Human Idiopathic Pulmonary Fibrosis Fibroblasts Research Articles

Transforming Growth Factor-β1 Regulates Peroxisomal Genes/Proteins via Smad Signaling in Idiopathic Pulmonary Fibrosis Fibroblasts and Transgenic Mouse Models

Idiopathic pulmonary fibrosis (IPF) is a chronic human disease with persistent destruction of lung parenchyma. Transforming growth factor-β1 (TGF-β1) signaling plays a pivotal role in the initiation and pathogenesis of IPF. As shown herein, TGF-β1 signaling down-regulated not only peroxisome biogenesis but also the metabolism of these organelles in human IPF fibroblasts. Invitro cell culture observations in human fibroblasts and human lung tissue indicated that peroxisomal biogenesis and metabolic proteins were significantly down-regulated in the lung of 1-month-old transgenic mice expressing a constitutively active TGF-β type I receptor kinase (ALK5). The peroxisome biogenesis protein peroxisomal membrane protein Pex13p (PEX13p) as well as the peroxisomal lipid metabolic enzyme peroxisomal acyl-coenzyme A oxidase 1 (ACOX1) and antioxidative enzyme catalase were highly up-regulated in TGF-β type II receptor and Smad3 knockout mice. This study reports a novel mechanism of peroxisome biogenesis and metabolic regulation via TGF-β1-Smad signaling: interaction of the Smad3 transcription factor with the PEX13 gene in chromatin immunoprecipitation-on-chip assay as well as in a bleomycin-induced pulmonary fibrosis model applied to TGF-β type II receptor knockout mice. Taken together, data from this study suggest that TGF-β1 participates in regulation of peroxisomal biogenesis and metabolism via Smad-dependent signaling, opening up novel strategies for the development of therapeutic approaches to inhibit progression of pulmonary fibrosis patients with IPF.

Inhibiting Fibroblast Mechanotransduction Modulates Severity of Idiopathic Pulmonary Fibrosis.

Objective: Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease that affects 63 in every 100,000 Americans. Its etiology remains unknown, although inflammatory pathways appear to be important. Given the dynamic environment of the lung, we examined the significance of mechanotransduction on both inflammatory and fibrotic signaling during IPF. Innovation: Mechanotransduction pathways have not been thoroughly examined in the context of lung disease, and pharmacologic approaches for IPF do not currently target these pathways. The interplay between mechanical strain and inflammation in pulmonary fibrosis remains incompletely understood. Approach: In this study, we used conditional KO mice to block mechanotransduction by knocking out Focal Adhesion Kinase (FAK) expression in fibroblasts, followed by induction of pulmonary fibrosis using bleomycin. We examined both normal human and human IPF fibroblasts and used immunohistochemistry, quantitative real-time polymerase chain reaction, and Western Blot to evaluate the effects of FAK inhibitor (FAK-I) on modulating fibrotic and inflammatory genes. Results: Our data indicate that the deletion of FAK in mice reduces expression of fibrotic and inflammatory genes in lungs. Similarly, mechanical straining in normal human lung fibroblasts activates inflammatory and fibrotic pathways. The FAK inhibition decreases these signals but has a less effect on IPF fibroblasts as compared with normal human fibroblasts. Conclusion: Administering FAK-I at early stages of fibrosis may attenuate the FAK-mediated fibrotic response pathway in IPF, potentially mediating disease progression.

Spontaneous Lung Fibrosis Resolution Reveals Novel Antifibrotic Regulators

Fibroblast activation is transient in successful wound repair but persistent in fibrotic pathologies. Understanding fibroblast deactivation during successful wound healing may provide new approaches to therapeutically reverse fibroblast activation. To characterize the gene programs that accompany fibroblast activation and reversal during lung fibrosis resolution, we used RNA sequencing analysis of flow sorted Col1α1-GFP-positive and CD45-, CD31-, and CD326-negative cells isolated from the lungs of young mice exposed to bleomycin. We compared fibroblasts isolated from control mice with those isolated at Days 14 and 30 after bleomycin exposure, representing the peak of extracellular matrix deposition and an early stage of fibrosis resolution, respectively. Bleomycin exposure dramatically altered fibroblast gene programs at Day 14. Principal component and differential gene expression analyses demonstrated the predominant reversal of these trends at Day 30. Upstream regulator and pathway analyses of reversing "resolution" genes identified novel candidate antifibrotic genes and pathways. Two genes from these analyses that were decreased in expression at Day 14 and reversed at Day 30, Aldh2 and Nr3c1, were selected for further analysis. Enhancement of endogenous expression of either gene by CRISPR activation in cultured human idiopathic pulmonary fibrosis fibroblasts was sufficient to reduce profibrotic gene expression, fibronectin deposition, and collagen gel compaction, consistent with roles for these genes in fibroblast deactivation. This combination of RNA sequencing analysis of freshly sorted fibroblasts and hypothesis testing in cultured idiopathic pulmonary fibrosis fibroblasts offers a path toward identification of novel regulators of lung fibroblast deactivation, with potential relevance to understanding fibrosis resolution and its failure in human disease.

Targeting MAP3K19 prevents human lung myofibroblast activation both in vitro and in a humanized SCID model of idiopathic pulmonary fibrosis

Idiopathic Pulmonary Fibrosis (IPF) is a disease with a devastating prognosis characterized by unrelenting lung scarring. Aberrant activation of lung fibroblasts is a key feature of this disease, yet the key pathways responsible for this are poorly understood. Mitogen-activated protein kinase, kinase, kinase- 19 (MAP3K19) was recently shown to be upregulated in IPF and this MAPK has a key role in target gene transcription in the TGF-β pathway. Herein, we further investigate the role of MAP3K19 in cultured normal and IPF fibroblasts and in a humanized SCID mouse model of IPF employing both short interfering (si) RNA and novel small-molecule inhibitors directed at this kinase. Targeting MAP3K19 had significant inhibitory effects on the expression of both alpha smooth muscle actin and extracellular matrix in cultured human IPF fibroblasts. Quantitative protein and biochemical assays, as well as histological analysis, showed that MAP3K19 was required for the development of lung fibrosis in SCID mice humanized with IPF lung fibroblasts. MAP3K19 was required for IPF myofibroblast differentiation, and targeting its activity attenuated the profibrotic activity of these cells both in vitro and in an adoptive transfer SCID model of pulmonary fibrosis.

NINTEDANIB AND GREEN TEA EXTRACTS (ELLAGIC ACID AND EPIGALLOCATECHIN GALLATE): A COST-EFFECTIVE, COMBINATION THERAPY TO REDUCE PULMONARY FIBROSIS IN HUMAN TGF-BETA STIMULATED FIBROBLASTS

SESSION TITLE: Tuesday Electronic Posters 1 SESSION TYPE: Original Inv Poster Discussion PRESENTED ON: 10/22/2019 01:00 PM - 02:00 PM PURPOSE: Cost-effective, combination therapies are sorely needed for idiopathic pulmonary fibrosis (IPF). Current FDA approved therapies are limited to nintedanib and pirfenidone, drugs which only slow FEV1 decline but with significant costs, exceeding $100,000 per year [1]. These monotherapies may have only tempering effects on IPF clinically because of the multiple pathways of lung injury involved [2]. Ellagic Acid (EA) and Epigallocatechin Gallate (EGCG) are naturally occurring, low cost polyphenol compounds found abundantly in fruits, nuts, and green tea. As strong anti-oxidants, they have clinical benefits in pulmonary toxicity from chemotherapy, radiation induced lung fibrosis, and inflammation in asthma [3, 4]. Recently, EA and EGCG were shown to have pulmonary anti-fibrotic effect in vitro and in vivo. Bleomycin mice fed with extract rich EA showed markedly lower levels of lung collagen, fibronectin, Snail1, and p-Smad3 [4]. METHODS: The objective of this study was to demonstrate whether the combination of EA or EGCG and low dose nintedanib can reduce fibrosis in vitro. Human IPF fibroblasts were incubated with 2uM EA, 1uM EA, 2uM EGCG, and 1uM EGCG. Cells were then stimulated with TGF-β, a key cytokine in the development of pulmonary fibrosis, for 24 hours. ELISAs (enzyme-linked immunosorbent assays) were performed for fibronectin protein production quantification. Cellular fibronectin is one of first extracellular matrix proteins to be deposited in fibroblasts upon TGFβ stimulation. RESULTS: Pooled ELISA analysis confirmed the anti-fibrotic effect of both EA and EGCG. 1uM EA lowered fibronectin levels to average 49.0% (+/- 17.13, p=0.01) of the TGF-β stimulated fibroblasts. 2uM EGCG and 1uM EGCG lowered fibronectin levels to average 42.6% (+/- 3.3, p=0.0002) and 63.9% (+/-13.5,p=0.06) respectively of the unstimulated fibroblasts. When combined with 0.5uM and 0.25 uM nintedanib, 1uM EA lowered fibronectin levels to an average 0.04% (+/-26.4) and 9.19% (+/-12.9) respectively of the TGF-β stimulated fibroblasts. This was an increased effect from 0.5uM and 0.25 uM nintedanib alone, which lowered fibronectin levels to an average of 26.3% and 39.7% respectively. 1uM EGCG, when combined with 0.5uM and 0.25 uM nintedanib also showed a fibronectin lowering effect, to an average of 47.7% (+/-3.6, p=0.03) and 49.6% (+/-6.09, p=0.09) respectively of the unstimulated fibroblasts. This was higher than the combined effect of either drug alone. CONCLUSIONS: Polyphenol compounds with natural anti-oxidant properties, such as Ellagic Acid and Epigallocatechin Gallate, have been shown to have pulmonary anti-fibrotic effect and demonstrate potential as novel therapies for IPF. CLINICAL IMPLICATIONS: With additional research, perhaps these widely available compounds could be added to current standard therapies to offset their significant costs to patients. DISCLOSURES: no disclosure on file for Theodore Kottom; No relevant relationships by Andrew Limper, source=Web Response No relevant relationships by Kyle Schaefbauer, source=Web Response No relevant relationships by Ann Vu, source=Web Response

Anti-fibrotic Effects of CXCR4-Targeting i-body AD-114 in Preclinical Models of Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a chronic fibrotic lung disease that is prevalent in individuals >50 years of age, with a median survival of 3–5 years and limited therapeutic options. The disease is characterized by collagen deposition and remodeling of the lung parenchyma in a process that is thought to be driven by collagen-expressing immune and structural cells. The G-protein coupled C-X-C chemokine receptor 4, CXCR4, is a candidate therapeutic target for IPF owing to its role in the recruitment of CXCR4+ fibrocytes from the bone marrow to fibrotic lung tissue and its increased expression levels by structural cells in fibrotic lung tissue. We have engineered a novel fully human single domain antibody “i-body” called AD-114 that binds with high affinity to human CXCR4. We demonstrate here that AD-114 inhibits invasive wound healing and collagen 1 secretion by human IPF fibroblasts but not non-diseased control lung fibroblasts. Furthermore, in a murine bleomycin model of pulmonary fibrosis, AD-114 reduced the accumulation of fibrocytes (CXCR4+/Col1+/CD45+) in fibrotic murine lungs and ameliorated the degree of lung injury. Collectively, these studies demonstrate that AD-114 holds promise as a new biological therapeutic for the treatment of IPF.

Histone Deacetylase Inhibition Downregulates Collagen 3A1 in Fibrotic Lung Fibroblasts

Idiopathic pulmonary fibrosis (IPF) is a deadly disease characterized by chronic inflammation and excessive collagen accumulation in the lung. Myofibroblasts are the primary collagen-producing cells in pulmonary fibrosis. Histone deacetylase inhibitor (HDACi) can affect gene expression, and some, such as suberoylanilide hydroxamic acid (SAHA), are US FDA approved for cancer treatment. In this study, we investigated SAHA’s effects on the expression of collagen III alpha 1 (COL3A1) in primary human IPF fibroblasts and in a murine model of pulmonary fibrosis. We observed that increased COL3A1 expression in IPF fibroblasts can be substantially reduced by SAHA treatment at the level of transcription as detected by RT-PCR; collagen III protein level was also reduced, as detected by Western blots and immunofluorescence. The deacetylation inhibitor effect of SAHA was verified by observing higher acetylation levels of both histone H3 and H4 in treated IPF cells. Chromatin immunoprecipitation (ChIP) experiments demonstrated that the reduced expression of COL3A1 by SAHA is with increased association of the repressive chromatin marker, H3K27Me3, and decreased association of the active chromatin marker, H3K9Ac. In our murine model of bleomycin-induced pulmonary fibrosis, the SAHA treated group demonstrated significantly less collagen III, as detected by immunohistochemistry. Our data indicate that the HDACi SAHA alters the chromatin associated with COL3A1, resulting in its decreased expression.

Decreased expression of autophagic beclin 1 protein in idiopathic pulmonary fibrosis fibroblasts

Autophagy is the main cellular pathway for degradation of long-lived proteins and organelles and regulates cell fate in response to stress. Beclin 1 is a key regulator of this process. In some settings autophagy and apoptosis seem to be interconnected. Recent reports indicate that fibroblasts in idiopathic pulmonary fibrosis (IPF) acquire resistance to apoptosis. Here, we examined the expression of beclin 1, and of the anti apoptotic protein Bcl-2 in human IPF fibroblasts using immunohistochemistry and molecular biology in bioptic sections, in primary cultures of fibroblasts taken from patients with IPF and in fibroblast cell lines. Expression of beclin 1 in fibroblasts from IPF was down-regulated in comparison with fibroblasts from normal lungs while the anti-apoptotic protein Bcl-2 expression was over-expressed. Treatment of fibroblast cell cultures with cisplatin induced a significant increase in beclin 1 and caspase 3 protein levels but a reduction in Bcl-2 expression. These observations were confirmed by the analysis of acid compartments and transmission electron microscopy. Our results demonstrate a modified expression of the apoptotic beclin 1 Bcl-2 proteins in human IPF fibroblasts suggesting the existence of an autophagy/apoptosis system dysfunction.

Aberrant innate immune sensing leads to the rapid progression of idiopathic pulmonary fibrosis

Novel approaches are needed to define subgroups of patients with Idiopathic pulmonary fibrosis (IPF) at risk for acute exacerbations and/or accelerated progression of this generally fatal disease. Progression of disease is an integral component of IPF with a median survival of 3 to 5 years. Conversely, a high degree of variability in disease progression has been reported among series. The characteristics of patients at risk of earlier death predominantly rely on baseline HRCT appearance, but this concept that has been challenged. Disparate physiological approaches have also been taken to identify patients at risk of mortality, with varying results. We hypothesized that the rapid decline in lung function in IPF may be a consequence of an abnormal host response to pathogen-associated molecular patterns (PAMPs), leading to aberrant activation in fibroblasts and fibrosis. Analysis of upper and lower lobe surgical lung biopsies (SLBs) indicated that TLR9, a hypomethylated CpG DNA receptor, is prominently expressed at the transcript and protein level, most notably in biopsies from rapidly progressive IPF patients. Surprisingly, fibroblasts appeared to be a major cellular source of TLR9 expression in IPF biopsies from this group of progressors. Further, CpG DNA promoted profibrotic cytokine and chemokine synthesis in isolated human IPF fibroblasts, most markedly again in cells from patients with the rapidly progressive IPF phenotype, in a TLR9-dependent manner. Finally, CpG DNA exacerbated fibrosis in an in vivo model initiated by the adoptive transfer of primary fibroblasts derived from patients who exhibited rapidly progressing fibrosis. Together, these data suggested that TLR9 activation via hypomethylated DNA might be an important mechanism in promoting fibrosis particularly in patients prone to rapidly progressing IPF.