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Idiopathic pulmonary fibrosis is a fatal lung disease characterized by progressive and excessive accumulation of myofibroblasts and in the lung. Connective-tissue growth factor (CTGF) exacerbates pulmonary fibrosis in radiation-induced lung fibrosis, and in this study, we demonstrate upregulation of CTGF in a rat lung fibrosis model induced by an adenovirus vector encoding active TGF-β1 (AdTGF-β1). We show that CTGF is also upregulated in patients with idiopathic pulmonary fibrosis. Expression of CTGF was upregulated in vascular smooth muscle cells cultured from fibrotic lungs on Days 7 and 14 as well as endothelial cells sorted from fibrotic lungs on Days 14 and 28. These findings suggest contributions of different cells in maintaining the fibrotic phenotype during fibrogenesis. Treatment of fibroblasts with recombinant CTGF along with TGF-β increases profibrotic markers in fibroblasts, confirming the synergistic effect of recombinant CTGF with TGF-β in inducing pulmonary fibrosis. Also, the fibrotic extracellular matrix upregulated CTGF expression, compared with the normal extracellular matrix, suggesting that not only profibrotic mediators but also a profibrotic environment contributes to fibrogenesis. We also showed that pamrevlumab, a CTGF inhibitory antibody, partially attenuates fibrosis in the model. These results suggest that pamrevlumab could be an option for treatment of pulmonary fibrosis.

The prevalence and contribution of cardiotropic viruses to various expressions of heart failure are increasing, yet primarily underappreciated and underreported due to variable clinical syndromes, a lack of consensus diagnostic standards and insufficient clinical laboratory tools. In this study, we developed an advanced methodology for identifying viruses across a spectrum of heart failure patients. We designed a custom tissue microarray from 78 patients with conditions commonly associated with virus-related heart failure, conditions where viral contribution is typically uncertain, or conditions for which the etiological agent remains suspect but elusive. Subsequently, we employed advanced, highly sensitive in situ hybridization to probe for common cardiotropic viruses: adenovirus 2, coxsackievirus B3, cytomegalovirus, Epstein–Barr virus, hepatitis C and E, influenza B and parvovirus B19. Viral RNA was detected in 46.4% (32/69) of heart failure patients, with 50% of virus-positive samples containing more than one virus. Adenovirus 2 was the most prevalent, detected in 27.5% (19/69) of heart failure patients, while in contrast to previous reports, parvovirus B19 was detected in only 4.3% (3/69). As anticipated, viruses were detected in 77.8% (7/9) of patients with viral myocarditis and 37.5% (6/16) with dilated cardiomyopathy. Additionally, viruses were detected in 50% of patients with coronary artery disease (3/6) and hypertrophic cardiomyopathy (2/4) and in 28.6% (2/7) of transplant rejection cases. We also report for the first time viral detection within a granulomatous lesion of cardiac sarcoidosis and in giant cell myocarditis, conditions for which etiological agents remain unknown. Our study has revealed a higher than anticipated prevalence of cardiotropic viruses within cardiac muscle tissue in a spectrum of heart failure conditions, including those not previously associated with a viral trigger or exacerbating role. Our work forges a path towards a deeper understanding of viruses in heart failure pathogenesis and opens possibilities for personalized patient therapeutic approaches., The prevalence and contribution of viruses to heart failure phenotypes are increasingly recognized, while still underappreciated and underreported. We designed a tissue microarray with cardiac muscle tissue from 78 heart failure patients and probed for common cardiotropic viruses via in situ hybridization. Viral RNA was detected in 46.4% of patients, higher than anticipated for these heart failure conditions that include those not previously associated with a viral trigger or an exacerbating role.

Introduction Idiopathic pulmonary fibrosis (IPF) is a chronic progressive lung disease with high associated morbidity and mortality. The therapeutic landscape has significantly changed in the last 20 years with two drugs currently approved that have demonstrated the ability to slow disease progression. Despite these developments, survival in IPF is limited, so there is a major interest in therapeutic targets which could serve to open up new therapeutic avenues. Areas covered We review the most recent information regarding drug targets and therapies currently being investigated in preclinical and early-stage clinical trials. Expert opinion The complex pathogenesis of IPF and variability in disease course and response to therapy highlights the importance of a precision approach to therapy. Novel technologies including transcriptomics and the use of serum biomarkers, will become essential tools to guide future drug development and therapeutic decision making particularly as it pertains to combination therapy.

Background The Spontaneously Hypertensive Rat (SHR) Colony was established in 1963 and is the most commonly used rodent model for studying heart failure (HF). Ideally, animal models should recapitulate the clinical disease as closely as possible. Any drift in a genetic model may create a new model that no longer adequately represents the human pathology. Further, instability overtime may lead to conflicting data between laboratories and/or irreproducible results. While systolic blood pressure (SBP) is closely monitored during inbreeding, the sequelae of HF (e.g., cardiac hypertrophy) are not. Thus, the object of this review was to investigate whether the hypertension-induced sequelae of HF in the SHR have remained stable after decades of inbreeding. Methods A systematic review was performed to evaluate indices of cardiovascular health in the SHR over the past 60 years. For post hoc statistical analyses, studies were separated into 2 cohorts: Initial (mid to late 1900s) and Current (early 2000s to present) Colony SHRs. Wistar-Kyoto rats (WKY) were used as controls. Results SBP was consistent between Initial and Current Colony SHRs. However, Current Colony SHRs presented with increased concentric hypertrophy (i.e., elevated heart weight and posterior wall thickness) while cardiac output remained consistent. Since these changes were not observed in the WKY controls, cardiac-derived changes in Current Colony SHRs were unlikely due to differences in environmental conditions. Conclusions Together, these data firmly establish a cardiac-based phenotypic shift in the SHR model and provide important insights into the beneficial function of concentric hypertrophy in hypertension-induced HF.

Pulmonary fibrosis is a progressive lung disease characterized by myofibroblast accumulation and excessive extracellular matrix deposition. Endoplasmic reticulum (ER) stress initiates the unfolded protein response (UPR), a cellular stress response pathway that has been implicated in both inflammatory and fibrotic processes. Here, we sought to investigate the role of the 13 kDa FK506-binding protein (FKBP13), an ER stress-inducible molecular chaperone, in various forms of pulmonary fibrosis. We first characterized the gene and protein expression of FKBP13 in lung biopsy samples from 24 patients with idiopathic pulmonary fibrosis (IPF) and 17 control subjects. FKBP13 expression was found to be elevated in the fibrotic regions of IPF lung tissues, and within this cohort, was correlated with declining forced vital capacity and dyspnea severity. FKBP13 expression was also increased in lung biopsies of patients with hypersensitivity pneumonitis, rheumatoid arthritis, and sarcoidosis-associated interstitial lung disease. We next evaluated the role of this protein using FKBP13-/- mice in a bleomycin model of pulmonary fibrosis. Animals were assessed for lung function and histopathology at different stages of lung injury including the inflammatory (Day 7), fibrotic (Day 21) and resolution (Day 50) phase. FKBP13-/- mice showed increased infiltration of inflammatory cells and cytokines at Day 7, increased lung elastance and fibrosis at Day 21, and impaired resolution of fibrosis at Day 50. These changes were associated with an increased number of cells that stained positive for TUNEL and cleaved caspase 3 in the FKBP13-/- lungs, indicating a heightened cellular sensitivity to bleomycin. Our findings suggest that FKBP13 is a potential biomarker for severity or progression of interstitial lung diseases, and that it has a biologically relevant role in protecting mice against bleomycin-induced injury, inflammation and fibrosis.

Pulmonary fibrosis includes several lung disorders characterized by progressive fibrosis, of which idiopathic pulmonary fibrosis (IPF) is a particularly severe form with a median survival time of 3-5 years after diagnosis. Although numerous compounds have shown efficacy in attenuating pulmonary fibrosis using animal models, only a few compounds have shown their beneficial effects for IPF in clinical trials. Thus, there is an emergent need to improve the preclinical development process to better identify, characterize and select clinically useful targets.In this review, the authors extensively describe current models of pulmonary fibrosis, including rodent models,Based upon our current understanding, improving the identification and characterization of clinically relevant molecules or pathways responsible for progressive fibrotic diseases and use of the appropriate preclinical model system to test these will likely be required to improve the drug development pipeline for pulmonary fibrosis. Combination with appropriate preclinical models with

Chronic lung disease accounts for a significant global burden with respect to death, disability, and health-care costs. Due to the heterogeneous nature and limited treatment options for these diseases, it is imperative that the cellular and molecular mechanisms underlying the disease pathophysiology are further understood. The lung is a complex organ with a diverse cell population, and each cell type will likely have different roles in disease initiation, progression, and resolution. The effectiveness of a given therapeutic agent may depend on the net effect on each of these cell types. Over the past decade, it has been established that endoplasmic reticulum stress and the unfolded protein response are involved in the development of several chronic lung diseases. These conserved cellular pathways are important for maintaining cellular proteostasis, but their aberrant activation can result in pathology. This review discusses the current understanding of endoplasmic reticulum stress and the unfolded protein response at the cellular level in the development and progression of various chronic lung diseases. We highlight the need for increased understanding of the specific cellular contributions of unfolded protein response activation to these pathologies and suggest that the development of cell-specific targeted therapies is likely required to further decrease disease progression and to promote resolution of chronic lung disease.

Background: Ischemia induced acute kidney injury (AKI) resulting in tubular damage can often progress to chronic kidney disease (CKD) and is a common cause of nephrology consultation. Following renal tubular epithelial damage, molecular and cellular mechanisms are activated to repair and regenerate the damaged epithelium. If these mechanisms are impaired, AKI can progress to CKD. Even in patients whose kidney function returns to normal baseline are more likely to develop CKD. Genome-wide association studies have provided robust evidence that genetic variants in SHROOM3, which encodes an actin-associated protein, are associated with CKD and poor outcomes in transplanted kidneys. Here, we sought to further understand the associations of Shroom3 in CKD. Methods: Kidney ischemia was induced in wild-type and Shroom3 heterozygous null mice (Shroom3Gt/+) and the mechanisms of cellular recovery and repair were examined. Results: A 28-minute bilateral ischemia in Shroom3Gt/+ mice resulted in 100% mortality within 24 hours. After 22-minute ischemic injury, Shroom3Gt/+ mice had a 16% increased mortality, worsened kidney function, and significantly worse histopathology, apoptosis, proliferation, inflammation, and fibrosis after injury. The cortical tubular damage in Shroom3Gt/+ was associated with disrupted epithelial redifferentiation, disrupted Rho-kinase/myosin signaling, and disorganized apical F-actin. Analysis of Madin Darby Canine Kidney Cells showed the levels of Shroom3 are directly correlated to apical organization of actin and actomyosin regulators. Conclusion: These findings establish that Shroom3 is required for epithelial repair and redifferentiation through the organization of actomyosin regulators and could explain why genetic variants in Shroom3 are associated with CKD and allograft rejection.

Introduction Over 300 million people in the world live with asthma, resulting in 500,000 annual global deaths with future increases expected. It is estimated that around 50–80% of asthma exacerbations are due to viral infections. Currently, a combination of long-acting beta agonists (LABA) for bronchodilation and glucocorticoids (GCS) to control lung inflammation represent the dominant strategy for the management of asthma, however, it is still sub-optimal in 35–50% of moderate-severe asthmatics resulting in persistent lung inflammation, impairment of lung function, and risk of mortality. Mechanistically, LABA/GCS combination therapy results in synergistic efficacy mediated by intracellular cyclic adenosine monophosphate (cAMP). Hypothesis Increasing intracellular cAMP during LABA/GCS combination therapy via inhibiting phosphodiesterase 4 (PDE4) and/or blocking the export of cAMP by ATP Binding Cassette Transporter C4 (ABCC4), will potentiate anti-inflammatory responses of mainstay LABA/GCS therapy. Methods Expression and localization experiments were performed using in situ hybridization and immunohistochemistry in human lung tissue from healthy subjects, while confirmatory transcript and protein expression analyses were performed in primary human airway epithelial cells and cell lines. Intervention experiments were performed on the human airway epithelial cell line, HBEC-6KT, by pre-treatment with combinations of LABA/GCS with PDE4 and/or ABCC4 inhibitors followed by Poly I:C or imiquimod challenge as a model for viral stimuli. Cytokine readouts for IL-6, IL-8, CXCL10/IP-10, and CCL5/RANTES were quantified by ELISA. Results Using archived human lung and human airway epithelial cells, ABCC4 gene and protein expression were confirmed in vitro and in situ. LABA/GCS attenuation of Poly I:C or imiquimod-induced IL-6 and IL-8 were potentiated with ABCC4 and PDE4 inhibition, which was greater when ABCC4 and PDE4 inhibition was combined. Modulation of cAMP levels had no impact on LABA/GCS modulation of Poly I:C-induced CXCL10/IP-10 or CCL5/RANTES. Conclusion Modulation of intracellular cAMP levels by PDE4 or ABCC4 inhibition potentiates LABA/GCS efficacy in human airway epithelial cells challenged with viral stimuli. The data suggest further exploration of the value of adding cAMP modulators to mainstay LABA/GCS therapy in asthma for potentiated anti-inflammatory efficacy.

Survivors of severe SARS-CoV-2 infections frequently suffer from a range of post-infection sequelae. Whether survivors of mild or asymptomatic infections can expect any long-term health consequences is not yet known. Herein we investigated lasting changes to soluble inflammatory factors and cellular immune phenotype and function in individuals who had recovered from mild SARS-CoV-2 infections (n=22) compared to those that had recovered from other mild respiratory infections (n=11). Individuals who had mild SARS-CoV-2 infections had elevated levels of C-reactive protein 1-3 months after symptom onset, and changes in phenotype and function of circulating T cells that were not apparent in individuals 6-9 months post-symptom onset. Markers of monocyte activation and expression of adherence and chemokine receptors indicative of altered migratory capacity were also higher at 1-3 months post-infection in individuals who had mild SARS-CoV-2, but these were no longer elevated by 6-9 months post-infection. Perhaps most surprisingly, polyclonal activation of T cells was higher in individuals who had recently experienced a mild SARS-CoV-2 infection compared to individuals with other recent respiratory infections. These data are indicative of prolonged immune activation and systemic inflammation that persists for up to three months after mild or asymptomatic SARS-CoV-2 infections.

A number of previous studies have observed greater levels of circulating fibrocytes in interstitial lung disease compared to healthy controls, and suggest a prognostic role in idiopathic pulmonary fibrosis (IPF) [1–4]. Fibrocytes are circulating mesenchymal progenitor cells that differentiate into tissue specific fibroblasts and contribute to multiple wound healing processes, including secretion of inflammatory cytokines, contractile wound closure and promotion of angiogenesis [5]. However, the contribution of fibrocytes to the pathogenesis of progressive pulmonary fibrosis remains unclear and clinical observations require independent validation in prospective cohorts., In people with idiopathic pulmonary fibrosis, circulating fibrocytes ≥2.2% were associated with a greater risk of mortality over a median 3 years of follow-up, but were not associated with disease-related decline in lung function or short-term progression. https://bit.ly/3bWydwQ

BackgroundThe role of mast cells accumulating in idiopathic pulmonary fibrosis (IPF) lungs is unknown.ObjectivesWe investigated the effect of fibrotic extracellular matrix (ECM) on mast cells in experimental and human pulmonary fibrosis.ResultsIn IPF lungs, mast cell numbers were increased and correlated with disease severity (control vs 60%90% vs 60%90% vs FVCConclusionThis study suggests that mast cells may contribute to the progression of pulmonary fibrosis.

Since the discovery of the myofibroblast over 50 years ago, much has been learned about its role in wound healing and fibrosis. Its origin, however, remains controversial, with a number of progenitor cells being proposed. Macrophage-myofibroblast transition (MMT) is a recent term coined in 2014 that describes the mechanism through which macrophages, derived from circulating monocytes originating in the bone marrow, transformed into myofibroblasts and contributed to kidney fibrosis. Over the past years, several studies have confirmed the existence of MMT in various systems, suggesting that MMT could potentially occur in all fibrotic conditions and constitute a reasonable therapeutic target to prevent progressive fibrotic disease. In this perspective, we examined recent evidence supporting the notion of MMT in both human disease and experimental models across organ systems. Mechanistic insight from these studies and information from in vitro studies is provided. The findings substantiating plausible MMT showcased the co-expression of macrophage and myofibroblast markers, including CD68 or F4/80 (macrophage) and α-SMA (myofibroblast), in fibroblast-like cells. Furthermore, fate-mapping experiments in murine models exhibiting myeloid-derived myofibroblasts in the tissue further provide direct evidence for MMT. Additionally, we provide some evidence from single cell RNA sequencing experiments confirmed by fluorescent in situ hybridisation studies, showing monocyte/macrophage and myofibroblast markers co-expressed in lung tissue from patients with fibrotic lung disease. In conclusion, MMT is likely a significant contributor to myofibroblast formation in wound healing and fibrotic disease across organ systems. Circulating precursors including monocytes and the molecular mechanisms governing MMT could constitute valid targets and provide insight for the development of novel antifibrotic therapies; however, further understanding of these processes is warranted.

Patient-reported outcome measures (PROMs), tools to assess patient self-report of health status, are now increasingly used in research, care and policymaking. While there are two well-developed disease-specific PROMs for interstitial lung diseases (ILD) and idiopathic pulmonary fibrosis (IPF), many unmet and urgent needs remain. In December 2019, 64 international ILD experts convened in Erice, Italy to deliberate on many topics, including PROMs in ILD. This review summarises the history of PROMs in ILD, shortcomings of the existing tools, challenges of development, validation and implementation of their use in clinical trials, and the discussion held during the meeting. Development of disease-specific PROMs for ILD including IPF with robust methodology and validation in concordance with guidance from regulatory authorities have increased user confidence in PROMs. Minimal clinically important difference for bidirectional changes may need to be developed. Cross-cultural validation and linguistic adaptations are necessary in addition to robust psychometric properties for effective PROM use in multinational clinical trials. PROM burden of use should be reduced through appropriate use of digital technologies and computerised adaptive testing. Active patient engagement in all stages from development, testing, choosing and implementation of PROMs can help improve probability of success and further growth.

Background a Objective: Idiopathic pulmonary fibrosis (IPF) is a common and severe form of pulmonary fibrosis. Nintedanib, a triple angiokinase inhibitor, is approved for treating IPF and other progressive interstitial lung diseases (ILD). Galectin 3 (Gal-3) activates a variety of pro-fibrotic processes. Currently, the Gal-3 inhibitor (TD139 by Galecto) is being tested in phase-II clinical trials 9on-top9 of Nintedanib. Therefore, we evaluated the impact of Nintedanib on Gal-3 expression using in-vitro and in-vivo models, in addition to IPF patient samples. Methods: Gal-3 levels were evaluated in IPF and control tissue samples, primary human lung fibroblasts (HLFs) following Nintedanib treatment (10-100nM, qPCR and Western blot) and in a silica-induced-fibrosis mouse model with/without Nintedanib (0.021-0.21 mg/kg) by immunohistochemistry (IHC). Additionally, Gal-3 levels were analyzed in serum samples from 41 ILD patients with/without Nintedanib treatment by ELISA. Results: Nintedanib addition to HLFs resulted in significant elevations in Gal-3, pSTAT3, as well as IL-8 mRNA levels (pl0.05). Gal-3 expression was elevated in IPF tissue samples compared with non-IPF controls (pl0.05). IHC staining demonstrated stromal involvement with strong staining mainly in the alveolar macrophages. In the in-vivo mouse model, Gal-3 levels were increased following fibrosis induction and even further increased with the addition of Nintedanib, mostly in macrophages (pl0.05). Patients receiving Nintedanib presented with higher Gal-3 serum levels in comparison to those who did not (pl0.05). Conclusion: Nintedanib elevates Gal-3 levels in both experimental models, along with patient samples. These findings highlight the possibility of using combined inhibition therapy.

The drug discovery pipeline, from discovery of therapeutic targets through preclinical and clinical development phases, to an approved product by health authorities, is a time-consuming and costly process, where a lead candidates' success at reaching the final stage is rare. Although the time from discovery to final approval has been reduced over the last decade, there is still potential to further optimize and streamline the evaluation process of each candidate as it moves through the different development phases. In this book chapter, we describe our preclinical strategies and overall decision-making process designed to evaluate the tolerability and efficacy of therapeutic candidates suitable for patients diagnosed with fibrotic lung disease. We also describe the benefits of conducting preliminary discovery trials, to aid in the selection of suitable primary and secondary outcomes to be further evaluated and assessed in subsequent internal and external validation studies. We outline all relevant research methodologies and protocols routinely performed by our research group and hope that these strategies and protocols will be a useful guide for biomedical and translational researchers aiming to develop safe and beneficial therapies for patients with fibrotic lung disease.

Cannabis smoking is the dominant route of delivery, with the airway epithelium functioning as the site of first contact. The endocannabinoid system is responsible for mediating the physiological effects of inhaled phytocannabinoids. The expression of the endocannabinoid system in the airway epithelium and contribution to normal physiological responses remains to be defined. To begin to address this knowledge gap, a curated dataset of 1090 unique human bronchial brushing gene expression profiles was created. The dataset included 616 healthy subjects, 136 subjects with asthma, and 338 subjects with COPD. A 32-gene endocannabinoid signature was analysed across all samples with sex and disease-specific analyses performed. Immunohistochemistry and immunoblots were performed to probe in situ and in vitro protein expression. CB1, CB2, and TRPV1 protein signal is detectable in human airway epithelial cells in situ and in vitro, justifying examining the downstream endocannabinoid pathway. Sex status was associated with differential expression of 7 of 32 genes. In contrast, disease status was associated with differential expression of 21 of 32 genes in people with asthma and 26 of 32 genes in people with COPD. We confirm at the protein level that TRPV1, the most differentially expressed candidate in our analyses, was upregulated in airway epithelial cells from people with asthma relative to healthy subjects. Our data demonstrate that the endocannabinoid system is expressed in human airway epithelial cells with expression impacted by disease status and minimally by sex. The data suggest that cannabis consumers may have differential physiological responses in the respiratory mucosa., The endocannabinoid system is differentially expressed in human airway epithelial cells from healthy subjects compared to those with asthma or COPD, which may be relevant for population-specific responses to inhaled cannabis smoke https://bit.ly/3cEWc2h

IntroductionOver 300 million people in the world live with asthma, resulting in 500,000 annual global deaths with future increases expected. It is estimated that around 50-80% of asthma exacerbations are due to viral infections. Currently, a combination of long-acting beta agonists (LABA) for bronchodilation and glucocorticoids (GCS) to control lung inflammation represent the dominant strategy for the management of asthma, however it is still sub-optimal in 35-50% of moderate-severe asthmatics resulting in persistent lung inflammation, impairment of lung function, and risk of mortality. Mechanistically, LABA/GCS combination therapy results in synergistic efficacy mediated by intracellular cyclic adenosine monophosphate (cAMP).HypothesisIncreasing intracellular cAMP during LABA/GCS combination therapy via inhibiting phosphodiesterase 4 (PDE4) and/or blocking export of cAMP by ATP Binding Cassette Transporter C4 (ABCC4), will potentiate anti-inflammatory responses of mainstay LABA/GCS therapy.MethodsExpression and localization experiments were performed usingin situhybridization and immunohistochemistry in human lung tissue from healthy subjects, while confirmatory transcript and protein expression analyses were performed in primary human airway epithelial cells and cell lines. Intervention experiments were performed on the human airway epithelial cell line, HBEC-6KT, by pre-treatment with combinations of LABA/GCS with PDE4 and/or ABCC4 inhibitors followed by Poly I:C or imiquimod challenge as a model for viral stimuli. Cytokine readouts for IL-6, IL-8, CXCL10/IP-10, and CCL5/RANTES were quantified by ELISA.ResultsUsing archived human lung and human airway epithelial cells, ABCC4 gene and protein expression were confirmedin vitroandin situ. LABA/GCS attenuation of Poly I:C or imiquimod-induced IL-6 and IL-8 was potentiated with ABCC4 and PDE4 inhibition, which was greater when ABCC4 and PDE4 inhibition was combined. Modulation of cAMP levels had no impact on LABA/GCS modulation of Poly I:C-induced CXCL10/IP-10 or CCL5/RANTES.ConclusionModulation of intracellular cAMP levels by PDE4 or ABCC4 inhibition is able to potentiate LABA/GCS efficacy in human airway epithelial cells challenged with viral stimuli. The data suggests further exploration of the value of adding cAMP modulators to mainstay LABA/GCS therapy in asthma for potentiated anti-inflammatory efficacy.

Background and Aims: Idiopathic pulmonary fibrosis (IPF) is a common and severe form of pulmonary fibrosis. Nintedanib, a triple angiokinase inhibitor, is approved for treating IPF. Galectin 3 (Gal-3) activates a variety of profibrotic processes. Currently, the Gal-3 inhibitor TD139 is being tested in phase II clinical trials. Since this treatment is given ‘on top’ of nintedanib, it is important to estimate its effect on Gal-3 levels. Therefore, we evaluated the impact of nintedanib on Gal-3 expression using both in vitro and in vivo models, in addition to serum samples from patients with IPF. Methods: Gal-3 levels were evaluated in IPF and control tissue samples, primary human lung fibroblasts (HLFs) following nintedanib treatment (10–100 nM, quantitative polymerase chain reaction), and in a silica-induced fibrosis mouse model with/without nintedanib (0.021–0.21 mg/kg) by immunohistochemistry. In addition, Gal-3 levels were analyzed in serum samples from 41 patients with interstitial lung disease patients with/without nintedanib treatment by ELISA. Results: Nintedanib addition to HLFs resulted in significant elevations in Gal-3, phospho-signal transducer and activator of transcription 3 (pSTAT3), as well as IL-8 mRNA levels ( p Conclusion: Nintedanib elevates Gal-3 levels in both experimental models, along with patient samples. These findings highlight the possibility of using combined inhibition therapy for patients with IPF.

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by progressive and excessive accumulation of myofibroblasts and extracellular matrix in the lung. Connective-tissue growth factor (CTGF) is known to exacerbate pulmonary fibrosis in radiation-induced lung fibrosis, and in this study, we show the upregulation of CTGF from a rat lung fibrosis model induced by adenovirus vector encoding active TGF-β1 (AdTGF-β1), and also in patients with IPF. The expression of CTGF was upregulated in vascular smooth muscle cells cultured from fibrotic lungs on days 7 or 14 as well as endothelial cells sorted from fibrotic lungs on day 14 or 28 respectively. These findings suggest the role of different cells in maintaining the fibrotic phenotype during fibrogenesis. Treatment of fibroblasts with recombinant CTGF along with TGF-β increases pro-fibrotic markers in fibroblasts, confirming the synergistic effect of recombinant CTGF with TGF-β in inducing pulmonary fibrosis. Also, fibrotic extracellular matrix upregulated the expression of CTGF, as compared to normal extracellular matrix, suggesting that not only profibrotic mediators but also a profibrotic environment contributes to fibrogenesis. We also showed that pamrevlumab, a CTGF inhibitory antibody, partially attenuates fibrosis in the model. These results suggest that pamrevlumab could be an option for the treatment of pulmonary fibrosis.

ObjectiveCirculating fibrocytes are elevated in idiopathic pulmonary fibrosis, but the relationship between fibrocyte level with lung function decline and outcomes is lacking replication in prospective clinical study. We aim to validate the utility of circulating fibrocyte levels as a prognostic biomarker in idiopathic pulmonary fibrosis.MethodsWe tested associations between circulating fibrocyte levels, mortality, disease progression and longitudinal lung function in a well-defined prospective observational study of pulmonary fibrosis (PROFILE; NCT01134822). A subset of recruited participants had blood samples processed for fibrocyte measurement, with flow cytometry based on CD45 and collagen-I gating. Associations were tested using univariable and multivariable generalised linear models. Mortality data were subsequently combined with an independent cohort in a mixed-effect multilevel analysis.ResultsIn 102 participants with idiopathic pulmonary fibrosis, an empirically defined cutpoint of 2.22% was associated with a greater risk of overall mortality in adjusted analysis (Hazard Ratio 2.24 95% CI 1.06-4.72). A 2.5 fold greater risk of mortality was supported in a pooled analysis with a historic cohort for a larger sample of 162 participants of idiopathic pulmonary fibrosis, specifically (Hazard Ratio 2.49 95% CI 2.41-2.56). A previously defined mortality risk threshold of 5% circulating fibrocytes was not reproducible in this cohort, circulating fibrocytes were not significantly elevated above non-specific interstitial pneumonia or healthy controls.. We found no association of fibrocytes with lung function or disease progression.ConclusionsIn a prospective clinical cohort of idiopathic pulmonary fibrosis, circulating fibrocytes of 2.22% or above are associated with greater mortality, but do not associate with disease related decline in lung function.What is the key question?Can circulating fibrocytes provide reproducible prognostic biomarker value in fibrotic lung disease?What is the bottom line?Greater proportions of fibrocytes isolated from circulating leukocyte populations are associated with an increase risk of mortality in idiopathic pulmonary fibrosis, but no association was observed with disease related decline in lung function.Why read on?We present associations and limitations of circulating fibrocytes in the largest sample of individuals with pulmonary fibrosis, recruited into a prospective observational study, and replicate prognositic insights from a historic cohort.

Oral nintedanib is marketed for the treatment of idiopathic pulmonary fibrosis (IPF). While effective slowing fibrosis progression, as an oral medicine nintedanib is limited. To reduce side effects and maximize efficacy, nintedanib was reformulated as a solution for nebulization and inhaled administration. To predict effectiveness treating IPF, the nintedanib pharmacokinetic/pharmacodynamic relationship was dissected. Pharmacokinetic analysis indicated oral-delivered nintedanib plasma exposure and lung tissue partitioning were not dose-proportional and resulting lung levels were substantially higher than blood. Although initial-oral absorbed nintedanib efficiently partitioned into the lung, only a quickly eliminated fraction appeared available to epithelial lining fluid (ELF). Because IPF disease appears to initiate and progress near the epithelial surface, this observation suggests short duration nintedanib exposure (oral portion efficiently partitioned to ELF) is sufficient for IPF efficacy. To test this hypothesis, exposure duration required for nintedanib activity was explored.In vitro, IPF-cellular matrix (IPF-CM) increased primary normal human fibroblast (nHLF) aggregate size and reduced nHLF cell count. IPF-CM also increased nHLF ACTA2 and COL1A expression. Whether short duration (inhalation mimic) or continuous exposure (oral mimic), nintedanib (1-100 nM) reversed these effects.In vivo, intubated silica produced a strong pulmonary fibrotic response. Once-daily (QD) 0.021, 0.21 and 2.1 mg/kg intranasal (IN; short duration inhaled exposure) and twice daily (BID) 30 mg/kg oral (PO; long duration oral exposure) showed that at equivalent-delivered lung concentrations, QD short duration inhaled nintedanib exposure (0.21 mg/kg IN vs. 30 mg/kg PO) exhibited equivalent-to-superior activity as BID oral (reduced silica-induced elastance, alpha-smooth muscle actin, interleukin-1 beta (IL-1β) and soluble collagen, and lung macrophage and neutrophils). An increased lung dose (2.1 mg/kg IN vs. 30 mg/kg PO) exhibited increased effect by further reducing silica-induced elastance, IL-1β and soluble collagen. Neither oral nor inhaled nintedanib reduced silica-induced parenchymal collagen. Both QD inhaled and BID oral nintedanib reduced silica-induced inflammatory index with oral achieving significance. In summary, nintedanib pulmonary anti-fibrotic activity can be achieved using small, infrequent inhaled doses to deliver oral equivalent-to-superior therapeutic effect.

Recreational and medicinal cannabis consumption in the past 12 months has been reported in 1/5th of Canadians, with greater use in males relative to females. Cannabis smoking is the dominant route of delivery in consumers, with the airway epithelium functioning as the site of first contact for inhaled phytocannabinoids. The endocannabinoid system is responsible for mediating the physiological effects of inhaled phytocannabinoids. Acute cannabis smoke inhalation can result in bronchodilation, which may have applications in chronic respiratory disease management. In contrast, chronic cannabis smoke inhalation is associated with reduced lung function and bronchitis, which challenges potential applications in the lung. The contribution of the endocannabinoid system in the airway epithelium to either beneficial or harmful physiological responses remains to be clearly defined in males and females and those with underlying chronic respiratory disease.To begin to address this knowledge gap, a curated dataset of 1090 unique human bronchial brushing gene expression profiles was created from Gene Expression Omnibus deposited microarray datasets. The dataset included 616 healthy subjects, 136 subjects with asthma, and 338 subjects with COPD. A 27-gene endocannabinoid signature was analyzed across all samples with sex and disease specific-analyses performed. Immunohistochemistry and immunoblots were performed to confirm in situ and in vitro protein expression of select genes in human airway epithelial cells.We confirm three receptors for cannabinoids, CB1, CB2, and TRPV1, are expressed at the protein level in human airway epithelial cells in situ and in vitro, justifying examining the downstream endocannabinoid pathway more extensively at the gene expression level. Sex status was associated with differential expression of 6/27 genes. In contrast, disease status was associated with differential expression of 18/27 genes in asthmatics and 22/27 genes in COPD subjects. We confirm at the protein level that TRPV1, the most differentially expressed candidate in our analyses, was up-regulated in airway epithelial cells from asthmatics relative to healthy subjects.Our data demonstrate that endocannabinoid system is expressed in human airway epithelial cells with expression impacted by disease status and minimally by sex. The data suggest that cannabis consumers may have differential physiological responses in the respiratory mucosa, which could impact both acute and chronic effects of cannabis smoke inhalation.

Survivors of severe SARS-CoV-2 infections frequently suffer from a range of post-infection sequelae. Whether survivors of mild or asymptomatic infections can expect any long-term health consequences is not yet known. Herein we investigated lasting changes to soluble inflammatory factors and cellular immune phenotype and function in individuals who had recovered from mild SARS-CoV-2 infections (n = 22), compared to those that had recovered from other mild respiratory infections (n = 11). Individuals who had experienced mild SARS-CoV-2 infections had elevated levels of C-reactive protein 1–3 months after symptom onset, and changes in phenotype and function of circulating T-cells that were not apparent in individuals 6–9 months post-symptom onset. Markers of monocyte activation, and expression of adherence and chemokine receptors indicative of altered migratory capacity, were also higher at 1–3 months post-infection in individuals who had mild SARS-CoV-2, but these were no longer elevated by 6–9 months post-infection. Perhaps most surprisingly, significantly more T-cells could be activated by polyclonal stimulation in individuals who had recently experienced a mild SARS-CoV-2, infection compared to individuals with other recent respiratory infections. These data are indicative of prolonged immune activation and systemic inflammation that persists for at least three months after mild or asymptomatic SARS-CoV-2 infections.

Non-targeted drug delivery systems have several limitations including the decreased bioavailability of the drug, poor stability and rapid clearance in addition to off-target distribution. Cell-specific targeted delivery approaches promise to overcome some of these limitations and enhance therapeutic selectivity. In this review, we aim to discuss cell-specific targeted approaches in the lung at the biochemical and molecular levels. These approaches include; a) directly administered small molecule drugs with intracellular action; b) targeted biologics and synthetic hybrids with extracellular action; c) site activated drugs; and d) delivery systems. We discuss the pharmaceutical and biochemical parameters that govern the fate of drug molecules at delivery sites while presenting an overview of relevant literature surrounding this area of research and current advancements.

Proteinuria is one of the primary risk factors for the progression of chronic kidney disease (CKD) and has been implicated in the induction of endoplasmic reticulum (ER) stress. We hypothesized that the suppression of ER stress with a low molecular weight chemical chaperone, 4-phenylbutyric acid (4-PBA), would reduce the severity of CKD and proteinuria in the Dahl salt-sensitive (SS) hypertensive rat. To induce hypertension and CKD, 12-wk-old male rats were placed on a high-salt (HS) diet for 4 wk with or without 4-PBA treatment. We assessed blood pressure and markers of CKD, including proteinuria, albuminuria, and renal pathology. Furthermore, we determined if HS feeding resulted in an impaired myogenic response, subsequent to ER stress. 4-PBA treatment reduced salt-induced hypertension, proteinuria, and albuminuria and preserved myogenic constriction. Furthermore, renal pathology was reduced with 4-PBA treatment, as indicated by lowered expression of profibrotic markers and fewer intratubular protein casts. In addition, ER stress in the glomerulus was reduced, and the integrity of the glomerular filtration barrier was preserved. These results suggest that 4-PBA treatment protects against proteinuria in the SS rat by preserving the myogenic response and by preventing ER stress, which led to a breakdown in the glomerular filtration barrier. As such, alleviating ER stress serves as a viable therapeutic strategy to preserve kidney function and to delay the progression of CKD in the animal model under study.

Rationale: Idiopathic pulmonary fibrosis (IPF) causes pulmonary vascular remodelling and can develop pulmonary hypertension (PH), resulting in a poorer prognosis. We hypothesized that endothelial dysfunction and vascular remodelling in PH secondary to IPF enhance pro-fibrotic signals in the fibrotic lung and thereby actively contribute to the progression of interstitial fibrosis. Methods: The fibrosis model using adenoviral vector coding active TGF-I²1 was established by our group, and it induces persistent pulmonary fibrosis as well as substantial PH without much inflammation, different from bleomycin. Vascular endothelial cells (ECs) were sorted from rat lungs using flow cytometry. Primary vascular smooth muscle cells (VSMCs) were cultured from rat lungs. Results: The number of ECs was decreased in the fibrotic lung. VSMCs from remodeled pulmonary arteries (fVSMCs) had increased abilities to migrate, invade, and proliferate. Co-culture rat fibroblasts with fVSMCs induced fibrotic phenotype in fibroblasts. Conditioned media from fVSMCs also induced fibrotic phenotype in fibroblasts. The levels of Bmpr2 and its signaling, crucial for maintaining the viability of ECs and inhibition of VSMC proliferation, were decreased in ECs and VSMCs from the fibrotic lung. While the level of Ctgf, a key fibrogenic molecule that affects several signaling including Tgfb and Bmp, was increased. Administration of Tacrolimus to activate Bmpr2 signaling attenuated lung fibrosis as well as PH in rats. Conclusions: Endothelial dysfunction and vascular remodeling in PH secondary to pulmonary fibrosis enhance pro-fibrotic effects in the fibrotic lung through Bmpr2 and Ctgf signaling.

The mononuclear phagocytic system is a key player in fibrotic diseasehttps://bit.ly/3nVIlbQ

Objective Our purpose was to determine if endoplasmic reticulum stress inhibition lowers blood pressure (BP) in hypertension by correcting vascular dysfunction. Methods The spontaneously hypertensive rat (SHR) was used as a model of human essential hypertension with its normotensive control, the Wistar Kyoto rat. Animals were subjected to endoplasmic reticulum stress inhibition with 4-phenylbutyric acid (4-PBA; 1 g/kg per day, orally) for 5 weeks from 12 weeks of age. BP was measured weekly noninvasively and at endpoint with carotid arterial cannulation. Small mesenteric arteries were removed for vascular studies. Function was assessed with a Mulvany-Halpern style myograph, and structure was assessed by measurement of medial-to-lumen ratio in perfusion fixed vessels as well as three-dimensional confocal reconstruction of vessel wall components. Endoplasmic reticulum stress was assessed by quantitative real time-PCR and western blotting; oxidative stress was assessed by 3-nitrotyrosine and dihydroethidium staining. Results 4-PBA significantly lowered BP in SHR (vehicle 206.1 ± 4.3 vs. 4-PBA 178.9 ± 3.1, systolic) but not Wistar Kyoto. 4-PBA diminished contractility and augmented endothelial-dependent vasodilation in SHR small mesenteric arteries, as well as reducing media-to-lumen ratio. 4-PBA significantly reduced endoplasmic reticulum stress in SHR resistance vessels. Normotensive resistance vessels, treated with the endoplasmic reticulum stress-inducing agent, tunicamycin, show decreased endothelial-dependent vasodilation; this was improved with 4-PBA treatment. 3-Nitrotyrosine and dihydroethidium staining indicated that endoplasmic reticulum stress leads to reactive oxygen species generation resolvable by 4-PBA treatment. Conclusion Endoplasmic reticulum stress caused endothelial-mediated vascular dysfunction contributing to elevated BP in the SHR model of human essential hypertension.

Recent findings suggesting transforming growth factor (TGF)-β1 activation by mechanical stimuli in vitro raised the question of whether this phenomenon was relevant in vivo in the context of pulmonary fibrosis.To explore the effect of mechanical stress on TGF-β1 activation and its signaling pathway in rat and human fibrotic lung tissue using a novel ex vivo model.Rat lung fibrosis was induced using transient gene expression of active TGF-β1. Lungs were harvested at Day 14 or 21 and submitted to various stimuli in a tissue bath equipped with a force transducer and servo-controlled arm.Fibrotic lung strips responded to tensile force by releasing active TGF-β1 from latent stores with subsequent increase in tissue phospho-Smad2/3. In contrast, measurable active TGF-β1 and phospho-Smad2/3 were not induced by mechanical stress in nonfibrotic lungs. Protease inhibition did not affect the release of active TGF-β1. A TGF-β1 receptor inhibitor, Rho-associated protein kinase inhibitor, and αv integrin inhibitor all attenuated mechanical stretch-induced phospho-Smad2/3 in fibrotic lung strips. Furthermore, the induction of phospho-Smad2/3 was enhanced in whole fibrotic rat lungs undergoing ventilation pressure challenge compared with control lungs. Last, tissue slices from human lung with usual interstitial pneumonia submitted to mechanical force showed an increase in TGF-β1 activation and induction of phospho-Smad2/3 in contrast with human nonfibrotic lungs.Mechanical tissue stretch contributes to the development of pulmonary fibrosis via mechanotransduced activation of TGF-β1 in rodent and human pulmonary fibrosis.

In December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged, causing the coronavirus disease 2019 (COVID-19) pandemic. SARS-CoV, the agent responsible for the 2003 SARS outbreak, utilises angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) host molecules for viral entry. ACE2 and TMPRSS2 have recently been implicated in SARS-CoV-2 viral infection. Additional host molecules including ADAM17, cathepsin L, CD147 and GRP78 may also function as receptors for SARS-CoV-2.To determine the expression and in situ localisation of candidate SARS-CoV-2 receptors in the respiratory mucosa, we analysed gene expression datasets from airway epithelial cells of 515 healthy subjects, gene promoter activity analysis using the FANTOM5 dataset containing 120 distinct sample types, single cell RNA sequencing (scRNAseq) of 10 healthy subjects, proteomic datasets, immunoblots on multiple airway epithelial cell types, and immunohistochemistry on 98 human lung samples.We demonstrate absent to low ACE2 promoter activity in a variety of lung epithelial cell samples and low ACE2 gene expression in both microarray and scRNAseq datasets of epithelial cell populations. Consistent with gene expression, rare ACE2 protein expression was observed in the airway epithelium and alveoli of human lung, confirmed with proteomics. We present confirmatory evidence for the presence of TMPRSS2, CD147 and GRP78 protein in vitro in airway epithelial cells and confirm broad in situ protein expression of CD147 and GRP78 in the respiratory mucosa.Collectively, our data suggest the presence of a mechanism dynamically regulating ACE2 expression in human lung, perhaps in periods of SARS-CoV-2 infection, and also suggest that alternative receptors for SARS-CoV-2 exist to facilitate initial host cell infection.

BackgroundIn clinical trials, the two anti-interleukin (IL)-5 monoclonal antibodies (mAbs: mepolizumab and reslizumab) approved to treat severe eosinophilic asthma reduce exacerbations by ∼50–60%.ObjectiveTo observe response to anti-IL-5 mAbs in a real-life clinical setting, and to evaluate predictors of suboptimal response.MethodsIn four Canadian academic centres, predefined clinical end-points in 250 carefully characterised moderate-to-severe asthmatic patients were collected prospectively to assess response to the two anti-IL-5 mAbs. Suboptimal response was determined based on failure to reduce maintenance corticosteroid (MCS) or asthma symptoms scores (Asthma Control Questionnaire (ACQ)) or exacerbations, in addition to persistence of sputum/blood eosinophils. Worsening in suboptimal responders was assessed based on reduced lung function by 25% or increase in MCS/ACQ. A representative subset of 39 patients was evaluated for inflammatory mediators, autoantibodies and complement activation in sputum (by ELISA) and for immune-complex deposition by immunostaining formalin-fixed paraffin-embedded sputum plugs.ResultsSuboptimal responses were observed in 42.8% (107 out of 250) patients treated with either mepolizumab or reslizumab. Daily prednisone requirement, sinus disease and late-onset asthma diagnoses were the strongest predictors of suboptimal response. Asthma worsened in 13.6% (34 out of 250) of these patients. The majority (79%) of them were prednisone-dependent. Presence of sputum anti-eosinophil peroxidase immunoglobulin (Ig)G was a predictor of suboptimal response to an anti-IL-5 mAb. An increase in sputum C3c (marker of complement activation) and deposition of C1q-bound/IL-5-bound IgG were observed in the sputa of those patients who worsened on therapy, suggesting an underlying autoimmune-mediated pathology.ConclusionA significant number of patients who meet currently approved indications for anti-IL5 mAbs show suboptimal response to them in real-life clinical practice, particularly if they are on high doses of prednisone. Monitoring blood eosinophil count is not helpful to identify these patients. The concern of worsening of symptoms associated with immune-complex mediated complement activation in a small proportion of these patients highlights the relevance of recognising airway autoimmune phenomena and this requires further evaluation.

Idiopathic pulmonary fibrosis (IPF) is a complex disease of unknown aetiology, which makes drug development challenging. Single administration of bleomycin directly to the lungs of mice is a widely used experimental model for studying pulmonary fibrogenesis and evaluating the effect of therapeutic antifibrotic strategies. The model works by inducing an early inflammatory phase, which transitions into fibrosis after 5–7 days. This initial inflammation makes therapeutic timing crucial. To accurately assess antifibrotic efficacy, the intervention should inhibit fibrosis without impacting early inflammation.Studies published between 2008 and 2019 using the bleomycin model to investigate pulmonary fibrosis were retrieved from PubMed, and study characteristics were analysed. Intervention-based studies were classified as either preventative (starting 7 days). In addition, studies were cross-referenced with current major clinical trials to assess the availability of preclinical rationale.A total of 976 publications were evaluated. 726 investigated potential therapies, of which 443 (61.0%) were solely preventative, 166 (22.9%) were solely therapeutic and 105 (14.5%) were both. Of the 443 preventative studies, only 70 (15.8%) characterised inflammation during the model's early inflammatory phase. In the reported 145 IPF clinical trials investigating 93 compounds/combinations, only 25 (26.9%) interventions had any preclinical data on bleomycin available on PubMed.Since 2008, we observed a shift (from

IF 12.242; International audience; Idiopathic pulmonary fibrosis (IPF) is a progressive disease with an unknown cause. Two drugs, nintedanib and pirfenidone, have been shown to slow, but not stop, disease progression. Pulmonary hypertension (PH) is a frequent complication in IPF patients and is associated with poor prognosis. Macitentan is a dual endothelin receptor antagonist that is approved for pulmonary arterial hypertension treatment. We hypothesised that using macitentan to treat animals with pulmonary fibrosis induced by adenoviral vector encoding biologically active transforming growth factor-β1 (AdTGF-β1) would improve the PH caused by chronic lung disease and would limit the progression of fibrosis.Rats (Sprague Dawley) which received AdTGF-β1 were treated by daily gavage of macitentan (100 mg·kg-1·day-1), pirfenidone (0.5% food admix) or a combination from day 14 to day 28. Pulmonary artery pressure (PAP) was measured before the rats were killed, and fibrosis was subsequently evaluated by morphometric measurements and hydroxyproline analysis.AdTGF-β1 induced pulmonary fibrosis associated with significant PH. Macitentan reduced the increase in PAP and both macitentan and pirfenidone stopped fibrosis progression from day 14 to day 28. Macitentan protected endothelial cells from myofibroblast differentiation and apoptosis whereas pirfenidone only protected against fibroblast-to-myofibroblast differentiation. Both drugs induced apoptosis of differentiated myofibroblasts in vitro and in vivoOur results demonstrate that dual endothelin receptor antagonism was effective in both PH and lung fibrosis whereas pirfenidone only affected fibrosis.

Interstitial lung disease (ILD) covers a large spectrum of lung disorders that affects the parenchyma and is often associated with inflammation and/or fibrosis. Clinically, there is a great need for biomarker development for these disorders, to help diagnosis, treatment selection and assessment of efficacy as well as to predict progression. Thus far, no broadly validated biomarker exists for ILD, due to the existence of a very large number of disorders of often-unknown etiology, overlapping symptoms and disorders associated with a spectrum of multi-morbidities involving similar chronic inflammatory and fibrotic biochemical processes. We discuss here the development of biomarkers in IPF, sarcoidosis, connective tissue disease-associated ILD (CTD-ILD), and chronic hypersensitivity pneumonitis. We further discuss the need and opportunity to develop a multimarker approach that would be clinically meaningful for patients with ILD. Such composite index could include clinical symptoms, pathological assessment, and lung physiology measurements added to molecular information derived from bronchoalveolar lavage and serum. We also discuss the increased opportunity for patients to be involved in research as recent technological advances now allow the serial measurement of lung function using handheld spirometers, combined with handheld devices allowing measurement of patient reported outcomes, mobility, exercise, and sleep.

Chronic kidney disease (CKD) is a major healthcare problem with increasing prevalence in the population. CKD leads to end stage renal disease and increases the risk of cardiovascular disease. As such, it is important to study the mechanisms underlying CKD progression. To this end, an animal model was developed to allow the testing of new treatment strategies or molecular targets for CKD prevention. Many underlying risk factors result in CKD but the disease itself has common features, including renal interstitial fibrosis, tubular epithelial cell loss through apoptosis, glomerular damage, and renal inflammation. Further, CKD shows differences in prevalence between the genders with premenopausal women being relatively resistant to CKD. We sought to develop and characterize an animal model with these common features of human CKD in the C57BL/6 mouse. Mice of this genetic background have been used to produce transgenic strains that are commercially available. Thus, a CKD model in this strain would allow the testing of the effects of numerous genes on the severity or progression of CKD with minimal cost. This paper describes such a mouse model of CKD utilizing angiotensin II and deoxycorticosterone acetate as inducers.

Although recent evidence indicates that gp130 cytokines, Oncostatin M (OSM) and IL-6 are involved in alternative programming of macrophages, their role in lung fibrogenesis is poorly understood. Here, we investigated the effect of transient adenoviral overexpression of OSM or IL-6 in mice during bleomycin-induced lung fibrosis. Lung fibrosis and M2-like macrophage accumulation were assessed by immunohistochemistry, western blotting, gene expression and flow cytometry. Ex-vivo isolated alveolar and bone marrow-derived macrophages were examined for M2-like programming and signalling. Airway physiology measurements at day 21 demonstrated that overexpression of OSM or IL-6 exacerbated bleomycin-induced lung elastance, consistent with histopathological assessment of extracellular matrix and myofibroblast accumulation. Flow cytometry analysis at day 7 showed increased numbers of M2-like macrophages in lungs of mice exposed to bleomycin and OSM or IL-6. These macrophages expressed the IL-6Rα, but were deficient for OSMRβ, suggesting that IL-6, but not OSM, may directly induce alternative macrophage activation. In conclusion, the gp130 cytokines IL-6 and OSM contribute to the accumulation of profibrotic macrophages and enhancement of bleomycin-induced lung fibrosis. This study suggests that therapeutic strategies targeting these cytokines or their receptors may be beneficial to prevent the accumulation of M2-like macrophages and the progression of fibrotic lung disease.

Background: Increased tissue stiffness is a consequence and a driver of fibrosis. Tissue stiffness is modulated by the lysyl oxidase (LOX) family of enzymes, which cross-link collagen and elastin fibres. Increased expression of LOX-like-2 (LOXL2) is associated with the development of fibrosis and therefore a therapeutic target. We have profiled novel orally bioavailable LOXL2-selective small molecule inhibitors in in vitro and in vivo models of lung fibrosis. Methods: In the in vitro model, lung fibroblasts from IPF patients were cultured under optimised conditions for mature collagen matrix deposition. Following TGF-β1 treatment, multicellular foci formed which were histochemically similar in organization to fibroblastic foci in vivo (Jones et al., AJRCCM 191;2015:A4912). Transient overexpression of active TGF-β1 by adenovector gene transfer in rat lungs results in severe progressive fibrosis (Sime et al JCI 1997;100:768–776). The effects of treatment with a LOXL2-selective inhibitor (days 2-28) on histology and lung function were assessed at day 28. Results: Treatment with LOXL2-selective inhibitors dose-dependently reduced cross-link formation, altered the organisation of collagen matrix and reduced matrix stiffness (assessed by parallel plate compression) in the in-vitro model. In vivo, treatment at 15 or 30 mg/kg significantly lowered lung elastance compared to vehicle control from 2.2 (0.39) to 1.7 (0.31) or 1.6 (0.31) respectively (mean (SD) cmH2O/ml; p Conclusions The results suggest that inhibition of LOXL2 using these novel inhibitors has the potential to improve lung function in patients with lung fibrosis by reducing tissue stiffness.

Idiopathic pulmonary fibrosis (IPF) is a progressive disease of the lung parenchyma, causing significant morbidity through worsening dyspnoea and overall functional decline. IPF is characterised by apoptosis-resistant myofibroblasts, which are a major source for the excessive production of extracellular matrix (ECM) overtaking normal lung tissue. We sought to study the role of heat shock protein (HSP) isoforms HSP90α and HSP90β, whose distinct roles in lung fibrogenesis remain elusive.We determined the level of circulating HSP90α in IPF patients (n=31) and age-matched healthy controls (n=9) by ELISA. The release of HSP90α and HSP90β was evaluated in vitro in primary IPF and control lung fibroblasts and ex vivo after mechanical stretch on fibrotic lung slices from rats receiving adenovector-mediated transforming growth factor-β1.We demonstrate that circulating HSP90α is upregulated in IPF patients in correlation with disease severity. The release of HSP90α is enhanced by the increase in mechanical stress of the fibrotic ECM. This increase in extracellular HSP90α signals through low-density lipoprotein receptor-related protein 1 (LRP1) to promote myofibroblast differentiation and persistence. In parallel, we demonstrate that the intracellular form of HSP90β stabilises LRP1, thus amplifying HSP90α extracellular action.We believe that the specific inhibition of extracellular HSP90α is a promising therapeutic strategy to reduce pro-fibrotic signalling in IPF.

Granzyme B (GzmB) is a serine protease with intracellular and extracellular activities capable of regulating inflammation through cytokine processing and the apoptosis of effector cells. We tested the hypothesis that GzmB expression in T regulatory cells (Tregs) is required for the control of inflammatory responses and pathology during acute lung injury. To substantiate the clinical relevance of GzmB during lung injury, we performed GzmB immunohistochemistry on lung tissue from patients with acute respiratory distress syndrome (ARDS) and healthy control subjects. We also performed in vivo experiments with wild-type (WT) C57BL/6 and GzmB(-/-) mice exposed to a single intranasal instillation of bleomycin to model lung injury. Our results demonstrate that the expression of GzmB was elevated in ARDS lung sections, relative to healthy control samples. Bleomycin-exposed GzmB(-/-) mice exhibited greater morbidity and mortality, which was associated with increased numbers of lung lymphocytes. Bleomycin induced an equal increase in CD4(+)/CD25(+)/FoxP3(+) Treg populations in WT and GzmB(-/-) mice. GzmB expression was not significant in Tregs, with the majority of the expression localized to natural killer (NK)-1.1(+) cells. The expression of GzmB in NK cells of bleomycin-exposed WT mice was associated with greater lymphocyte apoptosis, reduced total lymphocyte numbers, and reduced pathology relative to GzmB(-/-) mice. Our data demonstrate that GzmB deficiency results in the exacerbation of lymphocytic inflammation during bleomycin-induced acute lung injury, which is associated with pathology, morbidity, and mortality.

Endoplasmic reticulum (ER) stress is implicated in chronic kidney disease (CKD) development in patients and in animal models. Here we show that ER stress inhibition through 4-phenylbutyric acid (4-PBA) administration decreases blood pressure, albuminuria, and tubular casts in an angiotensin II/deoxycorticosterone acetate/salt murine model of CKD. Lower albuminuria in 4-PBA-treated mice was associated with higher levels of cubilin protein in renal tissue membrane fractions. 4-PBA decreased renal interstitial fibrosis, renal CD3+ T-cell and macrophage infiltration, mRNA expression of TGFβ1, Wnt signaling molecules, and ER stress-induced pro-inflammatory genes. CHOP deficient mice that underwent this model of CKD developed hypertension comparable to wild type mice, but had less albuminuria and tubular casts. CHOP deficiency resulted in higher nephrin levels and decreased glomerulosclerosis compared to wild type mice; this effect was accompanied by lower macrophage infiltration and fibrosis. Our findings portray ER stress inhibition as a means to alleviate hypertensive CKD by preserving glomerular barrier integrity and tubular function. These results demonstrate ER stress modulation as a novel target for preserving renal function in hypertensive CKD.