Acute respiratory distress syndrome (ARDS) is a severe lung condition without targeted therapy that is characterized by the disruption of epithelial and endothelial barriers. The role of the tight junction protein occludin in the pathogenesis of this disease is unknown, although it has previously been deemed redundant in some tissues. The aim of the present study is to determine whether occludin is required for lung function by controlling alveolar barrier integrity in mouse models. Immunofluorescence staining of lungs from ARDS patients revealed a significant decrease in occludin expression compared to controls. Gene delivery of shRNA against occludin in the mouse lung reduced occludin levels and induced lung injury, as assessed by wet-to-dry-ratio, histology, and cellularity and protein content of bronchial alveolar lavage fluid. Conversely, gene delivery of an occludin-expressing plasmid increased occludin expression and dampened endotoxin-induced lung injury. In primary rat alveolar epithelial cells, occludin levels were positively correlated with barrier integrity, as well as membrane localization of claudin-18, another tight junction protein. Collectively, our data demonstrate that occludin plays a significant role in alveolar barrier function and that targeting occludin may provide a new therapeutic approach for ARDS.

Abstract Rationale Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive interstitial lung disease that has no cure. Many current research efforts center on diagnostic and therapeutic modalities for IPF while other risk factors affecting disease pathogenesis receive less attention. Emerging data support the clinical importance of weight loss in patients with IPF. However, factors associated with weight loss and the impact of weight loss on mortality remain incompletely explored. Objectives Explore the association between weight loss and transplant-free survival in patients with IPF and identify clinical variables associated with weight loss in this population. Methods Kaplan–Meier and Cox proportional hazard regression analyses were generated and stratified by weight loss or use of antifibrotic medications. Conditional logistic regression was used to evaluate for factors associated with weight loss. Results There was a significant increase in mortality in patients who lost ≥ 5% of their body weight loss (HR 2.21, [1.29, 4.43] p = .021). The use of supplemental oxygen (adjusted OR 13.16), and ≥ 200 mL loss of FVC over 1 year (adjusted OR 5.44) were both associated with a ≥ 5% weight loss in the year following a diagnosis of IPF. The use of antifibrotic medication did not significantly change median transplant-free survival in patients who lost more than ≥ 5% of their body mass. Conclusions Weight loss over the first year following a diagnosis of IPF is strongly associated with decreased transplant-free survival. More research is needed to determine the mechanisms surrounding weight loss in patients with IPF.

Transforming growth factor beta (TGF-β) induced myofibroblast differentiation is central to the pathological scarring observed in Idiopathic Pulmonary Fibrosis (IPF) and other fibrotic diseases. Our lab has recently identified expression of GPR68 (Ovarian Cancer Gene Receptor 1, OGR1), a pH sensing G-protein coupled receptor, as a negative regulator of TGF-β induced profibrotic effects in primary human lung fibroblasts (PHLFs). We therefore hypothesized that small molecule activators of GPR68 would inhibit myofibroblast differentiation. Ogerin is a positive allosteric modulator (PAM) of GPR68, inducing a leftward shift of the dose response curve to proton induced signaling. Using PHLFs derived from patients with both non-fibrotic and IPF diagnoses, we show that Ogerin inhibits, and partially reverses TGF-β induced myofibroblast differentiation in a dose dependent manner. This occurs at the transcriptional level without inhibition of canonical TGF-β induced SMAD signaling. Ogerin induces PKA dependent CREB phosphorylation, a marker of Gαs pathway activation. The ability of Ogerin to inhibit both basal and TGF-β induced collagen gene transcription, and induction of Gαs signaling is enhanced at an acidic pH (pH 6.8). Similar findings were also found using fibroblasts derived from dermal, intestinal, and orbital tissue. The biological role of GPR68 in different tissues, cell types, and disease states is an evolving and emerging field. This work adds to the understanding of Gαs coupled GPCRs in fibrotic lung disease, the ability to harness the pH sensing properties of GPR68, and conserved mechanisms of fibrosis across different organ systems.

Idiopathic pulmonary fibrosis (IPF) is a chronic interstitial lung disease. The pathogenesis of IPF is not completely understood. However, numerous genes are associated with the development and progression of pulmonary fibrosis, indicating there is a significant genetic component to the pathogenesis of IPF. Epigenetic influences on the development of human disease, including pulmonary fibrosis, remain to be fully elucidated. In this paper, we identify miR-338-3p as a microRNA severely downregulated in the lungs of patients with pulmonary fibrosis and in experimental models of pulmonary fibrosis. Treatment of primary human lung fibroblasts with miR-338-3p inhibits myofibroblast differentiation and matrix protein production. Published and proposed targets of miR-338-3p such as TGFβ receptor 1, MEK/ERK 1/2, Cdk4, and Cyclin D are also not responsible for the regulation of pulmonary fibroblast behavior by miR-338-3p. miR-338-3p inhibits myofibroblast differentiation by preventing TGFβ-mediated downregulation of phosphatase and tensin homolog (PTEN), a known antifibrotic mediator.

Idiopathic pulmonary fibrosis (IPF) is a progressive, chronic, interstitial lung disease with a poor prognosis. Although specific anti-fibrotic medications are now available, the median survival time following diagnosis remains very low, and new therapies are urgently needed. To uncover novel therapeutic targets, we examined how biochemical properties of the fibrotic lung are different from the healthy lung. Previous work identified lactate as a metabolite that is upregulated in IPF lung tissue. Importantly, inhibition of the enzyme responsible for lactate production prevents fibrosis in vivo. Further studies revealed that fibrotic lesions of the lung experience a significant decline in tissue pH, likely due to the overproduction of lactate. It is not entirely clear how cells in the lung respond to changes in extracellular pH, but a family of proton sensing G-protein coupled receptors has been shown to be activated by reductions in extracellular pH. This work examines the expression profiles of proton sensing GPCRs in non-fibrotic and IPF-derived primary human lung fibroblasts. We identify TDAG8 as a proton sensing GPCR that is upregulated in IPF fibroblasts and that knockdown of TDAG8 dampens myofibroblast differentiation. To our surprise, BTB, a proposed positive allosteric modulator of TDAG8, inhibits myofibroblast differentiation. Our data suggest that BTB does not require TDAG8 to inhibit myofibroblast differentiation, but rather inhibits myofibroblast differentiation through suppression of RhoA mediated signaling. Our work highlights the therapeutic potential of BTB as an anti-fibrotic treatment and expands upon the importance of RhoA-mediated signaling pathways in the context of myofibroblast differentiation. Furthermore, this works also suggests that TDAG8 inhibition may have therapeutic relevance in the treatment of IPF.

Idiopathic pulmonary fibrosis (IPF) is a disease characterized by irreversible lung scarring. The pathophysiology is not fully understood, but the working hypothesis postulates that a combination of epithelial injury and myofibroblast differentiation drives progressive pulmonary fibrosis. We previously demonstrated that a reduction in extracellular pH activates latent TGF-β1, and that TGF-β1 then drives its own activation, creating a feed-forward mechanism that propagates myofibroblast differentiation. Given the important roles of extracellular pH in the progression of pulmonary fibrosis, we sought to identify whether pH mediates other cellular phenotypes independent of TGF-β1. Proton-sensing G-protein coupled receptors are activated by acidic environments, but their role in fibrosis has not been studied. Here, we report that the Ovarian Cancer G-Protein Coupled Receptor1 (OGR1 or GPR68) has dual roles in both promoting and mitigating pulmonary fibrosis. We demonstrate that OGR1 protein expression is significantly reduced in lung tissue from patients with IPF and that TGF-β1 decreases OGR1 expression. In fibroblasts, OGR1 inhibits myofibroblast differentiation and does not contribute to inflammation. However, in epithelial cells, OGR1 promotes epithelial to mesenchymal transition (EMT) and inflammation. We then demonstrate that sub-cellular localization and alternative signaling pathways may be responsible for the differential effect of OGR1 in each cell type. Our results suggest that strategies to selectively target OGR1 expression may represent a novel therapeutic strategy for pulmonary fibrosis.

Combustion related particulate matter air pollution (PM) is associated with an increased risk of respiratory infections in adults. The exact mechanism underlying this association has not been determined. We hypothesized that increased concentrations of combustion related PM would result in dysregulation of the innate immune system. This epidemiological study includes 111 adult patients hospitalized with respiratory infections who underwent transcriptional analysis of their peripheral blood. We examined the association between gene expression at the time of hospitalization and ambient measurements of particulate air pollutants in the 28 days prior to hospitalization. For each pollutant and time lag, gene-specific linear models adjusting for infection type were fit using LIMMA (Linear Models For Microarray Data), and pathway/gene set analyses were performed using the CAMERA (Correlation Adjusted Mean Rank) program. Comparing patients with viral and/or bacterial infection, the expression patterns associated with air pollution exposure differed. Adjusting for the type of infection, increased concentrations of Delta-C (a marker of biomass smoke) and other PM were associated with upregulation of iron homeostasis and protein folding. Increased concentrations of black carbon (BC) were associated with upregulation of viral related gene pathways and downregulation of pathways related to antigen presentation. The pollutant/pathway associations differed by lag time and by type of infection. This study suggests that the effect of air pollution on the pathogenesis of respiratory infection may be pollutant, timing, and infection specific.

Rationale: Idiopathic Pulmonary fibrosis (IPF) is a chronic interstitial lung disease with limited treatment options.Within the context of IPF fibroblasts differentiate into alpha smooth muscle (αSMA) positive myofibroblasts that are highly proliferative, relatively resistant to apoptosis and produce increased matrix proteins.A central profibrotic mediator, transforming growth factor β (TGFβ), is responsible for myofibroblast differentiation.In fibroblasts TGFβ induces proliferation that may contribute to IPF pathogenesis.However, other growth factors such as fibroblast growth factor (FGF) are upregulated in IPF and induce proliferation.Central to our treatment of IPF is the identifying mechanisms that block proliferation by myofibroblasts.We have begun to investigate how we can inhibit TGFβ and FGF induced proliferation in human lung fibroblasts.Our lab has been working with a novel ligand, BTB, which has shown efficacy in blocking proliferation.Our initial investigation has focused on how BTB effects cell cycle related proteins and growth factor-cell cycle interactions.I hypothesize the novel ligand BTB blocks TGFβ and FGF induced proliferation via alteration of cell cycle dynamics, specifically through regulation of cyclin associated kinase 2. Methods: Primary human lung fibroblasts isolated from patients with and without IPF were treated with 1 ng TGFβ and/or 50 uM BTB for the indicated timeframe.Protein was harvested for analysis by Western blot.Blots were analyzed for the following cell cycle proteins: cyclin B, cyclin D, cyclin E, Cdk2, Cdk4 and Cdk6.Proliferation was assayed through ATP based assays as well as the incorporation of EdU.Phase analysis of cell cycle dynamics was conducted with the Cell Clock Assay.Results: BTB blocks TGFβ and FGF induced proliferation as demonstrated by both thymidine incorporation and ATP production assays.Cell phase analysis indicates a larger proportion of cells remain in S phase after BTB treatment compared to vehicle treated cells.Our protein expression data confirms this result, showing BTB inhibits Cdk2 activity (via an inhibitory phosphorylation) which would result in S phase arrest.Conclusion: BTB effectively blocks TGFβ and FGF induced proliferation via alteration of cell cycle dynamics.BTB arrests cells in S phase, blocking the activity of Cdk2 to slow cell cycle progression.Intriguingly, BTB is able to block the proliferative effects of more than one cytokine, indicating BTB may block shared pro-fibrotic pathways.This represents an exciting new therapeutic which can specifically target fibroblast proliferation by blocking multiple pro-fibrotic stimuli.

Rationale: Pulmonary lymphangioleiomyomatosis (LAM) is a rare disease characterized by excess smooth muscle-like cells in the lung which ultimately results in the formation of cysts and destruction of lung architecture.LAM is a devastating disease that often leads to loss of pulmonary function and death, and treatment options are currently limited.LAM is most often diagnosed women of reproductive age, and estrogen is thought to promote LAM progression in humans as well as mediate LAM cell metastasis in mouse models.Many mechanistic studies surrounding the cell biology of LAM have relied upon existing cancer literature.However, there are several pathological changes seen in LAM that parallel the pathology of lung fibrosis.Similar to myofibroblasts in pulmonary fibrosis, LAM cells express alpha smooth muscle actin (αSMA) and are highly proliferative.Additionally, lung tissue remodeling in LAM may also be mediated by the extracellular matrix.Fibrosis associated signaling pathways, notably transforming growth factor beta 1 (TGFβ1) signaling pathways, are understudied in LAM.Studies have shown TGFβ1 is highly expressed in regions of matrix deposition and smooth muscle cells in LAM lung tissues.However, ex-vivo mechanistic studies of TGFβ1 in LAM cells have not yet been conducted.We hypothesize TGFβ1 signaling pathways have direct relevance to the pathophysiology of LAM, and that stimulation of LAM cells with the pro-fibrotic cytokine TGFβ1 will result in increased αSMA expression as well as increased matrix protein production and these effects will synergize with estradiol.Methods: ELT3 cells, a Tsc-2 null myometrial cell line frequently utilized in LAM research, were cultured with 10 nM estradiol (E2) and/or the pro-fibrotic cytokine TGFβ1 (1 ng /mL) for 72 hours.. Expression of αSMA and the matrix proteins collagen 1, fibronectin and calponin were measured by Western blot.Results: Estradiol induced expression of αSMA, calponin, fibronectin and collagen 1 protein in ELT3 cells.TGFβ also induced αSMA protein expression.Importantly we observed additive and synergistic effects of co-treatment with TGFβ1 and estradiol with respect to αSMA and calponin protein expression.Conclusion: Our data suggest that TGFβ1 induces smooth muscle protein and matrix proteins in ELT3 cells.We observed that TGFβ1 acts similarly to estradiol, which promote survival and metastasis of smooth muscle-like cells in LAM models and possibly in LAM patients.Importantly, we have identified that TGFβ1 and estradiol induce synergistic production of smooth muscle and matrix proteins.Thus, we hypothesize that TGFβ1 may contribute to the pathogenesis and progression of LAM.

Rationale: Extracellular matrix remodeling is a key component in the development of pulmonary fibrosis.Transforming Growth Factor Beta (TGF-β) is a crucial cytokine that promotes myofibroblast differentiation and enhances matrix turnover.Elastin fibers are degraded by elastases, and mice that are deficient in neutrophil elastase are protected from bleomycin-induced pulmonary fibrosis.We have demonstrated that patients with IPF have an increased collagen:elastin ratio using second harmonic generation microscopy.TGF-β has been shown to stabilize elastin mRNA and increase expression of alpha 1 anti-trypsin (A1AT), a protein that inhibits elastase activity.However despite TGF-β's actions, there is less mature elastin present in fibrotic areas and TGF-β induced A1AT demonstrates less activity than control conditions.We and others have shown that elastin degradation products (EDP) are increased in people with IPF and other chronic lung diseases.Therefore, we hypothesize that EDP promote myofibroblast in a TGF-β dependent manner.Methods: Healthy human lung fibroblasts were obtained in accordance with an IRB-approved protocol at URMC.Fibroblasts were cultured Modified Eagle Media (MEM) with 10% fetal bovine serum.Fibroblasts were then treated with either full-length elastin (100 µg), EDP (100 μg), TGF-β (1 ng/mL), and/or TGF-β neutralizing antibody (1 μg/mL) for 72 hours.Cells were harvested, and either western blot or qRT-PCR was performed.As a preliminary study, we performed oropharyngeal aspiration on eight week old C57Bl/6J mice (Jackson Labs) with full-length elastin or EDP (200 µg/Kg).After 7 days, mice were sacrificed and lung tissue was harvested.Quantitative RT-PCR was then performed to assess for changes in profibrotic gene expression.Statistical analysis involved ANOVA or student t-test and statistical significance was considered when p < 0.05.Results: In healthy human fibroblasts, treatment with EDP induced myofibroblast differentiation as measured by changes in mRNA and protein expression.However, similar changes were not seen with full-length elastin.Interestingly, myofibroblast differentiation was completely inhibited when treated with EDP and a neutralizing TGF-β antibody.When mice were administered EDP, they demonstrated increased profibrotic gene expression at 7 days.Conclusions: TGF-β signaling is complex, and here we demonstrate another potential feed forward loop involving TGF-β and elastin degradation.Specifically, TGF-β promotes elastin degradation and these degraded products stimulate myofibroblast differentiation in a TGF-β dependent manner.Interestingly, EDP alone can promote myofibroblast differentiation both in vitro and in vivo.These data add to the complexity of TGF-β signaling and may have therapeutic potential for the treatment of pulmonary fibrosis.

Pulmonary fibrosis is a devastating, progressive disease and carries a prognosis worse than most cancers. Despite ongoing research, the mechanisms that underlie disease pathogenesis remain only partially understood. However, the self-perpetuating nature of pulmonary fibrosis has led several researchers to propose the existence of pathological signalling loops. According to this hypothesis, the normal wound-healing process becomes corrupted and results in the progressive accumulation of scar tissue in the lung. In addition, several negative regulators of pulmonary fibrosis are downregulated and, therefore, are no longer capable of inhibiting these feed-forward loops. The combination of pathological signalling loops and loss of a checks and balances system ultimately culminates in a process of unregulated scar formation. This review details specific signalling pathways demonstrated to play a role in the pathogenesis of pulmonary fibrosis. The evidence of detrimental signalling loops is elucidated with regard to epithelial cell injury, cellular senescence and the activation of developmental and ageing pathways. We demonstrate where these loops intersect each other, as well as common mediators that may drive these responses and how the loss of pro-resolving mediators may contribute to the propagation of disease. By focusing on the overlapping signalling mediators among the many pro-fibrotic pathways, it is our hope that the pulmonary fibrosis community will be better equipped to design future trials that incorporate the redundant nature of these pathways as we move towards finding a cure for this unrelenting disease.