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Activation of TGF-β1 initiates a program of temporary collagen accumulation important to wound repair in many organs. However, the outcome of temporary extracellular matrix strengthening all too frequently morphs into progressive fibrosis, contributing to morbidity and mortality worldwide. To avoid this maladaptive outcome, TGF-β1 signaling is regulated at numerous levels and intimately connected to feedback signals that limit accumulation. Here, we examine the current understanding of the core functions of TGF-β1 in promoting collagen accumulation, parallel pathways that promote physiological repair, and pathological triggers that tip the balance toward progressive fibrosis. Implicit in better understanding of these processes is the identification of therapeutic opportunities that will need to be further advanced to limit or reverse organ fibrosis.

Fibrosis is a multicellular process leading to excessive extracellular matrix deposition. Factors that affect lung epithelial cell proliferation and activation may be important regulators of the extent of fibrosis after injury. We and others have shown that activated alveolar epithelial cells (AECs) directly contribute to fibrogenesis by secreting mesenchymal proteins, such as type I collagen. Recent evidence suggests that epithelial cell acquisition of mesenchymal features during carcinogenesis and fibrogenesis is regulated by several mesenchymal transcription factors. Induced expression of direct inhibitors to these mesenchymal transcription factors offers a potentially novel therapeutic strategy. Inhibitor of DNA-binding 2 (Id2) is an inhibitory helix-loop-helix transcription factor that is highly expressed by lung epithelial cells during development and has been shown to coordinate cell proliferation and differentiation of cancer cells. We found that overexpression of Id2 in primary AECs promotes proliferation by inhibiting a retinoblastoma protein/c-Abl interaction leading to greater c-Abl activity. Id2 also blocks transforming growth factor β1-mediated expression of type I collagen by inhibiting Twist, a prominent mesenchymal basic helix-loop-helix transcription factor. In vivo, Id2 induced AEC proliferation and protected mice from lung fibrosis. By using a high-throughput screen, we found that histone deacetylase inhibitors induce Id2 expression by adult AECs. Collectively, these findings suggest that Id2 expression by AECs can be induced, and overexpression of Id2 affects AEC phenotype, leading to protection from fibrosis.

Fibrosis is characterized by accumulation of activated fibroblasts and pathological deposition of fibrillar collagens. Activated fibroblasts overexpress matrix proteins and release factors that promote further recruitment of activated fibroblasts, leading to progressive fibrosis. The contribution of epithelial cells to this process remains unknown. Epithelium-directed injury may lead to activation of epithelial cells with phenotypes and functions similar to activated fibroblasts. Prior reports that used a reporter gene fate-mapping strategy are limited in their ability to investigate the functional significance of epithelial cell-derived mesenchymal proteins during fibrogenesis. We found that lung epithelial cell-derived collagen I activates fibroblast collagen receptor discoidin domain receptor-2, contributes significantly to fibrogenesis, and promotes resolution of lung inflammation. Alveolar epithelial cells undergoing transforming growth factor-β-mediated mesenchymal transition express several other secreted profibrotic factors and are capable of activating lung fibroblasts. These studies provide direct evidence that activated epithelial cells produce mesenchymal proteins that initiate a cycle of fibrogenic effector cell activation, leading to progressive fibrosis. Therapy targeted at epithelial cell production of type I collagen offers a novel pathway for abrogating this progressive cycle and for limiting tissue fibrosis but may lead to sustained lung injury/inflammation.

Injury-initiated epithelial to mesenchymal transition (EMT) depends on contextual signals from the extracellular matrix, suggesting a role for integrin signaling. Primary epithelial cells deficient in their prominent laminin receptor, alpha3beta1, were found to have a markedly blunted EMT response to TGF-beta1. A mechanism for this defect was explored in alpha3-null cells reconstituted with wild-type (wt) alpha3 or point mutants unable to engage laminin 5 (G163A) or epithelial cadherin (E-cadherin; H245A). After TGF-beta1 stimulation, wt epithelial cells but not cells expressing the H245A mutant internalize complexes of E-cadherin and TGF-beta1 receptors, generate phospho-Smad2 (p-Smad2)-pY654-beta-catenin complexes, and up-regulate mesenchymal target genes. Although Smad2 phosphorylation is normal, p-Smad2-pY654-beta-catenin complexes do not form in the absence of alpha3 or when alpha3beta1 is mainly engaged on laminin 5 or E-cadherin in adherens junctions, leading to attenuated EMT. These findings demonstrate that alpha3beta1 coordinates cross talk between beta-catenin and Smad signaling pathways as a function of extracellular contact cues and thereby regulates responses to TGF-beta1 activation.

Pulmonary fibrosis, especially idiopathic pulmonary fibrosis (IPF), portends significant morbidity and mortality, and current therapeutic options are suboptimal. We have previously shown that type I collagen signaling through discoidin domain receptor 2 (DDR2), a receptor tyrosine kinase expressed by fibroblasts, is critical for the regulation of fibroblast apoptosis and progressive fibrosis. However, the downstream signaling pathways for DDR2 remain poorly defined and could also be attractive potential targets for therapy. A recent phosphoproteomic approach indicated that PIK3C2α, a poorly studied member of the PI3 kinase family, could be a downstream mediator of DDR2 signaling. We hypothesized that collagen I/DDR2 signaling through PIK3C2α regulates fibroblast activity during progressive fibrosis. To test this hypothesis, we found that primary murine fibroblasts and IPF‐derived fibroblasts stimulated with endogenous or exogenous type I collagen led to the formation of a DDR2/PIK3C2α complex, resulting in phosphorylation of PIK3C2α. Fibroblasts treated with an inhibitor of PIK3C2α or with deletion of PIK3C2α had fewer markers of activation after stimulation with TGFβ and more apoptosis after stimulation with a Fas‐activating antibody. Finally, mice with fibroblast‐specific deletion of PIK3C2α had less fibrosis after bleomycin treatment than did littermate control mice with intact expression of PIK3Cα. Collectively, these data support the notion that collagen/DDR2/PIK3C2α signaling is critical for fibroblast function during progressive fibrosis, making this pathway a potential target for antifibrotic therapy. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland. [ABSTRACT FROM AUTHOR]

Injury-initiated epithelial to mesenchymal transition (EMT) depends on contextual signals from the extracellular matrix, suggesting a role for integrin signaling. Primary epithelial cells deficient in their prominent laminin receptor, [alpha]3[beta]1, were found to have a markedly blunted EMT response to TGF-[beta]1. A mechanism for this defect was explored in [alpha]3-null cells reconstituted with wild-type (wt) [alpha]3 or point mutants unable to engage laminin 5 (G163A) or epithelial cadherin (E-cadherin; H245A). After TGF-[beta]1 stimulation, wt epithelial cells but not cells expressing the H245A mutant internalize complexes of E-cadherin and TGF-[beta]1 receptors, generate phospho-Smad2 (p-Smad2)-pY654-[beta]-catenin complexes, and upregulate mesenchymal target genes. Although Smad2 phosphorylation is normal, p-Smad2--pY654--[beta]-catenin complexes do not form in the absence of [alpha]3 or when [alpha]3[beta]1 is mainly engaged on laminin 5 or E-cadherin in adherens junctions, leading to attenuated EMT. These findings demonstrate that [alpha]3[beta]1 coordinates cross talk between [beta]-catenin and Smad signaling pathways as a function of extracellular contact cues and thereby regulates responses to TGF-[beta]1 activation.

Introduction Progressive fibrosis of the lung, especially that of idiopathic pulmonary fibrosis (IPF), is thought to be a consequence of aberrant wound healing resulting in progressive scarification (1), (2). Fibroblast [...], Pulmonary fibrosis, in particular idiopathic pulmonary fibrosis (IPF), results from aberrant wound healing and scarification. One population of fibroblasts involved in the fibrotic process is thought to originate from lung epithelial cells via epithelial-mesenchymal transition (EMT). Indeed, alveolar epithelial cells (AECs) undergo EMT in vivo during experimental fibrosis and ex vivo in response to TGF-β1. As the ECM critically regulates AEC responses to TGF-β1, we explored the role of the prominent epithelial integrin α3β1 in experimental fibrosis by generating mice with lung epithelial cell--specific loss of α3 integrin expression. These mice had a normal acute response to bleomycin injury, but they exhibited markedly decreased accumulation of lung myofibroblasts and type I collagen and did not progress to fibrosis. Signaling through β-catenin has been implicated in EMT; we found that in primary AECs, α3 integrin was required for β-catenin phosphorylation at tyrosine residue 654 (Y654), formation of the pY654-β-catenin/pSmad2 complex, and initiation of EMT, both in vitro and in vivo during the fibrotic phase following bleomycin injury. Finally, analysis of lung tissue from IPF patients revealed the presence of pY654-β-catenin/pSmad2 complexes and showed accumulation of pY654-β-catenin in myofibroblasts. These findings demonstrate epithelial integrin--dependent profibrotic crosstalk between β-catenin and Smad signaling and support the hypothesis that EMT is an important contributor to pathologic fibrosis.

Urokinase-type plasminogen activator receptors (uPARs), up-regulated during tumor progression, associate with [beta]1 integrins, localizing urokinase to sites of cell attachment. Binding of uPAR to the [beta]-propeller of [alpha]3[beta]1 empowers vitronectin adhesion by this integrin. How uPAR modifies other [beta]1 integrins remains unknown. Using recombinant proteins, we found uPAR directly binds [alpha]5[beta]1 and rather than blocking, renders fibronectin (Fn) binding by [alpha]5[beta]1 Arg-Gly-Asp (RGD) resistant. This resulted from RGD-independent binding of [alpha]5[beta]1-uPAR to Fn type III repeats 12-15 in addition to type III repeats 9-11 bound by [alpha]5[beta]1. Suppression of endogenous uPAR by small interfering RNA in tumor cells promoted weaker, RGD-sensitive Fn adhesion and altered overall [alpha]5[beta]1 conformation. A [beta]1 peptide (res 224NLDSPEGGF232) that models near the known [alpha]-chain uPAR-binding region, or a [beta]1-chain Ser227Ala point mutation, abrogated effects of uPAR on [alpha]5[beta]1. Direct binding and regulation of [alpha]5[beta]1 by uPAR implies a modified 'bent' integrin conformation can function in an alternative activation state with this and possibly other cis-acting membrane ligands.

Background: The United States currently has more confirmed cases of COVID-19 than any other country in the world. Given the variability in COVID-19 testing and prevention capability, identifying factors associated with mortality in patients requiring mechanical ventilation is critical. This study aimed to identify which demographics, comorbidities, markers of disease progression, and interventions are associated with 30-day mortality in COVID-19 patients requiring mechanical ventilation. Methods: Adult patients with a confirmed diagnosis of COVID-19 admitted to one of the health system's intensive care units and requiring mechanical ventilation between March 9, 2020 and April 1, 2020, were included in this observational cohort study. We used Chi-Square and Mann-Whitney U tests to compare patient characteristics between deceased and living patients and multiple logistic regression to assess the association between independent variables and the likelihood of 30-day mortality. Results: We included 85 patients, of which 20 died (23.5%) within 30 days of the first hospital admission. In the univariate analysis, deceased patients were more likely ≥60 years of age (p < 0.001), non-Hispanic (p = 0.026), and diagnosed with a solid malignant tumor (p = 0.003). Insurance status also differed between survivors and non-survivors (p = 0.019). Age ≥60 and malignancy had a 9.5-fold (95% confidence interval 1.4-62.3, p = 0.020) and 5.8-fold higher odds ratio (95% confidence interval 1.2-28.4, p = 0.032) for 30-day mortality after adjusted analysis using multivariable logistic regression, while other independent variables were no longer significant. Conclusions: In our observational cohort study of 85 mechanically ventilated COVID-19 patients, age, and a diagnosis of a solid malignant tumor were associated with 30-day mortality. Our findings validate concerns for the survival of elderly and cancer patients in the face of the COVID-19 pandemic in the United States, where testing capabilities and preventative measures have been inconsistent. Preventative efforts geared to patients at risk for intensive care unit mortality from COVID-19 should be explored. [ABSTRACT FROM AUTHOR]

Fibrosis is characterized by fibroblast activation, leading to matrix remodeling culminating in a stiff, type I collagen-rich fibrotic matrix. Alveolar epithelial cell (AEC) apoptosis is also a major feature of fibrogenesis, and AEC apoptosis is sufficient to initiate a robust lung fibrotic response. TGF-β (transforming growth factor-β) is a major driver of fibrosis and can induce both AEC apoptosis and fibroblast activation. We and others have previously shown that changes in extracellular matrix stiffness and composition can regulate the cellular response to TGF-β. In the present study, we find that type I collagen signaling promotes TGF-β-mediated fibroblast activation and inhibits TGF-β-induced AEC death. Fibroblasts cultured on type I collagen or fibrotic decellularized lung matrix had augmented activation in response to TGF-β, whereas AECs on cultured on type I collagen or fibrotic lung matrix were more resistant to TGF-β-induced apoptosis. Both of these responses were mediated by integrin α2β1, a major collagen receptor. AECs treated with an α2 integrin inhibitor or with deletion of α2 integrin had loss of collagen-mediated protection from apoptosis. We found that mice with fibroblast-specific deletion of α2 integrin were protected from fibrosis whereas mice with AEC-specific deletion of α2 integrin had more lung injury and a greater fibrotic response to bleomycin. Intrapulmonary delivery of an α2 integrin-activating collagen peptide inhibited AEC apoptosis in vitro and in vivo and attenuated the fibrotic response. These studies underscore the need for a thorough understanding of the divergent response to matrix signaling. [ABSTRACT FROM AUTHOR]

Accumulating evidence suggests that fibrosis is a multicellular process with contributions from alveolar epithelial cells (AECs), recruited monocytes/macrophages, and fibroblasts. We have previously shown that AEC injury is sufficient to induce fibrosis, but the precise mechanism remains unclear. Several cell types, including AECs, can produce CCL2 and CCL12, which can promote fibrosis through CCR2 activation. CCR2 signaling is critical for the initiation and progression of pulmonary fibrosis, in part through recruitment of profibrotic bone marrow-derived monocytes. Attempts at inhibiting CCL2 in patients with fibrosis demonstrated a marked upregulation of CCL2 production and no therapeutic response. To better understand the mechanisms involved in CCL2/CCR2 signaling, we generated mice with conditional deletion of CCL12, a murine homolog of human CCL2. Surprisingly, we found that mice with complete deletion of CCL12 had markedly increased concentrations of other CCR2 ligands and were not protected from fibrosis after bleomycin injury. In contrast, mice with lung epithelial cell-specific deletion of CCL12 were protected from bleomycininduced fibrosis and had expression of CCL2 and CCL7 similar to that of control mice treated with bleomycin. Deletion of CCL12 within AECs led to decreased recruitment of exudate macrophages. Finally, injury to murine and human primary AECs resulted in increased production of CCL2 and CCL12, in part through activation of the mTOR pathway. In conclusion, these data suggest that targeting CCL2 may be a viable antifibrotic strategy once the pathways involved in the production and function of CCL2 and other CCR2 ligands are better defined. [ABSTRACT FROM AUTHOR]

Progressive fibrosis is a complication of many chronic diseases, and collectively, organ fibrosis is the leading cause of death in the United States. Fibrosis is characterized by accumulation of activated fibroblasts and excessive deposition of extracellular matrix proteins, especially type I collagen. Extensive research has supported a role for matrix signaling in propagating fibrosis, but type I collagen itself is often considered an end product of fibrosis rather than an important regulator of continued collagen deposition. Type I collagen can activate several cell surface receptors, including α2β1 integrin and discoidin domain receptor 2 (DDR2). We have previously shown that mice deficient in type I collagen have reduced activation of DDR2 and reduced accumulation of activated myofibroblasts. In the present study, we found that DDR2-null mice are protected from fibrosis. Surprisingly, DDR2-null fibroblasts have a normal and possibly exaggerated activation response to transforming growth factor-b and do not have diminished proliferation compared with wild-type fibroblasts. DDR2-null fibroblasts are significantly more prone to apoptosis, in vitro and in vivo, than wild-type fibroblasts, supporting a paradigm in which fibroblast resistance to apoptosis is critical for progression of fibrosis. We have identified a novel molecular mechanism by which DDR2 can promote the activation of a PDK1 (3-phosphoinositide dependent protein kinase-1)/Akt survival pathway, and we have found that inhibition of PDK1 can augment fibroblast apoptosis. Furthermore, our studies demonstrate that DDR2 expression is heavily skewed to mesenchymal cells compared with epithelial cells and that idiopathic pulmonary fibrosis cells and tissue demonstrate increased activation of DDR2 and PDK1. Collectively, these findings identify a promising target for fibrosis therapy. [ABSTRACT FROM AUTHOR]