Genetically modified ZIKA virus as a microRNA-sensitive oncolytic virus against central nervous system tumors.

Here we introduce a first-in-class microRNA-sensitive oncolytic Zika virus (ZIKV) for virotherapy application against central nervous system (CNS) tumors. The described methodology produced two synthetic modified ZIKV strains that are safe in normal cells, including neural stem cells, while preserving brain tropism and oncolytic effects in tumor cells. The microRNA-sensitive ZIKV introduces genetic modifications in two different virus sites: first, in the established 3'UTR region, and secondly, in the ZIKV protein coding sequence, demonstrating for the first time that the miRNA inhibition systems can be functional outside the UTR RNA sites. The total tumor remission in mice bearing human CNS tumors, including metastatic tumor growth, after intraventricular and systemic modified ZIKV administration, confirms the promise of this virotherapy as a novel agent against brain tumors-highly deadly diseases in urgent need of effective advanced therapies.

Challenges and Opportunities for Commercializing Technologies in the Pulmonary Arena: An Official American Thoracic Society Report.

"Translational medicine" has been a buzzword for over two decades. The concept was intended to be lofty, to reflect a new "bench-to-bedside" approach to basic and clinical research that would bridge fields, close gaps, accelerate innovation, and shorten the time and effort it takes to bring novel technologies from basic discovery to clinical application. Has this approach been successful and lived up to its promise? Despite incredible scientific advances and innovations developed within academia, successful clinical translation into real-world solutions has been difficult. This has been particularly challenging within the pulmonary field, because there have been fewer U.S. Food and Drug Administration-approved drugs and higher failure rates for pulmonary therapies than with other common disease areas. The American Thoracic Society convened a working group with the goal of identifying major challenges related to the commercialization of technologies within the pulmonary space and opportunities to enhance this process. A survey was developed and administered to 164 participants within the pulmonary arena. This report provides a summary of these survey results. Importantly, this report identifies a number of poorly recognized challenges that exist in pulmonary academic settings, which likely contribute to diminished efficiency of commercialization efforts, ultimately hindering the rate of successful clinical translation. Because many innovations are initially developed in academic settings, this is a global public health issue that impacts the entire American Thoracic Society community. This report also summarizes key resources and opportunities and provides recommendations to enhance successful commercialization of pulmonary technologies.

Myocardin-related transcription factor contributes to renal fibrosis through the regulation of extracellular microenvironment surrounding fibroblasts.

Fibroblast accumulation and extracellular matrix (ECM) deposition are common critical steps for the progression of organ fibrosis, but the precise molecular mechanisms remain to be fully investigated. We have previously demonstrated that lysophosphatidic acid contributes to organ fibrosis through the production of connective tissue growth factor (CTGF) via actin cytoskeleton-dependent signaling, myocardin-related transcription factor family (MRTF) consisting of MRTF-A and MRTF-B-serum response factor (SRF) pathway. In this study, we investigated the role of the MRTF-SRF pathway in the development of renal fibrosis, focusing on the regulation of ECM-focal adhesions (FA) in renal fibroblasts. Here we showed that both MRTF-A and -B were required for the expressions of ECM-related molecules such as lysyl oxidase family members, type I procollagen and fibronectin in response to transforming growth factor (TGF)-ß1 . TGF-ß1 -MRTF-SRF pathway induced the expressions of various components of FA such as integrin α subunits (αv , α2 , α11 ) and ß subunits (ß1 , ß3 , ß5 ) as well as integrin-linked kinase (ILK). On the other hand, the blockade of ILK suppressed TGF-ß1 -induced MRTF-SRF transcriptional activity, indicating a mutual relationship between MRTF-SRF and FA. Myofibroblast differentiation along with CTGF expression was also dependent on MRTF-SRF and FA components. Finally, global MRTF-A deficient and inducible fibroblast-specific MRTF-B deficient mice (MRTF-AKO BiFBKO mice) are protected from renal fibrosis with adenine administration. Renal expressions of ECM-FA components and CTGF as well as myofibroblast accumulation were suppressed in MRTF-AKO BiFBKO mice. These results suggest that the MRTF-SRF pathway might be a therapeutic target for renal fibrosis through the regulation of components forming ECM-FA in fibroblasts.

Sanglifehrin A mitigates multi-organ fibrosis in vivo by inducing secretion of the collagen chaperone cyclophilin B.

Pathological deposition and crosslinking of collagen type I by activated myofibroblasts drives progressive tissue fibrosis. Therapies that inhibit collagen synthesis by myofibroblasts have clinical potential as anti-fibrotic agents. Lysine hydroxylation by the prolyl-3-hydroxylase complex, comprised of cartilage associated protein, prolyl 3-hydroxylase 1, and cyclophilin B, is essential for collagen type I crosslinking and formation of stable fibers. Here, we identify the collagen chaperone cyclophilin B as a major cellular target of the macrocyclic natural product sanglifehrin A (SfA) using photo-affinity labeling and chemical proteomics. Our studies reveal a unique mechanism of action in which SfA binding to cyclophilin B in the endoplasmic reticulum (ER) induces the secretion of cyclophilin B to the extracellular space, preventing TGF-ß1-activated myofibroblasts from synthesizing collagen type I in vitro without inhibiting collagen type I mRNA transcription or inducing ER stress. In addition, SfA prevents collagen type I secretion without affecting myofibroblast contractility or TGF-ß1 signaling. In vivo, we provide chemical, molecular, functional, and translational evidence that SfA mitigates the development of lung and skin fibrosis in mouse models by inducing cyclophilin B secretion, thereby inhibiting collagen synthesis from fibrotic fibroblasts in vivo . Consistent with these findings in preclinical models, SfA reduces collagen type I secretion from fibrotic human lung fibroblasts and precision cut lung slices from patients with idiopathic pulmonary fibrosis, a fatal fibrotic lung disease with limited therapeutic options. Our results identify the primary liganded target of SfA in cells, the collagen chaperone cyclophilin B, as a new mechanistic target for the treatment of organ fibrosis.

Recent Advances in Molecular Diagnosis of Pulmonary Fibrosis for Precision Medicine.

Pulmonary fibrosis is a serious, progressive lung disease characterized by scarring and stiffening lung tissues, affecting the respiratory system and leading to organ failure. It is a complex disease consisting of alveolar damage, chronic inflammation, and a varying degree of lung fibrosis. Significant challenges with pulmonary fibrosis include the lack of effective means to diagnose the disease at early stages, identify patients at higher risks of progress, and assess disease progression and treatment response. Precision medicine powered by accurate molecular profiling and phenotyping could significantly improve our understanding of the disease's heterogeneity, potential biomarkers for diagnosis and prognosis, and molecular targets for treatment development. This Review discusses various translational model systems, including organoids and lung-on-a-chip systems, biomarkers in single cells and extracellular vesicles, and functional pharmacodynamic markers. We also highlight emerging sensing technologies for molecular characterization of pulmonary fibrosis and biomarker detection.

A Novel Protective Role for Matrix Metalloproteinase-8 in the Pulmonary Vasculature.

Rationale: Mechanical signaling through cell-matrix interactions plays a major role in progressive vascular remodeling in pulmonary arterial hypertension (PAH). MMP-8 (matrix metalloproteinase-8) is an interstitial collagenase involved in regulating inflammation and fibrosis of the lung and systemic vasculature, but its role in PAH pathogenesis remains unexplored. Objectives: To evaluate MMP-8 as a modulator of pathogenic mechanical signaling in PAH. Methods: MMP-8 levels were measured in plasma from patients with pulmonary hypertension (PH) and controls by ELISA. MMP-8 vascular expression was examined in lung tissue from patients with PAH and rodent models of PH. MMP-8-/- and MMP-8+/+ mice were exposed to normobaric hypoxia or normoxia for 4-8 weeks. PH severity was evaluated by right ventricular systolic pressure, echocardiography, pulmonary artery morphometry, and immunostaining. Proliferation, migration, matrix component expression, and mechanical signaling were assessed in MMP-8-/- and MMP-8+/+ pulmonary artery smooth muscle cells (PASMCs). Measurements and Main Results: MMP-8 expression was significantly increased in plasma and pulmonary arteries of patients with PH compared with controls and induced in the pulmonary vasculature in rodent PH models. Hypoxia-exposed MMP-8-/- mice had significant mortality, increased right ventricular systolic pressure, severe right ventricular dysfunction, and exaggerated vascular remodeling compared with MMP-8+/+ mice. MMP-8-/- PASMCs demonstrated exaggerated proliferation and migration mediated by altered matrix protein expression, elevated integrin-ß3 levels, and induction of FAK (focal adhesion kinase) and downstream YAP (Yes-associated protein)/TAZ (transcriptional coactivator with PDZ-binding motif) activity. Conclusions: MMP-8 is a novel protective factor upregulated in the pulmonary vasculature during PAH pathogenesis. MMP-8 opposes pathologic mechanobiological feedback by altering matrix composition and disrupting integrin-ß3/FAK and YAP/TAZ-dependent mechanical signaling in PASMCs.

Myofibroblast fate plasticity in tissue repair and fibrosis: Deactivation, apoptosis, senescence and reprogramming.

In response to tissue injury, fibroblasts differentiate into professional repair cells called myofibroblasts, which orchestrate many aspects of the normal tissue repair programme including synthesis, deposition and contraction of extracellular matrix proteins, leading to wound closure. Successful tissue repair responses involve termination of myofibroblast activities in order to prevent pathologic fibrotic scarring. Here, we discuss the cellular and molecular mechanisms limiting myofibroblast activities during physiological tissue repair, including myofibroblast deactivation, apoptosis, reprogramming and immune clearance of senescent myofibroblasts. In addition, we summarize pathological mechanisms leading to myofibroblast persistence and survival, a hallmark of fibrotic diseases. Finally, we discuss emerging anti-fibrotic therapies aimed at targeting myofibroblast fate such as senolytics, gene therapy, cellular immunotherapy and CAR-T cells.

Methods for Studying Myofibroblast Apoptotic Pathways.

Evasion of apoptosis by myofibroblasts is a hallmark of fibrotic diseases, ultimately leading to persistent myofibroblast activation, extracellular matrix (ECM) deposition, and remodeling. Targeting myofibroblast apoptosis is emerging as a novel therapeutic strategy to reverse established fibrosis. We have recently discovered that in the process of fibroblast-to-myofibroblast transdifferentiation driven by matrix stiffness, the "mitochondrial priming" (readiness to undergo apoptosis) is dramatically increased in stiffness-activated myofibroblasts. Thus, myofibroblasts, traditionally viewed as apoptosis-resistant cells, appear poised to die when survival pathways are blocked, a cellular state we call "primed for death." This apoptosis-prone phenotype is driven by high levels of pro-apoptotic proteins loaded in myofibroblast's mitochondria, which require concomitant upregulation of pro-survival BCL-2 proteins to suppress mitochondrial apoptosis and ensure survival. Here, we describe a method called BH3 profiling which measures myo/fibroblast apoptotic priming as well as their antiapoptotic dependencies for survival. In addition, we describe how BH3 profiling can be used to predict myofibroblast responses to therapeutic agents targeting pro-survival BCL-2 proteins, also known as BH3 mimetic drugs. Finally, we describe methods to assess myofibroblast sensitivity to extrinsic apoptosis via Annexin V staining.

Method for Investigating Fibroblast Durotaxis.

Durotaxis is the phenomena of directed cell migration driven by gradients of extracellular matrix stiffness. Durotaxis has been recently involved in the development of fibrosis by promoting the recruitment of pathological fibroblasts to areas of established fibrosis, thus amplifying the fibrotic response. Here, we describe the fabrication of mechanically patterned hydrogels that can be used to investigate molecular mechanisms controlling durotaxis of fibroblasts and other cells with mechanosensing properties. This method effectively creates a stiffness gradient of 275 Pa/µm, mimicking the natural spatial stiffness variations we observed in fibrotic tissues from mouse models of fibrosis and human fibrotic diseases.

Animal and Human Models of Tissue Repair and Fibrosis: An Introduction.

Reductionist cell culture systems are not only convenient but essential to understand molecular mechanisms of myofibroblast activation and action in carefully controlled conditions. However, tissue myofibroblasts do not act in isolation and the complexity of tissue repair and fibrosis in humans cannot be captured even by the most elaborate culture models. Over the past five decades, numerous animal models have been developed to study different aspects of myofibroblast biology and interactions with other cells and extracellular matrix. The underlying principles can be broadly classified into: (1) organ injury by trauma such as prototypical full thickness skin wounds or burns; (2) mechanical challenges, such as pressure overload of the heart by ligature of the aorta or the pulmonary vein; (3) toxic injury, such as administration of bleomycin to lungs and carbon tetrachloride to the liver; (4) organ infection with viruses, bacteria, and parasites, such as nematode infections of liver; (5) cytokine and inflammatory models, including local delivery or viral overexpression of active transforming growth factor beta; (6) "lifestyle" and metabolic models such as high-fat diet; and (7) various genetic models. We will briefly summarize the most widely used mouse models used to study myofibroblasts in tissue repair and fibrosis as well as genetic tools for manipulating myofibroblast repair functions in vivo.

Targeting Runt-Related Transcription Factor 1 Prevents Pulmonary Fibrosis and Reduces Expression of Severe Acute Respiratory Syndrome Coronavirus 2 Host Mediators.

Pulmonary fibrosis (PF) can arise from unknown causes, as in idiopathic PF, or as a consequence of infections, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Current treatments for PF slow, but do not stop, disease progression. We report that treatment with a runt-related transcription factor 1 (RUNX1) inhibitor (Ro24-7429), previously found to be safe, although ineffective, as a Tat inhibitor in patients with HIV, robustly ameliorates lung fibrosis and inflammation in the bleomycin-induced PF mouse model. RUNX1 inhibition blunted fundamental mechanisms downstream pathologic mediators of fibrosis and inflammation, including transforming growth factor-ß1 and tumor necrosis factor-α, in cultured lung epithelial cells, fibroblasts, and vascular endothelial cells, indicating pleiotropic effects. RUNX1 inhibition also reduced the expression of angiotensin-converting enzyme 2 and FES Upstream Region (FURIN), host proteins critical for SARS-CoV-2 infection, in mice and in vitro. A subset of human lungs with SARS-CoV-2 infection overexpress RUNX1. These data suggest that RUNX1 inhibition via repurposing of Ro24-7429 may be beneficial for PF and to battle SARS-CoV-2, by reducing expression of viral mediators and by preventing respiratory complications.

Induction of ADAM10 by Radiation Therapy Drives Fibrosis, Resistance, and Epithelial-to-Mesenchyal Transition in Pancreatic Cancer.

Stromal fibrosis activates prosurvival and proepithelial-to-mesenchymal transition (EMT) pathways in pancreatic ductal adenocarcinoma (PDAC). In patient tumors treated with neoadjuvant stereotactic body radiation therapy (SBRT), we found upregulation of fibrosis, extracellular matrix (ECM), and EMT gene signatures, which can drive therapeutic resistance and tumor invasion. Molecular, functional, and translational analysis identified two cell-surface proteins, a disintegrin and metalloprotease 10 (ADAM10) and ephrinB2, as drivers of fibrosis and tumor progression after radiation therapy (RT). RT resulted in increased ADAM10 expression in tumor cells, leading to cleavage of ephrinB2, which was also detected in plasma. Pharmacologic or genetic targeting of ADAM10 decreased RT-induced fibrosis and tissue tension, tumor cell migration, and invasion, sensitizing orthotopic tumors to radiation killing and prolonging mouse survival. Inhibition of ADAM10 and genetic ablation of ephrinB2 in fibroblasts reduced the metastatic potential of tumor cells after RT. Stimulation of tumor cells with ephrinB2 FC protein reversed the reduction in tumor cell invasion with ADAM10 ablation. These findings represent a model of PDAC adaptation that explains resistance and metastasis after RT and identifies a targetable pathway to enhance RT efficacy. SIGNIFICANCE: Targeting a previously unidentified adaptive resistance mechanism to radiation therapy in PDAC tumors in combination with radiation therapy could increase survival of the 40% of PDAC patients with locally advanced disease.See related commentary by Garcia Garcia et al., p. 3158 GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/12/3255/F1.large.jpg.

A FAK/HDAC5 signaling axis controls osteocyte mechanotransduction.

Osteocytes, cells ensconced within mineralized bone matrix, are the primary skeletal mechanosensors. Osteocytes sense mechanical cues by changes in fluid flow shear stress (FFSS) across their dendritic projections. Loading-induced reductions of osteocytic Sclerostin (encoded by Sost) expression stimulates new bone formation. However, the molecular steps linking mechanotransduction and Sost suppression remain unknown. Here, we report that class IIa histone deacetylases (HDAC4 and HDAC5) are required for loading-induced Sost suppression and bone formation. FFSS signaling drives class IIa HDAC nuclear translocation through a signaling pathway involving direct HDAC5 tyrosine 642 phosphorylation by focal adhesion kinase (FAK), a HDAC5 post-translational modification that controls its subcellular localization. Osteocyte cell adhesion supports FAK tyrosine phosphorylation, and FFSS triggers FAK dephosphorylation. Pharmacologic FAK catalytic inhibition reduces Sost mRNA expression in vitro and in vivo. These studies demonstrate a role for HDAC5 as a transducer of matrix-derived cues to regulate cell type-specific gene expression.

Mechano-therapeutics: Targeting Mechanical Signaling in Fibrosis and Tumor Stroma.

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

Assessing the progression of systemic sclerosis by monitoring the tissue optic axis using PS-OCT.

The clinical assessment of fibrosis is critical to the diagnosis and management of patients with systemic sclerosis. Current clinical standards for patient assessment is to use skin fibrosis as an indicator of organ involvement, though this approach is highly subjective and relies on manual palpation. The development of a new method for accurately quantifying collagen content may therefore significantly improve the accuracy of the traditional skin score in patients with systemic sclerosis and may additionally aid in the monitoring of anti-fibrotic therapies in clinical practice. Polarization-sensitive optical coherence tomography (PS-OCT) is a high-speed volumetric imaging modality that can be used to assess birefringent tissues including collagen. In this work we demonstrate a novel computational approach using PS-OCT for the assessment of fibrosis. This approach, based on the measured distribution of optic axis values associated with a given volume of collagen orientation, characterizes fibrotic changes independently from the depth of the region of interest in the tissue. This approach has the potential to accurately quantify collagen content and orientation faster and more robustly compared to traditional PS-OCT metrics. We investigate the viability of this approach for assessing the development of fibrosis in a bleomycin induced skin fibrosis mouse model.

Evasion of apoptosis by myofibroblasts: a hallmark of fibrotic diseases.

Organ fibrosis is a lethal outcome of autoimmune rheumatic diseases such as systemic sclerosis. Myofibroblasts are scar-forming cells that are ultimately responsible for the excessive synthesis, deposition and remodelling of extracellular matrix proteins in fibrosis. Advances have been made in our understanding of the mechanisms that keep myofibroblasts in an activated state and control myofibroblast functions. However, the mechanisms that help myofibroblasts to persist in fibrotic tissues remain poorly understood. Myofibroblasts evade apoptosis by activating molecular mechanisms in response to pro-survival biomechanical and growth factor signals from the fibrotic microenvironment, which can ultimately lead to the acquisition of a senescent phenotype. Growing evidence suggests that myofibroblasts and senescent myofibroblasts, rather than being resistant to apoptosis, are actually primed for apoptosis owing to concomitant activation of cell death signalling pathways; these cells are poised to apoptose when survival pathways are inhibited. This knowledge of apoptotic priming has paved the way for new therapies that trigger apoptosis in myofibroblasts by blocking pro-survival mechanisms, target senescent myofibroblast for apoptosis or promote the reprogramming of myofibroblasts into scar-resolving cells. These novel strategies are not only poised to prevent progressive tissue scarring, but also have the potential to reverse established fibrosis and to regenerate chronically injured tissues.

Senotherapeutics: Targeting senescence in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a fatal chronic lung disease characterized by progressive scarring of the lung tissue, leading to respiratory failure. There is no cure for IPF, and current anti-fibrotic treatments modestly arrest its further progression. IPF prevalence and incidence increase with age, which is a recognized risk factor. Intense clinical and basic research over the last fifteen years has shown that hallmarks of accelerated aging are present in the lungs of patients with IPF. Different cell types in IPF lungs exhibit premature hallmarks of aging, including telomere attrition and cellular senescence. In this Review, we discuss recent insights into the mechanisms behind these age-related alterations and their contribution to the development of lung fibrosis. We focus on the genetic and molecular basis of telomere attrition in alveolar type II epithelial cells, which promote cellular senescence and lung fibrosis. Mechanistically, senescent cells secrete pro-fibrotic factors that activate scar-forming myofibroblasts. Ultimately, senescent alveolar epithelial cells lose their regenerative capacity, impeding fibrosis resolution. In addition, mitochondrial dysfunction is strongly associated with the appearance of senescent epithelial cells and senescent myofibroblasts in IPF, which persist in the fibrotic tissue by adapting their metabolic pathways and becoming resistant to apoptosis. We discuss emerging novel therapeutic strategies to treat IPF by targeting cellular senescence with the so-called senotherapeutics.

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

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