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Activity of voltage-gated K+(KV) channels in pulmonary artery smooth muscle cells (PASMC) plays an important role in control of apoptosis and proliferation in addition to regulating membrane potential and pulmonary vascular tone. Bone morphogenetic proteins (BMPs) inhibit proliferation and induce apoptosis in normal human PASMC, whereas dysfunctional BMP signaling and downregulated KVchannels are involved in pulmonary vascular medial hypertrophy associated with pulmonary hypertension. This study evaluated the effect of BMP-2 on KVchannel function and expression in normal human PASMC. BMP-2 (100 nM for 18–24 h) significantly (>2-fold) upregulated mRNA expression of KCNA5, KCNA7, KCNA10, KCNC3, KCNC4, KCNF1, KCNG3, KCNS1, and KCNS3 but downregulated (at least 2-fold) KCNAB1, KCNA2, KCNG2, and KCNV2. The most dramatic change was the >10-fold downregulation of KCNG2 and KCNV2, two electrically silent γ-subunits that form heterotetramers with functional KVchannel α-subunits (e.g., KCNB1–2). Furthermore, the amplitude and current density of whole cell KVcurrents were significantly increased in PASMC treated with BMP-2. It has been demonstrated that K+currents generated by KCNB1 and KCNG1 (or KCNG2) or KCNB1 and KCNV2 heterotetramers are smaller than those generated by KCNB1 homotetramers, indicating that KCNG2 and KCNV2 (2 subunits that were markedly downregulated by BMP-2) are inhibitors of functional KVchannels. These results suggest that BMP-2 divergently regulates mRNA expression of various KVchannel α-, β-, and γ-subunits and significantly increases whole cell KVcurrents in human PASMC. Finally, we present evidence that attenuation of c-Myc expression by BMP-2 may be involved in BMP-2-mediated increase in KVchannel activity and regulation of KVchannel expression. The increased KVchannel activity may be involved in the proapoptotic and/or antiproliferative effects of BMP-2 on PASMC.

Acute hypoxia causes pulmonary vasoconstriction in part by inhibiting voltage-gated K+ (Kv) channel activity in pulmonary artery smooth muscle cells (PASMC). The hypoxia-mediated decrease in Kv currents [ IK(V)] is selective to PASMC; hypoxia has little effect on IK(V) in mesenteric artery smooth muscle cells (MASMC). Functional Kv channels are homo- and/or heterotetramers of pore-forming α-subunits and regulatory β-subunits. KCNA5 is a Kv channel α-subunit that forms functional Kv channels in PASMC and regulates resting membrane potential. We have shown that acute hypoxia selectively inhibits IK(V) through KCNA5 channels in PASMC. Overexpression of the human KCNA5 gene increased IK(V) and caused membrane hyperpolarization in HEK-293, COS-7, and rat MASMC and PASMC. Acute hypoxia did not affect IK(V) in KCNA5-transfected HEK-293 and COS-7 cells. However, overexpression of KCNA5 in PASMC conferred its sensitivity to hypoxia. Reduction of Po2 from 145 to 35 mmHg reduced IK(V) by ∼40% in rat PASMC transfected with human KCNA5 but had no effect on IK(V) in KCNA5-transfected rat MASMC (or HEK and COS cells). These results indicate that KCNA5 is an important Kv channel that regulates resting membrane potential and that acute hypoxia selectively reduces KCNA5 channel activity in PASMC relative to MASMC and other cell types. Because Kv channels (including KCNA5) are ubiquitously expressed in PASMC and MASMC, the observation from this study indicates that a hypoxia-sensitive mechanism essential for inhibiting KCNA5 channel activity is exclusively present in PASMC. The divergent effect of hypoxia on IK(V) in PASMC and MASMC also may be due to different expression levels of KCNA5 channels.

Transient receptor potential (TRP) genes represent a novel class of genes that are generally believed to encode for nonselective cation channels. A subfamily of TRP channels, canonical TRP (TRPC), which are highly permeable to Ca2+ (and Na+), co-assembles with each other to form functional store- and receptor-operated Ca2+ channels. TRPC mRNA and protein have been identified in pulmonary arterial smooth muscle and endothelial cells. The currents generated by Ca2+ influx through store- and receptor-operated Ca2+ channels have also been extensively characterized in these cells. More recently, the attention has shifted to identify the TRP subunits that underlie the function of native channels in the pulmonary vasculature, with the understanding that TRP channels assemble as either homo-or heterotetramers in vivo. This work in progress has yielded exciting information regarding the involvement of TRP channels in the control of smooth muscle contraction, and cell proliferation and migration. In this review, the authors focus on describing the function and transcriptional regulation of TRP proteins, and the store- and receptor-operated Ca2+ channels for which they are responsible, in pulmonary artery smooth muscle cells (PASMC). They also identify some key TRP proteins whose role in the pulmonary vasculature has been established, as well as some more novel subunits whose role, although intriguing, can only be inferred from other vascular studies. Finally, they describe the involvement of TRP channels in regulating pulmonary vasoconstriction, PASMC proliferation, and pulmonary endothelial barrier function.

ANG-(1–7) improves the function of the remodeling heart. Although this peptide is generated directly within the myocardium, the effects of ANG-(1–7) on cardiac fibroblasts that play a critical role in cardiac remodeling are largely unknown. We tested the hypothesis that specific binding of ANG-(1–7) to cardiac fibroblasts regulates cellular functions that are involved in cardiac remodeling. 125I-labeled ANG-(1–7) binding assays identified specific binding sites of ANG-(1–7) on adult rat cardiac fibroblasts (ARCFs) with an affinity of 11.3 nM and a density of 131 fmol/mg protein. At nanomolar concentrations, ANG-(1–7) interacted with specific sites that were distinct from ANG II type 1 and type 2 receptors without increasing cytosolic Ca2+ concentration. At these concentrations, ANG-(1–7) had inhibitory effects on collagen synthesis as assessed by [3H]proline incorporation and decreased mRNA expression of growth factors in ARCFs. These effects of ANG-(1–7) contrasted with effects of ANG II. Pretreatment of ARCFs with ANG-(1–7) inhibited ANG II-induced increases in collagen synthesis and in mRNA expression of growth factors, including endothelin-1 and leukemia inhibitory factor. ANG-(1–7) pretreatment also inhibited the stimulatory effects of conditioned medium from ANG II-treated ARCFs on [3H]leucine incorporation and atrial natriuretic factor mRNA expression, markers of hypertrophy, in cardiomyocytes. Thus ANG-(1–7) interacted with specific receptors on ARCFs to exert potential antifibrotic and antitrophic effects that could reverse ANG II effects. These results suggest that ANG-(1–7) may play an important role in the heart in regulating cardiac remodeling.

most membrane receptors in mammalian systems are seven-transmembrane domain G protein-coupled receptors. Proteinase (or protease)-activated receptors (PARs) are a novel family of G protein-coupled receptors. Unlike other G protein-coupled receptors that require ligand binding for activation, PARs

Bone morphogenetic proteins (BMPs) inhibit proliferation and induce apoptosis in pulmonary artery smooth muscle cells (PASMCs) from normal subjects. Dysfunction of BMP signaling due to mutations in and/or down-regulation of BMP receptors has been implicated in idiopathic pulmonary arterial hypertension (IPAH). The authors examined whether BMP differentially regulates gene expression in PASMCs from normal subjects and IPAH patients using the Affymetrix microarray analysis. BMP-2 treatment (200 nM for 24 hours) altered expression levels of 6206 genes in normal and IPAH PASMCs. Of these genes, 1063 were regulated oppositely by BMP-2: 523 genes were down-regulated by BMP-2 in normal PASMCs but up-regulated in IPAH PASMCs, whereas 540 genes were up-regulated by BMP-2 in normal PASMCs but down-regulated in IPAH PASMCs. The divergent effects of BMP-2 on gene expression profiles indicate that PASMCs may undergo significant phenotypic changes in IPAH patients during development of the disease. The transition of the antiproliferative effect of BMP-2 in normal PASMCs to its proliferative effect in IPAH patients is attributed potentially to its differential effect on expression patterns of various genes that are involved in cell proliferation and apoptosis. Among the 6206 BMP-2-sensitive genes, there are more than 1800 genes whose expression levels were negatively (correlation coefficient, r,-0.9) or positively (with r+ 0.9) correlated with the pulmonary arterial pressure. These results suggest that BMP-mediated gene regulation is significantly altered in PASMCs from IPAH patients and mRNA expression changes in BMP-regulated genes may be involved in the development of IPAH.

Hereditary long QT syndrome is a fatal arrhythmia associated with gene mutations in potassium and sodium channels that are expressed in ventricle. By employing heterologous expression and making comparisons to cells expressing wild-type human-ether-a-go-go-related protein (HERG), a potassium channel that contributes to I(Kr) current in ventricular cardiomyocytes, we demonstrate activation of an elevated endoplasmic reticulum (ER) stress response by the mutant I593R HERG potassium channel implicated in long QT syndrome type 2. I593R HERG is trafficking-impaired and forms Triton-insoluble aggregates. Expression of I593R HERG activates the unfolded protein response pathway and, separately, NF-kappaB signaling. ATF6, the activating transcription factor of the unfolded protein response pathway, is processed into the active transcription factor in the cells expressing I593R HERG. Consistent with ATF6 activation, the ER chaperones/calcium-binding proteins Grp78, Grp94, and calreticulin are elevated in I593R HERG-expressing cells. Coexpression of I593R HERG with wild-type HERG also results in ER stress pathway activation. By eliciting downstream links in signal transduction pathways associated with ER stress, expression of mutant trafficking impaired ion channels may contribute to disease etiology mechanisms that augment those associated with attenuated ion flux.

KCNH2 encodes Kv11.1 and underlies the rapidly activating delayed rectifier K+ current (IKr) in the heart. Loss-of-function KCNH2 mutations cause the type 2 long QT syndrome (LQT2), and most LQT2-linked missense mutations inhibit the trafficking of Kv11.1 channels. Drugs that bind to Kv11.1 and block IKr (e.g., E-4031) can act as pharmacological chaperones to increase the trafficking and functional expression for most LQT2 channels (pharmacological correction). We previously showed that LQT2 channels are selectively stored in a microtubule-dependent compartment within the endoplasmic reticulum (ER). We tested the hypothesis that pharmacological correction promotes the trafficking of LQT2 channels stored in this compartment. Confocal analyses of cells expressing the trafficking-deficient LQT2 channel G601S showed that the microtubule-dependent ER compartment is the transitional ER. Experiments with E-4031 and the protein synthesis inhibitor cycloheximide suggested that pharmacological correction promotes the trafficking of G601S stored in this compartment. Treating cells in E-4031 or ranolazine (a drug that blocks IKr and has a short half-life) for 30 min was sufficient to cause pharmacological correction. Moreover, the increased functional expression of G601S persisted 4–5 h after drug washout. Coexpression studies with a dominant-negative form of Rab11B, a small GTPase that regulates Kv11.1 trafficking, prevented the pharmacological correction of G601S trafficking from the transitional ER. These data suggest that pharmacological correction quickly increases the trafficking of LQT2 channels stored in the transitional ER via a Rab11B-dependent pathway, and we conclude that the pharmacological chaperone activity of drugs like ranolazine might have therapeutic potential.

Genetic alterations in pulmonary arterial hypertension (PAH) have become increasingly recognized in both known familial or heritable and sporadic or idiopathic PAH (IPAH). Unrecognized genetic alterations have now been found in up to 40% of the IPAH in which no familial predisposition is apparent.1 Bone morphogenetic protein (BMP) receptor II (BMPR-II) is the most common gene implicated in this hereditary form of PAH; furthermore, it is implicated in the pathogenesis of nonhereditary forms of PAH with a significant reduction in the expression of BMPR-II in both IPAH and experimental animal models of pulmonary hypertension (PH).2 BMPs represent the largest group of cytokines in the transforming growth factor-β superfamily and regulate growth, differentiation, and apoptosis in multiple cell types, whereas BMPR-II has been shown to have unique roles in differing cells. Article, see p 1159 BMPR-II is constitutively active at the cell membrane, and ligand stimulation initiates cross-linking with BMPR-I to form a receptor complex that is necessary to activate intracellular signaling. BMPR-II is most highly expressed in endothelial cells in the pulmonary vasculature, and BMPR-II activation leads to the increased proliferation and decreased apoptosis through Smad signaling.3,4 This is in contrast to pulmonary arterial smooth muscle cells, where BMP activation leads to the inhibition of proliferation and increased apoptosis through Smad signaling in large vessels, although in small pulmonary arteries, a proliferative effect is seen through activation of extracellular signal–regulated kinase and mitogen-activated protein kinase, which inhibits Smad signaling.5,6 It is these unique yet complementary functions that make BMPR-II mutations particularly damaging in the pulmonary circulation, leading to development of PAH. A dysfunctional mutation of BMPR-II, as in heritable PAH, or downregulation of protein expression, as in IPAH and animal models, can lead to endothelial dysfunction hallmarked by abnormal barrier function through …

Transient receptor potential (TRP) cation channels are a critical pathway for Ca2+entry during pulmonary artery (PA) smooth muscle contraction. However, whether canonical TRP (TRPC) subunits and which TRP channel isoforms are involved in store depletion-induced pulmonary vasoconstriction in vivo remain unclear. This study was designed to test whether overexpression of the human TRPC1 gene ( hTRPC1) in rat PA enhances pulmonary vasoconstriction due to store depletion-mediated Ca2+influx. The hTRPC1 was infected into rat PA rings with an adenoviral vector. RT-PCR and Western blot analyses confirmed the mRNA and protein expression of hTRPC1 in the arterial rings. The amplitude of active tension induced by 40 mM K+(40K) in PA rings infected with an empty adenoviral vector (647 ± 88 mg/mg) was similar to that in PA rings infected with hTRPC1 (703 ± 123 mg/mg, P = 0.3). However, the active tension due to capacitative Ca2+entry (CCE) induced by cyclopiazonic acid was significantly enhanced in PA rings overexpressing hTRPC1 (91 ± 13% of 40K-induced contraction) compared with rings infected with an empty adenoviral vector (61 ± 14%, P < 0.001). Endothelial expression of hTRPC1 was not involved since the CCE-induced vasoconstriction was also enhanced in endothelium-denuded PA rings infected with the adenoviral vector carrying hTRPC1. These observations demonstrate that hTRPC1 is an important Ca2+-permeable channel that mediates pulmonary vasoconstriction when PA smooth muscle cell intracellular Ca2+stores are depleted.

Primary pulmonary hypertension (PPH) is a rare but often fatal condition characterized by pulmonary artery remodeling leading to chronic elevation of pulmonary artery pressure in the absence of causes. The pathophysiology of PPH is not completely understood, but a number of recent studies have elucidated many possible gentic, hormonal, and environmental factors. Current treatment options slow the progression of the disease but do not halt it. The study of molecular mechanisms that result from mutations in onmental and hormonal modifiers holds great promise for the development of novel therapies that may halt the progression of the disease.

Human ether-a-go-go-related gene (hERG) channels conduct delayed rectifier K(+) current. However, little information is available on physiological situations affecting hERG channel protein and function. In the present study we examined the effects of intermittent hypoxia (IH), which is a hallmark manifestation of sleep apnea, on hERG channel protein and function. Experiments were performed on SH-SY5Y neuroblastoma cells, which express hERG protein. Cells were exposed to IH consisting of alternating cycles of 30 s of hypoxia (1.5% O2) and 5 min of 20% O2. IH decreased hERG protein expression in a stimulus-dependent manner. A similar reduction in hERG protein was also seen in adrenal medullary chromaffin cells from IH-exposed neonatal rats. The decreased hERG protein was associated with attenuated hERG K(+) current. IH-evoked hERG protein degradation was not due to reduced transcription or increased proteosome/lysomal degradation. Rather it was mediated by calcium-activated calpain proteases. Both COOH- and NH2-terminal sequences of the hERG protein were the targets of calpain-dependent degradation. IH increased reactive oxygen species (ROS) levels, intracellular Ca(2+) concentration ([Ca(2+)]i), calpain enzyme activity, and hERG protein degradation, and all these effects were prevented by manganese-(111)-tetrakis-(1-methyl-4-pyridyl)-porphyrin pentachloride, a membrane-permeable ROS scavenger. These results demonstrate that activation of calpains by ROS-dependent elevation of [Ca(2+)]i mediates hERG protein degradation by IH.

Rationale We have previously shown that Nampt, which regulates intracellular NAD levels and cellular redox state, regulates histone deacetylases and inhibits apoptosis, is significantly upregulated in patients with pulmonary arterial hypertension (PAH). The aims of this study were to determine (1) whether Nampt+/− mice are protected from hypoxia-mediated pulmonary hypertension (HPH), (2) whether pharmacological inhibition of Nampt could attenuate monocrotaline (MCT)-induced pulmonary hypertension (PH) in rats. In addition, we hypothesized that Nampt secreted from pulmonary artery endothelial cells (PAECs) or overexpressing Nampt in pulmonary artery smooth muscle cells (PASMCs) may promote PASMC proliferation via upregulation of calcium signaling pathway, which plays a role in cell proliferation and vascular constriction. Methods Nampt+/− mice and their WT siblings (male, 7-wk old) were exposed to a hypoxia chamber with 10% O2 for four weeks. Male Sprague-Dawley rats (n=6 per group) received one dose of MCT (60 mg/kg), IP. They were administrated with FK866 (an inhibitor of Nampt enzymatic activity) (2.5 mg/kg, IP, twice daily for 2wks) two weeks after MCT. Right ventricular systolic pressure (RVSP) was determined with a pressure transducer catheter. The right ventricle: left ventricle+septum (RV/LV+S) ratio was calculated. In a cell culture model, hPASMCs were stimulated with recombinant Nampt (25 mg/ml) for 6 hrs and 48 hrs. [Ca2+]cyt was measured in PASMC loaded with flura-2/AM (4mM) in a fluorescence microscope and cyclepiazonic acid (CPA, a specific Ca2+-ATPase inhibitor) was used to induce store-operated calcium entry (SOCE). In addition, BrdU assays were conducted to examine rNampt or overexpressing Nampt can promote PASMC proliferation or Nampt secreted from PAECs isolated from PAH patients stimulates more PASMC proliferation than from healthy controls. Results Administration of FK866 reversed established PH (RVSP [mm Hg] 19.77±0.80 [control] vs 51.24±4.35 [MCT] vs 34.45±3.49 [MCT+FK866], p Conclusion Inhibition of Nampt attenuates hypoxia-mediated PH in mice or MCT-induced PH in rats. Nampt may play a role in vascular remodeling via regulation of calcium signaling pathway. These data suggest that Nampt inhibition could be a potential therapeutic target for PH.

Activating protein (AP)-1 transcription factors modulate expression of genes involved in cell proliferation and migration. Chronic hypoxia increases pulmonary artery smooth muscle cell proliferation by upregulating AP-1-responsive genes encoding for endothelium-derived vasoactive and mitogenic factors implicated in pulmonary hypertension development. The expression of AP-1 transcription factors is sensitive to changes in cytosolic free [Ca2+] ([Ca2+]cyt). Capacitative Ca2+entry (CCE) via store-operated Ca2+channels (SOC) is an important mechanism for raising [Ca2+]cytin pulmonary artery endothelial cells (PAEC). Using combined molecular biological, fluorescence microscopy, and biophysical approaches, we examined the effect of chronic hypoxia (3% O2, 72 h) on AP-1 DNA binding activity, CCE, and transient receptor potential (TRP) gene expression in human (h) PAEC. EMSA showed that AP-1 binding to hPAEC nuclear protein extracts was significantly enhanced by hypoxia, the increase being dependent on store-operated Ca2+influx and sensitive to La3+, an SOC inhibitor. Hypoxia also increased basal [Ca2+]cyt, the amount of CCE produced by store depletion with cyclopiazonic acid, and the amplitude of SOC-mediated currents ( ISOC). The increases of CCE amplitude and ISOCcurrent density by hypoxia were paralleled by enhanced TRPC4 mRNA and protein expression. Hypoxia-enhanced CCE and TRPC4 expression were also attenuated by La3+. These data suggest that hypoxia increases AP-1 binding activity by enhancing Ca2+influx via La3+-sensitive TRP-encoded SOC channels in hPAEC. The Ca2+-mediated increase in AP-1 binding may play an important role in upregulating AP-1-responsive gene expression, in stimulating pulmonary vascular cell proliferation and, ultimately, in pulmonary vascular remodeling in patients with hypoxia-mediated pulmonary hypertension.

Cell shrinkage is an early prerequisite in programmed cell death, and cytoplasmic K+ is a dominant cation that controls intracellular ion homeostasis and cell volume. Blockade of K+ channels inhibits apoptotic cell shrinkage and attenuates apoptosis. We examined whether apoptotic repressor with caspase recruitment domain (ARC), an antiapoptotic protein, inhibits cardiomyocyte apoptosis by reducing K+ efflux through voltage-gated K+ (Kv) channels. In heart-derived H9c2 cells, whole cell Kv currents ( I K(V)) were isolated by using Ca2+-free extracellular (bath) solution and including 5 mM ATP and 10 mM EGTA in the intracellular (pipette) solution. Extracellular application of 5 mM 4-aminopyridine (4-AP), a blocker of Kv channels, reversibly reduced I K(V)by 50–60% in H9c2 cells. The remaining currents during 4-AP treatment may be generated by K+ efflux through 4-AP-insensitive K+ channels. Overexpression of ARC in heart-derived H9c2 cells significantly decreased I K(V), whereas treatment with staurosporine, a potent apoptosis inducer, enhanced I K(V)in wild-type cells. The staurosporine-induced increase in I K(V) was significantly suppressed and the staurosporine-mediated apoptosis was markedly inhibited in cells overexpressing ARC compared with cells transfected with the control neomycin vector. These results suggest that the antiapoptotic effect of ARC is, in part, due to inhibition of Kv channels in cardiomyocytes.

Polyamines are required for the early phase of mucosal restitution that occurs as a consequence of epithelial cell migration. Our previous studies have shown that polyamines increase RhoA activity by elevating cytosolic free Ca2+ concentration ([Ca2+]cyt) through controlling voltage-gated K+ channel expression and membrane potential ( E m) during intestinal epithelial restitution. The current study went further to determine whether increased RhoA following elevated [Ca2+]cyt activates Rho-kinase (ROK/ROCK) resulting in myosin light chain (MLC) phosphorylation. Studies were conducted in stable Cdx2-transfected intestinal epithelial cells (IEC-Cdx2L1), which were associated with a highly differentiated phenotype. Reduced [Ca2+]cyt, by either polyamine depletion or exposure to the Ca2+-free medium, decreased RhoA protein expression, which was paralleled by significant decreases in GTP-bound RhoA, ROCK-1, and ROKα proteins, Rho-kinase activity, and MLC phosphorylation. The reduction of [Ca2+]cyt also inhibited cell migration after wounding. Elevation of [Ca2+]cyt induced by the Ca2+ ionophore ionomycin increased GTP-bound RhoA, ROCK-1, and ROKα proteins, Rho-kinase activity, and MLC phosphorylation. Inhibition of RhoA function by a dominant negative mutant RhoA decreased the Rho-kinase activity and resulted in cytoskeletal reorganization. Inhibition of ROK/ROCK activity by the specific inhibitor Y-27632 not only decreased MLC phosphorylation but also suppressed cell migration. These results indicate that increase in GTP-bound RhoA by polyamines via [Ca2+]cytcan interact with and activate Rho-kinase during intestinal epithelial restitution. Activation of Rho-kinase results in increased MLC phosphorylation, leading to the stimulation of myosin stress fiber formation and cell migration.

Platelet-derived growth factor (PDGF) signaling is implicated in a wide range of diseases, and PDGF drives the pathological responses in many vascular disorders and fibrotic diseases such as atherosclerosis, restenosis, pulmonary arterial hypertension, and pulmonary fibrosis. In vascular smooth muscle cells (VSMC), PDGF is a potent mitogen which promotes proliferation and migration and contributes to pathological vascular remodeling. Recent studies have demonstrated a role for PDGF in stromal interaction molecule 1 (STIM1)/Orai1-mediated Ca2+ influx in VSMC.1 Regulation of cytosolic Ca2+ concentration ([Ca2+]cyt) is critical for many cellular processes. A rise in [Ca2+]cyt is a major trigger for VSMC proliferation and contraction. One of the major routes of Ca2+ entry into cells is through store-operated Ca2+ (SOC) channels. On depletion of Ca2+ from the stores (sarcoplasmic or endoplasmic reticulum, SR/ER), a Ca2+ deficiency signal is transmitted to the SOC channels in the plasma membrane via STIM1. This causes the SOC to open, allowing Ca2+ to flow into the cytosol, a process referred to as store-operated Ca2+ entry (SOCE). The cytosolic Ca2+ is then sequestered into the stores by the sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA), thus replenishing the stores. Studies have demonstrated a role for Ca2+ release-activated Ca2+ (CRAC) channels in SOCE, with STIM1 and Orai1 as the major components.2 Article, see p 66 The majority of STIM1 is expressed in the SR/ER membrane, where it senses decreased Ca2+ concentration in the SR/ER when inositol 1,4,5-triphosphate (IP3)-mediated activation of IP3 receptor induces Ca2+ release. STIM1 then undergoes a conformational change allowing STIM1 to multimerize and translocate …

Pulmonary arterial hypertension (PAH) is a hemodynamic abnormality that ultimately results in mortality due to right heart failure. Although the clinical manifestations of primary and secondary PAH are diverse, medial hypertrophy and arterial vasoconstriction are key components in the vascular remodeling leading to PAH. Abnormalities in the homeostasis of intracellular Ca(2+), transmembrane flux of ions, and membrane potential may play significant roles in the processes leading to pulmonary vascular remodeling. Decreased activity of K(+) channels causes membrane depolarization, leading to Ca(2+) influx. The elevated cytoplasmic Ca(2+) is a major trigger for pulmonary vasoconstriction and an important stimulus for vascular smooth muscle proliferation. Dysfunctional K(+) channels have also been linked to inhibition of apoptosis and contribute further to the medial hypertrophy. This review focuses on the relative role of K(+) and Ca(2+) ions and channels in human pulmonary artery smooth muscle cells in the development of PAH.

Pulmonary vascular medial hypertrophy due to proliferation of pulmonary artery smooth muscle cells (PASMC) greatly contributes to the increased pulmonary vascular resistance in pulmonary hypertension patients. A rise in cytosolic free Ca2+concentration ([Ca2+]cyt) is an important stimulus for cell growth in PASMC. Resting [Ca2+]cyt, intracellularly stored [Ca2+], capacitative Ca2+entry (CCE), and store-operated Ca2+currents ( ISOC) are greater in proliferating human PASMC than in growth-arrested cells. Expression of TRP1, a transient receptor potential gene proposed to encode the channels responsible for CCE and ISOC, was also upregulated in proliferating PASMC. Our aim was to determine if inhibition of endogenous TRP1 gene expression affects ISOCand CCE and regulates cell proliferation in human PASMC. Cells were treated with an antisense oligonucleotide (AS, for 24 h) specifically designed to cleave TRP1 mRNA and then returned to normal growth medium for 40 h before the experiments. Then, mRNA and protein expression of TRP1 was downregulated, and amplitudes of ISOCand CCE elicited by passive depletion of Ca2+from the sarcoplasmic reticulum using cyclopiazonic acid were significantly reduced in the AS-treated PASMC compared with control. Furthermore, the rate of cell growth was decreased by 50% in AS-treated PASMC. These results indicate that TRP1 may encode a store-operated Ca2+channel that plays a critical role in PASMC proliferation by regulating CCE and intracellular [Ca2+]cyt.

Nitric oxide (NO) is an endogenous endothelium-derived relaxing factor that regulates vascular smooth muscle cell proliferation and apoptosis. This study investigated underlying mechanisms involved in NO-induced apoptosis in human and rat pulmonary artery smooth muscle cells (PASMC). Exposure of PASMC to NO, which was derived from the NO donor S-nitroso- N-acetyl-penicillamine, increased the percentage of cells undergoing apoptosis. Increasing extracellular K+concentration to 40 mM or blocking K+channels with 1 mM tetraethylammonia (TEA), 100 nM iberiotoxin (IBTX), and 5 mM 4-aminopyridine (4-AP) significantly inhibited the NO-induced apoptosis. In single PASMC, NO reversibly increased K+currents through the large-conductance Ca2+-activated K+(KCa) channels, whereas TEA and IBTX markedly decreased the KCacurrents. In the presence of TEA, NO also increased K+currents through voltage-gated K+(Kv) channels, whereas 4-AP significantly decreased the Kvcurrents. Opening of KCachannels with 0.3 mM dehydroepiandrosterone increased KCacurrents, induced apoptosis, and further enhanced the NO-mediated apoptosis. Furthermore, NO depolarized the mitochondrial membrane potential. These observations indicate that NO induces PASMC apoptosis by activating KCaand Kvchannels in the plasma membrane. The resulting increase in K+efflux leads to cytosolic K+loss and eventual apoptosis volume decrease and apoptosis. NO-induced apoptosis may also be related to mitochondrial membrane depolarization in PASMC.

Ventilator-induced lung injury (VILI) is an acute lung injury that develops during mechanical ventilation with high tidal volume in patients in the intensive care unit (ICU). VILI is a common entity with an unacceptable mortality rate (~20%). As no current therapies exist for VILI, novel therapeutic targets for VILI and mechanical stress-induced tissue injury are needed. In this study, we defined Nrf2 signaling in VILI and mechanical stress-induced endothelial cell pathobiology. Nrf2 is a transcription factor that alters ROS balance and is a novel therapeutic target for VILI. First, we defined Nrf2 as a sensory marker for mechanical stress-induced endothelial cell injury. Antioxidant response element (ARE) luciferase plasmids were transfected to both endothelial cells and murine lung tissues. EC exposed to 18% cyclic stretch (18% CS) or mice exposed to excessive tidal volume ventilation (40 ml/kg) resulted in ARE activation, site of Nrf2 binding. We next confirmed Nrf2 signaling is essential for gene expression dysregulation by 18% CS via Nrf2 knockdown that significantly ameliorates 18% CS-induced gene expression pattern, including signaling pathways regulating endothelial barrier function including MYLK. Finally, we validated that Nrf2 is a novel and effective therapeutic target for VILI in pre-clinical models of VILI. Nrf2 activator sulforaphane (SF, 11.5 mg/kg) significantly attenuated VILI (40 ml/kg, 4 hrs)-induced lung inflammation, including BAL protein leakage and white blood cell infiltration. These novel findings characterized Nrf2 as a sensor for mechanical stress, a central mediator of mechanical stress induced gene expression dysregulation, and a novel therapeutic target for VILI.

Sphingosine kinases (SphKs) 1 and 2 regulate the synthesis of the bioactive sphingolipid sphingosine-1-phosphate (S1P), an important lipid mediator that promotes cell proliferation, migration, and angiogenesis.We aimed to examine whether SphKs and their product, S1P, play a role in the development of pulmonary arterial hypertension (PAH).SphK1(-/-), SphK2(-/-), and S1P lyase heterozygous (Sgpl1(+/-)) mice, a pharmacologic SphK inhibitor (SKI2), and a S1P receptor 2 (S1PR2) antagonist (JTE013) were used in rodent models of hypoxia-mediated pulmonary hypertension (HPH). S1P levels in lung tissues from patients with PAH and pulmonary arteries (PAs) from rodent models of HPH were measured.mRNA and protein levels of SphK1, but not SphK2, were significantly increased in the lungs and isolated PA smooth muscle cells (PASMCs) from patients with PAH, and in lungs of experimental rodent models of HPH. S1P levels were increased in lungs of patients with PAH and PAs from rodent models of HPH. Unlike SphK2(-/-) mice, SphK1(-/-) mice were protected against HPH, whereas Sgpl1(+/-) mice were more susceptible to HPH. Pharmacologic SphK1 and S1PR2 inhibition prevented the development of HPH in rodent models of HPH. Overexpression of SphK1 and stimulation with S1P potentially via ligation of S1PR2 promoted PASMC proliferation in vitro, whereas SphK1 deficiency inhibited PASMC proliferation.The SphK1/S1P axis is a novel pathway in PAH that promotes PASMC proliferation, a major contributor to pulmonary vascular remodeling. Our results suggest that this pathway is a potential therapeutic target in PAH.

Background— Pulmonary veno-occlusive disease is caused by excessive cell proliferation and fibrosis, which obliterate the lumen of pulmonary venules, leading to pulmonary hypertension, right ventricular failure, and death. This condition has no effective treatment and a 5-year survival of Methods and Results— We show that mice with homozygous deletion of the Ets transcription factor Erg die between embryonic day 16.5 and 3 months of age as a result of pulmonary veno-occlusive disease, capillary hemorrhage, and pancytopenia. We demonstrate that Erg binds to and serves as a transcriptional activator of the G-protein–coupled receptor gene Aplnr , the expression of which is uniquely specific for venous endothelium and that knockout of either Erg or Aplnr results in pulmonary venule–specific endothelial proliferation in vitro. We show that mice with either homozygous-global or endothelium-directed deletion of Aplnr manifest pulmonary veno-occlusive disease and right heart failure, detectable at 8 months of age. Levels of pulmonary ERG and APLNR in patients with pulmonary veno-occlusive disease undergoing lung transplantation were significantly lower than those of control subjects. Conclusions— Our results suggest that ERG and APLNR are essential for endothelial homeostasis in venules in the lung and that perturbation in ERG-APLNR signaling is crucial for the development of pulmonary veno-occlusive disease. We identify this pathway as a potential therapeutic target for the treatment of this incurable disease.

To explore the effects of gender on pathology and pulmonary vascular reactivity in chronic hypoxic pulmonary hypertension (HPH) in mice.HPH was induced by a 4-week exposure of male and female mice (8 weeks old C57BL/6, n = 15 each) to chronic hypoxia (10%O2). Normoxic controls were established both in male and female groups (n = 15 each). Right ventricular systolic pressure (RVSP) and Fulton index were detected.Isolated pulmonary artery ring contraction induced by high potassium (K(+)) and U-46619 was recorded and compared between two groups.Chronic hypoxia induced pulmonary hypertension after 4 weeks.RVSP significantly increased both in hypoxic female and male groups (normoxic vs hypoxic: (23.40 ± 0.16) vs (31.46 ± 2.33) and (24.71 ± 0.44) vs (33.66 ± 1.38) mmHg (1 mmHg = 0.133 kPa), P = 0.029 and P0.001).However, there were no inter-group differences (P = 0.447). The Fulton index of hypoxic female and male mice was higher than normoxic counterparts (P = 0.001, P0.001). But there were no gender differences (P = 0.471). Contraction of high K(+) and U-46619 was calcium ion (Ca(2+))-dependent and decreased markedly without calcium (P0.001, P = 0.007). High K(+) and U-46619-induced contraction of pulmonary artery was concentration dependent and greater in chronic hypoxic female (P = 0.002, P0.001) and male mice (both P0.001). But no differences existed between male and female mice in high K(+) (P = 0.657) or U-46619 (P = 0.751)-induced contraction.No significant correlation exists between gender and occurrence and severity of pulmonary hypertension or pulmonary artery vascular reactivity in HPH mice.

When Pulmonary Circulation was first launched in 2011, no one knew what to expect. A journal focused exclusively on research around the pulmonary circulation and pulmonary vascular disease? Are there enough people interested to sustain the journal’s readership? Who will submit to yet another new, unknown medical journal? And how will we establish and differentiate ourselves from the other journals in the field? After three years, and after more than 350 submissions, more than 200 published articles, and more than 57,000 PDF article downloads, Pulmonary Circulation has proven its ability to capture some of the best original research articles and comprehensive reviews in the field. The voice of the journal is made up of a diverse group of both new and established board members ranging from clinicians to physician scientists to investigators; it is heard in the authors from more than 35 different countries across the globe; and it is seen in the various types of articles we publish. Our goal was that Pulmonary Circulation would become the valued journal in the field. We believe that we are well on the way to achieving this. In order to maintain and exceed this goal, we recently had to make several changes to the production of the journal. Our biggest, and most valued, change is our new publisher. We are very excited to formally introduce our readers to the University of Chicago Press (UCP). With this change in publisher, we expect to see enhanced visibility for the journal, improved visual quality of articles published, and most importantly, a heightened efficiency of publication. UCP has been around for more than 100 years and publishes around 50 journals every year. They have recently established a medical journals division within the Press, which we are honored to join. The Press offers many new features to both its readers and its authors, including prompt ahead-of-print publication and a just-accepted link where new articles will be posted online, unedited, within 3 days of acceptance. Next, we have decided to go digital. We have seen over the past few years that, overwhelmingly, readers were accessing our articles online and not in print. In an effort to increase efficiency, we decided to make the journal available online only, with the option for readers to request any print-on-demand issue they may be interested in receiving. In addition, UCP offers an e-book format of the journal, which can be downloaded to all versions of e-readers and iPads, smart phones, and iPhones. Again, we look to you, our readers and contributors, to help sustain the journal and take us to the next level. We eagerly await the announcement of our impact factor, which will be another major step for us. Please submit to us your most novel and significant original research articles in basic, translational, clinical, and population health research; your most comprehensive review articles on scientifically impactful and clinically relevant topics related to the pulmonary circulation and pulmonary vascular disease; your highly novel (and rare) case reports; and your rapid communications/research reports on novel findings. We also continue to be interested in considering the publication of high-quality conference abstracts of selected scientific conferences in the pulmonary circulation field. We are grateful to all who support the journal—members of the Pulmonary Vascular Research Institute, editorial board members, authors, reviewers, and readers—for helping establish our unique voice within the world of medical publishing.

Pulmonary arterial hypertension (PAH) is a progressive disease that, if left untreated, eventually leads to right heart failure and death. Elevated pulmonary arterial pressure (PAP) in patients with PAH is mainly caused by an increase in pulmonary vascular resistance (PVR). Sustained vasoconstriction and excessive pulmonary vascular remodeling are two major causes for elevated PVR in patients with PAH. Excessive pulmonary vascular remodeling is mediated by increased proliferation of pulmonary arterial smooth muscle cells (PASMC) due to PASMC dedifferentiation from a contractile or quiescent phenotype to a proliferative or synthetic phenotype. Increased cytosolic Ca2+concentration ([Ca2+]cyt) in PASMC is a key stimulus for cell proliferation and this phenotypic transition. Voltage-dependent Ca2+entry (VDCE) and store-operated Ca2+entry (SOCE) are important mechanisms for controlling [Ca2+]cyt. Stromal interacting molecule proteins (e.g., STIM2) and Orai2 both contribute to SOCE and we have previously shown that STIM2 and Orai2, specifically, are upregulated in PASMC from patients with idiopathic PAH and from animals with experimental pulmonary hypertension in comparison to normal controls. In this study, we show that STIM2 and Orai2 are upregulated in proliferating PASMC compared with contractile phenotype of PASMC. Additionally, a switch in Ca2+regulation is observed in correlation with a phenotypic transition from contractile PASMC to proliferative PASMC. PASMC in a contractile phenotype or state have increased VDCE, while in the proliferative phenotype or state PASMC have increased SOCE. The data from this study indicate that upregulation of STIM2 and Orai2 is involved in the phenotypic transition of PASMC from a contractile state to a proliferative state; the enhanced SOCE due to upregulation of STIM2 and Orai2 plays an important role in PASMC proliferation.

Introduction: Recent studies suggest that microRNAs (miRNAs) play important roles in development and pathogenesis of human diseases. We sought to identify novel miRNAs that may be involved in the pathogenesis of pulmonary arterial hypertension (PAH) and to investigate underlying mechanisms. Methods and Results: We performed miRNA qPCR array using pulmonary arterial smooth muscle cells (PASMC) isolated from lungs of normal donor and PAH patients. We found that all of the six members of the miR-17~92 cluster were down-regulated in PAH PASMCs. In hypoxia-induced PAH mouse model and cultured human PASMCs, we found that short term hypoxia induced miR-17~92 whereas prolonged hypoxia suppressed miR-17~92. We generated a strain of SMC-specific miR-17~92 knockout (sm-17~92-/-) mouse by crossbreeding miR-17~92 fl/fl mice with sm22α-Cre mice and subsequent backcrossing. We exposed 8-10 week old sm-17~92-/- mice along with their wild type littermates to hypoxia (10% O2) or normoxia for five weeks. We showed that knoc...

Sodium-glucose cotransporter 2 (SGLT2) inhibitors are a new class of oral drugs for the treatment of type 2 diabetes and they reduce plasma blood glucose levels by inhibiting renal glucose reabsorp...

Idiopathic pulmonary arterial hypertension (IPAH) is a fatal and progressive disease which is attributed to elevated pulmonary arterial pressure and pulmonary vascular resistance due in part to sustained pulmonary vasoconstriction and vascular remodeling. Pulmonary arterial smooth muscle cells (PASMC) are normally shielded from exposure to shear stress and sense small flow shear through transmural interstitial flow driven by transvascular pressure differential. However, in the context of endothelial injury or enhanced vascular permeability caused by increased pulmonary arterial pressure, PASMC are exposed to shear stress or to enhanced transmural interstitial flow. Shear stress can increase cytosolic Ca2+ concentration ([Ca2+]cyt). An increase in [Ca2+]cyt in PASMC is a major trigger for vasoconstriction and a key stimulus for cell proliferation, which contribute to vascular remodeling. We show here that shear stress induced increased [Ca2+]cyt in PASMC from patients with IPAH compared with control. Fluid...

Rationale: An increase in cytosolic free Ca2+ in pulmonary arterial smooth muscle cells (PASMC) is not only a major trigger for pulmonary vasoconstriction but also an important stimulus for PASMC p...

hypoxic pulmonary vasoconstriction(HPV) serves as an important regulatory mechanism to match perfusion to ventilation by directing blood flow away from poorly ventilated regions of the lung to ensure maximal oxygenation of the venous blood. Persistent HPV, however, causes pulmonary hypertension that

The balance between apoptosis and proliferation in pulmonary artery smooth muscle cells (PASMCs) is important in maintaining normal pulmonary vascular structure. Activity of voltage-gated K+(KV) channels has been demonstrated to regulate cell apoptosis and proliferation. Treatment of PASMCs with staurosporine (ST) induced apoptosis in PASMCs, augmented KVcurrent [ IK(V)], and induced mitochondrial membrane depolarization. High K+(40 mM) negligibly affected the ST-induced mitochondrial membrane depolarization but inhibited the ST-induced IK(V)increase and apoptosis. Blockade of KVchannels with 4-aminopyridine diminished IK(V)and markedly decreased the ST-mediated apoptosis. Furthermore, the ST-induced apoptosis was preceded by the increase in IK(V). These results indicate that ST induces PASMC apoptosis by activation of plasmalemmal KVchannels and mitochondrial membrane depolarization. The increased IK(V)would result in an apoptotic volume decrease due to a loss of cytosolic K+and induce apoptosis. The mitochondrial membrane depolarization would cause cytochrome c release, activate the cytosolic caspases, and induce apoptosis. Inhibition of KVchannels would thus attenuate PASMC apoptosis.

Background Activity of voltage-gated K + (K v ) channels controls membrane potential (E m ) that regulates cytosolic free Ca 2+ concentration ([Ca 2+ ] cyt ) by regulating voltage-dependent Ca 2+ channel function. A rise in [Ca 2+ ] cyt in pulmonary artery smooth muscle cells (PASMCs) triggers vasoconstriction and stimulates PASMC proliferation. Whether c-Jun, a transcription factor that stimulates cell proliferation, affects K v channel activity in PASMCs was investigated. Methods and Results Infection of primary cultured PASMCs with an adenoviral vector expressing c-jun increased the protein level of c-Jun and reduced K v currents ( I K(V) ) compared with control cells (infected with an empty adenovirus). Using single-cell reverse transcription–polymerase chain reaction, we observed that the mRNA level of Kv1.5 and the current density of I K(V) were both attenuated in c-jun –infected PASMCs compared with control cells and cells infected with antisense c-jun . Overexpression of c-Jun also upregulated protein expression of Kvβ 2 and accelerated I K(V) inactivation. Furthermore, E m was more depolarized and [ 3 H]thymidine incorporation was greater in PASMCs infected with c-jun than in control cells and cells infected with antisense c-jun . Conclusions These results suggest that c-Jun–mediated PASMC proliferation is associated with a decrease in I K(V) . The resultant membrane depolarization increases [Ca 2+ ] cyt and enhances PASMC growth.

Agonist-induced increases in cytosolic Ca2+concentration ([Ca2+]cyt) in pulmonary artery (PA) smooth muscle cells (SMCs) consist of a transient Ca2+release from intracellular stores followed by a sustained Ca2+influx. Depletion of intracellular Ca2+stores triggers capacitative Ca2+entry (CCE), which contributes to the sustained increase in [Ca2+]cytand the refilling of Ca2+into the stores. In isolated PAs superfused with Ca2+-free solution, phenylephrine induced a transient contraction, apparently by a rise in [Ca2+]cytdue to Ca2+release from the intracellular stores. The transient contraction lasted for 3–4 min until the Ca2+store was depleted. Restoration of extracellular Ca2+in the presence of phentolamine produced a contraction potentially due to a rise in [Ca2+]cytvia CCE. The store-operated Ca2+channel blocker Ni2+reduced the store depletion-activated Ca2+currents, decreased CCE, and inhibited the CCE-mediated contraction. In single PASMCs, we identified, using RT-PCR, five transient receptor potential gene transcripts. These results suggest that CCE, potentially through transient receptor potential-encoded Ca2+channels, plays an important role in agonist-mediated PA contraction.

Expression of voltage-gated K+(Kv) channel genes is regulated by polyamines in intestinal epithelial cells (IEC-6 line), and Kv channel activity is involved in the regulation of cell migration during early restitution by controlling membrane potential ( Em) and cytosolic free Ca2+concentration ([Ca2+]cyt). This study tests the hypothesis that RhoA of small GTPases is a downstream target of elevated [Ca2+]cytfollowing activation of K+channels by increased polyamines in IEC-6 cells. Depletion of cellular polyamines by α-difluoromethylornithine (DFMO) reduced whole cell K+currents [ IK(v)] through Kv channels and caused membrane depolarization, which was associated with decreases in [Ca2+]cyt, RhoA protein, and cell migration. Exogenous polyamine spermidine reversed the effects of DFMO on IK(v ), Em, [Ca2+]cyt, and RhoA protein and restored cell migration to normal. Elevation of [Ca2+]cytinduced by the Ca2+ionophore ionomycin increased RhoA protein synthesis and stimulated cell migration, while removal of extracellular Ca2+decreased RhoA protein synthesis, reduced protein stability, and inhibited cell motility. Decreased RhoA activity due to Clostridium botulinum exoenzyme C3transferase inhibited formation of myosin II stress fibers and prevented restoration of cell migration by exogenous spermidine in polyamine-deficient cells. These findings suggest that polyamine-dependent cell migration is partially initiated by the formation of myosin II stress fibers as a result of Ca2+-activated RhoA activity.

Pulmonary arterial hypertension (PAH) is a rare, progressive, and fetal disease. The five-year survival rate after diagnosis is ～50%. In Japan, PAH is listed in the Specified Rare and Intractable Diseases. Pulmonary vascular remodeling and sustained pulmonary vasoconstriction are the major causes for the elevated pulmonary vascular resistance (PVR) in PAH. The pathogenic mechanisms involved in the pulmonary vascular abnormalities in PAH remain unclear. Sustained vasoconstriction and vascular remodeling owing to proliferation of pulmonary arterial smooth muscle cells (PASMCs) are key pathogenic events that lead to early morbidity and mortality. These events have been closely linked to Ca(2+) mobilization and signaling in PASMCs. An increase in cytosolic Ca(2+) concentration ([Ca(2+)]cyt) in PASMCs is an important stimulus for pulmonary vasoconstriction and cell proliferation which subsequently cause pulmonary vascular wall thickening followed by the increase in PVR. Increased resting [Ca(2+)]cyt and enhanced Ca(2+) influx have been implicated in PASMCs from PAH patients, but precise therapeutic targets to interrupt these signal pathways have not been identified. We recently found that the extracellular Ca(2+)-sensing receptor (CaSR), a G protein-coupled receptor (GPCR), is upregulated in PASMCs from patients with idiopathic pulmonary arterial hypertension (IPAH). In addition, blockage of the CaSR with an antagonist (NPS2143) prevents the development of pulmonary hypertension and right ventricular hypertrophy in animal models of pulmonary hypertension. The functionally upregulated CaSR in PASMCs is a novel pathogenic mechanism contributing to the augmented Ca(2+) signaling and excessive cell proliferation in IPAH. Targeting CaSR in PASMCs may help develop novel therapeutic approach for PAH.

Pulmonary vasoconstriction and vascular medial hypertrophy greatly contribute to the elevated pulmonary vascular resistance in patients with pulmonary hypertension. A rise in cytosolic free Ca2+([Ca2+]cyt) in pulmonary artery smooth muscle cells (PASMC) triggers vasoconstriction and stimulates cell growth. Membrane potential ( Em) regulates [Ca2+]cytby governing Ca2+influx through voltage-dependent Ca2+channels. Thus intracellular Ca2+may serve as a shared signal transduction element that leads to pulmonary vasoconstriction and vascular remodeling. In PASMC, activity of voltage-gated K+(Kv) channels regulates resting Em. In this study, we investigated whether changes of Kv currents [ IK(V)], Em, and [Ca2+]cytaffect cell growth by comparing these parameters in proliferating and growth-arrested PASMC. Serum deprivation induced growth arrest of PASMC, whereas chelation of extracellular Ca2+abolished PASMC growth. Resting [Ca2+]cytwas significantly higher, and resting Emwas more depolarized, in proliferating PASMC than in growth-arrested cells. Consistently, whole cell IK(V)was significantly attenuated in PASMC during proliferation. Furthermore, Emdepolarization significantly increased resting [Ca2+]cytand augmented agonist-mediated rises in [Ca2+]cytin the absence of extracellular Ca2+. These results demonstrate that reduced IK(V), depolarized Em, and elevated [Ca2+]cytmay play a critical role in stimulating PASMC proliferation. Pulmonary vascular medial hypertrophy in patients with pulmonary hypertension may be partly caused by a membrane depolarization-mediated increase in [Ca2+]cytin PASMC.

Although infants with persistent pulmonary hypertension of the newborn (PPHN) experience some relief and therapeutic benefit from current therapies, over 50% have a limited or transient response and significant morbidity. There is no consistency in the best first line treatment throughout hospitals in the United States. Ventilation with high levels of oxygen or inhaled nitric oxide (NO) are typical strategies for improving the extracorporeal membrane oxygen, although they remain unproven to increase survival rates. While oxygen may stimulate endothelial nitric oxide synthase (eNOS) and NO production dilating the pulmonary vasculature, it also fuels the production of reactive oxygen species (ROS). ROS is likely to have counterproductive effects; in addition to stimulating vascular smooth muscle cell proliferation and increasing vascular tone, ROS may directly regulate eNOS and NO. The recent article by Farrow and colleagues (3) in AJP-Lung investigates the role of ROS on eNOS. By using recombinant human superoxide dismutase (rhSOD), they observed 1) increased eNOS activity and expression, 2) increased tetrahydrobiopterin (BH4), a cofactor critical to the function of eNOS, and 3 )a decrease in oxidative stress, in addition to the stimulation of NO production and ultimately pulmonary vasodilatation. The observations they made may be paramount to increasing the survival of infants with PPHN and may lead to an adapted treatment regimen that addresses the pitfalls of current therapeutic approaches. PPHN When the pulmonary circulation fails to respond to natural stimuli, including increased oxygen tension, ventilation, and shear stress, it does not undergo the shift from the high resistance state in utero to a postnatal low resistance system, enabling efficient pulmonary gas exchange and oxygenation. Impaired NO-cGMP signaling has been shown to be critical to the regulation of pulmonary circulation in the newborn, and clinical strategies have involved administration of inhaled NO since the early 1990s (6, 10). While effective in immediate relief due to vasodilatation, the infants can enter an inhaled NO dependency state, and thus inhaled NO proffers poor long-term relief. The necessity for extensive research into the regulation of perinatal circulation and the changes that occur upon ventilation have led to improved and more specific therapeutic approaches for infants with PPHN over the past 30 years. Despite this, PPHN is still associated with significant shortterm and long-term morbidity. Farrow et al. (3) strive to dissect the signaling pathways, determining the impact of ROS and elucidating the potential of decreasing oxidative stress as a therapeutic approach in PPHN. This study is published in a milieu of recent publications exploring the functional abnor

PULMONARY ARTERIAL HYPERTENSION (PAH) is a progressive disease manifested by maladaptation of the pulmonary vasculature. The development of PAH can be influenced by genetic predisposition and/or by diverse endogenous or environmental stimuli. Regardless of the initial pathogenic factors, pulmonary vascular remodeling, sustained pulmonary vasoconstriction, in situ thrombosis, and increased pulmonary vascular wall stiffness are the major contributors to elevated pulmonary vascular resistance (PVR). The increase in PVR can lead to right ventricular failure and death in patients with PAH. While treatments for this disease are improving, it continues to be a life-threatening condition. MicroRNAs (miRNAs) have been implicated in the development and progression of PAH. MiRNAs are small, noncoding RNAs that regulate gene and protein expression by promoting degradation or suppressing translation of target mRNAs. Several studies have demonstrated aberrant expres

Pulmonary vascular remodeling and increased arterial wall stiffness are two major causes for the elevated pulmonary vascular resistance and pulmonary arterial pressure in patients and animals with pulmonary hypertension. Cellular copper (Cu) plays an important role in angiogenesis and extracellular matrix remodeling; increased Cu in vascular smooth muscle cells has been demonstrated to be associated with atherosclerosis and hypertension in animal experiments. In this study, we show that the Cu-uptake transporter 1, CTR1, and the Cu-efflux pump, ATP7A, were both upregulated in the lung tissues and pulmonary arteries of mice with hypoxia-induced pulmonary hypertension. Hypoxia also significantly increased expression and activity of lysyl oxidase (LOX), a Cu-dependent enzyme that causes crosslinks of collagen and elastin in the extracellular matrix. In vitro experiments show that exposure to hypoxia or treatment with cobalt (CoCl2) also increased protein expression of CTR1, ATP7A, and LOX in pulmonary arterial smooth muscle cells (PASMC). In PASMC exposed to hypoxia or treated with CoCl2, we also confirmed that the Cu transport is increased using 64Cu uptake assays. Furthermore, hypoxia increased both cell migration and proliferation in a Cu-dependent manner. Downregulation of hypoxia-inducible factor 1α (HIF-1α) with siRNA significantly attenuated hypoxia-mediated upregulation of CTR1 mRNA. In summary, the data from this study indicate that increased Cu transportation due to upregulated CTR1 and ATP7A in pulmonary arteries and PASMC contributes to the development of hypoxia-induced pulmonary hypertension. The increased Cu uptake and elevated ATP7A also facilitate the increase in LOX activity and thus the increase in crosslink of extracellular matrix, and eventually leading to the increase in pulmonary arterial stiffness.

Human pulmonary artery smooth muscle cells (HPASMCs) express both adenosine monophosphate-activated protein kinase (AMPK) α1 and α2. We investigated the distinct roles of AMPK α1 and α2 in the survival of HPASMCs during hypoxia and hypoxia-induced pulmonary hypertension (PH). The exposure of HPASMCs to hypoxia (3% O2) increased AMPK activation and phosphorylation, and the inhibition of AMPK with Compound C during hypoxia decreased their viability and increased lactate dehydrogenase activity and apoptosis. Although the suppression of either AMPK α1 or α2 expression led to increased cell death, the suppression of AMPK α2 alone increased caspase-3 activity and apoptosis in HPASMCs exposed to hypoxia. It also resulted in the decreased expression of myeloid cell leukemia sequence 1 (MCL-1). The knockdown of MCL-1 or MCL-1 inhibitors increased caspase-3 activity and apoptosis in HPASMCs exposed to hypoxia. On the other hand, the suppression of AMPK α1 expression alone prevented hypoxia-mediated autophagy. The inhibition of autophagy induced cell death in HPASMCs. Our results suggest that AMPK α1 and AMPK α2 play differential roles in the survival of HPASMCs during hypoxia. The activation of AMPK α2 maintains the expression of MCL-1 and prevents apoptosis, whereas the activation of AMPK α1 stimulates autophagy, promoting HPASMC survival. Moreover, treatment with Compound C, which inhibits both isoforms of AMPK, prevented and partly reversed hypoxia-induced PH in mice. Taking these results together, our study suggests that AMPK plays a key role in the pathogenesis of pulmonary arterial hypertension, and AMPK may represent a novel therapeutic target for the treatment of pulmonary arterial hypertension.

The National Heart, Lung, and Blood Institute (NHLBI) of the National Institutes of Health convened the Cell Therapy for Lung Disease Working Group on November 13–14, 2012, to review and formulate recommendations for future research directions. The workshop brought together investigators studying basic mechanisms and the roles of cell therapy in preclinical models of lung injury and pulmonary vascular disease, with clinical trial experts in cell therapy for cardiovascular diseases and experts from the NHLBI Production Assistance for Cell Therapy program. The purpose of the workshop was to discuss the current status of basic investigations in lung cell therapy, to identify some of the scientific gaps in current knowledge regarding the potential roles and mechanisms of cell therapy in the treatment of lung diseases, and to develop recommendations to the NHLBI and the research community on scientific priorities and practical steps that would lead to first-in-human trials of lung cell therapy.

The pulmonary circulation is a low resistance and low pressure system. Sustained pulmonary vasoconstriction and excessive vascular remodeling often occur under pathophysiological conditions such as in patients with pulmonary hypertension. Pulmonary vasoconstriction is a consequence of smooth muscle contraction. Many factors released from the endothelium contribute to regulating pulmonary vascular tone, while the extracellular matrix in the adventitia is the major determinant of vascular wall compliance. Pulmonary vascular remodeling is characterized by adventitial and medial hypertrophy due to fibroblast and smooth muscle cell proliferation, neointimal proliferation, intimal, and plexiform lesions that obliterate the lumen, muscularization of precapillary arterioles, and in situ thrombosis. A rise in cytosolic free Ca(2+) concentration ([Ca(2+)]cyt) in pulmonary artery smooth muscle cells (PASMC) is a major trigger for pulmonary vasoconstriction, while increased release of mitogenic factors, upregulation (or downregulation) of ion channels and transporters, and abnormalities in intracellular signaling cascades are key to the remodeling of the pulmonary vasculature. Changes in the expression, function, and regulation of ion channels in PASMC and pulmonary arterial endothelial cells play an important role in the regulation of vascular tone and development of vascular remodeling. This article will focus on describing the ion channels and transporters that are involved in the regulation of pulmonary vascular function and structure and illustrating the potential pathogenic role of ion channels and transporters in the development of pulmonary vascular disease.

Over the past decade a wealth of information has been divulged on stem cells present in the lung both in the pulmonary vasculature and the respiratory tract. Cells have been identified with the capability of repopulating the lung and others that contribute to the pathogenesis or, conversely, have therapeutic benefit in pulmonary vascular disease. The isolation of a single-resident lung stem cell capable of repopulating any lung epithelium still remains elusive. What is currently known about stem and progenitor cells in the lung suggests that a non-classical stem cell hierarchy exists with a novel array of cellular mechanisms controlling proliferation and differentiation of such cells. This chapter serves to provide an up-to-date review of what is currently known about stem and progenitor cells within the lung.