Substrate stiffness engineered to replicate disease conditions influence senescence and fibrotic responses in primary lung fibroblasts.

In fibrosis remodelling of ECM leads to changes in composition and stiffness. Such changes can have a major impact on cell functions including proliferation, secretory profile and differentiation. Several studies have reported that fibrosis is characterised by increased senescence and accumulating evidence suggests that changes to the ECM including altered composition and increased stiffness may contribute to premature cellular senescence. This study investigated if increased stiffness could modulate markers of senescence and/or fibrosis in primary human lung fibroblasts. Using hydrogels representing stiffnesses that fall within healthy and fibrotic ranges, we cultured primary fibroblasts from non-diseased lung tissue on top of these hydrogels for up to 7 days before assessing senescence and fibrosis markers. Fibroblasts cultured on stiffer (±15 kPa) hydrogels showed higher Yes-associated protein-1 (YAP) nuclear translocation compared to soft hydrogels. When looking at senescence-associated proteins we also found higher secretion of receptor activator of nuclear factor kappa-B ligand (RANKL) but no change in transforming growth factor-ß1 (TGF-ß1) or connective tissue growth factor (CTGF) expression and higher decorin protein deposition on stiffer matrices. With respect to genes associated with fibrosis, fibroblasts on stiffer hydrogels compared to soft had higher expression of smooth muscle alpha (α)-2 actin (ACTA2), collagen (COL) 1A1 and fibulin-1 (Fbln1) and higher Fbln1 protein deposition after 7 days. Our results show that exposure of lung fibroblasts to fibrotic stiffness activates genes and secreted factors that are part of fibrotic responses and part of the Senescence-associated secretory phenotype (SASP). This overlap may contribute to the creation of a feedback loop whereby fibroblasts create a perpetuating cycle reinforcing progression of a fibrotic response.

A cGAS-dependent response links DNA damage and senescence in alveolar epithelial cells: a potential drug target in IPF.

Alveolar epithelial cell (AEC) senescence is implicated in the pathogenesis of idiopathic pulmonary fibrosis (IPF). Mitochondrial dysfunction including release of mitochondrial DNA (mtDNA) is a feature of senescence, which led us to investigate the role of the DNA-sensing guanine monophosphate-adenine monophosphate (GMP-AMP) synthase (cGAS) in IPF, with a focus on AEC senescence. cGAS expression in fibrotic tissue from lungs of patients with IPF was detected within cells immunoreactive for epithelial cell adhesion molecule (EpCAM) and p21, epithelial and senescence markers, respectively. Submerged primary cultures of AECs isolated from lung tissue of patients with IPF (IPF-AECs, n = 5) exhibited higher baseline senescence than AECs from control donors (Ctrl-AECs, n = 5-7), as assessed by increased nuclear histone 2AXγ phosphorylation, p21 mRNA, and expression of senescence-associated secretory phenotype (SASP) cytokines. Pharmacological cGAS inhibition using RU.521 diminished IPF-AEC senescence in culture and attenuated induction of Ctrl-AEC senescence following etoposide-induced DNA damage. Short interfering RNA (siRNA) knockdown of cGAS also attenuated etoposide-induced senescence of the AEC line, A549. Higher levels of mtDNA were detected in the cytosol and culture supernatants of primary IPF- and etoposide-treated Ctrl-AECs when compared with Ctrl-AECs at baseline. Furthermore, ectopic mtDNA augmented cGAS-dependent senescence of Ctrl-AECs, whereas DNAse I treatment diminished IPF-AEC senescence. This study provides evidence that a self-DNA-driven, cGAS-dependent response augments AEC senescence, identifying cGAS as a potential therapeutic target for IPF.

A Senescence Bystander Effect in Human Lung Fibroblasts.

Idiopathic pulmonary fibrosis (IPF) is a chronic disease characterised by a dense fibrosing of the lung parenchyma. An association between IPF and cellular senescence is well established and several studies now describe a higher abundance of senescent fibroblasts and epithelial cells in the lungs of IPF patients compared with age-matched controls. The cause of this abnormal accumulation of senescent cells is unknown but evidence suggests that, once established, senescence can be transferred from senescent to non-senescent cells. In this study, we investigated whether senescent human lung fibroblasts (LFs) and alveolar epithelial cells (AECs) could induce a senescent-like phenotype in "naïve" non-senescent LFs in vitro. Primary cultures of LFs from adult control donors (Ctrl-LFs) with a low baseline of senescence were exposed to conditioned medium (CM) from: (i) Ctrl-LFs induced to become senescent using H2O2 or etoposide; (ii) LFs derived from IPF patients (IPF-LFs) with a high baseline of senescence; or (iii) senescence-induced A549 cells, an AEC line. Additionally, ratios of non-senescent Ctrl-LFs and senescence-induced Ctrl-LFs (100:0, 0:100, 50:50, 90:10, 99:1) were co-cultured and their effect on induction of senescence measured. We demonstrated that exposure of naïve non-senescent Ctrl-LFs to CM from senescence-induced Ctrl-LFs and AECs and IPF-LFs increased the markers of senescence including nuclear localisation of phosphorylated-H2A histone family member X (H2AXγ) and expression of p21, IL-6 and IL-8 in Ctrl-LFs. Additionally, co-cultures of non-senescent and senescence-induced Ctrl-LFs induced a senescent-like phenotype in the non-senescent cells. These data suggest that the phenomenon of "senescence-induced senescence" can occur in vitro in primary cultures of human LFs, and provides a possible explanation for the abnormal abundance of senescent cells in the lungs of IPF patients.

Regulation of Cellular Senescence Is Independent from Profibrotic Fibroblast-Deposited ECM.

Idiopathic pulmonary fibrosis (IPF) is a devastating lung disease with poor survival. Age is a major risk factor, and both alveolar epithelial cells and lung fibroblasts in this disease exhibit features of cellular senescence, a hallmark of ageing. Accumulation of fibrotic extracellular matrix (ECM) is a core feature of IPF and is likely to affect cell function. We hypothesize that aberrant ECM deposition augments fibroblast senescence, creating a perpetuating cycle favouring disease progression. In this study, primary lung fibroblasts were cultured on control and IPF-derived ECM from fibroblasts pretreated with or without profibrotic and prosenescent stimuli, and markers of senescence, fibrosis-associated gene expression and secretion of cytokines were measured. Untreated ECM derived from control or IPF fibroblasts had no effect on the main marker of senescence p16Ink4a and p21Waf1/Cip1. However, the expression of alpha smooth muscle actin (ACTA2) and proteoglycan decorin (DCN) increased in response to IPF-derived ECM. Production of the proinflammatory cytokines C-X-C Motif Chemokine Ligand 8 (CXCL8) by lung fibroblasts was upregulated in response to senescent and profibrotic-derived ECM. Finally, the profibrotic cytokines transforming growth factor ß1 (TGF-ß1) and connective tissue growth factor (CTGF) were upregulated in response to both senescent- and profibrotic-derived ECM. In summary, ECM deposited by IPF fibroblasts does not induce cellular senescence, while there is upregulation of proinflammatory and profibrotic cytokines and differentiation into a myofibroblast phenotype in response to senescent- and profibrotic-derived ECM, which may contribute to progression of fibrosis in IPF.

Regulation of cellular senescence by extracellular matrix during chronic fibrotic diseases.

The extracellular matrix (ECM) is a complex network of macromolecules surrounding cells providing structural support and stability to tissues. The understanding of the ECM and the diverse roles it plays in development, homoeostasis and injury have greatly advanced in the last three decades. The ECM is crucial for maintaining tissue homoeostasis but also many pathological conditions arise from aberrant matrix remodelling during ageing. Ageing is characterised as functional decline of tissue over time ultimately leading to tissue dysfunction, and is a risk factor in many diseases including cardiovascular disease, diabetes, cancer, dementia, glaucoma, chronic obstructive pulmonary disease (COPD) and fibrosis. ECM changes are recognised as a major driver of aberrant cell responses. Mesenchymal cells in aged tissue show signs of growth arrest and resistance to apoptosis, which are indicative of cellular senescence. It was recently postulated that cellular senescence contributes to the pathogenesis of chronic fibrotic diseases in the heart, kidney, liver and lung. Senescent cells negatively impact tissue regeneration while creating a pro-inflammatory environment as part of the senescence-associated secretory phenotype (SASP) favouring disease progression. In this review, we explore and summarise the current knowledge around how aberrant ECM potentially influences the senescent phenotype in chronic fibrotic diseases. Lastly, we will explore the possibility for interventions in the ECM-senescence regulatory pathways for therapeutic potential in chronic fibrotic diseases.

Senescence of IPF Lung Fibroblasts Disrupt Alveolar Epithelial Cell Proliferation and Promote Migration in Wound Healing.

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease marked by excessive accumulation of lung fibroblasts (LFs) and collagen in the lung parenchyma. The mechanisms that underlie IPF pathophysiology are thought to reflect repeated alveolar epithelial injury leading to an aberrant wound repair response. Recent work has shown that IPF-LFs display increased characteristics of senescence including growth arrest and a senescence-associated secretory phenotype (SASP) suggesting that senescent LFs contribute to dysfunctional wound repair process. Here, we investigated the influence of senescent LFs on alveolar epithelial cell repair responses in a co-culture system. Alveolar epithelial cell proliferation was attenuated when in co-culture with cells or conditioned media from, senescence-induced control LFs or IPF-LFs. Cell-cycle analyses showed that a larger number of epithelial cells were arrested in G2/M phase when co-cultured with IPF-LFs, than in monoculture. Paradoxically, the presence of LFs resulted in increased A549 migration after mechanical injury. Our data suggest that senescent LFs may contribute to aberrant re-epithelialization by inhibiting proliferation in IPF.

Self DNA perpetuates IPF lung fibroblast senescence in a cGAS-dependent manner.

Senescence and mitochondrial stress are mutually reinforcing age-related processes that contribute to idiopathic pulmonary fibrosis (IPF); a lethal disease that manifests primarily in the elderly. Whilst evidence is accumulating that GMP-AMP synthase (cGAS) is crucial in perpetuating senescence by binding damaged DNA released into the cytosol, its role in IPF is not known. The present study examines the contributions of cGAS and self DNA to the senescence of lung fibroblasts from IPF patients (IPF-LFs) and age-matched controls (Ctrl-LFs). cGAS immunoreactivity was observed in regions of fibrosis associated with fibroblasts in lung tissue of IPF patients. Pharmacological inhibition of cGAS or its knockdown by silencing RNA (siRNA) diminished the escalation of IPF-LF senescence in culture over 7 days as measured by decreased p21 and p16 expression, histone 2AXγ phosphorylation and/or IL-6 production (P < 0.05, n = 5-8). The targeting of cGAS also attenuated etoposide-induced senescence in Ctrl-LFs (P < 0.05, n = 5-8). Levels of mitochondrial DNA (mDNA) detected by qPCR in the cytosol and medium of IPF-LFs or senescence-induced Ctrl-LFs were higher than Ctrl-LFs at baseline (P < 0.05, n = 5-7). The addition of DNAse I (100 U/ml) deaccelerated IPF-LF senescence (P < 0.05, n = 5), whereas ectopic mDNA or the induction of endogenous mDNA release augmented Ctrl-LF senescence in a cGAS-dependent manner (P < 0.05, n = 5). In conclusion, we provide evidence that cGAS reinforces lung fibroblast senescence involving damaged self DNA. The targeting of cGAS to supress senescent-like responses may have potential important therapeutic implications in the treatment of IPF.

Extracellular vesicles in lung health, disease, and therapy.

Both physiological homeostasis and pathological disease processes in the lung typically result from complex, yet coordinated multicellular responses that are synchronized via paracrine and endocrine intercellular communication pathways. Of late, extracellular vesicles have emerged as important information shuttles that can coordinate and disseminate homeostatic and disease signals. In parallel, extracellular vesicles in biological fluids such as sputum, mucus, epithelial lining fluid, edema fluid, the pulmonary circulation, pleural fluid, and lymphatics have emerged as promising candidate biomarkers for diagnosis and prognosis in lung disease. Extracellular vesicles are small, subcellular, membrane-bound vesicles containing cargos from parent cells such as lipids, proteins, genetic information, or entire organelles. These cargos endow extracellular vesicles with biologically active information or functions by which they can reprogram their respective target cells. Recent studies show that extracellular vesicles found in lung-associated biological fluids play key roles as biomarkers and effectors of disease. Conversely, administration of naïve or engineered extracellular vesicles with homeostatic or reparative effects may provide a promising novel protective and regenerative strategy to treat lung disease. To highlight this rapidly developing field, the American Journal of Physiology-Lung Cellular and Molecular Physiology is now launching a special Call for Papers on extracellular vesicles in lung health, disease, and therapy. This review aims to set the stage for this call by introducing extracellular vesicles and their emerging roles in lung physiology and pathobiology.

STAT3 Regulates the Onset of Oxidant-induced Senescence in Lung Fibroblasts.

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease of unknown cause with a median survival of only 3 years. Other investigators and we have shown that fibroblasts derived from IPF lungs display characteristics of senescent cells, and that dysregulated activation of the transcription factor signal transducer and activator of transcription 3 (STAT3) correlates with IPF progression. The question of whether STAT3 activation is involved in fibroblast senescence remains unanswered. We hypothesized that inhibiting STAT3 activation after oxidant-induced senescence would attenuate characteristics of the senescent phenotype. We aimed to characterize a model of oxidant-induced senescence in human lung fibroblasts and to determine the effect of inhibiting STAT3 activity on the development of senescence. Exposing human lung fibroblasts to 150 µM hydrogen peroxide (H2O2) resulted in increased senescence-associated ß-galactosidase content and expression of p21 and IL-6, all of which are features of senescence. The shift into senescence was accompanied by an increase of STAT3 translocation to the nucleus and mitochondria. Additionally, Seahorse analysis provided evidence of increased mitochondrial respiration characterized by increased basal respiration, proton leak, and an associated increase in superoxide (O2-) production in senescent fibroblasts. Targeting STAT3 activity using the small-molecule inhibitor STA-21 attenuated IL-6 production, reduced p21 levels, decreased senescence-associated ß-galactosidase accumulation, and restored normal mitochondrial function. The results of this study illustrate that stress-induced senescence in lung fibroblasts involves the activation of STAT3, which can be pharmacologically modulated.

Sustained and regulated gene expression by Tet-inducible "all-in-one" retroviral vectors containing the HNRPA2B1-CBX3 UCOE®.

Genetic modification of induced pluripotent stem (iPS) cells may be necessary for the generation of effector cells for cellular therapies. Hereby, it can be important to induce transgene expression at restricted and defined time windows, especially if it interferes with pluripotency or differentiation. To achieve this, inducible expression systems can be used such as the tetracycline-inducible retroviral vector system, however, retroviral expression can be subjected to epigenetic silencing or to position-effect variegation. One strategy to overcome this is the incorporation of ubiquitous chromatin opening elements (UCOE®'s) into retroviral vectors to maintain a transcriptionally permissive chromatin state at the integration site. In this study, we developed Tet-inducible all-in-one gammaretroviral vectors carrying different sized UCOE®'s derived from the A2UCOE. The ability to prevent vector silencing by preserving the Tet-regulatory potential was investigated in different cell lines, and in murine and human iPS cells. A 670-bp fragment spanning the CBX3 promoter region of A2UCOE (U670) was the most potent element in preventing silencing, and conferred the strongest expression from the vector in the induced state. While longer fragments of A2UCOEs also sustained expression, vector titers and induction efficiencies were impaired. Finally, we demonstrate that U670 can be used for constitutive expression of the transactivator in the all-in-one vector for faithful regulation of transgenes by doxycycline, including the thrombopoietin receptor Mpl conferring cytokine-dependent cell growth.

Mitochondrial dysfunction contributes to the senescent phenotype of IPF lung fibroblasts.

Increasing evidence highlights that senescence plays an important role in idiopathic pulmonary fibrosis (IPF). This study delineates the specific contribution of mitochondria and the superoxide they form to the senescent phenotype of lung fibroblasts from IPF patients (IPF-LFs). Primary cultures of IPF-LFs exhibited an intensified DNA damage response (DDR) and were more senescent than age-matched fibroblasts from control donors (Ctrl-LFs). Furthermore, IPF-LFs exhibited mitochondrial dysfunction, exemplified by increases in mitochondrial superoxide, DNA, stress and activation of mTORC1. The DNA damaging agent etoposide elicited a DDR and augmented senescence in Ctrl-LFs, which were accompanied by disturbances in mitochondrial homoeostasis including heightened superoxide production. However, etoposide had no effect on IPF-LFs. Mitochondrial perturbation by rotenone involving sharp increases in superoxide production also evoked a DDR and senescence in Ctrl-LFs, but not IPF-LFs. Inhibition of mTORC1, antioxidant treatment and a mitochondrial targeting antioxidant decelerated IPF-LF senescence and/or attenuated pharmacologically induced Ctrl-LF senescence. In conclusion, increased superoxide production by dysfunctional mitochondria reinforces lung fibroblast senescence via prolongation of the DDR. As part of an auto-amplifying loop, mTORC1 is activated, altering mitochondrial homoeostasis and increasing superoxide production. Deeper understanding the mechanisms by which mitochondria contribute to fibroblast senescence in IPF has potentially important therapeutic implications.

Fibroblast senescence in the pathology of idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic fibrosing interstitial pneumonia of unknown cause with a median survival of only three years. Little is known about the mechanisms that precede the excessive collagen deposition seen in IPF, but cellular senescence has been strongly implicated in disease pathology. Senescence is a state of irreversible cell-cycle arrest accompanied by an abnormal secretory profile and is thought to play a critical role in both development and wound repair. Normally, once a senescent cell has contributed to wound repair, it is promptly removed from the environment via infiltrating immune cells. However, if immune clearance fails, the persistence of senescent cells is thought to drive disease pathology through their altered secretory profile. One of the major cell types involved in wound healing is fibroblasts, and senescent fibroblasts have been identified in the lungs of patients with IPF and in fibroblast cultures from IPF lungs. The question of what is driving abnormally high numbers of fibroblasts into senescence remains unanswered. The transcription factor signal transducer and activator of transcription 3 (STAT3) plays a role in a myriad of processes, including cell-cycle progression, gene transcription, as well as mitochondrial respiration, all of which are dysregulated during senescence. Activation of STAT3 has previously been shown to correlate with IPF progression and therefore is a potential molecular target to modify early-stage senescence and restore normal fibroblast function. This review summarizes what is presently known about fibroblast senescence in IPF and how STAT3 may contribute to this phenotype.