Our findings reveal altered perivascular inflammatory cell infiltration in pulmonary vascular lesions of patients with idiopathic pulmonary arterial hypertension. Targeting attraction of inflammatory cells by blocking stromal-derived factor-1 may be a novel approach for treatment of PAH.

Epithelial-mesenchymal transition (EMT) has emerged as a critical event in the pathogenesis of organ fibrosis and cancer and is typically induced by the multifunctional cytokine transforming growth factor (TGF)-␤1. The present study was undertaken to evaluate the potential role of phosphodiesterases (PDEs) in TGF-␤1-induced EMT in the human alveolar epithelial type II cell line A549. Stimulation of A549 with TGF-␤1 induced EMT by morphological alterations and by expression changes of the epithelial phenotype markers E-cadherin, cytokeratin-18, zona occludens-1, and the mesenchymal phenotype markers, collagen I, fibronectin, and ␣-smooth muscle actin. Interestingly, TGF-␤1 stimulation caused twofold increase in total cAMP-PDE activity, contributed mostly by PDE4. Furthermore, mRNA and protein expression demonstrated up-regulation of PDE4A and PDE4D isoforms in TGF-␤1-stimulated cells. Most importantly, treatment of TGF-␤1 stimulated epithelial cells with the PDE4-selective inhibitor rolipram or PDE4 small interfering RNA potently inhibited EMT changes in a Smad-independent manner by decreasing reactive oxygen species, p38, and extracellular signal-regulated kinase phosphorylation. In contrast, the ectopic overexpression of PDE4A and/or PDE4D resulted in a significant loss of epithelial marker E-cadherin but did not result in changes of mesenchymal markers. In addition, Rho kinase signaling activated by TGF-␤1 during EMT demonstrated to be a positive regulator of PDE4. Collectively, the findings presented herein suggest that TGF-␤1 mediated up-regulation of PDE4 promotes EMT in alveolar epithelial cells. Thus, targeting PDE4 isoforms may be a novel approach to attenuate EMT-associated lung diseases such as pulmonary fibrosis and lung cancer.

Lung cancer is the leading cause of cancer death worldwide. Recent data suggest that cyclic nucleotide phosphodiesterases (PDEs) are relevant in various cancer pathologies. Pathophysiological role of phosphodiesterase 4 (PDE4) with possible therapeutic prospects in lung cancer was investigated. We exposed 10 different lung cancer cell lines (adenocarcinoma, squamous and large cell carcinoma) to hypoxia and assessed expression and activity of PDE4 by real-time PCR, immunocytochemistry, western blotting and PDE activity assays. Expression and activity of distinct PDE4 isoforms (PDE4A and PDE4D) increased in response to hypoxia in eight of the studied cell lines. Furthermore, we analyzed various in silico predicted hypoxia-responsive elements (p-HREs) found in PDE4A and PDE4D genes. Performing mutation analysis of the p-HRE in luciferase reporter constructs, we identified four functional HRE sites in the PDE4A gene and two functional HRE sites in the PDE4D gene that mediated hypoxic induction of the reporter. Silencing of hypoxia-inducible factor subunits (HIF1a and HIF2a) by small interfering RNA reduced hypoxic induction of PDE4A and PDE4D. Vice versa, using a PDE4 inhibitor (PDE4i) as a cyclic adenosine monophosphate (cAMP) -elevating agent, cAMP analogs or protein kinase A (PKA)-modulating drugs and an exchange protein directly activated by cAMP (EPAC) activator, we demonstrated that PDE4-cAMP-PKA/EPAC axis enhanced HIF signaling as measured by HRE reporter gene assay, HIF and HIF target genes expression ((lactate dehydrogenase A), LDHA, (pyruvate dehydrogenase kinase 1) PDK1 and (vascular endothelial growth factor A) VEGFA). Notably, inhibition of PDE4 by PDE4i or silencing of PDE4A and PDE4D reduced human lung tumor cell proliferation and colony formation. On the other hand, overexpression of PDE4A or PDE4D increased human lung cancer proliferation. Moreover, PDE4i treatment reduced hypoxia-induced VEGF secretion in human cells. In vivo, PDE4i inhibited tumor xenograft growth in nude mice by attenuating proliferation and angiogenesis. Our findings suggest that PDE4 is expressed in lung cancer, crosstalks with HIF signaling and promotes lung cancer progression. Thus, PDE4 may represent a therapeutic target for lung cancer therapy.

Quantitative evaluation of lung tumor angiogenesis using immunohistochemical techniques has been limited by difficulties in generating reproducible data. To analyze intrapulmonary tumor angiogenesis, we used high-resolution micro-computed tomography (micro-CT) of lung tumors of mice inoculated with mouse Lewis lung carcinoma (LLC1) or human adenocarcinoma (A549) cell lines. The lung vasculature was filled with the radiopaque silicone rubber, Microfil, through the jugular vein (in vivo application) or pulmonary artery (ex vivo application). In addition, human adenocarcinoma lung tumor-bearing mice treated site-specifically with humanized monoclonal antibody (bevacizumab) against vascular endothelial growth factor. Quantitative analysis of lung tumor microvessels imaged with micro-CT showed that more vessels (mainly small, <0.02 mm(2)) were filled using the in vivo (5.4%) compared with the ex vivo (2.1%) method. Furthermore, bevacizumab-treated lung tumor-bearing mice showed significantly reduced lung tumor volume and lung tumor angiogenesis compared with untreated mice as assessed by micro-CT. Interestingly, microvascularization of mainly the smaller vessels (<0.02 mm(2)) was reduced after bevacizumab treatment. This observation with micro-CT was nicely correlated with immunohistochemical measurement of microvessels. Therefore, micro-CT is a novel method for investigating lung tumor angiogenesis, and this might be considered as an additional complementary tool for precise quantification of angiogenesis.

Lung cancer is frequently complicated by pulmonary infections which may impair prognosis of this disease. Therefore, we investigated the effect of bacterial lipopolysaccharides (LPS) on tumor proliferation in vitro in the non-small cell lung cancer (NSCLC) cell line A549, ex vivo in a tissue culture model using human NSCLC specimens and in vivo in the A549 adenocarcinoma mouse model. LPS induced a time- and dose-dependent increase in proliferation of A549 cells as quantified by MTS activity and cell counting. In parallel, an increased expression of the proliferation marker Ki-67 and cyclooxygenase (COX)-2 was detected both in A549 cells and in ex vivo human NSCLC tissue. Large amounts of COX-2-derived prostaglandin (PG)E2 were secreted from LPS-stimulated A549 cells. Pharmacological interventions revealed that the proliferative effect of LPS was dependent on CD14 and Toll-like receptor (TLR)4. Moreover, blocking of the epidermal growth factor receptor (EGFR) also decreased LPS-induced proliferation of A549 cells. Inhibition of COX-2 activity in A549 cells severely attenuated both PGE2 release and proliferation in response to LPS. Synthesis of PGE2 was also reduced by inhibiting CD14, TLR4 and EGFR in A549 cells. The proliferative effect of LPS on A549 cells could be reproduced in the A549 adenocarcinoma mouse model with enhancement of tumor growth and Ki-67 expression in implanted tumors. In summary, LPS induces proliferation of NSCLC cells in vitro, ex vivo in human NSCLC specimen and in vivo in a mouse model of NSCLC. Pulmonary infection may thus directly induce tumor progression in NSCLC.