Enhancing Extracellular Adenosine Levels Restores Barrier Function in Acute Lung Injury Through Expression of Focal Adhesion Proteins.

Background: Acute respiratory distress syndrome (ARDS) is a clinical presentation of acute lung injury (ALI) with often fatal lung complication. Adenosine, a nucleoside generated following cellular stress provides protective effects in acute injury. The levels of extracellular adenosine can be depleted by equilibrative nucleoside transporters (ENTs). ENT inhibition by pharmaceutical agent dipyridamole promotes extracellular adenosine accumulation and is protective in ARDS. However, the therapeutic potential of dipyridamole in acute lung injury has not yet been evaluated. Methods: Adenosine acts on three adenosine receptors, the adenosine A1 (Adora1), A2a (Adora2a), the A2b (Adora2b) or the adenosine A3 (Adora 3) receptor. Accumulation of adenosine is usually required to stimulate the low-affinity Adora2b receptor. In order to investigate the effect of adenosine accumulation and the contribution of epithelial-specific ENT2 or adora2b expression in experimental ALI, dipyridamole, and epithelial specific ENT2 or Adora2b deficient mice were utilized. MLE12 cells were used to probe downstream Adora2b signaling. Adenosine receptors, transporters, and targets were determined in ARDS lungs. Results: ENT2 is mainly expressed in alveolar epithelial cells and is negatively regulated by hypoxia following tissue injury. Enhancing adenosine levels with ENT1/ENT2 inhibitor dipyridamole at a time when bleomycin-induced ALI was present, reduced further injury. Mice pretreated with the ADORA2B agonist BAY 60-6583 were protected from bleomycin-induced ALI by reducing vascular leakage (558.6 ± 50.4 vs. 379.9 ± 70.4, p < 0.05), total bronchoalveolar lavage fluid cell numbers (17.9 ± 1.8 to 13.4 ± 1.4 e4, p < 0.05), and neutrophil infiltration (6.42 ± 0.25 vs. 3.94 ± 0.29, p < 0.05). While mice lacking Adora2b in AECs were no longer protected by dipyridamole. We also identified occludin and focal adhesion kinase as downstream targets of ADORA2B, thus providing a novel mechanism for adenosine-mediated barrier protection. Similarly, we also observed similar enhanced ADORA2B (3.33 ± 0.67 to 16.12 ± 5.89, p < 0.05) and decreased occludin (81.2 ± 0.3 to 13.3 ± 0.4, p < 0.05) levels in human Acute respiratory distress syndrome lungs. Conclusion: We have highlighted a role of dipyridamole and adenosine signaling in preventing or treating ALI and identified Ent2 and Adora2b as key mediators in important for the resolution of ALI.

MicroRNA-98 reduces nerve growth factor expression in nicotine-induced airway remodeling.

Evolving evidence suggests that nicotine may contribute to impaired asthma control by stimulating expression of nerve growth factor (NGF), a neurotrophin associated with airway remodeling and airway hyperresponsiveness. We explored the hypothesis that nicotine increases NGF by reducing lung fibroblast (LF) microRNA-98 (miR-98) and PPARγ levels, thus promoting airway remodeling. Levels of NGF, miR-98, PPARγ, fibronectin 1 (FN1), endothelin-1 (EDN1, herein referred to as ET-1), and collagen (COL1A1 and COL3A1) were measured in human LFs isolated from smoking donors, in mouse primary LFs exposed to nicotine (50 µg/ml), and in whole lung homogenates from mice chronically exposed to nicotine (100 µg/ml) in the drinking water. In selected studies, these pathways were manipulated in LFs with miR-98 inhibitor (anti-miR-98), miR-98 overexpression (miR-98 mimic), or the PPARγ agonist rosiglitazone. Compared with unexposed controls, nicotine increased NGF, FN1, ET-1, COL1A1, and COL3A1 expression in human and mouse LFs and mouse lung homogenates. In contrast, nicotine reduced miR-98 levels in LFs in vitro and in lung homogenates in vivo Treatment with anti-miR-98 alone was sufficient to recapitulate increases in NGF, FN1, and ET-1, whereas treatment with a miR-98 mimic significantly suppressed luciferase expression in cells transfected with a luciferase reporter linked to the putative seed sequence in the NGF 3'UTR and also abrogated nicotine-induced increases in NGF, FN1, and ET-1 in LFs. Similarly, rosiglitazone increased miR-98 and reversed nicotine-induced increases in NGF, FN1, and ET-1. Taken together, these findings demonstrate that nicotine-induced increases in NGF and other markers of airway remodeling are negatively regulated by miR-98.

Downregulation of CFIm25 amplifies dermal fibrosis through alternative polyadenylation.

Systemic sclerosis (SSc; scleroderma) is a multisystem fibrotic disease. The mammalian cleavage factor I 25-kD subunit (CFIm25; encoded by NUDT21) is a key regulator of alternative polyadenylation, and its depletion causes predominantly 3'UTR shortening through loss of stimulation of distal polyadenylation sites. A shortened 3'UTR will often lack microRNA target sites, resulting in increased mRNA translation due to evasion of microRNA-mediated repression. Herein, we report that CFlm25 is downregulated in SSc skin, primary dermal fibroblasts, and two murine models of dermal fibrosis. Knockdown of CFIm25 in normal skin fibroblasts is sufficient to promote the 3'UTR shortening of key TGFß-regulated fibrotic genes and enhance their protein expression. Moreover, several of these fibrotic transcripts show 3'UTR shortening in SSc skin. Finally, mice with CFIm25 deletion in fibroblasts show exaggerated skin fibrosis upon bleomycin treatment, and CFIm25 restoration attenuates bleomycin-induced skin fibrosis. Overall, our data link this novel RNA-processing mechanism to dermal fibrosis and SSc pathogenesis.

Transforming growth factor ß1 alters the 3'-UTR of mRNA to promote lung fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic disease characterized by the pathological remodeling of air sacs as a result of excessive accumulation of extracellular matrix (ECM) proteins, but the mechanism governing the robust protein expression is poorly understood. Our recent findings demonstrate that alternative polyadenylation (APA) caused by NUDT21 reduction is important for the increased expression of fibrotic mediators and ECM proteins in lung fibroblasts by shortening the 3'-untranslated regions (3'-UTRs) of mRNAs and stabilizing their transcripts, therefore activating pathological signaling pathways. Despite the importance of NUDT21 reduction in the regulation of fibrosis, the underlying mechanisms for the depletion are unknown. We demonstrate here that NUDT21 is depleted by TGFß1. We found that miR203, which is increased in IPF, was induced by TGFß1 to target the NUDT21 3'-UTR, thus depleting NUDT21 in human and mouse lung fibroblasts. TGFß1-mediated NUDT21 reduction was attenuated by the miR203 inhibitor antagomiR203 in fibroblasts. TGFß1 transgenic mice revealed that TGFß1 down-regulates NUDT21 in fibroblasts in vivo Furthermore, TGFß1 promoted differential APA of fibrotic genes, including FGF14, RICTOR, TMOD2, and UCP5, in association with increased protein expression. This unique differential APA signature was also observed in IPF fibroblasts. Altogether, our results identified TGFß1 as an APA regulator through NUDT21 depletion amplifying pulmonary fibrosis.

Comprehensive characterization of circular RNAs in ~ 1000 human cancer cell lines.

BACKGROUND: Human cancer cell lines are fundamental models for cancer research and therapeutic strategy development. However, there is no characterization of circular RNAs (circRNAs) in a large number of cancer cell lines. METHODS: Here, we apply four circRNA identification algorithms to heuristically characterize the expression landscape of circRNAs across ~ 1000 human cancer cell lines from CCLE polyA-enriched RNA-seq data. By using integrative analysis and experimental approaches, we explore the expression landscape, biogenesis, functional consequences, and drug response of circRNAs across different cancer lineages. RESULTS: We revealed highly lineage-specific expression patterns of circRNAs, suggesting that circRNAs may be powerful diagnostic and/or prognostic markers in cancer treatment. We also identified key genes involved in circRNA biogenesis and confirmed that TGF-ß signaling may promote biogenesis of circRNAs. Strikingly, we showed that clinically actionable genes are more likely to generate circRNAs, potentially due to the enrichment of RNA-binding protein (RBP) binding sites. Among these, circMYC can promote cell proliferation. We observed strong association between the expression of circRNAs and the response to drugs, especially those targeting chromatin histone acetylation. Finally, we developed a user-friendly data portal, CircRNAs in cancer cell lines (CircRiC, https://hanlab.uth.edu/cRic ), to benefit the biomedical research community. CONCLUSIONS: Our study provides the characterization of circRNAs in cancer cell lines and explored the potential mechanism of circRNA biogenesis as well as its therapeutic implications. We also provide a data portal to facilitate the related biomedical researches.

Interferon regulatory factor 7 (IRF7) represents a link between inflammation and fibrosis in the pathogenesis of systemic sclerosis.

OBJECTIVES: There is considerable evidence that implicates dysregulation of type I interferon signalling (or type I IFN signature) in the pathogenesis of systemic sclerosis (SSc). Interferon regulatory factor 7 (IRF7) has been recognised as a master regulator of type I IFN signalling. The objective of this study was to elucidate the role of IRF7 in dermal fibrosis and SSc pathogenesis. METHODS: SSc and healthy control skin biopsies were investigated to determine IRF7 expression and activation. The role of IRF7 in fibrosis was investigated using IRF7 knockout (KO) mice in the bleomycin-induced and TSK/+mouse models. In vitro experiments with dermal fibroblasts from patients with SSc and healthy controls were performed. RESULTS: IRF7 expression was significantly upregulated and activated in SSc skin tissue and explanted SSc dermal fibroblasts compared with unaffected, matched controls. Moreover, IRF7 expression was stimulated by IFN-α in dermal fibroblasts. Importantly, IRF7 co-immunoprecipitated with Smad3, a key mediator of transforming growth factor (TGF)-ß signalling, and IRF7 knockdown reduced profibrotic factors in SSc fibroblasts. IRF7 KO mice demonstrated attenuated dermal fibrosis and inflammation compared with wild-type mice in response to bleomycin. Specifically, hydroxyproline content, dermal thickness as well as Col1a2, ACTA2 and interleukin-6 mRNA levels were significantly attenuated in IRF7 KO mice skin tissue. Furthermore, IRF7 KO in TSK/+mice attenuated hydroxyproline content, subcutaneous hypodermal thickness, Col1a2 mRNA as well as α-smooth muscle actin and fibronectin expression. CONCLUSIONS: IRF7 is upregulated in SSc skin, interacts with Smad3 and potentiates TGF-ß-mediated fibrosis, and therefore may represent a promising therapeutic target in SSc.

Adenosine and hyaluronan promote lung fibrosis and pulmonary hypertension in combined pulmonary fibrosis and emphysema.

Combined pulmonary fibrosis and emphysema (CPFE) is a syndrome that predominantly affects male smokers or ex-smokers and it has a mortality rate of 55% and a median survival of 5 years. Pulmonary hypertension (PH) is a frequently fatal complication of CPFE. Despite this dismal prognosis, no curative therapies exist for patients with CPFE outside of lung transplantation and no therapies are recommended to treat PH. This highlights the need to develop novel treatment approaches for CPFE. Studies from our group have demonstrated that both adenosine and its receptor ADORA2B are elevated in chronic lung diseases. Activation of ADORA2B leads to elevated levels of hyaluronan synthases (HAS) and increased hyaluronan, a glycosaminoglycan that contributes to chronic lung injury. We hypothesize that ADORA2B and hyaluronan contribute to CPFE. Using isolated CPFE lung tissue, we characterized expression levels of ADORA2B and HAS. Next, using a unique mouse model of experimental lung injury that replicates features of CPFE, namely airspace enlargement, PH and fibrotic deposition, we investigated whether 4MU, a HAS inhibitor, was able to inhibit features of CPFE. Increased protein levels of ADORA2B and HAS3 were detected in CPFE and in our experimental model of CPFE. Treatment with 4MU was able to attenuate PH and fibrosis but not airspace enlargement. This was accompanied by a reduction of HAS3-positive macrophages. We have generated pre-clinical data demonstrating the capacity of 4MU, an FDA-approved drug, to attenuate features of CPFE in an experimental model of chronic lung injury.This article has an associated First Person interview with the first author of the paper.

Cleavage factor 25 deregulation contributes to pulmonary fibrosis through alternative polyadenylation.

Idiopathic pulmonary fibrosis (IPF) is a chronic and deadly disease with a poor prognosis and few treatment options. Pathological remodeling of the extracellular matrix (ECM) by myofibroblasts is a key factor that drives disease pathogenesis, although the underlying mechanisms remain unknown. Alternative polyadenylation (APA) has recently been shown to play a major role in cellular responses to stress by driving the expression of fibrotic factors and ECMs through altering microRNA sensitivity, but a connection to IPF has not been established. Here, we demonstrate that CFIm25, a global regulator of APA, is down-regulated in the lungs of patients with IPF and mice with pulmonary fibrosis, with its expression selectively reduced in alpha-smooth muscle actin (α-SMA) positive fibroblasts. Following the knockdown of CFIm25 in normal human lung fibroblasts, we identified 808 genes with shortened 3'UTRs, including those involved in the transforming growth factor-ß signaling pathway, the Wnt signaling pathway, and cancer pathways. The expression of key pro-fibrotic factors can be suppressed by CFIm25 overexpression in IPF fibroblasts. Finally, we demonstrate that deletion of CFIm25 in fibroblasts or myofibroblast precursors using either the Col1a1 or the Foxd1 promoter enhances pulmonary fibrosis after bleomycin exposure in mice. Taken together, our results identified CFIm25 down-regulation as a novel mechanism to elevate pro-fibrotic gene expression in pulmonary fibrosis.

Coordination of ENT2-dependent adenosine transport and signaling dampens mucosal inflammation.

Intestinal epithelial barrier repair is vital for remission in inflammatory bowel disease (IBD). Extracellular adenosine signaling has been implicated in promoting restoration of epithelial barrier function. Currently, no clinically approved agents target this pathway. Adenosine signaling is terminated by uptake from the extracellular space via equilibrative nucleoside transporters (ENTs). We hypothesized that ENT inhibition could dampen intestinal inflammation. Initial studies demonstrated transcriptional repression of ENT1 and ENT2 in IBD biopsies or in murine IBD models. Subsequent studies in mice with global Ent1 or Ent2 deletion revealed selective protection of Ent2-/- mice. Elevated intestinal adenosine levels in conjunction with abolished protection following pharmacologic blockade of A2B adenosine receptors implicate adenosine signaling as the mechanism of gut protection in Ent2-/- mice. Additional studies in mice with tissue-specific deletion of Ent2 uncovered epithelial Ent2 as the target. Moreover, intestinal protection provided by a selective Ent2 inhibitor was abolished in mice with epithelium-specific deletion of Ent2 or the A2B adenosine receptor. Taken together, these findings indicate that increased mucosal A2B signaling following repression or deletion of epithelial Ent2 coordinates the resolution of intestinal inflammation. This study suggests the presence of a targetable purinergic network within the intestinal epithelium designed to limit tissue inflammation.

Elevated ecto-5'-nucleotidase: a missing pathogenic factor and new therapeutic target for sickle cell disease.

Although excessive plasma adenosine is detrimental in sickle cell disease (SCD), the molecular mechanism underlying elevated circulating adenosine remains unclear. Here we report that the activity of soluble CD73, an ectonucleotidase producing extracellular adenosine, was significantly elevated in a murine model of SCD and correlated with increased plasma adenosine. Mouse genetic studies demonstrated that CD73 activity contributes to excessive induction of plasma adenosine and thereby promotes sickling, hemolysis, multiorgan damage, and disease progression. Mechanistically, we showed that erythrocyte adenosine 5'-monophosphate-activated protein kinase (AMPK) was activated both in SCD patients and in the murine model of SCD. AMPK functions downstream of adenosine receptor ADORA2B signaling and contributes to sickling by regulating the production of erythrocyte 2,3-bisphosphoglycerate (2,3-BPG), a negative allosteric regulator of hemoglobin-O2 binding affinity. Preclinically, we reported that treatment of α,ß-methylene adenosine 5'-diphosphate, a potent CD73 specific inhibitor, significantly decreased sickling, hemolysis, multiorgan damage, and disease progression in the murine model of SCD. Taken together, both human and mouse studies reveal a novel molecular mechanism contributing to the pathophysiology of SCD and identify potential therapeutic strategies to treat SCD.

Short-Term Hypoxia Dampens Inflammation in vivo via Enhanced Adenosine Release and Adenosine 2B Receptor Stimulation.

Hypoxia and inflammation are closely intertwined phenomena. Critically ill patients often suffer from systemic inflammatory conditions and concurrently experience short-lived hypoxia. We evaluated the effects of short-term hypoxia on systemic inflammation, and show that it potently attenuates pro-inflammatory cytokine responses during murine endotoxemia. These effects are independent of hypoxia-inducible factors (HIFs), but involve augmented adenosine levels, in turn resulting in an adenosine 2B receptor-mediated post-transcriptional increase of interleukin (IL)-10 production. We translated our findings to humans using the experimental endotoxemia model, where short-term hypoxia resulted in enhanced plasma concentrations of adenosine, augmentation of endotoxin-induced circulating IL-10 levels, and concurrent attenuation of the pro-inflammatory cytokine response. Again, HIFs were shown not to be involved. Taken together, we demonstrate that short-term hypoxia dampens the systemic pro-inflammatory cytokine response through enhanced purinergic signaling in mice and men. These effects may contribute to outcome and provide leads for immunomodulatory treatment strategies for critically ill patients.

Suppression of cleavage factor Im 25 promotes the proliferation of lung cancer cells through alternative polyadenylation.

BACKGROUND: Non-small cell lung cancer (NSCLC) is a life-threatening disease that has a poor prognosis and low survival rate. Cleavage factor Im 25 (CFIm25) is a RNA-binding protein that if down-regulated causes 3'UTR shortening and thus promotes the transcript stability of target genes. It is not clear whether CFIm25 and alternative polyadenylation (APA) play a role during cancer development. The purpose of this study is to explore the role of CFIm25 in lung cancer cell proliferation. METHODS: CFIm25 was knocked down in A549 cells. Western blots were carried out to determine the protein expression of CFIm25, insulin growth factor 1 receptor (IGF1R), CyclinD1 (CCND1) and TP53. Real-time qRT PCR was performed to determine the total transcript levels of CFIm25 targets and the normalized fold changes in their distal PAS (dPAS) usage. Immunofluorescence was carried out to check the expression of CFIm25, IGF1R and CCND1. Cell proliferation over time was determined using the WST-1 reagent. RESULTS: The transcript levels of CCND1 and GSK3ß were significantly increased and the dPAS usage of several oncogenes (IGF1R, CCND1 and GSK3ß) were decreased after CFIm25 knockdown. The protein level of IGF1R was increased, and we detected increased percentage of CCND1 positive cells and cell proliferation over time in CFIm25 knockdown cells. In addition, the mRNA and APA analysis of IGF1R using patient RNA-seq data from the Cancer Genome Atlas indicated that IGF1R is shortened in both lung adenocarcinoma and lung squamous cell carcinoma compared to normal controls. CONCLUSIONS: Our findings suggest that CFIm25 plays an important role in lung cancer cell proliferation through regulating the APA of oncogenes, including IGF1R, and promoting their protein expression.

Switching-Off Adora2b in Vascular Smooth Muscle Cells Halts the Development of Pulmonary Hypertension.

Background: Pulmonary hypertension (PH) is a devastating and progressive disease characterized by excessive proliferation of pulmonary artery smooth muscle cells (PASMCs) and remodeling of the lung vasculature. Adenosine signaling through the ADORA2B receptor has previously been implicated in disease progression and tissue remodeling in chronic lung disease. In experimental models of PH associated with chronic lung injury, pharmacological or genetic inhibition of ADORA2B improved markers of chronic lung injury and hallmarks of PH. However, the contribution of ADORA2B expression in the PASMC was not fully evaluated. Hypothesis: We hypothesized that adenosine signaling through the ADORA2B receptor in PASMC mediates the development of PH. Methods: PASMCs from controls and patients with idiopathic pulmonary arterial hypertension (iPAH) were characterized for expression levels of all adenosine receptors. Next, we evaluated the development of PH in ADORA2Bf/f-Transgelin (Tagln)cre mice. These mice or adequate controls were exposed to a combination of SUGEN (SU5416, 20 mg/kg/b.w. IP) and hypoxia (10% O2) for 28 days (HX-SU) or to chronic low doses of bleomycin (BLM, 0.035U/kg/b.w. IP). Cardiovascular readouts including right ventricle systolic pressures (RVSPs), Fulton indices and vascular remodeling were determined. Using PASMCs we identified ADORA2B-dependent mediators involved in vascular remodeling. These mediators: IL-6, hyaluronan synthase 2 (HAS2) and tissue transglutaminase (Tgm2) were determined by RT-PCR and validated in our HX-SU and BLM models. Results: Increased levels of ADORA2B were observed in PASMC from iPAH patients. ADORA2Bf/f-Taglncre mice were protected from the development of PH following HX-SU or BLM exposure. In the BLM model of PH, ADORA2Bf/f- Taglncre mice were not protected from the development of fibrosis. Increased expression of IL-6, HAS2 and Tgm2 was observed in PASMC in an ADORA2B-dependent manner. These mediators were also reduced in ADORA2Bf/f- Taglncre mice exposed to HX-SU or BLM. Conclusions: Our studies revealed ADORA2B-dependent increased levels of IL-6, hyaluronan and Tgm2 in PASMC, consistent with reduced levels in ADORA2Bf/f- Taglncre mice exposed to HX-SU or BLM. Taken together, our data indicates that ADORA2B on PASMC mediates the development of PH through the induction of IL-6, hyaluronan and Tgm2. These studies point at ADORA2B as a therapeutic target to treat PH.

The Antifibrotic Effect of A2B Adenosine Receptor Antagonism in a Mouse Model of Dermal Fibrosis.

OBJECTIVE: Systemic sclerosis (SSc; scleroderma) is a chronic disease that affects the skin and various internal organs. Dermal fibrosis is a major component of this disease. The mechanisms that promote dermal fibrosis remain elusive. Elevations in tissue adenosine levels and the subsequent engagement of the profibrotic A2B adenosine receptor (ADORA2B) have been shown to regulate fibrosis in multiple organs including the lung, kidney, and penis; however, the role of ADORA2B in dermal fibrosis has not been investigated. We undertook this study to test our hypothesis that elevated expression of ADORA2B in the skin drives the development of dermal fibrosis. METHODS: We assessed the involvement of ADORA2B in the regulation of dermal fibrosis using a well-established mouse model of dermal fibrosis. Using an orally active ADORA2B antagonist, we demonstrated how inhibition of ADORA2B results in reduced dermal fibrosis in 2 distinct experimental models. Finally, using human dermal fibroblasts, we characterized the expression of adenosine receptors. RESULTS: We demonstrated that levels of ADORA2B were significantly elevated in dermal fibrosis and that the therapeutic blockade of this receptor in vivo using an ADORA2B antagonist could reduce the production of profibrotic mediators in the skin and attenuate dermal fibrosis. Antagonism of ADORA2B resulted in reduced numbers of arginase-expressing macrophages and myofibroblasts and in reduced levels of the extracellular matrix proteins fibronectin, collagen, and hyaluronan. CONCLUSION: These findings identify ADORA2B as a potential profibrotic regulator in dermal fibrosis and suggest that ADORA2B antagonism may be a useful approach for the treatment of SSc.

Capturing the multifactorial nature of ARDS - "Two-hit" approach to model murine acute lung injury.

Severe acute respiratory distress syndrome (ARDS) presents typically with an initializing event, followed by the need for mechanical ventilation. Most animal models of ALI are limited by the fact that they focus on a singular cause of acute lung injury (ALI) and therefore fail to mimic the complex, multifactorial pathobiology of ARDS. To better capture this scenario, we provide a comprehensive characterization of models of ALI combining two injuries: intra tracheal (i.t.) instillation of LPS or hypochloric acid (HCl) followed by ventilator-induced lung injury (VILI). We hypothesized, that mice pretreated with LPS or HCl prior to VILI and thus receiving a ("two-hit injury") will sustain a superadditive lung injury when compared to VILI. Mice were allocated to following treatment groups: control with i.t. NaCl, ventilation with low peak inspiratory pressure (PIP), i.t. HCl, i.t. LPS, VILI (high PIP), HCl i.t. followed by VILI and LPS i.t. followed by VILI. Severity of injury was determined by protein content and MPO activity in bronchoalveolar lavage (BAL), the expression of inflammatory cytokines and histopathology. Mice subjected to VILI after HCl or LPS instillation displayed augmented lung injury, compared to singular lung injury. However, mice that received i.t. LPS prior to VILI showed significantly increased inflammatory lung injury compared to animals that underwent i.t. HCl followed by VILI. The two-hit lung injury models described, resulting in additive but differential acute lung injury recaptures the clinical relevant multifactorial etiology of ALI and could be a valuable tool in translational research.

Comprehensive Characterization of Alternative Polyadenylation in Human Cancer.

Background: Alternative polyadenylation (APA) is emerging as a major post-transcriptional mechanism for gene regulation, and dysregulation of APA contributes to several human diseases. However, the functional consequences of APA in human cancer are not fully understood. Particularly, there is no large-scale analysis in cancer cell lines. Methods: We characterized the global APA profiles of 6398 patient samples across 17 cancer types from The Cancer Genome Atlas and 739 cancer cell lines from the Cancer Cell Line Encyclopedia. We built a linear regression model to explore the correlation between APA factors and APA events across different cancer types. We used Spearman correlation to assess the effects of APA events on drug sensitivity and the Wilcoxon rank-sum test or Cox proportional hazards model to identify clinically relevant APA events. Results: We revealed a striking global 3'UTR shortening in cancer cell lines compared with tumor samples. Our analysis further suggested PABPN1 as the master regulator in regulating APA profile across different cancer types. Furthermore, we showed that APA events could affect drug sensitivity, especially of drugs targeting chromatin modifiers. Finally, we identified 1971 clinically relevant APA events, as well as alterations of APA in clinically actionable genes, suggesting that analysis of the complexity of APA profiles could have clinical utility. Conclusions: Our study highlights important roles for APA in human cancer, including reshaping cellular pathways and regulating specific gene expression, exemplifying the complex interplay between APA and other biological processes and yielding new insights into the action mechanism of cancer drugs.

Neutrophil transfer of miR-223 to lung epithelial cells dampens acute lung injury in mice.

Intercellular transfer of microRNAs can mediate communication between critical effector cells. We hypothesized that transfer of neutrophil-derived microRNAs to pulmonary epithelial cells could alter mucosal gene expression during acute lung injury. Pulmonary-epithelial microRNA profiling during coculture of alveolar epithelial cells with polymorphonuclear neutrophils (PMNs) revealed a selective increase in lung epithelial cell expression of microRNA-223 (miR-223). Analysis of PMN-derived supernatants showed activation-dependent release of miR-223 and subsequent transfer to alveolar epithelial cells during coculture in vitro or after ventilator-induced acute lung injury in mice. Genetic studies indicated that miR-223 deficiency was associated with severe lung inflammation, whereas pulmonary overexpression of miR-223 in mice resulted in protection during acute lung injury induced by mechanical ventilation or by infection with Staphylococcus aureus Studies of putative miR-223 gene targets implicated repression of poly(adenosine diphosphate-ribose) polymerase-1 (PARP-1) in the miR-223-dependent attenuation of lung inflammation. Together, these findings suggest that intercellular transfer of miR-223 from neutrophils to pulmonary epithelial cells may dampen acute lung injury through repression of PARP-1.

P2Y6 Receptor Activation Promotes Inflammation and Tissue Remodeling in Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a disease with a poor prognosis and very few available treatment options. The involvement of the purinergic receptor subtypes P2Y2 and P2X7 in fibrotic lung disease has been demonstrated recently. In this study, we investigated the role of P2Y6 receptors in the pathogenesis of IPF in humans and in the animal model of bleomycin-induced lung injury. P2Y6R expression was upregulated in lung structural cells but not in bronchoalveolar lavage (BAL) cells derived from IPF patients as well as in animals following bleomycin administration. Furthermore, BAL fluid levels of the P2Y6R agonist uridine-5'-diphosphate were elevated in animals with bleomycin-induced pulmonary fibrosis. Inflammation and fibrosis following bleomycin administration were reduced in P2Y6R-deficient compared to wild-type animals confirming the pathophysiological relevance of P2Y6R subtypes for fibrotic lung diseases. Experiments with bone marrow chimeras revealed the importance of P2Y6R expression on lung structural cells for pulmonary inflammation and fibrosis. Similar effects were obtained when animals were treated with the P2Y6R antagonist MRS2578. In vitro studies demonstrated that proliferation and secretion of the pro-inflammatory/pro-fibrotic cytokine IL-6 by lung fibroblasts are P2Y6R-mediated processes. In summary, our results clearly demonstrate the involvement of P2Y6R subtypes in the pathogenesis of pulmonary fibrosis. Thus, blocking pulmonary P2Y6 receptors might be a new target for the treatment of IPF.

Loss of CD73-mediated extracellular adenosine production exacerbates inflammation and abnormal alveolar development in newborn mice exposed to prolonged hyperoxia.

BackgroundHyperoxic lung injury is characterized by cellular damage from high oxygen concentrations that lead to an inflammatory response and it disrupts normal alveolarization in the developing newborn lung. Adenosine is a signaling molecule that is generated extracellularly by ecto-5'-nucleotidase (CD73) in response to injury. Extracellular adenosine signals through cell surface receptors and has been found to have a protective role in acute injury situations; however, chronic elevations have been associated with detrimental changes in chronic lung diseases. We hypothesized that hyperoxia-induced lung injury leads to CD73-mediated increases in extracellular adenosine, which are detrimental to the newborn lung.MethodsC57Bl/6 and CD73-/- mice were exposed to 95% oxygen, 70% oxygen, or room air. Adenosine concentration and markers of pulmonary inflammation and lung development were measured.ResultsExposure to hyperoxia caused pulmonary inflammation and disrupted normal alveolar development in association with increased pulmonary adenosine levels. Loss of CD73-mediated extracellular adenosine production led to decreased survival with exposure to 95% oxygen, and exacerbated pulmonary inflammation and worsened lung development with 70% oxygen exposure.ConclusionExposure to hyperoxia causes lung injury associated with an increase in adenosine concentration, and loss of CD73-mediated adenosine production leads to worsening of hyperoxic lung injury.Pediatric Research advance online publication, 23 August 2017; doi:10.1038/pr.2017.176.