The extracellular matrix protein fibulin-1 in idiopathic pulmonary fibrosis

One of the most pressing issues in the clinical management of patients with idiopathic pulmonary fibrosis (IPF) is how a clinician can respond when asked “how long do I have?” IPF has a varied clinical course and the only available treatments, aside from lung transplantation, are corticosteroids and immunosuppressants. The side effects of both corticosteroids and immunosuppressants may actually outweigh the benefits for use in IPF. Biomarkers of disease progression are often unable to predict acute lung function decline. This is possibly because the underlying mechanisms driving the disease are poorly understood and little attention has been paid to how intrinsic differences in resident lung fibroblasts may be contributing to this disease. In this thesis, extracellular matrix (ECM) molecules that are both found in the blood and released by resident lung fibroblasts were investigated for their utility as biomarkers of disease progression in IPF. The primary focus was on the ECM protein fibulin-1, an essential constituent of elastic fibres, which has not previously been studied in the context of interstitial lung disease. This thesis investigated the relationship between fibulin-1, and disease severity in patients with and without pulmonary fibrosis. In addition, the utility of fibulin-1 as a biomarker of disease progression was compared against other previously described components of the ECM, namely periostin, tenascin-C and fibronectin, in the same patients. Lastly, the effect of the profibrotic cytokine transforming growth factor-beta-1 (TGFβ1) on fibulin-1 levels in resident lung fibroblasts from patients with and without IPF was interrogated

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 2AX? 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

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

BIBF1120 inhibits fibroblasts proliferation and production of the extracellular matrix protein fibulin-1

Introduction: In patients with idiopathic pulmonary fibrosis (IPF) transforming growth factorbeta 1 (TGFβ1) induces excessive extracellular matrix (ECM) protein deposition leading to fibrosis. Our recent studies have shown that the glycoprotein fibulin-1 is increased in serum and lung tissue from patients with IPF and that their fibroblasts, which are the main fibrosis effector cells, produce more fibulin-1 than those from healthy people. Aim: To examine the modulation of fibroblast derived fibulin-1 from people with and without IPF. Methods: Primary parenchymal fibroblasts were derived from 5 patients with IPF and 5 without IPF (nonIPF). The production of fibulin-1 in the presence of cigarette smoke extract (CSE), foetal bovine serum (FBS) or TGFβ1 in the presence or absence of BIBF1120 (a triple kinase inhibitor the same as nintedanib) was measured by QPCR or ELISA. Proliferation was measured by MTT and CyQuant and cell attachment assessed by toluidine blue absorbance in fibroblasts from both groups. Results: CSE did not modulate fibulin-1 expression. Fibroblasts from IPF patients proliferated faster in 10% FBS and deposited more fibulin-1 in the presence of TGFβ1, compared to nonIPF fibroblasts, however attachment did not differ between the groups. BIBF1120 reduced proliferation and fibulin-1 deposition in the ECM, but did not influence attachment of fibroblasts. There were no differences in the degree of response to BIBF1120 between fibroblasts from patients with and without IPF. Conclusion: BIBF1120 inhibits fibroblastic behaviours that contribute to the pathogenesis of IPF. This new inhibitor may be effective in targeting the development of fibrosis in IPF patients

Establishing CREATE: Lessons learned in setting up a training environment for early-career researchers in respiratory medicine

The purpose of the National Health and Medical Research Council Centre of Research Excellence in Pulmonary Fibrosis (CRE-PF) is to improve and extend the lives of patients living with pulmonary fibrosis through the development of a comprehensive and integrated program of basic and clinical research and education across Australia. A key objective of the CRE-PF was establishment of a unique national training scheme, CREATE, for early-career researchers (ECRs) in respiratory research. CREATE ECRs are broadly drawn from two main fields of researchers: clinicians and scientists, where clinicians tend to be involved in part-time translational research and scientists are involved in broad scientific research including laboratory or genetic research, health economics or population research. We describe the CREATE Program which, with limited budget and the assistance of key organisations, has provided funding opportunities (scholarships, fellowships, prizes, travel and collaboration grants), professional development (mentoring program, symposia, presentation opportunities and on-line training) and fostered a connected, supportive research community for respiratory ECRs. The CREATE program has successfully fostered the development of the supported researchers, contributing substantially to the future of pulmonary fibrosis research in Australia. During the life of the program the CRE-PF has offered 10 PhD scholarships and five postdoctoral fellowships, awarded 13 travel grants and three grants to promote collaboration between ECRs from different institutes. A mentoring program has been established and CREATE Symposia have been held in association with key meetings. During COVID-19 restrictions, a series of virtual research meetings has offered 12 CREATE ECRs from seven universities the opportunity to present their research to a national audience. CREATE research-related achievements are impressive, including over 80 first-author publications by ECRs, and many conference presentations. Contributions to the research community, measured by committee membership, is also strong. In spite of a very limited budget, wide geographic distribution of participants and the multi-disciplinary nature of the cohort, we have succeeded in providing a unique, supportive academic development environment for CREATE ECRs. Lessons learned in the process of developing this program include the importance of leveraging funding, being flexible, building networks and seeking and responding to ECR input

Identification Of Potential Candidate Biomarkers In Lymphangioleiomyomatosis Using ITRAQ Proteomic Technology

Study Rational: Lympangioleiomyomatosis (LAM) is a rare and progressive cystic lung condition that affects between 3-7/million women. It manifests primarily in women of child-bearing age, with an average lag time between symptom onset and definitive diagnosis of upwards of 4 years. Currently no reliable biomarker exists for patients with LAM, therefore, identifying a predictive or prognostic disease biomarker could reduce diagnosis lag time, as well as increase our knowledge of the underlying disease mechanisms in this multifactorial condition. This study hypothesized that there were significant alterations in the protein content in serum between LAM patients and healthy aged-matched controls. Study Aim: Identification of potential candidate biomarkers in LAM patient serum. Methods: Serum from LAM patient volunteers (n= 3, n= 5) and healthy aged matched control volunteers (n= 4, n= 5) were pooled and two runs of high-throughput protein analysis were carried out using 4-plex iTRAQ technology on a Triple TOF 5600 (AB Sciex). After network pathway analysis, using Ingenuity Pathway Analysis software™, a number of the differentially expressed proteins (Fibronectin, von Willebrand Factor and Kallikrein III) were validated using ELISA in a wider individual patient cohort (n= 18) and an age matched healthy control group (n= 12). Results: A total of 14 proteins were differentially expressed in LAM serum compared to healthy age-matched control serum (p<0.05). It was found that an intricate network of cell trafficking proteins was altered. Differential expression of the remodelling proteins fibronectin (decreased expression of 30% in LAM sera, p = 0.03), von Willebrand Factor (vWF, 40% reduction in LAM, p = 0.03) and the serum protease Kallikrein III (KLK3, 25% increase in LAM, p = 0.027) were identified in pooled circulating LAM sera and subsequently validated in individual samples. Conclusion: This study demonstrates, for the first time, that in LAM a substantial imbalance exists in protein networks important for remodelling, particularly in the circulating levels of the structural proteins; fibronectin, vWF and KLK3. We have also revealed an intricate network of significantly altered proteins involved in cell trafficking, an integral component of LAM disease progression. These results provide a set of novel candidate biomarkers which may be useful for the development of a feasible approach to diagnosis, and potentially lead to a new effective targeted treatment for LAM

Matrix metalloproteinase-7 is increased in lung bases but not apices in idiopathic pulmonary fibrosis

Introduction Idiopathic pulmonary fibrosis (IPF) is a progressively fibrotic lung condition with poor prognosis. Matrix metalloproteinase-7 (MMP7) is a protein secreted by epithelial cells in IPF lungs. It is not known if MMP7 expression correlates with fibrotic changes in lung tissue. Methods Tissue samples from lung apices and bases were obtained from 20 IPF patients and 14 non-diseased control (NDC) donors. In formalin-fixed paraffin-embedded sections, histological assessment of fibrosis was performed; overall MMP7 positivity was assessed by immunohistochemistry and MMP7+ cells were quantified using multiplex immunohistochemistry. Protein expression of MMP7 in whole lung lysates was quantified by Western blotting. Bulk tissue transcriptomic profiles of 101 samples were analysed using RNA sequencing technologies. Results Lung tissue from IPF bases was more fibrotic than in apices. MMP7 protein is elevated in IPF lung base tissue. In IPF whole lung lysates, MMP7 protein levels are increased compared to NDC donors and was increased in IPF lung bases compared to apices. MMP7 protein levels correlated with MMP7 gene expression levels in lung tissue. MMP7 transcript levels were increased in IPF base compared to NDC base lung tissue and increased in IPF base tissue compared to IPF apex tissue. Conclusions Our cross-sectional study suggests that lung epithelial MMP7 expression increases as the tissue becomes more fibrotic and identifies a potentially nonepithelial or immune-cell source. Mechanisms of disease progression in IPF are still unclear, and our study suggests aberrant MMP7 production may be a histological starting point of lung tissue fibrosis