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Extracellular signal-regulated kinase (ERK), also known as classical mitogen-activated protein kinase, plays critical roles in cell regulation. ERK is activated through phosphorylation by a cascade of protein kinases including MEK. Various ligands activate the MEK/ERK pathway through receptor-dependent cell signaling. In cultured cells, many ligands such as growth factors, hormones, cytokines and vasoactive peptides elicit transient activation of MEK/ERK, often peaking at ~10 min after the cell treatment. Here, we describe a novel biological event, in which ligand-mediated cell signaling results in the dephosphorylation of MEK/ERK. Neuromedin N and neurotensin, peptides derived from the same precursor polypeptide, elicit cell signaling through the neurotensin receptors. In cultured human pulmonary artery smooth muscle cells (PASMCs), but not in human pulmonary artery endothelial cells (PAECs), we found that both neuromedin N and neurotensin promoted the dephosphorylation of ERK and MEK. Human PASMCs were found to express neurotensin receptor (NTR)-1, -2 and -3, while human PAECs only express NTR3. Neuromedin N-mediated dephosphorylation was suppressed by small chemical inhibitors of protein phosphatase 1/2A and peptidyl-prolyl isomerase. Transmission electron microscopy showed the formation of endocytic vesicles in response to neuromedin N treatment, and dephosphorylation did not occur when sorting nexin 9, a critical regulator of the endocytic vesicle formation, was knocked down. We conclude that neuromedin N and neurotensin elicit a unique dephosphorylation signaling in the MEK/ERK pathway that is regulated by endocytosis. Considering the pathophysiological importance of the MEK/ERK pathway, this discovery of the dephosphorylation mechanism should advance the field of cell signaling., (Copyright © 2018. Published by Elsevier Inc.)

Pulmonary artery hyperplasia is the result of proliferation of the pulmonary arterial smooth muscles (PASM). Hypoxia-induced PASM proliferation in the fetus and the newborn is the primary cause of persistent pulmonary hypertension of the newborn (PPHN). This study was performed to characterize the utility of the Laser Scanning Cytometry (LSC) method in elucidating arterial cytoskeletal remodeling in an in vitro model of PPHN. The aim was to demonstrate the following: (a) LSC is a valid method for the analysis of nuclear and cytosolic fluorescence and (b) the cumulative effects of mechanical stretch together with hypoxia promote reactive oxygen species (ROS) formation. The molecular events in response to hypoxia and the mechanical overload of the pulmonary circuit were demonstrated in vitro by subjecting hypoxic cultured primary PASM or human airway smooth muscles (hASM) to repetitive stretch-relaxation cycles at rates comparable to dynamic stretch in vivo. The altered cytoskeleton in the form of filamentous to globular actin (F:G actin) ratio was imaged and quantified at the cellular level by LSC as an endpoint. LSC can remove the nuclear G-actin fluorescence from the total G-actin fluorescence. Pulsatile stretch was found to significantly increase the total endogenous ROS and superoxide anion release in normoxic and hypoxic conditions in primary PASM fibers. The effect of stretch was predominant in increasing superoxide anion release, only under hypoxic conditions. These findings, obtained by LSC in vitro are amenable to validation in any in vivo model of interest. The in vitro model is clinically relevant to human pulmonary vascular remodeling.

Rationale: Forkhead box M1 (FoxM1) is a transcription factor that promotes cell proliferation by regulating a broad spectrum of genes that participate in cell cycle regulation, such as Cyclin B, CDC25B, and Aurora B Kinase. We have shown that hypoxia, a well-known stimulus for pulmonary hypertension (PH), induces FoxM1 in pulmonary artery smooth muscle cells (PASMC) in a HIF-dependent pathway, resulting in PASMC proliferation, while the suppression of FoxM1 prevents hypoxia-induced PASMC proliferation. However, the implications of FoxM1 in the development of PH remain less known., Methods: We determined FoxM1 levels in the lung samples of idiopathic PAH (pulmonary arterial hypertension) (IPAH) patients and hypoxia-induced PH mice. We generated constitutive and inducible smooth muscle cell (SMC)-specific FoxM1 knockdown or knockout mice as well as FoxM1 transgenic mice which overexpress FoxM1, and exposed them to hypoxia (10% O 2 , 90% N 2 ) or normoxia (Room air, 21% oxygen) for four weeks, and measured PH indices. We also isolated mouse PASMC (mPASMC) and mouse embryonic fibroblasts (MEF) from these mice to examine the cell proliferation and expression levels of SMC contractile proteins., Results: We showed that in hypertensive human lungs or mouse lungs, FoxM1 levels were elevated. Constitutive knockout of FoxM1 in mouse SMC caused early lethality, whereas constitutive knockdown of FoxM1 in mouse SMC prevented hypoxia-induced PH and PASMC proliferation. Inducible knockout of FoxM1 in SMC reversed hypoxia-induced pulmonary artery wall remodeling in existing PH. Overexpression of FoxM1 enhanced hypoxia-induced pulmonary artery wall remodeling and right ventricular hypertrophy in mice. Alteration of FoxM1 status did not affect hypoxia-induced hypoxia-inducible factor (HIF) activity in mice. Knockout of FoxM1 decreased PASMC proliferation and induced expression of SMC contractile proteins and TGF-β/Smad3 signaling., Conclusions: Our studies provide clear evidence that altered FoxM1 expression in PASMC contributes to PH and uncover a correlation between Smad3-dependent signaling in FoxM1-mediated proliferation and de-differentiation of PASMC., (Copyright © 2018 Elsevier Inc. All rights reserved.)

MicroRNAs (miRNAs) have a key role in the pathogenesis of pulmonary arterial hypertension (PAH), a disease characterized by enhanced proliferation and reduced apoptosis of pulmonary artery smooth muscle cells. In the present study, miR‑760 was demonstrated to be downregulated in PAH lung tissues compared with normal lung tissues, an effect that may be associated with the development of PAH. Hypoxia is an important stimulus for human pulmonary artery smooth muscle cell (hPASMC) proliferation and the occurrence of PAH. Therefore, the effect of miR‑760 in hypoxia‑treated and normal hPASMCs was investigated. Expression of exogenous miR‑760 decreased cell proliferation in hypoxia‑induced hPASMCs, and promoted cell apoptosis with an increase in the BCL2 associated X/BCL2 ratio and the expression levels of Caspase‑3 and Caspase‑9. In addition, overexpression of miR‑760 suppressed the migration of hPASMCs under hypoxic conditions. Furthermore, miR‑760 was demonstrated to mediate its anti‑proliferation effect via the regulation of toll‑like receptor 4 (TLR4), a direct target of miR‑760. The results revealed that knockdown of TLR4 restrained the hypoxia‑induced hPASMC proliferation and induced cell apoptosis. The present study uncovered a novel regulatory pathway involving miR‑760 and suggested that miR‑760 may be explored as a potential therapy for PAH in the future.

Pulmonary arterial hypertension is caused by an imbalance of pulmonary vasoconstriction and vasodilation. Pulmonary arteriolar remodeling is a primary pathological change and proliferation of pulmonary arterial smooth muscle cells (PASMC) is an important pathological basis for pulmonary arteriolar remodeling. Vasoactive substances, such as 5-HT, may play a role in proliferation of PASMC via unknown mechanisms. In vitro experiments with PASMC showed that the TRPC channel inhibitor SKF96365 inhibited the effects 5-HT and DOI on PASMC proliferation and G2M percentage increase, and decreased expression of TRPC1, TRPC6 and calcineurin A/NFATc3 induced by 5-HT and DOI. SKF96365 inhibited binding of NFATc3 and DNA promoted by 5-HT and DOI. Therefore, 5-HT may affect the TRPC channel to promote proliferation of PASMC; upregulate expression of TRPC1, TRPC6, and calcineurin A/NFATc3; and therefore promote NFATc3 nuclear translocation. There may be crosstalk between 5-HT and TRPC, which may contribute to the pathogeneis of pulmonary arterial hypertension and this may be a novel therapeutic target for treating pulmonary arterial hypertension.

Up-regulation of mammalian COP9 signalosome subunit 6 (CSN6) and consequent reduction of SCF ubiquitin ligase substrate receptor β-transduction repeat-containing protein (β-TrCP) have been shown to be associated with cancer cells proliferation. However, it is unclear whether CSN6 and β-TrCP are also involved in PDGF-induced pulmonary arterial smooth muscle cells (PASMCs) proliferation. This study aims to address this issue and further explore its potential mechanisms. Our results indicated that PDGF phosphorylated Akt, stimulated PASMCs proliferation; while inhibition of PDGF receptor (PDGFR) by imatinib prevented these effects. PDGF further up-regulated CSN6 protein expression, this was accompanied with β-TrCP reduction and increase of Cdc25A. Inhibition of PDGFR/PI3K/Akt signaling pathway reversed PDGF-induced such changes and cell proliferation. Prior transfection of CSN6 siRNA blocked PDGF-induced β-TrCP down-regulation, Cdc25A up-regulation and cell proliferation. Furthermore, pre-treatment of cells with MG-132 also abolished PDGF-induced β-TrCP reduction, Cdc25A elevation and cell proliferation. In addition, pre-depletion of Cdc25A by siRNA transfection suppressed PDGF-induced PASMCs proliferation. Taken together, our study indicates that up-regulation of CSN6 by PDGFR/PI3K/Akt signaling pathway decreases β-TrCP by increasing its ubiquitinated degradation, and thereby increases the expression of Cdc25A, which promotes PDGF-induced PASMCs proliferation., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: The nitric oxide-soluble guanylate cyclase-cyclic guanosine monophosphate (NO-sGC-cGMP) signaling pathway, plays a critical role in the pathogenesis of pulmonary arterial hypertension (PAH); however, its exact molecular mechanism remains undefined., Methods: Biotin-cGMP pull-down assay was performed to search for proteins regulated by cGMP. The interaction between cGMP and tropomyosin was analyzed with antibody dependent pull-down in vivo. Tropomyosin fragments were constructed to explore the tropomyosin-cGMP binding sites. The expression level and subcellular localization of tropomyosin were detected with Real-time PCR, Western blot and immunofluorescence assay after the 8-Br-cGMP treatment. Finally, isothermal titration calorimetry (ITC) was utilized to detect the binding affinity of actin-tropomyosin-myosin in the existence of cGMP-tropomyosin interaction., Results: cGMP interacted with tropomyosin. Isoform 4 of TPM1 gene was identified as the only isoform expressed in the human pulmonary artery smooth muscle cells (HPASMCs). The region of 68-208aa of tropomyosin was necessary for the interaction between tropomyosin and cGMP. The expression level and subcellular localization of tropomyosin showed no change after the stimulation of NO-sGC-cGMP pathway. However, cGMP-tropomyosin interaction decreased the affinity of tropomyosin to actin., Conclusions: We elucidate the downstream signal pathway of NO-sGC-cGMP. This work will contribute to the detection of innovative targeted agents and provide novel insights into the development of new therapies for PAH.

Background: Pulmonary arterial smooth muscle cell (PASMC) proliferation in response to hypoxia plays an important role in the vascular remodelling that occurs in hypoxic pulmonary hypertension. MicroRNAs (miRs) are emerging as important regulators in the progression of pulmonary hypertension. In this study, we investigated whether the expression of miR-17-5p is modulated by hypoxia and is involved in the hypoxia-induced proliferation of PASMCs., Methods: Human PASMCs were cultured under hypoxic conditions. miR-17-5p expression was determined by real-time RT-PCR. A BrdU incorporation assay and time-lapse recording were utilized to determine cell proliferation and migration., Results: PASMC proliferation was increased by moderate hypoxia (3% oxygen) but was reduced by severe hypoxia (0.1% oxygen) after 48 h. Moderate hypoxia induced miR-17-5p expression. Overexpression of miR-17-5p by transfection with miR-17-5p enhanced cell proliferation and migration in normoxia, whereas knockdown of miR-17-5p with anti-miR-17-5p inhibitors significantly reduced cell proliferation and migration. The expression of miR-17-5p target genes, specifically phosphatase and tensin homologue (PTEN) and cyclin-dependent kinase inhibitor 1 (p21WAF1/Cip1, p21), was reduced under moderate hypoxia in PASMCs. Under normoxia, overexpression of miR-17-5p in PASMCs reduced the expression of PTEN and p21., Conclusion: Our data indicate that miR-17-5p might play a significant role in hypoxia-induced pulmonary vascular smooth muscle cell proliferation by regulating multiple gene targets, including PTEN and p21, and that miR-17-5p could be a novel therapeutic target for the management of hypoxia-induced PH.

Hypoxic pulmonary vasoconstriction (HPV), which aids ventilation-perfusion matching in the lungs, is triggered by mechanisms intrinsic to pulmonary arterial smooth muscles. The unique sensitivity of these muscles to hypoxia is conferred by mitochondrial cytochrome c oxidase subunit 4 isoform 2, the inhibition of which has been proposed to trigger HPV through increased generation of mitochondrial reactive oxygen species. Contrary to this model, we have shown that the LKB1-AMPK-α1 signaling pathway is critical to HPV. Spectral Doppler ultrasound revealed that deletion of the AMPK-α1 catalytic subunit blocked HPV in mice during mild (8% O 2 ) and severe (5% O 2 ) hypoxia, whereas AMPK-α2 deletion attenuated HPV only during severe hypoxia. By contrast, neither of these genetic manipulations affected serotonin-induced reductions in pulmonary vascular flow. HPV was also attenuated by reduced expression of LKB1, a kinase that activates AMPK during energy stress, but not after deletion of CaMKK2, a kinase that activates AMPK in response to increases in cytoplasmic Ca 2+ Fluorescence imaging of acutely isolated pulmonary arterial myocytes revealed that AMPK-α1 or AMPK-α2 deletion did not affect mitochondrial membrane potential during normoxia or hypoxia. However, deletion of AMPK-α1, but not of AMPK-α2, blocked hypoxia from inhibiting K V 1.5, the classical "oxygen-sensing" K + channel in pulmonary arterial myocytes. We conclude that LKB1-AMPK-α1 signaling pathways downstream of mitochondria are critical for the induction of HPV, in a manner also supported by AMPK-α2 during severe hypoxia., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Acute hypoxia changes the redox-state of pulmonary arterial smooth muscle cells (PASMCs). This might influence the activity of redox-sensitive voltage-gated K⁺-channels (Kv-channels) whose inhibition initiates hypoxic pulmonary vasoconstriction (HPV). However, the molecular mechanism of how hypoxia-or the subsequent change in the cellular redox-state-inhibits Kv-channels remains elusive. For this purpose, a new multifunctional gas-tight microfluidic system was developed enabling simultaneous single-cell Raman spectroscopic studies (to sense the redox-state under normoxic/hypoxic conditions) and patch-clamp experiments (to study the Kv-channel activity). The performance of the system was tested by optically recording the O₂-content and taking Raman spectra on murine PASMCs under normoxic/hypoxic conditions or in the presence of H₂O₂. Oxygen sensing showed that hypoxic levels in the gas-tight microfluidic system were achieved faster, more stable and significantly lower compared to a conventional open system (1.6 ± 0.2%, respectively 6.7 ± 0.7%, n = 6, p < 0.001). Raman spectra revealed that the redistribution of biomarkers (cytochromes, FeS, myoglobin and NADH) under hypoxic/normoxic conditions were improved in the gas-tight microfluidic system ( p -values from 0.00% to 16.30%) compared to the open system ( p -value from 0.01% to 98.42%). In conclusion, the new redox sensor holds promise for future experiments that may elucidate the role of Kv-channels during HPV.

MiR-92b-3p has been shown to take part in several disease by regulate proliferation, apoptosis, differentiation and metastasis. However, the role of miR-92b-3p in pulmonary arterial hypertension (PAH) has not been illustrated clearly. Here, we found the level of miR-92b-3p which mainly located in the smooth muscle layer was down-regulation under hypoxic condition. It can inhibit pulmonary artery smooth muscle cells (PASMCs) proliferation and cell cycle progression. Through luciferase assay, miR-92b-3p bound to the 3'-UTR of USP28. we found that there was a significant negative relation between the level of miR-92b-3p and USP28 at protein level and reversed the down regulation of miR-92b-3p by hypoxia can suppress the proliferation of pulmonary artery smooth muscle cells by targeting USP28. These results suggested that miR-92b-3p acted a potential proliferation regulator in PASMCs and it maybe a novel treatment target of PAH., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Pulmonary hypertension (PH) is a disease with a developmental origin characterized by obstructive vascular remodelling that is partially due to excessive pulmonary arterial smooth muscle cells (PASMCs) proliferation. Paeonol has important effects on vascular cell proliferation, migration, and inflammation, but researchers have not determined whether paeonol participates in the development and progression of pulmonary vascular remodelling. We explored the remarkable anti-proliferative effects of paeonol on hypoxic PASMCs, which are postulated to be mediated by the extracellular signal-regulated kinase 1 and 2 (ERK1/2) signalling pathway. In this study, hypoxic rodent PH models, Western blotting, flow cytometry, immunochemistry, and morphometric analyses of the lung vasculature and right ventricle (RV) vessels were performed. Paeonol reversed hypoxia-induced increases in right ventricular function, right ventricular systolic pressure and thickening of medial walls. Meanwhile, paeonol ameliorated the hypoxia-induced PASMCs proliferation. Furthermore, paeonol modulated cell proliferation and cell cycle transitions from G 0 /G 1 phase to S phase and G 2 /M phase in an ERK1/2-dependent manner. Our findings emphasize the central function of paeonol in regulating PASMCs proliferation in subjects with PH. Therefore, paeonol represents a potential novel therapeutic approach for the treatment of PH., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The imbalance between the proliferation and apoptosis of pulmonary artery smooth muscle cells (PASMCs) is of importance in pulmonary vascular remodeling. Shikonin, a naphthoquinone compound extracted from the Chinese medicinal herb Lithospermum erythrorhizon, inhibits the proliferation of rat smooth muscle cells (SMCs). The present study was designed to investigate the effects of shikonin on the proliferation of rat PASMCs and the possible mechanisms involved. Rat PASMCs were cultured under the following five treatment conditions: Normal control; hypoxia for 24 h; hypoxia + 1 µM shikonin for 24 h; hypoxia + 2 µM shikonin for 24 h; and hypoxia + 4 µM shikonin for 24 h. The viability of PASMCs was measured using the Cell Counting Kit‑8 assay, the mRNA expression of nestin (NES) in each group was measured by reverse transcription‑polymerase chain reaction and the protein expression of NES was measured by western blotting. The proliferation of hypoxic PASMCs transfected with NES‑specific small interfering (si)RNA decreased compared with the non‑transfected group. These results indicated that hypoxia induced the proliferation of PASMCs through the enhancement of NES expression. The treatment of hypoxic PASMCs with shikonin resulted in a significant downregulation of NES expression and the inhibition of PASMC proliferation.

There is accumulating evidence indicating that the growth inhibitory effect of dichloroacetate (DCA) on pulmonary arterial smooth muscle cells (PASMCs) may be associated with the reversal of the Warburg effect and initiation of the mitochondria‑dependent apoptotic pathway. Previous studies indicated that platelet‑derived growth factor (PDGF) promoted the Warburg effect and resulted in apoptotic resistance of PASMCs, which was attributed to activation of the phosphatidylinositol 3‑kinase (PI3K)/protein kinase B (Akt) signalling pathway. However, the mechanism underlying the pro‑apoptotic effect of DCA on PDGF‑treated PASMCs has not been thoroughly elucidated, and the effect of the Akt/glycogen synthase kinase‑3β (GSK‑3β) pathway inhibition concomitant with the effect of DCA on PASMC proliferation remains unclear. The growth of human PASMCs and the lactate concentration in extracellular medium of PASMCs were detected by Cell Counting Kit‑8 assays and a Lactate Colorimetric Assay kit, respectively. Cell apoptosis was evaluated by fluorescence activated cell sorting. The mitochondrial membrane potential (ΔΨm) was assessed with 5,5',6,6'‑tetrachloro‑1,1',3,3'‑tetraethylbenzimidazol‑carbocyanine iodide assays. The expression levels of phosphorylated Akt and GSK‑3β, pyruvate dehydrogenase, cleaved caspase‑3, pyruvate dehydrogenase kinase‑1 (PDK‑1), hypoxia inducible factor‑1α (HIF‑1α) and hexokinase‑2 (HK‑2) were measured with western blot analysis. Confocal analyses were employed to determine HK‑2 co‑localisation with the mitochondria. The results indicated that DCA inhibited human PASMC proliferation in a dose‑dependent manner. DCA at 10 mM promoted apoptosis and the upregulation of activated caspase‑3 in PASMCs pre‑treated with 20 ng/ml PDGF‑homeodimer BB (BB). Treatment with 5 µM LY294002 produced minimal anti‑proliferative effects on human PASMCs and barely induced cellular apoptosis and caspase‑3 activation. However, co‑administration of 10 mM DCA with LY294002 significantly decreased the cell proliferation index and induced cell apoptosis and caspase‑3 activation. The combined administration of LY294002 with DCA significantly decreased lactate concentration, promoted the depolarisation of the ΔΨm and repressed HIF‑1α upregulation and HK‑2 activation in PASMCs treated with PDGF, which was attributed to the potentiation of DCA‑induced PDK‑1 inhibition by LY294002 via blockade of the Akt/GSK‑3β/HIF‑1α signalling pathway. In conclusion, inhibition of the Akt/GSK‑3β pathway improved the pro‑apoptotic effect of DCA on human PASMCs, which may be attributed to a reversal of the Warburg effect by blocking the mutual interaction between HIF‑1α and PDK‑1, consequently downregulating HK‑2. Therefore, combinatory treatment with DCA and PI3K inhibitors may represent a novel therapeutic strategy for the reversal of apoptosis resistance exhibited by PASMCs as a result of mitochondrial bioenergetic abnormalities, as well as the treatment of pulmonary vascular remodelling in pulmonary arterial hypertension.

Background: Endothelial barrier dysfunction characterized by hyperpermeability of the vascular endothelium is a key factor in the pathogenesis of chronic inflammatory diseases and affects clinical outcomes. In states of chronic inflammation, mediators secreted by activated immune cells or vascular endothelium may affect the barrier function and permeability of the vascular endothelium. The matricellular R-spondin family member RSPO3 is produced by inflammatory-activated human monocytes and vascular endothelial cells, but its effects in the regulation of vascular endothelial barrier function remains elusive., Methods: The present study investigates the effects of RSPO3 on the barrier function of adult human primary macro- and micro- vascular endothelial monolayers. Tight monolayers of primary endothelial cells from human coronary and pulmonary arteries, and cardiac, brain, and dermal microvascular beds were treated with RSPO3 either alone or in combination with pro-inflammatory mediator IL-1β. Endothelial barrier function was assessed non-invasively in real-time using Electric Cell-substrate Impedance Sensing., Results: RSPO3 treatment critically affected barrier function by enhancing the permeability of all vascular endothelial monolayers investigated. To confer hyperpermeable phenotype in vascular endothelial monolayers, RSPO3 induced inter-endothelial gap formation by disrupting the β-catenin and VE-cadherin alignment at adherens junctions. RSPO3 synergistically enhanced the barrier impairing properties of the pro-inflammatory mediator IL-1β., Conclusion: Here, we show that the matricellular protein RSPO3 is a mediator of endothelial hyperpermeability that can act in synergy with the inflammatory mediator IL-1β. This finding stimulates further studies to delineate the endothelial barrier impairing properties of RSPO3 and its synergistic interaction with IL-1β in chronic inflammatory diseases.

Studies were undertaken to examine any role for the hepcidin/ferroportin axis in proliferative responses of human pulmonary artery smooth muscle cells (hPASMCs). Entirely novel findings have demonstrated the presence of ferroportin in hPASMCs. Hepcidin treatment caused increased proliferation of these cells most likely by binding ferroportin resulting in internalisation and cellular iron retention. Cellular iron content increased with hepcidin treatment. Stabilisation of ferroportin expression and activity via intervention with the therapeutic monoclonal antibody LY2928057 reversed proliferation and cellular iron accumulation. Additionally, IL-6 treatment was found to enhance proliferation and iron accumulation in hPASMCs; intervention with LY2928057 prevented this response. IL-6 was also found to increase hepcidin transcription and release from hPASMCs suggesting a potential autocrine response. Hepcidin or IL-6 mediated iron accumulation contributes to proliferation in hPASMCs; ferroportin mediated cellular iron excretion limits proliferation. Haemoglobin also caused proliferation of hPASMCs; in other novel findings, CD163, the haemoglobin/haptoglobin receptor, was found on these cells and offers a means for cellular uptake of iron via haemoglobin. Il-6 was also found to modulate CD163 on these cells. These data contribute to a better understanding of how disrupted iron homeostasis may induce vascular remodelling, such as in pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is characterized by occlusion of lung arterioles, leading to marked increases in pulmonary vascular resistance. Although heritable forms of PAH are known to be driven by genetic mutations that share some commonality of function, the extent to which these effectors converge to regulate shared processes in this disease is unknown. We have causally connected extracellular matrix (ECM) remodeling and mechanotransduction to the miR-130/301 family in a feedback loop that drives vascular activation and downstream PAH. However, the molecular interconnections between factors genetically associated with PAH and this mechano-driven feedback loop remain undefined. We performed systematic manipulation of matrix stiffness, the miR-130/301 family, and factors genetically associated with PAH in primary human pulmonary arterial cells and assessed downstream and reciprocal consequences on their expression. We found that a network of factors linked to heritable PAH converges upon the matrix stiffening-miR-130/301-PPARγ-LRP8 axis in order to remodel the ECM. Furthermore, we leveraged a computational network biology approach to predict a number of additional molecular circuits functionally linking this axis to the ECM. These results demonstrate that multiple genes associated with heritable PAH converge to control the miR-130/301 circuit, triggering a self-amplifying feedback process central to pulmonary vascular stiffening and disease.

Pulmonary vascular remodeling is an important pathological feature of pulmonary arterial hypertension (PAH), which is characterized by thickening of the medial smooth muscle layer. Hypertrophy of pulmonary artery smooth muscle cells (PASMCs) participates in the development of medial thickening. Genistein can attenuate PAH and inhibit medial thickening of pulmonary arteries. Since hypoxia is one of the main causes of pulmonary hypertension, this study aims to investigate the mechanism of genistein in inhibiting hypertrophic responses in PASMCs induced by hypoxia. Cells isolated from the chick embryo were cultured with or without genistein and subjected to hypoxia or not. The increase of cell surface area and α-smooth muscle actin (α-SMA) of PASMCs was significantly suppressed by genistein during hypoxia. This result was confirmed by the incorporation of puromycin into peptide chains and flow cytometry analysis. Constrained mRNA and protein hypoxia-inducible factor (HIF)-1α expression was improved by genistein under hypoxia condition. Genistein restored redox homeostasis by fluorescent probe determination. The effect of genistein on hypertrophic response was blocked by estrogen receptor inhibitor, β1-adrenoceptor agonist and β2-adrenoceptor antagonist. In conclusion, genistein potently attenuates hypoxia-induced hypertrophy of PASMCs, which may enable a novel therapy for PAH., (Copyright © 2018. Published by Elsevier Inc.)

Pulmonary hypertension in the neonate requires treatment with oxygen and positive pressure ventilation, both known to induce lung injury. The direct response of pulmonary artery smooth muscle cells, the most abundant cells in the artery wall, to the stress of positive pressure and hyperoxia has not been previously studied. Pulmonary artery smooth muscle cells were cultured in temperature- and pressure-controlled air-tight chambers under conditions of positive pressure or hyperoxia for 24 h. Control cells were cultured in room air under atmospheric pressure. After the exposure period, culture medium was collected and samples were analyzed by ELISA, Human Cytokine 25-Plex Panel using a Luminex 200 analyzer and Western blot. Secretion of various inflammatory mediators, specifically IL-6, IL-8, IL-2R, MIP-1β, MCP-1, IP-10, IL-7, IL-1RA, and IFN-α, was higher in the positive pressure and hyperoxia groups compared with control. The level of cyclin D1 was decreased in the hyperoxia and positive pressure group compared with control. Levels of fibronectin and α-smooth muscle actin were not different among the groups. Pulmonary artery smooth muscle cells directly produce multiple inflammatory mediators in response to oxidative and biophysical stress in vitro, which may be part of a cascade that leads to the vascular and perivascular changes in pulmonary hypertension.

We have previously shown that 15-lipoxygenase (15-LOX) and its metabolite 15-hydroxyeicosanoid (15-HETE) play a critical role on hypoxia-triggered pulmonary artery smooth muscle cell (PASMC) phenotype alteration through multifactorial pathways, like extracellular signal-regulated kinase and p38 mitogen-activated protein kinases. Here, we hypothesize that activator protein 1 (AP-1) was also involved in the PASMC phenotype alteration. Hypoxia elevated AP-1 expression in pulmonary arterials and in cultured PASMCs with a time-dependent manner. Both the gene disruption and pharmacological inactivation of 15-lipoxygenase (15-LOX) significantly attenuated the hypoxia-elevated AP-1 expression. Silencing of AP-1 with small interference RNA abrogated the hypoxia- and 15-HETE-increased cell viability, proliferating cell nuclear antigen expression, and Ki67 and α-tubulin staining. Moreover, AP-1 knockdown prevented hypoxia- and 15-HETE-promoted cyclin D1 expression and subsequent cell cycle progression into G2/M+S phase. Interestingly, AP-1 knockdown also inhibited the expression of 15-LOX, indicating a feedback regulation of 15-LOX/15-HETE signaling by AP-1. These findings shed light on the involvement of AP-1 in the PASMCs phenotype alteration via the hypoxia/15-LOX/15-HETE signaling., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Objective: To study the changes of cytoskeleton during the phenotypic transition of rat pulmonary artery smooth muscle cells (PASMCs) induced by platelet-derived growth factor (PDGF-BB), and to explore the mechanism involved in the process of phenotypic transition of PASMCs., Methods: PASMCs of Sprague Dawley (SD) rats were cultured and identified by immunohistochemistry (IHC) method. The cells were randomly divided into control group and PDGF-BB treated group (10 ng/mL). RT-qPCR and Western blot were used to detect the mRNA and protein level of marker genes (α-SMA and SM22α) during the process of phenotypic transition of PASMCs. The changes of cytoskeleton were observed by fluorescent microscopy, cell proliferation was measured by CCK-8 method; and cell migration was observed by wound healing assay., Results: Compared with control group, PDGF-BB down-regulated the mRNA and protein expression of α-SMA and SM22α. The fluorescence intensity of cytoskeletal protein was significantly reduced after the treatment of PDGF-BB. In addition, the structure of F-actin was disorganized with a burr-like appearance, and the structures of α-tubulin and β-tubulin were irregular with co-location appearance. PDGF-BB significantly enhanced the proliferation and migration of PASMCs ., Conclusion: PDGF-BB could induce a conformation change in cytoskeletal proteins for PASMCs phenotypic transition, and enhance the ability of proliferation and migration of PASMCs., (Copyright© by Editorial Board of Journal of Sichuan University (Medical Science Edition).)

Objective: NKX2-5 is a homeobox transcription factor that is required for the formation of the heart and vessels during development, with significant postnatal down-regulation and reactivation in disease states, characterized by vascular remodeling. The purpose of this study was to investigate mechanisms that activate NKX2-5 expression in diseased vessels, such as systemic sclerosis (scleroderma; SSc)-associated pulmonary hypertension (PH), and to identify genetic variability that potentially underlies susceptibility to specific vascular complications., Methods: We explored NKX2-5 expression in biopsy samples from patients with SSc-associated PH and in pulmonary artery smooth muscle cells (PASMCs) from patients with scleroderma. Disease-associated putative functional single-nucleotide polymorphisms (SNPs) at the NKX2-5 locus were cloned and studied in reporter gene assays. SNP function was further examined through protein-DNA binding assays, chromatin immunoprecipitation assays, and RNA silencing analyses., Results: Increased NKX2-5 expression in biopsy samples from patients with SSc-associated PH was localized to remodeled vessels and PASMCs. Meta-analysis of 2 independent scleroderma cohorts revealed an association of rs3131917 with scleroderma (P = 0.029). We demonstrated that disease-associated SNPs are located in a novel functional enhancer, which increases NKX2-5 transcriptional activity through the binding of GATA-6, c-Jun, and myocyte-specific enhancer factor 2C. We also characterized an activator/coactivator transcription-enhancer factor domain 1 (TEAD1)/Yes-associated protein 1 (YAP1) complex, which was bound at rs3095870, another functional SNP, with TEAD1 binding the risk allele and activating the transcription of NKX2-5., Conclusion: NKX2-5 is genetically associated with scleroderma, pulmonary hypertension, and fibrosis. Functional evidence revealed a regulatory mechanism that results in NKX2-5 transcriptional activation in PASMCs through the interaction of an upstream promoter and a novel downstream enhancer. This mechanism can act as a model for NKX2-5 activation in cardiovascular disease characterized by vascular remodeling., (© 2018 The Authors. Arthritis & Rheumatology published by Wiley Periodicals, Inc. on behalf of American College of Rheumatology.)

Objective: Energy metabolism shift from oxidative phosphorylation toward glycolysis in pulmonary artery smooth muscle cells (PASMCs) is suggested to be involved in their hyperproliferation in pulmonary arterial hypertension (PAH). Here, we studied the role of the deacetylase sirtuin1 (SIRT1) in energy metabolism regulation in PASMCs via various pathways including activation of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), master regulator of mitochondrial biogenesis., Approach and Results: Contents of PGC-1α and its downstream targets as well as markers of mitochondrial mass (voltage-dependent anion channel and citrate synthase) were diminished in human PAH PASMCs. These cells and platelet-derived growth factor-stimulated rat PASMCs demonstrated a shift in cellular acetylated/deacetylated state, as evidenced by the increase of the acetylated forms of SIRT1 targets: histone H1 and Forkhead box protein O1. Rat and human PASMC proliferation was potentiated by SIRT1 pharmacological inhibition or specific downregulation via short-interfering RNA. Moreover, after chronic hypoxia exposure, SIRT1 inducible knock out mice displayed a more intense vascular remodeling compared with their control littermates, which was associated with an increase in right ventricle pressure and hypertrophy. SIRT1 activator Stac-3 decreased the acetylation of histone H1 and Forkhead box protein O1 and strongly inhibited rat and human PASMC proliferation without affecting cell mortality. This effect was associated with the activation of mitochondrial biogenesis evidenced by higher expression of mitochondrial markers and downstream targets of PGC-1α., Conclusion: Altered acetylation/deacetylation balance as the result of SIRT1 inactivation is involved in the pathogenesis of PAH, and this enzyme could be a promising therapeutic target for PAH treatment.

The present study was designed to examine whether hypoxia induces the proliferation, migration and differentiation of pulmonary arterial fibroblasts (PAFs) via the PI3K/Akt/p70S6K signaling pathway. PAFs were subjected to normoxia (21% O2) or hypoxia (1% O2). The proliferation, migration, differentiation and cellular p110α, p‑Akt, and p‑p70S6K expression levels of the PAFs were examined in vitro. In addition, rats were maintained under hypoxic conditions, and the right ventricular systolic pressure (RVSP), right ventricular hypertrophy index (RVHI) and right ventricular weight/body weight ratio (RV/BW) were examined. The expression levels of p110α, p‑Akt, p70S6K, fibronectin and α‑SMA in the rat pulmonary vessels were also examined. Hypoxia significantly elevated the proliferation, migration and differentiation of rat PAFs. It also strongly elevated the expression of p110α, p‑Akt and p‑p70S6K in PAFs in vitro. NVP‑BEZ235 was revealed to significantly reduce the hypoxia‑induced proliferation, migration and differentiation. In vivo experiments demonstrated that hypoxia significantly induced the elevation of RVSP, RVHI, RV/BW, medial thickening, adventitious thickening, and fibronectin and collagen deposition around pulmonary artery walls. The expression of p110α, p‑Akt and p70S6K was evident in the pulmonary arteries of the hypoxic rats. NVP‑BEZ235 significantly reduced the hypoxia‑induced hypoxic pulmonary vascular remodeling, as well as fibronectin and collagen deposition in the pulmonary arteries. Therefore, hypoxia was demonstrated to induce the proliferation, migration and differentiation of PAFs and the hypoxic pulmonary vascular remodeling of rats via the PI3K/Akt/p70S6K signaling pathway.

We have previously shown that hypoxia-induced proliferation of human pulmonary microvascular endothelial cells (hPMVEC) depends on arginase II, and that epidermal growth factor receptor (EGFR) is necessary for hypoxic-induction of arginase II. However, it remains unclear how hypoxia activates EGFR-mediated signaling in hPMVEC. We hypothesized that hypoxia results in epidermal growth factor (EGF) production and that EGF binds to EGFR to activate the signaling cascade leading to arginase II induction and proliferation in hPMVEC. We found that hypoxia significantly increased the mRNA levels of EGF, EGFR, and arginase in hPMVEC. Hypoxia significantly increased pEGFR(Tyr845) protein levels and an EGF neutralizing antibody prevented the hypoxic induction of pEGFR. Inhibiting EGFR activation prevented hypoxia-induced arginase II mRNA and protein induction. Treatment of hPMVEC with exogenous EGF resulted in greater levels of arginase II protein both in normoxia and hypoxia. An EGF neutralizing antibody diminished hypoxic induction of arginase II and resulted in fewer viable cells in hPMVEC. Similarly, siRNA against EGF prevented hypoxic induction of arginase II and resulted in fewer viable cells. Finally, conditioned media from hypoxic hPMVEC induced proliferation in human pulmonary artery smooth muscle cells (hPASMC), however, conditioned media from a group of hypoxic hPMVEC in which EGF were knocked down did not promote hPASMC proliferation. These findings demonstrate that hypoxia-induced arginase II expression and cellular proliferation depend on autocrine EGF production leading to EGFR activation in hPMVEC. We speculate that EGF-EGFR signaling may be a novel therapeutic target for pulmonary hypertensive disorders associated with hypoxia., (© 2018 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

Pulmonary vascular remodeling due to excessive proliferation and resistance to apoptosis of pulmonary artery smooth muscle cells (PASMCs) is the hallmark feature of pulmonary arterial hypertension (PAH). Recent evidence suggests that miR-125a-5p plays a role in a rat model of monocrotaline-induced PAH (MCT-PAH); however, the underlying mechanism is currently unknown. Here, we examined the expression profile of miR-125a-5p in MCT-PAH rats and investigated the putative therapeutic effect of miR-125a-5p using the miR-125a-5p agomir. In addition, the miR-125a-5p agomir or antagomir was transfected into rat PASMCs, and proliferation and apoptosis were measured. Activity of the miR-125a-5p target STAT3 was measured using a luciferase reporter assay, and the expression of downstream molecules was measured using RT-qPCR and/or western blot analysis. Importantly, inducing miR-125a-5p expression in vivo slowed the progression of MCT-PAH by reducing systolic pulmonary arterial pressure, the Fulton index, and pulmonary vascular remodeling. Moreover, overexpressing miR-125a-5p inhibited the proliferation and promoted the apoptosis of PASMCs. In addition, stimulating PASMCs with TGF-β1 or IL-6 upregulated miR-125a-5p expression, whereas overexpressing miR-125a-5p reduced TGF-β1 and IL-6 production, as well as the expression of their downstream targets STAT3 and Smad2/3; in contrast, downregulating miR-125a-5p increased TGF-β1 and IL-6 production. Finally, a dual-luciferase reporter assay revealed that miR-125a-5p targets the 3'-UTR of STAT3, suppressing the downstream molecules PCNA, Bcl-2, and Survivin. Taken together, these findings suggest that miR-125a-5p ameliorates MCT-PAH in rats, has a negative feedback regulation with TGF-β1 and IL-6, and regulates the proliferation and apoptosis of PASMCs by directly targeting STAT3.

Two-pore segment channel 2 (TPC2) is a ubiquitously expressed, lysosomally targeted ion channel that aids in terminating autophagy and is inhibited upon its association with mechanistic target of rapamycin (mTOR). It is controversial whether TPC2 mediates lysosomal Ca 2+ release or selectively conducts Na + and whether the binding of nicotinic acid adenine dinucleotide phosphate (NAADP) or phosphatidylinositol 3,5-bisphosphate [PI(3,5)P 2 ] is required for the activity of this ion channel. We show that TPC2 is required for intracellular Ca 2+ signaling in response to NAADP or to mTOR inhibition by rapamycin. In pulmonary arterial myocytes, rapamycin and NAADP evoked global Ca 2+ transients that were blocked by depletion of lysosomal Ca 2+ stores. Preincubation of cells with high concentrations of rapamycin resulted in desensitization and blocked NAADP-evoked Ca 2+ signals. Moreover, rapamycin and NAADP did not evoke discernable Ca 2+ transients in myocytes derived from Tpcn2 knockout mice, which showed normal responses to other Ca 2+ -mobilizing signals. In HEK293 cells stably overexpressing human TPC2, shRNA-mediated knockdown of mTOR blocked rapamycin- and NAADP-evoked Ca 2+ signals. Confocal imaging of a genetically encoded Ca 2+ indicator fused to TPC2 demonstrated that rapamycin-evoked Ca 2+ signals localized to lysosomes and were in close proximity to TPC2. Therefore, inactivation of mTOR may activate TPC2 and consequently lysosomal Ca 2+ release., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Application of H 2 S ("sulfide") elicits a complex contraction in rat pulmonary arteries (PAs) comprising a small transient contraction (phase 1; Ph1) followed by relaxation and then a second, larger, and more sustained contraction (phase 2; Ph2). We investigated the mechanisms causing this response using isometric myography in rat second-order PAs, with Na 2 S as a sulfide donor. Both phases of contraction to 1,000 μM Na 2 S were attenuated by the pan-PKC inhibitor Gö6983 (3 μM) and by 50 μM ryanodine; the Ca 2+ channel blocker nifedipine (1 μM) was without effect. Ph2 was attenuated by the mitochondrial complex III blocker myxothiazol (1 μM), the NADPH oxidase (NOX) blocker VAS2870 (10 μM), and the antioxidant TEMPOL (3 mM) but was unaffected by the complex I blocker rotenone (1 μM). The bath sulfide concentration, measured using an amperometric sensor, decreased rapidly following Na 2 S application, and the peak of Ph2 occurred when this had fallen to ~50 μM. Sulfide caused a transient increase in NAD(P)H autofluorescence, the offset of which coincided with development of the Ph2 contraction. Sulfide also caused a brief mitochondrial hyperpolarization (assessed using tetramethylrhodamine ethyl ester), followed immediately by depolarization and then a second more prolonged hyperpolarization, the onset of which was temporally correlated with the Ph2 contraction. Sulfide application to cultured PA smooth muscle cells increased reactive oxygen species (ROS) production (recorded using L012); this was absent when the mitochondrial flavoprotein sulfide-quinone oxoreductase (SQR) was knocked down using small interfering RNA. We propose that the Ph2 contraction is largely caused by SQR-mediated sulfide metabolism, which, by donating electrons to ubiquinone, increases electron production by complex III and thereby ROS production.

Autophagy is an evolutionarily conserved cellular process that facilitates the continuous recycling of intracellular components (organelles and proteins) and provides an alternative source of energy when nutrients are scarce. Recent studies have implicated autophagy in many disorders, including pulmonary diseases. However, the role of autophagy in endothelial cell (EC) barrier dysfunction and its relevance in the context of acute lung injury (ALI) remain uncertain. Here, we provide evidence that autophagy is a critical component of EC barrier disruption in ALI. Using an aerosolized bacterial lipopolysaccharide (LPS) inhalation mouse model of ALI, we found that administration of the autophagy inhibitor 3-methyladenine (3-MA), either prophylactically or therapeutically, markedly reduced lung vascular leakage and tissue edema. 3-MA was also effective in reducing the levels of proinflammatory mediators and lung neutrophil sequestration induced by LPS. To test the possibility that autophagy in EC could contribute to lung vascular injury, we addressed its role in the mechanism of EC barrier disruption. Knockdown of ATG5, an essential regulator of autophagy, attenuated thrombin-induced EC barrier disruption, confirming the involvement of autophagy in the response. Similarly, exposure of cells to 3-MA, either before or after thrombin, protected against EC barrier dysfunction by inhibiting the cleavage and loss of vascular endothelial cadherin at adherens junctions, as well as formation of actin stress fibers. 3-MA also reversed LPS-induced EC barrier disruption. Together, these data imply a role of autophagy in lung vascular injury and reveal the protective and therapeutic utility of 3-MA against ALI.

Prostacyclin agonists that bind the prostacyclin receptor (IP) to stimulate cAMP synthesis are effective vasodilators for the treatment of idiopathic pulmonary arterial hypertension (IPAH), but this signaling may occur through nuclear peroxisome proliferator-activated receptor-γ (PPARγ). There is evidence of scant IP and PPARγ expression but stable prostanoid EP 4 receptor (EP 4 ) expression in IPAH patients. Both IP and EP 4 functionally couple with stimulatory G protein (G s ), which activates signal transduction. We investigated the effect of an EP 4 -specific agonist on pulmonary arterial remodeling and its regulatory mechanisms in pulmonary arterial smooth muscle cells (PASMCs). Immunoblotting evealed IP, EP 4 , and PPARγ expression in human pulmonary arterial hypertension (PAH) and monocrotaline (MCT)-induced PAH rat lung tissue. Isolated PASMCs from MCT-induced PAH rats (MCT-PASMCs) were treated with L-902,688, a selective EP 4 agonist, to investigate the anti-vascular remodeling effect. Scant expression of IP and PPARγ but stable expression of EP 4 was observed in IPAH patient lung tissues and MCT-PASMCs. L-902,688 inhibited IP-insufficient MCT-PASMC proliferation and migration by activating PPARγ in a time- and dose-dependent manner, but these effects were reversed by AH-23848 (an EP 4 antagonist) and H-89 [a protein kinase A (PKA) inhibitor], highlighting the crucial role of PPARγ in the activity of this EP 4 agonist. L-902,688 attenuated pulmonary arterial remodeling in hypoxic PAH mice and MCT-induced PAH rats; therefore, we conclude that the selective EP 4 agonist L-902,688 reverses vascular remodeling by activating PPARγ. This study identified a novel EP 4 -PKA-PPARγ pathway, and we propose EP 4 as a potential therapeutic target for PAH.

Effective re-endothelialization is critical for the use of decellularized scaffolds for ex vivo lung engineering. Current approaches yield insufficiently re-endothelialized scaffolds that haemorrhage and become thrombogenic upon implantation. Herein, gravity-driven seeding coupled with bioreactor culture facilitated widespread distribution and engraftment of endothelial cells throughout rat lung scaffolds. Initially, human umbilical vein endothelial cells were seeded into the pulmonary artery by either gravity-driven, variable flow perfusion seeding or pump-driven, pulsatile flow perfusion seeding. Gravity seeding evenly distributed cells and supported cell survival and re-lining of the vascular walls while perfusion pump-driven seeding led to increased cell fragmentation and death. Using gravity seeding, rat pulmonary artery endothelial cells and rat pulmonary vein endothelial cells attached in intermediate and large vessels, while rat pulmonary microvascular endothelial cells deposited mostly in microvessels. Combination seeding of these cells led to positive vascular endothelial cadherin staining. In addition, combination seeding improved barrier function as assessed by serum albumin extravasation; however, leakage was observed in the distal portions of the re-endothelialized tissue suggesting that recellularization of the alveoli is necessary to complete barrier function of the capillary-alveolar network. Overall, these data indicate that vascular recellularization of rat lung scaffolds is achieved through gravity seeding. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Infants with lung disease may be exposed to high O 2 concentrations, and may have transient hypoxic episodes due to worsening lung pathophysiology, aggravating pulmonary arterial (PA) oxidative stress. NADPH oxidase (NOX) converts O 2 to superoxide. Mitochondrial antioxidants such as superoxide dismutase (SOD) are sensitive to O 2 tension. We previously reported decreased SOD2 activity in hypoxic PA myocytes due to nitration. In this study, we examined whether a transient hypoxic episode exposes antioxidant defense defects in hyperoxic PA myocytes. PA myocytes of term newborn piglets were cultured in hyperoxia (60% O 2 ) or normoxia (21% O 2 ) for 72 h; cells from both groups were challenged with transient hypoxia (10% O 2 ) for 2 h. We measured NOX activity, SOD activities (fractionated by centrifugation and concavalin A- Sepharose chromatography), total ROS and superoxide generation, 8-isoprostane, and calcium responses to thromboxane mimetic. NOX activity increased in hyperoxic myocytes. Hyperoxia increased SOD1 activity but decreased SOD2 activity. Total ROS were reduced in hyperoxia, and hyperoxia + hypoxia groups. While hyperoxia alone did not alter superoxide content, superoxide increased after a hypoxic challenge of both normoxic and hyperoxic myocytes. Increased 8-isoprostane was seen only in hyperoxic myocytes challenged by transient hypoxia. We conclude that hyperoxic PA myocytes can limit total ROS despite increased NOX activity, but with inhibited SOD2 activity. Transient hypoxia increases superoxide formation; in the face of impaired SOD2, despite induction of SOD1, this oxidative stress causes increased 8-isoprostane generation. This may contribute to the mechanism of pulmonary arterial reactivity in infants with severe lung disease., (Copyright © 2017. Published by Elsevier Ltd.)

Pulmonary arterial hypertension (PAH) is a progressive vascular remodeling disease characterized by a persistent elevation of pulmonary artery pressure, leading to right heart failure and premature death. Exaggerated proliferation and resistance to apoptosis of pulmonary artery smooth muscle cells (PASMCs) is a key component of vascular remodeling. Despite major advances in the field, current therapies for PAH remain poorly effective in reversing the disease or significantly improving long-term survival. Because the transcription factor FOXM1 is necessary for PASMC proliferation during lung morphogenesis and its overexpression stimulates proliferation and evasion of apoptosis in cancer cells, we thus hypothesized that upregulation of FOXM1 in PAH-PASMCs promotes cell expansion and vascular remodeling. Our results showed that FOXM1 was markedly increased in distal pulmonary arteries and isolated PASMCs from PAH patients compared to controls as well as in two preclinical models. In vitro, we showed that miR-204 expression regulates FOXM1 levels and that inhibition of FOXM1 reduced cell proliferation and resistance to apoptosis through diminished DNA repair mechanisms and decreased expression of the pro-remodeling factor survivin. Accordingly, inhibition of FOXM1 with thiostrepton significantly improved established PAH in two rat models. Thus, we show for the first time that FOXM1 is implicated in PAH development and represents a new promising target., Key Messages: FOXM1 is overexpressed in human PAH-PASMCs and PAH animal models. FOXM1 promotes PAH-PASMC proliferation and resistance to apoptosis. Pharmacological inhibition of FOXM1 improves established PAH in the MCT and Su/Hx rat models. FOXM1 may be a novel therapeutic target in PAH.

MicroRNA (miR)‑138 serves an important role in the proliferation, differentiation and apoptosis of human pulmonary artery smooth muscle cells (HPASMCs), indi-cating the involvement of miR‑138 in the development and progression of pulmonary artery hypertension (PAH). Potassium channel subfamily K member 3 (TASK‑1), a two‑pore domain K+ channel, is expressed in HPASMCs and is associated with hypoxic PAH. However, whether miR‑138 mediates PAH through targeting TASK‑1 is not known. In the present study, HPASMCs were transfected with miR‑138 mimic to establish a PAH model in vitro, and the effects of a miR‑138 inhibitor and a TASK‑1 inhibitor (A293) were examined. Cell proliferation and mitochondrial membrane potential (MMP) were measured by CCK‑8 assay and flow cytometry, respectively. Reverse transcription-quantitative polymerase chain reaction and western blotting were performed to examine the expression of miR‑138, TASK‑1, Bcl‑2, caspase‑3 and activation of extracellular signal‑regulated kinase 1/2 (ERK1/2). A dual‑luciferase reporter assay was also used to analyse the expression level of TASK‑1 in HPASMCs. The results of the present study demonstrated that the miR‑138 mimic promoted proliferation and MMP level, which was similar to the effect of A293 treatment on HPASMCs. However, the miR‑138 inhibitor inhibited the effects induced by miR‑138 mimic or A293 treatment, as demonstrated by a decrease in proliferation and MMP level in HPASMCs, accompanied by a decrease of Bcl‑2 and an increase of caspase‑3 expression levels, as well as ERK1/2 activation. The dual‑luciferase reporter assay indicated that TASK‑1 expression was negatively regulated by miR‑138. The results of the present study suggested that miR‑138 promoted proliferation and suppressed mitochondrial depolarization of HPASMCs by targeting TASK‑1.

Loss of peroxisome proliferator-activated receptor γ (PPARγ) has been found to contribute to pulmonary artery smooth muscle cell (PASMC) proliferation and pulmonary arterial remodeling therefore the development of pulmonary hypertension (PH). Yet, the molecular mechanisms underlying PPARγ reduction in PASMC remain poorly understood. Here, we demonstrated that leptin dose- and time-dependently inducued PPARγ down-regulation and proliferation of primary cultured rat PASMC, this was accompanied with the activation of extracellular regulated kinase1/2 (ERK1/2) signaling pathway and subsequent induction of early growth response-1 (Egr-1) expression. The presence of MEK inhibitors U0126 or PD98059, or prior silencing Egr-1 with small interfering RNA suppressed leptin-induced PPARγ reduction. In addition, activation of PPARγ by pioglitazone or targeting ERK1/2/Egr-1 suppressed leptin-induced PASMC proliferation. Taken together, our study indicates that ERK1/2 signaling pathway-mediated leptin-induced PPARγ reduction and PASMC proliferation through up-regulation of Egr-1 and suggests that targeting leptin/ERK1/2/Egr-1 pathway might have potential value in ameliorating vascular remodeling and benefit PH., (© 2018 Xie et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Pulmonary artery hypertension (PAH) is a rare and fatal disorder that involves extensive remodeling of the pulmonary arteries mediated by hyperproliferation of pulmonary artery smooth muscle cells (PASMCs). Aberrant platelet-derived growth factor (PDGF) activity can lead to hyperproliferation of PASMCs; however, little is known about the role of long noncoding RNA (lncRNA) in this process. Using RNA sequencing, we identified 725 lncRNAs in rat PASMCs, 95 of which were expressed differentially in response to PDGF-BB treatment. Depletion of four lncRNAs affected the proliferation of rat PASMCs as measured by 5-ethynyl-2'-deoxyuridine incorporation assay. Among these, one lncRNA, named LnRPT (lncRNA regulated by PDGF and transforming growth factor β), was found to be the most potent in promoting the proliferation of PASMCs when knocked down. In contrast, proliferation of PASMCs was repressed when LnRPT was overexpressed. Mechanistically, LnRPT inhibited the expression of two genes involved in the Notch signaling pathway (notch3 and jag1) as well as the cell-cycle-regulating gene ccna2. In addition, downregulation of LnRPT induced by PDGF-BB was abrogated when phosphatidylinositol 3'-kinase activity was inhibited with pictilisib. Downregulation of LnRPT was also observed in the pulmonary arteries of rats with monocrotaline-induced PAH. This study provides novel insights into the effects of PDGF-BB on lncRNA expression in PASMCs, and identifies one lncRNA, LnRPT, that plays a role in PAH development as a regulator of PASMC proliferation by mediating the Notch signaling pathway and cell cycle.

Exploration of human pulmonary artery endothelial cell (EC) as a prototypical biomechanical system has important pathophysiologic relevance because this cell type plays a key role in the development of a wide variety of clinical conditions. The complex hierarchical organization ranging from the molecular scale up to the cellular level has an intimate and intricate relationship to the barrier function between lung tissue and blood. To understand the innate molecule-cell-tissue relationship across varied length-scales, the functional role of c-Abl kinase in the cytoskeletal nano-biomechanics of ECs in response to barrier-altering agonists was investigated using atomic force microscopy. Concurrently, the spatially specific arrangement of cytoskeleton structure and dynamic distribution of critical proteins were examined using scanning electron microscopy and immunofluorescence. Reduction in c-Abl expression by siRNA attenuates both thrombin- and sphingosine 1-phosphate (S1P)-mediated structural changes in ECs, specifically spatially-defined changes in elastic modulus and distribution of critical proteins. These results indicate that c-Abl kinase is an important determinant of cortical actin-based cytoskeletal rearrangement. Our findings directly bridge the gap between kinase activity, structural complexity, and functional connectivity across varied length-scales, and suggest that manipulation of c-Abl kinase activity may be a potential target for the treatment of pulmonary barrier disorders.

Hypoxia-induced pulmonary hypertension is a life-threatening disease arising from a progressive increase in pulmonary vascular resistance, irreversible pulmonary vascular remodeling and resulting in right ventricular failure. Recent studies suggested that pulmonary artery smooth muscle cell proliferation and migration played an important role in the pathogenesis of hypoxia-induced pulmonary hypertension. However, the mechanisms of hypoxia-induced pulmonary hypertension are complicated and largely unclear. In this study, we discovered that lncRNA MEG3 was down-regulated in human pulmonary artery smooth muscle cell in hypoxia, and inhibition of MEG3 promoted the cell proliferation and cell migration in both normal and hypoxia condition. Further study demonstrated that MEG3 exerted its function via regulation of miR-21 expression in both normal and hypoxia condition. In addition, we displayed the modulation of PTEN by miR-21 and their role in hypoxia. Ultimately, our study illustrated that MEG3 exerts its role via miR-21/PTEN axis in human pulmonary artery smooth muscle cell under both normal and hypoxia conditions., (Copyright © 2017. Published by Elsevier Inc.)

Background/aims: The underlying molecular mechanisms involved in sphingosine kinase 1 (SphK1)/sphingosine 1-phosphate (S1P) mediation of platelet-derived growth factor (PDGF)-induced pulmonary arterial smooth muscle cell (PASMC) proliferation are still unclear, and the present study aims to address this issue., Methods: Small interfering RNA (siRNA) and microRNA inhibitor transfection was performed to block the expression of SphK1, bone morphogenetic protein receptor II (BMPRII) and microRNA-21 (miR-21). Gene expression levels of SphK1, BMPRII and inhibitor of DNA binding 1 (Id1) were detected by immunoblotting, miR-21 expression level was examined with qRT-PCR, and S1P production was measured by ELISA. Additionally, PASMC proliferation was determined by BrdU incorporation assay., Results: Our results indicated that PDGF increased the expression of SphK1 protein and S1P production, up-regulated miR-21 expression, reduced BMPRII and Id1 expression, and promoted PASMCs proliferation. Pre-silencing of SphK1 with siRNA reversed PDGF-induced S1P production, miR-21 up-regulation, BMPRII and Id1 down-regulation, as well as PASMC proliferation. Pre-inhibition of miR-21 also blocked BMPRII and Id1 down-regulation as well as PASMC proliferation caused by PDGF. Knockdown of BMPRII down-regulated Id1 expression in PASMCs. We further found that inhibition of PI3K/Akt and ERK signaling pathways, particularly ERK cascade, suppressed PDGF-induced above changes., Conclusion: Our study indicates that SphK1/S1P pathway plays an important role in PDGF-induced PASMC proliferation via miR-21/BMPRII/Id1 axis and targeting against SphK1/S1P axis might be a novel strategy in the prevention and treatment of pulmonary arterial hypertension (PAH)., (© 2018 The Author(s). Published by S. Karger AG, Basel.)

Endothelium plays an important role in maintaining the vascular barrier and physiological homeostasis. Endothelium also is fundamental to the initiation and regulation of inflammation. Endothelium demonstrates phenotypic and functional heterogeneity not only among various organs but also within an organ. One of the striking examples would be the pulmonary endothelium that participates in creating blood-air barrier. Endothelium in large pulmonary blood vessels is distinct in structure and function from that lining of the pulmonary capillaries. This chapter focuses on the comparative aspects of pulmonary endothelium and highlight unique differences such as the presence of pulmonary intravascular macrophages among select species.

Background/aims: Pulmonary arterial hypertension (PAH) is a severe and debilitating disease characterized by remodeling of the pulmonary vessels, which is driven by excessive proliferation and migration and apoptosis resistance in pulmonary artery smooth muscle cells (PASMCs). The calcineurin (CaN)/nuclear factor of activated T-cells (NFAT) signaling pathway is the most important downstream signaling pathway of store-operated Ca2+ entry (SOCE), which is increased in PAH. CaN/NFAT has been reported to contribute to abnormal proliferation in chronic hypoxia (CH)-induced PAH. However, the effect of CaN/NFAT signaling on PASMC proliferation, migration and apoptosis in monocrotaline (MCT)-induced PAH remains unclear., Methods: PAH rats were established by a single intraperitoneal injection of MCT for 21 days. PASMCs were isolated and cultured in normal and MCT-induced PAH Sprague-Dawley rat. PASMCs were treated with CsA targeting CaN and siRNA targeting NFATc2-4 gene respectively by liposome. We investigated the expression of calcineurin/NFAT signaling by immunofluorescence, qRT-PCR and Western blotting methods. Cell proliferation was monitored using MTS reagent or by assessing proliferating cell nuclear antigen (PCNA) expression. Cell apoptosis was evaluated with an Annexin V - FITC/propidium iodide (PI) apoptosis kit by flow cytometry. PASMC migration was assessed with a Transwell chamber., Results: MCT successfully induced PAH and pulmonary vascular remodeling in rats. CaN phosphatase activity and nuclear translocation of NFATc2-4 were increased in PASMCs derived from MCT-treated rats. In addition, CaNBβ/NFATc2-4 expression was amplified at the mRNA and protein levels. PASMC proliferation and migration were markedly inhibited in a dosedependent manner by cyclosporin A (CsA). Furthermore, siRNA targeting NFATc2 and NFATc4 attenuated the excessive proliferation and migration and apoptosis resistance in PASMCs derived from both CON and MCT-treated rats, while NFATc3 knockdown specifically affected MCT-PASMCs., Conclusion: Our results demonstrate that CaN/NFAT signaling is activated and involved in the modulation of PASMC proliferation, migration and apoptosis in MCT-induced PAH., (© 2018 The Author(s). Published by S. Karger AG, Basel.)

We recently demonstrated that blue light induces vasorelaxation in the systemic mouse circulation, a phenomenon mediated by the nonvisual G protein-coupled receptor melanopsin (Opsin 4; Opn4). Here we tested the hypothesis that nonvisual opsins mediate photorelaxation in the pulmonary circulation. We discovered Opsin 3 (Opn3), Opn4, and G protein-coupled receptor kinase 2 (GRK2) in rat pulmonary arteries (PAs) and in pulmonary arterial smooth muscle cells (PASMCs), where the opsins interact directly with GRK2, as demonstrated with a proximity ligation assay. Light elicited an intensity-dependent relaxation of PAs preconstricted with phenylephrine (PE), with a maximum response between 400 and 460 nm (blue light). Wavelength-specific photorelaxation was attenuated in PAs from Opn4 -/- mice and further reduced following shRNA-mediated knockdown of Opn3. Inhibition of GRK2 amplified the response and prevented physiological desensitization to repeated light exposure. Blue light also prevented PE-induced constriction in isolated PAs, decreased basal tone, ablated PE-induced single-cell contraction of PASMCs, and reversed PE-induced depolarization in PASMCs when GRK2 was inhibited. The photorelaxation response was modulated by soluble guanylyl cyclase but not by protein kinase G or nitric oxide. Most importantly, blue light induced significant vasorelaxation of PAs from rats with chronic pulmonary hypertension and effectively lowered pulmonary arterial pressure in isolated intact perfused rat lungs subjected to acute hypoxia. These findings show that functional Opn3 and Opn4 in PAs represent an endogenous "optogenetic system" that mediates photorelaxation in the pulmonary vasculature. Phototherapy in conjunction with GRK2 inhibition could therefore provide an alternative treatment strategy for pulmonary vasoconstrictive disorders.

Phospholipase D (PLD) catalyzes the hydrolysis of phosphatidylcholine to produce phosphatidic acid (PA) which in some cell types play a pivotal role in agonist-induced increase in NADPH oxidase-derived [Formula: see text]production. Involvement of ADP ribosylation factor (Arf) in agonist-induced activation of PLD is known for smooth muscle cells of systemic arteries, but not in pulmonary artery smooth muscle cells (PASMCs). Additionally, role of cytohesin in this scenario is unknown in PASMCs. We, therefore, determined the involvement of Arf and cytohesin in U46619-induced stimulation of PLD in PASMCs, and the probable mechanism by which curcumin, a natural phenolic compound, inhibits the U46619 response. Treatment of PASMCs with U46619 stimulated PLD activity in the cell membrane, which was inhibited upon pretreatment with SQ29548 (Tp receptor antagonist), FIPI (PLD inhibitor), SecinH3 (inhibitor of cytohesins), and curcumin. Transfection of the cells with Tp, Arf-6, and cytohesin-1 siRNA inhibited U46619-induced activation of PLD. Upon treatment of the cells with U46619, Arf-6 and cytohesin-1 were translocated and associated in the cell membrane, which were not inhibited upon pretreatment of the cells with curcumin. Cytohesin-1 appeared to be necessary for in vitro binding of GTPγS with Arf-6; however, addition of curcumin inhibited binding of GTPγS with Arf-6 even in the presence of cytohesin-1. Our computational study suggests that although curcumin to some extent binds with Tp receptor, yet the inhibition of Arf6 GDP to Arf6 GTP conversion appeared to be an important mechanism by which curcumin inhibits U46619-induced increase in PLD activity in PASMCs.

Background/aims: Vascular muscularity is a key event in vessel remodeling during pulmonary artery hypertension (PAH). Endothelial-mesenchymal transdifferentiation (EndMT) has been increasingly reported to play a role in disease occurrence. Galectin-3, a carbohydrate-binding protein regulates cell proliferation, differentiation, migration and neovascularization. However, whether galectin-3 controls endothelial cell transdifferentiation during the development of PAH is unknown., Methods: Rats were exposed to normoxic or hypoxic conditions (fraction of inspired O2 0.10) for 21 d to establish PAH models. Hemodynamic changes were evaluated through surgery of the right jugular vein and ultrasound biomicroscopy inviVue. And vessel pathological alterations were detected by H&E staining. Galectin-3 (Gal-3)-induced pulmonary artery endothelium cell (PAEC) dynamic alterations were measured by MTT assays, Cell immunofluorescence, Flow cytometry, Real-time PCR and Western blot., Results: Our study demonstrated that Gal-3 was expressed in hypoxic pulmonary vascular adventitia and intima. The increased Gal-3 expression was responsible for hypoxic vessel remodeling and PAH development in vivo. Gal-3 was found to inhibit cell proliferation and apoptosis in cultured endothelial cells. Meanwhile endothelial cell morphology was altered and exhibited smooth muscle-like cell features as demonstrated by the expression of α-SMA after Gal-3 treatment. Gal-3 activated Jagged1/Notch1 pathways and induced MyoD and SRF. When MyoD or SRF were silenced with siRNAs, Gal-3-initiated transdifferentiation in endothelial cells was blocked as indicated by a lack of α-SMA., Conclusion: These results suggest that Gal-3 induces PAECs to acquire an α-SMA phenotype via a transdifferentiation process which depends on the activation of Jagged1/Notch1 pathways that mediate MyoD and SRF expression., (© 2018 The Author(s). Published by S. Karger AG, Basel.)

Pulmonary blood vessels act as a well-regulated barrier to the flux of fluid and solutes between the lumen and the air space. Perturbation of the barrier function results in excessive fluid leak into the interstitium and alveoli, and impairs gas exchange. Recent studies provide deeper insight into the precise control mechanisms involved in the regulation of the barrier. This chapter will highlight these mechanisms and discuss the current understanding on the fluid and solute transport pathways across the vascular endothelial layer. In addition, the chapter summarizes the contributions of extra-endothelial structures such as pericytes and glycocalyx in regulating fluid flux across pulmonary vessels. The chapter concludes with an analysis on the impact of pulmonary endothelial heterogeneity and experimental models on current interpretations of barrier function and regulatory mechanisms.

In this study, the effect of four xyloketals 1-4 on store-operated calcium entry (SOCE) was investigated in primary distal pulmonary arterial smooth muscle cells (PASMCs) isolated from mice. The results showed that xyloketal A (1), an unusual ketal with C-3 symmetry, exhibited strong SOCE blocking activity. Secretion of interleukin-8 (IL-8) was also inhibited by xyloketal A. The parallel artificial membrane permeability assay (PAMPA) of 1-4 suggested that these xyloketals penetrated easily through the cell membrane. Moreover, the molecular docking study of xyloketal A with activation region of the stromal interaction molecule (STIM) 1 and the calcium release-activated calcium modulator (ORAI) 1 (STIM1-ORAI1) protein complex, the key domain of SOCE, revealed that xyloketal A exhibited a noncovalent interaction with the key residue lysine 363 (LYS363) in the identified cytosolic regions in STIM1-C. These findings provided useful information about xyloketal A as a SOCE inhibitor for further evaluation.

Recent studies have revealed that particulate matter (PM) exert deleterious effects on vascular function. Pulmonary artery endothelial cells (HPAEC), which are involved in the vasomotricity regulation, can be a direct target of inhaled particles. Modifications in calcium homeostasis and oxidative stress are critical events involved in the physiopathology of vascular diseases. The objectives of this study were to assess the effects of PM 2.5 on oxidative stress and calcium signaling in HPAEC. Different endpoints were studied, (i) intrinsic and intracellular production of reactive oxygen species (ROS) by the H 2 DCF-DA probe, (ii) intrinsic, intracellular and mitochondrial production of superoxide anion (O 2 - ) by electronic paramagnetic resonance spectroscopy and MitoSOX probe, (iii) reactive nitrosative species (RNS) production by Griess reaction, and (vi) calcium signaling by the Fluo-4 probe. In acellular conditions, PM 2.5 leads to an intrinsic free radical production (ROS, O 2 - ) and a 4h-exposure to PM 2.5 (5-15μg/cm 2 ), induced, in HPAEC, an increase of RNS, of global ROS and of cytoplasmic and mitochondrial O 2 - levels. The basal intracellular calcium ion level [Ca 2+ ]i was also increased after 4h-exposure to PM 2.5 and a pre-treatment with superoxide dismutase and catalase significantly reduced this response. This study provides evidence that the alteration of intracellular calcium homeostasis induced by PM 2.5 is closely correlated to an increase of oxidative stress., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Hypoxic pulmonary vasoconstriction (HPV) in combination with hypercapnic pulmonary vasoconstriction redistributes pulmonary blood flow from poorly aerated to better ventilated lung regions by an active process of local vasoconstriction. Impairment of HPV results in ventilation-perfusion mismatch and is commonly associated with various lung diseases including pneumonia, sepsis, or cystic fibrosis. Although several regulatory pathways have been identified, considerable knowledge gaps persist, and a unifying concept of the signaling pathways that underlie HPV and their impairment in lung diseases has not yet emerged. In the past, conceptual models of HPV have focused on pulmonary arterial smooth muscle cells (PASMC) acting as sensor and effector of hypoxia in the pulmonary vasculature. In contrast, the endothelium was considered a modulating bystander in this scenario. For an ideal design, however, the oxygen sensor in HPV should be located in the region of gas exchange, i.e., in the alveolar capillary network. This concept requires the retrograde propagation of the hypoxic signal along the endothelial layer of the vascular wall and subsequent contraction of PASMC in upstream arterioles that is elicited via temporospatially tightly controlled endothelial-smooth muscle cell crosstalk. The present review summarizes recent work that provides proof-of-principle for the existence and functional relevance of such signaling pathway in HPV that involves important roles for connexin 40, epoxyeicosatrienoic acids, sphingolipids, and cystic fibrosis transmembrane conductance regulator. Of translational relevance, implication of these molecules provides for novel mechanistic explanations for impaired ventilation/perfusion matching in patients with pneumonia, sepsis, cystic fibrosis, and presumably various other lung diseases.