Background The current classification of pulmonary hypertension (PH), based largely on expert opinion, has limitations in prognostication and guiding therapies. We hypothesize that novel PH clusters that predict survival will reveal mechanistic phenotypes associated with biomarkers of vascular health across all PH groups. Methods We first identify novel PH clinical clusters by performing unsupervised clustering analysis on the CC‐PH (Cleveland Clinic PH) registry (N=1529). We develop classification models to predict the new PH clusters and then apply them to the multicenter PVDOMICS (Pulmonary Vascular Diseases Phenomics) cohort (N=853) for validation. We compare transplantation‐free survival across the new PH clusters. We quantify metabolites of the arginine‐nitric oxide pathway and D‐dimer levels and calculate global arginine bioavailability (arginine/[ornithine+citrulline]) to assess endothelial function and activation in the new clusters and link these biomarkers to clinical outcomes. Results Clustering analysis identify 3 clear clusters in CC‐PH that are validated in PVDOMICS and outperform conventional classifications in predicting transplantation‐free survival. The phenotype associated with the worst survival is characterized by reduced lung diffusion capacity, decreased arginine bioavailability and nitrate levels, and elevated D‐dimer levels, consistent with loss of pulmonary microcirculation and endothelial dysfunction. Conclusions We identify new informative PH phenotypes associated with mortality and defined by biomarkers of endothelial function and activation. Loss of endothelial health and pronounced pulmonary vascular rarefication contribute more substantially to mortality across the spectrum of PH than right heart function. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique identifier: NCT02980887.

Emerging evidence suggests that macrophage-fibroblast interactions can drive organ fibrosis. Myofibroblast differentiation is a key step in the pathogenesis of pulmonary fibrosis that requires both a soluble (e.g., TGF-β) and mechanical signal. We have previously implicated the fibroblast mechanosensitive cation channel, transient receptor potential vanilloid 4 (TRPV4), as a mediator of myofibroblast differentiation and experimental pulmonary fibrogenesis in response to matrix biophysical signals. Less is understood regarding how or if the matrix drives macrophage activation to mediate fibrosis. We demonstrate that loss of TRPV4 specifically in myeloid cells protects against experimental pulmonary fibrosis in vivo. Mechanistically, macrophage TRPV4 responds to matrix substrate stiffness in the pathophysiologic range, thereby optimizing TGF-β activation. Macrophage conditioned media transfer and coculture systems demonstrate a profound effect of TRPV4-dependent TGF-β activation in inducing myofibroblast differentiation in fibroblasts. This TGF-β activating effect was dependent on the actinomyosin binding domain within the C-terminal intracytoplasmic tail of TRPV4 and on assembly of actinomyosin cytoskeleton and its force generation. Our current study identifies a novel TRPV4-TGF-β axis in macrophages that drives myofibroblast differentiation and experimental pulmonary fibrosis through optimal activation of TGF-β. As TGF-β is a critical pro-fibrotic factor, these findings are broadly applicable to many fibrotic diseases.

Respiratory pathogens, such as Pseudomonas aeruginosa damage the alveolar-capillary barrier leading to lung injury and stiffness. Lung stiffness is a key macrophage signal for bacterial clearance, but it remains unknown how stiffness-sensing mechanosensitive ion channels in macrophages are regulated during pneumonia. Macrophage Piezo1 is critical to bacterial clearance in experimental pneumonia in vivo; however, identification of putative matrix-derived signals and the mechanism of their effects remain to be determined. To our knowledge, our work is the first to show that during pneumonia, transcription of the mechanosensitive ion channel Piezo1 is increased in macrophages by the NF-κB transcription factor, p65, through its signaling adaptor protein, MyD88, leading to increased Piezo1 Ca2+ channel activity. Piezo1 mRNA abundance is increased in association with open chromatin at the Piezo1 promoter in macrophages. The enhanced level of Piezo1 increases the abundance of transcription factor EB (Tfeb) resulting in lysosome biogenesis and stiffness-dependent phagolysosome maturation, a critical step for macrophage bacterial clearance. Our data support the mechanism whereby transcription of macrophage Piezo1 is enhanced by p65 to augment bacterial clearance on an injured, stiffened lung matrix during pneumonia. Therefore, Piezo1 is a future therapeutic target against pneumonia-induced lung injury.

RATIONALE: Pulmonary arterial microthrombi are well documented in post-mortem analyses of patients across all etiologic forms of pulmonary hypertension (PH). However, it remains unclear whether specific PH patients are disproportionately affected by microthrombi and therefore represent a distinct hypercoagulable endotype. This study was undertaken to determine if elevated levels of fibrin D-dimer can identify a hypercoagulable endotype, and thereby support further refinement of anticoagulant therapy indications within PH. METHODS: Data from the Pulmonary Vascular Disease Phenomics (PVDomics) study were analyzed. Fibrin D-dimer levels were measured by enzyme-linked immunosorbent assay (ELISA) on venous blood samples (n = 808) from patients with documented pulmonary hypertension, disease comparators, and healthy controls. Non-parametric statistical analyses tested for differences in D-dimer levels across PH groups (groups 1-4), comparators, and healthy controls. Spearman's rank correlation evaluated associations between D-dimer and N-terminal pro-B-type natriuretic peptide (NT-proBNP) and 6-minute walk distance (6-MWD). Data are reported as median, interquartile range [IQR] (25%-75%), and considered significant at p ≤ 0.05. RESULTS: D-dimer levels were 44% higher in all PH patients compared to healthy controls (median 396.4 [IQR 445] vs 273.9 [IQR 192.5] ng/mL, p = 0.01). D-dimer levels in PH groups 2 and 3 were the highest, relative to both disease comparators (Grp 2, by 44%; p<0.001; Grp 3, by 12%; p <0.01) and relative to PH groups 1 and 4 combined (Grp 2, by 60%, p < 0.001; Grp 3, by 42%, p < 0.001). D-dimer levels in Groups 2 and 3 remained disproportionately elevated even after the exclusion of patients with mixed group PH. D-dimer was positively correlated with NT-proBNP in PH groups 2 (r = 0.2, p-value = 0.04) and 3 (r = 0.39, p-value < 0.001), and negatively correlated with 6-MWD in groups 2 (r = -0.23, p-value = 0.017) and 3 (r = -0.34, p-value < 0.001. D-dimer levels for patients in PH groups 2 and 3 that were on anticoagulants were indistinguishable from those not on anticoagulants. CONCLUSIONS: All PH patients have evidence of over-exuberant thrombosis/fibrinolysis, regardless of etiology. The correlation of higher D-dimer levels in groups 2 and 3 with key markers of disease severity supports the need for further investigation into the role of D-dimer, and of multi-omics based analyses in defining a novel hypercoagulable endotype. Elucidating a novel hypercoagulable endotype would allow for further refinement of anticoagulant indications in PH.

RATIONALE: Pulmonary macrophages are important for clearing bacterial infections but can induce lung tissue injury through pro-inflammatory cytokine secretion, leading to acute respiratory distress syndrome (ARDS). We have previously reported that the macrophage inflammatory response to pathogens depends on a soluble and mechanical signal. We undertook this study to define the intracellular mechanism whereby the mechanosensitive cation channel, Transient receptor potential vanilloid 4 (TRPV4), regulates the macrophage pro-inflammatory response to pathogens. METHODS: The lung injury response was assessed by bronchoalveolar lavage fluid inflammatory cell infiltration and macrophage phagocytosis in control (Trpv4fl/fl) and myeloid-specific Trpv4-deficient mice (Trpv4LysMcre) after intratracheal instillation of Pseudomonas aeruginosa. Cytokine, mRNA and protein levels were analyzed by qPCR and ELISA, in BMDMs and alveolar macrophages from WT/Trpv4(-/-)(Trpv4 knockout) mice and healthy/ARDS patient tissue with ±TLR agonism, ±TRPV4 inhibitor, and ±NF-ĸB/p65-knockdown. Co-immunoprecipitation of TRPV4 and NF-ĸB/p65 proteins were performed in 293T, Hela, and BMDMs using expression/deletion/overexpression systems. Co-localization of TRPV4 with p65 was assessed via bimolecular fluorescence complementation assay (BiFC). A NF-ĸB luciferase reporter assay evaluated the transcriptional activity of the NF-ĸB pathway. Structural predictions of protein-protein interactions were performed using AlphaFold and confirmed using Nanoluc Binary Technology (NanoBiT) assays in 293T cells. RESULTS:Trpv4LysMcre mice recapitulated the lung response to P. aeruginosa seen previously in global Trpv4(-/-) mice, including impaired phagocytosis and increased lung injury compared with Trpv4fl/fl. Trpv4(-/-)BMDMs revealed an enrichment of pro-inflammatory genes compared to WT BMDMs ± TLR1, 2 and 4 agonism, suggesting broad TRPV4-dependent inhibition of the NF-κB pathway. Conversely, overexpression of TRPV4 suppressed NF-κB promoter transcriptional activity. Functional loss of TRPV4 (pharmacologic inhibition or genetic deletion) enhanced IL-1β secretion in murine and human ARDS-derived macrophages, this was alleviated by knockdown of the NF-ĸB pathway (p65 siRNA). TRPV4 binding to p65 limits its translocation to the nucleus, paralleling canonical NF-ĸB inhibitors (e.g. IκBα/β). Stimulation with LPS yields NF-κB translocation to the nucleus and TRPV4 translocation from the endoplasmic reticulum to the plasma membrane. In silico modeling implicates TRPV4's Ankyrin repeat (AR) domain as the site of NF-κB interaction. NanoBiT experiments confirm that TRPV4 AR domain is critical for its binding to NF-ĸB/p65, with TRPV4 AR domain-deleted constructs exhibiting a significant loss of binding in vitro. CONCLUSIONS: Collectively, these data point to a critical interaction between TRPV4 and p65 that regulates NF-ĸB signaling, and thereby identifying a novel target for therapeutics in ARDS.

Rationale: Bacteria, such as P. aeruginosa, cause lung injury and stiffening through their virulence factors (i.e. flagellin) during pneumonia, leading to respiratory failure and death. Our group has identified that mechanosensitive ion channels (e.g. TRPV4, Piezo1) integrate the bacterial and stiffness signals leading to effective bacterial clearance. However, regulation of macrophage mechanosensitive ion channel expression and function in response to pneumonia is poorly understood. Piezo1 is a macrophage mechanosensitive ion channel which increases Ca2+ influx upon mechanical stiffness. Therefore, we wondered how Piezo1 is regulated during pneumonia and its role in augmenting macrophage stiffness sensing and Ca2+-dependent bacterial clearance. Methods: Bone marrow-derived macrophages (BMDMs) from C57BL/6J (WT) mice were treated ± P. aeruginosa flagellin. Piezo1 mRNA transcription was measured using qPCR. Binding of the NF-κB transcription factor, p65, to the Piezo1 enhancer region was measured using chromatin immunoprecipitation. Piezo1 Ca2+ channel activity in response to agonist, Yoda1, was measured on pathophysiologic-range lung stiffnesses (1-25 kPa) using the FLIPR 5 Ca2+ assay. Phagolysosome maturation, a key step in bacterial clearance, was measured using pHrodo bioparticles. The role of Piezo1 was evaluated in vivo after pneumonia was induced by intratracheal administration of a clinical strain, P. aeruginosa (PAM 57-15) in WT mice ± the Piezo1 inhibitor, GsMTx4. To extend our findings to humans, phagolysosome maturation was measured in response to P. aeruginosa flagellin in human alveolar macrophages isolated from healthy human donor lungs. Results: Flagellin increased Piezo1 mRNA levels by > 20-fold in association with increased binding of p65 to the Piezo1 enhancer region by 1.8-fold in WT BMDMs. Piezo1 surface expression increased by 25% and Piezo1 Ca2+ channel activity increased by 10-fold, as evidenced by a shift in the Yoda1 EC50 from 10 μM to 1 μM, upon flagellin stimulation. Consistent with its role as a macrophage mechanosensor, Piezo1 Ca2+ channel activity required matrix stiffness of ≥ 25 kPa, as seen in infected lung. Piezo1 activation increased flagellin-induced phagolysosome maturation > 2-fold, demonstrating the importance of Piezo1 for bacterial clearance. Piezo1 reduced inflammatory cell infiltration and enhanced bacterial clearance in the lungs during P. aeruginosa pneumonia. Finally, Piezo1 activation in human alveolar macrophages increased flagellin-induced phagolysosome maturation by 3-fold. Conclusion: NF-κB upregulates Piezo1 upon P. aeruginosa flagellin. Piezo1 augments intracellular Ca2+ and phagolysosome maturation in response to increased lung matrix stiffness during pneumonia. These data support targeting macrophage Piezo1 as a potential therapy for pneumonia-induced lung injury.

RATIONALE: Burkholderia cenocepacia is an intracellular pathogen that can cause a fatal, rapid onset of necrotizing pneumonia in patients with cystic fibrosis (CF). B. cenocepacia utilizes a combination of virulence factors to evade key innate immune effector cells such as lung macrophages. Autophagy is a specialized form of intracellular phagocytosis and is a primary mechanism macrophages use to clear B. cenocepacia. We have previously reported that the macrophage transient receptor potential vanilloid 4 (TRPV4), a mechanosensitive cation channel, is required for bacterial clearance of an extracellular CF pathogen, Pseudomonas aeruginosa. However, TRPV4's role in the clearance of the intracellular pathogen B. cenocepacia has not been studied. Here, we investigate the role of TRPV4 in autophagy to control clearance of B. cenocepacia in macrophages in vitro and in vivo. METHODS: To assess the role of TRPV4 in B. cenocepacia pneumonia in vivo, we intranasally inoculated WT C57BL/6 and Trpv4-/- mice with ∼108 CFU of B. cenocepacia (J2315). Lung homogenate bacterial burden and bronchoalveolar lavage (BAL) cell differential were assessed. To examine macrophage-specific mechanisms of B. cenocepacia clearance, we incubated B. cenocepacia with bone-marrow derived macrophages (BMDMs) from WT ± TRPV4 inhibitor (HC-067047) and Trpv4-/- mice. To investigate the role of TRPV4 in autophagy, expression of endogenous autophagy-initiating proteins (Beclin1, ATG12-ATG5, ATG16L), autophagosome-components (LC3BI/II), and a cargo marker (p62) were measured. Immunofluorescence microscopy of LC3BI/II and p62 in WT ± TRPV4 inhibitor (HC), Trpv4-/- BMDMs, and of LC3B-reporter RAW macrophages ± TRPV4 siRNA was used to measure autophagosome formation. RESULTS: WT mice had a 1.4x log10 reduction in lung bacteria compared to Trpv4-/- mice after intranasal infection with B. cenocepacia. Cell differentials from BAL were not significantly different between genotypes, implying bacteria burden is due to cell function rather than cell number. Trpv4-/- BMDMs had a 5x increase in intracellular B. cenocepacia compared with WT, indicating either enhanced uptake or decreased clearance. Trpv4-/- BMDMs also had defective autophagosome formation, as shown by a decrease in autophagy-initiating proteins (i.e., ATG12-ATG5, ATG16L, Beclin1) and autophagosome-components (i.e., LC3BI/II). Furthermore, p62 was increased in Trpv4-/- BMDMs, suggesting defective cargo degradation. We additionally confirmed with reporter macrophages that TRPV4-downregulation inhibited autophagy. CONCLUSIONS: These data indicate that TRPV4 is required for clearance of B. cenocepacia through autophagy in vitro and in vivo. Thus, TRPV4 is a potential therapeutic target for improving bacterial clearance in pneumonia.

The nuclear factor κB (NF-κB) signaling pathway plays a critical role in activating macrophages in the pathogenesis of many inflammatory diseases. Tissue mechanical properties are important in modulating key cellular proinflammatory responses. Here, we investigated how the mechanosensitive membrane cation channel TRPV4 (transient receptor potential vanilloid 4) limits macrophage proinflammatory responses in bacterial pneumonia. We found that TRPV4 suppressed proinflammatory gene expression in alveolar macrophages in response to Pseudomonas aeruginosa pneumonia in mice and in response to agonists of various Toll-like receptors (TLRs) in vitro. TRPV4 suppressed proinflammatory gene expression in macrophages by decreasing the activity of the NF-κB subunit p65. Upon stimulation of macrophages with bacterial lipopolysaccharide, a fraction of TRPV4 translocated from the endoplasmic reticulum to the plasma membrane, releasing p65 for nuclear translocation. TRPV4 interacted with p65 through an N-terminal cytoplasmic ankyrin repeat domain (ANKRD) that shares sequence homology with the p65-binding ANKRD of the NF-κB inhibitor IκBα. Given the diverse roles of TRPV4 and NF-κB in various cell types, our identification of cross-talk between a mechanosensitive channel and p65 in macrophages suggests application to many NF-κB-dependent diseases, such as cancer and atherosclerosis.

OBJECTIVE:Intestinal fibrosis is considered an inevitable consequence of chronic IBD, leading to stricture formation and need for surgery. During the process of fibrogenesis, extracellular matrix (ECM) components critically regulate the function of mesenchymal cells. We characterised the composition and function of ECM in fibrostenosing Crohn's disease (CD) and control tissues. DESIGN:Decellularised full-thickness intestinal tissue platforms were tested using three different protocols, and ECM composition in different tissue phenotypes was explored by proteomics and validated by quantitative PCR (qPCR) and immunohistochemistry. Primary human intestinal myofibroblasts (HIMFs) treated with milk fat globule-epidermal growth factor 8 (MFGE8) were evaluated regarding the mechanism of their antifibrotic response, and the action of MFGE8 was tested in two experimental intestinal fibrosis models. RESULTS:We established and validated an optimal decellularisation protocol for intestinal IBD tissues. Matrisome analysis revealed elevated MFGE8 expression in CD strictured (CDs) tissue, which was confirmed at the mRNA and protein levels. Treatment with MFGE8 inhibited ECM production in normal control HIMF but not CDs HIMF. Next-generation sequencing uncovered functionally relevant integrin-mediated signalling pathways, and blockade of integrin αvβ5 and focal adhesion kinase rendered HIMF non-responsive to MFGE8. MFGE8 prevented and reversed experimental intestinal fibrosis in vitro and in vivo. CONCLUSION:MFGE8 displays antifibrotic effects, and its administration may represent a future approach for prevention of IBD-induced intestinal strictures.

Sepsis is a systemic inflammatory response that requires effective macrophage metabolic functions to resolve ongoing inflammation. Previous work showed that the mechanosensitive cation channel, transient receptor potential vanilloid 4 (TRPV4), mediates macrophage phagocytosis and cytokine production in response to lung infection. Here, we show that TRPV4 regulates glycolysis in a stiffness-dependent manner by augmenting macrophage glucose uptake by GLUT1. In addition, TRPV4 is required for LPS-induced phagolysosome maturation in a GLUT1-dependent manner. In a cecal slurry mouse model of sepsis, TRPV4 regulates sepsis-induced glycolysis as measured by BAL fluid (BALF) lactate and sepsis-induced lung injury as measured by BALF total protein and lung compliance. TRPV4 is necessary for bacterial clearance in the peritoneum to limit sepsis-induced lung injury. It is interesting that BALF lactate is increased in patients with sepsis compared with healthy control participants, supporting the relevance of lung cell glycolysis to human sepsis. These data show that macrophage TRPV4 is required for glucose uptake through GLUT1 for effective phagolysosome maturation to limit sepsis-induced lung injury. Our work presents TRPV4 as a potential target to protect the lung from injury in sepsis.

Rationale: Chronic obstructive pulmonary disease (COPD) is the third leading cause of death worldwide. Our previous studies have identified that nocturnal hypoxemia causes skeletal muscle loss (i.e., sarcopenia) in in vitro models of COPD. Objectives: We aimed to extend our preclinical mechanistic findings by analyzing a large sleep registry to determine whether nocturnal hypoxemia is associated with sarcopenia in patients with COPD. Methods: Sleep studies from patients with COPD (n = 479) and control subjects without COPD (n = 275) were analyzed. Patients with obstructive sleep apnea, as defined by apnea-hypopnea index ⩾ 5, were excluded. Pectoralis muscle cross-sectional area (PMcsa) was quantified using computed tomography scans performed within 1 year of the sleep study. We defined sarcopenia as less than the lowest 20% residuals for PMcsa of control subjects, which was adjusted for age and body mass index (BMI) and stratified by sex. Youden's optimal cut-point criteria were used to predict sarcopenia based on mean oxygen saturation during sleep. Additional measures of nocturnal hypoxemia were analyzed. The pectoralis muscle index (PMI) was defined as PMcsa normalized to BMI. Results: On average, males with COPD had a 16.6% lower PMI than control males (1.41 ± 0.44 vs. 1.69 ± 0.56 cm2/BMI; P < 0.001), whereas females with COPD had a 9.4% lower PMI than control females (0.96 ± 0.27 vs. 1.06 ± 0.33 cm2/BMI; P < 0.001). Males with COPD with nocturnal hypoxemia had a 9.5% decrease in PMI versus COPD with normal O2 (1.33 ± 0.39 vs. 1.47 ± 0.46 cm2/BMI; P < 0.05) and a 23.6% decrease compared with control subjects (1.33 ± 0.39 vs. 1.74 ± 0.56 cm2/BMI; P < 0.001). Females with COPD with nocturnal hypoxemia had an 11.2% decrease versus COPD with normal O2 (0.87 ± 0.26 vs. 0.98 ± 0.28 cm2/BMI; P < 0.05) and a 17.9% decrease compared with control subjects (0.87 ± 0.26 vs. 1.06 ± 0.33 cm2/BMI; P < 0.001). These findings were largely replicated using multiple measures of nocturnal hypoxemia. Conclusions: We defined sarcopenia in the pectoralis muscle using residuals that take into account age, BMI, and sex. We found that patients with COPD have a lower PMI than patients without COPD and that nocturnal hypoxemia was associated with an additional decrease in the PMI of patients with COPD. Additional prospective analyses are needed to determine a protective threshold of oxygen saturation to prevent or reverse sarcopenia due to nocturnal hypoxemia in COPD.

PURPOSE: Diffusing capacity for carbon monoxide (DLCO) is often impaired in pulmonary vascular disease, but its importance among varied pulmonary hypertension (PH) groups is not fully defined.We sought to further describe the relationship of DLCO to characteristics and outcomes across the spectrum of phenotypes captured in the Pulmonary Vascular Disease Phenomics (PVDOMICS) cohort. METHODS:Of the 1193 patients (incident and prevalent) enrolled prospectively from 11/30/2016 to 10/18/2019 at 7 centers in the US, DLCO data was available in 1042 patients.Data in WHO Group 1-5 PH patients [by the 2013 World Symposium on Pulmonary Hypertension criteria], (n¼327, 116, 135, 50, 29)], comparators with mild PH [mPAP <25, PVR <3 WU], (n¼50, 121, 97, 19, 6)] and 92 healthy controls were analyzed.Low DLCO was defined as < 40% predicted.Categorical variables were summarized as count (%) and continuous variables as median (IQR).Chi-squared and Fisher Exact tests were used to compare categorical variables across groups, and Wilcoxon rank sum test for continuous variables.Age and sex adjusted transplant-free survival by DLCO status was assessed using Cox regression methods. RESULTS:Across Group 1-5, low DLCO occurred in 26% patients.Prevalence of low DLCO in Group 1-5 was 22.9%, 24.1%, 71.9%, 12%, and 37.9%, respectively and in comparators 4%, 8.3%, 41.2%, 5.3% and 16.7%.DLCO<40% was significantly associated with older age, ever smoking, CT abnormalities (Interstitial lung disease [ILD], Emphysema, Ground glass opacities [GGOs]), higher NTpro-BNP, lower 6-minute walk distance (6MWD), worse echocardiographic parameters (Right ventricular systolic pressure[RVSP], right ventricular strain) and hemodynamics (mPAP, PVR; p<0.001).Age and sex adjusted risk of transplant/death was significantly higher with DLCO<40% (HR 2.52, 95% CI 1. 86-3.43; p<0.0001)In Group 1 PAH, low DLCO was associated with older age, incident disease, shorter duration of PH, certain etiologies (Connective tissue disease, Pulmonary veno-occlusive disease), smoking, higher NTpro-BNP, CT abnormalities, restrictive pulmonary function tests, higher RVSP and lower left ventricular ejection fraction, worse peak oxygen consumption and ventilatory efficiency on Cardiopulmonary exercise testing and a lower 6MWD.Pulmonary hemodynamics were similar in high vs low DLCO and significantly fewer patients with low DLCO were on vasodilator therapy including prostanoids.Low DLCO was associated with worse transplant-free survival (HR 2.80, 95% CI 1.67-4.68;p<0.001).A 1% decline in DLCO was associated with a 3% increase in the risk of transplant/death.CONCLUSIONS: Low DLCO is present in a substantial percentage of patients across the spectrum of pulmonary vascular disease.Low DLCO correlates with markers of disease severity and outcomes including transplant free survival.CLINICAL IMPLICATIONS: DLCO is an important correlate of disease intensity in PH with implications on outcomes.

Fibroblast to myofibroblast transdifferentiation mediates numerous fi brotic disorders, such as idiopathic pulmonary fi brosis (IPF). We have previously demonstrated that non-muscle myosin II (NMII) is activated in response to fi brotic lung extracellular matrix, thereby mediating myofibroblast trans- differentiation. NMII-A is known to interact with the calcium- binding protein S100A4, but the mechanism by which S100A4 regulates fi brotic disorders is unclear. In this study, we show that fi broblast S100A4 is a calcium-dependent, mechanoeffector protein that is uniquely sensitive to pathophysiologic-range lung stiffness (8-25 kPa) and thereby mediates myofibroblast trans- differentiation. Re-expression of endogenous fi broblast S100A4 rescues the myofibroblastic phenotype in S100A4 KO fi broblasts. Analysis of NMII-A/actin dynamics reveals that S100A4 mediates the unraveling and redistribution of peripheral actomyosin to a central location, resulting in a contractile myofibroblast. Furthermore, S100A4 loss protects against murine in vivo pulmonary fi brosis, and S100A4 expression is dysregulated in IPF. Our data reveal a novel mechanosensor/effector role for endogenous fi broblast S100A4 in inducing cytoskeletal redistribution in fi brotic disorders such as IPF.

Pulmonary fibrosis is a devastating disease with available pharmacologic therapy that only slows progression and lacks curative potential (1). Fibroblasts differentiating into myofibroblasts play a vital role in the pathogenesis of lung fibrosis (2). Hence, specific myofibroblast targets to reverse or resolve fibrosis are desperately needed. Transient receptor potential (TRP) channels are a family of plasma membrane cation channels with unique functions in a variety of cell types (3). Seven TRP families have been discovered, which are categorized on the basis of sequence homology, including TRPV (vanilloid) and TRPA (ankyrin) (3, 4). We and others have shown that myofibroblast differentiation is dependent on cations (e.g., calcium) (5). Others have implicated calcium through non-TRP channels, such as L-type and T-type calcium channels, to mediate pulmonary fibrosis (6). Our group has discovered that the mechanosensitive cation channel TRPV4 (TRP vanilloid 4) is important in myofibroblast differentiation and experimental pulmonary fibrosis in vivo (5). In contrast to the mechanosensitive properties of TRPV4, TRPA1 is activated by extreme temperatures or compounds with spicy or pungent scents, including cinnamon, onion, garlic, and mustard (7). TRPA1 is well-known to play a role in the pathobiology of respiratory diseases, including cough and asthma (e.g., neurons and epithelial cells); however, its role in parenchymal lung disease is less certain (7, 8). In this issue of the Journal, Geiger and colleagues (pp. 314–325) show that TRPA1 plays a role in profibrotic signaling in human lung fibroblasts (HLFs) (9). Specifically, an reverse transcriptase–polymerase chain reaction screen revealed that TRPA1mRNAwas downregulated by TGFb in primary HLFs from normal subjects. Concordantly, TGF-b1 downregulated TRPA1mRNA expression and channel function as measured by an intracellular calcium-sensitive dye. Basal downregulation of TRPA1 (with TRPA1 siRNA), increased mRNA expression of genes encoding several fibrosis markers (ACTA2 [a-smooth muscle actin], SERPINE1 [plasminogen activator inhibitor 1], FN1 [fibronectin], and COL1A1 [type I collagen]) in HLFs. Importantly, treatment of HLFs with a TRPA1 agonist (AITC and J7010) blocked the TGF-b induction of a smoothmuscle actin and collagen I proteins. Collectively, these data suggest that TRPA1 downregulation by TGFb results in a loss of TRPA1’s inhibitory function on fibrotic gene expression. The findings, which address the mechanism by which TRPA1 exerts its inhibitory effect on TGFb signaling, remain somewhat open to interpretation.Without either loss of function and/or gain of function studies of all steps in the TGFb signaling pathway, it is difficult to put these latter findings into the fibrosis context. Calcium entry into cells via ion channels is highly cited in the literature as an essential process underlying fibroblast transition to a myofibroblast (10). Geiger and colleagues show that calcium is necessary but not sufficient to make a myofibroblast. Therefore, it remains possible that other mono or bivalent cations (Mg, Na, and K) through ion channels are drivers of fibrosis or that other calcium channels contribute to a greater extent to the fibrotic process (11). In addition, it is not just a net increase in intracellular calcium, which drives myofibroblast differentiation, as difficult-to-detect timing of calcium increases, either transient, persistent, or cyclical, may condition the response (10). Similarly, the subcellular location of calcium oscillations, the extent of concomitant soluble profibrotic signals, and/or cross-talk with other channel-dependent and/or independent signals may also drive the fibrotic process (10, 12). Furthermore, TRP channels have intracellular domains, which may interact with other signaling cascades, as we have shown for TRPV4 in fibroblasts and macrophages (13–15). Future work that uncovers these key mechanisms will allow for more targeted therapy. TRP channel cross-talk has been shown to modulate intracellular processes through channel–channel and/or protein–protein interactions as well as through posttranslational modifications in neurons (7). For example, coexpression of TRPA1 and TRPV1 is required for proper calcium-dependent neuronal functions (16, 17). Cofactors (e.g., Tmem100 [transmembrane protein 100] and prokineticins [PK1 and PK2]) have also been identified, which modulate both TRPA1 and TRPV1 neuropathic function (18). This suggests that targeting a binding partner to TRPA1–TRPV1 can potentially alter ion channel function or sensitivity. Although the phosphorylation of TRPA1 is less well described, there is evidence that TRPA1 interacts with a G-protein–coupled bradykinin receptor through activation of PKA and PKC (protein kinase A and C) (19–21). The spectrum of nonspecific ion channel simulators spans the gamut, from varying temperature, osmolality, mechanical stress, endogenous inflammatory ligands, and synthetic and/or naturally occurring chemical stimuli. TRPA1 in the lung has primarily been implicated in airway diseases such as chronic cough, asthma, rhinorrhea, and chronic obstructive pulmonary disease. TRPA1 is ubiquitously expressed but primarily in sensory afferent nerves, which detect irritants and/or inhaled (e.g., cigarette smoke, pollutants, and chlorine) in the airway. Byproducts of pollutants, specifically exhaust particles, have been shown to agonize TRPA1 as a mechanism of cough exacerbation. Furthermore, asthmatic smooth muscle TRPA1 activation limits smooth muscle proliferation in the airway, implicating TRPA1 in associative studies or patients with asthma (22). Activation of TRPA1 has been directly associated with chronic rhinitis (23). The current study by Gieger and colleagues highlights the role of TRPA1 in parenchymal cells such as the

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.

BACKGROUND PVDOMICS (Pulmonary Vascular Disease Phenomics) is a precision medicine initiative to characterize pulmonary vascular disease (PVD) using deep phenotyping. PVDOMICS tests the hypothesis that integration of clinical metrics with omic measures will enhance understanding of PVD and facilitate an updated PVD classification. OBJECTIVES The purpose of this study was to describe clinical characteristics and transplant-free survival in the PVDOMICS cohort. METHODS Subjects with World Symposium Pulmonary Hypertension (WSPH) group 1-5 PH, disease comparators with similar underlying diseases and mild or no PH and healthy control subjects enrolled in a cross-sectional study. PH groups, comparators were compared using standard statistical tests including log-rank tests for comparing time to transplant or death. RESULTS A total of 1,193 subjects were included. Multiple WSPH groups were identified in 38.9% of PH subjects. Nocturnal desaturation was more frequently observed in groups 1, 3, and 4 PH vs comparators. A total of 50.2% of group 1 PH subjects had ground glass opacities on chest computed tomography. Diffusing capacity for carbon monoxide was significantly lower in groups 1-3 PH than their respective comparators. Right atrial volume index was higher in WSPH groups 1-4 than comparators. A total of 110 participants had a mean pulmonary artery pressure of 21-24 mm Hg. Transplant-free survival was poorest in group 3 PH. CONCLUSIONS PVDOMICS enrolled subjects across the spectrum of PVD, including mild and mixed etiology PH. Novel findings include low diffusing capacity for carbon monoxide and enlarged right atrial volume index as shared features of groups 1-3 and 1-4 PH, respectively; unexpected, frequent presence of ground glass opacities on computed tomography; and sleep alterations in group 1 PH, and poorest survival in group 3 PH. PVDOMICS will facilitate a new understanding of PVD and refine the current PVD classification. (Pulmonary Vascular Disease Phenomics Program PVDOMICS [PVDOMICS]; NCT02980887) (J Am Coll Cardiol 2022;80:697-718) (C) 2022 by the American College of Cardiology Foundation.

The transient receptor potential vanilloid 1 (TRPV1) channel is expressed in human bronchial epithelium (HBE), where it transduces Ca2+ in response to airborne irritants. TRPV1 activation results in bronchoconstriction, cough, and mucus production, and may therefore contribute to the pathophysiology of obstructive airway disease. Since children with asthma face the greatest risk of developing virus-induced airway obstruction, we hypothesized that changes in TRPV1 expression, localization, and function in the airway epithelium may play a role in bronchiolitis and asthma in childhood. We sought to measure TRPV1 protein expression, localization, and function in HBE cells from children with versus without asthma, both at baseline and after RSV infection. We determined changes in TRPV1 protein expression, subcellular localization, and function both at baseline and after RSV infection in primary HBE cells from normal children and children with asthma. Basal TRPV1 protein expression was higher in HBE from children with versus without asthma and primarily localized to plasma membranes (PMs). During RSV infection, TRPV1 protein increased more in the PM of asthmatic HBE as compared with nonasthmatic cells. TRPV1-mediated increase in intracellular Ca2+ was greater in RSV-infected asthmatic cells, but this increase was attenuated when extracellular Ca2+ was removed. Nerve growth factor (NGF) recapitulated the effect of RSV on TRPV1 activation in HBE cells. Our data suggest that children with asthma have intrinsically hyperreactive airways due in part to higher TRPV1-mediated Ca2+ influx across epithelial membranes, and this abnormality is further exacerbated by NGF overexpression during RSV infection driving additional Ca2+ from intracellular stores.

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-1 alpha, 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.