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.

Inhibition of discoidin domain receptor 2 reveals kinase-dependent and kinase-independent functions in regulating fibroblast activity.

Progressive pulmonary fibrosis is a devastating condition and current treatment is suboptimal. There has been considerable interest in the role of tyrosine kinase signaling as mediators of pro- and antifibrotic processes. Nintedanib is a nonspecific tyrosine kinase that has been shown to have therapeutic benefit in lung fibrosis. However, the precise mechanism of action remains unclear because nintedanib inhibits several tyrosine kinases, which are often expressed on multiple cell types with different activities during fibrosis. Discoidin domain receptor 2 (DDR2) has been suggested as a potential target of nintedanib. DDR2 is a receptor tyrosine kinase that is activated by fibrillar collagens such as type I collagen. DDR2 is primarily expressed by fibroblasts. The effectiveness of specifically targeting DDR2 signaling during fibrosis remains undefined. In the present study, we show that nintedanib acts as a direct and indirect inhibitor of DDR2. We then utilize a novel allosteric inhibitor of DDR2, WRG-28, which blocks ligand binding and activation of DDR2. We find that WRG-28 augments fibroblast apoptosis and attenuates fibrosis. Finally, we show that fibroblast type I collagen autocrine signaling is regulated by DDR2 through both kinase-dependent and kinase-independent functions of DDR2. These findings highlight the importance of type I collagen autocrine signaling by fibroblasts during fibrosis and demonstrate that DDR2 has a central role in this pathway making it a potential therapeutic target.NEW & NOTEWORTHY Type I collagen is a major component of fibrosis and can signal through cell surface receptors such as discoidin domain receptor 2 (DDR2). DDR2 activation can lead to further collagen deposition by fibroblasts setting up a profibrotic positive feedback loop. In this report, we find that inhibition of DDR2 with nintedanib or a specific DDR2 inhibitor, WRG-28, can disrupt this cycle and prevent fibrosis through augmented fibroblast apoptosis and inhibited activation.

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.

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.

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.

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.

Type I Collagen Signaling Regulates Opposing Fibrotic Pathways through α2ß1 Integrin.

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-ß (transforming growth factor-ß) 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-ß. In the present study, we find that type I collagen signaling promotes TGF-ß-mediated fibroblast activation and inhibits TGF-ß-induced AEC death. Fibroblasts cultured on type I collagen or fibrotic decellularized lung matrix had augmented activation in response to TGF-ß, whereas AECs on cultured on type I collagen or fibrotic lung matrix were more resistant to TGF-ß-induced apoptosis. Both of these responses were mediated by integrin α2ß1, 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.

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.