Role of STIM1 in Stretch-induced Signaling in Human Airway Smooth Muscle.

Alteration in the normal mechanical forces of breathing can contribute to changes in contractility and remodeling characteristic of airway diseases, but the mechanisms that mediate these effects in airway cells are still under investigation. Airway smooth muscle (ASM) cells contribute to both contractility and extracellular matrix (ECM) remodeling. In this study, we explored ASM mechanisms activated by mechanical stretch, focusing on mechanosensitive piezo channels and the key Ca2+ regulatory protein stromal interaction molecule 1 (STIM1). Expression of Ca2+ regulatory proteins, including STIM1, Orai1 and caveolin-1, mechanosensitive ion channels Piezo-1 and Piezo-2, and NLRP3 inflammasomes were upregulated by 10% static stretch superimposed on 5% cyclic stretch. These effects were blunted by STIM1 siRNA. Histamine-induced [Ca2+]i responses and inflammasome activation were similarly blunted by STIM1 knockdown. These data show that the effects of mechanical stretch in human ASM cells are mediated through STIM1, which activates multiple pathways including Piezo channels and the inflammasome, leading to potential downstream changes in contractility and ECM remodeling.

Endoplasmic Reticulum Stress Induced Senescence in Human Lung Fibroblasts.

Loss of proteostasis and cellular senescence have been previously established as characteristics of aging, however their interaction in the context of lung aging and potential contributions to aging-associated lung remodeling remains understudied. In this study we aimed to characterize endoplasmic reticulum (ER) stress response, cellular senescence, and their interaction in relation to extracellular matrix (ECM) production in lung fibroblasts from young (25-45 years) and old (>60 years) humans. Fibroblasts from young and old patients without significant preexisting lung disease were exposed to vehicle, MG132, etoposide or salubrinal. Afterwards, cells and cell lysates or supernatants were analyzed for ER stress, cellular senescence and ECM changes using protein analysis, proliferation assay and senescence-associated beta galactosidase (SA-ß-Gal) staining. At baseline, fibroblasts from aging individuals showed increased levels of ER stress (ATF6 and PERK), senescence (p21 and McL-1) and ECM marker (COL1A1) compared to those from young individuals. Upon ER stress induction and etoposide exposure, fibroblasts showed an increase in senescence (SA-ß-Gal, p21, Cav-1), ER stress (PERK) and ECM markers (COL1A1 and LUM) compared to vehicle. Additionally, CXCL8 and IL-6 levels were increase in the supernatants of MG132 and etoposide-treated fibroblasts, respectively. Finally, the ER stress inhibitor salubrinal decreased the expression of p21 compared to vehicle and MG132 treatments, however salubrinal inhibited COL1A1 but not p21 expression in MG132-treated fibroblasts. Our study suggests that ER stress response plays an important role in establishment and maintenance of a senescence phenotype in lung fibroblasts and therefore contributes to altered remodeling in the aging lung.

Collagen type XIV is proportionally lower in the lung tissue of patients with IPF.

Abnormal deposition of extracellular matrix (ECM) in lung tissue is a characteristic of idiopathic pulmonary fibrosis (IPF). Increased collagen deposition is also accompanied by altered collagen organization. Collagen type XIV, a fibril-associated collagen, supports collagen fibril organization. Its status in IPF has not been described at the protein level yet. In this study, we utilized publicly available datasets for single-cell RNA-sequencing for characterizing collagen type XIV expression at the gene level. For protein level comparison, we applied immunohistochemical staining for collagen type XIV on lung tissue sections from IPF patients and compared it to lung tissue sections from never smoking and ex-smoking donors. Analyzing the relative amounts of collagen type XIV at the whole tissue level, as well as in parenchyma, airway wall and bronchial epithelium, we found consistently lower proportions of collagen type XIV in all lung tissue compartments across IPF samples. Our study suggests proportionally lower collagen type XIV in IPF lung tissues may have implications for the assembly of the ECM fibers potentially contributing to progression of fibrosis.

Age-associated differences in the human lung extracellular matrix.

Extracellular matrix (ECM) remodeling has been associated with chronic lung diseases. However, information about specific age-associated differences in lung ECM is currently limited. In this study, we aimed to identify and localize age-associated ECM differences in human lungs using comprehensive transcriptomic, proteomic, and immunohistochemical analyses. Our previously identified age-associated gene expression signature of the lung was re-analyzed limiting it to an aging signature based on 270 control patients (37-80 years) and focused on the Matrisome core geneset using geneset enrichment analysis. To validate the age-associated transcriptomic differences on protein level, we compared the age-associated ECM genes (false discovery rate, FDR < 0.05) with a profile of age-associated proteins identified from a lung tissue proteomics dataset from nine control patients (49-76 years) (FDR < 0.05). Extensive immunohistochemical analysis was used to localize and semi-quantify the age-associated ECM differences in lung tissues from 62 control patients (18-82 years). Comparative analysis of transcriptomic and proteomic data identified seven ECM proteins with higher expression with age at both gene and protein levels: COL1A1, COL6A1, COL6A2, COL14A1, FBLN2, LTBP4, and LUM. With immunohistochemistry, we demonstrated higher protein levels with age for COL6A2 in whole tissue, parenchyma, airway wall, and blood vessel, for COL14A1 and LUM in bronchial epithelium, and COL1A1 in lung parenchyma. Our study revealed that higher age is associated with lung ECM remodeling, with specific differences occurring in defined regions within the lung. These differences may affect lung structure and physiology with aging and as such may increase susceptibility to developing chronic lung diseases.NEW & NOTEWORTHY We identified seven age-associated extracellular matrix (ECM) proteins, i.e., COL1A1, COL6A1, COL6A2 COL14A1, FBLN2, LTBP4, and LUM with higher transcript and protein levels in human lung tissue with age. Extensive immunohistochemical analysis revealed significant age-associated differences for COL6A2 in whole tissue, parenchyma, airway wall, and vessel, for COL14A1 and LUM in bronchial epithelium, and COL1A1 in parenchyma. Our findings lay a new foundation for the investigation of ECM differences in age-associated chronic lung diseases.

Asthmatic lung fibroblasts promote type 2 immune responses via endoplasmic reticulum stress response dependent thymic stromal lymphopoietin secretion.

Lung fibroblasts contribute to asthma pathology partly through modulation of the immune environment in the airway. Tumor necrosis factor-α (TNFα) expression is upregulated in asthmatic lungs. How asthmatic lung fibroblasts respond to TNFα stimulation and subsequently regulate immune responses is not well understood. Endoplasmic reticulum (ER) stress and unfolded protein responses (UPR) play important roles in asthma, but their functional roles are still under investigation. In this study, we investigated TNFα-induced cytokine production in primary lung fibroblasts from asthmatic vs. non-asthmatic human subjects, and downstream effects on type 2 immune responses. TNFα significantly upregulated IL-6, IL-8, C-C motif chemokine ligand 5 (CCL5), and thymic stromal lymphopoietin (TSLP) mRNA expression and protein secretion by lung fibroblasts. Asthmatic lung fibroblasts secreted higher levels of TSLP which promoted IL-33-induced IL-5 and IL-13 production by peripheral blood mononuclear cells. TNFα exposure enhanced expression of ER stress/UPR pathways in both asthmatic and non-asthmatic lung fibroblasts, especially inositol-requiring protein 1α in asthmatics. ER stress/UPR inhibitors decreased IL-6, CCL5, and TSLP protein secretion by asthmatic lung fibroblasts. Our data suggest that TNFα and lung fibroblasts form an important axis in asthmatic lungs to promote asthmatic inflammation that can be attenuated by inhibiting ER stress/UPR pathway.

Cellular Senescence in Aging Lungs and Diseases.

Cellular senescence represents a state of irreversible cell cycle arrest occurring naturally or in response to exogenous stressors. Following the initial arrest, progressive phenotypic changes define conditions of cellular senescence. Understanding molecular mechanisms that drive senescence can help to recognize the importance of such pathways in lung health and disease. There is increasing interest in the role of cellular senescence in conditions such as chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF) in the context of understanding pathophysiology and identification of novel therapies. Herein, we discuss the current knowledge of molecular mechanisms and mitochondrial dysfunction regulating different aspects of cellular senescence-related to chronic lung diseases to develop rational strategies for modulating the senescent cell phenotype in the lung for therapeutic benefit.