Pathophysiology and pathogenic mechanisms of pulmonary hypertension: role of membrane receptors, ion channels, and Ca2+ signaling.

The pulmonary circulation is a low-resistance, low-pressure, and high-compliance system that allows the lungs to receive the entire cardiac output. Pulmonary arterial pressure is a function of cardiac output and pulmonary vascular resistance, and pulmonary vascular resistance is inversely proportional to the fourth power of the intraluminal radius of the pulmonary artery. Therefore, a very small decrease of the pulmonary vascular lumen diameter results in a significant increase in pulmonary vascular resistance and pulmonary arterial pressure. Pulmonary arterial hypertension is a fatal and progressive disease with poor prognosis. Regardless of the initial pathogenic triggers, sustained pulmonary vasoconstriction, concentric vascular remodeling, occlusive intimal lesions, in situ thrombosis, and vascular wall stiffening are the major and direct causes for elevated pulmonary vascular resistance in patients with pulmonary arterial hypertension and other forms of precapillary pulmonary hypertension. In this review, we aim to discuss the basic principles and physiological mechanisms involved in the regulation of lung vascular hemodynamics and pulmonary vascular function, the changes in the pulmonary vasculature that contribute to the increased vascular resistance and arterial pressure, and the pathogenic mechanisms involved in the development and progression of pulmonary hypertension. We focus on reviewing the pathogenic roles of membrane receptors, ion channels, and intracellular Ca2+ signaling in pulmonary vascular smooth muscle cells in the development and progression of pulmonary hypertension.

eNAMPT is a novel therapeutic target for mitigation of coronary microvascular disease in type 2 diabetes.

AIMS/HYPOTHESIS: Individuals with diabetes are at high risk of cardiovascular complications, which significantly increase morbidity/mortality. Coronary microvascular disease (CMD) is recognised as a critical contributor to the increased cardiac mortality observed in people with diabetes. Therefore, there is an urgent need for treatments that are specific to CMD. eNAMPT (extracellular nicotinamide phosphoribosyltransferase) is a damage-associated molecular pattern and TLR4 ligand, whose plasma levels are elevated in people with diabetes. This study was thus designed to investigate the pathogenic role of intracellular nicotinamide phosphoribosyltransferase (iNAMPT) and eNAMPT in promoting the development of CMD in a preclinical murine model of type 2 diabetes. METHODS: An inducible type 2 diabetic mouse model was generated by a single injection of low-dose streptozocin (75 mg/kg, i.p.) combined with a high-fat diet for 16 weeks. The in vivo effects of i/eNAMPT inhibition on cardiac endothelial cell (CEC) function were evaluated by using Nampt+/- heterozygous mice, chronic administration of eNAMPT-neutralising monoclonal antibody (mAb) or use of an NAMPT enzymatic inhibitor (FK866). RESULTS: As expected, diabetic wild-type mice exhibited significantly lower coronary flow velocity reserve (CFVR), a determinant of coronary microvascular function, compared with control wild-type mice. eNAMPT plasma levels or expression in CECs were significantly greater in diabetic mice than in control mice. Furthermore, in comparison with diabetic wild-type mice, diabetic Nampt+/- heterozygous mice showed markedly improved CFVR, accompanied by increased left ventricular capillary density and augmented endothelium-dependent relaxation (EDR) in the coronary artery. NAMPT inhibition by FK866 or an eNAMPT-neutralising mAb significantly increased CFVR in diabetic mice. Furthermore, administration of the eNAMPT mAb upregulated expression of angiogenesis- and EDR-related genes in CECs from diabetic mice. Treatment with either eNAMPT or NAD+ significantly decreased CEC migration and reduced EDR in coronary arteries, partly linked to increased production of mitochondrial reactive oxygen species. CONCLUSIONS/INTERPRETATION: These data indicate that increased i/eNAMPT expression contributes to the development of diabetic coronary microvascular dysfunction, and provide compelling support for eNAMPT inhibition as a novel and effective therapeutic strategy for CMD in diabetes.

Enhanced lung endothelial glycolysis is implicated in the development of severe pulmonary hypertension in type 2 diabetes.

Metabolic abnormalities in pulmonary endothelial cells are implicated in pulmonary hypertension (PH) while increasing evidence shows the influence of diabetes on progressing PH. In this study, we examined the effect of type 2 diabetes on hypoxia-induced PH and investigated its molecular mechanisms using hypoxia-induced diabetic male mice. Chronic hypoxia led to a more severe PH in type 2 diabetic mice than in control mice. Next, we compared gene expression patterns in isolated pulmonary endothelial cells (MPECs) from control mice in normoxia (CN), diabetic mice in normoxia (DN), control mice exposed to hypoxia (CH), and diabetic mice exposed to hypoxia (DH). The results showed that expression levels of 27 mRNAs, out of 92 mRNAs, were significantly different among the four groups. Two glycolysis-related proteins, GAPDH and HK2, were increased in MPECs of DH mice compared to those in DN or CH mice. In addition, the levels of pyruvate and lactate (glycolysis end products) were significantly increased in MPECs of DH mice, but not in CH mice, compared to MPECs of CN mice. Augmentation of glycolysis by terazosin exacerbated hypoxia-induced PH in CH mice but not in DH mice. On the contrary, inhibiting GAPDH (a key enzyme of the glycolytic pathway) by koningic acid ameliorated hypoxia-induced PH in DH mice but had no effect in CH mice. These data suggest that enhanced glycolysis in diabetic mice is involved in severe hypoxia-induced PH, and glycolysis inhibition is a potential target to reduce the severe progression of PH in diabetic patients.

Mechanosensitive cation currents through TRPC6 and Piezo1 channels in human pulmonary arterial endothelial cells.

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.

Mechanosensitive channel Piezo1 is required for pulmonary artery smooth muscle cell proliferation.

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.

Endothelial upregulation of mechanosensitive channel Piezo1 in pulmonary hypertension.

Piezo is a mechanosensitive cation channel responsible for stretch-mediated Ca2+ and Na+ influx in multiple types of cells. Little is known about the functional role of Piezo1 in the lung vasculature and its potential pathogenic role in pulmonary arterial hypertension (PAH). Pulmonary arterial endothelial cells (PAECs) are constantly under mechanic stretch and shear stress that are sufficient to activate Piezo channels. Here, we report that Piezo1 is significantly upregulated in PAECs from patients with idiopathic PAH and animals with experimental pulmonary hypertension (PH) compared with normal controls. Membrane stretch by decreasing extracellular osmotic pressure or by cyclic stretch (18% CS) increases Ca2+-dependent phosphorylation (p) of AKT and ERK, and subsequently upregulates expression of Notch ligands, Jagged1/2 (Jag-1 and Jag-2), and Delta like-4 (DLL4) in PAECs. siRNA-mediated downregulation of Piezo1 significantly inhibited the stretch-mediated pAKT increase and Jag-1 upregulation, whereas downregulation of AKT by siRNA markedly attenuated the stretch-mediated Jag-1 upregulation in human PAECs. Furthermore, the mRNA and protein expression level of Piezo1 in the isolated pulmonary artery, which mainly contains pulmonary arterial smooth muscle cells (PASMCs), from animals with severe PH was also significantly higher than that from control animals. Intraperitoneal injection of a Piezo1 channel blocker, GsMTx4, ameliorated experimental PH in mice. Taken together, our study suggests that membrane stretch-mediated Ca2+ influx through Piezo1 is an important trigger for pAKT-mediated upregulation of Jag-1 in PAECs. Upregulation of the mechanosensitive channel Piezo1 and the resultant increase in the Notch ligands (Jag-1/2 and DLL4) in PAECs may play a critical pathogenic role in the development of pulmonary vascular remodeling in PAH and PH.

TRPC6, a therapeutic target for pulmonary hypertension.

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 ∼50% 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.

mTOR Signaling in Pulmonary Vascular Disease: Pathogenic Role and Therapeutic Target.

Pulmonary arterial hypertension (PAH) is a progressive and fatal disease without a cure. The exact pathogenic mechanisms of PAH are complex and poorly understood, yet a number of abnormally expressed genes and regulatory pathways contribute to sustained vasoconstriction and vascular remodeling of the distal pulmonary arteries. Mammalian target of rapamycin (mTOR) is one of the major signaling pathways implicated in regulating cell proliferation, migration, differentiation, and protein synthesis. Here we will describe the canonical mTOR pathway, structural and functional differences between mTOR complexes 1 and 2, as well as the crosstalk with other important signaling cascades in the development of PAH. The pathogenic role of mTOR in pulmonary vascular remodeling and sustained vasoconstriction due to its contribution to proliferation, migration, phenotypic transition, and gene regulation in pulmonary artery smooth muscle and endothelial cells will be discussed. Despite the progress in our elucidation of the etiology and pathogenesis of PAH over the two last decades, there is a lack of effective therapeutic agents to treat PAH patients representing a significant unmet clinical need. In this review, we will explore the possibility and therapeutic potential to use inhibitors of mTOR signaling cascade to treat PAH.

Notch enhances Ca2+ entry by activating calcium-sensing receptors and inhibiting voltage-gated K+ channels.

The increase in cytosolic Ca2+ concentration ([Ca2+]cyt) and upregulation of calcium-sensing receptor (CaSR) and stromal interaction molecule 2 (STIM2) along with inhibition of voltage-gated K+ (KV) channels in pulmonary arterial smooth muscle cells (PASMC) have been implicated in the development of pulmonary arterial hypertension; however, the precise upstream mechanisms remain elusive. Activation of CaSR, a G protein-coupled receptor (GPCR), results in Ca2+ release from the endoplasmic/sarcoplasmic reticulum (ER/SR) and Ca2+ influx through receptor-operated and store-operated Ca2+ channels (SOC). Upon Ca2+ depletion from the SR, STIM forms clusters to mediate store-operated Ca2+ entry. Activity of KV channels, like KCNA5/KV1.5 and KCNA2/KV1.2, contributes to regulating membrane potential, and inhibition of KV channels results in membrane depolarization that increases [Ca2+]cyt by opening voltage-dependent Ca2+ channels. In this study, we show that activation of Notch by its ligand Jag-1 promotes the clustering of STIM2, and clustered STIM2 subsequently enhances the CaSR-induced Ca2+ influx through SOC channels. Extracellular Ca2+-mediated activation of CaSR increases [Ca2+]cyt in CASR-transfected HEK293 cells. Treatment of CASR-transfected cells with Jag-1 further enhances CaSR-mediated increase in [Ca2+]cyt. Moreover, CaSR-mediated increase in [Ca2+]cyt was significantly augmented in cells co-transfected with CASR and STIM2. CaSR activation results in STIM2 clustering in CASR/STIM2-cotransfected cells. Notch activation also induces significant clustering of STIM2. Furthermore, activation of Notch attenuates whole cell K+ currents in KCNA5- and KCNA2-transfected cells. Together, these results suggest that Notch activation enhances CaSR-mediated increases in [Ca2+]cyt by enhancing store-operated Ca2+ entry and inhibits KCNA5/KV1.5 and KCNA2/KV1.2, ultimately leading to voltage-activated Ca2+ entry.

MicroRNA-mediated downregulation of K+ channels in pulmonary arterial hypertension.

Downregulated expression of K+ channels and decreased K+ currents in pulmonary artery smooth muscle cells (PASMC) have been implicated in the development of sustained pulmonary vasoconstriction and vascular remodeling in patients with idiopathic pulmonary arterial hypertension (IPAH). However, it is unclear exactly how K+ channels are downregulated in IPAH-PASMC. MicroRNAs (miRNAs) are small non-coding RNAs that are capable of posttranscriptionally regulating gene expression by binding to the 3'-untranslated regions of their targeted mRNAs. Here, we report that specific miRNAs are responsible for the decreased K+ channel expression and function in IPAH-PASMC. We identified 3 miRNAs (miR-29b, miR-138, and miR-222) that were highly expressed in IPAH-PASMC in comparison to normal PASMC (>2.5-fold difference). Selectively upregulated miRNAs are correlated with the decreased expression and attenuated activity of K+ channels. Overexpression of miR-29b, miR-138, or miR-222 in normal PASMC significantly decreased whole cell K+ currents and downregulated voltage-gated K+ channel 1.5 (KV1.5/KCNA5) in normal PASMC. Inhibition of miR-29b in IPAH-PASMC completely recovered K+ channel function and KV1.5 expression, while miR-138 and miR-222 had a partial or no effect. Luciferase assays further revealed that KV1.5 is a direct target of miR-29b. Additionally, overexpression of miR-29b in normal PASMC decreased large-conductance Ca2+-activated K+ (BKCa) channel currents and downregulated BKCa channel ß1 subunit (BKCaß1 or KCNMB1) expression, while inhibition of miR-29b in IPAH-PASMC increased BKCa channel activity and BKCaß1 levels. These data indicate upregulated miR-29b contributes at least partially to the attenuated function and expression of KV and BKCa channels in PASMC from patients with IPAH.

Pulmonary vascular dysfunction in metabolic syndrome.

Metabolic syndrome is a critically important precursor to the onset of many diseases, such as cardiovascular disease, and cardiovascular disease is the leading cause of death worldwide. The primary risk factors of metabolic syndrome include hyperglycaemia, abdominal obesity, dyslipidaemia, and high blood pressure. It has been well documented that metabolic syndrome alters vascular endothelial and smooth muscle cell functions in the heart, brain, kidney and peripheral vessels. However, there is less information available regarding how metabolic syndrome can affect pulmonary vascular function and ultimately increase an individual's risk of developing various pulmonary vascular diseases, such as pulmonary hypertension. Here, we review in detail how metabolic syndrome affects pulmonary vascular function.

Divergent changes of p53 in pulmonary arterial endothelial and smooth muscle cells involved in the development of pulmonary hypertension.

The tumor-suppressive role of p53, a transcription factor that regulates the expression of many genes, has been linked to cell cycle arrest, apoptosis, and senescence. The noncanonical function or the pathogenic role of p53 has more recently been implicated in pulmonary vascular disease. We previously reported that rapid nuclear accumulation of hypoxia-inducible factor (HIF)-1α in pulmonary arterial smooth muscle cells (PASMCs) upregulates transient receptor potential channels and enhances Ca2+ entry to increase cytosolic Ca2+ concentration ([Ca2+]cyt). Also, we observed differences in HIF-1α/2α expression in PASMCs and pulmonary arterial endothelial cells (PAECs). Here we report that p53 is increased in PAECs, but decreased in PASMCs, isolated from mice with hypoxia-induced pulmonary hypertension (PH) and rats with monocrotaline (MCT)-induced PH (MCT-PH). The increased p53 in PAECs from rats with MCT-PH is associated with an increased ratio of Bax/Bcl-2, while the decreased p53 in PASMCs is associated with an increased HIF-1α. Furthermore, p53 is downregulated in PASMCs isolated from patients with idiopathic pulmonary arterial hypertension compared with PASMCs from normal subjects. Overexpression of p53 in normal PASMCs inhibits store-operated Ca2+ entry (SOCE) induced by passive depletion of intracellularly stored Ca2+ in the sarcoplasmic reticulum, while downregulation of p53 enhances SOCE. These data indicate that differentially regulated expression of p53 and HIF-1α/2α in PASMCs and PAECs and the cross talk between p53 and HIF-1α/2α in PASMCs and PAECs may play an important role in the development of PH via, at least in part, induction of PAEC apoptosis and PASMC proliferation.

Akt2 (Protein Kinase B Beta) Stabilizes ATP7A, a Copper Transporter for Extracellular Superoxide Dismutase, in Vascular Smooth Muscle: Novel Mechanism to Limit Endothelial Dysfunction in Type 2 Diabetes Mellitus.

OBJECTIVE: Copper transporter ATP7A (copper-transporting/ATPase) is required for full activation of SOD3 (extracellular superoxide dismutase), which is secreted from vascular smooth muscle cells (VSMCs) and anchors to endothelial cell surface to preserve endothelial function by scavenging extracellular superoxide. We reported that ATP7A protein expression and SOD3 activity are decreased in insulin-deficient type 1 diabetes mellitus vessels, thereby, inducing superoxide-mediated endothelial dysfunction, which are rescued by insulin treatment. However, it is unknown regarding the mechanism by which insulin increases ATP7A expression in VSMCs and whether ATP7A downregulation is observed in T2DM (type2 diabetes mellitus) mice and human in which insulin-Akt (protein kinase B) pathway is selectively impaired. APPROACH AND RESULTS: Here we show that ATP7A protein is markedly downregulated in vessels isolated from T2DM patients, as well as those from high-fat diet-induced or db/db T2DM mice. Akt2 (protein kinase B beta) activated by insulin promotes ATP7A stabilization via preventing ubiquitination/degradation as well as translocation to plasma membrane in VSMCs, which contributes to activation of SOD3 that protects against T2DM-induced endothelial dysfunction. Downregulation of ATP7A in T2DM vessels is restored by constitutive active Akt or PTP1B-/- (protein-tyrosine phosphatase 1B-deficient) T2DM mice, which enhance insulin-Akt signaling. Immunoprecipitation, in vitro kinase assay, and mass spectrometry analysis reveal that insulin stimulates Akt2 binding to ATP7A to induce phosphorylation at Ser1424/1463/1466. Furthermore, SOD3 activity is reduced in Akt2-/- vessels or VSMCs, which is rescued by ATP7A overexpression. CONCLUSION: Akt2 plays a critical role in ATP7A protein stabilization and translocation to plasma membrane in VSMCs, which contributes to full activation of vascular SOD3 that protects against endothelial dysfunction in T2DM.

Overexpression of hexokinase 2 reduces mitochondrial calcium overload in coronary endothelial cells of type 2 diabetic mice.

Coronary microvascular rarefaction, due to endothelial cell (EC) dysfunction, is one of the causes of increased morbidity and mortality in diabetes. Coronary ECs in diabetes are more apoptotic due partly to mitochondrial calcium overload. This study was designed to investigate the role of hexokinase 2 (HK2, an endogenous inhibitor of voltage-dependent anion channel) in coronary endothelial dysfunction in type 2 diabetes. We used mouse coronary ECs (MCECs) isolated from type 2 diabetic mice and human coronary ECs (HCECs) from type 2 diabetic patients to examine protein levels and mitochondrial function. ECs were more apoptotic and capillary density was lower in the left ventricle of diabetic mice than the control. MCECs from diabetic mice exhibited significant increase in mitochondrial Ca2+ concentration ([Ca2+]mito) compared with the control. Among several regulatory proteins for [Ca2+]mito, hexokinase 1 (HK1) and HK2 were significantly lower in MCECs from diabetic mice than control MCECs. We also found that the level of HK2 ubiquitination was higher in MCECs from diabetic mice than in control MCECs. In line with the data from MCECs, HCECs from diabetic patients showed lower HK2 protein levels than HCECs from nondiabetic patients. High-glucose treatment, but not high-fat treatment, significantly decreased HK2 protein levels in MCECs. HK2 overexpression in MCECs of diabetic mice not only lowered the level of [Ca2+]mito, but also reduced mitochondrial reactive oxygen species production toward the level seen in control MCECs. These data suggest that HK2 is a potential therapeutic target for coronary microvascular disease in diabetes by restoring mitochondrial function in coronary ECs.

Hypoxia selectively upregulates cation channels and increases cytosolic [Ca2+] in pulmonary, but not coronary, arterial smooth muscle cells.

Ca2+ signaling, particularly the mechanism via store-operated Ca2+ entry (SOCE) and receptor-operated Ca2+ entry (ROCE), plays a critical role in the development of acute hypoxia-induced pulmonary vasoconstriction and chronic hypoxia-induced pulmonary hypertension. This study aimed to test the hypothesis that chronic hypoxia differentially regulates the expression of proteins that mediate SOCE and ROCE [stromal interacting molecule (STIM), Orai, and canonical transient receptor potential channel TRPC6] in pulmonary (PASMC) and coronary (CASMC) artery smooth muscle cells. The resting cytosolic [Ca2+] ([Ca2+]cyt) and the stored [Ca2+] in the sarcoplasmic reticulum were not different in CASMC and PASMC. Seahorse measurement showed a similar level of mitochondrial bioenergetics (basal respiration and ATP production) between CASMC and PASMC. Glycolysis was significantly higher in PASMC than in CASMC. The amplitudes of cyclopiazonic acid-induced SOCE and OAG-induced ROCE in CASMC are slightly, but significantly, greater than in PASMC. The frequency and the area under the curve of Ca2+ oscillations induced by ATP and histamine were also larger in CASMC than in PASMC. Na+/Ca2+ exchanger-mediated increases in [Ca2+]cyt did not differ significantly between CASMC and PASMC. The basal protein expression levels of STIM1/2, Orai1/2, and TRPC6 were higher in CASMC than in PASMC, but hypoxia (3% O2 for 72 h) significantly upregulated protein expression levels of STIM1/STIM2, Orai1/Orai2, and TRPC6 and increased the resting [Ca2+]cyt only in PASMC, but not in CASMC. The different response of essential components of store-operated and receptor-operated Ca2+ channels to hypoxia is a unique intrinsic property of PASMC, which is likely one of the important explanations why hypoxia causes pulmonary vasoconstriction and induces pulmonary vascular remodeling, but causes coronary vasodilation.

Endothelial HIF-2α contributes to severe pulmonary hypertension due to endothelial-to-mesenchymal transition.

Pulmonary vascular remodeling characterized by concentric wall thickening and intraluminal obliteration is a major contributor to the elevated pulmonary vascular resistance in patients with idiopathic pulmonary arterial hypertension (IPAH). Here we report that increased hypoxia-inducible factor 2α (HIF-2α) in lung vascular endothelial cells (LVECs) under normoxic conditions is involved in the development of pulmonary hypertension (PH) by inducing endothelial-to-mesenchymal transition (EndMT), which subsequently results in vascular remodeling and occlusive lesions. We observed significant EndMT and markedly increased expression of SNAI, an inducer of EndMT, in LVECs from patients with IPAH and animals with experimental PH compared with normal controls. LVECs isolated from IPAH patients had a higher level of HIF-2α than that from normal subjects, whereas HIF-1α was upregulated in pulmonary arterial smooth muscle cells (PASMCs) from IPAH patients. The increased HIF-2α level, due to downregulated prolyl hydroxylase domain protein 2 (PHD2), a prolyl hydroxylase that promotes HIF-2α degradation, was involved in enhanced EndMT and upregulated SNAI1/2 in LVECs from patients with IPAH. Moreover, knockdown of HIF-2α (but not HIF-1α) with siRNA decreases both SNAI1 and SNAI2 expression in IPAH-LVECs. Mice with endothelial cell (EC)-specific knockout (KO) of the PHD2 gene, egln1 (egln1EC-/-), developed severe PH under normoxic conditions, whereas Snai1/2 and EndMT were increased in LVECs of egln1EC-/- mice. EC-specific KO of the HIF-2α gene, hif2a, prevented mice from developing hypoxia-induced PH, whereas EC-specific deletion of the HIF-1α gene, hif1a, or smooth muscle cell (SMC)-specific deletion of hif2a, negligibly affected the development of PH. Also, exposure to hypoxia for 48-72 h increased protein level of HIF-1α in normal human PASMCs and HIF-2α in normal human LVECs. These data indicate that increased HIF-2α in LVECs plays a pathogenic role in the development of severe PH by upregulating SNAI1/2, inducing EndMT, and causing obliterative pulmonary vascular lesions and vascular remodeling.

Mitochondrial connexin40 regulates mitochondrial calcium uptake in coronary endothelial cells.

Connexins (Cxs) are a group of integral membrane proteins that can form gap junctions between adjacent cells. Recently, it was reported that Cx43 is expressed not only in the plasma membrane but also in the inner mitochondrial membrane and that it regulates mitochondrial functions. Cx40 is predominantly expressed in vascular endothelial cells (ECs) and plays an important role in the electrical propagation between ECs and endothelial/smooth muscle cells. However, it is unknown whether Cx40 is expressed in the mitochondria and what the role of mitochondrial Cx40 is in endothelial functions. We observed in coronary ECs that Cx40 protein was expressed in the mitochondria, as determined by Western blot and immunofluorescence studies. We found that mouse coronary ECs (MCECs) isolated from Cx40 knockout (Cx40 KO) mice exhibited significantly lower resting mitochondrial calcium concentration ([Ca2+]mito) than MCECs from wild-type (WT) mice. After increase in cytosolic Ca2+ concentration ([Ca2+]cyto) with cyclopiazonic acid, calcium uptake into the mitochondria was significantly attenuated in MCECs from Cx40 KO mice compared with WT MCECs. There was no difference in resting [Ca2+]cyto and store-operated calcium entry in MCECs from WT and Cx40 KO mice. We also detected a significant decrease in the concentration of mitochondrial reactive oxygen species (ROS) in Cx40 KO MCECs. Cx40 overexpression in ECs significantly increased resting [Ca2+]mito level and calcium uptake by mitochondria in response to increased [Ca2+]cyto and augmented mitochondrial ROS production. These data suggest that mitochondrial Cx40 contributes to the regulation of mitochondrial calcium homeostasis.

Capsaicin-induced Ca2+ signaling is enhanced via upregulated TRPV1 channels in pulmonary artery smooth muscle cells from patients with idiopathic PAH.

Capsaicin is an active component of chili pepper and a pain relief drug. Capsaicin can activate transient receptor potential vanilloid 1 (TRPV1) channels to increase cytosolic Ca2+ concentration ([Ca2+]cyt). A rise in [Ca2+]cyt in pulmonary artery smooth muscle cells (PASMCs) is an important stimulus for pulmonary vasoconstriction and vascular remodeling. In this study, we observed that a capsaicin-induced increase in [Ca2+]cyt was significantly enhanced in PASMCs from patients with idiopathic pulmonary arterial hypertension (IPAH) compared with normal PASMCs from healthy donors. In addition, the protein expression level of TRPV1 in IPAH PASMCs was greater than in normal PASMCs. Increasing the temperature from 23 to 43°C, or decreasing the extracellular pH value from 7.4 to 5.9 enhanced capsaicin-induced increases in [Ca2+]cyt; the acidity (pH 5.9)- and heat (43°C)-mediated enhancement of capsaicin-induced [Ca2+]cyt increases were greater in IPAH PASMCs than in normal PASMCs. Decreasing the extracellular osmotic pressure from 310 to 200 mOsmol/l also increased [Ca2+]cyt, and the hypo-osmolarity-induced rise in [Ca2+]cyt was greater in IPAH PASMCs than in healthy PASMCs. Inhibition of TRPV1 (with 5'-IRTX or capsazepine) or knockdown of TRPV1 (with short hairpin RNA) attenuated capsaicin-, acidity-, and osmotic stretch-mediated [Ca2+]cyt increases in IPAH PASMCs. Capsaicin induced phosphorylation of CREB by raising [Ca2+]cyt, and capsaicin-induced CREB phosphorylation were significantly enhanced in IPAH PASMCs compared with normal PASMCs. Pharmacological inhibition and knockdown of TRPV1 attenuated IPAH PASMC proliferation. Taken together, the capsaicin-mediated [Ca2+]cyt increase due to upregulated TRPV1 may be a critical pathogenic mechanism that contributes to augmented Ca2+ influx and excessive PASMC proliferation in patients with IPAH.

ATP promotes cell survival via regulation of cytosolic [Ca2+] and Bcl-2/Bax ratio in lung cancer cells.

Adenosine triphosphate (ATP) is a ubiquitous extracellular messenger elevated in the tumor microenvironment. ATP regulates cell functions by acting on purinergic receptors (P2X and P2Y) and activating a series of intracellular signaling pathways. We examined ATP-induced Ca(2+) signaling and its effects on antiapoptotic (Bcl-2) and proapoptotic (Bax) proteins in normal human airway epithelial cells and lung cancer cells. Lung cancer cells exhibited two phases (transient and plateau phases) of increase in cytosolic [Ca(2+)] ([Ca(2+)]cyt) caused by ATP, while only the transient phase was observed in normal cells. Removal of extracellular Ca(2+) eliminated the plateau phase increase of [Ca(2+)]cyt in lung cancer cells, indicating that the plateau phase of [Ca(2+)]cyt increase is due to Ca(2+) influx. The distribution of P2X (P2X1-7) and P2Y (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11) receptors was different between lung cancer cells and normal cells. Proapoptotic P2X7 was nearly undetectable in lung cancer cells, which may explain why lung cancer cells showed decreased cytotoxicity when treated with high concentration of ATP. The Bcl-2/Bax ratio was increased in lung cancer cells following treatment with ATP; however, the antiapoptotic protein Bcl-2 demonstrated more sensitivity to ATP than proapoptotic protein Bax. Decreasing extracellular Ca(2+) or chelating intracellular Ca(2+) with BAPTA-AM significantly inhibited ATP-induced increase in Bcl-2/Bax ratio, indicating that a rise in [Ca(2+)]cyt through Ca(2+) influx is the critical mediator for ATP-mediated increase in Bcl-2/Bax ratio. Therefore, despite high ATP levels in the tumor microenvironment, which would induce cell apoptosis in normal cells, the decreased P2X7 and elevated Bcl-2/Bax ratio in lung cancer cells may enable tumor cells to survive. Increasing the Bcl-2/Bax ratio by exposure to high extracellular ATP may, therefore, be an important selective pressure promoting transformation and cancer progression.

Pathogenic role of calcium-sensing receptors in the development and progression of pulmonary hypertension.

An increase in cytosolic free Ca(2+) concentration ([Ca(2+)]cyt) in pulmonary arterial smooth muscle cells (PASMC) is a major trigger for pulmonary vasoconstriction and a critical stimulation for PASMC proliferation and migration. Previously, we demonstrated that expression and function of calcium sensing receptors (CaSR) in PASMC from patients with idiopathic pulmonary arterial hypertension (IPAH) and animals with experimental pulmonary hypertension (PH) were greater than in PASMC from normal subjects and control animals. However, the mechanisms by which CaSR triggers Ca(2+) influx in PASMC and the implication of CaSR in the development of PH remain elusive. Here, we report that CaSR functionally interacts with TRPC6 to regulate [Ca(2+)]cyt in PASMC. Downregulation of CaSR or TRPC6 with siRNA inhibited Ca(2+)-induced [Ca(2+)]cyt increase in IPAH-PASMC (in which CaSR is upregulated), whereas overexpression of CaSR or TRPC6 enhanced Ca(2+)-induced [Ca(2+)]cyt increase in normal PASMC (in which CaSR expression level is low). The upregulated CaSR in IPAH-PASMC was also associated with enhanced Akt phosphorylation, whereas blockade of CaSR in IPAH-PASMC attenuated cell proliferation. In in vivo experiments, deletion of the CaSR gene in mice (casr(-/-)) significantly inhibited the development and progression of experimental PH and markedly attenuated acute hypoxia-induced pulmonary vasoconstriction. These data indicate that functional interaction of upregulated CaSR and upregulated TRPC6 in PASMC from IPAH patients and animals with experimental PH may play an important role in the development and progression of sustained pulmonary vasoconstriction and pulmonary vascular remodeling. Blockade or downregulation of CaSR and/or TRPC6 with siRNA or miRNA may be a novel therapeutic strategy to develop new drugs for patients with pulmonary arterial hypertension.