Discoidin domain receptor 2 signaling through PIK3C2α in fibroblasts promotes lung fibrosis.

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 TGFß 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.

CD36/Lyn kinase interactions within macrophages promotes pulmonary fibrosis in response to oxidized phospholipid.

Recent data from human studies and animal models have established roles for type II alveolar epithelial cell (AEC2) injury/apoptosis and monocyte/macrophage accumulation and activation in progressive lung fibrosis. Although the link between these processes is not well defined, we have previously shown that CD36-mediated uptake of apoptotic AEC2s by lung macrophages is sufficient to drive fibrosis. Importantly, apoptotic AEC2s are rich in oxidized phospholipids (oxPL), and amongst its multiple functions, CD36 serves as a scavenger receptor for oxPL. Recent studies have established a role for oxPLs in alveolar scarring, and we hypothesized that uptake and accrual of oxPL by CD36 would cause a macrophage phenotypic change that promotes fibrosis. To test this hypothesis, we treated wild-type and CD36-null mice with the oxPL derivative oxidized phosphocholine (POVPC) and found that CD36-null mice were protected from oxPL-induced scarring. Compared to WT mice, fewer macrophages accumulated in the lungs of CD36-null animals, and the macrophages exhibited a decreased accumulation of intracellular oxidized lipid. Importantly, the attenuated accrual of oxPL in CD36-null macrophages was associated with diminished expression of the profibrotic mediator, TGFß. Finally, the pathway linking oxPL uptake and TGFß expression was found to require CD36-mediated activation of Lyn kinase. Together, these observations elucidate a causal pathway that connects AEC2 injury with lung macrophage activation via CD36-mediated uptake of oxPL and suggest several potential therapeutic targets.

Spatial heterogeneity of bone marrow endothelial cells unveils a distinct subtype in the epiphysis.

Bone marrow endothelial cells (BMECs) play a key role in bone formation and haematopoiesis. Although recent studies uncovered the cellular taxonomy of stromal compartments in the bone marrow (BM), the complexity of BMECs is not fully characterized. In the present study, using single-cell RNA sequencing, we defined a spatial heterogeneity of BMECs and identified a capillary subtype, termed type S (secondary ossification) endothelial cells (ECs), exclusively existing in the epiphysis. Type S ECs possessed unique phenotypic characteristics in terms of structure, plasticity and gene expression profiles. Genetic experiments showed that type S ECs atypically contributed to the acquisition of bone strength by secreting type I collagen, the most abundant bone matrix component. Moreover, these cells formed a distinct reservoir for haematopoietic stem cells. These findings provide the landscape for the cellular architecture in the BM vasculature and underscore the importance of epiphyseal ECs during bone and haematopoietic development.

Overexpression of Decay Accelerating Factor Mitigates Fibrotic Responses to Lung Injury.

CD55 or decay accelerating factor (DAF), a ubiquitously expressed glycosylphosphatidylinositol (GPI)-anchored protein, confers a protective threshold against complement dysregulation which is linked to the pathogenesis of idiopathic pulmonary fibrosis (IPF). Since lung fibrosis is associated with downregulation of DAF, we hypothesize that overexpression of DAF in fibrosed lungs will limit fibrotic injury by restraining complement dysregulation. Normal primary human alveolar type II epithelial cells (AECs) exposed to exogenous complement 3a or 5a, and primary AECs purified from IPF lungs demonstrated decreased membrane-bound DAF expression with concurrent increase in the endoplasmic reticulum (ER) stress protein, ATF6. Increased loss of extracellular cleaved DAF fragments was detected in normal human AECs exposed to complement 3a or 5a, and in lungs of IPF patients. C3a-induced ATF6 expression and DAF loss was inhibited using pertussis toxin (an enzymatic inactivator of G-protein coupled receptors), in murine AECs. Treatment with soluble DAF abrogated tunicamycin-induced C3a secretion and ER stress (ATF6 and BiP expression) and restored epithelial cadherin. Bleomycin-injured fibrotic mice subjected to lentiviral overexpression of DAF demonstrated diminished levels of local collagen deposition and complement activation. Further analyses showed diminished release of DAF fragments, as well as reduction in apoptosis (TUNEL and caspase 3/7 activity), and ER stress-related transcripts. Loss-of-function studies using Daf1 siRNA demonstrated worsened lung fibrosis detected by higher mRNA levels of Col1a1 and epithelial injury-related Muc1 and Snai1, with exacerbated local deposition of C5b-9. Our studies provide a rationale for rescuing fibrotic lungs via DAF induction that will restrain complement dysregulation and lung injury.

Transesophageal echocardiography for perioperative management in thoracic surgery.

PURPOSE OF REVIEW: Perioperative transesophageal echocardiography (TEE) is most often employed during cardiac surgery. This review will summarize some of the recent findings relevant to TEE utilization during thoracic surgical procedures. RECENT FINDINGS: Hemodynamic monitoring is a key component of goal-directed fluid therapy, which is also becoming more common for management of thoracic surgical procedures. Although usually not required for the anesthetic management of common thoracic surgeries, TEE is frequently used during lung transplantation and pulmonary thromboendarterectomy. Few clinical studies support current practice patterns, and most recommendations are based on expert opinion. SUMMARY: Currently, routine use of TEE in thoracic surgery is often limited to specific high-risk patients and/or procedures. As in other perioperative settings, TEE may be utilized to elucidate the reasons for acute hemodynamic instability without apparent cause. Contraindications to TEE apply and have to be taken into consideration before performing a TEE on a thoracic surgical patient.

Characteristics and Outcomes of Mechanically Ventilated COVID-19 Patients-An Observational Cohort Study.

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.

Diverse Injury Pathways Induce Alveolar Epithelial Cell CCL2/12, Which Promotes Lung Fibrosis.

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 bleomycin-induced 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.

Telomerase reverse transcriptase ameliorates lung fibrosis by protecting alveolar epithelial cells against senescence.

Mutations in the genes encoding telomerase reverse transcriptase (TERT) and telomerase's RNA components as well as shortened telomeres are risk factors for idiopathic pulmonary fibrosis, where repetitive injury to the alveolar epithelium is considered a key factor in pathogenesis. Given the importance of TERT in stem cells, we hypothesized that TERT plays an important role in epithelial repair and that its deficiency results in exacerbation of fibrosis by impairing this repair/regenerative process. To evaluate the role of TERT in epithelial cells, we generated type II alveolar epithelial cell (AECII)-specific TERT conditional knockout (SPC-Tert cKO) mice by crossing floxed Tert mice with inducible SPC-driven Cre mice. SPC-Tert cKO mice did not develop pulmonary fibrosis spontaneously up to 9 months of TERT deficiency. However, upon bleomycin treatment, they exhibited enhanced lung injury, inflammation, and fibrosis compared with control mice, accompanied by increased pro-fibrogenic cytokine expression but without a significant effect on AECII telomere length. Moreover, selective TERT deficiency in AECII diminished their proliferation and induced cellular senescence. These findings suggest that AECII-specific TERT deficiency enhances pulmonary fibrosis by heightening susceptibility to bleomycin-induced epithelial injury and diminishing epithelial regenerative capacity because of increased cellular senescence. We confirmed evidence for increased AECII senescence in idiopathic pulmonary fibrosis lungs, suggesting potential clinical relevance of the findings from our animal model. Our results suggest that TERT has a protective role in AECII, unlike its pro-fibrotic activity, observed previously in fibroblasts, indicating that TERT's role in pulmonary fibrosis is cell type-specific.

Loss of myeloid-specific protein phosphatase 2A enhances lung injury and fibrosis and results in IL-10-dependent sensitization of epithelial cell apoptosis.

Protein phosphatase 2A (PP2A), a ubiquitously expressed Ser/Thr phosphatase is an important regulator of cytokine signaling and cell function. We previously showed that myeloid-specific deletion of PP2A (LysMcrePP2A-/-) increased mortality in a murine peritoneal sepsis model. In the current study, we assessed the role of myeloid PP2A in regulation of lung injury induced by lipopolysaccharide (LPS) or bleomycin delivered intratracheally. LysMcrePP2A-/- mice experienced increased lung injury in response to both LPS and bleomycin. LysMcrePP2A-/- mice developed more exuberant fibrosis in response to bleomycin, elevated cytokine responses, and chronic myeloid inflammation. Bone marrow-derived macrophages (BMDMs) from LysMcrePP2A-/- mice showed exaggerated inflammatory cytokine release under conditions of both M1 and M2 activation. Notably, secretion of IL-10 was elevated under all stimulation conditions, including activation of BMDMs by multiple Toll-like receptor ligands. Supernatants collected from LPS-stimulated LysMcrePP2A-/- BMDMs induced epithelial cell apoptosis in vitro but this effect was mitigated when IL-10 was also depleted from the BMDMs by crossing LysMcrePP2A-/- mice with systemic IL-10-/- mice (LysMcrePP2A-/- × IL-10-/-) or when IL-10 was neutralized. Despite these findings, IL-10 did not directly induce epithelial cell apoptosis but sensitized epithelial cells to other mediators from the BMDMs. Taken together our results demonstrate that myeloid PP2A regulates production of multiple cytokines but that its effect is most pronounced on IL-10 production. Furthermore, IL-10 sensitizes epithelial cells to apoptosis in response to myeloid-derived mediators, which likely contributes to the pathogenesis of lung injury and fibrosis in this model.

Efferocytosis of apoptotic alveolar epithelial cells is sufficient to initiate lung fibrosis.

Type II alveolar epithelial cell (AEC) apoptosis is a prominent feature of fibrotic lung diseases and animal models of pulmonary fibrosis. While there is growing recognition of the importance of AEC injury and apoptosis as a causal factor in fibrosis, the underlying mechanisms that link these processes remain unknown. We have previously shown that targeting the type II alveolar epithelium for injury by repetitively administering diphtheria toxin to transgenic mice expressing the diphtheria toxin receptor off of the surfactant protein C promoter (SPC-DTR) develop lung fibrosis, confirming that AEC injury is sufficient to cause fibrosis. In the present study, we find that SPC-DTR mice develop increased activation of caspase 3/7 after initiation of diphtheria toxin treatment consistent with apoptosis within AECs. We also find evidence of efferocytosis, the uptake of apoptotic cells, by alveolar macrophages in this model. To determine the importance of efferocytosis in lung fibrosis, we treated cultured alveolar macrophages with apoptotic type II AECs and found that the uptake induced pro-fibrotic gene expression. We also found that the repetitive intrapulmonary administration of apoptotic type II AEC or MLE-12 cells induces lung fibrosis. Finally, mice lacking a key efferocytosis receptor, CD36, developed attenuated fibrosis in response to apoptotic MLE-12 cells. Collectively, these studies support a novel mechanism linking AEC apoptosis with macrophage pro-fibrotic activation via efferocytosis and reveal previously unrecognized therapeutic targets.

Discoidin Domain Receptor 2 Signaling Regulates Fibroblast Apoptosis through PDK1/Akt.

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 α2ß1 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-ß 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.

Lung epithelial cell focal adhesion kinase signaling inhibits lung injury and fibrosis.

Progressive pulmonary fibrosis is a devastating consequence of many acute and chronic insults to the lung. Lung injury leads to alveolar epithelial cell (AEC) death, destruction of the basement membrane, and activation of transforming growth factor-ß (TGF-ß). There is subsequent resolution of the injury and a coordinated and concurrent initiation of fibrosis. Both of these processes may involve activation of similar intracellular signaling pathways regulated in part by dynamic changes to the extracellular matrix. Matrix signaling can augment the profibrotic fibroblast response to TGF-ß. However, similar matrix/integrin signaling pathways may also be involved in the inhibition of ongoing TGF-ß-induced AEC apoptosis. Focal adhesion kinase (FAK) is an integrin-associated signaling molecule expressed by many cell types. We used mice with AEC-specific FAK deletion to isolate the epithelial aspect of integrin signaling in the bleomycin model of lung injury and fibrosis. Mice with AEC-specific deletion of FAK did not exhibit spontaneous lung injury but did have significantly greater terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling-positive cells (18.6 vs. 7.1) per ×200 field, greater bronchoalveolar lavage protein (3.2 vs. 1.8 mg/ml), and significantly greater death (77 vs. 19%) after bleomycin injury compared with littermate control mice. Within primary AECs, activated FAK directly associates with caspase-8 and inhibits activation of the caspase cascade resulting in less apoptosis in response to TGF-ß. Our studies support a model in which dynamic changes to the extracellular matrix after injury promote fibroblast activation and inhibition of epithelial cell apoptosis in response to TGF-ß through FAK activation potentially complicating attempts to nonspecifically target this pathway for antifibrotic therapy.

Fibroblast growth factors and pulmonary fibrosis: it's more complex than it sounds.

Lung fibrosis results from the cumulative effect of dysfunctional wound repair involving multiple cell types, including fibroblasts, epithelial cells, and macrophages responding to an array of soluble and matrix-mediated stimuli. Recent studies have shown that a tyrosine kinase inhibitor that targets FGF, VEGF, and PDGF receptors can slow the rate of decline in pulmonary function in patients with idiopathic pulmonary fibrosis. However, each of these growth factor families is comprised of multiple ligands and receptors with pleiotropic activities on different cell types such that their broad inhibition might have both pro-fibrotic and anti-fibrotic effects, limiting the potential therapeutic efficacy. Continued investigation and delineation of specific roles of individual proteins and receptors on different cell types hold promise for targeting specific pathways with precision and optimizing the potential efficacy of future approaches to lung fibrosis therapy. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Overexpression of inhibitor of DNA-binding 2 attenuates pulmonary fibrosis through regulation of c-Abl and Twist.

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 ß1-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.

Inhibition of myocardin-related transcription factor/serum response factor signaling decreases lung fibrosis and promotes mesenchymal cell apoptosis.

Myofibroblasts are crucial to the pathogenesis of tissue fibrosis. Their formation of stress fibers results in the release of myocardin-related transcription factor (MRTF), a transcriptional coactivator of serum response factor (SRF). MRTF-A (Mkl1)-deficient mice are protected from lung fibrosis. We hypothesized that the SRF/MRTF pathway inhibitor CCG-203971 would modulate myofibroblast function in vitro and limit lung fibrosis in vivo. Normal and idiopathic pulmonary fibrosis lung fibroblasts were treated with/without CCG-203971 (N-[4-chlorophenyl]-1-[3-(2-furanyl)benzoyl]-3-piperidine carboxamide) and/or Fas-activating antibody in the presence/absence of transforming growth factor (TGF)-ß1, and apoptosis was assessed. In vivo studies examined the effect of therapeutically administered CCG-203971 on lung fibrosis in two distinct murine models of fibrosis induced by bleomycin or targeted type II alveolar epithelial injury. In vitro, CCG-203971 prevented nuclear localization of MRTF-A; increased the apoptotic susceptibility of normal and idiopathic pulmonary fibrosis fibroblasts; blocked TGF-ß1-induced myofibroblast differentiation; and inhibited TGF-ß1-induced expression of fibronectin, X-linked inhibitor of apoptosis, and plasminogen activator inhibitor-1. TGF-ß1 did not protect fibroblasts or myofibroblasts from apoptosis in the presence of CCG-203971. In vivo, CCG-203971 significantly reduced lung collagen content in both murine models while decreasing alveolar plasminogen activator inhibitor-1 and promoting myofibroblast apoptosis. These data support a central role of the SRF/MRTF pathway in the pathobiology of lung fibrosis and suggest that its inhibition can help resolve lung fibrosis by promoting fibroblast apoptosis.

Fibrocytes are not an essential source of type I collagen during lung fibrosis.

Progressive fibrosis involves accumulation of activated collagen-producing mesenchymal cells. Fibrocytes are hematopoietic-derived cells with mesenchymal features that potentially have a unique and critical function during fibrosis. Fibrocytes have been proposed as an important direct contributor of type I collagen deposition during fibrosis based largely on fate-mapping studies. To determine the functional contribution of hematopoietic cell-derived type I collagen to fibrogenesis, we use a double-transgenic system to specifically delete the type I collagen gene across a broad population of hematopoietic cells. These mice develop a robust fibrotic response similar to littermate genotype control mice injured with bleomycin indicating that fibrocytes are not a necessary source of type I collagen. Using collagen-promoter GFP mice, we find that fibrocytes express type I collagen. However, fibrocytes with confirmed deletion of the type I collagen gene have readily detectable intracellular type I collagen indicating that uptake of collagen from neighboring cells account for much of the fibrocyte collagen. Collectively, these results clarify several seemingly conflicting reports regarding the direct contribution of fibrocytes to collagen deposition.

Activated alveolar epithelial cells initiate fibrosis through autocrine and paracrine secretion of connective tissue growth factor.

Fibrogenesis involves a pathological accumulation of activated fibroblasts and extensive matrix remodeling. Profibrotic cytokines, such as TGF-ß, stimulate fibroblasts to overexpress fibrotic matrix proteins and induce further expression of profibrotic cytokines, resulting in progressive fibrosis. Connective tissue growth factor (CTGF) is a profibrotic cytokine that is indicative of fibroblast activation. Epithelial cells are abundant in the normal lung, but their contribution to fibrogenesis remains poorly defined. Profibrotic cytokines may activate epithelial cells with protein expression and functions that overlap with the functions of active fibroblasts. We found that alveolar epithelial cells undergoing TGF-ß-mediated mesenchymal transition in vitro were also capable of activating lung fibroblasts through production of CTGF. Alveolar epithelial cell expression of CTGF was dramatically reduced by inhibition of Rho signaling. CTGF reporter mice demonstrated increased CTGF promoter activity by lung epithelial cells acutely after bleomycin in vivo. Furthermore, mice with lung epithelial cell-specific deletion of CTGF had an attenuated fibrotic response to bleomycin. These studies provide direct evidence that epithelial cell activation initiates a cycle of fibrogenic effector cell activation during progressive fibrosis. Therapy targeted at epithelial cell production of CTGF offers a novel pathway for abrogating this progressive cycle and limiting tissue fibrosis.

Paracrine functions of fibrocytes to promote lung fibrosis.

Fibrocytes are derived from the bone marrow and are found in the circulation. They can be recruited to sites of injury and contribute to repair/remodeling. In vitro evidence suggests that fibrocytes may differentiate into fibroblasts to promote lung fibrosis. However, in vivo evidence for this is sparse. This review summarizes recent literature which may suggest that fibrocytes function to promote fibrosis via paracrine actions. In this way, secretion of growth factors, proteases and matricellular proteins may strongly influence the actions of resident epithelial and mesenchymal cells to promote repair and resolution or to tip the scale toward pathologic remodeling.

Axin pathway activity regulates in vivo pY654-ß-catenin accumulation and pulmonary fibrosis.

Epithelial to mesenchymal transition (EMT) and pulmonary fibrogenesis require epithelial integrin α3ß1-mediated cross-talk between TGFß1 and Wnt signaling pathways. One hallmark of this cross-talk is pY654-ß-catenin accumulation, but whether pY654-ß-catenin is a biomarker of fibrogenesis or functionally important is unknown. To clarify further the role of ß-catenin in fibrosis, we explored pY654-ß-catenin generation and function. α3ß1 was required for TGFß1-mediated activation of Src family kinases, and Src inhibition blocked both pY654 and EMT in primary alveolar epithelial cells (AECs). TGFß1 stimulated ß-catenin/Lef1-dependent promoter activity comparably in immortalized AECs stably expressing WT ß-catenin as well as Y654E or Y654F ß-catenin point mutants. But EMT was abrogated in the Tyr to Phe mutant. pY654-ß-catenin was sensitive to the axin ß-catenin turnover pathway as inhibition of tankyrase 1 led to high AEC axin levels, loss of pY654-ß-catenin, and inhibition of EMT ex vivo. Mice given a tankyrase inhibitor (50 mg/kg orally) daily for 7 days beginning 10 days after intratracheal bleomycin had improved survival over controls. Treated mice developed raised axin levels in the lung that abrogated pY654-ß-catenin and attenuated lung Snail1, Twist1, α-smooth muscle actin, and type I collagen accumulation. Total ß-catenin levels were unaltered. These findings identify Src kinase(s) as a mediator of TGFß1-induced pY654-ß-catenin, provide evidence that pY654-ß-catenin levels are a critical determinant of EMT and fibrogenesis, and suggest regulation of axin levels as a novel therapeutic approach to fibrotic disorders.

Alveolar epithelial cells express mesenchymal proteins in patients with idiopathic pulmonary fibrosis.

Prior work has shown that transforming growth factor-ß (TGF-ß) can mediate transition of alveolar type II cells into mesenchymal cells in mice. Evidence this occurs in humans is limited to immunohistochemical studies colocalizing epithelial and mesenchymal proteins in sections of fibrotic lungs. To acquire further evidence that epithelial-to-mesenchymal transition occurs in the lungs of patients with idiopathic pulmonary fibrosis (IPF), we studied alveolar type II cells isolated from fibrotic and normal human lung. Unlike normal type II cells, type II cells isolated from the lungs of patients with IPF express higher levels of mRNA for the mesenchymal proteins type I collagen, α-smooth muscle actin (α-SMA), and calponin. When cultured on Matrigel/collagen, human alveolar type II cells maintain a cellular morphology consistent with epithelial cells and expression of surfactant protein C (SPC) and E-cadherin. In contrast, when cultured on fibronectin, the human type II cells flatten, spread, lose expression of pro- SPC, and increase expression of vimentin, N-cadherin, and α-SMA; markers of mesenchymal cells. Addition of a TGF-ß receptor kinase inhibitor (SB431542) to cells cultured on fibronectin inhibited vimentin expression and maintained pro-SPC expression, indicating persistence of an epithelial phenotype. These data suggest that alveolar type II cells can acquire features of mesenchymal cells in IPF lungs and that TGF-ß can mediate this process.