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Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease of unknown etiology characterized by distorted distal lung architecture, inflammation, and fibrosis. The molecular mechanisms involved in the pathophysiology of IPF are incompletely defined. Several lung cell types including alveolar epithelial cells, fibroblasts, monocyte-derived macrophages, and endothelial cells have been implicated in the development and progression of fibrosis. Regardless of the cell types involved, changes in gene expression, disrupted glycolysis, and mitochondrial oxidation, dysregulated protein folding, and altered phospholipid and sphingolipid metabolism result in activation of myofibroblast, deposition of extracellular matrix proteins, remodeling of lung architecture and fibrosis. Lipid mediators derived from phospholipids, sphingolipids, and polyunsaturated fatty acids play an important role in the pathogenesis of pulmonary fibrosis and have been described to exhibit pro- and anti-fibrotic effects in IPF and in preclinical animal models of lung fibrosis. This review describes the current understanding of the role and signaling pathways of prostanoids, lysophospholipids, and sphingolipids and their metabolizing enzymes in the development of lung fibrosis. Further, several of the lipid mediators and enzymes involved in their metabolism are therapeutic targets for drug development to treat IPF.

We have shown that both reactive oxygen species (ROS) and paxillin tyrosine phosphorylation regulate LPS-induced human lung endothelial permeability. Mitochondrial ROS (mtROS) is known to increase endothelial cell (EC) permeability which requires dynamic change in mitochondrial morphology, events that are likely to be regulated by paxillin. Here, we investigated the role of paxillin and its tyrosine phosphorylation in regulating LPS-induced mitochondrial dynamics, mtROS production and human lung microvascular EC (HLMVEC) dysfunction. LPS, in a time-dependent manner, induced higher levels of ROS generation in the mitochondria compared to cytoplasm or nucleus. Down-regulation of paxillin expression with siRNA or ecto-expression of paxillin Y31F or Y118F mutant plasmids attenuated LPS-induced mtROS in HLMVECs. Pre-treatment with MitoTEMPO, a scavenger of mtROS, attenuated LPS-induced mtROS, endothelial permeability and VE-cadherin phosphorylation. Further, LPS-induced mitochondrial fission in HLMVECs was attenuated by both a paxillin siRNA, and paxillin Y31F/Y118F mutant. LPS stimulated phosphorylation of dynamin-related protein (DRP1) at S616, which was also attenuated by paxillin siRNA, and paxillinY31/Y118 mutants. Inhibition of DRP1 phosphorylation by P110 attenuated LPS-induced mtROS and endothelial permeability. LPS challenge of HLMVECs enhanced interaction between paxillin, ERK, and DRP1, and inhibition of ERK1/2 activation with PD98059 blocked mitochondrial fission. Taken together, these results suggest a key role for paxillin tyrosine phosphorylation in LPS-induced mitochondrial fission, mtROS generation and EC barrier dysfunction.

The sphingosine kinase 1 (SPHK1)/sphingosine–1–phosphate (S1P) signaling axis is emerging as a key player in the development of idiopathic pulmonary fibrosis (IPF) and bleomycin (BLM)-induced lung fibrosis in mice. Recent evidence implicates the involvement of the Hippo/Yes-associated protein (YAP) 1 pathway in lung diseases, including IPF, but its plausible link to the SPHK1/S1P signaling pathway is unclear. Herein, we demonstrate the increased co-localization of YAP1 with the fibroblast marker FSP1 in the lung fibroblasts of BLM-challenged mice, and the genetic deletion of Sphk1 in mouse lung fibroblasts (MLFs) reduced YAP1 localization in fibrotic foci. The PF543 inhibition of SPHK1 activity in mice attenuated YAP1 co-localization with FSP1 in lung fibroblasts. In vitro, TGF-β stimulated YAP1 translocation to the nucleus in primary MLFs, and the deletion of Sphk1 or inhibition with PF543 attenuated TGF-β-mediated YAP1 nuclear localization. Moreover, the PF543 inhibition of SPHK1, or the verteporfin inhibition of YAP1, decreased the TGF-β- or BLM-induced mitochondrial reactive oxygen species (mtROS) in human lung fibroblasts (HLFs) and the expression of fibronectin (FN) and alpha-smooth muscle actin (α-SMA). Furthermore, scavenging mtROS with MitoTEMPO attenuated the TGF-β-induced expression of FN and α-SMA. The addition of the S1P antibody to HLFs reduced TGF-β- or S1P-mediated YAP1 activation, mtROS, and the expression of FN and α-SMA. These results suggest a role for SPHK1/S1P signaling in TGF-β-induced YAP1 activation and mtROS generation, resulting in fibroblast activation, a critical driver of pulmonary fibrosis.

Insulin-like growth factor (IGF)-1 is a potent mitogen of vascular smooth muscle cells (SMCs), but its role in pulmonary vascular remodeling associated with pulmonary hypertension (PH) is not clear. In an earlier study, we implicated IGF-1 in the pathogenesis of hypoxia-induced PH in neonatal mice. In this study, we hypothesized that hypoxia-induced up-regulation of IGF-1 in vascular smooth muscle is directly responsible for pulmonary vascular remodeling and PH. We studied neonatal and adult mice with smooth muscle-specific deletion of IGF-1 and also used an inhibitor of IGF-1 receptor (IGF-1R), OSI-906, in neonatal mice. We found that, in neonatal mice, SMC-specific deletion of IGF-1 or IGF-1R inhibition with OSI-906 attenuated hypoxia-induced pulmonary vascular remodeling in small arteries, right ventricular hypertrophy, and right ventricular systolic pressure. Pulmonary arterial SMCs from IGF-1-deleted mice or after OSI-906 treatment exhibited reduced proliferative potential. However, in adult mice, smooth muscle-specific deletion of IGF-1 had no effect on hypoxia-induced PH. Our data suggest that vascular smooth muscle-derived IGF-1 plays a critical role in hypoxia-induced PH in neonatal mice but not in adult mice. We speculate that the IGF-1/IGF-1R axis is important in pathogenesis of PH in the developing lung and may be amenable to therapeutic manipulation in this age group.

There is growing evidence that microRNAs are implicated in pulmonary arterial hypertension (PAH), but underlying mechanisms remain elusive. Here, we identified that miR-223 was significantly downregulated in chronically hypoxic mouse and rat lungs, as well as in pulmonary artery and pulmonary artery smooth muscle cells (PASMC) exposed to hypoxia. Knockdown of miR-223 increased PASMC proliferation. In contrast, miR-223 overexpression abrogated cell proliferation, migration and stress fiber formation. Administering miR-223 agomir in vivo antagonized hypoxia-induced increase in pulmonary artery pressure and distal arteriole muscularization. RhoB, which was increased by hypoxia, was identified as one of the targets of miR-223. Overexpressed miR-223 suppressed RhoB and inhibited the consequent phosphorylation of myosin phosphatase target subunit (MYPT1) and the expression of myosin light chain of myosin II (MLC2), which was identified as another target of miR-223. Furthermore, serum miR-223 levels were decreased in female patients with PAH associated with congenital heart disease. Our study provides the first evidence that miR-223 can regulate PASMC proliferation, migration and actomyosin reorganization through its novel targets, RhoB and MLC2, resulting in vascular remodeling and the development of PAH. It also highlights miR-223 as a potential circulating biomarker and a small molecule drug for diagnosis and treatment of PAH.

Cigarette smoke is the primary cause of Chronic Obstructive Pulmonary Disorder (COPD). Cigarette smoke extract (CSE)-induced oxidative damage of the lungs results in mitochondrial dysfunction and apoptosis of epithelium. Mitochondrial cardiolipin (CL) present in the inner mitochondrial membrane plays an important role in mitochondrial function, wherein its fatty acid composition is regulated by lysocardiolipin acyltransferase (LYCAT). In this study, we investigated the role of LYCAT expression and activity in mitochondrial oxidative stress, mitochondrial dynamics, and lung epithelial cell apoptosis. LYCAT expression was increased in human lung specimens from smokers, and cigarette smoke-exposed-mouse lung tissues. Cigarette smoke extract (CSE) increased LYCAT mRNA levels and protein expression, modulated cardiolipin fatty acid composition, and enhanced mitochondrial fission in the bronchial epithelial cell line, BEAS-2B in vitro. Inhibition of LYCAT activity with a peptide mimetic, attenuated CSE-mediated mitochondrial (mt) reactive oxygen species (ROS), mitochondrial fragmentation, and apoptosis, while MitoTEMPO attenuated CSE-induced MitoROS, mitochondrial fission and apoptosis of BEAS-2B cells. Collectively, these findings suggest that increased LYCAT expression promotes MitoROS, mitochondrial dynamics and apoptosis of lung epithelial cells. Given the key role of LYCAT in mitochondrial cardiolipin remodeling and function, strategies aimed at inhibiting LYCAT activity and ROS may offer an innovative approach to minimize lung inflammation caused by cigarette smoke.

Sphingosine-1-phosphate (S1P), a bioactive lipid mediator, is generated from sphingosine by sphingosine kinases (SPHKs) 1 and 2 and is metabolized to ∆2-hexadecenal (∆2-HDE) and ethanolamine phosphate by S1P lyase (S1PL) in mammalian cells. We have recently demonstrated the activation of nuclear SPHK2 and the generation of S1P in the nucleus of lung epithelial cells exposed to Pseudomonas aeruginosa. Here, we have investigated the nuclear localization of S1PL and the role of ∆2-HDE generated from S1P in the nucleus as a modulator of histone deacetylase (HDAC) activity and histone acetylation. Electron micrographs of the nuclear fractions isolated from MLE-12 cells showed nuclei free of ER contamination, and S1PL activity was detected in nuclear fractions isolated from primary lung bronchial epithelial cells and alveolar epithelial MLE-12 cells. Pseudomonasaeruginosa-mediated nuclear ∆2-HDE generation, and H3/H4 histone acetylation was attenuated by S1PL inhibitors in MLE-12 cells and human bronchial epithelial cells. In vitro, the addition of exogenous ∆2-HDE (100–10,000 nM) to lung epithelial cell nuclear preparations inhibited HDAC1/2 activity, and increased acetylation of Histone H3 and H4, whereas similar concentrations of S1P did not show a significant change. In addition, incubation of ∆2-HDE with rHDAC1 generated five different amino acid adducts as detected by LC-MS/MS; the predominant adduct being ∆2-HDE with lysine residues of HDAC1. Together, these data show an important role for the nuclear S1PL-derived ∆2-HDE in the modification of HDAC activity, histone acetylation, and chromatin remodeling in lung epithelial cells.

To kill pathogens, neutrophils must be able to transmigrate into tissues. Xu and colleagues show that the kinase MYLK, by phosphorylating Pyk2, is required for neutrophils to reach sites of infection. Nonmuscle myosin light-chain kinase (MYLK) mediates increased lung vascular endothelial permeability in lipopolysaccharide-induced lung inflammatory injury, the chief cause of the acute respiratory distress syndrome. In a lung injury model, we demonstrate here that MYLK was also essential for neutrophil transmigration, but that this function was mostly independent of myosin II regulatory light chain, the only known substrate of MYLK. Instead, MYLK in neutrophils was required for the recruitment and activation of the tyrosine kinase Pyk2, which mediated full activation of β 2 integrins. Our results demonstrate that MYLK-mediated activation of β 2 integrins through Pyk2 links β 2 integrin signaling to the actin motile machinery of neutrophils.

Pulmonary arterial hypertension (PAH) is a devastating disease, and no effective treatments are available. Hypoxia-induced pulmonary artery remodeling, including smooth muscle cell proliferation, contributes to PAH, but the exact mechanisms underlying this abnormal process are largely undefined. The forkhead box M1 (FoxM1) transcription factor regulates cancer cell growth by modulating gene expression critical for cell cycle progression. Here, we report for the first time, to the best of our knowledge, a novel function of FoxM1 in the hypoxia-stimulated proliferation of human pulmonary artery smooth muscle cells (HPASMCs). Exposure to hypoxia caused a marked up-regulation of FoxM1 gene expression, mainly at the transcription level, and this induction correlated with HPASMC cell proliferation. The knockdown of FoxM1 inhibited the hypoxia-stimulated proliferation of HPASMCs. We found that the knockdown of HIF-2α, but not HIF-1α, diminished FoxM1 induction in response to hypoxia. However, the knockdown of FoxM1 did not alter expression levels of HIF-2α or HIF-1α, suggesting that HIF-2α is an upstream regulator of FoxM1. Furthermore, the knockdown of FoxM1 prevented the hypoxia-induced expression of aurora A kinase and cyclin D1. Collectively, our results suggest that hypoxia induces FoxM1 gene expression in an HIF-2α-dependent pathway, thereby promoting HPASMC proliferation.

In the lung, communication between pulmonary vascular endothelial cells (PVEC) and pulmonary artery smooth muscle cells (PASMC) is essential for the maintenance of vascular homeostasis. In pulmonary hypertension (PH), the derangement in their cell–cell communication plays a major role in the pathogenesis of pulmonary vascular remodeling. In this study, we focused on the role of PVEC‐derived extracellular vesicles (EV), specifically their microRNA (miRNA, miR‐) cargo, in the regulation of PASMC proliferation and vascular remodeling in PH. We found that the amount of pro‐proliferative miR‐210‐3p was increased in PVEC‐derived EV in hypoxia (H‐EV), which contributes to the H‐EV‐induced proliferation of PASMC and the development of PH.