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Pulmonary fibrosis is characterized by the accumulation of myofibroblasts in the lung and progressive tissue scarring. Fibroblasts exist across a spectrum of states, from quiescence in health to activated myofibroblasts in the setting of injury. Highly activated myofibroblasts have a critical role in the establishment of fibrosis as the predominant source of type 1 collagen and profibrotic mediators. Myofibroblasts are also highly contractile cells and can alter lung biomechanical properties through tissue contraction. Inhibiting signaling pathways involved in myofibroblast activation could therefore have significant therapeutic value. One of the ways myofibroblast activation occurs is through activation of the Rho/myocardin-related transcription factor (MRTF)/serum response factor (SRF) pathway, which signals through intracellular actin polymerization. However, concerns surrounding the pleiotropic and ubiquitous nature of these signaling pathways have limited the translation of inhibitory drugs. Herein, we demonstrate a novel therapeutic antifibrotic strategy using myofibroblast-targeted nanoparticles containing a MTRF/SRF pathway inhibitor (CCG-1423), which has been shown to block myofibroblast activation in vitro. Myofibroblasts were preferentially targeted via the angiotensin 2 receptor, which has been shown to be selectively upregulated in animal and human studies. These nanoparticles were nontoxic and accumulated in lung myofibroblasts in the bleomycin-induced mouse model of pulmonary fibrosis, reducing the number of these activated cells and their production of profibrotic mediators. Ultimately, in a murine model of lung fibrosis, a single injection of these drugs containing targeted nanoagents reduced fibrosis as compared with control mice. This approach has the potential to deliver personalized therapy by precisely targeting signaling pathways in a cell-specific manner, allowing increased efficacy with reduced deleterious off-target effects.

Ritanserin inhibits macropinocytosis and acidic organelle homeostasis in TSC2-deficient cells

Lymphangioleiomyomatosis is a rare destructive lung disease affecting primarily women and is the primary lung manifestation of tuberous sclerosis complex (TSC). In lymphangioleiomyomatosis, biallelic loss of TSC1/2 leads to hyperactivation of mTORC1 and inhibition of autophagy. To determine how the metabolic vulnerabilities of TSC2-deficient cells can be targeted, we performed a high-throughput screen utilizing the “Repurposing” library at the Broad Institute of MIT and Harvard (Cambridge, MA), with or without the autophagy inhibitor chloroquine. Ritanserin, an inhibitor of diacylglycerol kinase alpha (DGKA), was identified as a selective inhibitor of proliferation of Tsc2−/− mouse embryonic fibroblasts (MEF), with no impact on Tsc2+/+ MEFs. DGKA is a lipid kinase that metabolizes diacylglycerol to phosphatidic acid, a key component of plasma membranes. Phosphatidic acid levels were increased 5-fold in Tsc2−/− MEFs compared with Tsc2+/+ MEFs, and treatment of Tsc2−/− MEFs with ritanserin led to depletion of phosphatidic acid as well as rewiring of phospholipid metabolism. Macropinocytosis is known to be upregulated in TSC2-deficient cells. Ritanserin decreased macropinocytic uptake of albumin, limited the number of lysosomes, and reduced lysosomal activity in Tsc2−/− MEFs. In a mouse model of TSC, ritanserin treatment decreased cyst frequency and volume, and in a mouse model of lymphangioleiomyomatosis, genetic downregulation of DGKA prevented alveolar destruction and airspace enlargement. Collectively, these data indicate that DGKA supports macropinocytosis in TSC2-deficient cells to maintain phospholipid homeostasis and promote proliferation. Targeting macropinocytosis with ritanserin may represent a novel therapeutic approach for the treatment of TSC and lymphangioleiomyomatosis.Significance:This study identifies macropinocytosis and phospholipid metabolism as novel mechanisms of metabolic homeostasis in mTORC1-hyperactive cells and suggest ritanserin as a novel therapeutic strategy for use in mTORC1-hyperactive tumors, including pancreatic cancer.

Myocardial fibrosis is a key pathologic feature of hypertrophic cardiomyopathy (HCM). However, the fibrotic pathways activated by HCM-causing sarcomere protein gene mutations are poorly defined. Because lysophosphatidic acid is a mediator of fibrosis in multiple organs and diseases, we tested the role of the lysophosphatidic acid pathway in HCM. Lysphosphatidic acid receptor 1 (LPAR1), a cell surface receptor, is required for lysophosphatidic acid mediation of fibrosis. We bred HCM mice carrying a pathogenic myosin heavy-chain variant (403 +/− ) with Lpar1 -ablated mice to create mice carrying both genetic changes (403 +/− LPAR1 −/− ) and assessed development of cardiac hypertrophy and fibrosis. Compared with 403 +/− LPAR1 WT , 403 +/− LPAR1 −/− mice developed significantly less hypertrophy and fibrosis. Single-nucleus RNA sequencing of left ventricular tissue demonstrated that Lpar1 was predominantly expressed by lymphatic endothelial cells (LECs) and cardiac fibroblasts. Lpar1 ablation reduced the population of LECs, confirmed by immunofluorescence staining of the LEC markers Lyve1 and Ccl21a and, by in situ hybridization, for Reln and Ccl21a . Lpar1 ablation also altered the distribution of fibroblast cell states. FB1 and FB2 fibroblasts decreased while FB0 and FB3 fibroblasts increased. Our findings indicate that Lpar1 is expressed predominantly by LECs and fibroblasts in the heart and is required for development of hypertrophy and fibrosis in an HCM mouse model. LPAR1 antagonism, including agents in clinical trials for other fibrotic diseases, may be beneficial for HCM.

The lack of animal models for some human diseases precludes our understanding of disease mechanisms and our ability to test prospective therapies in vivo. Generation of kidney organoids from Tuberous Sclerosis Complex (TSC) patient-derived-hiPSCs allows us to recapitulate a rare kidney tumor called angiomyolipoma (AML). Organoids derived from TSC2−/− hiPSCs but not from isogenic TSC2+/− or TSC2+/+ hiPSCs share a common transcriptional signature and a myomelanocytic cell phenotype with kidney AMLs, and develop epithelial cysts, replicating two major TSC-associated kidney lesions driven by genetic mechanisms that cannot be consistently recapitulated with transgenic mice. Transplantation of multiple TSC2−/− renal organoids into the kidneys of immunodeficient rats allows us to model AML in vivo for the study of tumor mechanisms, and to test the efficacy of rapamycin-loaded nanoparticles as an approach to rapidly ablate AMLs. Collectively, our experimental approaches represent an innovative and scalable tissue-bioengineering strategy for modeling rare kidney disease in vivo., The lack of animal models for some human diseases precludes our understanding of disease mechanisms and our ability to test new therapies in vivo. Here the authors present a tissue bioengineering strategy for the study of a rare kidney tumor called angiomyolipoma, in vitro and in vivo, using patient-derived hiPSCs.

Lymphangioleiomyomatosis is a rare destructive lung disease affecting primarily women and is the primary lung manifestation of tuberous sclerosis complex (TSC). In lymphangioleiomyomatosis, biallelic loss of TSC1/2 leads to hyperactivation of mTORC1 and inhibition of autophagy. To determine how the metabolic vulnerabilities of TSC2-deficient cells can be targeted, we performed a high-throughput screen utilizing the “Repurposing” library at the Broad Institute of MIT and Harvard (Cambridge, MA), with or without the autophagy inhibitor chloroquine. Ritanserin, an inhibitor of diacylglycerol kinase alpha (DGKA), was identified as a selective inhibitor of proliferation of Tsc2−/− mouse embryonic fibroblasts (MEF), with no impact on Tsc2+/+ MEFs. DGKA is a lipid kinase that metabolizes diacylglycerol to phosphatidic acid, a key component of plasma membranes. Phosphatidic acid levels were increased 5-fold in Tsc2−/− MEFs compared with Tsc2+/+ MEFs, and treatment of Tsc2−/− MEFs with ritanserin led to depletion of phosphatidic acid as well as rewiring of phospholipid metabolism. Macropinocytosis is known to be upregulated in TSC2-deficient cells. Ritanserin decreased macropinocytic uptake of albumin, limited the number of lysosomes, and reduced lysosomal activity in Tsc2−/− MEFs. In a mouse model of TSC, ritanserin treatment decreased cyst frequency and volume, and in a mouse model of lymphangioleiomyomatosis, genetic downregulation of DGKA prevented alveolar destruction and airspace enlargement. Collectively, these data indicate that DGKA supports macropinocytosis in TSC2-deficient cells to maintain phospholipid homeostasis and promote proliferation. Targeting macropinocytosis with ritanserin may represent a novel therapeutic approach for the treatment of TSC and lymphangioleiomyomatosis. Significance: This study identifies macropinocytosis and phospholipid metabolism as novel mechanisms of metabolic homeostasis in mTORC1-hyperactive cells and suggest ritanserin as a novel therapeutic strategy for use in mTORC1-hyperactive tumors, including pancreatic cancer.

The lack of animal models for certain human diseases precludes our understanding of disease mechanisms and our ability to test new therapies in vivo. Here we generated kidney organoids from Tuberous Sclerosis Complex (TSC) patient-derived-hiPSCs to recapitulate a rare kidney tumor called angiomylipoma (AML). Organoids derived from TSC2-/- hiPSCs but not from isogenic TSC2+/- or TSC2+/+ hiPSCs shared a common transcriptional signature and a myomelanocytic cell phenotype with kidney AMLs, and developed epithelial cysts, replicating two major TSC-associated kidney lesions driven by genetic mechanisms that cannot be robustly and consistently recapitulated with transgenic mice. Transplantation of multiple TSC2-/- kidney organoids into the kidneys of immunodeficient rats allowed us to recapitulate AML and cystic kidney disease in vivo, in a scalable fashion and with fidelity, and to test the efficiency of rapamycin-loaded nanoparticles as a novel approach to ablate AMLs by inducing apoptosis triggered by mTOR-inhibition. Collectively, these methods represent a novel tissue-bioengineering strategy for rare disease modeling in vivo.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive disease which leads to significant morbidity and mortality from respiratory failure. The two drugs currently approved for clinical use slow the rate of decline in lung function but have not been shown to halt disease progression or reverse established fibrosis. Thus, new therapeutic targets are needed. Endothelial injury and the resultant vascular permeability are critical components in the response to tissue injury and are present in patients with IPF. However, it remains unclear how vascular permeability affects lung repair and fibrosis following injury. Lipid mediators such as sphingosine-1-phosphate (S1P) are known to regulate multiple homeostatic processes in the lung including vascular permeability. We demonstrate that endothelial cell–(EC) specific deletion of the S1P receptor 1 (S1PR1) in mice (EC-S1pr1(−/−)) results in increased lung vascular permeability at baseline. Following a low-dose intratracheal bleomycin challenge, EC-S1pr1(−/−) mice had increased and persistent vascular permeability compared with wild-type mice, which was strongly correlated with the amount and localization of resulting pulmonary fibrosis. EC-S1pr1(−/−) mice also had increased immune cell infiltration and activation of the coagulation cascade within the lung. However, increased circulating S1P ligand in ApoM-overexpressing mice was insufficient to protect against bleomycin-induced pulmonary fibrosis. Overall, these data demonstrate that endothelial cell S1PR1 controls vascular permeability in the lung, is associated with changes in immune cell infiltration and extravascular coagulation, and modulates the fibrotic response to lung injury.

Idiopathic pulmonary fibrosis is a lung disease with limited therapeutic options that is characterized by pathological fibroblast activation and aberrant lung remodeling with scar formation. YAP (Yes-associated protein) is a transcriptional coactivator that mediates mechanical and biochemical signals controlling fibroblast activation. In this study, we developed a high-throughput small-molecule screen for YAP inhibitors in primary human lung fibroblasts. Multiple HMG-CoA (hydroxymethylglutaryl-coenzyme A) reductase inhibitors (statins) were found to inhibit YAP nuclear localization via induction of YAP phosphorylation, cytoplasmic retention, and degradation. We further show that the mevalonate pathway regulates YAP activation, and that simvastatin treatment reduces fibrosis markers in activated human lung fibroblasts and in the bleomycin mouse model of pulmonary fibrosis. Finally, we show that simvastatin modulates YAP in vivo in mouse lung fibroblasts. Our results highlight the potential of small-molecule screens for YAP inhibitors and provide a mechanism for the antifibrotic activity of statins in idiopathic pulmonary fibrosis.

Pulmonary fibrosis is thought to result from dysregulated wound repair after repetitive lung injury. Many cellular responses to injury involve rearrangements of the actin cytoskeleton mediated by the two isoforms of the Rho-associated coiled-coil-forming protein kinase (ROCK), ROCK1 and ROCK2. In addition, profibrotic mediators such as transforming growth factor-β, thrombin, and lysophosphatidic acid act through receptors that activate ROCK. Inhibition of ROCK activation may be a potent therapeutic strategy for human pulmonary fibrosis. Pharmacological inhibition of ROCK using nonselective ROCK inhibitors has been shown to prevent fibrosis in animal models; however, the specific roles of each ROCK isoform are poorly understood. Furthermore, the pleiotropic effects of this kinase have raised concerns about on-target adverse effects of ROCK inhibition such as hypotension. Selective inhibition of one isoform might be a better-tolerated strategy. In the present study, we used a genetic approach to determine the roles of ROCK1 and ROCK2 in a mouse model of bleomycin-induced pulmonary fibrosis. Using ROCK1- or ROCK2-haploinsufficient mice, we found that reduced expression of either ROCK1 or ROCK2 was sufficient to protect them from bleomycin-induced pulmonary fibrosis. In addition, we found that both isoforms contribute to the profibrotic responses of epithelial cells, endothelial cells, and fibroblasts. Interestingly, ROCK1- and ROCK2-haploinsufficient mice exhibited similar protection from bleomycin-induced vascular leak, myofibroblast differentiation, and fibrosis; however, ROCK1-haploinsufficient mice demonstrated greater attenuation of epithelial cell apoptosis. These findings suggest that selective inhibition of either ROCK isoform has the potential to be an effective therapeutic strategy for pulmonary fibrosis.

Maladaptive wound healing responses to chronic tissue injury result in organ fibrosis. Fibrosis, which entails excessive extracellular matrix (ECM) deposition and tissue remodeling by activated myofibroblasts, leads to loss of proper tissue architecture and organ function; however, the molecular mediators of myofibroblast activation have yet to be fully identified. Here we identify soluble ephrin-B2 (sEphrin-B2) as a new profibrotic mediator in lung and skin fibrosis. We provide molecular, functional and translational evidence that the ectodomain of membrane-bound ephrin-B2 is shed from fibroblasts into the alveolar airspace after lung injury. Shedding of sEphrin-B2 promotes fibroblast chemotaxis and activation via EphB3 and/or EphB4 receptor signaling. We found that mice lacking ephrin-B2 in fibroblasts are protected from skin and lung fibrosis and that a disintegrin and metalloproteinase 10 (ADAM10) is the major ephrin-B2 sheddase in fibroblasts. ADAM10 expression is increased by transforming growth factor (TGF)-β1, and ADAM10-mediated sEphrin-B2 generation is required for TGF-β1-induced myofibroblast activation. Pharmacological inhibition of ADAM10 reduces sEphrin-B2 levels in bronchoalveolar lavage and prevents lung fibrosis in mice. Consistent with the mouse data, ADAM10-sEphrin-B2 signaling is upregulated in fibroblasts from human subjects with idiopathic pulmonary fibrosis. These results uncover a new molecular mechanism of tissue fibrogenesis and identify sEphrin-B2, its receptors EphB3 and EphB4 and ADAM10 as potential therapeutic targets in the treatment of fibrotic diseases.

Objective We previously implicated the lipid mediator lysophosphatidic acid (LPA) as having a role in dermal fibrosis in systemic sclerosis (SSc). The aim of this study was to identify the role of the LPA-producing enzyme autotaxin (ATX), and to connect the ATX/LPA and interleukin-6 (IL-6) pathways in SSc. Methods We evaluated the effect of a novel ATX inhibitor, PAT-048, on fibrosis and IL-6 expression in the mouse model of bleomycin-induced dermal fibrosis. We used dermal fibroblasts from SSc patients and control subjects to evaluate LPA-induced expression of IL-6, and IL-6–induced expression of ATX. We next evaluated whether LPA-induced ATX expression is dependent on IL-6, and whether baseline IL-6 expression in fibroblasts from SSc patients is dependent on ATX. Finally, we compared ATX and IL-6 expression in the skin of patients with SSc and healthy control subjects. Results PAT-048 markedly attenuated bleomycin-induced dermal fibrosis when treatment was initiated before or after the development of fibrosis. LPA stimulated expression of IL-6 in human dermal fibroblasts, and IL-6 stimulated fibroblast expression of ATX, connecting the ATX/LPA and IL-6 pathways in an amplification loop. IL-6 knockdown abrogated LPA-induced ATX expression in fibroblasts, and ATX inhibition attenuated IL-6 expression in fibroblasts and the skin of bleomycin-challenged mice. Expression of both ATX and IL-6 was increased in SSc skin, and LPA-induced IL-6 levels and IL-6–induced ATX levels were increased in fibroblasts from SSc patients compared with controls. Conclusion ATX is required for the development and maintenance of dermal fibrosis in a mouse model of bleomycin-induced SSc and enables 2 major mediators of SSc fibrogenesis, LPA and IL-6, to amplify the production of each other. Our results suggest that concurrent inhibition of these 2 pathways may be an effective therapeutic strategy for dermal fibrosis in SSc.

An amplifiable magnetic resonance imaging (MRI) probe that combines the stability of the macrocyclic Gd-DOTAGA core with a peroxidase-reactive 5-hydroxytryptamide (5-HT) moiety is reported. The incubation of the complex under enzymatic oxidative conditions led to a 1.7-fold increase in r1 at 1.4 T that was attributed to an oligomerization of the probe upon oxidation. This probe, Gd-5-HT-DOTAGA, provided specific detection of lung inflammation by MRI in bleomycin-injured mice.

Background: Kidney angiomyolipomas (AML) are benign mesenchymal tumors that are commonly seen in Tuberous Sclerosis Complex (TSC), a rare genetic neurocutaneous disorder, but also occur sporadically. Kidney AML are due to either TSC1 or TSC2 biallelic loss, whereas other somatic genetic events are rare and do not contribute to tumor development. We hypothesized that the chromatin state and master transcription factors are also drivers of kidney angiomyolipoma growth, alongside mTORC1 hyperactivation. Material and Methods: We performed whole transcriptome RNA-sequencing on 28 kidney AML, and ChIP-seq for H3K27ac (a histone modification that marks open chromatin and regulates high transcription of nearby genes) on 25 kidney AML, a human kidney AML derived TSC2 null cell line (621-101), and a pigmented melanoma cell line (SK-MEL30). ChIP-Seq for MITF (Microphthalmia-associated transcription factor) was also carried out on three kidney AML tumors and SK-MEL30 cells. Functional studies were performed to assess the oncogenic role of MITF in vitro and in vivo. Results: Differential expression analyses of kidney AML compared to both The Cancer Genome Atlas (TCGA) tumors and GTEx normal tissues revealed 347 differentially expressed genes (DEGs), including 18 transcription factors (TFs; FDR/adjusted p-value=3 sections per tumor) compared to adjacent normal tissue. Knock out of MITF-A by CRISPR/Cas9 showed reduction in cell growth (82%, p Citation Format: Krinio Giannikou, Clemens K. Probst, Mahsa Zarei, Xintao Qiu, Melissa Duarte, Nikolas Kesten, Zachary Hebert, Raga Vadhi, Alba Font-Tello, Paloma Cejas, Charles H. Yoon, Chin-Lee Wu, Myles Brown, Elizabeth P. Henske, Henry Long, David J. Kwiatkowski. Kidney angiomyolipomas are defined by a unique transcriptomic profile and H3K27ac chromatin state [abstract]. In: Abstracts: AACR Special Virtual Conference on Epigenetics and Metabolism; October 15-16, 2020; 2020 Oct 15-16. Philadelphia (PA): AACR; Cancer Res 2020;80(23 Suppl):Abstract nr PO-010.

Idiopathic pulmonary fibrosis (IPF) is thought to result from aberrant tissue repair processes in response to chronic or repetitive lung injury. The origin and nature of the injury, as well as its cellular and molecular targets, are likely heterogeneous, which complicates accurate pre-clinical modelling of the disease and makes therapeutic targeting a challenge. Efforts are underway to identify central pathways in fibrogenesis which may allow targeting of aberrant repair processes regardless of the initial injury stimulus. Dysregulated endothelial permeability and vascular leak have long been studied for their role in acute lung injury and repair. Evidence that these processes are of importance to the pathogenesis of fibrotic lung disease is growing. Endothelial permeability is increased in non-fibrosing lung diseases, but it resolves in a self-limited fashion in conditions such as bacterial pneumonia and acute respiratory distress syndrome. In progressive fibrosing diseases such as IPF, permeability appears to persist, however, and may also predict mortality. In this hypothesis-generating review, we summarise available data on the role of endothelial permeability in IPF and focus on the deleterious consequences of sustained endothelial hyperpermeability in response to and during pulmonary inflammation and fibrosis. We propose that persistent permeability and vascular leak in the lung have the potential to establish and amplify the pro-fibrotic environment. Therapeutic interventions aimed at recognising and “plugging” the leak may therefore be of significant benefit for preventing the transition from lung injury to fibrosis and should be areas for future research.

Persistent myofibroblast activation distinguishes pathological fibrosis from physiological wound healing, suggesting that therapies selectively inducing myofibroblast apoptosis could prevent progression and potentially reverse established fibrosis in diseases such as scleroderma, a heterogeneous autoimmune disease characterized by multiorgan fibrosis. We demonstrate that fibroblast-to-myofibroblast differentiation driven by matrix stiffness increases the mitochondrial priming (proximity to the apoptotic threshold) of these activated cells. Mitochondria in activated myofibroblasts, but not quiescent fibroblasts, are primed by death signals such as the proapoptotic BH3-only protein BIM, which creates a requirement for tonic expression of the antiapoptotic protein BCL-XL to sequester BIM and ensure myofibroblast survival. Myofibroblasts become particularly susceptible to apoptosis induced by “BH3 mimetic” drugs inhibiting BCL-XL such as ABT-263. ABT-263 displaces BCL-XL binding to BIM, allowing BIM to activate apoptosis on stiffness-primed myofibroblasts. Therapeutic blockade of BCL-XL with ABT-263 (navitoclax) effectively treats established fibrosis in a mouse model of scleroderma dermal fibrosis by inducing myofibroblast apoptosis. Using a BH3 profiling assay to assess mitochondrial priming in dermal fibroblasts derived from patients with scleroderma, we demonstrate that the extent of apoptosis induced by BH3 mimetic drugs correlates with the extent of their mitochondrial priming, indicating that BH3 profiling could predict apoptotic responses of fibroblasts to BH3 mimetic drugs in patients with scleroderma. Together, our findings elucidate the potential efficacy of targeting myofibroblast antiapoptotic proteins with BH3 mimetic drugs in scleroderma and other fibrotic diseases.

Maladaptive wound healing responses to chronic tissue injury result in organ fibrosis. Fibrosis, which entails excessive extracellular matrix (ECM) deposition and tissue remodelling by activated myofibroblasts, leads to loss of proper tissue architecture and organ function; however the molecular mediators of myofibroblast activation remain to be fully identified. Here we identify soluble ephrin-B2 as a novel pro-fibrotic mediator in lung and skin fibrosis. We provide molecular, functional and translational evidence that the ectodomain of membrane-bound ephrin-B2 is shed from fibroblasts into the alveolar airspace after lung injury. Shedding of soluble ephrin-B2 (sEphrin-B2) promotes fibroblast chemotaxis and activation via EphB3/EphB4 receptor signaling. We found that mice lacking ephrin-B2 in fibroblasts are protected from skin and lung fibrosis and that a distintegrin and metalloproteinase 10 (ADAM10) is the major ephrin-B2 sheddase in fibroblasts. ADAM10 is induced by transforming growth factor-β1 (TGF-β1), and ADAM10-mediated sEphrin-B2 generation is required for TGF-β1–induced myofibroblast activation. Pharmacological inhibition of ADAM10 reduces sEphrin-B2 levels in bronchoalveolar lavage and prevents lung fibrosis in mice. Consistent with the mouse data, ADAM10/sEphrin-B2 signaling is upregulated in fibroblasts from human subjects with idiopathic pulmonary fibrosis. These results uncover a new molecular mechanism of tissue fibrogenesis and identify sEphrin-B2, its receptors Eph3/Eph4, and ADAM10 as potential therapeutic targets in the treatment of fibrotic diseases.

Pathological fibrosis is driven by a feedback loop in which the fibrotic extracellular matrix is both a cause and consequence of fibroblast activation. However, the molecular mechanisms underlying this process remain poorly understood. Here we identify yes-associated protein (YAP) (homolog of drosophila Yki) and transcriptional coactivator with PDZ-binding motif (TAZ) (also known as Wwtr1), transcriptional effectors of the Hippo pathway, as key matrix stiffness-regulated coordinators of fibroblast activation and matrix synthesis. YAP and TAZ are prominently expressed in fibrotic but not healthy lung tissue, with particularly pronounced nuclear expression of TAZ in spindle-shaped fibroblastic cells. In culture, both YAP and TAZ accumulate in the nuclei of fibroblasts grown on pathologically stiff matrices but not physiologically compliant matrices. Knockdown of YAP and TAZ together in vitro attenuates key fibroblast functions, including matrix synthesis, contraction, and proliferation, and does so exclusively on pathologically stiff matrices. Profibrotic effects of YAP and TAZ operate, in part, through their transcriptional target plasminogen activator inhibitor-1, which is regulated by matrix stiffness independent of transforming growth factor-β signaling. Immortalized fibroblasts conditionally expressing active YAP or TAZ mutant proteins overcome soft matrix limitations on growth and promote fibrosis when adoptively transferred to the murine lung, demonstrating the ability of fibroblast YAP/TAZ activation to drive a profibrotic response in vivo. Together, these results identify YAP and TAZ as mechanoactivated coordinators of the matrix-driven feedback loop that amplifies and sustains fibrosis.

Fibrosis results from the dysregulation of tissue repair mechanisms affecting major organ systems, leading to chronic extracellular matrix buildup, and progressive, often fatal, organ failure. Current diagnosis relies on invasive biopsies. Noninvasive methods today cannot distinguish actively progressive fibrogenesis from stable scar, and thus are insensitive for monitoring disease activity or therapeutic responses. Collagen oxidation is a universal signature of active fibrogenesis that precedes collagen crosslinking. Biochemically targeting oxidized lysine residues formed by the action of lysyl oxidase on collagen with a small-molecule gadolinium chelate enables targeted molecular magnetic resonance imaging. This noninvasive direct biochemical elucidation of the fibrotic microenvironment specifically and robustly detected and staged pulmonary and hepatic fibrosis progression, and monitored therapeutic response in animal models. Furthermore, this paradigm is translatable and generally applicable to diverse fibroproliferative disorders.

Fibrogenesis is the active production of extracellular matrix in response to tissue injury. In many chronic diseases persistent fibrogenesis results in the accumulation of scar tissue, which can lead to organ failure and death. However, no non-invasive technique exists to assess this key biological process. All tissue fibrogenesis results in the formation of allysine, which enables collagen cross-linking and leads to tissue stiffening and scar formation. We report herein a novel allysine-binding gadolinium chelate (GdOA), that can non-invasively detect and quantify the extent of fibrogenesis using magnetic resonance imaging (MRI). We demonstrate that GdOA signal enhancement correlates with the extent of the disease and is sensitive to a therapeutic response.

Fibrotic lung disease, most notably idiopathic pulmonary fibrosis (IPF), is thought to result from aberrant wound-healing responses to repetitive lung injury. Increased vascular permeability is a cardinal response to tissue injury, but whether it is mechanistically linked to lung fibrosis is unknown. We previously described a model in which exaggeration of vascular leak after lung injury shifts the outcome of wound-healing responses from normal repair to pathological fibrosis. Here we report that the fibrosis produced in this model is highly dependent on thrombin activity and its downstream signaling pathways. Direct thrombin inhibition with dabigatran significantly inhibited protease-activated receptor-1 (PAR1) activation, integrin αvβ6 induction, TGF-β activation, and the development of pulmonary fibrosis in this vascular leak-dependent model. We used a potentially novel imaging method - ultashort echo time (UTE) lung magnetic resonance imaging (MRI) with the gadolinium-based, fibrin-specific probe EP-2104R - to directly visualize fibrin accumulation in injured mouse lungs, and to correlate the antifibrotic effects of dabigatran with attenuation of fibrin deposition. We found that inhibition of the profibrotic effects of thrombin can be uncoupled from inhibition of hemostasis, as therapeutic anticoagulation with warfarin failed to downregulate the PAR1/αvβ6/TGF-β axis or significantly protect against fibrosis. These findings have direct and important clinical implications, given recent findings that warfarin treatment is not beneficial in IPF, and the clinical availability of direct thrombin inhibitors that our data suggest could benefit these patients.

Pulmonary fibrosis is scarring of the lungs that can arise from radiation injury, drug toxicity, environmental or genetic causes, and for unknown reasons [idiopathic pulmonary fibrosis (IPF)]. Overexpression of collagen is a hallmark of organ fibrosis. We describe a peptide-based positron emission tomography (PET) probe (68Ga-CBP8) that targets collagen type I. We evaluated 68Ga-CBP8 in vivo in the bleomycin-induced mouse model of pulmonary fibrosis. 68Ga-CBP8 showed high specificity for pulmonary fibrosis and high target/background ratios in diseased animals. The lung PET signal and lung 68Ga-CBP8 uptake (quantified ex vivo) correlated linearly (r2 = 0.80) with the amount of lung collagen in mice with fibrosis. We further demonstrated that the 68Ga-CBP8 probe could be used to monitor response to treatment in a second mouse model of pulmonary fibrosis associated with vascular leak. Ex vivo analysis of lung tissue from patients with IPF supported the animal findings. These studies indicate that 68Ga-CBP8 is a promising candidate for noninvasive imaging of human pulmonary fibrosis.

Background and Rationale: The actin cytoskeleton directs many pro-fibrotic cellular responses in pulmonary fibrosis, including epithelial cell apoptosis, endothelial cell barrier disruption and fibroblast migration and differentiation. The Rho kinases ROCK1 and ROCK2 regulate the actin cytoskeleton, making them logical targets for anti-fibrotic therapy. We used a genetic approach to identify the …

Lysophosphatidic acid (LPA) signaling through one of its receptors, LPA1, contributes to both the development and the pathological remodeling after injury of many organs. Because we found previously that LPA-LPA1 signaling contributes to pulmonary fibrosis, here we investigated whether this pathway is also involved in lung development. Quantitative assessment of lung architecture of LPA1-deficient knock-out (KO) and wild-type (WT) mice at 3, 12, and 24 weeks of age using design-based stereology suggested the presence of an alveolarization defect in LPA1 KO mice at 3 weeks, which persisted as alveolar numbers increased in WT mice into adulthood. Across the ages examined, the lungs of LPA1 KO mice exhibited decreased alveolar numbers, septal tissue volumes, and surface areas, and increased volumes of the distal airspaces. Elastic fibers, critical to the development of alveolar septa, appeared less organized and condensed and more discontinuous in KO alveoli starting at P4. Tropoelastin messenger RNA expression was decreased in KO lungs, whereas expression of matrix metalloproteinases degrading elastic fibers was either decreased or unchanged. These results are consistent with the abnormal lung phenotype of LPA1 KO mice, being attributable to reduced alveolar septal formation during development, rather than to increased septal destruction as occurs in the emphysema of chronic obstructive pulmonary disease. Peripheral septal fibroblasts and myofibroblasts, which direct septation in late alveolarization, demonstrated reduced production of tropoelastin and matrix metalloproteinases, and diminished LPA-induced migration, when isolated from LPA1 KO mice. Taken together, our data suggest that LPA-LPA1 signaling is critically required for septation during alveolarization.

We previously implicated the lipid mediator lysophosphatidic acid (LPA) as having a role in dermal fibrosis in systemic sclerosis (SSc). The aim of this study was to identify the role of the LPA-producing enzyme autotaxin (ATX), and to connect the ATX/LPA and interleukin-6 (IL-6) pathways in SSc.We evaluated the effect of a novel ATX inhibitor, PAT-048, on fibrosis and IL-6 expression in the mouse model of bleomycin-induced dermal fibrosis. We used dermal fibroblasts from SSc patients and control subjects to evaluate LPA-induced expression of IL-6, and IL-6-induced expression of ATX. We next evaluated whether LPA-induced ATX expression is dependent on IL-6, and whether baseline IL-6 expression in fibroblasts from SSc patients is dependent on ATX. Finally, we compared ATX and IL-6 expression in the skin of patients with SSc and healthy control subjects.PAT-048 markedly attenuated bleomycin-induced dermal fibrosis when treatment was initiated before or after the development of fibrosis. LPA stimulated expression of IL-6 in human dermal fibroblasts, and IL-6 stimulated fibroblast expression of ATX, connecting the ATX/LPA and IL-6 pathways in an amplification loop. IL-6 knockdown abrogated LPA-induced ATX expression in fibroblasts, and ATX inhibition attenuated IL-6 expression in fibroblasts and the skin of bleomycin-challenged mice. Expression of both ATX and IL-6 was increased in SSc skin, and LPA-induced IL-6 levels and IL-6-induced ATX levels were increased in fibroblasts from SSc patients compared with controls.ATX is required for the development and maintenance of dermal fibrosis in a mouse model of bleomycin-induced SSc and enables 2 major mediators of SSc fibrogenesis, LPA and IL-6, to amplify the production of each other. Our results suggest that concurrent inhibition of these 2 pathways may be an effective therapeutic strategy for dermal fibrosis in SSc.

Lysophosphatidic acid (LPA) is an important mediator of pulmonary fibrosis. In blood and multiple tumor types, autotaxin produces LPA from lysophosphatidylcholine (LPC) via lysophospholipase D activity, but alternative enzymatic pathways also exist for LPA production. We examined the role of autotaxin (ATX) in pulmonary LPA production during fibrogenesis in a bleomycin mouse model. We found that bleomycin injury increases the bronchoalveolar lavage (BAL) fluid levels of ATX protein 17-fold. However, the LPA and LPC species that increase in BAL of bleomycin-injured mice were discordant, inconsistent with a substrate-product relationship between LPC and LPA in pulmonary fibrosis. LPA species with longer chain polyunsaturated acyl groups predominated in BAL fluid after bleomycin injury, with 22:5 and 22:6 species accounting for 55 and 16% of the total, whereas the predominant BAL LPC species contained shorter chain, saturated acyl groups, with 16:0 and 18:0 species accounting for 56 and 14% of the total. Further, administration of the potent ATX inhibitor PAT-048 to bleomycin-challenged mice markedly decreased ATX activity systemically and in the lung, without effect on pulmonary LPA or fibrosis. Therefore, alternative ATX-independent pathways are likely responsible for local generation of LPA in the injured lung. These pathways will require identification to therapeutically target LPA production in pulmonary fibrosis.-Black, K. E., Berdyshev, E., Bain, G., Castelino, F. V., Shea, B. S., Probst, C. K., Fontaine, B. A., Bronova, I., Goulet, L., Lagares, D., Ahluwalia, N., Knipe, R. S., Natarajan, V., Tager, A. M. Autotaxin activity increases locally following lung injury, but is not required for pulmonary lysophosphatidic acid production or fibrosis.

Despite the well-established role of arginine vasopressin (AVP) in adult social behavior, its role in social development is relatively unexplored. In this paper, we focus on the most prominent social behavior of juvenile rats, social play. Previous pharmacological experiments in our laboratory suggested that AVP regulates play in a sex- and brain region-specific manner in juvenile rats. Here we investigate the role of specific AVP systems in the emergence of social play. We first characterize the development of play in male and female Wistar rats and then ask whether the development of AVP mRNA expression correlates with the emergence of play. Unexpectedly, play emerged more rapidly in weanling-aged females than in males, resulting in a sex difference opposite of that typically reported for older, juvenile rats. AVP mRNA and play were correlated in males only, with a negative correlation in the bed nucleus of the stria terminalis (BNST) and a positive correlation in the paraventricular nucleus of the hypothalamus (PVN). These findings support the hypothesis that AVP acts differentially on multiple systems in a sex-specific manner to regulate social play and suggest a role for PVN and BNST AVP systems in the development of play. Differential neuropeptide regulation of male and female social development may underlie well-documented sex differences in incidence, progression, and symptom severity of behavioral disorders during development.

Background Fibrotic diseases including systemic sclerosis (SSc) or scleroderma are characterized by fibroblast differentiation into myofibroblasts and extracellular matrix deposition. The mechanisms driving fibroblast activation are not fully known, and the identification of new profibrotic mediators will hopefully lead to the development of new rational anti- fibrotic treatments. Microarray studies show that ephrin-B2 is overexpressed in SSc fibroblasts, suggesting that this cell- membrane-anchored ligand may be a novel profibrotic mediator. Objectives To identify the role of ephrin B2 in skin and lung fibrosis. Methods Mice were injected subcutaneously with recombinant mouse ephrin-B2/Fc (100μg/kg/mouse) daily for two weeks and assessed for the development of skin fibrosis. Fibroblast-specific ephrin-B2 knockout (KO) mice were generated and assessed for the development of lung and skin fibrosis induced by intratracheal or subcutaneous injection of bleomycin respectively, by histology, hydroxyproline levels, q-PCR and western blot. Ephrin-B2 levels were determined by western blot and ELISA in broncoalveolar lavage (BAL) fluids from these mice, and from humans with SSc. Cultured mouse and human skin fibroblasts were treated with recombinant ephrin-B2-Fc (0.1-5 mg/mL) and the expression of profibrotic genes was assessed by qPCR and western blot. 96-Multiwell Insert Systems (BD Biosciences) were used to measure the chemotaxis of mouse skin fibroblasts from mice treated with either PBS or bleomycin for 7 days. Fibroblasts in these chemotaxis experiments were transfected with 20nM siRNA (Dharmacon) targeting EphB2, EphB3 or EphB4 receptors. Non-targeting siRNA was used as a control. Results Treatment of normal human skin fibroblasts with recombinant ephrin-B2 in vitro induced myofibroblast differentiation, as indicated by increased stress fiber formation, increased expression of α-SMA and collagen type I. Mice treated sub-cutaneously with recombinant mouse ephrin-B2/Fc exhibited significant skin fibrosis indicated by increased dermal thickness, collagen deposition, hydroxyproline content and α-SMA-expressing myofibroblasts. Fibroblast-specific ephrin-B2 KO mice were significantly protected from bleomycin-induced lung and skin fibrosis, as indicated by significant reductions in hydroxyproline and TGF-b levels. Q-PCR and immunohistochemical analyses showed that ephrin-B2 expression was elevated in SSc fibroblasts and skin sections from IPF patients respectively. Soluble ephrin-B2 levels determined by ELISA and western blot were increased in BAL from SSc patients and bleomycin-challenged mice. Soluble ephrin-B2 induced fibroblast chemotaxis (through EphB3/EphB4 but not EphB2 receptors), suggesting that pathological shedding of soluble ephrin-B2 might account for the profibrotic effect of this ligand in lung and skin fibrosis. Conclusions Our study identifies ephrin-B2 as a novel mediator of fibrogenesis, and suggests that targeting ephrin-B2 ligand or its binding receptors EphB3/EphB4 could potentially be a new therapeutic strategy in SSc. Disclosure of Interest None declared