Toll-like Receptor 9 Inhibition Mitigates Fibroproliferative Responses in Translational Models of Pulmonary Fibrosis.

RATIONALE: Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease for which current treatment options only slow clinical progression. Previously, we identified a subset of patients with IPF with an accelerated disease course associated with fibroblast expression of Toll-Like Receptor 9 (TLR9) mediated by interactions with its ligand mitochondrial DNA (mtDNA). OBJECTIVES: We aimed to show that TLR9 activation induces fibroproliferative responses that are abrogated by its antagonism by using two commercially-available indirect inhibitors and a proprietary, selective direct small molecule inhibitor. METHODS: We employed two independent cohorts of patients with IPF, multiple in vitro fibroblast cell culture platforms, an in vivo mouse model, and an ex vivo human precision cut lung slices system to investigate the clinical and biologic significance of TLR9 in this disease. MEASUREMENTS AND MAIN RESULTS: In two independent IPF cohorts, plasma mtDNA activates TLR9 in a manner associated with the expression of MCP-1, IL-6, TNFα, and IP-10 and worsened transplant-free survival. Our cell culture platform showed that TLR9 mediates fibroblast activation via TGFß1 and stiff substrates, and that its antagonism, particularly direct inhibition, ameliorates this process, including production of these TLR9 associated pharmacodynamic endpoints. We further demonstrated that direct TLR9 inhibition mitigates these fibroproliferative responses in our in vivo and ex vivo models of pulmonary fibrosis. CONCLUSIONS: In this novel study, we found that direct TLR9 inhibition mitigates fibroproliferative responses in preclinical models of pulmonary fibrosis. Our work demonstrates the therapeutic potential of direct TLR9 antagonism in IPF and related fibrotic lung diseases.

α1 Adrenoreceptor antagonism mitigates extracellular mitochondrial DNA accumulation in lung fibrosis models and in patients with idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis is increasingly associated with nerve-driven processes and endogenous innate immune ligands such as mitochondrial DNA (mtDNA). Interestingly, a connection between these entities has not been explored. Here, we report that noradrenaline (NA) derived from the lung's adrenergic nerve supply drives α-smooth muscle actin (αSMA)-expressing fibroblast accumulation via mechanisms involving α1 adrenoreceptors and mtDNA. Using the bleomycin model, we compared ablation of the lung's adrenergic nerve supply with surgical adrenal resection and found that NA derived from local but not adrenal sources contributes to experimentally induced lung fibrosis and the emergence of an αSMA+ve fibroblast population expressing adrenoreceptor α-1D (ADRA1D). Therapeutic delivery of the α1 adrenoreceptor antagonist terazosin reversed these changes and suppressed extracellular mtDNA accumulation. Cultured normal human lung fibroblasts displayed α1 adrenoreceptors and in response to costimulation with TGFß1 and NA adopted ACTA2 expression and extracellular mtDNA release. These findings were opposed by terazosin. Evaluation of a previously studied IPF cohort revealed that patients prescribed α1 adrenoreceptor antagonists for nonpulmonary indications demonstrated improved survival and reduced concentrations of plasma mtDNA. Our observations link nerve-derived NA, α1 adrenoreceptors, extracellular mtDNA, and lung fibrogenesis in mouse models, cultured cells, and humans with IPF. Further study of this neuroinnate connection may yield new avenues for investigation in the clinical and basic science realms.

Extracellular Mitochondrial DNA Is Generated by Fibroblasts and Predicts Death in Idiopathic Pulmonary Fibrosis.

RATIONALE: Idiopathic pulmonary fibrosis (IPF) involves the accumulation of α-smooth muscle actin-expressing myofibroblasts arising from interactions with soluble mediators such as transforming growth factor-ß1 (TGF-ß1) and mechanical influences such as local tissue stiffness. Whereas IPF fibroblasts are enriched for aerobic glycolysis and innate immune receptor activation, innate immune ligands related to mitochondrial injury, such as extracellular mitochondrial DNA (mtDNA), have not been identified in IPF. OBJECTIVES: We aimed to define an association between mtDNA and fibroblast responses in IPF. METHODS: We evaluated the response of normal human lung fibroblasts (NHLFs) to stimulation with mtDNA and determined whether the glycolytic reprogramming that occurs in response to TGF-ß1 stimulation and direct contact with stiff substrates, and spontaneously in IPF fibroblasts, is associated with excessive levels of mtDNA. We measured mtDNA concentrations in bronchoalveolar lavage (BAL) from subjects with and without IPF, as well as in plasma samples from two longitudinal IPF cohorts and demographically matched control subjects. MEASUREMENTS AND MAIN RESULTS: Exposure to mtDNA augments α-smooth muscle actin expression in NHLFs. The metabolic changes in NHLFs that are induced by interactions with TGF-ß1 or stiff hydrogels are accompanied by the accumulation of extracellular mtDNA. These findings replicate the spontaneous phenotype of IPF fibroblasts. mtDNA concentrations are increased in IPF BAL and plasma, and in the latter compartment, they display robust associations with disease progression and reduced event-free survival. CONCLUSIONS: These findings demonstrate a previously unrecognized and highly novel connection between metabolic reprogramming, mtDNA, fibroblast activation, and clinical outcomes that provides new insight into IPF.

Matrix-driven Myosin II Mediates the Pro-fibrotic Fibroblast Phenotype.

Pro-fibrotic mesenchymal cells are known to be the key effector cells of fibroproliferative disease, but the specific matrix signals and the induced cellular responses that drive the fibrogenic phenotype remain to be elucidated. The key mediators of the fibroblast fibrogenic phenotype were characterized using a novel assay system that measures fibroblast behavior in response to actual normal and fibrotic lung tissue. Using this system, we demonstrate that normal lung promotes fibroblast motility and polarization, while fibrotic lung immobilizes the fibroblast and promotes myofibroblast differentiation. These context-specific phenotypes are surprisingly both mediated by myosin II. The role of myosin II is supported by the observation of an increase in myosin phosphorylation and a change in intracellular distribution in fibroblasts on fibrotic lung, as compared with normal lung. Moreover, loss of myosin II activity has opposing effects on protrusive activity in fibroblasts on normal and fibrotic lung. Loss of myosin II also selectively inhibits myofibroblast differentiation in fibroblasts on fibrotic lung. Importantly, these findings are recapitulated by varying the matrix stiffness of polyacrylamide gels in the range of normal and fibrotic lung tissue. Comparison of the effects of myosin inhibition on lung tissue with that of polyacrylamide gels suggests that matrix fiber organization drives the fibroblast phenotype under conditions of normal/soft lung, while matrix stiffness drives the phenotype under conditions of fibrotic/stiff lung. This work defines novel roles for myosin II as a key regulatory effector molecule of the pro-fibrotic phenotype, in response to biophysical properties of the matrix.

Plexin C1 deficiency permits synaptotagmin 7-mediated macrophage migration and enhances mammalian lung fibrosis.

Pulmonary fibrosis is a progressive and often fatal condition that is believed to be partially orchestrated by macrophages. Mechanisms that control migration of these cells into and within the lung remain undefined. We evaluated the contributions of the semaphorin receptor, plexin C1 (PLXNC1), and the exocytic calcium sensor, synaptotagmin 7 (Syt7), in these processes. We evaluated the role of PLXNC1 in macrophage migration by using Boyden chambers and scratch tests, characterized its contribution to experimentally induced lung fibrosis in mice, and defined the mechanism for our observations. Our findings reveal that relative to control participants, patients with idiopathic pulmonary fibrosis demonstrate excessive monocyte migration and underexpression of PLXNC1 in the lungs and circulation, a finding that is recapitulated in the setting of scleroderma-related interstitial lung disease. Relative to wild type, PLXNC1-/- mouse macrophages are excessively migratory, and PLXNC1-/- mice show exacerbated collagen accumulation in response to either inhaled bleomycin or inducible lung targeted TGF-ß1 overexpression. These findings are ameliorated by replacement of PLXNC1 on bone marrow-derived cells or by genetic deletion of Syt7. These data demonstrate the previously unrecognized observation that PLXNC1 deficiency permits Syt7-mediated macrophage migration and enhances mammalian lung fibrosis.-Peng, X., Moore, M., Mathur, A., Zhou, Y., Sun, H., Gan, Y., Herazo-Maya, J. D., Kaminski, N., Hu, X., Pan, H., Ryu, C., Osafo-Addo, A., Homer, R. J., Feghali-Bostwick, C., Fares, W. H., Gulati, M., Hu, B., Lee, C.-G., Elias, J. A., Herzog, E. L. Plexin C1 deficiency permits synaptotagmin 7-mediated macrophage migration and enhances mammalian lung fibrosis.

Fibroblast engraftment in the decellularized mouse lung occurs via a ß1-integrin-dependent, FAK-dependent pathway that is mediated by ERK and opposed by AKT.

Creation of bioartificial organs has been enhanced by the development of strategies involving decellularized mammalian lung. Because fibroblasts critically support lung function through a number of mechanisms, study of these cells in the context of the decellularized lung has the potential to improve the structure and function of tissue-engineered lungs. We characterized the engraftment and survival of a mouse fibroblast cell line in decellularized rat lung slices and found a time-dependent increase in cell numbers assessed by hematoxylin and eosin staining, cell proliferation assessed by Ki67 staining, and minimal cell death assessed by TUNEL staining. We developed a repopulation index to allow quantification of cell survival that accounts for variation in cell density throughout the seeded scaffold. We then applied this method to the study of mouse lung scaffolds and found that decellularization of presliced mouse lungs produced matrices with preserved alveolar architecture and proteinaceous components including fibronectin, collagens I and IV, laminin, and elastin. Treatment with a ß1-integrin-neutralizing antibody significantly reduced the repopulation index after 24 h of culture. Treatment with focal adhesion kinase (FAK) inhibitor and extracellular signal-regulated kinase (ERK) inhibitor further reduced initial repopulation scores while treatment with AKT inhibitor increased initial scores. Rho-associated kinase inhibitor had no discernible effect. These data indicate that initial adhesion and survival of mouse fibroblasts in the decellularized mouse lung occur in a ß1-integrin-dependent, FAK/ERK-dependent manner that is opposed by AKT.

Proteolysis and Contractility Regulate Tissue Opening and Wound Healing by Lung Fibroblasts in 3D Microenvironments.

Damage and repair are recurring processes in tissues, with fibroblasts playing key roles by remodeling extracellular matrices (ECM) through protein synthesis, proteolysis, and cell contractility. Dysregulation of fibroblasts can lead to fibrosis and tissue damage, as seen in idiopathic pulmonary fibrosis (IPF). In advanced IPF, tissue damage manifests as honeycombing, or voids in the lungs. This study explores how transforming growth factor-beta (TGF-ß), a crucial factor in IPF, induces lung fibroblast spheroids to create voids in reconstituted collagen through proteolysis and cell contractility, a process is termed as hole formation. These voids reduce when proteases are blocked. Spheroids mimic fibroblast foci observed in IPF. Results indicate that cell contractility mediates tissue opening by stretching fractures in the collagen meshwork. Matrix metalloproteinases (MMPs), including MMP1 and MT1-MMP, are essential for hole formation, with invadopodia playing a significant role. Blocking MMPs reduces hole size and promotes wound healing. This study shows how TGF-ß induces excessive tissue destruction and how blocking proteolysis can reverse damage, offering insights into IPF pathology and potential therapeutic interventions.

A Nerve-Fibroblast Axis in Mammalian Lung Fibrosis.

Tissue fibrosis contributes to pathology in vital organs including the lung. Curative therapies are scant. Myofibroblasts, pivotal effector cells in tissue fibrosis, accumulate via incompletely understood interactions with their microenvironment. In an investigative platform grounded in experimental lung biology, we find that sympathetic innervation stimulates fibrotic remodeling via noradrenergic α1-adrenergic receptor engagement in myofibroblasts. We demonstrate the anti-fibrotic potential of targeted sympathetic denervation and pharmacological disruption of noradrenergic neurotransmitter functions mediated by α1-adrenoreceptors (α1-ARs). Using the α1-adrenoreceptor subtype D as a representative α1-AR, we discover direct noradrenergic input from sympathetic nerves to lung myofibroblasts utilizing established mouse models, genetic denervation, pharmacologic interventions, a newly invented transgenic mouse line, advanced tissue mimetics, and samples from patients with diverse forms of pulmonary fibrosis. The discovery of this previously unappreciated nerve-fibroblast axis in the lung demonstrates the crucial contribution of nerves to tissue repair and heralds a novel paradigm in fibrosis research.

cGAS Expression is Enhanced in Systemic Sclerosis Associated Interstitial Lung Disease and Stimulates Inflammatory Myofibroblast Activation.

Objective: The lungs of patients with Systemic Sclerosis Associated Interstitial Lung Disease (SSc-ILD) contain inflammatory myofibroblasts arising in association with fibrotic stimuli and perturbed innate immunity. The innate immune DNA binding receptor Cyclic GMP-AMP synthase (cGAS) is implicated in inflammation and fibrosis, but its involvement in SSc-ILD remains unknown. We examined cGAS expression, activity, and therapeutic potential in SSc-ILD using cultured fibroblasts, precision cut lung slices (PCLS), and a well-accepted animal model. Methods: Expression and localization of cGAS, cytokines, and type 1 interferons were evaluated in SSc-ILD lung tissues, bronchoalveolar lavage (BAL), and isolated lung fibroblasts. CGAS activation was assessed in a publicly available SSc-ILD single cell RNA sequencing dataset. Production of cytokines, type 1 interferons, and αSMA elicited by TGFß1 or local substrate stiffness were measured in normal human lung fibroblasts (NHLFs) via qRT-PCR, ELISA, and immunofluorescence. Small molecule cGAS inhibition was tested in cultured fibroblasts, human PCLS, and the bleomycin pulmonary fibrosis model. Results: SSc-ILD lung tissue and BAL are enriched for cGAS, cytokines, and type 1 interferons. The cGAS pathway shows constitutive activation in SSc-ILD fibroblasts and is inducible in NHLFs by TGFß1 or mechanical stimuli. In these settings, and in human PCLS, cGAS expression is paralleled by the production of cytokines, type 1 interferons, and αSMA that are mitigated by a small molecule cGAS inhibitor. These findings are recapitulated in the bleomycin mouse model. Conclusion: cGAS signaling contributes to pathogenic inflammatory myofibroblast phenotypes in SSc-ILD. Inhibiting cGAS or its downstream effectors represents a novel therapeutic approach.

Small molecular modulation of macrophage migration inhibitory factor in the hyperoxia-induced mouse model of bronchopulmonary dysplasia.

BACKGROUND: The role and mechanism of action of MIF in bronchopulmonary dysplasia (BPD) are not known. We hypothesized that increased MIF signaling would ameliorate the pulmonary phenotype of BPD in the mouse lung. METHODS: We studied newborn wild type (WT), MIF knockout (MIFKO), and lung MIF transgenic (MIFTG) mice in room air and a BPD model, and examined the effects of administering a small molecule MIF agonist and antagonist. Lung morphometry was performed and mRNA and protein expression of vascular mediators were analyzed. RESULTS: The pulmonary phenotype of MIFKO and MIFTG mice lungs in room air (RA) and BPD model were comparable to the WT-BPD mice at postnatal (PN) day 14. Vascular endothelial growth factor (VEGF)-A, -R1 and Angiopoietin (Ang)1 mRNA were decreased, and Ang2 increased in the WT-BPD, MIFKO-RA, MIFKO-BPD, MIFTG-RA and MIFTG-BPD mice lungs, compared to appropriate controls. The protein expression of Ang1 in the MIFKO-RA was similar to WT-RA, but decreased in MIFTG-RA, and decreased in all the BPD groups. Ang2 was increased in MIFKO-RA, MIFTG-RA and in all 3 BPD groups. Tie2 was increased in WT-BPD compared to WT-RA, but decreased in MIFKO- and MIFTG- RA and BPD groups. VEGFR1 was uniformly decreased in MIFKO-RA, MIFTG-RA and in all 3 BPD groups. VEGF-A had a similar expression across all RA and BPD groups. There was partial recovery of the pulmonary phenotype in the WT-BPD model treated with the MIF agonist, and in the MIFTG mice treated with the MIF antagonist. CONCLUSIONS: These data point to the careful regulatory balance exerted by MIF in the developing lung and response to hyperoxia and support the potential therapeutic value of small molecule MIF modulation in BPD.

Increased hyperoxia-induced lung injury in nitric oxide synthase 2 null mice is mediated via angiopoietin 2.

Supplemental oxygen is frequently prescribed. However, prolonged exposure to high concentrations of oxygen causes hyperoxic acute lung injury (HALI), which manifests as acute respiratory distress syndrome in adults and leads to bronchopulmonary dysplasia in newborns (NBs). Nitric oxide (NO), NO synthases (NOSs), and angiopoietin (Ang) 2 have been implicated in the pathogenesis of HALI. However, the mechanisms of the contributions of NOS/NO and the relationship(s) between NOS/NO and Ang2 have not been addressed. In addition, the relevance of these moieties in adults and NBs has not been evaluated. To address these issues, we compared the responses in hyperoxia of wild-type (NOS [+/+]) and NOS null (-/-) young adult and NB mice. When compared with NOS2(+/+) adult controls, NOS2(-/-) animals manifest exaggerated alveolar-capillary protein leak and premature death. These responses were associated with enhanced levels of structural cell death, enhanced expression of proapoptotic regulatory proteins, and Ang2. Importantly, silencing RNA knockdown of Ang2 decreased the levels of cell death and the expression of proapoptotic mediators. These effects were at least partially NOS2 specific, and were development dependent, because survival was similar in adult NOS3(+/+) and NOS3(-/-) mice and NB NOS2(+/+) and NOS2(-/-) mice, respectively. These studies demonstrate that NOS2 plays an important protective role in HALI in adult animals. They also demonstrate that this response is mediated, at least in part, by the ability of NOS2 to inhibit hyperoxia-induced Ang2 production and thereby decrease Ang2-induced tissue injury.

A potential role of the JNK pathway in hyperoxia-induced cell death, myofibroblast transdifferentiation and TGF-ß1-mediated injury in the developing murine lung.

BACKGROUND: Transforming growth factor-beta 1 (TGF-ß1) has been implicated in hyperoxia-induced cell death and impaired alveolarization in the developing lung. In addition, the c-JunNH2-terminal kinase (JNK) pathway has been shown to have a role for TGF-ß1-mediated effects. We hypothesized that the JNK pathway is an important regulator of hyperoxia-induced pulmonary responses in the developing murine lung. RESULTS: We used cultured human lung epithelial cells, fetal rat lung fibroblasts and a neonatal TGF-ß1 transgenic mouse model. We demonstrate that hyperoxia inhibits cell proliferation, activates cell death mediators and causes cell death, and promotes myofibroblast transdifferentiation, in a dose-dependent manner. Except for fibroblast proliferation, the effects were mediated via the JNK pathway. In addition, since we observed increased expression of TGF-ß1 by epithelial cells on exposure to hyperoxia, we used a TGF-ß1 transgenic mouse model to determine the role of JNK activation in TGF-ß1 induced effects on lung development and on exposure to hyperoxia. We noted that, in this model, inhibition of JNK signaling significantly improved the spontaneously impaired alveolarization in room air and decreased mortality on exposure to hyperoxia. CONCLUSIONS: When viewed in combination, these studies demonstrate that hyperoxia-induced cell death, myofibroblast transdifferentiation, TGF-ß1- and hyperoxia-mediated pulmonary responses are mediated, at least in part, via signaling through the JNK pathway.

Macrophage-derived netrin-1 drives adrenergic nerve-associated lung fibrosis.

Fibrosis is a macrophage-driven process of uncontrolled extracellular matrix accumulation. Neuronal guidance proteins such as netrin-1 promote inflammatory scarring. We found that macrophage-derived netrin-1 stimulates fibrosis through its neuronal guidance functions. In mice, fibrosis due to inhaled bleomycin engendered netrin-1-expressing macrophages and fibroblasts, remodeled adrenergic nerves, and augmented noradrenaline. Cell-specific knockout mice showed that collagen accumulation, fibrotic histology, and nerve-associated endpoints required netrin-1 of macrophage but not fibroblast origin. Adrenergic denervation; haploinsufficiency of netrin-1's receptor, deleted in colorectal carcinoma; and therapeutic α1 adrenoreceptor antagonism improved collagen content and histology. An idiopathic pulmonary fibrosis (IPF) lung microarray data set showed increased netrin-1 expression. IPF lung tissues were enriched for netrin-1+ macrophages and noradrenaline. A longitudinal IPF cohort showed improved survival in patients prescribed α1 adrenoreceptor blockade. This work showed that macrophages stimulate lung fibrosis via netrin-1-driven adrenergic processes and introduced α1 blockers as a potentially new fibrotic therapy.

A 21-Year-Old Immune-Competent Man With Recurrent Cough.

CASE PRESENTATION: A 21-year-old Chinese man presented with a nonproductive cough for the past 5 months. He denied fevers, chills, night sweats, chest pain, dyspnea, hemoptysis, or weight loss. He was an undergraduate with an unremarkable medical history. He denied any sick contacts and he never smoked. Laboratory tests showed a leukocyte count of 11,200/µL (normal range, 3,500-9,500/µL) with a high neutrophil count and a raised erythrocyte sedimentation rate of 81 mm/h. The purified protein derivative skin test result was positive, and a TB test (T.SPOT.TB; Oxford Immunotec) produced a positive result. The HIV test result was negative. The lung window of the patient's thoracic CT scan showed mottled, patchy opacification in the right lower lobe, and enlarged mediastinal and right hilar lymph nodes (Fig 1A). Bronchoscopy showed mucosal swelling and congestion (Fig 1B). A lymph node (station 11R) biopsy, obtained by endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) (Fig 1C), showed nonspecific necrosis. An acid-fast bacillus smear of bronchial secretion produced negative results. He was administered empiric anti-TB therapy (ethambutol, isoniazid, pyrazinamide, and rifapentine). But his cough had not improved by 4 months later. Thus he came to our hospital for a second opinion.

Bioactive Plasma Mitochondrial DNA Is Associated With Disease Progression in Scleroderma-Associated Interstitial Lung Disease.

OBJECTIVE: Systemic sclerosis-associated interstitial lung disease (SSc-ILD) is characterized by variable clinical outcomes, activation of innate immune pattern-recognition receptors (PRRs), and accumulation of α-smooth muscle actin (α-SMA)-expressing myofibroblasts. The aim of this study was to identify an association between these entities and mitochondrial DNA (mtDNA), an endogenous ligand for the intracellular DNA-sensing PRRs Toll-like receptor 9 (TLR-9) and cyclic GMP-AMP synthase/stimulator of interferon genes (cGAS/STING), which has yet to be determined. METHODS: Human lung fibroblasts (HLFs) from normal donors and SSc-ILD explants were treated with synthetic CpG DNA and assayed for α-SMA expression and extracellular mtDNA using quantitative polymerase chain reaction for the human MT-ATP6 gene. Plasma MT-ATP6 concentrations were evaluated in 2 independent SSc-ILD cohorts and demographically matched controls. The ability of SSc-ILD and control plasma to induce TLR-9 and cGAS/STING activation was evaluated with commercially available HEK 293 reporter cells. Plasma concentrations of type I interferons (IFNs), interleukin-6 (IL-6), and oxidized DNA were measured using electrochemiluminescence and enzyme-linked immunosorbent assay-based methods. Extracellular vesicles (EVs) precipitated from plasma were evaluated for MT-ATP6 concentrations and proteomics via liquid chromatography mass spectrometry. RESULTS: Normal HLFs and SSc-ILD fibroblasts developed increased α-SMA expression and MT-ATP6 release following CpG stimulation. Plasma mtDNA concentrations were increased in the 2 SSc-ILD cohorts, reflective of ventilatory decline, and were positively associated with both TLR-9 and cGAS/STING activation as well as type I IFN and IL-6 expression. Plasma mtDNA was not oxidized and was conveyed by EVs displaying a proteomics profile consistent with a multicellular origin. CONCLUSION: These findings demonstrate a previously unrecognized connection between EV-encapsulated mtDNA, clinical outcomes, and intracellular DNA-sensing PRR activation in SSc-ILD. Further study of these interactions could catalyze novel mechanistic and therapeutic insights into SSc-ILD and related disorders.

Dual knockdown of N-ras and epiregulin synergistically suppressed the growth of human hepatoma cells.

Hepatocellular carcinoma (HCC) is a major challenge because of its resistance to conventional cytotoxic chemotherapy and radiotherapy. Multi-targeted therapy might be a new option for HCC treatment. Our previous study showed that N-ras gene was activated in HCC and was inhibited by RNA interference. In the present study, we investigated the alternation of gene expression by microarray in N-Ras-siRNA-treated HepG2 cells. The results revealed that the EREG gene, encoding epiregulin, was dramatically up-regulated in response to silence of N-ras. We speculated that the up-regulation of epiregulin was involved in the compensatory mechanism of N-ras knockdown for cell growth. Therefore, we evaluated whether dual silence of N-ras and epiregulin display a greater suppression of cell growth. The results confirmed that dual knockdown of N-ras and epiregulin synergistically inhibited cell growth. Our results also showed that dual knockdown of N-ras and epiregulin significantly induced cell arrest at G0/G1 phase. Furthermore, Western blot assay showed that dual knockdown of N-ras and epiregulin markedly reduced the phosphorylations of ERK1/2, Akt and Rb, and inhibited the expression of cyclin D1. Our findings imply that multi-targeted silence of oncogenes might be an effective treatment for HCC.

Human pericytes adopt myofibroblast properties in the microenvironment of the IPF lung.

Idiopathic pulmonary fibrosis (IPF) is a fatal disease of unknown etiology characterized by a compositionally and mechanically altered extracellular matrix. Poor understanding of the origin of α-smooth muscle actin (α-SMA) expressing myofibroblasts has hindered curative therapies. Though proposed as a source of myofibroblasts in mammalian tissues, identification of microvascular pericytes (PC) as contributors to α-SMA-expressing populations in human IPF and the mechanisms driving this accumulation remain unexplored. Here, we demonstrate enhanced detection of α-SMA+ cells coexpressing the PC marker neural/glial antigen 2 in the human IPF lung. Isolated human PC cultured on decellularized IPF lung matrices adopt expression of α-SMA, demonstrating that these cells undergo phenotypic transition in response to direct contact with the extracellular matrix (ECM) of the fibrotic human lung. Using potentially novel human lung-conjugated hydrogels with tunable mechanical properties, we decoupled PC responses to matrix composition and stiffness to show that α-SMA+ PC accumulate in a mechanosensitive manner independent of matrix composition. PC activated with TGF-ß1 remodel the normal lung matrix, increasing tissue stiffness to facilitate the emergence of α-SMA+ PC via MKL-1/MTRFA mechanotranduction. Nintedanib, a tyrosine-kinase inhibitor approved for IPF treatment, restores the elastic modulus of fibrotic lung matrices to reverse the α-SMA+ phenotype. This work furthers our understanding of the role that microvascular PC play in the evolution of IPF, describes the creation of an ex vivo platform that advances the study of fibrosis, and presents a potentially novel mode of action for a commonly used antifibrotic therapy that has great relevance for human disease.

Comparative biology of decellularized lung matrix: Implications of species mismatch in regenerative medicine.

Lung engineering is a promising technology, relying on re-seeding of either human or xenographic decellularized matrices with patient-derived pulmonary cells. Little is known about the species-specificity of decellularization in various models of lung regeneration, or if species dependent cell-matrix interactions exist within these systems. Therefore decellularized scaffolds were produced from rat, pig, primate and human lungs, and assessed by measuring residual DNA, mechanical properties, and key matrix proteins (collagen, elastin, glycosaminoglycans). To study intrinsic matrix biologic cues, human endothelial cells were seeded onto acellular slices and analyzed for markers of cell health and inflammation. Despite similar levels of collagen after decellularization, human and primate lungs were stiffer, contained more elastin, and retained fewer glycosaminoglycans than pig or rat lung scaffolds. Human endothelial cells seeded onto human and primate lung tissue demonstrated less expression of vascular cell adhesion molecule and activation of nuclear factor-κB compared to those seeded onto rodent or porcine tissue. Adhesion of endothelial cells was markedly enhanced on human and primate tissues. Our work suggests that species-dependent biologic cues intrinsic to lung extracellular matrix could have profound effects on attempts at lung regeneration.

Netrin-1 Regulates Fibrocyte Accumulation in the Decellularized Fibrotic Sclerodermatous Lung Microenvironment and in Bleomycin-Induced Pulmonary Fibrosis.

OBJECTIVE: Fibrocytes are collagen-producing leukocytes that accumulate in patients with systemic sclerosis (SSc; scleroderma)-related interstitial lung disease (ILD) via unknown mechanisms that have been associated with altered expression of neuroimmune proteins. The extracellular matrix (ECM) influences cellular phenotypes. However, a relationship between the lung ECM and fibrocytes in SSc has not been explored. The aim of this study was to use a novel translational platform based on decellularized human lungs to determine whether the lung ECM of patients with scleroderma controls the development of fibrocytes from peripheral blood mononuclear cells. METHODS: We performed biomechanical evaluation of decellularized scaffolds prepared from lung explants from healthy control subjects and patients with scleroderma, using tensile testing and biochemical and proteomic analysis. Cells obtained from healthy controls and patients with SSc-related ILD were cultured on these scaffolds, and CD45+pro-ColIα1+ cells meeting the criteria for fibrocytes were quantified. The contribution of the neuromolecule netrin-1 to fibrosis was assessed using neutralizing antibodies in this system and by administering bleomycin via inhalation to netrin-1(+/-) mice. RESULTS: Compared with control lung scaffolds, lung scaffolds from patients with SSc-related ILD showed aberrant anatomy, enhanced stiffness, and abnormal ECM composition. Culture of control cells in lung scaffolds from patients with SSc-related ILD increased production of pro-ColIα1+ cells, which was stimulated by enhanced stiffness and abnormal ECM composition. Cells from patients with SSc-related ILD demonstrated increased pro-ColIα1 responsiveness to lung scaffolds from scleroderma patients but not enhanced stiffness. Enhanced detection of netrin-1-expressing CD14(low) cells in patients with SSc-related ILD was observed, and antibody-mediated netrin-1 neutralization attenuated detection of CD45+pro-ColIα1+ cells in all settings. Netrin-1(+/-) mice were protected against bleomycin-induced lung fibrosis and fibrocyte accumulation. CONCLUSION: Factors present in the lung matrices of patients with scleroderma regulate fibrocyte accumulation via a netrin-1-dependent pathway. Netrin-1 regulates bleomycin-induced pulmonary fibrosis in mice. Netrin-1 might be a novel therapeutic target in SSc-related ILD.

CD4+CD25+FoxP3+ Regulatory Tregs inhibit fibrocyte recruitment and fibrosis via suppression of FGF-9 production in the TGF-ß1 exposed murine lung.

Pulmonary fibrosis is a difficult to treat, often fatal disease whose pathogenesis involves dysregulated TGF-ß1 signaling. CD4+CD25+FoxP3+ Regulatory T cells ("Tregs") exert important effects on host tolerance and arise from naïve CD4+ lymphocytes in response to TGF-ß1. However, the precise contribution of Tregs to experimentally induced murine lung fibrosis remains unclear. We sought to better understand the role of Tregs in this context. Using a model of fibrosis caused by lung specific, doxycycline inducible overexpression of the bioactive form of the human TGF-ß1 gene we find that Tregs accumulate in the lung parenchyma within 5 days of transgene activation and that this enhancement persists to at least 14 days. Anti-CD25 Antibody mediated depletion of Tregs causes increased accumulation of soluble collagen and of intrapulmonary CD45+Col Iα1 fibrocytes. These effects are accompanied by enhanced local concentrations of the classical inflammatory mediators CD40L, TNF-α, and IL-1α, along with the neuroimmune molecule fibroblast growth factor 9 (FGF-9, also known as "glial activating factor"). FGF-9 expression localizes to parenchymal cells and alveolar macrophages in this model and antibody mediated neutralization of FGF-9 results in attenuated detection of intrapulmonary collagen and fibrocytes without affecting Treg quantities. These data indicate that CD4+CD25+FoxP3+ Tregs attenuate TGF-ß1 induced lung fibrosis and fibrocyte accumulation in part via suppression of FGF-9.