Thy-1-Integrin Interactions in cis and Trans Mediate Distinctive Signaling.

Thy-1 is a cell surface glycosylphosphatidylinositol (GPI)-anchored glycoprotein that bears a broad mosaic of biological roles across various cell types. Thy-1 displays strong physiological and pathological implications in development, cancer, immunity, and tissue fibrosis. Quite uniquely, Thy-1 is capable of mediating integrin-related signaling through direct trans- and cis-interaction with integrins. Both interaction types have shown distinctive roles, even when interacting with the same type of integrin, where binding in trans or in cis often yields divergent signaling events. In this review, we will revisit recent progress and discoveries of Thy-1-integrin interactions in trans and in cis, highlight their pathophysiological consequences and explore other potential binding partners of Thy-1 within the integrin regulation/signaling paradigm.

Interstitial lung disease in children with Rubinstein-Taybi syndrome.

INTRODUCTION: Rubinstein-Taybi syndrome (RSTS) is a rare genetic syndrome caused primarily by a mutation in the CREBBP gene found on chromosome 16. Patients with RSTS are at greater risk for a variety of medical problems, including upper airway obstruction and aspiration. Childhood interstitial lung disease (ILD) thus far has not been definitively linked to RSTS. Here we present three patients with RSTS who developed ILD and discuss possible mechanisms by which a mutation in CREBBP may be involved in the development of ILD. METHODS: Routine hematoxylin and eosin staining was performed on lung biopsy tissue for histological analysis. Immunofluorescent staining was performed on lung biopsy tissue for markers of fibrosis, surfactant deficiency and histone acetylation. Cases 1 and 2 had standard clinical microarray analysis. Case 3 had whole exome sequencing. Bioinformatics analyses were performed to identify possible causative genes using ToppGene. RESULTS: Computed tomography images in all cases showed consolidated densities overlying ground glass opacities. Lung histopathology revealed accumulation of proteinaceous material within alveolar spaces, evidence of fibrosis, and increased alveolar macrophages. Immunofluorescent staining showed increase in surfactant protein C staining, patchy areas of increased anti-smooth muscle antibody staining, and increased staining for acetylated histone 2 and histone 3 lysine 9. DISCUSSION: Clinical characteristics, radiographic imaging, lung histopathology, and immunofluorescent staining results shared by all cases demonstrated findings consistent with ILD. Immunofluorescent staining suggests two possible mechanisms for the development of ILD: abnormal surfactant metabolism and/or persistent activation of myofibroblasts. These two pathways could be related to dysfunctional CREBBP protein.

Cooperative signaling between integrins and growth factor receptors in fibrosis.

Fibrosis is a pathological process characterized by accumulation of fibrous connective tissue in organs, leading to organ malfunction and failure. At the cellular level, tissue injury or cellular stress results in aberrant and/or sustained fibroblast "activation" leading to excessive extracellular matrix (ECM) accumulation and remodeling, as well as abnormal crosstalk with other cell types. Fibroblast functions within the fibrotic milieu are broad and complex, but among the most prominent are regulation of tissue architecture via modulation of ECM deposition and synthesis, and production of, activation of, and response to growth factors. Thus, both integrins and growth factor receptors (GFRs) play critical roles in fibroblast orchestration of tissue remodeling. However, the interplay between integrins and GFRs in this context is not fully understood. Their interaction has been described for other diseases, such as cancer. Here, we review the literature relevant to integrin/GFR interactions in the context of fibrosis, classify the known interactions into broad categories, and discuss research opportunities that may yield novel therapeutic targets for a broad range of debilitating chronic diseases.

Loss of Thy-1 may reduce lung regeneration after pneumonectomy in mice.

BACKGROUND: Lung regeneration plays an important role in lung repair after injury. It is reliant upon proliferation of multiple cell types in the lung, including endothelium, epithelium, and fibroblasts, as well as remodeling of the extracellular matrix. METHODS: Lung regeneration following injury progresses via an initial inflammatory response during which macrophages clear the tissue of cellular debris. This process continues through cellular proliferation when existing cells and progenitors act to repopulate cells lost during injury, followed by tissue maturation in which newly formed cells achieve a differentiated phenotype. RESULTS: Signaling pathways critical for lung regeneration include FGF, EGF, WNT, and NOTCH. In addition, HDACs, miRNAs, ELASTIN, and MMP14 have been shown to regulate lung regeneration. Partial pneumonectomy (PNX) has been used as a therapeutic and investigational tool for several decades. Following PNX the remaining lung increases in size to compensate for loss of volume and respiratory capacity. CONCLUSIONS: Much has been learned about the triggers and mechanisms regulating pulmonary regeneration. However, the role of thymocyte differentiation antigen-1 (Thy-1) in post-PNX lung growth remains incompletely characterized. Thy-1 is a phosphatidylinositol glycoprotein with a relative molecular weight of 25000~37000 Da, which is expressed in almost all types of fibroblasts and regulates many biological functions. It not only supports the structure of fibroblasts, but also can balance cell proliferation, migration and regulate the synthesis of immune inflammatory mediators.

Pulmonary Complications of Pediatric Hematopoietic Cell Transplantation. A National Institutes of Health Workshop Summary.

Approximately 2,500 pediatric hematopoietic cell transplants (HCTs), most of which are allogeneic, are performed annually in the United States for life-threatening malignant and nonmalignant conditions. Although HCT is undertaken with curative intent, post-HCT complications limit successful outcomes, with pulmonary dysfunction representing the leading cause of nonrelapse mortality. To better understand, predict, prevent, and/or treat pulmonary complications after HCT, a multidisciplinary group of 33 experts met in a 2-day National Institutes of Health Workshop to identify knowledge gaps and research strategies most likely to improve outcomes. This summary of Workshop deliberations outlines the consensus focus areas for future research.

Childhood rare lung disease in the 21st century: "-omics" technology advances accelerating discovery.

Childhood rare lung diseases comprise a large number of heterogeneous respiratory disorders that are individually rare but are collectively associated with substantial morbidity, mortality, and healthcare resource utilization. Although the genetic mechanisms for several of these disorders have been elucidated, the pathogenesis mechanisms for others remain poorly understood and treatment options remain limited. Childhood rare lung diseases are enriched for genetic etiologies; identification of the disease mechanisms underlying these rare disorders can inform the biology of normal human lung development and has implications for the treatment of more common respiratory diseases in children and adults. Advances in "-omics" technology, such as genomic sequencing, clinical phenotyping, biomarker discovery, genome editing, in vitro and model organism disease modeling, single-cell analyses, cellular imaging, and high-throughput drug screening have enabled significant progress for diagnosis and treatment of rare childhood lung diseases. The most striking example of this progress has been realized for patients with cystic fibrosis for whom effective, personalized therapies based on CFTR genotype are now available. In this chapter, we focus on recent technology advances in childhood rare lung diseases, acknowledge persistent challenges, and identify promising new technologies that will impact not only biological discovery, but also improve diagnosis, therapies, and survival for children with these rare disorders.

Therapeutic Use of Extracellular Vesicles for Acute and Chronic Lung Disease.

Multipotent mesenchymal stem cells (MSCs) possess regenerative properties and have been shown to improve outcomes and survival in acute and chronic lung diseases, but there have been some safety concerns raised related to MSC-based therapy. Subsequent studies have demonstrated that many of the regenerative effects of MSCs can be attributed to the MSC-derived secretome, which contains soluble factors and extracellular vesicles (EVs). MSC-derived extracellular vesicles (MSC-derived EVs) replicate many of the beneficial effects of MSCs and contain a variety of bioactive factors that are transferred to recipient cells, mediating downstream signaling. MSC-derived EV therapy holds promise as a safe and effective treatment for pulmonary disease, but there remain many scientific and clinical questions that will need to be addressed before EVs are widely applied as a therapy. To date, the use of MSC-derived EVs as a treatment for lung disease has been conducted primarily in in vitro or pre-clinical animal models. In this review, we will discuss the current published research investigating the use of EVs as a potential therapeutic for acute lung injury/acute respiratory distress syndrome (ALI/ARDS), bronchopulmonary dysplasia (BPD), idiopathic pulmonary fibrosis (IPF), pulmonary arterial hypertension (PAH), asthma, and silicosis.

Neuroendocrine Cell Hyperplasia of Infancy. Clinical Score and Comorbidities.

Rationale: Neuroendocrine cell hyperplasia of infancy (NEHI) is an important form of children's interstitial and diffuse lung disease for which the diagnostic strategy has evolved. The prevalence of comorbidities in NEHI that may influence treatment has not been previously assessed.Objectives: To evaluate a previously unpublished NEHI clinical score for assistance in diagnosis of NEHI and to assess comorbidities in NEHI.Methods: We performed a retrospective chart review of 199 deidentified patients with NEHI from 11 centers. Data were collected in a centralized Research Electronic Data Capture registry and we performed descriptive statistics.Results: The majority of patients with NEHI were male (66%). The sensitivity of the NEHI Clinical Score was 87% (95% confidence interval [CI], 0.82-0.91) for all patients from included centers and 93% (95% CI, 0.86-0.97) for those with complete scores (e.g., no missing data). Findings were similar when we limited the population to the 75 patients diagnosed by lung biopsy (87%; 95% CI, 0.77-0.93). Of those patients evaluated for comorbidities, 51% had gastroesophageal reflux, 35% had aspiration or were at risk for aspiration, and 17% had evidence of immune system abnormalities.Conclusions: The NEHI Clinical Score is a sensitive tool for clinically evaluating NEHI; however, its specificity has not yet been addressed. Clinicians should consider evaluating patients with NEHI for comorbidities, including gastroesophageal reflux, aspiration, and immune system abnormalities, because these can contribute to the child's clinical picture and may influence clinical course and treatment.

αvß3 Integrin drives fibroblast contraction and strain stiffening of soft provisional matrix during progressive fibrosis.

Fibrosis is characterized by persistent deposition of extracellular matrix (ECM) by fibroblasts. Fibroblast mechanosensing of a stiffened ECM is hypothesized to drive the fibrotic program; however, the spatial distribution of ECM mechanics and their derangements in progressive fibrosis are poorly characterized. Importantly, fibrosis presents with significant histopathological heterogeneity at the microscale. Here, we report that fibroblastic foci (FF), the regions of active fibrogenesis in idiopathic pulmonary fibrosis (IPF), are surprisingly of similar modulus as normal lung parenchyma and are nonlinearly elastic. In vitro, provisional ECMs with mechanical properties similar to those of FF activate both normal and IPF patient-derived fibroblasts, whereas type I collagen ECMs with similar mechanical properties do not. This is mediated, in part, by αvß3 integrin engagement and is augmented by loss of expression of Thy-1, which regulates αvß3 integrin avidity for ECM. Thy-1 loss potentiates cell contractility-driven strain stiffening of provisional ECM in vitro and causes elevated αvß3 integrin activation, increased fibrosis, and greater mortality following fibrotic lung injury in vivo. These data suggest a central role for αvß3 integrin and provisional ECM in overriding mechanical cues that normally impose quiescent phenotypes, driving progressive fibrosis through physical stiffening of the fibrotic niche.

Thy-1 dependent uptake of mesenchymal stem cell-derived extracellular vesicles blocks myofibroblastic differentiation.

Bone marrow-derived mesenchymal stem cells (MSC) have been promoted for multiple therapeutic applications. Many beneficial effects of MSCs are paracrine, dependent on extracellular vesicles (EVs). Although MSC-derived EVs (mEVs) are beneficial for acute lung injury and pulmonary fibrosis, mechanisms of mEV uptake by lung fibroblasts and their effects on myofibroblastic differentiation have not been established. We demonstrate that mEVs, but not fibroblast EVs (fEVs), suppress TGFß1-induced myofibroblastic differentiation of normal and idiopathic pulmonary fibrosis (IPF) lung fibroblasts. MEVs display increased time- and dose-dependent cellular uptake compared to fEVs. Removal or blocking of Thy-1, or blocking Thy-1-beta integrin interactions, decreased mEV uptake and prevented suppression of myofibroblastic differentiation. MicroRNAs (miRs) 199a/b-3p, 21-5p, 630, 22-3p, 196a-5p, 199b-5p, 34a-5p and 148a-3p are selectively packaged in mEVs. In silico analyses indicated that IPF lung fibroblasts have increased expression of genes that are targets of mEV-enriched miRs. MiR-630 mimics blocked TGFß1 induction of CDH2 in normal and IPF fibroblasts, and antagomiR-630 abrogated the effect of mEV on CDH2 expression. These data suggest that the interaction of Thy-1 with beta integrins mediates mEV uptake by lung fibroblasts, which blocks myofibroblastic differentiation, and that mEVs are enriched for miRs that target profibrotic genes up-regulated in IPF fibroblasts.

Rac2 is required for alternative macrophage activation and bleomycin induced pulmonary fibrosis; a macrophage autonomous phenotype.

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease characterized by cellular phenotype alterations and deposition of extracellular matrix proteins. The alternative activation of macrophages in the lungs has been associated as a major factor promoting pulmonary fibrosis, however the mechanisms underlying this phenomenon are poorly understood. In the present study, we have defined a molecular mechanism by which signals transmitted from the extracellular matrix via the α4ß1 integrin lead to the activation of Rac2 which regulates alternative macrophage differentiation, a signaling axis within the pulmonary macrophage compartment required for bleomycin induced pulmonary fibrosis. Mice deficient in Rac2 were protected against bleomycin-induced fibrosis and displayed diminished collagen deposition in association with lower expression of alternatively activated profibrotic macrophage markers. We have demonstrated a macrophage autonomous process by which the injection of M2 and not M1 macrophages restored the bleomycin induced pulmonary fibrosis susceptibility in Rac2-/- mice, establishing a critical role for a macrophage Rac2 signaling axis in the regulation of macrophage differentiation and lung fibrosis in vivo. We also demonstrate that markers of alternative macrophage activation are increased in patients with IPF. Taken together, these studies define an important role for an integrin-driven Rac2 signaling axis in macrophages, and reveal that Rac2 activation is required for polarization of macrophages towards a profibrotic phenotype and progression of pulmonary fibrosis in vivo.

Thy-1 interaction with Fas in lipid rafts regulates fibroblast apoptosis and lung injury resolution.

Thy-1-negative lung fibroblasts are resistant to apoptosis. The mechanisms governing this process and its relevance to fibrotic remodeling remain poorly understood. By using either sorted or transfected lung fibroblasts, we found that Thy-1 expression is associated with downregulation of anti-apoptotic molecules Bcl-2 and Bcl-xL, as well as increased levels of cleaved caspase-9. Addition of rhFasL and staurosporine, well-known apoptosis inducers, caused significantly increased cleaved caspase-3, -8, and PARP in Thy-1-transfected cells. Furthermore, rhFasL induced Fas translocation into lipid rafts and its colocalization with Thy-1. These in vitro results indicate that Thy-1, in a manner dependent upon its glycophosphatidylinositol anchor and lipid raft localization, regulates apoptosis in lung fibroblasts via Fas-, Bcl-, and caspase-dependent pathways. In vivo, Thy-1 deficient (Thy1-/-) mice displayed persistence of myofibroblasts in the resolution phase of bleomycin-induced fibrosis, associated with accumulation of collagen and failure of lung fibrosis resolution. Apoptosis of myofibroblasts is decreased in Thy1-/- mice in the resolution phase. Collectively, these findings provide new evidence regarding the role and mechanisms of Thy-1 in initiating myofibroblast apoptosis that heralds the termination of the reparative response to bleomycin-induced lung injury. Understanding the mechanisms regulating fibroblast survival/apoptosis should lead to novel therapeutic interventions for lung fibrosis.

Risk and impact of pulmonary complications in survivors of childhood cancer: A report from the Childhood Cancer Survivor Study.

BACKGROUND: Pulmonary complications after cancer therapy are varied. This study describes pulmonary outcomes among childhood cancer survivors and evaluates their impact on daily activities. METHODS: The incidence of pulmonary outcomes (asthma, chronic cough, emphysema, lung fibrosis, oxygen need, and recurrent pneumonia) reported among 5-year cancer survivors (n = 14,316) and the incidence of death due to pulmonary causes among all eligible survivors (n = 20,690) in the Childhood Cancer Survivor Study were compared with those for sibling controls (n = 4027) with cumulative incidence, standardized mortality ratio (SMR), and piecewise exponential models. Logistic regression with random effects was used to estimate odds ratios (ORs) and 95% confidence intervals (CIs) for activity limitations with pulmonary complications. RESULTS: By the age of 45 years, the cumulative incidence of any pulmonary condition was 29.6% (95% CI, 29.1%-30.0%) for cancer survivors and 26.5% (95% CI, 24.9%-28.0%) for siblings. Fewer survivors reported ever smoking (23.6% vs 36.4%, P < .001), but survivors were more likely to report chronic cough (rate ratio [RR], 1.6; 95% CI, 1.4-1.9), oxygen need (RR, 1.8; 95% CI, 1.5-2.2), lung fibrosis (RR, 3.5; 95% CI, 2.3-5.4), and recurrent pneumonia (RR, 2.0; 95% CI, 1.4-3.0). The SMR for death due to pulmonary causes was 5.9 (95% CI, 4.2-8.1), and it was associated with platinum exposure and lung radiation (P < .01). The impact of chronic cough on daily activities for survivors (OR vs survivors without chronic cough, 2.7) was greater than that for siblings (OR, 2.0; P = .04). CONCLUSIONS: Pulmonary complications are substantial among adult survivors of childhood cancer and can affect daily activities. Cancer 2016;122:3687-96. © 2016 American Cancer Society.

Cigarette Smoke Enhances the Expression of Profibrotic Molecules in Alveolar Epithelial Cells.

Idiopathic pulmonary fibrosis (IPF) is a progressive and lethal disease of unknown etiology. A growing body of evidence indicates that it may result from an aberrant activation of alveolar epithelium, which induces the expansion of the fibroblast population, their differentiation to myofibroblasts and the excessive accumulation of extracellular matrix. The mechanisms that activate the alveolar epithelium are unknown, but several studies indicate that smoking is the main environmental risk factor for the development of IPF. In this study we explored the effect of cigarette smoke on the gene expression profile and signaling pathways in alveolar epithelial cells. Lung epithelial cell line from human (A549), was exposed to cigarette smoke extract (CSE) for 1, 3, and 5 weeks at 1, 5 and 10% and gene expression was evaluated by complete transcriptome microarrays. Signaling networks were analyzed with the Ingenuity Pathway Analysis software. At 5 weeks of exposure, alveolar epithelial cells acquired a fibroblast-like phenotype. At this time, gene expression profile revealed a significant increase of more than 1000 genes and deregulation of canonical signaling pathways such as TGF-ß and Wnt. Several profibrotic genes involved in EMT were over-expressed, and incomplete EMT was observed in these cells, and corroborated in mouse (MLE-12) and rat (RLE-6TN) epithelial cells. The secretion of activated TGF-ß1 increased in cells exposed to cigarette smoke, which decreased when the integrin alpha v gene was silenced. These findings suggest that the exposure of alveolar epithelial cells to CSE induces the expression and release of a variety of profibrotic genes, and the activation of TGF-ß1, which may explain at least partially, the increased risk of developing IPF in smokers.

Conformational coupling of integrin and Thy-1 regulates Fyn priming and fibroblast mechanotransduction.

Progressive fibrosis is characterized by excessive deposition of extracellular matrix (ECM), resulting in gross alterations in tissue mechanics. Changes in tissue mechanics can further augment scar deposition through fibroblast mechanotransduction. In idiopathic pulmonary fibrosis, a fatal form of progressive lung fibrosis, previous work has shown that loss of Thy-1 (CD90) expression in fibroblasts correlates with regions of active fibrogenesis, thus representing a pathologically relevant fibroblast subpopulation. We now show that Thy-1 is a regulator of fibroblast rigidity sensing. Thy-1 physically couples to inactive αvß3 integrins via its RGD-like motif, altering baseline integrin avidity to ECM ligands and also facilitating preadhesion clustering of integrin and membrane rafts via Thy-1's glycophosphatidylinositol tether. Disruption of Thy-1-αvß3 coupling altered recruitment of Src family kinases to adhesion complexes and impaired mechanosensitive, force-induced Rho signaling, and rigidity sensing. Loss of Thy-1 was sufficient to induce myofibroblast differentiation in soft ECMs and may represent a physiological mechanism important in wound healing and fibrosis.

Matrix metalloproteinases as therapeutic targets for idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a restrictive lung disease that is associated with high morbidity and mortality. Current medical therapies are not fully effective at limiting mortality in patients with IPF, and new therapies are urgently needed. Matrix metalloproteinases (MMPs) are proteinases that, together, can degrade all components of the extracellular matrix and numerous nonmatrix proteins. MMPs and their inhibitors, tissue inhibitors of MMPs (TIMPs), have been implicated in the pathogenesis of IPF based upon the results of clinical studies reporting elevated levels of MMPs (including MMP-1, MMP-7, MMP-8, and MMP-9) in IPF blood and/or lung samples. Surprisingly, studies of gene-targeted mice in murine models of pulmonary fibrosis (PF) have demonstrated that most MMPs promote (rather than inhibit) the development of PF and have identified diverse mechanisms involved. These mechanisms include MMPs: (1) promoting epithelial-to-mesenchymal transition (MMP-3 and MMP-7); (2) increasing lung levels or activity of profibrotic mediators or reducing lung levels of antifibrotic mediators (MMP-3, MMP-7, and MMP-8); (3) promoting abnormal epithelial cell migration and other aberrant repair processes (MMP-3 and MMP-9); (4) inducing the switching of lung macrophage phenotypes from M1 to M2 types (MMP-10 and MMP-28); and (5) promoting fibrocyte migration (MMP-8). Two MMPs, MMP-13 and MMP-19, have antifibrotic activities in murine models of PF, and two MMPs, MMP-1 and MMP-10, have the potential to limit fibrotic responses to injury. Herein, we review what is known about the contributions of MMPs and TIMPs to the pathogenesis of IPF and discuss their potential as therapeutic targets for IPF.

Thy-1 modulates neurological cell-cell and cell-matrix interactions through multiple molecular interactions.

Thy-1, or CD90, is a glycosylphosphatidylinositol-linked cell surface glycoprotein expressed on multiple cell types, including neurons, thymocytes, fibroblasts, endothelial cells, mesangial cells, and some hematopoietic and stromal stem cells. Thy-1 is developmentally regulated and evolutionarily conserved. Its cellular effects vary between and in some cases within cell types, tissues, and species, indicating that its biological role is context dependent. However, it most often seems to affect cell-cell or cell-matrix interactions and cellular adhesion and migration. In the nervous system, Thy-1 mediates bidirectional cell-cell communication, which modulates cell-matrix adhesion. Neurons express high levels of Thy-1, which interacts with alpha(v)beta3 integrin present in astrocytes and stimulates increased astrocyte adhesion to the underlying surface (trans signaling) and in neurites, the same ligand-receptor association triggers neurite retraction and inhibition of axonal growth (cis signaling). Although Thy-1 lacks a cytoplasmic domain, it affects multiple intracellular signaling cascades through interaction with a number of molecules within lipid raft microdomains. Improved understanding of how this enigmatic adhesion molecule modulates signaling and cell phenotype may yield novel insights into neurodevelopment and nerve recovery after injury.