Regenerative Metaplastic Clones in COPD Lung Drive Inflammation and Fibrosis.

Chronic obstructive pulmonary disease (COPD) is a progressive condition of chronic bronchitis, small airway obstruction, and emphysema that represents a leading cause of death worldwide. While inflammation, fibrosis, mucus hypersecretion, and metaplastic epithelial lesions are hallmarks of this disease, their origins and dependent relationships remain unclear. Here we apply single-cell cloning technologies to lung tissue of patients with and without COPD. Unlike control lungs, which were dominated by normal distal airway progenitor cells, COPD lungs were inundated by three variant progenitors epigenetically committed to distinct metaplastic lesions. When transplanted to immunodeficient mice, these variant clones induced pathology akin to the mucous and squamous metaplasia, neutrophilic inflammation, and fibrosis seen in COPD. Remarkably, similar variants pre-exist as minor constituents of control and fetal lung and conceivably act in normal processes of immune surveillance. However, these same variants likely catalyze the pathologic and progressive features of COPD when expanded to high numbers.

The landscape of immune dysregulation in Crohn's disease revealed through single-cell transcriptomic profiling in the ileum and colon.

Crohn's disease (CD) is a chronic gastrointestinal disease that is increasing in prevalence worldwide. CD is multifactorial, involving the complex interplay of genetic, immune, and environmental factors, necessitating a system-level understanding of its etiology. To characterize cell-type-specific transcriptional heterogeneity in active CD, we profiled 720,633 cells from the terminal ileum and colon of 71 donors with varying inflammation status. Our integrated datasets revealed organ- and compartment-specific responses to acute and chronic inflammation; most immune changes were in cell composition, whereas transcriptional changes dominated among epithelial and stromal cells. These changes correlated with endoscopic inflammation, but small and large intestines exhibited distinct responses, which were particularly apparent when focusing on IBD risk genes. Finally, we mapped markers of disease-associated myofibroblast activation and identified CHMP1A, TBX3, and RNF168 as regulators of fibrotic complications. Altogether, our results provide a roadmap for understanding cell-type- and organ-specific differences in CD and potential directions for therapeutic development.

Dermal αSMA+ myofibroblasts orchestrate skin wound repair via ß1 integrin and independent of type I collagen production.

Skin wound repair is essential for organismal survival and failure of which leads to non-healing wounds, a leading health issue worldwide. However, mechanistic understanding of chronic wounds remains a major challenge due to lack of appropriate genetic mouse models. αSMA+ myofibroblasts, a unique class of dermal fibroblasts, are associated with cutaneous wound healing but their precise function remains unknown. We demonstrate that genetic depletion of αSMA+ myofibroblasts leads to pleiotropic wound healing defects, including lack of reepithelialization and granulation, dampened angiogenesis, and heightened hypoxia, hallmarks of chronic non-healing wounds. Other wound-associated FAP+ and FSP1+ fibroblasts do not exhibit such dominant functions. While type I collagen (COL1) expressing cells play a role in the repair process, COL1 produced by αSMA+ myofibroblasts is surprisingly dispensable for wound repair. In contrast, we show that ß1 integrin from αSMA+ myofibroblasts, but not TGFßRII, is essential for wound healing, facilitating contractility, reepithelization, and vascularization. Collectively, our study provides evidence for the functions of myofibroblasts in ß1 integrin-mediated wound repair with potential implications for treating chronic non-healing wounds.

mTOR signalling controls the formation of smooth muscle cell-derived luminal myofibroblasts during vasculitis.

The accumulation of myofibroblasts within the intimal layer of inflamed blood vessels is a potentially catastrophic complication of vasculitis, which can lead to arterial stenosis and ischaemia. In this study, we have investigated how these luminal myofibroblasts develop during Kawasaki disease (KD), a paediatric vasculitis typically involving the coronary arteries. By performing lineage tracing studies in a murine model of KD, we reveal that luminal myofibroblasts develop independently of adventitial fibroblasts and endothelial cells, and instead derive from smooth muscle cells (SMCs). Notably, the emergence of SMC-derived luminal myofibroblasts-in both mice and patients with KD, Takayasu's arteritis and Giant Cell arteritis-coincided with activation of the mechanistic target of rapamycin (mTOR) signalling pathway. Moreover, SMC-specific deletion of mTOR signalling, or pharmacological inhibition, abrogated the emergence of luminal myofibroblasts. Thus, mTOR is an intrinsic and essential regulator of luminal myofibroblast formation that is activated in vasculitis patients and therapeutically tractable. These findings provide molecular insight into the pathogenesis of coronary artery stenosis and identify mTOR as a therapeutic target in vasculitis.

Fibrosis in Pathology of Heart and Kidney: From Deep RNA-Sequencing to Novel Molecular Targets.

Diseases of the heart and the kidney, including heart failure and chronic kidney disease, can dramatically impair life expectancy and the quality of life of patients. The heart and kidney form a functional axis; therefore, functional impairment of 1 organ will inevitably affect the function of the other. Fibrosis represents the common final pathway of diseases of both organs, regardless of the disease entity. Thus, inhibition of fibrosis represents a promising therapeutic approach to treat diseases of both organs and to resolve functional impairment. However, despite the growing knowledge in this field, the exact pathomechanisms that drive fibrosis remain elusive. RNA-sequencing approaches, particularly single-cell RNA-sequencing, have revolutionized the investigation of pathomechanisms at a molecular level and facilitated the discovery of disease-associated cell types and mechanisms. In this review, we give a brief overview over the evolution of RNA-sequencing techniques, summarize most recent insights into the pathogenesis of heart and kidney fibrosis, and discuss how transcriptomic data can be used, to identify new drug targets and to develop novel therapeutic strategies.

Multi-organ phenotypes in mice lacking latent TGFß binding protein 2 (LTBP2).

BACKGROUND: Latent TGFß binding protein-2 (LTBP2) is a fibrillin 1 binding component of the microfibril. LTBP2 is the only LTBP protein that does not bind any isoforms of TGFß, although it may interfere with the function of other LTBPs or interact with other signaling pathways. RESULTS: Here, we investigate mice lacking Ltbp2 (Ltbp2-/- ) and identify multiple phenotypes that impact bodyweight and fat mass, and affect bone and skin development. The alterations in skin and bone development are particularly noteworthy since the strength of these tissues is differentially affected by loss of Ltbp2. Interestingly, some tissues that express high levels of Ltbp2, such as the aorta and lung, do not have a developmental or homeostatic phenotype. CONCLUSIONS: Analysis of these mice show that LTBP2 has complex effects on development through direct effects on the extracellular matrix (ECM) or on signaling pathways that are known to regulate the ECM.

Ventral body wall closure: Mechanistic insights from mouse models and translation to human pathology.

The ventral body wall (VBW) that encloses the thoracic and abdominal cavities arises by extensive cell movements and morphogenetic changes during embryonic development. These morphogenetic processes include embryonic folding generating the primary body wall; the initial ventral cover of the embryo, followed by directed mesodermal cell migrations, contributing to the secondary body wall. Clinical anomalies in VBW development affect approximately 1 in 3000 live births. However, the cell interactions and critical cellular behaviors that control VBW development remain little understood. Here, we describe the embryonic origins of the VBW, the cellular and morphogenetic processes, and key genes, that are essential for VBW development. We also provide a clinical overview of VBW anomalies, together with environmental and genetic influences, and discuss the insight gained from over 70 mouse models that exhibit VBW defects, and their relevance, with respect to human pathology. In doing so we propose a phenotypic framework for researchers in the field which takes into account the clinical picture. We also highlight cases where there is a current paucity of mouse models for particular clinical defects and key gaps in knowledge about embryonic VBW development that need to be addressed to further understand mechanisms of human VBW pathologies.

Novel mechanisms and clinical trial endpoints in intestinal fibrosis.

The incidence of inflammatory bowel diseases (IBD) worldwide has resulted in a global public health challenge. Intestinal fibrosis leading to stricture formation and bowel obstruction is a frequent complication in Crohn's disease (CD), and the lack of anti-fibrotic therapies makes elucidation of fibrosis mechanisms a priority. Progress has shown that mesenchymal cells, cytokines, microbial products, and mesenteric adipocytes are jointly implicated in the pathogenesis of intestinal fibrosis. This recent information puts prevention or reversal of intestinal strictures within reach through innovative therapies validated by reliable clinical trial endpoints. Here, we review the role of immune and non-immune components of the pathogenesis of intestinal fibrosis, including new cell clusters, cytokine networks, host-microbiome interactions, creeping fat, and their translation for endpoint development in anti-fibrotic clinical trials.

Fibrosis in IBD: from pathogenesis to therapeutic targets.

BACKGROUND: Intestinal fibrosis resulting in stricture formation and obstruction in Crohn's disease (CD) and increased wall stiffness leading to symptoms in ulcerative colitis (UC) is among the largest unmet needs in inflammatory bowel disease (IBD). Fibrosis is caused by a multifactorial and complex process involving immune and non-immune cells, their soluble mediators and exposure to luminal contents, such as microbiota and environmental factors. To date, no antifibrotic therapy is available. Some progress has been made in creating consensus definitions and measurements to quantify stricture morphology for clinical practice and trials, but approaches to determine the degree of fibrosis within a stricture are still lacking. OBJECTIVE: We herein describe the current state of stricture pathogenesis, measuring tools and clinical trial endpoints development. DESIGN: Data presented and discussed in this review derive from the past and recent literature and the authors' own research and experience. RESULTS AND CONCLUSIONS: Significant progress has been made in better understanding the pathogenesis of fibrosis, but additional studies and preclinical developments are needed to define specific therapeutic targets.

Nicotinamide N-methyltransferase mediates lipofibroblast-myofibroblast transition and apoptosis resistance.

Metabolism controls cellular phenotype and fate. In this report, we demonstrate that nicotinamide N-methyltransferase (NNMT), a metabolic enzyme that regulates developmental stem cell transitions and tumor progression, is highly expressed in human idiopathic pulmonary fibrosis (IPF) lungs, and is induced by the pro-fibrotic cytokine, transforming growth factor-ß1 (TGF-ß1) in lung fibroblasts. NNMT silencing reduces the expression of extracellular matrix proteins, both constitutively and in response to TGF-ß1. Furthermore, NNMT controls the phenotypic transition from homeostatic, pro-regenerative lipofibroblasts to pro-fibrotic myofibroblasts. This effect of NNMT is mediated, in part, by the downregulation of lipogenic transcription factors, TCF21 and PPARγ, and the induction of a less proliferative but more differentiated myofibroblast phenotype. NNMT confers an apoptosis-resistant phenotype to myofibroblasts that is associated with the downregulation of pro-apoptotic members of the Bcl-2 family, including Bim and PUMA. Together, these studies indicate a critical role for NNMT in the metabolic reprogramming of fibroblasts to a pro-fibrotic and apoptosis-resistant phenotype and support the concept that targeting this enzyme may promote regenerative responses in chronic fibrotic disorders such as IPF.

MEOX1 triggers myofibroblast apoptosis resistance, contributing to pulmonary fibrosis in mice.

The apoptosis resistance of myofibroblasts is a hallmark in the irreversible progression of pulmonary fibrosis (PF). While the underlying molecular mechanism remains elusive. In this study, we unveiled a previously unrecognized mechanism underlying myofibroblast apoptosis resistance during PF. Our investigation revealed heightened expression of mesenchyme homeobox 1 (MEOX1) in the lungs of idiopathic pulmonary fibrosis (IPF) patients and bleomycin-induced PF mice. Silencing MEOX1 significantly attenuated PF progression in mice. In vitro, we found a notable increase in MEOX1 expression in transforming growth factor-ß1 (TGF-ß1)-induced myofibroblasts. Silencing MEOX1 enhanced apoptosis of myofibroblasts. Mechanistically, we identified G-protein signaling pathway regulatory factor 4 (RGS4) as a critical downstream target of MEOX1, as predicted by bioinformatics analysis. MEOX1 enhanced apoptosis resistance by upregulating RGS4 expression in myofibroblasts. In conclusion, our study highlights MEOX1 as a promising therapeutic target for protecting against PF by modulating myofibroblast apoptosis resistance.

Continuous genetic monitoring of transient mesenchymal gene activities in distal tubule and collecting duct epithelial cells during renal fibrosis.

Epithelial cells (ECs) have been proposed to contribute to myofibroblasts or fibroblasts through epithelial-mesenchymal transition (EMT) during renal fibrosis. However, since EMT may occur dynamically, transiently, and reversibly during kidney fibrosis, conventional lineage tracing based on Cre-loxP recombination in renal ECs could hardly capture the transient EMT activity, yielding inconsistent results. Moreover, previous EMT research has primarily focused on renal proximal tubule ECs, with few reports of distal tubules and collecting ducts. Here, we generated dual recombinases-mediated genetic lineage tracing systems for continuous monitoring of transient mesenchymal gene expression in E-cadherin+ and EpCAM+ ECs of distal tubules and collecting ducts during renal fibrosis. Activation of key EMT-inducing transcription factor (EMT-TF) Zeb1 and mesenchymal markers αSMA, vimentin, and N-cadherin, were investigated following unilateral ureteral obstruction (UUO). Our data revealed that E-cadherin+ and EpCAM+ ECs did not transdifferentiate into myofibroblasts, nor transiently expressed these mesenchymal genes during renal fibrosis. In contrast, in vitro a large amount of cultured renal ECs upregulated mesenchymal genes in response to TGF-ß, a major inducer of EMT.

Th17 Cell-Derived Amphiregulin Promotes Colitis-Associated Intestinal Fibrosis Through Activation of mTOR and MEK in Intestinal Myofibroblasts.

BACKGROUND & AIMS: Intestinal fibrosis is a significant complication of Crohn's disease (CD). Gut microbiota reactive Th17 cells are crucial in the pathogenesis of CD; however, how Th17 cells induce intestinal fibrosis is still not completely understood. METHODS: In this study, T-cell transfer model with wild-type (WT) and Areg-/- Th17 cells and dextran sulfate sodium (DSS)-induced chronic colitis model in WT and Areg-/- mice were used. CD4+ T-cell expression of AREG was determined by quantitative reverse-transcriptase polymerase chain reaction and enzyme-linked immunosorbent assay. The effect of AREG on proliferation/migration/collagen expression in human intestinal myofibroblasts was determined. AREG expression was assessed in healthy controls and patients with CD with or without intestinal fibrosis. RESULTS: Although Th1 and Th17 cells induced intestinal inflammation at similar levels when transferred into Tcrßxδ-/- mice, Th17 cells induced more severe intestinal fibrosis. Th17 cells expressed higher levels of AREG than Th1 cells. Areg-/- mice developed less severe intestinal fibrosis compared with WT mice on DSS insults. Transfer of Areg-/- Th17 cells induced less severe fibrosis in Tcrßxδ-/- mice compared with WT Th17 cells. Interleukin (IL)6 and IL21 promoted AREG expression in Th17 cells by activating Stat3. Stat3 inhibitor suppressed Th17-induced intestinal fibrosis. AREG promoted human intestinal myofibroblast proliferation, motility, and collagen I expression, which was mediated by activating mammalian target of rapamycin and MEK. AREG expression was increased in intestinal CD4+ T cells in fibrotic sites compared with nonfibrotic sites from patients with CD. CONCLUSIONS: These findings reveal that Th17-derived AREG promotes intestinal fibrotic responses in experimental colitis and human patients with CD. Thereby, AREG might serve as a potential therapeutic target for fibrosis in CD.

Neointimal myofibroblasts contribute to maintaining Th1/Tc1 and Th17/Tc17 inflammation in giant cell arteritis.

Vascular smooth muscle cells (VSMCs) have been shown to play a role in the pathogenesis of giant cell arteritis (GCA) through their capacity to produce chemokines recruiting T cells and monocytes in the arterial wall and their ability to migrate and proliferate in the neointima where they acquire a myofibroblast (MF) phenotype, leading to vascular stenosis. This study aimed to investigate if MFs could also impact T-cell polarization. Confocal microscopy was used to analyze fresh fragments of temporal artery biopsies (TABs). Healthy TAB sections were cultured to obtain MFs, which were then treated or not with interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) and analyzed by immunofluorescence and RT-PCR. After peripheral blood mononuclear cells and MFs were co-cultured for seven days, T-cell polarization was analyzed by flow cytometry. In the neointima of GCA arteries, we observed a phenotypic heterogeneity among VSMCs that was consistent with a MF phenotype (α-SMA+CD90+desmin+MYH11+) with a high level of STAT1 phosphorylation. Co-culture experiments showed that MFs sustain Th1/Tc1 and Th17/Tc17 polarizations. The increased Th1 and Tc1 polarization was further enhanced following the stimulation of MFs with IFN-γ and TNF-α, which induced STAT1 phosphorylation in MFs. These findings correlated with increases in the production of IL-1ß, IL-6, IL-12 and IL-23 by MFs. Our study showed that MFs play an additional role in the pathogenesis of GCA through their ability to maintain Th17/Tc17 and Th1/Tc1 polarizations, the latter being further enhanced in case of stimulation of MF with IFN-γ and TNF-α.

Endometriotic tissue fragments are viable after cryopreservation in an ex vivo tissue model recapitulating the fibrotic microenvironment.

STUDY QUESTION: Is it possible to establish an ex vivo endometriosis model using cryopreserved endometriotic tissue fragments? SUMMARY ANSWER: Cryopreserved endometriotic tissue fragments remain viable after thawing and during at least 3 days of culture and can therefore be used to establish an ex vivo endometriosis model to efficiently test potential therapeutic agents. WHAT IS KNOWN ALREADY: Endometriosis is the most prevalent benign gynecologic disease with an enormous societal burden; however, curative therapies are still lacking. To efficiently test potential new therapies, an ex vivo model based on previously cryopreserved endometriotic tissue that recapitulates the different endometriosis subtypes and their microenvironment is highly desirable. STUDY DESIGN, SIZE, DURATION: Endometriotic tissue fragments of three different subtypes were obtained from 28 patients by surgical resection. After cryopreservation and thawing, viability and metabolic activity of these tissue fragments were assessed. Viability was compared with fresh fragments from 11 patients directly after surgical removal. Experimental intervention studies were performed in cryopreserved and thawed tissue fragments from two patients to confirm the usability of these tissues for ex vivo intervention studies. PARTICIPANTS/MATERIALS, SETTING, METHODS: Endometriotic tissue fragments (n = 45) were cryopreserved according to three different protocols. After thawing, fragments were cultured for 24 h. A resazurin-based assay was performed to assess the metabolic activity of the tissue fragments. In addition, cell type-specific viability was analyzed by VivaFix, Hoechst 33342, and α-smooth muscle actin immunofluorescence staining and confocal microscopy. The presence of endometriosis was histologically confirmed based on hematoxylin-eosin staining. Cryopreserved and thawed tissue fragments were treated for 72 h with pirfenidone or metformin and COL1A1 and CEMIP gene expressions were assessed using RT-PCR and RT-qPCR, either in the whole tissue fragments or in myofibroblasts isolated by laser capture microdissection. MAIN RESULTS AND THE ROLE OF CHANCE: Metabolic activity of endometriotic tissue fragments obtained from peritoneal (PER), ovarian (OMA), and deep (DE) endometriotic lesions was well preserved after cryopreservation in a dimethyl sulfoxide-based medium and was comparable with fresh tissue fragments. Relative metabolic activity compared to fresh tissue was 70% (CI: 92-47%) in PER, 43% (CI: 53-15%) in OMA and 94% (CI: 186-3%) in DE lesions. In fragments from PE lesions 92% (CI: 87-96%), from OMA lesions 95% (CI: 91-98%), and from DE lesions 88% (CI: 78-98%) of cells were viable after cryopreservation and thawing followed by a 24-h culture period. Differences in gene expression of fibrotic markers COL1A1 and CEMIP after 72-h treatment with pirfenidone or metformin could be detected in whole tissue fragments and in isolated myofibroblasts, indicating that cryopreserved and thawed endometriotic tissue fragments are suitable for testing anti-fibrotic interventions. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Viability and metabolic activity of the endometriotic tissue fragments may have been partially compromised by damage sustained during the surgical procedure, contributing to inter-sample variance. WIDER IMPLICATIONS OF THE FINDINGS: The storage of viable endometriotic tissue fragments for later usage in an ex vivo model creates the possibility to efficiently test potential new therapeutic strategies and facilitates the exchange of viable endometriotic tissue between different research laboratories. STUDY FUNDING/COMPETING INTEREST(S): This study was not financially supported by external funding. The authors declare no competing interest. TRIAL REGISTRATION NUMBER: N/A.

Preclinical targeting of liver fibrosis with a 89Zr-labeled Fibrobody® directed against platelet derived growth factor receptor-ß.

PURPOSE: Hepatic fibrosis develops as a response to chronic liver injury, resulting in the formation of fibrous scars. This process is initiated and driven by collagen-producing activated myofibroblasts which reportedly express high levels of platelet derived growth factor receptor-ß (PDGFRß). We therefore regard PDGFRß as an anchor for diagnosis and therapy. The Fibrobody® SP02SP26-ABD is a biparatopic VHH-construct targeting PDGFRß. Here, we explore its potential as a theranostic vector for liver fibrosis. METHODS: Specificity, cross-species binding, and cellular uptake of SP02SP26-ABD was assessed using human, mouse and rat PDGFRß ectodomains and PDGFRß-expressing cells. Cellular uptake by PDGFRß-expressing cells was also evaluated by equipping the Fibrobody® with auristatinF and reading out in vitro cytotoxicity. The validity of PDGFRß as a marker for active fibrosis was confirmed in human liver samples and 3 mouse models of liver fibrosis (DDC, CCl4, CDA-HFD) through immunohistochemistry and RT-PCR. After radiolabeling of DFO*-SP02SP26-ABD with 89Zr, its in vivo targeting ability was assessed in healthy mice and mice with liver fibrosis by PET-CT imaging, ex vivo biodistribution and autoradiography. RESULTS: SP02SP26-ABD shows similar nanomolar affinity for human, mouse and rat PDGFRß. Cellular uptake and hence subnanomolar cytotoxic potency of auristatinF-conjugated SP02SP26-ABD was observed in PDGFRß-expressing cell lines. Immunohistochemistry of mouse and human fibrotic livers confirmed co-localization of PDGFRß with markers of active fibrosis. In all three liver fibrosis models, PET-CT imaging and biodistribution analysis of [89Zr]Zr-SP02SP26-ABD revealed increased PDGFRß-specific uptake in fibrotic livers. In the DDC model, liver uptake was 12.15 ± 0.45, 15.07 ± 0.90, 20.23 ± 1.34, and 20.93 ± 4.35%ID/g after 1,2,3 and 4 weeks of fibrogenesis, respectively, compared to 7.56 ± 0.85%ID/g in healthy mice. Autoradiography revealed preferential uptake in the fibrotic (PDGFRß-expressing) periportal areas. CONCLUSION: The anti-PDGFRß Fibrobody® SP02SP26-ABD shows selective and high-degree targeting of activated myofibroblasts in liver fibrosis, and qualifies as a vector for diagnostic and therapeutic purposes.

Two-phase mechanism in the treatment of corneal stromal fibrosis with topical losartan.

Recent studies in rabbits and case reports in humans have demonstrated the efficacy of topical losartan in the treatment of corneal scarring fibrosis after a wide range of injuries, including chemical burns, infections, surgical complications, and some diseases. It is hypothesized that the effect of losartan on the fibrotic corneal stroma occurs through a two-phase process in which losartan first triggers the elimination of myofibroblasts by directing their apoptosis via inhibition of extracellular signal-regulated kinase (ERK)-mediated signal transduction, and possibly through signaling effects on the viability and development of corneal fibroblast and fibrocyte myofibroblast precursor cells. This first step likely occurs within a week or two in most corneas with fibrosis treated with topical losartan, but the medication must be continued for much longer until the epithelial basement membrane (EBM) is fully regenerated or new myofibroblasts will develop from precursor cells. Once the myofibroblasts are eliminated from the fibrotic stroma, corneal fibroblasts can migrate into the fibrotic tissue and reabsorb/reorganize the disordered extracellular matrix (ECM) previously produced by the myofibroblasts. This second stage is longer and more variable in different eyes of rabbits and humans, and accounts for most of the variability in the time it takes for the stromal opacity to be markedly reduced by topical losartan treatment. Eventually, keratocytes reemerge in the previously fibrotic stromal tissue to fine-tune the collagens and other ECM components and maintain the normal structure of the corneal stroma. The efficacy of losartan in the prevention and treatment of corneal fibrosis suggests that it acts as a surrogate for the EBM, by suppressing TGF beta-directed scarring of the wounded corneal stroma, until control over TGF beta action is re-established by a healed EBM, while also supporting regeneration of the EBM by allowing corneal fibroblasts to occupy the subepithelial stroma in the place of myofibroblasts.

Corneal stromal localization of TGF beta isoforms in spontaneous persistent epithelial defects after PRK in rabbits.

The purpose of this study was to evaluate transforming growth factor beta (TGFß) isoform localization in rabbit corneas with spontaneous persistent epithelial defects (PEDs) after photorefractive keratectomy (PRK). Four cryofixed corneas from a previously reported series of PEDs in rabbits that had PRK were evaluated with triplex immunohistochemistry (IHC) for TGFß3, myofibroblast marker alpha-smooth muscle actin (α-SMA) and mesenchymal marker vimentin. One cornea had sufficient remaining tissue for triplex IHC for TGFß1, TGFß2, or TGFß3 (each with α-SMA and vimentin) using isoform-specific antibodies. All three TGFß isoforms were detected in the subepithelial stroma at and surrounding the PED. Some of each TGFß isoform co-localized with α-SMA of myofibroblasts, which could be TGFß isoform autocrine production by myofibroblasts or TGFß-1, -2, and -3 binding to these myofibroblasts.

Lnc PVT1 facilitates TGF-ß1-induced human cardiac fibroblast activation in vitro and ISO-induced myocardial fibrosis in vivo through regulating MYC.

Cardiac fibrosis is a commonly seen pathophysiological process in various cardiovascular disorders, such as coronary heart disorder, hypertension, and cardiomyopathy. Cardiac fibroblast trans-differentiation into myofibroblasts (MFs) is a key link in myocardial fibrosis. LncRNA PVT1 participates in fibrotic diseases in multiple organs; however, its role and mechanism in cardiac fibrosis remain largely unknown. Human cardiac fibroblasts (HCFs) were stimulated with TGF-ß1 to induce myofibroblast; Immunofluorescent staining, Immunoblotting, and fluorescence in situ hybridization were used to detect the myofibroblasts phenotypes and lnc PVT1 expression. Cell biological phenotypes induced by lnc PVT1 knockdown or overexpression were detected by CCK-8, flow cytometry, and Immunoblotting. A mouse model of myocardial fibrosis was induced using isoproterenol (ISO), and the cardiac functions were examined by echocardiography measurements, cardiac tissues by H&E, and Masson trichrome staining. In this study, TGF-ß1 induced HCF transformation into myofibroblasts, as manifested as significantly increased levels of α-SMA, vimentin, collagen I, and collagen III; the expression level of lnc PVT1 expression showed to be significantly increased by TGF-ß1 stimulation. The protein levels of TGF-ß1, TGFBR1, and TGFBR2 were also decreased by lnc PVT1 knockdown. Under TGF-ß1 stimulation, lnc PVT1 knockdown decreased FN1, α-SMA, collagen I, and collagen III protein contents, inhibited HCF cell viability and enhanced cell apoptosis, and inhibited Smad2/3 phosphorylation. Lnc PVT1 positively regulated MYC expression with or without TGF-ß1 stimulation; MYC overexpression in TGF-ß1-stimulated HCFs significantly attenuated the effects of lnc PVT1 knockdown on HCF proliferation and trans-differentiation to MFs. In the ISO-induced myocardial fibrosis model, lnc PVT1 knockdown partially reduced fibrotic area, improved cardiac functions, and decreased the levels of fibrotic markers. In addition, lnc PVT1 knockdown decreased MYC and CDK4 levels but increased E-cadherin in mice heart tissues. lnc PVT1 is up-regulated in cardiac fibrosis and TGF-ß1-stimulated HCFs. Lnc PVT1 knockdown partially ameliorates TGF-ß1-induced HCF activation and trans-differentiation into MFs in vitro and ISO-induced myocardial fibrosis in vivo, potentially through interacting with MYC and up-regulating MYC.

Fibroblast GSK-3α Promotes Fibrosis via RAF-MEK-ERK Pathway in the Injured Heart.

BACKGROUND: Heart failure is the leading cause of mortality, morbidity, and health care expenditures worldwide. Numerous studies have implicated GSK-3 (glycogen synthase kinase-3) as a promising therapeutic target for cardiovascular diseases. GSK-3 isoforms seem to play overlapping, unique and even opposing functions in the heart. Previously, we have shown that of the 2 isoforms of GSK-3, cardiac fibroblast GSK-3ß acts as a negative regulator of myocardial fibrosis in the ischemic heart. However, the role of cardiac fibroblast-GSK-3α in the pathogenesis of cardiac diseases is completely unknown. METHODS: To define the role of cardiac fibroblast-GSK-3α in myocardial fibrosis and heart failure, GSK-3α was deleted from fibroblasts or myofibroblasts with tamoxifen-inducible Tcf21- or Postn-promoter-driven Cre recombinase. Control and GSK-3α KO mice were subjected to cardiac injury and heart parameters were evaluated. The fibroblast kinome mapping was carried out to delineate molecular mechanism followed by in vivo and in vitro analysis. RESULTS: Fibroblast-specific GSK-3α deletion restricted fibrotic remodeling and preserved function of the injured heart. We observed reductions in cell migration, collagen gel contraction, α-SMA protein levels, and expression of ECM genes in TGFß1-treated KO fibroblasts, indicating that GSK-3α is required for myofibroblast transformation. Surprisingly, GSK-3α deletion did not affect SMAD3 activation, suggesting the profibrotic role of GSK-3α is SMAD3 independent. The molecular studies confirmed decreased ERK signaling in GSK-3α-KO CFs. Conversely, adenovirus-mediated expression of a constitutively active form of GSK-3α (Ad-GSK-3αS21A) in fibroblasts increased ERK activation and expression of fibrogenic proteins. Importantly, this effect was abolished by ERK inhibition. CONCLUSIONS: GSK-3α-mediated MEK-ERK activation is a critical profibrotic signaling circuit in the injured heart, which operates independently of the canonical TGF-ß1-SMAD3 pathway. Therefore, strategies to inhibit the GSK-3α-MEK-ERK signaling circuit could prevent adverse fibrosis in diseased hearts.