junctions in a given area (mm-2). We found that the three parameters determined by angiogram were not significantly different among the apical, middle, and basal regions of the mouse lung from normal mice, and were not influenced by gender (no significant difference between female and male mice). We conducted RHC in mice to measure right ventricular systolic pressure, a surrogate measure for pulmonary artery systolic pressure and right ventricle (RV) contractility (RV ± dP/dtmax) to estimate RV function. RHC, a short time (4-6 min) procedure, did not alter the lung angiography measurements. In summary, utilizing ex vivo angiogram to determine peripheral vascular structure and density in the mouse lung and utilizing in vivo RHC to measure pulmonary hemodynamics are reliable readouts to phenotype normal mice and mice with experimental PH. Lung angiogram and RHC are also reliable approaches to examine pharmacological effects of new drugs on pulmonary vascular remodeling and hemodynamics.
RESUMEN
Excessive pulmonary artery (PA) smooth muscle cell (PASMC) proliferation and migration are implicated in the development of pathogenic pulmonary vascular remodeling characterized by concentric arterial wall thickening and arteriole muscularization in patients with pulmonary arterial hypertension (PAH). Pulmonary artery smooth muscle cell contractile-to-proliferative phenotypical transition is a process that promotes pulmonary vascular remodeling. A rise in cytosolic Ca2+ concentration [(Ca2+) cyt ] in PASMCs is a trigger for pulmonary vasoconstriction and a stimulus for pulmonary vascular remodeling. Here, we report that the calcium homeostasis modulator (CALHM), a Ca2+ (and ATP) channel that is allosterically regulated by voltage and extracellular Ca2+, is upregulated during the PASMC contractile-to-proliferative phenotypical transition. Protein expression of CALHM1/2 in primary cultured PASMCs in media containing serum and growth factors (proliferative PASMC) was significantly greater than in freshly isolated PA (contractile PASMC) from the same rat. Upregulated CALHM1/2 in proliferative PASMCs were associated with an increased ratio of pAKT/AKT and pmTOR/mTOR and an increased expression of the cell proliferation marker PCNA, whereas serum starvation and rapamycin significantly downregulated CALHM1/2. Furthermore, CALHM1/2 were upregulated in freshly isolated PA from rats with monocrotaline (MCT)-induced PH and in primary cultured PASMC from patients with PAH in comparison to normal controls. Intraperitoneal injection of CGP 37157 (0.6 mg/kg, q8H), a non-selective blocker of CALHM channels, partially reversed established experimental PH. These data suggest that CALHM upregulation is involved in PASMC contractile-to-proliferative phenotypical transition. Ca2+ influx through upregulated CALHM1/2 may play an important role in the transition of sustained vasoconstriction to excessive vascular remodeling in PAH or precapillary PH. Calcium homeostasis modulator could potentially be a target to develop novel therapies for PAH.