Endogenous PGI2 signaling through IP inhibits neutrophilic lung inflammation in LPS-induced acute lung injury mice model.

Endogenous prostaglandin I2 (PGI2) has inhibitory effects on immune responses against pathogens or allergens; however, the immunomodulatory activity of endogenous PGI2 signaling in endotoxin-induced inflammation is unknown. To test the hypothesis that endogenous PGI2 down-regulates endotoxin-induced lung inflammation, C57BL/6 wild type (WT) and PGI2 receptor (IP) KO mice were challenged intranasally with LPS. Urine 6-keto-PGF1α, a stable metabolite of PGI2, was significantly increased following the LPS-challenge, suggesting that endogenous PGI2 signaling modulates the host response to LPS-challenge. IPKO mice had a significant increase in neutrophils in the BAL fluid as well as increased proteins of KC, LIX, and TNF-α in lung homogenates compared with WT mice. In contrast, IL-10 was decreased in LPS-challenged IPKO mice compared with WT mice. The PGI2 analog cicaprost significantly decreased LPS-induced KC, and TNF-α, but increased IL-10 and AREG in bone marrow-derived dendritic cells (BMDCs) and bone marrow-derived macrophages (BMMs) compared with vehicle-treatment. These results indicated that endogenous PGI2 signaling attenuated neutrophilic lung inflammation through the reduced inflammatory cytokine and chemokine and enhanced IL-10.

Secretory IgA Deficiency in Individual Small Airways Is Associated with Persistent Inflammation and Remodeling.

RATIONALE: Maintenance of a surface immune barrier is important for homeostasis in organs with mucosal surfaces that interface with the external environment; however, the role of the mucosal immune system in chronic lung diseases is incompletely understood. OBJECTIVES: We examined the relationship between secretory IgA (SIgA) on the mucosal surface of small airways and parameters of inflammation and airway wall remodeling in chronic obstructive pulmonary disease (COPD). METHODS: We studied 1,104 small airways (<2 mm in diameter) from 50 former smokers with COPD and 39 control subjects. Small airways were identified on serial tissue sections and examined for epithelial morphology, SIgA, bacterial DNA, nuclear factor-κB activation, neutrophil and macrophage infiltration, and airway wall thickness. MEASUREMENTS AND MAIN RESULTS: Morphometric evaluation of small airways revealed increased mean airway wall thickness and inflammatory cell counts in lungs from patients with COPD compared with control subjects, whereas SIgA level on the mucosal surface was decreased. However, when small airways were classified as SIgA intact or SIgA deficient, we found that pathologic changes were localized almost exclusively to SIgA-deficient airways, regardless of study group. SIgA-deficient airways were characterized by (1) abnormal epithelial morphology, (2) invasion of bacteria across the apical epithelial barrier, (3) nuclear factor-κB activation, (4) accumulation of macrophages and neutrophils, and (5) fibrotic remodeling of the airway wall. CONCLUSIONS: Our findings support the concept that localized, acquired SIgA deficiency in individual small airways of patients with COPD allows colonizing bacteria to cross the epithelial barrier and drive persistent inflammation and airway wall remodeling, even after smoking cessation.

Endothelial HIF signaling regulates pulmonary fibrosis-associated pulmonary hypertension.

Pulmonary hypertension (PH) complicating chronic parenchymal lung disease, such as idiopathic pulmonary fibrosis, results in significant morbidity and mortality. Since the hypoxia-inducible factor (HIF) signaling pathway is important for development of pulmonary hypertension in chronic hypoxia, we investigated whether HIF signaling in vascular endothelium regulates development of PH related to pulmonary fibrosis. We generated a transgenic model in which HIF is deleted within vascular endothelial cells and then exposed these mice to chronic intraperitoneal bleomycin to induce PH associated with lung fibrosis. Although no differences in the degree of fibrotic remodeling were observed, we found that endothelial HIF-deficient mice were protected against development of PH, including right ventricle and pulmonary vessel remodeling. Similarly, endothelial HIF-deficient mice were protected from PH after a 4-wk exposure to normobaric hypoxia. In vitro studies of pulmonary vascular endothelial cells isolated from the HIF-targeted mice and controls revealed that endothelial HIF signaling increases endothelial cell expression of connective tissue growth factor, enhances vascular permeability, and promotes pulmonary artery smooth muscle cell proliferation and wound healing ability, all of which have the potential to impact the development of PH in vivo. Taken together, these studies demonstrate that vascular endothelial cell HIF signaling is necessary for development of hypoxia and pulmonary fibrosis associated PH. As such, HIF and HIF-regulated targets represent a therapeutic target in these conditions.

Extensive phenotyping of individuals at risk for familial interstitial pneumonia reveals clues to the pathogenesis of interstitial lung disease.

RATIONALE: Asymptomatic relatives of patients with familial interstitial pneumonia (FIP), the inherited form of idiopathic interstitial pneumonia, carry increased risk for developing interstitial lung disease. OBJECTIVES: Studying these at-risk individuals provides a unique opportunity to investigate early stages of FIP pathogenesis and develop predictive models of disease onset. METHODS: Seventy-five asymptomatic first-degree relatives of FIP patients (mean age, 50.8 yr) underwent blood sampling and high-resolution chest computed tomography (HRCT) scanning in an ongoing cohort study; 72 consented to bronchoscopy with bronchoalveolar lavage (BAL) and transbronchial biopsies. Twenty-seven healthy individuals were used as control subjects. MEASUREMENTS AND MAIN RESULTS: Eleven of 75 at-risk subjects (14%) had evidence of interstitial changes by HRCT, whereas 35.2% had abnormalities on transbronchial biopsies. No differences were noted in inflammatory cells in BAL between at-risk individuals and control subjects. At-risk subjects had increased herpesvirus DNA in cell-free BAL and evidence of herpesvirus antigen expression in alveolar epithelial cells (AECs), which correlated with expression of endoplasmic reticulum stress markers in AECs. Peripheral blood mononuclear cell and AEC telomere length were shorter in at-risk individuals than healthy control subjects. The minor allele frequency of the Muc5B rs35705950 promoter polymorphism was increased in at-risk subjects. Levels of several plasma biomarkers differed between at-risk subjects and control subjects, and correlated with abnormal HRCT scans. CONCLUSIONS: Evidence of lung parenchymal remodeling and epithelial dysfunction was identified in asymptomatic individuals at risk for FIP. Together, these findings offer new insights into the early pathogenesis of idiopathic interstitial pneumonia and provide an ongoing opportunity to characterize presymptomatic abnormalities that predict progression to clinical disease.

Rare variants in RTEL1 are associated with familial interstitial pneumonia.

RATIONALE: Up to 20% of cases of idiopathic interstitial pneumonia cluster in families, comprising the syndrome of familial interstitial pneumonia (FIP); however, the genetic basis of FIP remains uncertain in most families. OBJECTIVES: To determine if new disease-causing rare genetic variants could be identified using whole-exome sequencing of affected members from FIP families, providing additional insights into disease pathogenesis. METHODS: Affected subjects from 25 kindreds were selected from an ongoing FIP registry for whole-exome sequencing from genomic DNA. Candidate rare variants were confirmed by Sanger sequencing, and cosegregation analysis was performed in families, followed by additional sequencing of affected individuals from another 163 kindreds. MEASUREMENTS AND MAIN RESULTS: We identified a potentially damaging rare variant in the gene encoding for regulator of telomere elongation helicase 1 (RTEL1) that segregated with disease and was associated with very short telomeres in peripheral blood mononuclear cells in 1 of 25 families in our original whole-exome sequencing cohort. Evaluation of affected individuals in 163 additional kindreds revealed another eight families (4.7%) with heterozygous rare variants in RTEL1 that segregated with clinical FIP. Probands and unaffected carriers of these rare variants had short telomeres (<10% for age) in peripheral blood mononuclear cells and increased T-circle formation, suggesting impaired RTEL1 function. CONCLUSIONS: Rare loss-of-function variants in RTEL1 represent a newly defined genetic predisposition for FIP, supporting the importance of telomere-related pathways in pulmonary fibrosis.

Regulation of alveolar procoagulant activity and permeability in direct acute lung injury by lung epithelial tissue factor.

Tissue factor (TF) initiates the extrinsic coagulation cascade in response to tissue injury, leading to local fibrin deposition. Low levels of TF in mice are associated with increased severity of acute lung injury (ALI) after intratracheal LPS administration. However, the cellular sources of the TF required for protection from LPS-induced ALI remain unknown. In the current study, transgenic mice with cell-specific deletions of TF in the lung epithelium or myeloid cells were treated with intratracheal LPS to determine the cellular sources of TF important in direct ALI. Cell-specific deletion of TF in the lung epithelium reduced total lung TF expression to 39% of wild-type (WT) levels at baseline and to 29% of WT levels after intratracheal LPS. In contrast, there was no reduction of TF with myeloid cell TF deletion. Mice lacking myeloid cell TF did not differ from WT mice in coagulation, inflammation, permeability, or hemorrhage. However, mice lacking lung epithelial TF had increased tissue injury, impaired activation of coagulation in the airspace, disrupted alveolar permeability, and increased alveolar hemorrhage after intratracheal LPS. Deletion of epithelial TF did not affect alveolar permeability in an indirect model of ALI caused by systemic LPS infusion. These studies demonstrate that the lung epithelium is the primary source of TF in the lung, contributing 60-70% of total lung TF, and that lung epithelial, but not myeloid, TF may be protective in direct ALI.

Telomerase deficiency delays renal recovery in mice after ischemia-reperfusion injury by impairing autophagy.

The aged population suffers increased morbidity and higher mortality in response to episodes of acute kidney injury (AKI). Aging is associated with telomere shortening, and both telomerase reverse transcriptase (TerT) and RNA (TerC) are essential to maintain telomere length. To define a role of telomerase deficiency in susceptibility to AKI, we used ischemia/reperfusion injury in wild-type mice or mice with either TerC or TerT deletion. Injury induced similar renal impairment at day 1 in each genotype, as assessed by azotemia, proteinuria, acute tubular injury score, and apoptotic tubular epithelial cell index. However, either TerC or TerT knockout significantly delayed recovery compared with wild-type mice. Electron microscopy showed increased autophagosome formation in renal tubular epithelial cells in wild-type mice but a significant delay of their development in TerC and TerT knockout mice. There were also impeded increases in the expression of the autophagosome marker LC3 II, prolonged accumulation of the autophagosome protein P62, an increase of the cell cycle regulator p16, and greater activation of the mammalian target of rapamycin (mTOR) pathway. The mTORC1 inhibitor, rapamycin, partially restored the ischemia/reperfusion-induced autophagy response, without a significant effect on either p16 induction or tubule epithelial cell proliferation. Thus, muting the maintenance of normal telomere length in mice impaired recovery from AKI, owing to an increase in tubule cell senescence and impairment of mTOR-mediated autophagy.

Future directions in idiopathic pulmonary fibrosis research. An NHLBI workshop report.

The median survival of patients with idiopathic pulmonary fibrosis (IPF) continues to be approximately 3 years from the time of diagnosis, underscoring the lack of effective medical therapies for this disease. In the United States alone, approximately 40,000 patients die of this disease annually. In November 2012, the NHLBI held a workshop aimed at coordinating research efforts and accelerating the development of IPF therapies. Basic, translational, and clinical researchers gathered with representatives from the NHLBI, patient advocacy groups, pharmaceutical companies, and the U.S. Food and Drug Administration to review the current state of IPF research and identify priority areas, opportunities for collaborations, and directions for future research. The workshop was organized into groups that were tasked with assessing and making recommendations to promote progress in one of the following six critical areas of research: (1) biology of alveolar epithelial injury and aberrant repair; (2) role of extracellular matrix; (3) preclinical modeling; (4) role of inflammation and immunity; (5) genetic, epigenetic, and environmental determinants; (6) translation of discoveries into diagnostics and therapeutics. The workshop recommendations provide a basis for directing future research and strategic planning by scientific, professional, and patient communities and the NHLBI.

Endoplasmic reticulum stress as a pro-fibrotic stimulus.

Current evidence suggests a prominent role for endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR) in fibrotic conditions affecting a number of internal organs, including the lungs, liver, GI tract, kidney, and heart. ER stress enhances the susceptibility of structural cells, in most cases the epithelium, to pro-fibrotic stimuli. Studies suggest that ER stress facilitates fibrotic remodeling through activation of pro-apoptotic pathways, induction of epithelial-mesenchymal transition, and promotion of inflammatory responses. While genetic mutations that lead to ER stress underlie some cases of fibrosis, including lung fibrosis secondary to mutations in surfactant protein C (SFTPC), a variety of other factors can cause ER stress. These ER stress inducing factors include metabolic abnormalities, oxidative stress, viruses, and environmental exposures. Interestingly, the ability of the ER to maintain homeostasis under stress diminishes with age, potentially contributing to the fact that fibrotic disorders increase in incidence with aging. Taken together, underlying ER stress and UPR pathways are emerging as important determinants of fibrotic remodeling in different forms of tissue fibrosis. Further work is needed to better define the mechanisms by which ER stress facilitates progressive tissue fibrosis. In addition, it remains to be seen whether targeting ER stress and the UPR could have therapeutic benefit. This article is part of a Special Issue entitled: Fibrosis: Translation of basic research to human disease.

ß-catenin in the alveolar epithelium protects from lung fibrosis after intratracheal bleomycin.

RATIONALE: Alveolar epithelial cells (AECs) play central roles in the response to lung injury and the pathogenesis of pulmonary fibrosis. OBJECTIVES: We aimed to determine the role of ß-catenin in alveolar epithelium during bleomycin-induced lung fibrosis. METHODS: Genetically modified mice were developed to selectively delete ß-catenin in AECs and were crossed to cell fate reporter mice that express ß-galactosidase (ßgal) in cells of AEC lineage. Mice were given intratracheal bleomycin (0.04 units) and assessed for AEC death, inflammation, lung injury, and fibrotic remodeling. Mouse lung epithelial cells (MLE12) with small interfering RNA knockdown of ß-catenin underwent evaluation for wound closure, proliferation, and bleomycin-induced cytotoxicity. MEASUREMENTS AND MAIN RESULTS: Increased ß-catenin expression was noted in lung parenchyma after bleomycin. Mice with selective deletion of ß-catenin in AECs had greater AEC death at 1 week after bleomycin, followed by increased numbers of fibroblasts and enhanced lung fibrosis as determined by semiquantitative histological scoring and total collagen content. However, no differences in lung inflammation or protein levels in bronchoalveolar lavage were noted. In vitro, ß-catenin-deficient AECs showed increased bleomycin-induced cytotoxicity as well as reduced proliferation and impaired wound closure. Consistent with these findings, mice with AEC ß-catenin deficiency showed delayed recovery after bleomycin. CONCLUSIONS: ß-Catenin in the alveolar epithelium protects against bleomycin-induced fibrosis. Our studies suggest that AEC survival and wound healing are enhanced through ß-catenin-dependent mechanisms. Activation of the developmentally important ß-catenin pathway in AECs appears to contribute to epithelial repair after epithelial injury.

Ancestral mutation in telomerase causes defects in repeat addition processivity and manifests as familial pulmonary fibrosis.

The telomerase reverse transcriptase synthesizes new telomeres onto chromosome ends by copying from a short template within its integral RNA component. During telomere synthesis, telomerase adds multiple short DNA repeats successively, a property known as repeat addition processivity. However, the consequences of defects in processivity on telomere length maintenance are not fully known. Germline mutations in telomerase cause haploinsufficiency in syndromes of telomere shortening, which most commonly manifest in the age-related disease idiopathic pulmonary fibrosis. We identified two pulmonary fibrosis families that share two non-synonymous substitutions in the catalytic domain of the telomerase reverse transcriptase gene hTERT: V791I and V867M. The two variants fell on the same hTERT allele and were associated with telomere shortening. Genealogy suggested that the pedigrees shared a single ancestor from the nineteenth century, and genetic studies confirmed the two families had a common founder. Functional studies indicated that, although the double mutant did not dramatically affect first repeat addition, hTERT V791I-V867M showed severe defects in telomere repeat addition processivity in vitro. Our data identify an ancestral mutation in telomerase with a novel loss-of-function mechanism. They indicate that telomere repeat addition processivity is a critical determinant of telomere length and telomere-mediated disease.

Endoplasmic reticulum stress enhances fibrotic remodeling in the lungs.

Evidence of endoplasmic reticulum (ER) stress has been found in lungs of patients with familial and sporadic idiopathic pulmonary fibrosis. We tested whether ER stress causes or exacerbates lung fibrosis by (i) conditional expression of a mutant form of surfactant protein C (L188Q SFTPC) found in familial interstitial pneumonia and (ii) intratracheal treatment with the protein misfolding agent tunicamycin. We developed transgenic mice expressing L188Q SFTPC exclusively in type II alveolar epithelium by using the Tet-On system. Expression of L188Q SFTPC induced ER stress, as determined by increased expression of heavy-chain Ig binding protein (BiP) and splicing of X-box binding protein 1 (XBP1) mRNA, but no lung fibrosis was identified in the absence of a second profibrotic stimulus. After intratracheal bleomycin, L188Q SFTPC-expressing mice developed exaggerated lung fibrosis and reduced static lung compliance compared with controls. Bleomycin-treated L188Q SFTPC mice also demonstrated increased apoptosis of alveolar epithelial cells and greater numbers of fibroblasts in the lungs. With a complementary model, intratracheal tunicamycin treatment failed to induce lung remodeling yet resulted in augmentation of bleomycin-induced fibrosis. These data support the concept that ER stress produces a dysfunctional epithelial cell phenotype that facilitates fibrotic remodeling. ER stress pathways may serve as important therapeutic targets in idiopathic pulmonary fibrosis.

Association of antiplatelet therapy with clinical outcomes in patients with peripheral artery disease.

BACKGROUND: The beneficial role of dual anti-platelet therapy (DAPT) in coronary artery disease is well established. However, there is limited data describing the effects of DAPT in patients with atherosclerotic peripheral artery disease (PAD). The aim of this meta-analysis is to compare clinical outcomes associated with DAPT versus single anti-platelet therapy (SAPT) in patients with symptomatic PAD. METHODS: We performed a literature search for studies assessing the risk of adverse cardiovascular and limb events in cohorts receiving either DAPT or SAPT. The primary endpoint was all cause mortality. The secondary endpoints included graft failure, amputation, total bleeding, severe bleeding and fatal bleeding. The search included the following databases: Ovid MEDLINE, EMBASE, Web of Science, and Google Scholar. The search was not restricted to time or publication status. RESULTS: A total of 11 studies with 54,331 participants (24,449 on SAPT and 29,882 on DAPT) were included. Patients with PAD treated with SAPT had higher all-cause mortality compared to patients treated with DAPT (OR 1.37, 95 % CI 1.09-1.74; p < 0.01). There was no difference in risk of graft failure or amputation between patients treated with SAPT or DAPT (OR 0.9, 95 % CI 0.77-1.06; p = 0.19; OR 1.11, 95 % CI 0.88-1.41; p = 0.37). Patients treated with SAPT had lower total bleeds compared to patients treated with DAPT (OR 0.53, 95 % CI 0.36-0.77; p < 0.01). However, For SAPT plus AC vs SAPT, a total of 8 studies with 17,100 participants (3447 with SAPT plus AC and 8619 with only SAPT) were included. Patients on SAPT plus AC did not have a statistically significant difference in risk for all-cause mortality, (OR 0.91, 95 % CI 0.67-1.24; p = 0.56). SAPT plus AC had significantly lower risk of MI (OR 0.82, 95 % CI 0.69-0.97; p = 0.02), amputation (OR 0.72, 95 % CI 0.53-0.97; p = 0.03), and graft failure (OR 0.66, 95 % CI 0.48-0.93; p = 0.02). There was no significant different in risk of fatal bleeding be-tween the two groups (OR 1.60, 95 % CI 0.76-3.35; p = 0.22). CONCLUSIONS: In patients with symptomatic PAD, a strategy of DAPT may confer a mortality benefit when compared to SAPT without significantly increasing the risk of serious bleeding events.

Respiratory Health Associated with Systemic Metal Exposure in Post 9/11 Veterans in the Department of Veterans Affairs Toxic Embedded Fragment Registry.

OBJECTIVE: Adverse respiratory outcomes in post-9/11 Veterans with elevated urinary metal measures and enrolled in the VA's Toxic Embedded Fragment registry were compared to those without elevated urinary metals. METHODS: Veterans completed questionnaires, pulmonary physiology tests (pulmonary function and oscillometry) and provided urine samples for analysis of 13 metals. Respiratory symptoms, diagnoses and physiology measures were compared in Veterans with ≥1 urine metal elevation to those without metal elevations, adjusted for covariates, including smoking. RESULTS: Among 402 study participants, 24% had elevated urine metals, often just exceeding upper limits of reference values. Compared to Veterans without elevated metals, those with elevated metals had had higher FEV1 values but similar frequencies of respiratory symptoms and diagnoses and abnormalities on pulmonary physiology tests. CONCLUSIONS: Mild systemic metal elevations in post 9/11 Veterans are not associated with adverse respiratory health outcomes.

Animal models of fibrotic lung disease.

Interstitial lung fibrosis can develop as a consequence of occupational or medical exposure, as a result of genetic defects, and after trauma or acute lung injury leading to fibroproliferative acute respiratory distress syndrome, or it can develop in an idiopathic manner. The pathogenesis of each form of lung fibrosis remains poorly understood. They each result in a progressive loss of lung function with increasing dyspnea, and most forms ultimately result in mortality. To better understand the pathogenesis of lung fibrotic disorders, multiple animal models have been developed. This review summarizes the common and emerging models of lung fibrosis to highlight their usefulness in understanding the cell-cell and soluble mediator interactions that drive fibrotic responses. Recent advances have allowed for the development of models to study targeted injuries of Type II alveolar epithelial cells, fibroblastic autonomous effects, and targeted genetic defects. Repetitive dosing in some models has more closely mimicked the pathology of human fibrotic lung disease. We also have a much better understanding of the fact that the aged lung has increased susceptibility to fibrosis. Each of the models reviewed in this report offers a powerful tool for studying some aspect of fibrotic lung disease.

Aquaporin 11 insufficiency modulates kidney susceptibility to oxidative stress.

Aquaporin 11 (AQP11) is a newly described member of the protein family of transport channels. AQP11 associates with the endoplasmic reticulum (ER) and is highly expressed in proximal tubular epithelial cells in the kidney. Previously, we identified and characterized a recessive mutation of the highly conserved Cys227 to Ser227 in mouse AQP11 that caused proximal tubule (PT) injury and kidney failure in mutant mice. The current study revealed induction of ER stress, unfolded protein response, and apoptosis as molecular mechanisms of this PT injury. Cys227Ser mutation interfered with maintenance of AQP11 oligomeric structure. AQP11 is abundantly expressed in the S1 PT segment, a site of major renal glucose flux, and Aqp11 mutant mice developed PT-specific mitochondrial injury. Glucose increased AQP11 protein expression in wild-type kidney and upregulation of AQP11 expression by glucose in vitro was prevented by phlorizin, an inhibitor of sodium-dependent glucose transport across PT. Total AQP11 levels in heterozygotes were higher than in wild-type mice but were not further increased in response to glucose. In Aqp11 insufficient PT cells, glucose potentiated increases in reactive oxygen species (ROS) production. ROS production was also elevated in Aqp11 mutation carriers. Phenotypically normal mice heterozygous for the Aqp11 mutation repeatedly treated with glucose showed increased blood urea nitrogen levels that were prevented by the antioxidant sulforaphane or by phlorizin. Our results indicate an important role for AQP11 to prevent glucose-induced oxidative stress in proximal tubules.

Role of endoplasmic reticulum stress in age-related susceptibility to lung fibrosis.

The incidence of idiopathic pulmonary fibrosis (IPF) increases with age. The mechanisms that underlie the age-dependent risk for IPF are unknown. Based on studies that suggest an association of IPF and γherpesvirus infection, we infected young (2-3 mo) and old (≥18 mo) C57BL/6 mice with the murine γherpesvirus 68. Acute murine γherpesvirus 68 infection in aging mice resulted in severe pneumonitis and fibrosis compared with young animals. Progressive clinical deterioration and lung fibrosis in the late chronic phase of infection was observed exclusively in old mice with diminution of tidal volume. Infected aging mice showed higher expression of transforming growth factor-ß during the acute phase of infection. In addition, aging, infected mice showed elevation of proinflammatory cytokines and the fibrocyte recruitment chemokine, CXCL12, in bronchoalveolar lavage. Analyses of lytic virus infection and virus reactivation indicate that old mice were able to control chronic infection and elicit antivirus immune responses. However, old, infected mice showed a significant increase in apoptotic responses determined by in situ terminal deoxynucleotidyl transferase dUTP nick end labeling assay, levels of caspase-3, and expression of the proapoptotitc molecule, Bcl-2 interacting mediator. Apoptosis of type II lung epithelial cells in aging lungs was accompanied by up-regulation of endoplasmic reticulum stress marker, binding immunoglobulin protein, and splicing of X-box-binding protein 1. These results indicate that the aging lung is more susceptible to injury and fibrosis associated with endoplasmic reticulum stress, apoptosis of type II lung epithelial cells, and activation of profibrotic pathways.

Alveolar epithelial cells undergo epithelial-to-mesenchymal transition in response to endoplasmic reticulum stress.

Expression of mutant surfactant protein C (SFTPC) results in endoplasmic reticulum (ER) stress in type II alveolar epithelial cells (AECs). AECs have been implicated as a source of lung fibroblasts via epithelial-to-mesenchymal transition (EMT); therefore, we investigated whether ER stress contributes to EMT as a possible mechanism for fibrotic remodeling. ER stress was induced by tunicamyin administration or stable expression of mutant (L188Q) SFTPC in type II AEC lines. Both tunicamycin treatment and mutant SFTPC expression induced ER stress and the unfolded protein response. With tunicamycin or mutant SFTPC expression, phase contrast imaging revealed a change to a fibroblast-like appearance. During ER stress, expression of epithelial markers E-cadherin and Zonula occludens-1 decreased while expression of mesenchymal markers S100A4 and α-smooth muscle actin increased. Following induction of ER stress, we found activation of a number of pathways, including MAPK, Smad, ß-catenin, and Src kinase. Using specific inhibitors, the combination of a Smad2/3 inhibitor (SB431542) and a Src kinase inhibitor (PP2) blocked EMT with maintenance of epithelial appearance and epithelial marker expression. Similar results were noted with siRNA targeting Smad2 and Src kinase. Together, these studies reveal that induction of ER stress leads to EMT in lung epithelial cells, suggesting possible cross-talk between Smad and Src kinase pathways. Dissecting pathways involved in ER stress-induced EMT may lead to new treatment strategies to limit fibrosis.