Kamal A. Mohammed

Pleural mesothelial cell transformation into myofibroblasts and haptotactic migration in response to TGF-β1 in vitro

Idiopathic pulmonary fibrosis (IPF) is a disease of unknown etiology characterized by the development of subpleural foci of myofibroblasts that contribute to the exuberant fibrosis noted in the pulmonary parenchyma. Pleural mesothelial cells (PMC) are metabolically dynamic cells that cover the lung and chest wall as a monolayer and are in intimate proximity to the underlying lung parenchyma. The precise role of PMC in the pathogenesis of pulmonary parenchymal fibrosis remains to be identified. Transforming growth factor (TGF)-beta1, a cytokine known for its capacity to induce proliferative and transformative changes in lung cells, is found in significantly higher quantities in the lungs of patients with IPF. High levels of TGF-beta1 in the subpleural milieu may play a key role in the transition of normal PMC to myofibroblasts. Here we demonstrate that PMC activated by TGF-beta1 undergo epithelial-mesenchymal transition (EMT) and respond with haptotactic migration to a gradient of TGF-beta1 and that the transition of PMC to myofibroblasts is dependent on smad-2 signaling. The EMT of PMC was marked by upregulation of alpha-smooth muscle actin (alpha-SMA), fibroblast specific protein-1 (FSP-1), and collagen type I expression. Cytokeratin-8 and E-cadherin expression decreased whereas vimentin remained unchanged over time in transforming PMC. Knockdown of smad-2 gene by silencing small interfering RNA significantly suppressed the transition of PMC to myofibroblasts and significantly inhibited the PMC haptotaxis. We conclude that PMC undergo EMT when exposed to TGF-beta1, involving smad-2 signaling, and PMC may be a possible source of myofibroblasts in IPF.

Silencing the receptor EphA2 suppresses the growth and haptotaxis of malignant mesothelioma cells

The over‐expression of the ephrin‐A1 ligand receptor EphA2 is associated with the growth and metastatic potential of tumors. Although EphA2 is expressed in a variety of tumors, its expression and function in malignant mesothelioma (MM) remain unknown. The authors hypothesized that expression of the receptor EphA2 in MM cells (MMCs) plays a key role in the growth and haptotactic migration of MM. They also hypothesized that silencing EphA2 expression by using small‐interfering RNA (siRNA) inhibits the proliferation and haptotaxis of MMCs and induces apoptosis in MMCs.

Talc mediates angiostasis in malignant pleural effusions via endostatin induction

Talc remains the most effective sclerosing agent for pleurodesis. However, its mechanism of action in resolving pleural malignant disease remains unclear. The present study evaluated the angiogenic balance in the pleural space in patients with malignant pleural effusions (MPE) following talc insufflation. Patient pleural fluid samples were collected both before and after talc insufflation. The ability of pleural mesothelial cells (PMC) and malignant mesothelioma cells (MMC) to produce endostatin in vitro was compared. The biological effects of pleural fluids and conditioned media from talc-activated PMC on endothelial cells were evaluated by performing proliferation, invasion, tube formation and apoptosis assays. Pleural fluids from patients with MPE who received thoracoscopic talc insufflation contained significantly higher levels of endostatin (median 16.75 ng·mL−1) compared with pre-talc instillation (1.06 ng·mL−1). Talc-activated PMC released significantly greater amounts of endostatin (mean±sem 1052.39±38.66 pg·mL−1) when compared with a MMC line (134.73±8.72 pg·mL−1). In conlusion, talc alters the angiogenic balance in the pleural space from a biologically active and angiogenic environment to an angiostatic milieu. Functional improvement following talc poudrage in patients with malignant pleural effusions may, in part, reflect these alterations in the pleural space.

Innate immune responses in murine pleural mesothelial cells: Toll-like receptor-2 dependent induction of β-defensin-2 by staphylococcal peptidoglycan

The innate immune response is mediated in part by pattern recognition receptors including Toll-like receptors (TLRs). The pleural mesothelial cells (PMCs) that line the pleural surface are in direct contact with pleural fluid and accordingly carry the risk of exposure to infiltrating microorganisms or their components in an event of a complicated parapneumonic effusion. Here we show that murine primary PMCs constitutively express TLR-1 through TLR-9 and, upon activation with peptidoglycan (PGN), mouse PMC produce antimicrobial peptide beta-defensin-2 (mBD-2). Treatment of PMCs with staphylococcal PGN, a gram-positive bacterial cell wall component and a TLR-2 agonist, resulted in a significant increase in TLR-2 and mBD-2 expression. Silencing of TLR-2 expression by small interfering RNA led to the downregulation of PGN-induced mBD-2 expression, thereby establishing causal relationship between the activation of TLR-2 receptor and mBD-2 production. PMCs exposed to PGN showed increased p38 MAPK activity. In addition, PGN-induced mBD-2 expression was attenuated by SB203580, a p38 MAPK inhibitor, underlining the importance of p38 MAPK in mBD-2 induction. Inhibition of erk1/erk2 or phosphatidylinositol 3-kinase did not block PGN-induced mBD-2 expression in PMC. PGN-activated PMC-derived mBD-2 significantly killed Staphylococcus aureus, and mBD-2-neutralizing antibodies blunted this antimicrobial activity. Taken together, these data indicate that PMCs may contribute to host innate immune defense upon exposure to gram-positive bacteria or their products within the pleural space by upregulating TLR-2 and mBD-2 expression.

Parenchymal trafficking of pleural mesothelial cells in idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is characterised by myofibroblast proliferation leading to architectural destruction. Neither the origin nor the continued proliferation of myofibroblasts is well understood. Explanted human IPF lungs were stained by immunohistochemistry for calretinin, a marker of pleural mesothelial cells (PMCs). Chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF) lungs acted as controls. The number of PMCs per 100 nucleated cells and per photomicrograph was estimated along with the Ashcroft score of fibrosis. Mouse PMCs expressing green fluorescent protein (GFP) or labelled with nanoparticles were injected into the pleural space of mice given intranasal transforming growth factor (TGF)-&bgr;1. Mouse lungs were lavaged and examined for the presence of GFP, smooth muscle &agr;-actin (&agr;-SMA) and calretinin. Calretinin-positive PMCs were found throughout IPF lungs, but not in COPD or CF lungs. The number of PMCs correlated with the Ashcroft score. In mice, nanoparticle-laden PMCs were recoverable by bronchoalveolar lavage, depending on the TGF-&bgr;1 dose. Fluorescent staining showed &agr;-SMA expression in GFP-expressing PMCs, with co-localisation of GFP and &agr;-SMA. PMCs can traffic through the lung and show myofibroblast phenotypic markers. PMCs are present in IPF lungs, and their number correlates with IPF severity. Since IPF presumably begins subpleurally, PMCs could play a pathogenetic role via mesothelial–mesenchymal transition.

RSV causes HIF-1α stabilization via NO release in primary bronchial epithelial cells

RSV infection is characterized by airway edema. Stabilization of hypoxia inducible factor-1α (HIF-1α) is important in both inflammation and edema formation. In this study we evaluated whether RSV induced release of nitric oxide (NO) by bronchial airway epithelial cells leading to the stabilization of HIF-1α and subsequent transcription of VEGF165. Primary human bronchial epithelial cells (HBEpC) were used; cell supernatants were analyzed. Western blot analysis was used for the detection of HIF-1α. Bronchial airway epithelial monolayer permeability was assessed using electric cell-substrate impedance sensing (ECIS) in real time. There was increased stabilization of HIF-1α in RSV infected cells. Addition of an NO inhibitor blocked RSV mediated HIF-1α expression. Antagonism of NO also inhibited VEGF production and HBEpC monolayer permeability. Our results demonstrate that in HBEpC, RSV induced NO causes stabilization of HIF-1α in vitro.

FBXO11 promotes ubiquitination of the Snail family of transcription factors in cancer progression and epidermal development

The Snail family of transcription factors are core inducers of epithelial-to-mesenchymal transition (EMT). Here we show that the F-box protein FBXO11 recognizes and promotes ubiquitin-mediated degradation of multiple Snail family members including Scratch. The association between FBXO11 and Snai1 in vitro is independent of Snai1 phosphorylation. Overexpression of FBXO11 in mesenchymal cells reduces Snail protein abundance and cellular invasiveness. Conversely, depletion of endogenous FBXO11 in epithelial cancer cells causes Snail protein accumulation, EMT, and tumor invasion, as well as loss of estrogen receptor expression in breast cancer cells. Expression of FBXO11 is downregulated by EMT-inducing signals TGFβ and nickel. In human cancer, high FBXO11 levels correlate with expression of epithelial markers and favorable prognosis. The results suggest that FBXO11 sustains the epithelial state and inhibits cancer progression. Inactivation of FBXO11 in mice leads to neonatal lethality, epidermal thickening, and increased Snail protein levels in epidermis, validating that FBXO11 is a physiological ubiquitin ligase of Snail. Moreover, in C. elegans, the FBXO11 mutant phenotype is attributed to the Snail factors as it is suppressed by inactivation/depletion of Snail homologs. Collectively, these findings suggest that the FBXO11-Snail regulatory axis is evolutionarily conserved and critically governs carcinoma progression and mammalian epidermal development.

Targeted delivery of let-7a microRNA encapsulated ephrin-A1 conjugated liposomal nanoparticles inhibit tumor growth in lung cancer

MicroRNAs (miRs) are small noncoding RNA sequences that negatively regulate the expression of target genes by posttranscriptional repression. miRs are dysregulated in various diseases, including cancer. let-7a miR, an antioncogenic miR, is downregulated in lung cancers. Our earlier studies demonstrated that let-7a miR inhibits tumor growth in malignant pleural mesothelioma (MPM) and could be a potential therapeutic against lung cancer. EphA2 (ephrin type-A receptor 2) tyrosine kinase is overexpressed in most cancer cells, including MPM and non-small-cell lung cancer (NSCLC) cells. Ephrin-A1, a specific ligand of the EphA2 receptor, inhibits cell proliferation and migration. In this study, to enhance the delivery of miR, the miRs were encapsulated in the DOTAP (N-[1-(2.3-dioleoyloxy)propyl]-N,N,N-trimethyl ammonium)/Cholesterol/DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[cyanur(polyethylene glycol)-2000])-PEG (polyethylene glycol)-cyanur liposomal nanoparticles (LNP) and ephrin-A1 was conjugated on the surface of LNP to target receptor EphA2 on lung cancer cells. The LNP with an average diameter of 100 nm showed high stability, low cytotoxicity, and high loading efficiency of precursor let-7a miR and ephrin-A1. The ephrin-A1 conjugated LNP (ephrin-A1–LNP) and let-7a miR encapsulated LNP (miR–LNP) showed improved transfection efficiency against MPM and NSCLC. The effectiveness of targeted delivery of let-7a miR encapsulated ephrin-A1 conjugated LNP (miR–ephrin-A1–LNP) was determined on MPM and NSCLC tumor growth in vitro. miR–ephrin-A1–LNP significantly increased the delivery of let-7a miR in lung cancer cells when compared with free let-7a miR. In addition, the expression of target gene Ras was significantly repressed following miR–ephrin-A1–LNP treatment. Furthermore, the miR–ephrin-A1–LNP complex significantly inhibited MPM and NSCLC proliferation, migration, and tumor growth. Our results demonstrate that the engineered miR–ephrin-A1–LNP complex is an effective carrier for the targeted delivery of small RNA molecules to lung cancer cells. This could be a potential therapeutic approach against tumors overexpressing the EphA2 receptor.

EphrinA1 inhibits malignant mesothelioma tumor growth via let-7 microRNA-mediated repression of the RAS oncogene

EphrinA1 binding with receptor EphA2 suppresses malignant mesothelioma (MM) growth. The mechanisms whereby EphrinA1 attenuates the MM cell (MMC) growth are not clear. In this study, we report that the activation of MMCs with EphrinA1 leads to an induction of let-7 microRNA (miRNA) expression, repression of RAS proto-oncogene and the attenuation of MM tumor growth. The expression of miRNAs was determined by reverse transcription-quantitative polymerase chain reaction and in situ hybridization. RAS expression was determined by q-PCR, western blotting and immunofluorescence. MMC proliferation and tumor growth were determined by WST-1 and Matrigel assay, respectively. EphrinA1 activation induced several fold increases in let-7a1, let-7a3, let-7f1 and let-7f2 miRNA expression in MMCs. In contrast, EphrinA1 activation significantly downregulated H-RAS, K-RAS and N-RAS expression and inhibited MMC proliferation and tumor growth. In MMCs transfected with 2′-O-methyl antisense oligonucleotides to let-7 miRNA, EphrinA1 activation failed to inhibit the proliferative response and tumor growth. In mismatch antisense oligonucleotide-treated MMCs, the proliferation and tumor growth were comparable to untreated proliferating cells. Furthermore, the transfection of MMCs with let-7a miRNA precursor inhibited RAS expression and attenuated MMC tumor growth. Our data revealed that EphrinA1 signaling induces let-7 miRNA expression and attenuates MM tumor growth by targeting RAS proto-oncogene in MMCs.

α‐Defensins increase lung fibroblast proliferation and collagen synthesis via the β‐catenin signaling pathway

α‐defensins are released from granules of leukocytes and are implicated in inflammatory and fibrotic lung diseases. In the present study, the effects of α‐defensins on the proliferation and collagen synthesis of lung fibroblasts were examined. We found that α‐defensin‐1 and α‐defensin‐2 induced dose‐dependent increases in the incorporation of 5‐bromo‐2′‐deoxy‐uridine into newly synthesized DNA in two lines of human lung fibroblasts (HFL‐1 and LL‐86), suggesting that α‐defensin‐1 and α‐defensin‐2 stimulate the proliferation of lung fibroblasts. α‐defensin‐1 and α‐defensin‐2 also increased collagen‐I mRNA (COL1A1) levels and protein contents of collagen‐I and active/dephosphorylated β‐catenin without changes in total β‐catenin protein content in lung fibroblasts (HFL‐1 and LL‐86). Inhibition of the β‐catenin signaling pathway using quercetin prevented increases in cell proliferation and the protein content of collagen‐I and active/dephosphorylated β‐catenin in lung fibroblasts, and in COL1A1 mRNA levels and collagen release into culture medium induced by α‐defensin‐1 and α‐defensin‐2. Knocking‐down β‐catenin using small interfering RNA technology also prevented α‐defensin‐induced increases in cell proliferation and the protein content of collagen‐I and active/dephosphorylated β‐catenin in lung fibroblasts, and in COL1A1 mRNA levels. Moreover, increases in the phosphorylation of glycogen synthase kinase 3β, accumulation/activation of β‐catenin, and collagen synthesis induced by α‐defensin‐1 and α‐defensin‐2 were prevented by p38 mitogen‐activated protein kinase inhibitor SB203580 and phosphoinositide 3‐kinase inhibitor LY294002. These results indicate that α‐defensin‐1 and α‐defensin‐2 stimulate proliferation and collagen synthesis of lung fibroblasts. The β‐catenin signaling pathway mediates α‐defensin‐induced increases in cell proliferation and collagen synthesis of lung fibroblasts. α‐defensin‐induced activation of β‐catenin in lung fibroblasts might be caused by phosphorylation/inactivation of glycogen synthase kinase 3β as a result of the activation of the p38 mitogen‐activated protein kinase and phosphoinositide 3‐kinase/Akt pathways.