Transglutaminase 2 knockout mice are protected from bleomycin-induced lung fibrosis with preserved lung function and reduced metabolic derangements.

Pulmonary fibrosis is an interstitial scarring disease of the lung characterized by poor prognosis and limited treatment options. Tissue transglutaminase 2 (TG2) is believed to promote lung fibrosis by crosslinking extracellular matrix components and activating latent TGFß. This study assessed physiologic pulmonary function and metabolic alterations in the mouse bleomycin model with TG2 genetic deletion. TG2-deficient mice demonstrated attenuated the fibrosis and preservation of lung function, with significant reduction in elastance and increases in compliance and inspiratory capacity compared to control mice treated with bleomycin. Bleomycin induced metabolic changes in the mouse lung that were consistent with increased aerobic glycolysis, including increased expression of lactate dehydrogenase A and increased production of lactate, as well as increased glutamine, glutamate, and aspartate. TG2-deficient mice treated with bleomycin exhibited similar metabolic changes but with reduced magnitude. Our results demonstrate that TG2 is required for a typical fibrosis response to injury. In the absence of TG2, the fibrotic response is biochemically similar to wild-type, but lesions are smaller and lung function is preserved. We also show for the first time that profibrotic pathways of tissue stiffening and metabolic reprogramming are interconnected, and that metabolic disruptions in fibrosis go beyond glycolysis.

Is there a role for specialized pro-resolving mediators in pulmonary fibrosis?

Pulmonary fibrotic diseases are characterized by proliferation of lung fibroblasts and myofibroblasts and excessive deposition of extracellular matrix proteins. Depending on the specific form of lung fibrosis, there can be progressive scarring of the lung, leading in some cases to respiratory failure and/or death. Recent and ongoing research has demonstrated that resolution of inflammation is an active process regulated by families of small bioactive lipid mediators termed "specialized pro-resolving mediators." While there are many reports of beneficial effects of SPMs in animal and cell culture models of acute and chronic inflammatory and immune diseases, there have been fewer reports investigating SPMs and fibrosis, especially pulmonary fibrosis. Here, we will review evidence that resolution pathways are impaired in interstitial lung disease, and that SPMs and other similar bioactive lipid mediators can inhibit fibroblast proliferation, myofibroblast differentiation, and accumulation of excess extracellular matrix in cell culture and animal models of pulmonary fibrosis, and we will consider future therapeutic implications of SPMs in fibrosis.

Exhaled breath condensate identifies metabolic dysregulation in patients with radiation-induced lung injury.

Radiation-induced lung injury (RILI) is a consequence of therapeutic thoracic irradiation (TR) for many cancers, and there are no FDA-approved curative strategies. Studies report that 80% of patients who undergo TR will have CT-detectable interstitial lung abnormalities, and strategies to limit the risk of RILI may make radiotherapy less effective at treating cancer. Our lab and others have reported that lung tissue from patients with idiopathic pulmonary fibrosis (IPF) exhibits metabolic defects including increased glycolysis and lactate production. In this pilot study, we hypothesized that patients with radiation-induced lung damage will exhibit distinct changes in lung metabolism that may be associated with the incidence of fibrosis. Using liquid chromatography/tandem mass spectrometry to identify metabolic compounds, we analyzed exhaled breath condensate (EBC) in subjects with CT-confirmed lung lesions after TR for lung cancer, compared with healthy subjects, smokers, and cancer patients who had not yet received TR. The lung metabolomic profile of the irradiated group was significantly different from the three nonirradiated control groups, highlighted by increased levels of lactate. Pathway enrichment analysis revealed that EBC from the case patients exhibited concurrent alterations in lipid, amino acid, and carbohydrate energy metabolism associated with the energy-producing tricarboxylic acid (TCA) cycle. Radiation-induced glycolysis and diversion of lactate to the extracellular space suggests that pyruvate, a precursor metabolite, converts to lactate rather than acetyl-CoA, which contributes to the TCA cycle. This TCA cycle deficiency may be compensated by these alternate energy sources to meet the metabolic demands of chronic wound repair. Using an "omics" approach to probe lung disease in a noninvasive manner could inform future mechanistic investigations and the development of novel therapeutic targets.NEW & NOTEWORTHY We report that exhaled breath condensate (EBC) identifies cellular metabolic dysregulation in patients with radiation-induced lung injury. In this pilot study, untargeted metabolomics revealed a striking metabolic signature in EBC from patients with radiation-induced lung fibrosis compared to patients with lung cancer, at-risk smokers, and healthy volunteers. Patients with radiation-induced fibrosis exhibit specific changes in tricarboxylic acid (TCA) cycle energy metabolism that may be required to support the increased energy demands of fibroproliferation.

Altered TGFB1 regulated pathways promote accelerated tendon healing in the superhealer MRL/MpJ mouse.

To better understand the molecular mechanisms of tendon healing, we investigated the Murphy Roth's Large (MRL) mouse, which is considered a model of mammalian tissue regeneration. We show that compared to C57Bl/6J (C57) mice, injured MRL tendons have reduced fibrotic adhesions and cellular proliferation, with accelerated improvements in biomechanical properties. RNA-seq analysis revealed that differentially expressed genes in the C57 healing tendon at 7 days post injury were functionally linked to fibrosis, immune system signaling and extracellular matrix (ECM) organization, while the differentially expressed genes in the MRL injured tendon were dominated by cell cycle pathways. These gene expression changes were associated with increased α-SMA+ myofibroblast and F4/80+ macrophage activation and abundant BCL-2 expression in the C57 injured tendons. Transcriptional analysis of upstream regulators using Ingenuity Pathway Analysis showed positive enrichment of TGFB1 in both C57 and MRL healing tendons, but with different downstream transcriptional effects. MRL tendons exhibited of cell cycle regulatory genes, with negative enrichment of the cell senescence-related regulators, compared to the positively-enriched inflammatory and fibrotic (ECM organization) pathways in the C57 tendons. Serum cytokine analysis revealed decreased levels of circulating senescence-associated circulatory proteins in response to injury in the MRL mice compared to the C57 mice. These data collectively demonstrate altered TGFB1 regulated inflammatory, fibrosis, and cell cycle pathways in flexor tendon repair in MRL mice, and could give cues to improved tendon healing.

Mechanical Feed-Forward Loops Contribute to Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis is a progressive scarring disease characterized by extracellular matrix accumulation and altered mechanical properties of lung tissue. Recent studies support the hypothesis that these compositional and mechanical changes create a progressive feed-forward loop in which enhanced matrix deposition and tissue stiffening contribute to fibroblast and myofibroblast differentiation and activation, which further perpetuates matrix production and stiffening. The biomechanical properties of tissues are sensed and responded to by mechanotransduction pathways that facilitate sensing of changes in mechanical cues by tissue resident cells and convert the mechanical signals into downstream biochemical signals. Although our understanding of mechanotransduction pathways associated with pulmonary fibrosis remains incomplete, recent progress has allowed us to begin to elucidate the specific mechanisms supporting fibrotic feed-forward loops. The mechanosensors discussed here include integrins, Piezo channels, transient receptor potential channels, and nonselective ion channels. Also discussed are downstream transcription factors, including myocardin-related transcription factor and Yes-associated protein/transcriptional coactivator with PDZ-binding motif. This review describes mechanosensors and mechanotransduction pathways associated with fibrosis progression and highlights promising therapeutic insights.

The self-fulfilling prophecy of pulmonary fibrosis: a selective inspection of pathological signalling loops.

Pulmonary fibrosis is a devastating, progressive disease and carries a prognosis worse than most cancers. Despite ongoing research, the mechanisms that underlie disease pathogenesis remain only partially understood. However, the self-perpetuating nature of pulmonary fibrosis has led several researchers to propose the existence of pathological signalling loops. According to this hypothesis, the normal wound-healing process becomes corrupted and results in the progressive accumulation of scar tissue in the lung. In addition, several negative regulators of pulmonary fibrosis are downregulated and, therefore, are no longer capable of inhibiting these feed-forward loops. The combination of pathological signalling loops and loss of a checks and balances system ultimately culminates in a process of unregulated scar formation. This review details specific signalling pathways demonstrated to play a role in the pathogenesis of pulmonary fibrosis. The evidence of detrimental signalling loops is elucidated with regard to epithelial cell injury, cellular senescence and the activation of developmental and ageing pathways. We demonstrate where these loops intersect each other, as well as common mediators that may drive these responses and how the loss of pro-resolving mediators may contribute to the propagation of disease. By focusing on the overlapping signalling mediators among the many pro-fibrotic pathways, it is our hope that the pulmonary fibrosis community will be better equipped to design future trials that incorporate the redundant nature of these pathways as we move towards finding a cure for this unrelenting disease.

Transcriptomic Analysis of Cellular Pathways in Healing Flexor Tendons of Plasminogen Activator Inhibitor 1 (PAI-1/Serpine1) Null Mice.

Injuries to flexor tendons can be complicated by fibrotic adhesions, which severely impair the function of the hand. Plasminogen activator inhibitor 1 (PAI-1/SERPINE1), a master suppressor of fibrinolysis and protease activity, is associated with adhesions. Here, we used next-generation RNA sequencing (RNA-Seq) to assess genome-wide differences in messenger RNA expression due to PAI-1 deficiency after zone II flexor tendon injury. We used the ingenuity pathway analysis to characterize molecular pathways and biological drivers associated with differentially expressed genes (DEG). Analysis of hundreds of overlapping and DEG in PAI-1 knockout (KO) and wild-type mice (C57Bl/6J) during tendon healing revealed common and distinct biological processes. Pathway analysis identified cell proliferation, survival, and senescence, as well as chronic inflammation as potential drivers of fibrotic healing and adhesions in injured tendons. Importantly, we identified the activation of PTEN signaling and the inhibition of FOXO1-associated biological processes as unique transcriptional signatures of the healing tendon in the PAI-1/Serpine1 KO mice. Further, transcriptomic differences due to the genetic deletion of PAI-1 were mechanistically linked to PI3K/Akt/mTOR, PKC, and MAPK signaling cascades. These transcriptional observations provide novel insights into the biological roles of PAI-1 in tendon healing and could identify therapeutic targets to achieve scar-free regenerative healing of tendons. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:43-58, 2020.

Serpine1 Knockdown Enhances MMP Activity after Flexor Tendon Injury in Mice: Implications for Adhesions Therapy.

Injuries to flexor tendons can be complicated by fibrotic adhesions, which severely impair the function of the hand. Adhesions have been associated with TGF-ß1, which causes upregulation of PAI-1, a master suppressor of protease activity, including matrix metalloproteinases (MMP). In the present study, the effects of inhibiting PAI-1 in murine zone II flexor tendon injury were evaluated utilizing knockout (KO) mice and local nanoparticle-mediated siRNA delivery. In the PAI-1 KO murine model, reduced adherence of injured tendon to surrounding subcutaneous tissue and accelerated recovery of normal biomechanical properties compared to wild type controls were observed. Furthermore, MMP activity was significantly increased in the injured tendons of the PAI-1 KO mice, which could explain their reduced adhesions and accelerated remodeling. These data demonstrate that PAI-1 mediates fibrotic adhesions in injured flexor tendons by suppressing MMP activity. In vitro siRNA delivery to silence Serpine1 expression after treatment with TGF-ß1 increased MMP activity. Nanoparticle-mediated delivery of siRNA targeting Serpine1 in injured flexor tendons significantly reduced target gene expression and subsequently increased MMP activity. Collectively, the data demonstrate that PAI-1 can be a druggable target for treating adhesions and accelerating the remodeling of flexor tendon injuries.

Diblock Copolymer Hydrophobicity Facilitates Efficient Gene Silencing and Cytocompatible Nanoparticle-Mediated siRNA Delivery to Musculoskeletal Cell Types.

pH-responsive diblock copolymers provide tailorable nanoparticle (NP) architecture and chemistry critical for siRNA delivery. Here, diblock polymers varying in first (corona) and second (core) block molecular weight (Mn), corona/core ratio, and core hydrophobicity (%BMA) were synthesized to determine their effect on siRNA delivery in murine tenocytes (mTenocyte) and murine and human mesenchymal stem cells (mMSC and hMSCs, respectively). NP-mediated siRNA uptake, gene silencing, and cytocompatibility were quantified. Uptake is positively correlated with first block Mn in mTenocytes and hMSCs (p ≤ 0.0005). All NP resulted in significant gene silencing that was positively correlated with %BMA (p < 0.05) in all cell types. Cytocompatibility was reduced in mTenocytes compared to MSCs (p < 0.0001). %BMA was positively correlated with cytocompatibility in MSCs (p < 0.05), suggesting stable NP are more cytocompatible. Overall, this study shows that NP-siRNA cytocompatibility is cell type dependent, and hydrophobicity (%BMA) is the critical diblock copolymer property for efficient gene silencing in musculoskeletal cell types.