ERG Regulates Lymphatic Vessel Specification Genes and Its Deficiency Impairs Wound Healing-Associated Lymphangiogenesis.

OBJECTIVE: Rarefaction of blood and lymphatic vessels in the skin has been reported in systemic sclerosis (SSc) (scleroderma). E26 transformation-specific-related factor (ERG) and Friend leukemia virus-induced erythroleukemia 1 (FLI-1) are important regulators of angiogenesis, but their role in lymphatic vasculature is lesser known. The goal of this study was to determine the role of ERG and FLI-1 in postnatal lymphangiogenesis and SSc lymphatic system defects. METHODS: Immunofluorescence was used to detect ERG and FLI-1 in skin biopsy samples from patients with SSc and healthy controls. Transcriptional analysis of ERG or FLI-1-silenced human dermal lymphatic endothelial cells (LECs) was performed using microarrays. Effects of ERG and FLI-1 deficiency on in vitro tubulogenesis in human dermal LECs were examined using a Matrigel assay. ERG and FLI-1 endothelial-specific knockouts and ERG lymphatic-specific knockouts were generated to examine vessel regeneration in mice. RESULTS: ERG and FLI-1 protein levels were reduced in the blood and lymphatic vasculature in SSc skin biopsy samples. ERG levels were shown to regulate genes involved in lymphatic vessel specification, including vascular endothelial growth factor receptor 3/FLT-4, lymphatic vessel endothelial hyaluronan receptor 1, SOX-18, and prospero homeobox 1 (PROX-1), whereas FLI-1 enhanced the function of ERG. The ERG-FLT-4 pathway regulated in vitro tubulogenesis in human LECs. Deficiency of ERG or FLI-1 similarly impaired the function of blood vessels in mice. However, only ERG deficiency affected the regeneration of lymphatic vessels during wound healing. CONCLUSION: ERG and FLI-1 are essential regulators of blood and lymphatic vessel regeneration. Deficiency of ERG and FLI-1 in SSc endothelial cells may contribute to the impairment of blood and lymphatic vasculature in patients with SSc.

Lung injury-induced activated endothelial cell states persist in aging-associated progressive fibrosis.

Progressive lung fibrosis is associated with poorly understood aging-related endothelial cell dysfunction. To gain insight into endothelial cell alterations in lung fibrosis we performed single cell RNA-sequencing of bleomycin-injured lungs from young and aged mice. Analysis reveals activated cell states enriched for hypoxia, glycolysis and YAP/TAZ activity in ACKR1+ venous and TrkB+ capillary endothelial cells. Endothelial cell activation is prevalent in lungs of aged mice and can also be detected in human fibrotic lungs. Longitudinal single cell RNA-sequencing combined with lineage tracing demonstrate that endothelial activation resolves in young mouse lungs but persists in aged ones, indicating a failure of the aged vasculature to return to quiescence. Genes associated with activated lung endothelial cells states in vivo can be induced in vitro by activating YAP/TAZ. YAP/TAZ also cooperate with BDNF, a TrkB ligand that is reduced in fibrotic lungs, to promote capillary morphogenesis. These findings offer insights into aging-related lung endothelial cell dysfunction that may contribute to defective lung injury repair and persistent fibrosis.

Rapid Generation of hPSC-Derived High Endothelial Venule Organoids with In Vivo Ectopic Lymphoid Tissue Capabilities.

Bioengineering strategies for the fabrication of implantable lymphoid structures mimicking lymph nodes (LNs) and tertiary lymphoid structures (TLS) could amplify the adaptive immune response for therapeutic applications such as cancer immunotherapy. No method to date has resulted in the consistent formation of high endothelial venules (HEVs), which is the specialized vasculature responsible for naïve T cell recruitment and education in both LNs and TLS. Here orthogonal induced differentiation of human pluripotent stem cells carrying a regulatable ETV2 allele is used to rapidly and efficiently induce endothelial differentiation. Assembly of embryoid bodies combining primitive inducible endothelial cells and primary human LN fibroblastic reticular cells results in the formation of HEV-like structures that can aggregate into 3D organoids (HEVOs). Upon transplantation into immunodeficient mice, HEVOs successfully engraft and form lymphatic structures that recruit both antigen-presenting cells and adoptively-transferred lymphocytes, therefore displaying basic TLS capabilities. The results further show that functionally, HEVOs can organize an immune response and promote anti-tumor activity by adoptively-transferred T lymphocytes. Collectively, the experimental approaches represent an innovative and scalable proof-of-concept strategy for the fabrication of bioengineered TLS that can be deployed in vivo to enhance adaptive immune responses.

Vascular Contribution to Lung Repair and Fibrosis.

Lungs are constantly exposed to environmental perturbations and therefore have remarkable capacity to regenerate in response to injury. Sustained lung injuries, aging, and increased genomic instability, however, make lungs particularly susceptible to disrepair and fibrosis. Pulmonary fibrosis constitutes a major cause of morbidity and is often relentlessly progressive, leading to death from respiratory failure. The pulmonary vasculature, which is critical for gas exchanges and plays a key role during lung development, repair, and regeneration, becomes aberrantly remodeled in patients with progressive pulmonary fibrosis. Although capillary rarefaction and increased vascular permeability are recognized as distinctive features of fibrotic lungs, the role of vasculature dysfunction in the pathogenesis of pulmonary fibrosis has only recently emerged as an important contributor to the progression of this disease. This review summarizes current findings related to lung vascular repair and regeneration and provides recent insights into the vascular abnormalities associated with the development of persistent lung fibrosis.

Transbronchial lung cryobiopsy and pulmonary fibrosis: A never-ending story?

Background: The diagnostic process of pulmonary fibrosis (PF) is often challenging, requires a collaborative effort of several experts, and often requires bioptic material, which can be difficult to obtain, both in terms of quality and technique. The main procedures available to obtain such samples are transbronchial lung cryobiopsy (TBLC) and surgical lung biopsy (SLB). Objective: The purpose of this paper is to review the evidence for the role of TBLC in the diagnostic-therapeutic process of PF. Methods: A comprehensive review was performed to identify articles to date that addressed the role of TBLC in the diagnostic-therapeutic process of PF using the PubMed® database. Results: The reasoned search identified 206 papers, including 21 manuscripts (three reviews, one systematic review, two guidelines, two prospective studies, three retrospective studies, one cross-sectional study, one original article, three editorials, three clinical trials, and two unclassifiable studies), which were included in the final review. Conclusions: TBLC is gaining increasing efficacy and improving safety profile; however, there are currently no clear data demonstrating its superiority over SLB. Therefore, the two techniques should be considered with careful rationalization on a case-by-case basis. Further research is needed to further optimize and standardize the procedure and to thoroughly study the histological and molecular characteristics of PF.

Mesenchymal cells in the Lung: Evolving concepts and their role in fibrosis.

Mesenchymal cells in the lung are crucial during development, but also contribute to the pathogenesis of fibrotic disorders, including idiopathic pulmonary fibrosis (IPF), the most common and deadly form of fibrotic interstitial lung diseases. Originally thought to behave as supporting cells for the lung epithelium and endothelium with a singular function of producing basement membrane, mesenchymal cells encompass a variety of cell types, including resident fibroblasts, lipofibroblasts, myofibroblasts, smooth muscle cells, and pericytes, which all occupy different anatomic locations and exhibit diverse homeostatic functions in the lung. During injury, each of these subtypes demonstrate remarkable plasticity and undergo varying capacity to proliferate and differentiate into activated myofibroblasts. Therefore, these cells secrete high levels of extracellular matrix (ECM) proteins and inflammatory cytokines, which contribute to tissue repair, or in pathologic situations, scarring and fibrosis. Whereas epithelial damage is considered the initial trigger that leads to lung injury, lung mesenchymal cells are recognized as the ultimate effector of fibrosis and attempts to better understand the different functions and actions of each mesenchymal cell subtype will lead to a better understanding of why fibrosis develops and how to better target it for future therapy. This review summarizes current findings related to various lung mesenchymal cells as well as signaling pathways, and their contribution to the pathogenesis of pulmonary fibrosis.

The matricellular protein CCN3 supports lung endothelial homeostasis and function.

Aberrant vascular remodeling contributes to the progression of many aging-associated diseases, including idiopathic pulmonary fibrosis (IPF), where heterogeneous capillary density, endothelial transcriptional alterations, and increased vascular permeability correlate with poor disease outcomes. Thus, identifying disease-driving mechanisms in the pulmonary vasculature may be a promising strategy to limit IPF progression. Here, we identified Ccn3 as an endothelial-derived factor that is upregulated in resolving but not in persistent lung fibrosis in mice, and whose function is critical for vascular homeostasis and repair. Loss and gain of function experiments were carried out to test the role of CCN3 in lung microvascular endothelial function in vitro through RNAi and the addition of recombinant human CCN3 protein, respectively. Endothelial migration, permeability, proliferation, and in vitro angiogenesis were tested in cultured human lung microvascular endothelial cells (ECs). Loss of CCN3 in lung ECs resulted in transcriptional alterations along with impaired wound-healing responses, in vitro angiogenesis, barrier integrity as well as an increased profibrotic activity through paracrine signals, whereas the addition of recombinant CCN3 augmented endothelial function. Altogether, our results demonstrate that the matricellular protein CCN3 plays an important role in lung endothelial function and could serve as a promising therapeutic target to facilitate vascular repair and promote lung fibrosis resolution.

Pim-1 kinase is a positive feedback regulator of the senescent lung fibroblast inflammatory secretome.

Cellular senescence is emerging as a driver of idiopathic pulmonary fibrosis (IPF), a progressive and fatal disease with limited effective therapies. The senescence-associated secretory phenotype (SASP), involving the release of inflammatory cytokines and profibrotic growth factors by senescent cells, is thought to be a product of multiple cell types in IPF, including lung fibroblasts. NF-κB is a master regulator of the SASP, and its activity depends on the phosphorylation of p65/RelA. The purpose of this study was to assess the role of Pim-1 kinase as a driver of NF-κB-induced production of inflammatory cytokines from low-passage IPF fibroblast cultures displaying markers of senescence. Our results demonstrate that Pim-1 kinase phosphorylates p65/RelA, activating NF-κB activity and enhancing IL-6 production, which in turn amplifies the expression of PIM1, generating a positive feedback loop. In addition, targeting Pim-1 kinase with a small molecule inhibitor dramatically inhibited the expression of a broad array of cytokines and chemokines in IPF-derived fibroblasts. Furthermore, we provide evidence that Pim-1 overexpression in low-passage human lung fibroblasts is sufficient to drive premature senescence, in vitro. These findings highlight the therapeutic potential of targeting Pim-1 kinase to reprogram the secretome of senescent fibroblasts and halt IPF progression.

Dysfunctional ERG signaling drives pulmonary vascular aging and persistent fibrosis.

Vascular dysfunction is a hallmark of chronic diseases in elderly. The contribution of the vasculature to lung repair and fibrosis is not fully understood. Here, we performed an epigenetic and transcriptional analysis of lung endothelial cells (ECs) from young and aged mice during the resolution or progression of bleomycin-induced lung fibrosis. We identified the transcription factor ETS-related gene (ERG) as putative orchestrator of lung capillary homeostasis and repair, and whose function is dysregulated in aging. ERG dysregulation is associated with reduced chromatin accessibility and maladaptive transcriptional responses to injury. Loss of endothelial ERG enhances paracrine fibroblast activation in vitro, and impairs lung fibrosis resolution in young mice in vivo. scRNA-seq of ERG deficient mouse lungs reveales transcriptional and fibrogenic abnormalities resembling those associated with aging and human lung fibrosis, including reduced number of general capillary (gCap) ECs. Our findings demonstrate that lung endothelial chromatin remodeling deteriorates with aging leading to abnormal transcription, vascular dysrepair, and persistent fibrosis following injury.

Transcriptional analysis of lung fibroblasts identifies PIM1 signaling as a driver of aging-associated persistent fibrosis.

Idiopathic pulmonary fibrosis (IPF) is an aging-associated disease characterized by myofibroblast accumulation and progressive lung scarring. To identify transcriptional gene programs driving persistent lung fibrosis in aging, we performed RNA-Seq on lung fibroblasts isolated from young and aged mice during the early resolution phase after bleomycin injury. We discovered that, relative to injured young fibroblasts, injured aged fibroblasts exhibited a profibrotic state characterized by elevated expression of genes implicated in inflammation, matrix remodeling, and cell survival. We identified the proviral integration site for Moloney murine leukemia virus 1 (PIM1) and its target nuclear factor of activated T cells-1 (NFATc1) as putative drivers of the sustained profibrotic gene signatures in injured aged fibroblasts. PIM1 and NFATc1 transcripts were enriched in a pathogenic fibroblast population recently discovered in IPF lungs, and their protein expression was abundant in fibroblastic foci. Overexpression of PIM1 in normal human lung fibroblasts potentiated their fibrogenic activation, and this effect was attenuated by NFATc1 inhibition. Pharmacological inhibition of PIM1 attenuated IPF fibroblast activation and sensitized them to apoptotic stimuli. Interruption of PIM1 signaling in IPF lung explants ex vivo inhibited prosurvival gene expression and collagen secretion, suggesting that targeting this pathway may represent a therapeutic strategy to block IPF progression.

Wet-dry-wet drug screen leads to the synthesis of TS1, a novel compound reversing lung fibrosis through inhibition of myofibroblast differentiation.

Therapies halting the progression of fibrosis are ineffective and limited. Activated myofibroblasts are emerging as important targets in the progression of fibrotic diseases. Previously, we performed a high-throughput screen on lung fibroblasts and subsequently demonstrated that the inhibition of myofibroblast activation is able to prevent lung fibrosis in bleomycin-treated mice. High-throughput screens are an ideal method of repurposing drugs, yet they contain an intrinsic limitation, which is the size of the library itself. Here, we exploited the data from our "wet" screen and used "dry" machine learning analysis to virtually screen millions of compounds, identifying novel anti-fibrotic hits which target myofibroblast differentiation, many of which were structurally related to dopamine. We synthesized and validated several compounds ex vivo ("wet") and confirmed that both dopamine and its derivative TS1 are powerful inhibitors of myofibroblast activation. We further used RNAi-mediated knock-down and demonstrated that both molecules act through the dopamine receptor 3 and exert their anti-fibrotic effect by inhibiting the canonical transforming growth factor ß pathway. Furthermore, molecular modelling confirmed the capability of TS1 to bind both human and mouse dopamine receptor 3. The anti-fibrotic effect on human cells was confirmed using primary fibroblasts from idiopathic pulmonary fibrosis patients. Finally, TS1 prevented and reversed disease progression in a murine model of lung fibrosis. Both our interdisciplinary approach and our novel compound TS1 are promising tools for understanding and combating lung fibrosis.

Myofibroblast dedifferentiation proceeds via distinct transcriptomic and phenotypic transitions.

Myofibroblasts are the major cellular source of collagen, and their accumulation - via differentiation from fibroblasts and resistance to apoptosis - is a hallmark of tissue fibrosis. Clearance of myofibroblasts by dedifferentiation and restoration of apoptosis sensitivity has the potential to reverse fibrosis. Prostaglandin E2 (PGE2) and mitogens such as FGF2 have each been shown to dedifferentiate myofibroblasts, but - to our knowledge - the resultant cellular phenotypes have neither been comprehensively characterized or compared. Here, we show that PGE2 elicited dedifferentiation of human lung myofibroblasts via cAMP/PKA, while FGF2 utilized MEK/ERK. The 2 mediators yielded transitional cells with distinct transcriptomes, with FGF2 promoting but PGE2 inhibiting proliferation and survival. The gene expression pattern in fibroblasts isolated from the lungs of mice undergoing resolution of experimental fibrosis resembled that of myofibroblasts treated with PGE2 in vitro. We conclude that myofibroblast dedifferentiation can proceed via distinct programs exemplified by treatment with PGE2 and FGF2, with dedifferentiation occurring in vivo most closely resembling the former.

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.

Muscularis macrophages establish cell-to-cell contacts with telocytes/PDGFRα-positive cells and smooth muscle cells in the human and mouse gastrointestinal tract.

BACKGROUND AND AIM: Muscularis macrophages (MMs) not only mediate the innate immunity, but also functionally interact with cells important for gastrointestinal motility. The aim of this study was to determine the spatial relationship and types of contacts between the MMs and neighboring cells in the muscularis propria of human and mouse stomach, small intestine, and large intestine. METHODS: The distribution and morphology of MMs and their contacts with other cells were investigated by immunohistochemistry and transmission electron microscopy. KEY RESULTS: Immunohistochemistry showed variable shape and number of MMs according to their location in different portions of the muscle coat. By double labeling, a close association between MMs and neighboring cells, that is, neurons, smooth muscle cells, interstitial cells of Cajal (ICCs), telocytes (TCs)/PDGFRα-positive cells, was seen. Electron microscopy demonstrated that in the muscle layers of both animal species, MMs have similar ultrastructural features and have specialized cell-to-cell contacts with smooth muscle cells and TCs/PDGFRα-positive cells but not with ICCs and enteric neurons. CONCLUSION & INFERENCES: This study describes varying patterns of distribution of MMs between different regions of the gut, and reports the presence of distinct and extended cell-to-cell contacts between MMs and smooth muscle cells and between MMs and TCs/PDGFRα-positive cells. In contrast, MMs, although close to ICCs and nerve elements, did not make contact with them. These findings indicate specialized and variable roles for MMs in the modulation of gastrointestinal motility whose significance should be more closely investigated in normal and pathological conditions.

Role of the Transcription Factor Yin Yang 1 and Its Selectively Identified Target Survivin in High-Grade B-Cells Non-Hodgkin Lymphomas: Potential Diagnostic and Therapeutic Targets.

B-cell non-Hodgkin lymphomas (B-NHLs) are often characterized by the development of resistance to chemotherapeutic drugs and/or relapse. During drug-induced apoptosis, Yin Yang 1 (YY1) transcription factor might modulate the expression of apoptotic regulators genes. The present study was aimed to: (1) examine the potential oncogenic role of YY1 in reversing drug resistance in B-NHLs; and (2) identify YY1 transcriptional target(s) that regulate the apoptotic pathway in B-NHLs. Predictive analyses coupled with database-deposited data suggested that YY1 binds the promoter of the BIRC5/survivin anti-apoptotic gene. Gene Expression Omnibus (GEO) analyses of several B-NHL repositories revealed a conserved positive correlation between YY1 and survivin, both highly expressed, especially in aggressive B-NHLs. Further validation experiments performed in Raji Burkitt's lymphomas cells, demonstrated that YY1 silencing was associated with survivin downregulation and sensitized the cells to apoptosis. Overall, our results revealed that: (1) YY1 and survivin are positively correlated and overexpressed in B-NHLs, especially in BLs; (2) YY1 strongly binds to the survivin promoter, hence survivin may be suggested as YY1 transcriptional target; (3) YY1 silencing sensitizes Raji cells to drug-induced apoptosis via downregulation of survivin; (4) both YY1 and survivin are potential diagnostic markers and therapeutic targets for the treatment of resistant/relapsed B-NHLs.

Vascular dysfunction in aged mice contributes to persistent lung fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive disease thought to result from impaired lung repair following injury and is strongly associated with aging. While vascular alterations have been associated with IPF previously, the contribution of lung vasculature during injury resolution and fibrosis is not well understood. To compare the role of endothelial cells (ECs) in resolving and non-resolving models of lung fibrosis, we applied bleomycin intratracheally to young and aged mice. We found that injury in aged mice elicited capillary rarefaction, while injury in young mice resulted in increased capillary density. ECs from the lungs of injured aged mice relative to young mice demonstrated elevated pro-fibrotic and reduced vascular homeostasis gene expression. Among the latter, Nos3 (encoding the enzyme endothelial nitric oxide synthase, eNOS) was transiently upregulated in lung ECs from young but not aged mice following injury. Young mice deficient in eNOS recapitulated the non-resolving lung fibrosis observed in aged animals following injury, suggesting that eNOS directly participates in lung fibrosis resolution. Activation of the NO receptor soluble guanylate cyclase in human lung fibroblasts reduced TGFß-induced pro-fibrotic gene and protein expression. Additionally, loss of eNOS in human lung ECs reduced the suppression of TGFß-induced lung fibroblast activation in 2D and 3D co-cultures. Altogether, our results demonstrate that persistent lung fibrosis in aged mice is accompanied by capillary rarefaction, loss of EC identity, and impaired eNOS expression. Targeting vascular function may thus be critical to promote lung repair and fibrosis resolution in aging and IPF.

TBK1 regulates YAP/TAZ and fibrogenic fibroblast activation.

Idiopathic pulmonary fibrosis (IPF) results in scarring of the lungs by excessive extracellular matrix (ECM) production. Resident fibroblasts are the major cell type involved in ECM deposition. The biochemical pathways that facilitate pathological fibroblast activation leading to aberrant ECM deposition are not fully understood. Tank binding protein kinase-1 (TBK1) is a kinase that regulates multiple signaling pathways and was recently identified as a candidate regulator of fibroblast activation in a large-scale small-interfering RNA (siRNA) screen. To determine the effect of TBK1 on fibroblast activation, TBK1 was inhibited pharmacologically (MRT-68601) and genetically (siRNA) in normal and IPF human lung fibroblasts. Reducing the activity or expression of TBK1 led to reduction in α-smooth muscle actin stress fiber levels by 40-60% and deposition of ECM components collagen I and fibronectin by 50% in TGF-ß-stimulated normal and IPF fibroblasts. YAP and TAZ are homologous mechanoregulatory profibrotic transcription cofactors known to regulate fibroblast activation. TBK1 knockdown or inhibition decreased the total and nuclear protein levels of YAP/TAZ. Additionally, low cell-cell contact and increased ECM substrate stiffness augmented the phosphorylation and activation of TBK1, consistent with cues that regulate YAP/TAZ. The action of TBK1 toward YAP/TAZ activation was independent of LATS1/2 and canonical downstream TBK1 signaling mediator IRF3 but dependent on proteasomal machinery of the cell. This study identifies TBK1 as a fibrogenic activator of human pulmonary fibroblasts, suggesting TBK1 may be a novel therapeutic target in pulmonary fibrosis.

Targeted regulation of fibroblast state by CRISPR-mediated CEBPA expression.

BACKGROUND: Fibroblasts regulate tissue homeostasis and the balance between tissue repair and fibrosis. CCAAT/enhancer-binding protein alpha (CEBPA) is a key transcription factor that regulates adipogenesis. CEBPA has been shown to be essential for lung maturation, and deficiency of CEBPA expression leads to abnormal lung architecture. However, its specific role in lung fibroblast regulation and fibrosis has not yet been elucidated. METHODS: Lung fibroblast CEBPA expression, pro-fibrotic and lipofibroblast gene expression were assessed by qRT-PCR. CEBPA gain and loss of function experiments were carried out to evaluate the role of CEBPA in human lung fibroblast activation with and without TGF-ß1 treatment. Adipogenesis assay was used to measure the adiopogenic potential of lung fibroblasts. Finally, CRISPR activation system was used to enhance endogenous CEBPA expression. RESULTS: We found that CEBPA gene expression is significantly decreased in IPF-derived fibroblasts compared to normal lung fibroblasts. CEBPA knockdown in normal human lung fibroblasts enhanced fibroblast pro-fibrotic activation and ECM production. CEBPA over-expression by transient transfection in IPF-derived fibroblasts significantly reduced pro-fibrotic gene expression, ECM deposition and αSMA expression and promoted the formation of lipid droplets measured by Oil Red O staining and increased lipofibroblast gene expression. Inhibition of the histone methyl transferase G9a enhanced CEBPA expression, and the anti-fibrotic effects of G9a inhibition were partially mediated by CEBPA expression. Finally, targeted CRISPR-mediated activation of CEBPA resulted in fibroblasts switching from fibrogenic to lipofibroblast states. CONCLUSIONS: CEBPA expression is reduced in human IPF fibroblasts and its deficiency reduces adipogenic potential and promotes fibrogenic activation. CEBPA expression can be rescued via an inhibitor of epigenetic repression or by targeted CRISPR activation, leading to reduced fibrogenic activation.

Selective YAP/TAZ inhibition in fibroblasts via dopamine receptor D1 agonism reverses fibrosis.

Tissue fibrosis is characterized by uncontrolled deposition and diminished clearance of fibrous connective tissue proteins, ultimately leading to organ scarring. Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) have recently emerged as pivotal drivers of mesenchymal cell activation in human fibrosis. Therapeutic strategies inhibiting YAP and TAZ have been hindered by the critical role that these proteins play in regeneration and homeostasis in different cell types. Here, we find that the Gαs-coupled dopamine receptor D1 (DRD1) is preferentially expressed in lung and liver mesenchymal cells relative to other resident cells of these organs. Agonism of DRD1 selectively inhibits YAP/TAZ function in mesenchymal cells and shifts their phenotype from profibrotic to fibrosis resolving, reversing in vitro extracellular matrix stiffening and in vivo tissue fibrosis in mouse models. Aromatic l-amino acid decarboxylase [DOPA decarboxylase (DDC)], the enzyme responsible for the final step in biosynthesis of dopamine, is decreased in the lungs of subjects with idiopathic pulmonary fibrosis, and its expression inversely correlates with disease severity, consistent with an endogenous protective role for dopamine signaling that is lost in pulmonary fibrosis. Together, these findings establish a pharmacologically tractable and cell-selective approach to targeting YAP/TAZ via DRD1 that reverses fibrosis in mice.

PGC1α repression in IPF fibroblasts drives a pathologic metabolic, secretory and fibrogenic state.

Idiopathic pulmonary fibrosis (IPF) is a fatal ageing-related disease linked to mitochondrial dysfunction. The present study aimed to determine whether peroxisome proliferator activated receptor gamma co-activator 1-alpha (PPARGC1A, encoding PGC1α), a master regulator of mitochondrial biogenesis, is diminished in IPF and controls pathologic fibroblast activation. Primary human IPF, control lung fibroblasts and fibroblasts sorted from bleomycin-injured mice were used to evaluate the expression and function of PGC1α. In vitro PGC1α manipulation was performed by small interfering RNA knockdown or overexpression. Fibroblast activation was assessed by quantitative PCR, Western blotting, matrix deposition, secreted cytokine array, immunofluorescence and traction force microscopy. Mitochondrial function was assessed by Seahorse analyzer and mitochondria mass and number by flow cytometry, mitochondrial DNA quantification and transmission electron microscopy (TEM). We found that PGC1α levels are stably repressed in IPF fibroblasts. After bleomycin injury in young mice, PGC1α expression drops transiently but then increases prior to fibrosis resolution. In contrast, PGC1α expression fails to recover in aged mice with persistent fibrosis. PGC1α knockdown alone in normal human lung fibroblasts reduces mitochondrial mass and function while enhancing contractile and matrix synthetic fibroblast activation, senescence-related gene expression and soluble profibrotic and prosenescence signalling. Re-expression of PGC1α in IPF fibroblasts ameliorates all of these pathological cellular functions. Pharmacological treatment of IPF fibroblasts with rosiglitazone, but not thyroid hormone, elevated PGC1α expression and attenuated fibroblast activation. The sustained repression of PGC1α and beneficial effects of its rescue in IPF fibroblasts identifies PGC1α as an important regulator of the fibroblast's pathological state in IPF.