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Repair and regeneration of a diseased lung using stem cells or bioengineered tissues is an exciting therapeutic approach for a variety of lung diseases and critical illnesses. Over the past decade, increasing evidence from preclinical models suggests that mesenchymal stromal cells, which are not normally resident in the lung, can be used to modulate immune responses after injury, but there have been challenges in translating these promising findings to the clinic. In parallel, there has been a surge in bioengineering studies investigating the use of artificial and acellular lung matrices as scaffolds for three-dimensional lung or airway regeneration, with some recent attempts of transplantation in large animal models. The combination of these studies with those involving stem cells, induced pluripotent stem cell derivatives, and/or cell therapies is a promising and rapidly developing research area. These studies have been further paralleled by significant increases in our understanding of the molecular and cellular events by which endogenous lung stem and/or progenitor cells arise during lung development and participate in normal and pathological remodeling after lung injury. For the 2023 Stem Cells, Cell Therapies, and Bioengineering in Lung Biology and Diseases Conference, scientific symposia were chosen to reflect the most cutting-edge advances in these fields. Sessions focused on the integration of "omics" technologies with function, the influence of immune cells on regeneration, and the role of the extracellular matrix in regeneration. The necessity for basic science studies to enhance fundamental understanding of lung regeneration and to design innovative translational studies was reinforced throughout the conference.

Fibrosis in the lung is thought to be driven by epithelial cell dysfunction and aberrant cell-cell interactions. Unveiling the molecular mechanisms of cellular plasticity and cell-cell interactions is imperative to elucidating lung regenerative capacity and aberrant repair in pulmonary fibrosis. By mining publicly available RNA-Seq data sets, we identified loss of CCAAT enhancer-binding protein alpha (CEBPA) as a candidate contributor to idiopathic pulmonary fibrosis (IPF). We used conditional KO mice, scRNA-Seq, lung organoids, small-molecule inhibition, and potentially novel gene manipulation methods to investigate the role of CEBPA in lung fibrosis and repair. Long-term (6 months or more) of Cebpa loss in AT2 cells caused spontaneous fibrosis and increased susceptibility to bleomycin-induced fibrosis. Cebpa knockout (KO) in these mice significantly decreased AT2 cell numbers in the lung and reduced expression of surfactant homeostasis genes, while increasing inflammatory cell recruitment as well as upregulating S100a8/a9 in AT2 cells. In vivo treatment with an S100A8/A9 inhibitor alleviated experimental lung fibrosis. Restoring CEBPA expression in lung organoids ex vivo and during experimental lung fibrosis in vivo rescued CEBPA deficiency-mediated phenotypes. Our study establishes a direct mechanistic link between CEBPA repression, impaired AT2 cell identity, disrupted tissue homeostasis, and lung fibrosis.

Idiopathic pulmonary fibrosis (IPF) is an aggressive and thus far incurable disease, characterized by aberrant fibroblast-mediated extracellular matrix deposition. Our understanding of the disease etiology is incomplete; however, there is consensus that a reduction-oxidation (redox) imbalance plays a role. In this study we use the autofluorescent properties of two redox molecules, NAD(P)H and FAD, to quantify changes in their relative abundance in living lung tissue of mice with experimental lung fibrosis, and in freshly isolated cells from mouse lungs and humans with IPF. Our results identify cell population-specific intracellular redox changes in the lungs in experimental and human fibrosis. We focus particularly on redox changes within collagen producing cells, where we identified a bimodal distribution of NAD(P)H concentrations, establishing NAD(P)H high and NAD(P)H low sub-populations. NAD(P)H high fibroblasts exhibited elevated pro-fibrotic gene expression and decreased collagenolytic protease activity relative to NAD(P)H low fibroblasts. The NAD(P)H high population was present in healthy lungs but expanded with time after bleomycin injury suggesting a potential role in fibrosis progression. We identified a similar increased abundance of NAD(P)H high cells in freshly dissociated lungs of subjects with IPF relative to controls, and similar reductions in collagenolytic activity in this cell population. These data highlight the complexity of redox state changes in experimental and human pulmonary fibrosis and the need for selective approaches to restore redox imbalances in the fibrotic lung.

Pulmonary fibrosis is characterized by pathological accumulation of scar tissue in the lung parenchyma. Many of the processes that are implicated in fibrosis, including increased extracellular matrix synthesis, also occur following pneumonectomy (PNX), but PNX instead results in regenerative compensatory growth of the lung. As fibroblasts are the major cell type responsible for extracellular matrix production, we hypothesized that comparing fibroblast responses to PNX and bleomycin (BLM) would unveil key differences in the role they play during regenerative versus fibrotic lung responses. RNA-sequencing was performed on flow-sorted fibroblasts freshly isolated from mouse lungs 14 days after BLM, PNX, or sham controls. RNA-sequencing analysis revealed highly similar biological processes to be involved in fibroblast responses to both BLM and PNX, including TGF-β1 and TNF-α. Interestingly, we observed smaller changes in gene expression after PNX than BLM at Day 14, suggesting that the fibroblast response to PNX may be muted by expression of transcripts that moderate pro-fibrotic pathways. Itpkc, encoding inositol triphosphate kinase C, was a gene uniquely up-regulated by PNX and not BLM. ITPKC overexpression in lung fibroblasts antagonized the pro-fibrotic effect of TGF-β1. RNA-sequencing analysis has identified considerable overlap in transcriptional changes between fibroblasts following PNX and those overexpressing ITPKC., (© 2024 The Author(s). Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Objective: Phlpp1 inhibition is a potential therapeutic strategy for cartilage regeneration and prevention of post-traumatic osteoarthritis (PTOA). To understand how Phlpp1 loss affects cartilage structure, cartilage elastic modulus was measured with atomic force microscopy (AFM) in male and female mice after injury., Methods: Osteoarthritis was induced in male and female Wildtype (WT) and Phlpp1 -/- mice by destabilization of the medial meniscus (DMM). At various timepoints post-injury, activity was measured, and knee joints examined with AFM and histology. In another cohort of WT mice, the PHLPP inhibitor NSC117079 was intra-articularly injected 4 weeks after injury., Results: Male WT mice showed decreased activity and histological signs of cartilage damage at 12 but not 6-weeks post-DMM. Female mice showed a less severe response to DMM by comparison, with no histological changes seen at any time point. In both sexes the elastic modulus of medial condylar cartilage was decreased in WT mice but not Phlpp1 -/- mice after DMM as measured by AFM. By 6-weeks, cartilage modulus had decreased from 2 MPa to 1 MPa in WT mice. Phlpp1 -/- mice showed no change in modulus at 6-weeks and only a 25% decrease at 12-weeks. The PHLPP inhibitor NSC117079 protected cartilage structure and prevented signs of OA 6-weeks post-injury., Conclusions: AFM is a sensitive method for detecting early changes in articular cartilage post-injury. Phlpp1 suppression, either through genetic deletion or pharmacological inhibition, protects cartilage degradation in a model of PTOA, validating Phlpp1 as a therapeutic target for PTOA., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2024 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Pulmonary arterial hypertension (PAH) causes pulmonary vascular remodeling, increasing pulmonary vascular resistance (PVR) and leading to right heart failure and death. Matrix stiffening early in the disease promotes remodeling in pulmonary artery smooth muscle cells (PASMCs), contributing to PAH pathogenesis. Our research identified YAP and TAZ as key drivers of the mechanobiological feedback loop in PASMCs, suggesting targeting them could mitigate remodeling. However, YAP/TAZ are ubiquitously expressed and carry out diverse functions, necessitating a cell-specific approach. Our previous work demonstrated that targeting non-canonical IKB kinase TBK1 reduced YAP/TAZ activation in human lung fibroblasts. Here, we investigate non-canonical IKB kinases TBK1 and IKKε in pulmonary hypertension (PH) and their potential to modulate PASMC pathogenic remodeling by regulating YAP/TAZ. We show that TBK1 and IKKε are activated in PASMCs in a rat PH model. Inflammatory cytokines, elevated in PAH, activate these kinases in human PASMCs. Inhibiting TBK1/IKKε expression/activity significantly reduces PAH-associated PASMC remodeling, with longer-lasting effects on YAP/TAZ than treprostinil, an approved PAH therapy. These results show that non-canonical IKB kinases regulate YAP/TAZ in PASMCs and may offer a novel approach for reducing vascular remodeling in PAH., (© 2024 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Background: Dilated cardiomyopathy (DCM) is a major cause of heart failure and carries a high mortality rate. Myocardial recovery in DCM-related heart failure patients is highly variable, with some patients having little or no response to standard drug therapy. A genome-wide association study may agnostically identify biomarkers and provide novel insight into the biology of myocardial recovery in DCM., Methods: A genome-wide association study for change in left ventricular ejection fraction was performed in 686 White subjects with recent-onset DCM who received standard pharmacotherapy. Genome-wide association study signals were subsequently functionally validated and studied in relevant cellular models to understand molecular mechanisms that may have contributed to the change in left ventricular ejection fraction., Results: The genome-wide association study identified a highly suggestive locus that mapped to the 5'-flanking region of the CDCP1 (CUB [complement C1r/C1s, Uegf, and Bmp1] domain containing protein 1) gene (rs6773435; P =7.12×10 -7 ). The variant allele was associated with improved cardiac function and decreased CDCP1 transcription. CDCP1 expression was significantly upregulated in human cardiac fibroblasts (HCFs) in response to the PDGF (platelet-derived growth factor) signaling, and knockdown of CDCP1 significantly repressed HCF proliferation and decreased AKT (protein kinase B) phosphorylation. Transcriptomic profiling after CDCP1 knockdown in HCFs supported the conclusion that CDCP1 regulates HCF proliferation and mitosis. In addition, CDCP1 knockdown in HCFs resulted in significantly decreased expression of soluble ST2 (suppression of tumorigenicity-2), a prognostic biomarker for heart failure and inductor of cardiac fibrosis., Conclusions: CDCP1 may play an important role in myocardial recovery in recent-onset DCM and mediates its effect primarily by attenuating cardiac fibrosis., Competing Interests: Disclosures R.M. Weinshilboum is a cofounder of and stockholder in OneOme, LLC. The other authors report no conflicts.

The neutral amino acid glutamine plays a central role in TGF-β (transforming growth factor-β)-induced myofibroblast activation and differentiation. Cells take up glutamine mainly through a transporter expressed on the cell surface known as solute carrier SLC1A5 (solute carrier transporter 1A5). In the present work, we demonstrated that profibrotic actions of TGF-β are mediated, at least in part, through a metabolic maladaptation of SLC1A5 and that targeting SLC1A5 abrogates multiple facets of fibroblast activation. This approach could thus represent a novel therapeutic strategy to treat patients with fibroproliferative diseases. We found that SLC1A5 was highly expressed in fibrotic lung fibroblasts and fibroblasts isolated from idiopathic pulmonary fibrosis lungs. The expression of profibrotic targets, cell migration, and anchorage-independent growth by TGF-β required the activity of SLC1A5. Loss or inhibition of SLC1A5 function enhanced fibroblast susceptibility to autophagy; suppressed mTOR, HIF (hypoxia-inducible factor), and Myc signaling; and impaired mitochondrial function, ATP production, and glycolysis. Pharmacological inhibition of SLC1A5 by the small-molecule inhibitor V-9302 shifted fibroblast transcriptional profiles from profibrotic to fibrosis resolving and attenuated fibrosis in a bleomycin-treated mouse model of lung fibrosis. This is the first study, to our knowledge, to demonstrate the utility of a pharmacological inhibitor of glutamine transport in fibrosis, providing a framework for new paradigm-shifting therapies targeting cellular metabolism for this devastating disease.

Pulmonary fibroblasts are the primary producers of extracellular matrix (ECM) in the lungs, and their pathogenic activation drives scarring and loss of lung function in idiopathic pulmonary fibrosis (IPF). This uncontrolled production of ECM is stimulated by mechanosignaling and transforming growth factor beta 1 (TGF- β 1) signaling that together promote transcriptional programs including Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ). G protein-coupled receptors (GPCRs) that couple to G α s have emerged as pharmacological targets to inactivate YAP/TAZ signaling and promote lung fibrosis resolution. Previous studies have shown a loss of expression of "antifibrotic GPCRs"-receptors that couple to G α s, in IPF patient-derived fibroblasts compared with non-IPF samples. Of the 14 G α s GPCRs we found to be expressed in lung fibroblasts, the dopamine receptor D1 ( DRD1 ) was one of only two not repressed by TGF- β 1 signaling, with the β 2-adrenergic receptor being the most repressed. We compared the potency and efficacy of multiple D1 and β 2 receptor agonists +/- TGF- β 1 treatment in vitro for their ability to elevate cAMP, inhibit nuclear localization of YAP/TAZ, regulate expression of profibrotic and antifibrotic genes, and inhibit cellular proliferation and collagen deposition. Consistently, the activity of β 2 receptor agonists was lost, whereas D1 receptor agonists was maintained, after stimulating cultured lung fibroblasts with TGF- β 1. These data further support the therapeutic potential of the dopamine receptor D1 and highlight an orchestrated and pervasive loss of antifibrotic GPCRs mediated by TGF- β 1 signaling. SIGNIFICANCE STATEMENT: Idiopathic pulmonary fibrosis (IPF) is a deadly lung disease with limited therapies. GPCRs have emerged as a primary target for the development of novel antifibrotic drugs; however, a challenge to this approach is the dramatic changes in GPCR expression in response to profibrotic stimuli. Here, we investigate the impact of TGF- β 1 on the expression of antifibrotic GPCRs and show the D1 dopamine receptor expression is uniquely maintained in response to TGF- β 1, further implicating it as a compelling target to treat IPF., (Copyright © 2023 by The American Society for Pharmacology and Experimental Therapeutics.)

The mechanical properties of biological tissues influence their function and can predict degenerative conditions before gross histological or physiological changes are detectable. This is especially true for structural tissues such as articular cartilage, which has a primarily mechanical function that declines after injury and in the early stages of osteoarthritis. While atomic force microscopy (AFM) has been used to test the elastic modulus of articular cartilage before, there is no agreement or consistency in methodologies reported. For murine articular cartilage, methods differ in two major ways: experimental parameter selection and sample preparation. Experimental parameters that affect AFM results include indentation force and cantilever stiffness; these are dependent on the tip, sample, and instrument used. The aim of this project was to optimize these experimental parameters to measure murine articular cartilage elastic modulus by AFM micro-indentation. We first investigated the effects of experimental parameters on a control material, polydimethylsiloxane gel (PDMS), which has an elastic modulus on the same order of magnitude as articular cartilage. Experimental parameters were narrowed on this control material, and then finalized on wildtype C57BL/6J murine articular cartilage samples that were prepared with a novel technique that allows for cryosectioning of epiphyseal segments of articular cartilage and long bones without decalcification. This technique facilitates precise localization of AFM measurements on the murine articular cartilage matrix and eliminates the need to separate cartilage from underlying bone tissues, which can be challenging in murine bones because of their small size. Together, the new sample preparation method and optimized experimental parameters provide a reliable standard operating procedure to measure microscale variations in the elastic modulus of murine articular cartilage.

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.

Systemic sclerosis (SSc) is a clinically heterogeneous fibrotic disease with no effective treatment. Myofibroblasts are responsible for unresolving synchronous skin and internal organ fibrosis in SSc, but the drivers of sustained myofibroblast activation remain poorly understood. Using unbiased transcriptome analysis of skin biopsies, we identified the downregulation of SPAG17 in multiple independent cohorts of patients with SSc, and by orthogonal approaches, we observed a significant negative correlation between SPAG17 and fibrotic gene expression. Fibroblasts and endothelial cells explanted from SSc skin biopsies showed reduced chromatin accessibility at the SPAG17 locus. Remarkably, mice lacking Spag17 showed spontaneous skin fibrosis with increased dermal thickness, collagen deposition and stiffness, and altered collagen fiber alignment. Knockdown of SPAG17 in human and mouse fibroblasts and microvascular endothelial cells was accompanied by spontaneous myofibroblast transformation and markedly heightened sensitivity to profibrotic stimuli. These responses were accompanied by constitutive TGF-β pathway activation. Thus, we discovered impaired expression of SPAG17 in SSc and identified, to our knowledge, a previously unreported cell-intrinsic role for SPAG17 in the negative regulation of fibrotic responses. These findings shed fresh light on the pathogenesis of SSc and may inform the search for innovative therapies for SSc and other fibrotic conditions through SPAG17 signaling., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: During liver injury, liver sinusoidal endothelial cells (LSECs) dysfunction and capillarization promote liver fibrosis. We have previously reported that the LSEC vascular cell adhesion molecule 1 (VCAM1) plays a key role in liver inflammation in nonalcoholic steatohepatitis (NASH) and we now aim to uncover its role in LSEC capillarization and liver fibrosis., Methods: Wild-type C57BL/6J mice were fed either chow or high fat, fructose and cholesterol diet to induce NASH and treated with either anti-VCAM1 neutralizing antibody or control isotype antibody. Inducible endothelial cell-specific Vcam1 deleted mice ( Vcam1 Δend ) and control mice ( Vcam1 fl/fl ) were fed choline-deficient high-fat diet (CD-HFD) to induce NASH or injected with carbon tetrachloride to induce liver fibrosis. LSECs isolated from Vcam1 fl/fl or Vcam1 Δend and hepatic stellate cells (HSCs) isolated from wild-type mice were cocultured in a 3-D system or a μ-Slide 2 well co-culture system., Results: Immunostaining for Lyve1 (marker of differentiated LSECs) was reduced in Vcam1 fl/fl mice and restored in Vcam1 Δend mice in both NASH and liver fibrosis models. Co-immunostaining showed increased α-smooth muscle actin in the livers of Vcam1 fl/fl mice in areas lacking Lyve1. Furthermore, scanning electron microscopy showed reduced LSEC fenestrae in the Vcam1 fl/fl mice but not Vcam1 Δend mice in both injury models, suggesting that VCAM1 promotes LSEC capillarization during liver injury. HSCs profibrogenic markers were reduced when cocultured with LSECs from CD-HFD fed Vcam1 Δend mice compared to Vcam1 fl/fl mice. Furthermore, recombinant VCAM1 activated the Yes-associated protein 1 pathway and induced a fibrogenic phenotype in HSCs in vitro , supporting the profibrogenic role of LSEC VCAM1., Conclusion: VCAM1 is not just a scaffold for leukocyte adhesion during liver injury, but also a modulator of LSEC capillarization and liver fibrosis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Guo, Furuta, Islam, Caporarello, Kostallari, Dielis, Tschumperlin, Hirsova and Ibrahim.)

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., (© 2022. The Author(s).)

Idiopathic pulmonary fibrosis (IPF) and rheumatoid arthritis-associated interstitial lung disease (RA-ILD) are two fibrotic interstitial lung diseases that share the usual interstitial pneumonia (UIP) injury pattern. Here, we report that RNA sequencing of lung biopsies from patients with RA-ILD and IPF revealed shared and distinct disease-causing pathways. Analysis of transcriptomic data identified a JAK2 related JAK/STAT signaling pathway gene signature that distinguishes RA-UIP from idiopathic UIP. This was further confirmed by immunohistostaining, which identified JAK2 phosphorylation with two distinct forms of activation: a cytoplasmic form of JAK2 activation in most IPF cases (13/20) and a nuclear form of p-JAK2 in RA-UIP (5/5) and a minority of IPF (6/20) cases. Further immunohistostaining identified STAT5A&B as the downstream transcriptional activator for JAK2-mediated canonical signal transduction and phosphorylation of Tyr41 on histone H3 (H3Y41ph) as the downstream epigenetic regulation site for JAK2-mediated noncanonical signal transduction. Gene Set Enrichment Analysis (GSEA) of the RNA-Seq data further supported this shared pathogenic mechanism for the two diseases with the enrichment of STAT5A&B target gene sets as well as the JAK2 regulated H3Y41ph target gene set. This regulatory role of JAK2 in the pathogenesis of pulmonary fibrosis was further demonstrated by the attenuation of bleomycin-induced murine pulmonary fibrosis using a JAK2-selective pharmacological inhibitor CEP33779. In vitro studies with normal and IPF derived lung fibroblasts revealed a central role for JAK2 as an essential intermediary molecule in TGF-β-mediated myofibroblast trans-differentiation, proliferation, and extracellular matrix protein production. These observations support a crucial role for JAK2 as an intermediary molecule in fibrotic lung disease development., (© 2022 Federation of American Societies for Experimental Biology.)

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.

The fibrogenic wound-healing response in liver increases stiffness. Stiffness mechanotransduction, in turn, amplifies fibrogenesis. Here, we aimed to understand the distribution of stiffness in fibrotic liver, how it impacts hepatic stellate cell (HSC) heterogeneity, and identify mechanisms by which stiffness amplifies fibrogenic responses. Magnetic resonance elastography and atomic force microscopy demonstrated a heterogeneous distribution of liver stiffness at macroscopic and microscopic levels, respectively, in a carbon tetrachloride (CCl 4 ) mouse model of liver fibrosis as compared with controls. High stiffness was mainly attributed to extracellular matrix dense areas. To identify a stiffness-sensitive HSC subpopulation, we performed single-cell RNA sequencing (scRNA-seq) on primary HSCs derived from healthy versus CCl 4 -treated mice. A subcluster of HSCs was matrix-associated with the most upregulated pathway in this subpopulation being focal adhesion signaling, including a specific protein termed four and a half LIM domains protein 2 (FHL2). In vitro, FHL2 expression was increased in primary human HSCs cultured on stiff matrix as compared with HSCs on soft matrix. Moreover, FHL2 knockdown inhibited fibronectin and collagen 1 expression, whereas its overexpression promoted matrix production. In summary, we demonstrate stiffness heterogeneity at the whole organ, lobular, and cellular level, which drives an amplification loop of fibrogenesis through specific focal adhesion molecular pathways. NEW & NOTEWORTHY The fibrogenic wound-healing response in liver increases stiffness. Here, macro and microheterogeneity of liver stiffness correlate with HSC heterogeneity in a hepatic fibrosis mouse model. Fibrogenic HSCs localized in stiff collagen-high areas upregulate the expression of focal adhesion molecule FHL2, which, in turn, promotes extracellular matrix protein expression. These results demonstrate that stiffness heterogeneity at the whole organ, lobular, and cellular level drives an amplification loop of fibrogenesis through specific focal adhesion molecular pathways.

Aging is accompanied by declining lung function and increasing susceptibility to lung diseases. The role of endothelial dysfunction and vascular remodeling in these changes is supported by growing evidence, but underlying mechanisms remain elusive. In this review we summarize functional, structural, and molecular changes in the aging pulmonary vasculature and explore how interacting aging and mechanobiological cues may drive progressive vascular remodeling in the lungs.

Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) are transcription cofactors implicated in the contractile and profibrotic activation of fibroblasts. Fibroblast contractile function is important in alveologenesis and in lung wound healing and fibrosis. As paralogs, YAP and TAZ may have independent or redundant roles in regulating transcriptional programs and contractile function. Using IMR-90 lung fibroblasts, microarray analysis, and traction microscopy, we tested whether independent YAP or TAZ knockdown alone was sufficient to limit transcriptional activation and contraction in vitro. Our results demonstrate limited effects of knockdown of either YAP or TAZ alone, with more robust transcriptional and functional effects observed with combined knockdown, consistent with cooperation or redundancy of YAP and TAZ in transforming growth factor β1 (TGFβ1)-induced fibroblast activation and contractile force generation. The transcriptional responses to combined YAP/TAZ knockdown were focused on a relatively small subset of genes with prominent overrepresentation of genes implicated in contraction and migration. To explore potential disease relevance of our findings, we tested primary human lung fibroblasts isolated from patients with idiopathic pulmonary fibrosis and confirmed that YAP and TAZ combined knockdown reduced the expression of three cytoskeletal genes, ACTA2 , CNN1 , and TAGLN . We then compared the contribution of these genes, along with YAP and TAZ, to contractile function. Combined knockdown targeting YAP/TAZ was more effective than targeting any of the individual cytoskeletal genes in reducing contractile function. Together, our results demonstrate that YAP and TAZ combine to regulate a multigene program that is essential to fibroblast contractile function.

Epithelial-mesenchymal transition (EMT) creates an environment facilitating fibrosis following alveolar epithelial cell injury. IL-23 has important roles in chronic autoimmune conditions like rheumatoid arthritis (RA), but its role in the interstitial lung disease that affects patients with RA is unclear. This study aimed to determine the profibrogenic role of IL-23 on somatic alveolar type I (ATI) epithelial cells. Primary ATI cells were isolated from rats and cultured on plastic dishes for 1-3 wk. After prolonged culture (≥14 days) on rigid culture dishes, primary ATI cells gradually acquired a mesenchymal phenotype, identified by decreased expression of caveolin-1, and reorganization of F-actin cytoskeleton, indicating the initiation of EMT by matrix stiffness. To determine how IL-23 promotes EMT in vitro, transitioning ATI cells, cultured on a stiff substrate for ≥14 days were stimulated with IL-23. The EMT phenotype was significantly enhanced by IL-23, which upregulated α-smooth muscle actin (α-SMA), collagen I/III protein, and decreased caveolin-1. Furthermore, IL-23 significantly promoted cell invasion, as well as apoptotic resistance on transitioning ATI cells. Mechanistically, IL-23-induced EMT was mammalian target of rapamycin/ribosomal protein S6 (mTOR/S6) signaling dependent and reversible by rapamycin. Transcriptional sequencing analysis of human lung fibrosis biopsy tissue revealed key roles for IL-23 in rheumatoid arthritis-associated interstitial lung disease (RA-ILD). This result was further validated by significantly upregulated IL-23 expression at the mRNA level in RA-ILD lung sections. Notably, transitioning ATI epithelial cells were abundantly detected in RA-ILD tissue. Taken together, these data support a role for IL-23 in the pathogenesis of RA lung fibrosis by promoting EMT in alveolar epithelial cells through mTOR/S6 signaling.

Idiopathic pulmonary fibrosis (IPF) is a lethal lung disease targeting the alveolar gas exchange apparatus, leading to death by asphyxiation. IPF progresses on a tissue scale through aberrant matrix remodeling, enhanced cell contraction, and subsequent microenvironment densification. Although two pharmaceuticals modestly slow progression, IPF patient survival averages less than 5 years. A major impediment to therapeutic development is the lack of high-fidelity models that account for the fibrotic microenvironment. Our goal is to create a three-dimensional (3D) platform to enable lung fibrosis studies and recapitulate IPF tissue features. We demonstrate that normal lung fibroblasts encapsulated in collagen microspheres can be pushed toward an activated phenotype, treated with FDA-approved therapies, and their fibrotic function quantified using imaging assays (extracellular matrix deposition, contractile protein expression, and microenvironment compaction). Highlighting the system's utility, we further show that fibroblasts isolated from IPF patient lungs maintain fibrotic phenotypes and manifest reduced fibrotic function when treated with epigenetic modifiers. Our system enables enhanced screening due to improved predictability and fidelity compared to 2D systems combined with superior tractability and throughput compared to 3D systems., (© 2021 Author(s).)

Yes-associated protein (YAP) and PDZ-binding motif (TAZ) have emerged as important regulators of pathologic fibroblast activation in fibrotic diseases. Agonism of Gαs-coupled G protein coupled receptors (GPCRs) provides an attractive approach to inhibit the nuclear localization and function of YAP and TAZ in fibroblasts that inhibits or reverses their pathological activation. Agonism of the dopamine D1 GPCR has proven effective in preclinical models of lung and liver fibrosis. However, the molecular mechanisms coupling GPCR agonism to YAP and TAZ inactivation in fibroblasts remain incompletely understood. Here, using human lung fibroblasts, we identify critical roles for the cAMP effectors EPAC1/2, the small GTPase RAP2c, and the serine/threonine kinase MAP4K7 as the essential elements in the downstream signaling cascade linking GPCR agonism to LATS1/2-mediated YAP and TAZ phosphorylation and nuclear exclusion in fibroblasts. We further show that this EPAC/RAP2c/MAP4K7 signaling cascade is essential to the effects of dopamine D1 receptor agonism on reducing fibroblast proliferation, contraction, and extracellular matrix production. Targeted modulation of this cascade in fibroblasts may prove a useful strategy to regulate YAP and TAZ signaling and fibroblast activities central to tissue repair and fibrosis., (© 2021 Wiley Periodicals LLC.)

Matrix stiffness is a central regulator of fibroblast function. However, the transcriptional mechanisms linking matrix stiffness to changes in fibroblast phenotype are incompletely understood. Here, we evaluated the effect of matrix stiffness on genome-wide chromatin accessibility in freshly isolated lung fibroblasts using ATAC-seq. We found higher matrix stiffness profoundly increased global chromatin accessibility relative to lower matrix stiffness, and these alterations were in close genomic proximity to known profibrotic gene programs. Motif analysis of these regulated genomic loci identified ZNF416 as a putative mediator of fibroblast stiffness responses. Genome occupancy analysis using ChIP-seq confirmed that ZNF416 occupies a broad range of genes implicated in fibroblast activation and tissue fibrosis, with relatively little overlap in genomic occupancy with other mechanoresponsive and profibrotic transcriptional regulators. Using loss- and gain-of-function studies, we demonstrated that ZNF416 plays a critical role in fibroblast proliferation, extracellular matrix synthesis, and contractile function. Together, these observations identify ZNF416 as novel mechano-activated transcriptional regulator of fibroblast biology., (© 2021 Jones et al.)

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.

Fibroblasts and myofibroblasts are found throughout mechanically loaded tissues, where they take primary responsibility for generating and maintaining the extracellular matrix scaffold upon which organ structure and function depends. They are thus tasked with creating the appropriate mechanical environment in which cells and tissues function optimally, and constantly adapting this environment as needed in response to changing environmental cues. To carry out these functions, fibroblasts must not only deposit and resorb the extracellular matrix, they must adhere to and sense its physical characteristics, and exert the forces necessary to shape, distort, and remodel it as desired. It is thus only through a constant reciprocal sensing and exertion of stress that fibroblasts can carry out their key functions. This introductory chapter will introduce these aspects of fibroblast stress sensing and matrix remodeling during tissue homeostasis, wound repair and fibrotic disease as a lead in to the detailed method chapters to follow on myofibroblast mechanobiology.

Matrix resorption is essential to the clearance of the extracellular matrix (ECM) after normal wound healing. A disruption in these processes constitutes a main component of fibrotic diseases, characterized by excess deposition and diminished clearance of fibrillar ECM proteins, such as collagen type I. The mechanisms and stimuli regulating ECM resorption in the lung remain poorly understood. Recently, agonism of dopamine receptor D1 (DRD1), which is predominantly expressed on fibroblasts in the lung, has been shown to accelerate tissue repair and clearance of ECM following bleomycin injury in mice. Therefore, we investigated whether DRD1 receptor signaling promotes the degradation of collagen type I by lung fibroblasts. For cultured fibroblasts, we found that DRD1 agonism enhances extracellular cleavage, internalization and lysosomal degradation of collagen I mediated by cathepsin K, which results in reduced stiffness of cell-derived matrices, as measured by atomic force microscopy. In vivo agonism of DRD1 similarly enhanced fibrillar collagen degradation by fibroblasts, as assessed by tissue labeling with a collagen-hybridizing peptide. Together, these results implicate DRD1 agonism in fibroblast-mediated collagen clearance, suggesting an important role for this mechanism in fibrosis resolution.This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interestsA.J.H. and D.J.T. are co-inventors of a patent application (‘Methods of Treating Fibrotic Pathologies’ PCT/US2019/016178) related to the findings described in this article., (© 2020. Published by The Company of Biologists Ltd.)

DNA double-strand breaks (DSBs) are highly toxic lesions that can drive genetic instability. These lesions also contribute to the efficacy of radiotherapy and many cancer chemotherapeutics. DNA repair efficiency is regulated by both intracellular and extracellular chemical signals. However, it is largely unknown whether this process is regulated by physical stimuli such as extracellular mechanical signals. Here, we report that DSB repair is regulated by extracellular mechanical signals. Low extracellular matrix (ECM) stiffness impairs DSB repair and renders cells sensitive to genotoxic agents. Mechanistically, we found that the MAP4K4/6/7 kinases are activated and phosphorylate ubiquitin in cells at low stiffness. Phosphorylated ubiquitin impairs RNF8-mediated ubiquitin signaling at DSB sites, leading to DSB repair deficiency. Our results thus demonstrate that ECM stiffness regulates DSB repair efficiency and genotoxic sensitivity through MAP4K4/6/7 kinase-mediated ubiquitin phosphorylation, providing a previously unidentified regulation in DSB-induced ubiquitin signaling., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Pathological remodeling of the extracellular matrix (ECM) by activated myofibroblasts is a hallmark of fibrotic diseases and desmoplastic tumors. Activation of myofibroblasts occurs in response to fibrogenic tissue injury as well as in tumor-associated fibrotic reactions. The molecular determinants of myofibroblast activation in fibrosis and tumor stroma have traditionally been viewed to include biochemical agents, such as dysregulated growth factor and cytokine signaling, which profoundly alter the biology of fibroblasts, ultimately leading to overexuberant matrix deposition and fibrosis. More recently, compelling evidence has shown that altered mechanical properties of the ECM such as matrix stiffness are major drivers of tissue fibrogenesis by promoting mechano-activation of fibroblasts. In this Review, we discuss new insights into the role of the biophysical microenvironment in the amplified activation of fibrogenic myofibroblasts during the development and progression of fibrotic diseases and desmoplastic tumors. We also summarize novel therapeutic targets for anti-fibrotic therapy based on the mechanobiology of tissue fibrosis and tumor stroma, a class of drugs known as "mechano-therapeutics"., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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 TGFβ-induced pro-fibrotic gene and protein expression. Additionally, loss of eNOS in human lung ECs reduced the suppression of TGFβ-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., (© 2020 The Authors. Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

Objective: This study was designed to evaluate the roles of microRNAs (miRNAs) in slow transit constipation (STC)., Design: All human tissue samples were from the muscularis externa of the colon. Expression of 372 miRNAs was examined in a discovery cohort of four patients with STC versus three age/sex-matched controls by a quantitative PCR array. Upregulated miRNAs were examined by quantitative reverse transcription qPCR (RT-qPCR) in a validation cohort of seven patients with STC and age/sex-matched controls. The effect of a highly differentially expressed miRNA on a custom human smooth muscle cell line was examined in vitro by RT-qPCR, electrophysiology, traction force microscopy, and ex vivo by lentiviral transduction in rat muscularis externa organotypic cultures., Results: The expression of 13 miRNAs was increased in STC samples. Of those miRNAs, four were predicted to target SCN5A , the gene that encodes the Na + channel Na V 1.5. The expression of SCN5A mRNA was decreased in STC samples. Let-7f significantly decreased Na + current density in vitro in human smooth muscle cells. In rat muscularis externa organotypic cultures, overexpression of let-7f resulted in reduced frequency and amplitude of contraction., Conclusions: A small group of miRNAs is upregulated in STC, and many of these miRNAs target the SCN5A-encoded Na + channel Na V 1.5. Within this set, a novel Na V 1.5 regulator, let-7f, resulted in decreased Na V 1.5 expression, current density and reduced motility of GI smooth muscle. These results suggest Na V 1.5 and miRNAs as novel diagnostic and potential therapeutic targets in STC., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

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-β-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.

Uterine fibroids (UFs) are benign myometrial neoplasms. The mechanical environment activates signaling through the Hippo pathway effectors Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding domain (TAZ) in other fibrotic disorders. Here, we assess the differences in YAP/TAZ responsiveness to signals in UF compared with myometrium (Myo). Matched samples of UF and Myo were collected. Atomic force microscopy (AFM) was used to determine in situ stiffness. Cells were plated sparsely on hydrogels or at confluence. Ten nanomolars of estradiol (E2) and 100 nM progesterone (P4) were used. Immunostaining for YAP/TAZ and extracellular matrix (ECM) proteins was performed. Cells were incubated with control or YAP1 (YAP)/WWTR1 (TAZ) small interfering RNA (siRNA). Real time qPCR was completed for connective tissue growth factor (CTGF). Cells were treated with verteporfin (a YAP inhibitor) or Y27632 (a ROCK inhibitor), and ECM gene expression was analyzed. Paired t test and Wilcoxon sign-rank test were used. AFM-measured tissue stiffness and YAP/TAZ nuclear localization in situ and in confluent cells were higher in UF compared with Myo (p < 0.05). Decreasing substrate stiffness reduced YAP/TAZ nuclear localization for both Myo and UF (p = 0.05). Stimulating cells with E2 or P4 increased YAP/TAZ nuclear localization, but only in Myo (p = 0.01). UFs had increased FN, COLI, and COLIII deposition. Following siRNA targeting, CTGF was found to be statistically decreased. Verteporfin treatment reduced cell survival and reduced FN deposition. Treatment with Y27632 demonstrated better cell tolerance and a reduction in ECM deposition. The mechanosensitive pathway may be linked to YAP/TAZ function and involved in transducing fibroid growth.

A variety of G protein-coupled receptors (GPCRs) have been implicated in the pathogenesis of pulmonary fibrosis, largely through their promotion of profibrotic fibroblast activation. By contrast, recent work has highlighted the beneficial effects of Gαs-coupled GPCRs on reducing fibroblast activation and fibrosis. This review highlights how fibrosis-promoting and -inhibiting GPCR signaling converges on downstream signaling and transcriptional effectors, and how the diversity and dynamics of GPCR expression challenge efforts to identify effective therapies for idiopathic pulmonary fibrosis (IPF). Next-generation strategies to overcome these challenges, focusing on target selection, polypharmacology, and personalized medicine approaches, are discussed as a path towards more effective GPCR-targeted therapies for pulmonary fibrosis., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Gliosis and fibrosis after spinal cord injury (SCI) lead to formation of a scar that is thought to present both molecular and mechanical barriers to neuronal regeneration. The scar consists of a meshwork of reactive glia and deposited, cross-linked, extracellular matrix (ECM) that has long been assumed to present a mechanically "stiff" blockade. However, remarkably little quantitative information is available about the rheological properties of chronically injured spinal tissue. In this study we utilize atomic force microscopy microindentation to provide quantitative evidence of chronic mechanical stiffening after SCI. Using the results of this tissue characterization, we assessed the sensitivity of both mouse and human astrocytes in vitro and determined that they are exquisitely mechanosensitive within the relevant range of substrate stiffness observed in the injured/uninjured spinal cord. We then utilized a novel immune modifying nanoparticle (IMP) treatment as a tool to reveal fibrotic scarring as one of the key drivers of mechanical stiffening after SCI in vivo . We also demonstrate that glial scar-forming astrocytes form a highly aligned, anisotropic network of glial fibers after SCI, and that IMP treatment mitigates this pathological alignment. Taken together, our results identify chronic mechanical stiffening as a critically important aspect of the complex lesion milieu after SCI that must be considered when assessing and developing potential clinical interventions for SCI.

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-β1 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.

Introduction:  Impaired plasminogen activation (PA) is causally related to the development of lung fibrosis. Prior studies demonstrate that enhanced PA in the lung limits the severity of scarring following injury and in vitro studies indicate that PA promotes matrix degradation and fibroblast apoptosis. These findings led us to hypothesize that increased PA in an in vivo model would enhance the resolution of established lung fibrosis in conjunction with increased myofibroblast apoptosis., Methods:  Transgenic C57BL/6 mice with doxycycline inducible lung-specific urokinase plasminogen activator (uPA) expression or littermate controls were treated (day 0) with bleomycin or saline. Doxycycline was initiated on days 1, 9, 14, or 21. Lung fibrosis, stiffness, apoptosis, epithelial barrier integrity, and inflammation were assessed., Results:  Protection from fibrosis with uPA upregulation from day 1 through day 28 was associated with reduced parenchymal stiffness as determined by atomic force microscopy. Initiation of uPA expression beginning in the late inflammatory or the early fibrotic phase reduced stiffness and fibrosis at day 28. Induction of uPA activity in mice with established fibrosis decreased lung collagen and lung stiffness while increasing myofibroblast apoptosis. Upregulation of uPA did not alter lung inflammation but was associated with improved epithelial cell homeostasis., Conclusion:  Restoring intrapulmonary PA activity diminishes lung fibrogenesis and enhances the resolution of established lung fibrosis. This PA-mediated resolution is associated with increased myofibroblast apoptosis and improved epithelial cell homeostasis. These studies support the potential capacity of the lung to resolve existing scar in murine models., Competing Interests: J.C.H., T.H.S., K.K.K., and D.J.T. report grants from National Institutes of Health, during the conduct of the study., (Georg Thieme Verlag KG Stuttgart · New York.)

Reciprocal epithelial-mesenchymal interactions are pivotal in lung development, homeostasis, injury, and repair. Organoids have been used to investigate such interactions, but with a major focus on epithelial responses to mesenchyme and less attention to epithelial effects on mesenchyme. In the present study, we used nascent organoids composed of human and mouse lung epithelial and mesenchymal cells to demonstrate that healthy lung epithelium dramatically represses transcriptional, contractile, and matrix synthetic functions of lung fibroblasts. Repression of fibroblast activation requires signaling via the bone morphogenetic protein (BMP) pathway. BMP signaling is diminished after epithelial injury in vitro and in vivo , and exogenous BMP4 restores fibroblast repression in injured organoids. In contrast, inhibition of BMP signaling in healthy organoids is sufficient to derepress fibroblast matrix synthetic function. Our results reveal potent repression of fibroblast activation by healthy lung epithelium and a novel mechanism by which epithelial loss or injury is intrinsically coupled to mesenchymal activation via loss of repressive BMP signaling.

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 Gα s -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., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Tissue fibrosis is a chronic disease driven by persistent fibroblast activation that has recently been linked to epigenetic modifications. Here, we screened a small library of epigenetic small-molecule modulators to identify compounds capable of inhibiting or reversing TGFβ-mediated fibroblast activation. We identified pracinostat, an HDAC inhibitor, as a potent attenuator of lung fibroblast activation and confirmed its efficacy in patient-derived fibroblasts isolated from fibrotic lung tissue. Mechanistically, we found that HDAC-dependent transcriptional repression was an early and essential event in TGFβ-mediated fibroblast activation. Treatment of lung fibroblasts with pracinostat broadly attenuated TGFβ-mediated epigenetic repression and promoted fibroblast quiescence. We confirmed a specific role for HDAC-dependent histone deacetylation in the promoter region of the anti-fibrotic gene PPARGC1A ( PGC1α ) in response to TGFβ stimulation. Finally, we identified HDAC7 as a key factor whose siRNA-mediated knockdown attenuates fibroblast activation without altering global histone acetylation. Together, these results provide novel mechanistic insight into the essential role HDACs play in TGFβ-mediated fibroblast activation via targeted gene repression., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)