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ObjectiveCreeping fat, the wrapping of mesenteric fat around the bowel wall, is a typical feature of Crohn’s disease, and is associated with stricture formation and bowel obstruction. How creeping fat forms is unknown, and we interrogated potential mechanisms using novel intestinal tissue and cell interaction systems.DesignTissues from normal, UC, non-strictured and strictured Crohn’s disease intestinal specimens were obtained. The muscularis propria matrisome was determined via proteomics. Mesenteric fat explants, primary human preadipocytes and adipocytes were used in multiple ex vivo and in vitro cell migration systems on muscularis propria muscle cell derived or native extracellular matrix. Functional experiments included integrin characterisation via flow cytometry and their inhibition with specific blocking antibodies and chemicals.ResultsCrohn’s disease muscularis propria cells produced an extracellular matrix scaffold which is in direct spatial and functional contact with the immediately overlaid creeping fat. The scaffold contained multiple proteins, but only fibronectin production was singularly upregulated by transforming growth factor-β1. The muscle cell-derived matrix triggered migration of preadipocytes out of mesenteric fat, fibronectin being the dominant factor responsible for their migration. Blockade of α5β1 on the preadipocyte surface inhibited their migration out of mesenteric fat and on 3D decellularised intestinal tissue extracellular matrix.ConclusionCrohn’s disease creeping fat appears to result from the migration of preadipocytes out of mesenteric fat and differentiation into adipocytes in response to an increased production of fibronectin by activated muscularis propria cells. These new mechanistic insights may lead to novel approaches for prevention of creeping fat-associated stricture formation.

Aim: Pirfenidone is a novel anti-fibrotic agent utilized in the treatment of idiopathic pulmonary fibrosis (IPF). It has been implicated in mitigating myocardial fibrosis and left ventricular (LV) systolic and diastolic dysfunction in animal models. However, its impact on LV mechanics in humans remains unknown. The aim of this study was to retrospectively evaluate the effects of pirfenidone on echocardiographic parameters of LV function and structure in patients with IPF. Methods: A total of 124 patients with IPF were included in this study: 64 patients treated with pirfenidone (treatment group) and 60 patients not taking pirfenidone (control group), who had serial pretreatment/baseline and posttreatment/follow-up echocardiograms done within a time frame of four years. Changes in the means of parameters of LV function (systolic, diastolic, and global longitudinal strain) and LV structure (mass and volume indices) were compared between the treatment and control groups. This was followed by a subgroup analysis that included only 88 patients (47 treated, 41 controls) with echocardiographic evidence of myocardial dysfunction at baseline (defined as an ejection fraction of ≤45, or diastolic dysfunction stage 1 or more) in addition to a known clinical diagnosis of congestive heart failure. To account for potential confounders, a secondary adjusted analysis by way of 1:1 propensity score matching (PSM) was carried out. This yielded a sample consisting of 62 patients with 56 patients in the subgroup cohort. Results: Patients in the treatment group were significantly younger (69.4 vs. 77 years, p Conclusion: From an echocardiographic perspective, pirfenidone had no significant effects on LV structure and function in patients with IPF, even in patients with more overt cardiac dysfunction.

Background The diagnosis of cardiac sarcoidosis (CS) is challenging. Because of the current limitations of endomyocardial biopsy as a reference standard, physicians rely on advanced cardiac imaging, multidisciplinary evaluation, and diagnostic criteria to diagnose CS. Aims To compare the 3 main available diagnostic criteria in patients clinically judged to have CS. Methods We prospectively included patients clinically judged to have CS by a multidisciplinary sarcoidosis team from November 2016 to October 2017. We included only incident cases (diagnosis of CS within 1 year of inclusion). We applied retrospectively the following diagnostic criteria: the World Association of Sarcoidosis and Other Granulomatous Diseases (WASOG), the Heart Rhythm Society (HRS), and the Japanese Circulation Society (JCS) 2016 criteria. Results We identified 69 patients. Diagnostic criteria classified patients as follows: WASOG as highly probable (1.4%), probable (52.2%), possible (0%), some criteria (40.6%), and no criteria (5.8%); HRS as histological diagnosis (1.4%), probable (52.2%), some criteria (40.6%), and no criteria (5.8%); JCS as histological diagnosis (1.4%), clinical diagnosis (58%), some criteria (39.1%), and no criteria (1.4%). Concordance was high between WASOG and HRS (κ = 1) but low between JCS and the others (κ = 0.326). Conclusions A high proportion of patients clinically judged to have CS are unable to be classified according to the 3 main diagnostic criteria. There is low concordance between JCS criteria and the other 2 criteria (WASOG and HRS).

The importance of innate immune cells to sense and respond to their physical environment is becoming increasingly recognized. Innate immune cells (e.g. macrophages and neutrophils) are able to receive mechanical signals through several mechanisms. In this review, we discuss the role of mechanosensitive ion channels, such as Piezo1 and transient receptor potential vanilloid 4 (TRPV4), and cell adhesion molecules, such as integrins, selectins, and cadherins in biology and human disease. Furthermore, we explain that these mechanical stimuli activate intracellular signaling pathways, such as MAPK (p38, JNK), YAP/TAZ, EDN1, NF-kB, and HIF-1α, to induce protein conformation changes and modulate gene expression to drive cellular function. Understanding the mechanisms by which immune cells interpret mechanosensitive information presents potential targets to treat human disease. Important areas of future study in this area include autoimmune, allergic, infectious, and malignant conditions.

Purpose: Nintedanib is an approved treatment for idiopathic pulmonary fibrosis (IPF), which slows disease progression. Management of patients with IPF receiving nintedanib can be complicated by tolerability issues, comorbidities, and concomitant medications. We developed consensus recommendations on the management of dosing, adverse events and comorbidities in patients with IPF treated with nintedanib. Methods: A modified Delphi process using 3 questionnaires was used to survey 14 pulmonologists experienced in using nintedanib. Panelists rated their agreement with statements on a Likert scale from −5 (strongly disagree) to +5 (strongly agree). Consensus was predefined as a mean score of ⩽−2.5 or ⩾+2.5 with a standard deviation not crossing zero. Results: The panelists’ recommendations were largely aligned with clinical trial data, real-world evidence, and the prescribing information, and provided additional guidance regarding minimizing gastrointestinal effects, periodic monitoring for liver dysfunction, caution with respect to concomitant administration of cytochrome P450 3A4 and P-glycoprotein 1 inhibitors and inducers and anticoagulants, and management of comorbidities. The panelists unanimously agreed that adverse event management should be individualized, based on careful consideration of the risks and benefits of each possible intervention and discussion with the patient. Conclusions: These consensus recommendations provide additional guidance on the appropriate management of IPF with nintedanib, for use alongside evidence-based literature and the prescribing information.

Ion channels/pumps are essential regulators of innate immune cell function. Macrophages have been increasingly recognized to have phenotypic plasticity and location-specific functions in the lung. Transient receptor potential vanilloid 4 (TRPV4) function in lung injury has been shown to be stimulus- and cell-type specific. In the current review, we discuss the importance of TRPV4 in macrophages and its role in phagocytosis and cytokine secretion in acute lung injury/acute respiratory distress syndrome (ARDS). Furthermore, TRPV4 controls a MAPK molecular switch from predominately c-Jun N-terminal kinase, JNK activation, to that of p38 activation, that mediates phagocytosis and cytokine secretion in a matrix stiffness-dependent manner. Expanding knowledge regarding the downstream mechanisms by which TRPV4 acts to tailor macrophage function in pulmonary inflammatory diseases will allow for formulation of novel therapeutics.

Analyses of COVID-19 patients with preexisting interstitial lung disease (ILD) or pulmonary sarcoidosis is lacking but registries are ongoing. Treatment of COVID-19 in patients with underlying ILD or sarcoidosis may include hospital admission, possible drug treatment, caution with steroids, and avoidance of mechanical ventilation in acute exacerbation of ILD. Patients with COVID-19 respiratory illness are at risk for developing ILD.

Mechanical cell–matrix interactions can drive the innate immune responses to infection; however, the molecular underpinnings of these responses remain elusive. This study was undertaken to understand the molecular mechanism by which the mechanosensitive cation channel, transient receptor potential vanilloid 4 (TRPV4), alters the in vivo response to lung infection. For the first time, to our knowledge, we show that TRPV4 protects the lung from injury upon intratracheal Pseudomonas aeruginosa in mice. TRPV4 functions to enhance macrophage bacterial clearance and downregulate proinflammatory cytokine secretion. TRPV4 mediates these effects through a novel mechanism of molecular switching of LPS signaling from predominant activation of the MAPK, JNK, to that of p38. This is accomplished through the activation of the master regulator of inflammation, dual-specificity phosphatase 1. Further, TRPV4’s modulation of the LPS signal is mechanosensitive in that both upstream activation of p38 and its downstream biological consequences depend on pathophysiological range extracellular matrix stiffness. We further show the importance of TRPV4 on LPS-induced activation of macrophages from healthy human controls. These data are the first, to our knowledge, to demonstrate new roles for macrophage TRPV4 in regulating innate immunity in a mechanosensitive manner through the modulation of dual-specificity phosphatase 1 expression to mediate MAPK activation switching.

Myofibroblasts are key contributors to pathological fibrotic conditions of several major organs. The transdifferentiation of fibroblasts into myofibroblasts requires both a mechanical signal and transforming growth factor-β (TGF-β) signaling. The cation channel transient receptor potential vanilloid 4 (TRPV4) is a critical mediator of myofibroblast transdifferentiation and in vivo fibrosis through its mechanosensitivity to extracellular matrix stiffness. Here, we showed that TRPV4 promoted the transdifferentiation of human and mouse lung fibroblasts through its interaction with phosphoinositide 3-kinase γ (PI3Kγ), forming nanomolar-affinity, intracellular TRPV4-PI3Kγ complexes. TGF-β induced the recruitment of TRPV4-PI3Kγ complexes to the plasma membrane and increased the activities of both TRPV4 and PI3Kγ. Using gain- and loss-of-function approaches, we showed that both TRPV4 and PI3Kγ were required for myofibroblast transdifferentiation as assessed by the increased production of α-smooth muscle actin and its incorporation into stress fibers, cytoskeletal changes, collagen-1 production, and contractile force. Expression of various mutant forms of the PI3Kγ catalytic subunit (p110γ) in cells lacking PI3Kγ revealed that only the noncatalytic, amino-terminal domain of p110γ was necessary and sufficient for TGF-β-induced TRPV4 plasma membrane recruitment and myofibroblast transdifferentiation. These data suggest that TGF-β stimulates a noncanonical scaffolding action of PI3Kγ, which recruits TRPV4-PI3Kγ complexes to the plasma membrane, thereby increasing myofibroblast transdifferentiation. Given that both TRPV4 and PI3Kγ have pleiotropic actions, targeting the interaction between them could provide a specific therapeutic approach for inhibiting myofibroblast transdifferentiation.

As long-term smokers undergo computed tomography (CT) to screen for lung cancer, cases of interstitial lung disease are being discovered incidentally. This article explains how to distinguish among the most common forms of interstitial lung disease in this situation and the role of primary care physicians in managing them.

Background To date, there remains to be a lack of effective treatment modalities for HFpEF, which accounts for approximately 50% of all cases of congestive heart failure. Pirfenidone is a novel anti-fibrotic agent that has been shown to improve pulmonary function in patients with Idiopathic Pulmonary Fibrosis. In pre-clinical studies, pirfenidone has been implicated in decreasing myocardial fibrosis and diastolic dysfunction (Kuwahara et al, Circulation 2002). The effects of this medication on markers of LV function and structure in humans with a history of IPF and HFpEF has not been studied. Hypothesis We hypothesize that patients taking pirfenidone will have favorable changes in echocardiographic parameters of LV function and structure. Methodology We performed a retrospective review of 24 patients with a history of HFpEF and IPF being treated with pirfenidone. All patients had pre and post treatment echocardiograms. Changes in parameters of LV structure (indexed LV mass, end diastolic and end systolic volumes), diastolic function (indexed left atrial volume, trans-mitral Doppler flow patterns, tissue Doppler indices), systolic function (EF) and global LV strain were compared before and after pirfenidone administration. Results mean age of patients involved was 70±6.8 years and patients were predominantly males(76%). 34% patients had a history of CAD, 62% HTN, 16% Atrial fibrillation, 61% hyperlipidemia, 23% diabetes, 25% CKD and 46% obesity. 88% of patients had stage 1 diastolic dysfunction at baseline and 12% with stage 2 diastolic dysfunction. Mean NT pro BNP was 2287±3444 pg/ml . Post pirfenidone treatment, there was a significant reduction in indexed LV end diastolic(-11.9±1.1ml/m2) and end systolic (-6.2±2.3ml/m2) volumes. No significant changes were noted in other parameters. Conclusion Treatment with pirfenidone was associated with decreases in indexed LV end diastolic and end systolic volumes in patients with HFpEF and a known history of IPF. However, contrary to the rest of the hypothesis, no improvements were noted in markers of LV diastolic, systolic function and strain. Our study was retrospective in nature and limited by its small sample size.

Mechanical cell-matrix interactions can drive the innate immune responses to infection, however the molecular underpinnings of these responses remain elusive. We have discovered that the biophysical properties of the matrix in the range of injured fibrotic lung (≥ 25 kPa) conditions the macrophage response to LPS and infection respectively through the mechanosensitive cation channel, TRPV4 in vitro and in vivo. Studies suggest that LPS-induced macrophage activation is controlled in part by the MAPK pathway (i.e. p38, ERK, JNK). Thus, we investigated if TRPV4 plays a role in the macrophage activation response after LPS through alteration of the MAPK pathway. TRPV4 KO mice exhibited reduced lung bacterial clearance by macrophages (6-fold, p=0.012) after intratracheal P. aeruginosa administration and increased lung injury as measured by inflammatory cell infiltration (≥80±3%, p

Idiopathic pulmonary fibrosis (IPF) is a specific type of fibrosing interstitial pneumonia of unknown cause. It is usually chronic and progressive, tends to affect mainly adults over age 60, has a predilection for men, and is often fatal. The condition is still underappreciated by pulmonologists and primary care physicians. This article attempts to close that information gap by reviewing the natural course of IPF and presenting an algorithmic approach to diagnosis and treatment based on evidence-based international guidelines. New treatment options are briefly discussed, to raise awareness of new medications that target pulmonary fibrosis.

Ion channels/pumps are essential regulators of organ homeostasis and disease. In the present review, we discuss the role of the mechanosensitive cation channel, transient receptor potential vanilloid 4 (TRPV4), in cytokine secretion and pulmonary inflammatory diseases such as asthma, cystic fibrosis (CF), and acute lung injury/acute respiratory distress syndrome (ARDS). TRPV4 has been shown to play a role in lung diseases associated with lung parenchymal stretch or stiffness. TRPV4 indirectly mediates hypotonicity-induced smooth muscle contraction and airway remodeling in asthma. Further, the literature suggests that in CF TRPV4 may improve ciliary beat frequency enhancing mucociliary clearance, while at the same time increasing pro-inflammatory cytokine secretion/lung tissue injury. Currently it is understood that the role of TRPV4 in immune cell function and associated lung tissue injury/ARDS may depend on the injury stimulus. Uncovering the downstream mechanisms of TRPV4 action in pulmonary inflammatory diseases is likely important to understanding disease pathogenesis and may lead to novel therapeutics.

Idiopathic pulmonary fibrosis (IPF) is a fatal fibrotic lung disorder with no effective medical treatments available. The generation of myofibroblasts, which are critical for fibrogenesis, requires both a mechanical signal and activated TGF-β; however, it is not clear how fibroblasts sense and transmit the mechanical signal(s) that promote differentiation into myofibroblasts. As transient receptor potential vanilloid 4 (TRPV4) channels are activated in response to changes in plasma membrane stretch/matrix stiffness, we investigated whether TRPV4 contributes to generation of myofibroblasts and/or experimental lung fibrosis. We determined that TRPV4 activity is upregulated in lung fibroblasts derived from patients with IPF. Moreover, TRPV4-deficient mice were protected from fibrosis. Furthermore, genetic ablation or pharmacological inhibition of TRPV4 function abrogated myofibroblast differentiation, which was restored by TRPV4 reintroduction. TRPV4 channel activity was elevated when cells were plated on matrices of increasing stiffness or on fibrotic lung tissue, and matrix stiffness-dependent myofibroblast differentiation was reduced in response to TRVP4 inhibition. TRPV4 activity modulated TGF-β1-dependent actions in a SMAD-independent manner, enhanced actomyosin remodeling, and increased nuclear translocation of the α-SMA transcription coactivator (MRTF-A). Together, these data indicate that TRPV4 activity mediates pulmonary fibrogenesis and suggest that manipulation of TRPV4 channel activity has potential as a therapeutic approach for fibrotic diseases.

The urokinase-type plasminogen activator receptor (uPAR) is a glycosylphosphatidylinositol-linked membrane protein with no cytosolic domain that localizes to lipid raft microdomains. Our laboratory and others have documented that lung fibroblasts from patients with idiopathic pulmonary fibrosis (IPF) exhibit a hypermotile phenotype. This study was undertaken to elucidate the molecular mechanism whereby uPAR ligation with its cognate ligand, urokinase, induces a motile phenotype in human lung fibroblasts. We found that uPAR ligation with the urokinase receptor binding domain (amino-terminal fragment) leads to enhanced migration of fibroblasts on fibronectin in a protease-independent, lipid raft-dependent manner. Ligation of uPAR with the amino-terminal fragment recruited α5β1 integrin and the acylated form of the Src family kinase, Fyn, to lipid rafts. The biological consequences of this translocation were an increase in fibroblast motility and a switch of the integrin-initiated signal pathway for migration away from the lipid raft-independent focal adhesion kinase pathway and toward a lipid raft-dependent caveolin-Fyn-Shc pathway. Furthermore, an integrin homologous peptide as well as an antibody that competes with β1 for uPAR binding have the ability to block this effect. In addition, its relative insensitivity to cholesterol depletion suggests that the interactions of α5β1 integrin and uPAR drive the translocation of α5β1 integrin-acylated Fyn signaling complexes into lipid rafts upon uPAR ligation through protein-protein interactions. This signal switch is a novel pathway leading to the hypermotile phenotype of IPF patient-derived fibroblasts, seen with uPAR ligation. This uPAR dependent, fibrotic matrix-selective, and profibrotic fibroblast phenotype may be amenable to targeted therapeutics designed to ameliorate IPF.

Background Pirfenidone, an FDA-approved novel anti-fibrotic agent used in the treatment of idiopathic pulmonary fibrosis (IPF), has been implicated in mitigating myocardial fibrosis, systolic and diastolic dysfunction in animal models (Kuwahara et al, Circulation 2002). However to date, the effect of Pirfenidone on left ventricular (LV) structure and function in humans with IPF has not been studied. Hypothesis We hypothesize that changes in echocardiographic parameters of LV structure and function would be more favorable in responders compared to non-responders of pirfenidone in patients with IPF. Methods We performed a retrospective review of 27 consecutive patients with IPF on pirfenidone between 2014-2018 with serial baseline and follow up echocardiograms and pulmonary function testing. Treatment failure (non-response) was defined as an absolute decline in forced vital capacity (FVC) of more than 10% whilst being on the medication. Comprehensive echocardiographic data extraction and analysis was performed in all patients. Changes in parameters of LV structure (LV mass, interventricular septal and posterior wall thickness, indexed end systolic and diastolic volumes), systolic function (LVEF), diastolic function (Indexed left atrial volume, trans-mitral Doppler flow patterns, tissue Doppler indices) and global LV longitudinal strain were compared between responders and non-responders. Results In our study cohort, 18 responders and 9 non-responders were identified. Responders were significantly older than non-responders, yet sex, history of CAD, hypertension, diabetes, atrial fibrillation, CKD and cardiac medication use were not significantly different. Baseline echocardiographic parameters were comparable, except for septal e/e’, which was higher in responders (11.68 vs 10.1; p Conclusion In contrary to our hypothesis, we observed that response to pirfenidone therapy in patients with IPF did not associate with echocardiographic changes of left ventricle structure and function.

Obesity is characterized by chronic activation of the innate immune system. Leptin is an adipokine upregulated in obesity that has been shown to stimulate macrophage function through an unidentified signal. Our lab recently showed that the mechanosensitive calcium channel, transient receptor potential vanilloid 4 (TRPV4) mediates macrophage phagocytosis in response to infection. This study seeks to determine if TRPV4 also mediates the macrophage response to leptin. Bone marrow derived macrophages were isolated from 7–10 week old WT and TRPV4 KO mice and incubated in the presence of pathophysiological range leptin concentrations (0.1–1μg/mL; 24 hr) ± LPS (E coli 0111:B4 as a positive control) ± TRPV4 inhibitor. Macrophage phagocytosis was measured as uptake of fluorescently-labeled E coli particles. Intracellular calcium was measured using the FLIPR Calcium 5 Assay in the presence of a selective TRPV4 agonist (GSK). MIP-2 and IL-8 secretion was measured by ELISA. Leptin increases phagocytosis of E coli particles by 17% (59% of the LPS response) and TRPV4 activity by 24% (51% of the LPS response). The phagocytic response and calcium influx are abrogated by either pharmacologic inhibition or deletion of TRPV4. Secretion of MIP-2 and IL-8 in response to leptin increases by 8-fold after pharmacologic inhibition of TRPV4. Our data show that TRPV4 mediates macrophage phagocytosis of E coli particles, and suppresses proinflammatory cytokine release, in response to leptin. In addition, we show that TRPV4 function is increased by leptin. In vitro results predict that TRPV4 enhances host defense and suppresses inflammation in the setting of obesity-induced hyperleptinemia, thus potentially modulating pneumonia severity in obese patients.

Pro-fibrotic mesenchymal cells are known to be the key effector cells of fibroproliferative disease, but the specific matrix signals and the induced cellular responses that drive the fibrogenic phenotype remain to be elucidated. The key mediators of the fibroblast fibrogenic phenotype were characterized using a novel assay system that measures fibroblast behavior in response to actual normal and fibrotic lung tissue. Using this system, we demonstrate that normal lung promotes fibroblast motility and polarization, while fibrotic lung immobilizes the fibroblast and promotes myofibroblast differentiation. These context-specific phenotypes are surprisingly both mediated by myosin II. The role of myosin II is supported by the observation of an increase in myosin phosphorylation and a change in intracellular distribution in fibroblasts on fibrotic lung, as compared with normal lung. Moreover, loss of myosin II activity has opposing effects on protrusive activity in fibroblasts on normal and fibrotic lung. Loss of myosin II also selectively inhibits myofibroblast differentiation in fibroblasts on fibrotic lung. Importantly, these findings are recapitulated by varying the matrix stiffness of polyacrylamide gels in the range of normal and fibrotic lung tissue. Comparison of the effects of myosin inhibition on lung tissue with that of polyacrylamide gels suggests that matrix fiber organization drives the fibroblast phenotype under conditions of normal/soft lung, while matrix stiffness drives the phenotype under conditions of fibrotic/stiff lung. This work defines novel roles for myosin II as a key regulatory effector molecule of the pro-fibrotic phenotype, in response to biophysical properties of the matrix.

Macrophage phagocytosis and cytokine production are sensitive to the surrounding matrix and thereby mediate host defense and lung tissue injury. The consequences and mechanism of this matrix sensitivity are unknown. We have determined that the mechanosensitive channel, TRPV4, responds to extracellular matrix biophysical properties and thereby modulates the macrophage response to pathogens. We undertook this study to determine the in vivo consequences and the intracellular signaling pathway by which TRPV4 modulates macrophage responses. In order to evaluate the role of TRPV4 in chronic pneumonia/lung injury, WT and TRPV4 KO mice were administered agarose bead embedded-Pseudomonas aeruginosa. Loss of TRPV4 led to decreased phagocytosis by macrophages (6-fold), and increased lung injury as measured by inflammatory cell infiltration (≥ 80 ± 3%), vascular permeability (total protein ≥ 63 ± 6%), and cytokine secretion (IL-1β ≥ 71 ± 4%). In vivo, lung alveolar macrophages predominantly expressed TRPV4 and were the key phagocytic cell, as assessed by FACS and immunofluorescence. Known LPS signaling pathways were investigated in vitro. Loss of TRPV4 abrogates LPS-induced p38 activation and phagocytosis of E. coli particles. Additionally, loss of TRPV4 increased basal pro-/active IL-1β expression (2-fold), thereby enhancing IL-1β secretion independent of caspase 1 cleavage and blockade. These findings demonstrate that TRPV4 is important for bacterial clearance, lung injury and cytokine production in macrophages. TRPV4 mediates these effects through p38 and through upregulation of IL-1β protein expression in an inflammasome-independent manner. The results implicate macrophage TRPV4 as a key component of the host defense.

Macrophage phagocytosis of particles and pathogens is an essential aspect of innate host defense. Phagocytic function requires cytoskeletal rearrangements that depend on the interaction between macrophage surface receptors, particulates/pathogens, and the extracellular matrix. In the present study we determine the role of a mechanosensitive ion channel, transient receptor potential vanilloid 4 (TRPV4), in integrating the LPS and matrix stiffness signals to control macrophage phenotypic change for host defense and resolution from lung injury. We demonstrate that active TRPV4 mediates LPS-stimulated murine macrophage phagocytosis of nonopsonized particles (Escherichia coli) in vitro and opsonized particles (IgG-coated latex beads) in vitro and in vivo in intact mice. Intriguingly, matrix stiffness in the range seen in inflamed or fibrotic lung is required to sensitize the TRPV4 channel to mediate the LPS-induced increment in macrophage phagocytosis. Furthermore, TRPV4 is required for the LPS induction of anti-inflammatory/proresolution cytokines. These findings suggest that signaling through TRPV4, triggered by changes in extracellular matrix stiffness, cooperates with LPS-induced signals to mediate macrophage phagocytic function and lung injury resolution. These mechanisms are likely to be important in regulating macrophage function in the context of pulmonary infection and fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a fatal fibrotic lung disorder with marginally effective medical treatment. Myofibroblasts are critical to the fibrogenic lung repair process; however, the mechanism whereby the mechanical signal that leads to myofibroblast generation is sensed and transmitted remains to be determined. As transient receptor potential vanilloid 4 (TRPV4) ion channels are activated by plasma membrane stretch/matrix stiffness, we investigated whether TRPV4 plays a role in myofibroblast differentiation and/or in vivo lung fibrosis. TRPV4 inhibition by small-molecule inhibitors almost completely abrogated both the calcium influx response to TPRV4 agonist in a dose-dependent manner and the myofibroblast differentiation response to transforming growth factor-β. Similar findings were noted on TRPV4 deletion with small interfering RNA or in TRPV4 KO murine lung fibroblasts. Further, TRPV4 exhibited its mechanosensing and myofibroblast-differentiating effect under conditions of matrix stiffness in the pathophysiological range (1–25 kPa). Mechanistically, TRPV4 activity modulates transforming growth factor β actions in a Smad-independent manner, in part through enhanced actomyosin remodeling with resultant nuclear translocation of the α–smooth muscle actin transcription coactivator (myocardin-related transcription factor-A). The myofibroblast differentiation response to actual fibrotic lung tissue was also completely inhibited on TRPV4 blockade. Furthermore, TRPV4 deficiency protects mice from fibrosis-inducing effects of bleomycin (1 or 4 U/kg dose) at the biochemical (75% less hydroxyproline; P < 0.05), histologic, and physiologic levels (57% less impairment of compliance; P < 0.05). These data identify TRPV4 as a long-elusive mechanosensor/transducer that mediates myofibroblast differentiation and in vivo pulmonary fibrogenesis. Successful manipulation of TRPV4 channel activity may be a novel therapeutic approach for fibrotic diseases of the lung and other organs.

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease whose underlying molecular mechanisms are largely unknown. Herein, we show that focal adhesion kinase–related nonkinase (FRNK) plays a key role in limiting the development of lung fibrosis. Loss of FRNK function in vivo leads to increased lung fibrosis in an experimental mouse model. The increase in lung fibrosis is confirmed at the histological, biochemical, and physiological levels. Concordantly, loss of FRNK function results in increased fibroblast migration and myofibroblast differentiation and activation of signaling proteins that drive these phenotypes. FRNK-deficient murine lung fibroblasts also have an increased capacity to produce and contract matrix proteins. Restoration of FRNK expression in vivo and in vitro reverses these profibrotic phenotypes. These data demonstrate the multiple antifibrotic actions of FRNK. More important, FRNK expression is down-regulated in human IPF, and down-regulation of FRNK in normal human lung fibroblasts recapitulates the profibrotic phenotype seen in FRNK-deficient cells. The effect of loss and gain of FRNK in the experimental model, when taken together with its down-regulation in human IPF, suggests that FRNK acts as an endogenous negative regulator of lung fibrosis by repressing multiple profibrotic responses.