Myofibroblasts Research Articles

αvβ3 Integrin as a Link between the Development of Fibrosis and Thyroid Hormones in Systemic Sclerosis.

Systemic sclerosis (SSc) is an autoimmune disease characterized by fibrosis of the skin and internal organs. Key players mediating fibrosis are myofibroblasts (MF) that, following transforming growth factor β (TGFβ) exposure, produce a collagen-rich extracellular matrix (ECM) that induces myofibroblast differentiation. Myofibroblasts express αvβ3 integrin (a membrane receptor for thyroid hormones) and miRNA-21 that promotes deiodinase-type-3 expression (D3), causing the degradation of triiodothyronine (T3) that attenuates fibrosis. We hypothesized that αvβ3 affects the fibrotic processes through its thyroid hormones (THs) binding site. To test this, dermal fibroblasts (DF) were cultured with/without TGFβ and removed with a base, leaving only normal/fibrotic ECMs in wells. Then, DF were cultured on the ECMs with/without tetrac (αvβ3 ligand, T4 antagonist), and evaluated for pro-fibrotic characteristics, αvβ3, miRNA-21, and D3 levels. Blood free-T3 (fT3), miRNA-21 levels, and the modified Rodnan skin score (MRSS) were evaluated in SSc patients. We found that the "fibrotic-ECM" significantly increased the pro-fibrotic characteristics of DF and the levels of miRNA-21, D3, and αvβ3, compared to the "normal-ECM." Tetrac significantly inhibited the effects of the "fibrotic-ECM" on the cells. In accordance with tetrac's effect on D3/miRNA-21, a negative correlation was found between the patients' fT3 to miRNA-21 levels, and to the development of pulmonary arterial hypertension (PAH). We conclude that occupying the THs binding site of αvβ3 may delay the development of fibrosis.

Myocardial fibrosis and calcification are attenuated by microRNA-129-5p targeting Asporin and Sox9 in cardiac fibroblasts.

Myocardial fibrosis and calcification associate with adverse outcomes in nonischemic heart failure. Cardiac fibroblasts (CF) transition into myofibroblasts (MF) and osteogenic fibroblasts (OF) to promote myocardial fibrosis and calcification. However, common upstream mechanisms regulating both CF-to-MF transition and CF-to-OF transition remain unknown. microRNAs are promising targets to modulate CF plasticity. Our bioinformatics revealed downregulation of miR-129-5p and upregulation of its targets small leucine-rich proteoglycan Asporin (ASPN) and transcription factor SOX9 as common in mouse and human heart failure (HF). We experimentally confirmed decreased miR-129-5p and enhanced SOX9 and ASPN expression in CF in human hearts with myocardial fibrosis and calcification. miR-129-5p repressed both CF-to-MF and CF-to-OF transition in primary CF, as did knockdown of SOX9 and ASPN. Sox9 and Aspn are direct targets of miR-129-5p that inhibit downstream β-catenin expression. Chronic Angiotensin II infusion downregulated miR-129-5p in CF in WT and TCF21-lineage CF reporter mice, and it was restored by miR-129-5p mimic. Importantly, miR-129-5p mimic not only attenuated progression of myocardial fibrosis, calcification marker expression, and SOX9 and ASPN expression in CF but also restored diastolic and systolic function. Together, we demonstrate miR-129-5p/ASPN and miR-129-5p/SOX9 as potentially novel dysregulated axes in CF-to-MF and CF-to-OF transition in myocardial fibrosis and calcification and the therapeutic relevance of miR-129-5p.

Progress on role of ion channels of cardiac fibroblasts in fibrosis.

Cardiac fibrosis is defined as excessive deposition of extracellular matrix (ECM) in pathological conditions. Cardiac fibroblasts (CFs) activated by injury or inflammation differentiate into myofibroblasts (MFs) with secretory and contractile functions. In the fibrotic heart, MFs produce ECM which is composed mainly of collagen and is initially involved in maintaining tissue integrity. However, persistent fibrosis disrupts the coordination of excitatory contractile coupling, leading to systolic and diastolic dysfunction, and ultimately heart failure. Numerous studies have demonstrated that both voltage- and non-voltage-gated ion channels alter intracellular ion levels and cellular activity, contributing to myofibroblast proliferation, contraction, and secretory function. However, an effective treatment strategy for myocardial fibrosis has not been established. Therefore, this review describes the progress made in research related to transient receptor potential (TRP) channels, Piezo1, Ca2+ release-activated Ca2+ (CRAC) channels, voltage-gated Ca2+ channels (VGCCs), sodium channels, and potassium channels in myocardial fibroblasts with the aim of providing new ideas for treating myocardial fibrosis.

Spatiotemporal regulation of cholangiocarcinoma growth and dissemination by peritumoral myofibroblasts in a Vcam1-dependent manner

Intrahepatic cholangiocarcinoma (iCCA) is characterized by its highly desmoplastic stroma. Myofibroblasts (MFs) are present both within the tumor mass (intratumoral MFs, iMFs) and at the tumor border (peritumoral MFs, pMFs). Using a spheroid-based coculture system, we show that the initial iCCA-pMF contact is growth suppressive to the tumor cells. However, prolonged iCCA-pMF interaction elicits significant tumor cell invasion and dissemination. We find that vascular cell adhesion molecule-1 (Vcam1) level is elevated in tumor cells in contact with pMFs but low in disseminated tumor cells both in vitro and in vivo. A gene regulatory network analysis of mouse and patient iCCA tumors and Vcam1 knockout (Vcam1KO) demonstrate a heavy involvement of Vcam1 in epithelial-to-mesenchymal transition. While Vcam1KO has only a limited impact on tumor cell growth in their monoculture, Vcam1KO spheroids exhibit instant dissemination and a severe growth defect when cocultured with pMFs. When transplanted into the liver, Vcam1KO iCCA cells show a similar increase in dissemination but a significant defect in establishing primary and metastatic tumors. Incomplete blocking of Vcam1 in vivo reduces the size but increase the number of metastatic lesions. Overall, our study shows a spatiotemporal regulation of iCCA growth and dissemination by pMFs in a Vcam1-dependent manner.

Targeting YAP-mediated HSC death susceptibility and senescence for treatment of liver fibrosis.

Liver fibrosis results from the accumulation of myofibroblasts (MFs) derived from quiescent HSCs, and yes-associated protein (YAP) controls this state transition. Although fibrosis is also influenced by HSC death and senescence, whether YAP regulates these processes and whether this could be leveraged to treat liver fibrosis are unknown. YAP activity was manipulated in MF-HSCs to determine how YAP impacts susceptibility to pro-apoptotic senolytic agents or ferroptosis. Effects of senescence on YAP activity and susceptibility to apoptosis versus ferroptosis were also examined. CCl 4 -treated mice were treated with a ferroptosis inducer or pro-apoptotic senolytic to determine the effects on liver fibrosis. YAP was conditionally disrupted in MFs to determine how YAP activity in MF-HSC affects liver fibrosis in mouse models. Silencing YAP in cultured MF-HSCs induced HSC senescence and vulnerability to senolytics, and promoted ferroptosis resistance. Conversely, inducing HSC senescence suppressed YAP activity, increased sensitivity to senolytics, and decreased sensitivity to ferroptosis. Single-cell analysis of HSCs from fibrotic livers revealed heterogeneous sensitivity to ferroptosis, apoptosis, and senescence. In mice with chronic liver injury, neither the ferroptosis inducer nor senolytic improved fibrosis. However, selectively depleting YAP in MF-HSCs induced senescence and decreased liver injury and fibrosis. YAP determines whether MF-HSCs remain activated or become senescent. By regulating this state transition, Yap controls both HSC fibrogenic activity and susceptibility to distinct mechanisms for cell death. MF-HSC-specific YAP depletion induces senescence and protects injured livers from fibrosis. Clarifying determinants of HSC YAP activity may facilitate the development of novel anti-fibrotic therapies.

Pathomechanism of Liver Fibrosis and Mesenchymal Stem Cells in its Resolution Process

Liver fibrosis is a disease process that, without adequate treatment, can lead to liver failure and can be life-threatening. This disease is reversible and appropriate therapy can prevent further liver damage. Liver transplant therapy is the only treatment for an end-stage liver disease that works, but it has various obstacles and limitations in its implementation. Therefore, nowadays, mesenchymal stem cells (MSCs) have become a hope of therapy for liver fibrosis. Our literature review describes the pathomechanism of liver fibrosis and the steps of its resolution, accompanied by the possible role of MSCs in supporting the process. The activation of several complex pathways regulates liver fibrosis, and its resolution, involving Transforming Growth Factor (TGF)-β, signal transducer and activator of transcription-3, and Wnt/β-catenin signaling is involved in Hepatic Stellate Cells (HSCs) activation, which are precursors of myofibroblasts (MFs) and causes fibrosis. The presence of the High-mobility group box-1 pathway, which also induces the production of proinflammatory cytokines and the role of matrix metalloproteinases (MMPs)/tissue Inhibitors of MMPs s and Syndecan-1, is incorporated into the extracellular matrix (ECM). In repairing liver damage, four steps of liver fibrosis resolution are required, such as preventing further damage, restoring the intrahepatic balance of inflammation, removing and inactivating MFs, and ECM degradation associated with arresting the eight pathways of the fibrosis mechanism. MSCs can help resolve liver fibrosis and speed up wound healing, increase hepatocyte survival, and suppress HSCs activation by blocking fibrosis mechanism pathways such as TGF-β and pro-inflammatory factors such as tumor necrosis factor-alpha, interferon-gamma, IL-6, IL-17, and IL-23, in addition to an elevated level of an anti-inflammatory factor like IL-10.

Neuron-Glial Antigen 2 Participates in Liver Fibrosis via Regulating the Differentiation of Bone Marrow Mesenchymal Stem Cell to Myofibroblast

Neuron-glial antigen 2 (NG2, gene name: Cspg4) has been characterized as an important factor in many diseases. However, the pathophysiological relevance of NG2 in liver disease specifically regarding bone marrow mesenchymal stem cell (BMSC) differentiation to myofibroblast (MF) and the molecular details remain unknown. Human liver tissues were obtained from patients with different chronic liver diseases, and mouse liver injury models were induced by feeding a methionine-choline-deficient and high-fat diet, carbon tetrachloride administration, or bile duct ligation operation. NG2 expression was increased in human and mouse fibrotic liver and positively correlated with MF markers α-smooth muscle actin (αSMA) and other fibrotic markers in the liver. There was a co-localization between NG2 and αSMA, NG2 and EGFP (BMSC-derived MF) in the fibrotic liver determined by immunofluorescence analysis. In vitro, TGFβ1-treated BMSC showed a progressive increase in NG2 levels, which were mainly expressed on the membrane surface. Interestingly, there was a translocation of NG2 from the cell membrane into cytoplasm after the transfection of Cspg4 siRNA in TGFβ1-treated BMSC. siRNA-mediated inhibition of Cspg4 abrogated the TGFβ1-induced BMSC differentiation to MF. Importantly, inhibition of NG2 in vivo significantly attenuated the extent of liver fibrosis in methionine-choline-deficient and high fat (MCDHF) mice, as demonstrated by the decreased mRNA expression of fibrotic parameters, collagen deposition, serum transaminase levels, liver steatosis and inflammation after the administration of Cspg4 siRNA in MCDHF mice. We identify the positive regulation of NG2 in BMSC differentiation to MF during liver fibrosis, which may provide a promising target for the treatment of liver disease.

Isolation, Purification, and Culture of Primary Murine Hepatic Stellate Cells: An Update.

In the healthy liver, quiescent hepatic stellate cells (HSCs) are found in the perisinusoidal space (i.e., the space of Dissé) in close proximity to endothelial cells and hepatocytes. HSCs represent 5-8% of the total number of liver cells and are characterized by numerous fat vacuoles that store vitamin A in the form of retinyl esters. Upon liver injury caused by different etiologies, HSCs become activated and acquire a myofibroblast (MFB) phenotype in a process called transdifferentiation. In contrast to quiescent HSC, MFB become highly proliferative and are characterized by an imbalance in extracellular matrix (ECM) homeostasis, by producing an excess of collagen and blocking its turnover by synthesis of protease inhibitors. This leads to a net accumulation of ECM during fibrosis. In addition to HSC, there are fibroblasts in the portal fields (pF), which also have the potency to acquire a myofibroblastic phenotype (pMF). The contributions of these two fibrogenic cell types (i.e., MFB and pMF) vary based on the etiology of liver damage (parenchymal vs. cholestatic). Based on their importance to hepatic fibrosis, the isolation and purification protocols of these primary cells are in great demand. Moreover, established cell lines may offer only limited information about the in vivo behavior of HSC/MFB and pF/pMF.Here we describe a method for high-purity isolation of HSC from mice. In the first step, the liver is digested with pronase and collagenase, and the cells are dissociated from the tissue. In the second step, HSCs are enriched by density gradient centrifugation of the crude cell suspension using a Nycodenz gradient. The resulting cell fraction can be further optionally purified by flow cytometric enrichment to generate ultrapure HSC.

Новые маркеры генитального пролапса

Pelvic organ prolapse (POP) is a very considerable problem not only due to its high prevalence, but also because of the significant decrease in the quality of life of women suffering from this disease. Given the high prevalence, pelvic floor disorders requiring plastic surgery are becoming an economic problem for public health. In recent decades, techniques for the treatment of pelvic floor dysfunction (PFD) have evolved rapidly. However, the pathophysiology of this disease is still poorly understood, which may be the reason for inadequate, untimely, and sometimes ineffective treatment of POP. One of the important findings when analyzing the results of morphological and morphometric studies of the levator ani muscle in PFD and clinical forms of POP became the detection of significant fibrous degradation of the pelvic floor muscular compartment in all the examined samples. Objective. To search for promising biomarkers of muscular and connective tissue remodeling and the interaction of the muscular and fascial compartments of the pelvic floor to predict pelvic organ prolapse. According to the literature, we found at least three candidate markers for assessing the condition of the muscular-fascial compartment of the pelvic floor: tenascin C, hyaluronic acid (HA), and myofibroblasts (MFB). Tenascin C is a non-structural component of the extracellular matrix of both muscular and connective tissue. Its role in the regulation of inflammation pathways has been discovered in recent studies, but in our study, this protein is of interest as a marker of tissue remodeling in case of tissue damage. Hyaluronic acid is one of the most abundant glycosaminoglycans in the connective tissue of vertebrates. HA is ubiquitous in fascial tissue, but its content is particularly high in loose connective tissue and between muscles and deep fascia. In the fascia, HA is not only a key structural component of the extracellular matrix, but also a functional one. The qualitative and quantitative analysis of HA content in pelvic floor fasciae is likely to help more accurately assess the tissue state, identify the nature of myofascial interaction during the formation of the hernia gate and may be the key to determining the treatment tactics for the initial signs of PFD. Myofibroblasts are connective tissue cells that represent an intermediate link between fibroblasts and smooth muscle cells. Their main task is to increase the basal tone and rigidity of the fascia and ensure proper muscular-skeletal dynamics. Normally, this tension is not enough for significant changes in musculature biomechanics, but persistent “hypertonicity” of the fascia quite quickly led to the formation of pathological contractures, which limited the motor activity of the underlying muscles. Thus, it can be assumed that myofibroblasts participate in the process of healing and remodeling of the fascial compartment. And the search for MFB in biopsy specimens of the levator ani muscle in patients with PFD can shed light on connective tissue dysplasia as the most controversial component of POP development. Conclusion. To date, the problem of pelvic organ prolapse remains unresolved in modern gynecology. When studying the literature, it became evident that determining the biochemical and biomechanical properties of the myofascial complex of the pelvic floor is necessary to gain a comprehensive picture of the disease. Although the significance of markers in predicting pelvic organ prolapse has not yet been investigated, we express optimistic expectations to find an ideal biomarker of the rate of PFD and POP development, improving knowledge about the pathogenesis of the disease. Key words: biomarkers, hyaluronic acid, myofibroblasts, pelvic organ prolapse, connective tissue, tenascin C

Silencing of UTX Mitigates Aging-Associated Cardiac Fibrosis via Blocking Cardiac Fibroblasts-to-Myofibroblasts Trans-Differentiation.

Cardiac fibrosis increases with age. Fibroblast activation plays an essential role in cardiac fibrosis. Histone modifications are involved in various chromatin-dependent processes. Attenuation of the histone H3 trimethylation on lysine 27 demethylase UTX by RNA interference or heterozygous mutation extends lifespan in worm. The objective of this study was to explore whether epigenetic silencing of UTX mitigates aging-associated cardiac fibrosis. Middle-aged mice (15 months old) were used and started to receive adeno-associated virus-scrambled-small hairpin RNA and adeno-associated virus-UTX-small hairpin RNA every 3 months from 15 months to 21 months, respectively. The mice were euthanized at 24 months of age (length of the study). Adeno-associated virus-UTX-small hairpin RNA delivery significantly attenu-ated aging-associated increase in blood pressure, especially in diastolic blood pressure, indicating silencing of UTX rescued aging-associated cardiac dysfunction. Aging-associated cardiac fibrosis is characterized by fibroblast activation and abundant extracellular matrix deposition, including collagen deposition and alpha smooth muscle actin activation. Silencing of UTX abolished collagen deposition and alpha smooth muscle actin activation, decreased serum transforming growth factor β, blocked cardiac fibro blast s-to- myofi brobl asts trans-differentiation by elevation of cardiac resident mature fibroblast markers, TCF21, and platelet-derived growth factor receptor alpha, which are important proteins for maintaining cardiac fibroblast physiological function. In the mechanistic study, adeno-associated virus-UTX-small hairpin RNA blocked transforming growth factor β-induced cardiac fibro blast s-to- myofi brobl asts trans-differentiation in isolated fibroblasts from 24-month-old mouse heart. The same results demonstrated as the in vivo study. Silencing of UTX attenuates aging-associated cardiac fibrosis via blocking cardiac fibroblasts-to-myofibroblasts transdifferentiation and consequently attenuates aging-associated cardiac dysfunction and cardiac fibrosis.

EXPRESSION OF ALPHA-SMOOTH MUSCLE ACTIN IN ODONTOGENIC CYSTS AND TUMORS.

Odontogenic cysts and tumors exhibit different degrees of aggressiveness in their biological behavior. There has been evidence that thepresence of myofibroblasts (MFs) at theinvasion front promotes tumor invasion. Our study is based on thefact that MFs are important in thebiological behavior of odontogenic cysts and tumors. To assess immunohistochemically expression of alpha-smooth muscle actin (α-SMA) of MFs in odontogenic cysts and tumors and correlate this expression to their biological behavior. Thearchival tissues collected for 1.5years were obtained from theDepartment of Oral Pathology & Microbiology, People's Dental Academy, Bhopal (India). A total of 40cases consisting of 10cases each of odontogenic keratocysts, radicular cysts, dentigerous cysts and ameloblastomas formed thestudy group. An immunohistochemical analysis of α-SMA expression and localization was carried out. Mean MF counts were thehighest in odontogenic keratocysts which was followed by ameloblastomas, entigerous cysts and radicular cysts. Weak α-SMA-expression was found in 50% of cases, moderate in 22.5% of cases, and intense- in 10% cases. MFs were arranged in thespindle, focal, or network patterns in 35; 27.5 and 20% of cases, respectively. Theanalysis revealed that theMFs were distinctly heterogeneous in distribution and pattern of arrangement. This provided persuasive evidence that stroma of these lesions harbor MFs as reflected by α-SMA immunopositive cells.

Abstract 15800: Dissecting Cardiomyocyte-Fibroblast Communication During Homeostasis and Cardiac Fibrosis in a Compartmentalized Biomimetic Tissue Model

Introduction: Cardiac fibroblasts (CFs) maintain cardiac structure and function by synthesizing and remodeling the extracellular matrix (ECM) found throughout the heart. Homeostatic maintenance of the ECM required for healthy tissue function depends on CF communication with cardiomyocytes (CMs). During cardiac fibrosis, signals resulting from tissue injury and dysregulated CF-CM communication promote CF differentiation into α-smooth muscle actin + (αSMA) myofibroblasts (MFs), which deposit excess ECM that mechanically impairs heart function. Given the challenges involved in parsing the dynamic and multifactorial heterocellular interactions underlying tissue homeostasis versus fibrosis, we engineered a compartmentalized, bilayer cardiac tissues where iPSC-derived CMs and CFs can be cultured on either side of a shared fibrous ECM to study CM-CF communication. Methods: Dextran vinyl sulfone (DVS) fibers were electrospun onto arrays of microfabricated PDMS wells. Matrix stiffness and alignment were controlled by modulating the speed of the collecting surface and UV light exposure, respectively. iPSC-CMs and primary human CFs were seeded onto opposing sides of the matrix. Immunostaining for αSMA was conducted to assess CF differentiation to MFs. Results: In CF monocultures on matrices varying in alignment and stiffness, MF differentiation was greatest on stiff (17.3 kPa) aligned matrices with exogenous TGFβ1 compared to soft (0.68 kPa) aligned matrices and both soft/stiff random matrices. We also observed increased nuclear YAP expression on stiff/aligned matrices, indicating altered mechanosensing in response to matrix properties. However, the presence of co-cultured CMs inhibited MF differentiation despite exogenous TGFβ1. MF differentiation was also impaired in CFs cultured on glass below matrix-seeded CMs, implying the role of protective paracrine signaling from CMs to CFs. Conclusions: We developed a microfabricated biomimetic coculture platform to examine CM-CF communication driving tissue homeostasis versus fibrosis. Specifically, we find that fibrous mechanical cues modulate YAP-mediated signaling and αSMA expression. Further, CM-CF paracrine signaling mitigates MF differentiation on aligned fibrous matrices.

A Retrospective Analysis of NRG/RTOG 0522: Low Myofibroblast Content in the Tumor-Associated Stroma may Predict Response to Epidermal Growth Factor Receptor (EGFR) Inhibitors

<h3>Purpose/Objective(s)</h3> Most head and neck cancers (HNC) overexpress EGFR, a feature associated with poor clinical outcome. Preclinical and clinical data suggest that peritumoral myofibroblasts (MF) express EGFR ligands and the presence of a high MF density in the tumor stroma is associated with statistically worse survival in patients with locally advanced HNC. We evaluated whether peritumoral MF density was prognostic of outcome in patients treated on RTOG 0522, a negative phase III trial evaluating the addition of C225 to cisplatin-based chemoradiation (CRT). We also evaluated whether MF density was predictive of benefit to C225. <h3>Materials/Methods</h3> Specimens from HPV-negative patients treated on RTOG 0522 were obtained from the NRG Oncology Biospecimen Repository and evaluated by a blinded pathologist (JW). MF density was determined as a percentage and the skewed data were normalized using Box-Cox transformation, from which the mean was calculated and used to stratify patients into low- and high-density groups. Overall survival (OS), progression-free survival (PFS), and distant metastasis (DM) rates were estimated using the Kaplan-Meier method and a multivariable analysis (MVA) by Cox proportional hazards model was used to test for the prognostic effect of this biomarker for patients with low vs. high MF density. To test for predictive effect of MF density on clinical outcomes in patients treated with CRT vs. CRT+C225, a Cox model with a treatment-by-biomarker interaction term was used. <h3>Results</h3> A total of 186 patients were evaluated, 94 were treated with CRT and 92 were treated with CRT+C225. On MVA, including primary site, age, PS, smoking history, T-stage, N-stage, and EGFR level, there was no significant difference in OS (Hazard ratio [HR], 0.65; 95% CI, 0.41-1.02; P=0.06), PFS (HR, 0.75; 95% CI, 0.50-1.12; P=0.16), or DM (HR, 0.76; 95% CI, 0.38-1.54; P=0.44) based on MF density. However, MVA Cox model including biomarker, treatment arm and biomarker-treatment interaction demonstrated that this interaction was significant (P=0.01). Within the low MF density subgroup, patients treated with CRT+C225 had significantly higher PFS (HR, 0.42; 95% CI, 0.21 to 0.82; P=0.01) and lower incidence of DM (HR, 0.18; 95% CI, 0.04 to 0.82; P=0.01), as well as numerically higher OS (HR, 0.48; 95% CI, 0.22 to 1.05; P=0.06) compared to patients treated with CRT. Patients with high MF density demonstrated no difference in OS, PFS, or DM based on treatment arm. <h3>Conclusion</h3> Peritumoral MF density was not prognostic of OS, PFS, or DM among patients treated on RTOG 0522. However, patients with low peritumoral MF density who received CRT+C225 had significantly higher PFS and lower incidence of DM compared to patients who received CRT alone. These results suggest that MF density is not a prognostic biomarker but may be predictive of benefit to C225. Further investigations are needed to validate these exploratory findings.

The Molecular Mechanisms of Liver Fibrosis and Its Potential Therapy in Application.

Liver fibrosis results from repeated and persistent liver damage. It can start with hepatocyte injury and advance to inflammation, which recruits and activates additional liver immune cells, leading to the activation of the hepatic stellate cells (HSCs). It is the primary source of myofibroblasts (MFs), which result in collagen synthesis and extracellular matrix protein accumulation. Although there is no FDA and EMA-approved anti-fibrotic drug, antiviral therapy has made remarkable progress in preventing or even reversing the progression of liver fibrosis, but such a strategy remains elusive for patients with viral, alcoholic or nonalcoholic steatosis, genetic or autoimmune liver disease. Due to the complexity of the etiology, combination treatments affecting two or more targets are likely to be required. Here, we review the pathogenic mechanisms of liver fibrosis and signaling pathways involved, as well as various molecular targets for liver fibrosis treatment. The development of efficient drug delivery systems that target different cells in liver fibrosis therapy is also summarized. We highlight promising anti-fibrotic events in clinical trial and preclinical testing, which include small molecules and natural compounds. Last, we discuss the challenges and opportunities in developing anti-fibrotic therapies.

Cathepsin L was involved in vascular aging by mediating phenotypic transformation of vascular cells

Vascular media and adventitia-induced remodeling plays an important role in vascular aging. However, the mechanism remains unclear. This study aims to investigate the mechanisms underlying vascular aging. Transcriptome analysis revealed that the expression of cathepsin L (CTSL) significantly decreased in arteries of old mice (24 months old) compared with that in arteries of young mice (4 months old), which was confirmed by immunohistochemistry and Western blot. The expression of CTSL in adventitia fibroblasts (AFs) and vascular smooth muscle cells (VSMCs) of aged mice was lower than that of young mice. Compared with wild-type control mice, CTSL knockout (CTSL - /−) mice had increased collagen deposition (fibrosis) and decreased telomerase activity and LC3Ⅱ/ LC3Ⅰratio. The expression of mammalian target of rapamycin (mTOR) and osteopontin (OPN) increased in aortas of CTSL−/−mice compared with that in aortas of wild-type control mice. In vitro, lentivirus-mediated CTSL knockdown induced VSMCs senescence and AFs transformed into myofibroblasts (MFs). Rapamycin, a mTOR inhibitor, inhibited CTSL deficiency induced VSMCs senescence, osteopontin (OPN) secretion and AFs migration. In conclusion, the decreased level of CTSL with age may participate in vascular aging by promoting the phenotypic transformation of vascular cells.

Cellular sources of airway smooth muscle cells in asthmatic airway remodeling and their clinical relevance: a narrative review.

Background and ObjectiveAirway remodeling in asthma refers to numerous structural changes in the airway in asthmatic patients, with thickening of the airway smooth muscle layer as its core feature. However, the nature and sources of the abnormally increased airway smooth muscle cells (ASMCs) in airway remodeling remain unclear. ASMCs play a key role in the pathogenesis of fatal asthma; therefore, it is important to clarify the properties and sources of these ASMCs responsible for asthmatic airway remodeling, which may provide a new direction for the precise treatment for asthma.MethodsWe performed a narrative review of the literature on PubMed, Web of Science, and Google Scholar databases searching for the cellular sources of ASMCs in asthmatic airway remodeling and their clinical relevance.Key Content and FindingsIt has long been thought that ASMCs are the result of abnormal proliferation of the native ASMCs in asthma; however, increasing evidence suggests that increased “ASMCs” may be due to the differentiation/transdifferentiation of other cells including mesenchymal stem cells (MSCs), myofibroblasts (MYFs), pericytes, and epithelial-mesenchymal transition (EMT). Recently, several pharmacological and biological therapies aimed at “reducing asthmatic ASMCs” have been developed, among which gallopamil, JQ1 [an inhibitor of the bromodomain and extra-terminal domain (BET) protein family], and histone deacetylase (HDAC) inhibitors can alleviate asthma airway remodeling and hyperresponsiveness and improve asthma symptoms in both mouse models and preclinical experiments.ConclusionsAs one of the core features of asthma, ASMCs are an important effector of airway remodeling. It has become extremely important to develop therapies for the reduction and prevention of the “ASMCs” on the basis of the properties and sources of “ASMCs”. Many studies have shown that epigenetic regulation is closely related to the abnormal increase of ASMCs in asthma, and interfering with epigenetic regulation factors can reduce the increased smooth muscle cells. Although the epigenetic regulation of asthma is still in its nascent stage, epigenetic therapy targeting “ASMCs” may become another new strategy for asthma prevention and treatment.

Hepatic Myofibroblasts: A Heterogeneous and Redox-Modulated Cell Population in Liver Fibrogenesis.

During chronic liver disease (CLD) progression, hepatic myofibroblasts (MFs) represent a unique cellular phenotype that plays a critical role in driving liver fibrogenesis and then fibrosis. Although they could originate from different cell types, MFs exhibit a rather common pattern of pro-fibrogenic phenotypic responses, which are mostly elicited or sustained both by oxidative stress and reactive oxygen species (ROS) and several mediators (including growth factors, cytokines, chemokines, and others) that often operate through the up-regulation of the intracellular generation of ROS. In the present review, we will offer an overview of the role of MFs in the fibrogenic progression of CLD from different etiologies by focusing our attention on the direct or indirect role of ROS and, more generally, oxidative stress in regulating MF-related phenotypic responses. Moreover, this review has the purpose of illustrating the real complexity of the ROS modulation during CLD progression. The reader will have to keep in mind that a number of issues are able to affect the behavior of the cells involved: a) the different concentrations of reactive species, b) the intrinsic state of the target cells, as well as c) the presence of different growth factors, cytokines, and other mediators in the extracellular microenvironment or of other cellular sources of ROS.

Fibroblast fate determination during cardiac reprogramming by remodeling of actin filaments.

SummaryFibroblasts can be reprogrammed into induced cardiomyocyte-like cells (iCMs) by forced expression of cardiogenic transcription factors. However, it remains unknown how fibroblasts adopt a cardiomyocyte (CM) fate during their spontaneous ongoing transdifferentiation toward myofibroblasts (MFs). By tracing fibroblast lineages following cardiac reprogramming in vitro, we found that most mature iCMs are derived directly from fibroblasts without transition through the MF state. This direct conversion is attributable to mutually exclusive induction of cardiac sarcomeres and MF cytoskeletal structures in the cytoplasm of fibroblasts during reprogramming. For direct fate switch from fibroblasts to iCMs, significant remodeling of actin isoforms occurs in fibroblasts, including induction of α-cardiac actin and decrease of the actin isoforms predominant in MFs. Accordingly, genetic or pharmacological ablation of MF-enriched actin isoforms significantly enhances cardiac reprogramming. Our results demonstrate that remodeling of actin isoforms is required for fibroblast to CM fate conversion by cardiac reprogramming.

Expression of Matrix Metalloproteinases 7 and 9, Desmin, Alpha-Smooth Muscle Actin and Caldesmon, in Odontogenic Keratocyst Associated with NBCCS, Recurrent and Sporadic Keratocysts.

Nevoid basal cell carcinoma syndrome (NBCCS) associated odontogenic keratocysts (OKCs) show more aggressive behavior and it has a higher frequency of relapse than non-syndromic OKCs. Stromal myofibroblasts (MFs), characterized by α-smooth muscle actin (αSMA), desmin and caldesmon expression, and metalloproteinases (MMPs) have an essential role in the remodeling of the extracellular matrix (ECM). The aim of the study is to analyze the immunohistochemical expression of MMP-7, MMP-9, αSMA and other new markers in the study of OKCs MFs such as desmin and caldesmon in NBCCS-associated OKCs compared to recurrent and sporadic keratocysts. Fourty 40 patients (23 M and 17 F) underwent surgery to remove the OKCs. The histological sections in paraffin were incubated with markers antibodies and a semi-quantitative score was used to evaluate the immunoreactivity. Densitometric analysis showed a very significantly increased expression of αSMA, caldesmon, MMP-7 and MMP-9 in NBCCS-OKCs compared to non-syndromic OKCs (p < 0.001). However, desmin showed a not significant increased expression in non-syndromic OKC compared to NBCCS-OKCs specimens in which desmin was slightly or not at all expressed. NBCSS-OKCs showed a greater distribution of MFs compared to the other OKCs subtypes. Further studies will be needed to evaluate whether the different expressions of these markers can be correlated to a different clinical behavior.

BRD4‐MK2 signaling: target for Crohn’s Disease‐associated fibrosis

IntroductionFibrosis is the major complication of Crohn’s disease (CD). No targeted therapies are currently available to revert CD‐associated fibrosis. While inflammatory mechanisms in CD have been extensively investigated, understanding the pathogenesis of fibrosis is relatively limited. Mesenchymal cells are the major effectors in profibrotic processes in CD. A dramatic increase in α‐SMA expressing subsets of mesenchymal cells, also known as myofibroblasts (MF), is among the main hallmarks of profibrotic changes in CD. Mitogen‐activated protein kinase‐activated protein kinase 2 (MK2) is involved in the increase of α‐SMAexpression by MFs. Along with an increase in MK2 activity in CD we also observed an increase in the bromodomain‐containing Protein 4 (BRD4), an important epigenetic reader. BRD4 is critical for the expression of enhancer‐mediated profibrotic genes and, thus, is a potential novel regulator of MK2. However, the presence of mechanistic link between BRD4 and MK2 activity with respect to profibrotic activation of MFs in CD is unknown. Herein, we tested hypothesis that BRD4 mediated increase in MK2 activity is critical to the profibrotic reprogramming of MFs in CD and this pathway has potential for novel anti‐fibrotic therapy.MethodsHuman normal (N)‐ and CD‐derived MFs were analyzed using RNAseq, cytokine/chemokine multiplex array, FACS, and immunofluorescent microscopy. The T cell transfer murine colitis model relevant to CD was also used in this study.ResultsAn Increase in BRD4 and MK2 expression was observed in primary human CD‐ when compared to N‐MFs and were associated with the expression of the profibrotic genes including Col1α2, α‐SMA, TNC, Fn1, and TGF‐β1. Inhibition of MK2 activity in CD‐MFs with the MK2 specific inhibitor PF‐3644022 and murine MK2 KO MFs showed significantly reduced basal and TGF‐β1‐induced profibrotic gene expression. Use of the BRD4 specific inhibitor ZL0454 significantly reduced MK2 mRNA expression in CD‐MFs Additionally, ex vivo treatment of the CD tissues with ZL0454 also abrogated MK2 expression. Therapeutic use of the BRD4 inhibitor ZL0454 (10 mg/kg, daily for 10 days) in T cell transfer murine colitis significantly decreased MK2 expression and downstream profibrotic responses as measured by collagen deposition and expression of fibrosis‐related genes.ConclusionsOur data suggests that BRD4 positively regulates MK2 expression in CD. Further, BRD4‐MK2 signaling is critical to profibrotic activation of mesenchymal cells in CD and is a potential therapeutic target to reverse CD‐associated fibrosis.