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Acute respiratory distress syndrome constitutes a significant disease burden with regard to both morbidity and mortality. Current therapies are mostly supportive and do not address the underlying pathophysiologic mechanisms. Removal of protein-rich alveolar edema—a clinical hallmark of acute respiratory distress syndrome—is critical for survival. Here, we describe a transforming growth factor (TGF)-β–triggered mechanism, in which megalin, the primary mediator of alveolar protein transport, is negatively regulated by glycogen synthase kinase (GSK) 3β, with protein phosphatase 1 and nuclear inhibitor of protein phosphatase 1 being involved in the signaling cascade. Inhibition of GSK3β rescued transepithelial protein clearance in primary alveolar epithelial cells after TGF-β treatment. Moreover, in a bleomycin-based model of acute lung injury, megalin+/– animals (the megalin–/– variant is lethal due to postnatal respiratory failure) showed a marked increase in intra-alveolar protein and more severe lung injury compared with wild-type littermates. In contrast, wild-type mice treated with the clinically relevant GSK3β inhibitors, tideglusib and valproate, exhibited significantly decreased alveolar protein concentrations, which was associated with improved lung function and histopathology. Together, we discovered that the TGF-β–GSK3β–megalin axis is centrally involved in disturbances of alveolar protein clearance in acute lung injury and provide preclinical evidence for therapeutic efficacy of GSK3β inhibition.

Pulmonary hypertension (PH) is characterized by increased proliferation and apoptosis resistance of pulmonary artery smooth muscle cells (PASMCs). Forkhead box O (FoxO) transcription factors are key regulators of cellular proliferation. Here we show that in pulmonary vessels and PASMCs of human and experimental PH lungs, FoxO1 expression is downregulated and FoxO1 is inactivated via phosphorylation and nuclear exclusion. These findings could be reproduced using ex vivo exposure of PASMCs to growth factors and inflammatory cytokines. Pharmacological inhibition and genetic ablation of FoxO1 in smooth muscle cells reproduced PH features in vitro and in vivo. Either pharmacological reconstitution of FoxO1 activity using intravenous or inhaled paclitaxel, or reconstitution of the transcriptional activity of FoxO1 by gene therapy, restored the physiologically quiescent PASMC phenotype in vitro, linked to changes in cell cycle control and bone morphogenic protein receptor type 2 (BMPR2) signaling, and reversed vascular remodeling and right-heart hypertrophy in vivo. Thus, PASMC FoxO1 is a critical integrator of multiple signaling pathways driving PH, and reconstitution of FoxO1 activity offers a potential therapeutic option for PH.

Rationale Recent studies indicate that tumor-associated macrophages (MΦ) with an M2 phenotype can influence cancer progression and metastasis, but the regulatory pathways remain poorly characterized. Objectives This study investigated the role of tumor-associated MΦ in lung cancer. Methods Coculturing of MΦ with mouse Lewis lung carcinoma (LLC1) and 10 different human lung cancer cell lines (adenocarcinoma, squamous cell carcinoma, and large cell carcinoma) caused up-regulation of CCR2/CCL2 and CX3CR1/CX3CL1 in both the cancer cells and the MΦ. Measurements and main results In the MΦ-tumor cell system, IL-10 drove CCR2 and CX3CR1 up-regulation, whereas CCL1, granulocyte colony-stimulating factor, and MIP1α were required for the up-regulation of CCL2 and CX3CL1. Downstream phenotypic effects included enhanced LLC1 proliferation and migration and MΦ M2 polarization. In vivo, MΦ depletion (clodronate, MΦ Fas-induced apoptosis mice) and genetic ablation of CCR2 and CX3CR1 all inhibited LLC1 tumor growth and metastasis, shifted tumor-associated MΦ toward M1 polarization, suppressed tumor vessel growth, and enhanced survival (metastasis model). Furthermore, mice treated with CCR2 antagonist mimicked genetic ablation of CCR2, showing reduced tumor growth and metastasis. In human lung cancer samples, tumor MΦ infiltration and CCR2 expression correlated with tumor stage and metastasis. Conclusions Tumor-associated MΦ play a central role in lung cancer growth and metastasis, with bidirectional cross-talk between MΦ and cancer cells via CCR2 and CX3CR1 signaling as a central underlying mechanism. These findings suggest that the therapeutic strategy of blocking CCR2 and CX3CR1 may prove beneficial for halting lung cancer progression.

Lung Cancer is the leading cause of cancer deaths worldwide. It is increasingly appreciated that the tumor stroma, consistent of a heterogeneous population of non-neoplastic host cells, is an essential part of cancer initiation, growth and progression. Tumor-associated macrophages (TAMs) can influence cancer progression and metastasis, but the responsible mechanisms remain unclear. Here, we focus on the involvement of bone-marrow derived macrophages (BM- MM) and two of their major chemotactic pathways (CX3CR1-CX3CL1 and CCR2-CCL2 axis) in cancer proliferation and migration. We demonstrate that conditioned medium (CM) derived from co-cultures of lewis lung carcinoma (LLC1) cells with BM- MM (purity >99%) enhances proliferation of LLC1-cells significantly from 1.79±0.1717 to 2.43±0.2067 in an absorbance based BrdU-Assay and a higher colony-number was found in a colony-formation-assay compared to control. In addition LLC1-migration in presence of CM was 5 fold increased compared to controls (172.9±2.18 versus 33.93±2.29). Further, analysis of the cytokine profile of co-culture-derived CM showed regulation of pro-inflammatory cytokines (Interleukin-6 and Interleukin-1α), anti-inflammatory cytokines (Interleukin-10 and Interleukin-1RA) and several chemokines (CCL5 and CCL2). Importantly, evaluation of tumor growth in chemokine receptor knockout mice, CX3CR1-KO and CCR2-KO demonstrated reduced tumor size compared to wild type mice. CX3CR1-KO and CCR2-KO tumor showed reduced in vivo proliferation as assessed by PCNA immunostaining (12.46%±3.188 versus 46.45%±2.922). Further analysis of the tumor microenvironment in CX3CR1-KO and CCR2-KO demonstrated a significant decrease in monocyte/ macrophage accumulation compared to control tumors as assessed by FACS. Compared to the percentage of macrophages in control tumor (7.518±0.78%), a significant decrease was observed in CCR2-KO (2.96± 0.233%) and CX3CR1-KO (3.975±0.6019%) tumors. Based on these findings, we conclude that macrophage plays a crucial role in lung cancer progression and the knowledge of tumor-host interactions can provide novel therapeutic approaches for lung cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1484. doi:1538-7445.AM2012-1484

BackgroundPulmonary fibrosis (PF) is a group of devastating and largely irreversible diseases. Phosphodiesterase (PDE) 4 is involved in the processes of remodeling and inflammation, which play key role in tissue fibrosis. The aim of the study was, therefore, to investigate the effect of PDE4 inhibition in experimental model of PF.MethodsPF was induced in C57BL/6N mice by instillation of bleomycin. Pharmacological inhibition of PDE4 was achieved by using cilomilast, a selective PDE4 inhibitor. Changes in either lung inflammation or remodeling were evaluated at different stages of experimental PF. Lung inflammation was assessed by bronchoalveolar lavage fluid (BALF) differential cell count and reverse transcription quantitative polymerase chain reaction (RT-qPCR) for inflammatory cytokines. Changes in tissue remodeling were evaluated by pulmonary compliance measurement, quantified pathological examination, measurement of collagen deposition and RT-qPCR for late remodeling markers. Survival in all groups was analyzed as well.ResultsPDE4 inhibition significantly reduced the total number of alveolar inflammatory cells in BALF of mice with bleomycin-induced PF at early fibrosis stage (days 4 and 7). Number of macrophages and lymphocytes, but not neutrophils, was significantly reduced as well. Treatment decreased lung tumor necrosis factor (TNF)-α mRNA level and increased mRNA level of interleukin (IL)-6 but did not influence IL-1β. At later stage (days 14 and 24) cilomilast improved lung function, which was shown by increase in lung compliance. It also lowered fibrosis degree, as was shown by quantified pathological examination of Hematoxilin-Eosin stained lung sections. Cilomilast had no significant effect on the expression of late remodeling markers such as transforming growth factor (TGF)-β1 and collagen type Ia1 (COL(I)α1). However, it tended to restore the level of lung collagen, assessed by SIRCOL assay and Masson's trichrome staining, and to improve the overall survival.ConclusionsSelective PDE4 inhibition suppresses early inflammatory stage and attenuates the late stage of experimental pulmonary fibrosis.