Deposition Of Fibrotic Tissue Research Articles

Combined regenerative rehabilitation improves recovery following volumetric muscle loss injury in a rat model.

Volumetric muscle loss (VML) injury causes irreversible deficits in muscle mass and function, often resulting in permanent disability. The current standard of care is physical therapy, but it is limited in mitigating functional deficits. We have previously optimized a rehabilitation technique using electrically stimulated eccentric contraction training (EST) that improved muscle mass, strength, and size in VML-injured rats. A biosponge scaffold composed of extracellular matrix proteins has previously enhanced muscle function postVML. This study aimed to determine whether combining a regenerative therapy (i.e., biosponge) with a novel rehabilitation technique (i.e., EST) could enhance recovery in a rat model of VML. A VML defect was created by removing ~20% of muscle mass from the tibialis anterior muscle in adult male Lewis rats. Experimental groups included VML-injured rats treated with biosponge with EST or biosponge alone (n = 6/group). EST was implemented 2 weeks postinjury at 150 Hz and was continued for 4 weeks. A linear increase in eccentric torque over 4 weeks showed the adaptability of the VML-injured muscle to EST. Combining biosponge with EST improved peak isometric torque by ~52% compared with biosponge treatment alone at 6 weeks postinjury. Application of EST increased MyoD gene expression and the percentage of large (>2000 μm2) type 2B myofibers but reduced fibrotic tissue deposition in VML-injured muscles. Together, these changes may provide the basis for improved torque production. This study demonstrates the potential for combined regenerative and rehabilitative therapy to improve muscle recovery following VML.

In-Silico analysis of the structural and functional impact of SNPs in adhesion molecules associated with cardiovascular disease and the effect of potential phytochemicals on these molecules.

Adhesion molecules are one of the most important proteins playing a major role in the regulationof cell migration, apoptosis, and survival. While adhesion molecules play a pivotal role inpreserving cellular matrix and tissue integrity, these molecules are also involved in thepathogenesis of cardiovascular diseases. The cell adhesion molecules are involved inatherosclerotic plaque progression, deposition of fibrotic tissue, and thrombus formation. Selectins-E and -P and members of the immunoglobulin superfamily ICAM-1 and ICAM-3 are the majorgroups of the adhesion molecules involved in cardiovascular diseases.In the current study, computational analysis was performed to understand the effects of missensemutations on the structure and functions of the adhesion molecules. The computational studiescan lead to a better understanding of the role of single nucleotide polymorphic mutations on thefunctions of adhesion molecules. The single nucleotide polymorphism data of adhesion moleculeswas retrieved from gnomAD v2.1.1. Only the missense SNPs were selected, and the 3D proteinstructures of the adhesion molecules were retrieved from AlphaFold. The 3D protein structures ofthe adhesion molecules were mutated using USCF Chimera 1.14.The comparison of structural stability, structural change, and interaction with ligands wasperformed between the wild-type and mutant adhesion molecules by several bioinformatics tools.The R458W mutant of ICAM-1 was found to be destabilizing the structure of the protein.Similarly, the V88M mutant of ICAM-3 and H468Y mutant of E-selectin (SELE), along withG179R and G727Q mutants of P-selectin (SELP) have also shown structural deviations ascompared to wild-type molecules. The R367C mutant of ICAM-1, P15L mutant of ICAM-3, andT756P mutant of SELP showed a significant change in the hydrogen bonding interactions withtheir respective ligands as compared to the wild-type protein interactions with a deviation from theoriginal binding free energies. No significant changes in the hydrogen bonding interactions wereobserved in the G179R and G727Q mutants of SELP.Moreover, phytochemicals data of several medicinal plants which were effective againstcardiovascular diseases was retrieved from the Indian Medicinal Plants, Photochemistry, andTherapeutics database. The molecular docking was performed to analyze the effects ofphytochemicals on adhesion molecules. The 3,29-Dihydroxyolean-12-en-28-oic acid, aphytochemical of the Panax ginseng plant was found to have an inhibitory effect on the activesites of adhesion molecules ICAM-1, ICAM-3, SELE, and SELP. Furthermore, the results of thisstudy can be tested experimentally to confirm the effect of adhesion molecule mutations on theonset of cardiovascular diseases and the role of 3,29-Dihydroxyolean-12-en-28-oic acid as ageneral drug for the treatment of diseases caused by mutant adhesion molecules.

Potential role of SIRT-1 and SIRT-3 as biomarkers for the diagnosis and prognosis of idiopathic pulmonary fibrosis

BackgroundIdiopathic pulmonary fibrosis (IPF) is a debilitating and progressive lung disease of unknown aetiology, characterized by the relentless deposition of fibrotic tissue. Biomarkers may play a pivotal role as indicators of disease presence, progression, and treatment response. Sirtuins, a family of enzymes with ADP ribosyltransferase or deacetylase activity, have been implicated in several diseases, including pulmonary fibrosis.MethodsA cross-sectional, prospective, observational single-center study was conducted to investigate the potential role of serum SIRTs levels as biomarkers in patients with IPF. Demographic, clinical, and functional data and serological samples were collected from 34 patients with IPF followed at the Interstital Lung and Rare Diseases Outpatient Clinic of the Vanvitelli Pneumology Clinic, Monaldi Hospital, Naples, Italy and from 19 age-matched controls.ResultsSerum SIRT-1 levels were significantly reduced in IPF patients compared to controls (median IPF 667 [435–858] pg/mL versus controls 925 [794–1173] pg/mL; p < 0.001 ). In contrast, serum SIRT-3 levels were significantly increased in IPF patients compared to controls (median IPF 338 [230–500] pg/mL versus controls 154 [99.8–246] pg/mL; p < 0.001). There were no statistically significant differences in serum SIRT-6 and SIRT-7 levels between IPF and controls. In addition, we found a significant positive correlation between SIRT-1 and lung function parameters such as FEV1% (ϱ=0.417;p = 0.016), FVC% (ϱ=0.449;p = 0.009) and DLCO% (ϱ=0.393;p = 0.024), while a significant negative correlation was demonstrated between SIR-1 and GAP score, demonstrating a significant reduction in SIRT-1 in advanced Gender-Age-Physiology (GAP) stages 2–3 compared to GAP stage 1 (p = 0.008).ConclusionsThis prospective, cross-sectional study showed that SIRT-1 was associated with lung function and IPF severity and that both SIRT-1 and SIRT-3 could be considered as potential biomarkers of IPF, whereas SIRT-6 and SIRT-7 were not associated with IPF.

Abstract 665: MEN1703/SEL24 exhibits promising antitumoral activity in preclinical models of myelofibrosis both as single agent and combined with ruxolitinib

Abstract Myelofibrosis (MF) is a pathological chronic bone marrow (BM) condition characterized by abnormal growth of erythroid and megakaryocytic precursors and deposition of fibrotic tissue. The JAK/STAT pathway plays an important role in MF (Cardoso 2015), and multiple JAK1/2 inhibitors (JAKis) are approved for MF treatment. Despite initially limiting disease symptoms, JAKis often fail due to persistent JAK2 activation and survival of MF-driving cells, hence new treatment strategies are highly needed. PIM kinases also contribute to MF pathogenesis. PIM1 is highly overexpressed in granulocytes, peripheral blood mononuclear cells (PBMCs), and BM of patients with MF. PIM1/2 expression is dependent upon JAK2 signaling in myeloproliferative neoplasms (MPNs). Hence, PIM inhibitors represent potential therapeutic targets for MPNs and have been evaluated in several preclinical and clinical studies (Rampal 2021). MEN1703/SEL24 (MEN) is an oral, first-in-class, dual PIM/FLT3 kinase inhibitor in development for hematologic malignancies. This study aims to investigate the efficacy of MEN alone and in combination with the JAKi ruxolitinib (RUX) in preclinical MF models and to elucidate the underlying signaling pathways. MF cell lines (JAK2V617F and JAK2 wild type) were used in MTS assays to assess the in vitro cytotoxicity of MEN alone and in combination with RUX, compared with the PIM inhibitor TP-3654 (Dutta 2022). Cell cycle distribution and signaling pathways were analyzed via propidium iodide/FACS analysis and immunocapillary electrophoresis, respectively, upon treatment with the single agents and MEN + RUX. In vivo efficacy was assessed in a murine xenograft model treated with MEN +/- RUX. In vitro studies showed single agent MEN activity with an IC50 ranging from 0.3 to 2.3 µM. Moreover, a synergistic drug interaction was found with MEN + RUX in all tested cell lines (Combination Index [CI] range 0.41-0.80). MEN presented cytotoxic and cytostatic capacity, inducing apoptosis and arresting a proportion of cells in G2/M phase. This effect was maintained in combination with RUX. Signaling pathway analysis showed that MEN inhibits phosphorylation of STAT proteins and decreases Mcl1 protein expression. These effects were enhanced with MEN + RUX. Compared with TP-3654, MEN was more effective both as a single agent (IC50 2-3 fold lower than TP-3654) and in combination with RUX (mean CI of 0.77-0.80 compared to 0.79-0.98 with TP-3654). MEN showed anti-tumoral efficacy in MF preclinical models, moderately higher than TP-3654, exhibiting in vitro activity at clinically relevant concentrations. Importantly, MEN combined with the standard of care RUX was synergistic, and molecular analyses confirmed the role of PIM downstream target inhibition. Our results support the therapeutic potential and relevance of MEN in MF treatment strategies. Citation Format: Maria Luisa Iannitto, Diego Bisignano, Sonia Zicari, Francesco Belli, Alessio Fiascarelli, Simone Talucci, Giuseppe Merlino, Vittoria Nicolis di Robilant, Clelia Irrissuto, Mario Bigioni, Alessandro Bressan, Daniela Bellarosa, Monica Binaschi. MEN1703/SEL24 exhibits promising antitumoral activity in preclinical models of myelofibrosis both as single agent and combined with ruxolitinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 665.

Expression of the Pro-Fibrotic Marker Periostin in a Mouse Model of Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is characterised by fibrotic tissue deposition in skeletal muscle. We assessed the role of periostin in fibrosis using mdx mice, an established DMD murine model, for which we conducted a thorough examination of periostin expression over a year. RNA and protein levels in diaphragm (DIA) muscles were assessed and complemented by a detailed histological analysis at 5 months of age. In dystrophic DIAs, periostin (Postn) mRNA expression significantly exceeded that seen in wildtype controls at all timepoints analysed, with the highest expression at 5 months of age (p < 0.05). We found Postn to be more consistently highly expressed at the earlier timepoints compared to established markers of fibrosis like transforming growth factor-beta 1 (Tgf-β1) and connective tissue growth factor (Ctgf). Immunohistochemistry confirmed a significantly higher periostin protein expression in 5-month-old mdx mice compared to age-matched healthy controls (p < 0.01), coinciding with a significant fibrotic area percentage (p < 0.0001). RT-qPCR also indicated an elevated expression of Tgf-β1, Col1α1 (collagen type 1 alpha 1) and Ctgf in mdx DIAs compared to wild type controls (p < 0.05) at 8- and 12-month timepoints. Accordingly, immunoblot quantification demonstrated elevated periostin (3, 5 and 8 months, p < 0.01) and Tgf-β1 (8 and 12 months, p < 0.001) proteins in the mdx muscle. These findings collectively suggest that periostin expression is a valuable marker of fibrosis in this relevant model of DMD. They also suggest periostin as a potential contributor to fibrosis development, with an early onset of expression, thereby offering the potential for timely therapeutic intervention and its use as a biomarker in muscular dystrophies.

Renal and Urinary Tract Involvement in Fibrosclerosing or Fibroinflammatory Diseases: A Narrative Review

Fibroinflammatory diseases are a group of rare pathologies in which the hallmark is the exuberant deposition of fibrotic tissue and inflammatory cellular infiltrates, characteristic of the specific disease. A sclerotic mass develops within soft tissues and/or organs, damaging and replacing them, with effects ranging from asymptomatic to life-threatening clinical manifestations. The kidneys and urinary tract can be involved in some of these diseases, which can lead to acute kidney injury, chronic kidney disease, and even end-stage kidney disease. IgG4-related disease, retroperitoneal fibrosis, and Erdheim–Chester disease are the three fibroinflammatory disorders that can involve the kidneys. Only a timely and accurate collection of clinical, radiological, metabolic, laboratory, and histological data allows prompt diagnosis and targeted treatment of these pathologies, allowing the stoppage of the evolution of renal and systemic manifestations, which can lead to complete remission. The epidemiology, clinical and histological features, and management of these conditions are herein described in a narrative fashion.

Fibrotic Diseases of the Human Urinary and Genital Tract: Current Understanding and Potential Strategies for Treatment.

Fibrosis is a disease condition characterized by abnormalities of the extracellular matrix, such as accumulation of the transforming growth factor β, infiltration by myofibroblasts, deposition of collagen, and a generalized dysregulation of collagen maturation. It can severely impair the function of organs by replacing normal tissue with a highly collagenized matrix, thereby reducing the elasticity and compliance of tissues. Fibrotic diseases of the genitourinary tract present relevant problems in healthcare, and their principles of pathophysiology remain unclarified; hence, the armamentarium for prevention and treatment is limited. These diseases include renal fibrosis, Peyronie's disease and ureteral and urethral strictures due to perturbations in the process of wound healing in response to injuries. Such deteriorations may contribute to obstructive uropathies or sexual dysfunction. This review provides a brief overview of the most frequent fibrotic diseases of the genitourinary system and of how the pathophysiology is related to symptoms, and also highlights potential therapeutic strategies to address the abnormal deposition of collagen. Although the understanding of factors associated with fibrotic conditions of the urinary and genital tract is still limited, some beneficial advances have been made. Further research will serve to provide a more comprehensive insight into factors responsible for the development of fibrotic tissue deposition.

Endometrial gene expression of DEFB4B, LYZ, and SLPI in biopsy samples of mares resistant or susceptible to endometritis

Endometritis is one of the main causes of subfertility in mares and therefore one of the most common reasons for economic losses in the horse breeding industry. Multiparous older mares are more susceptible to the development of persistent endometritis after mating, artificial insemination, or foaling since aging is associated with an altered systemic immune response and anatomical defects that interfere with physiological uterine clearance (Katila et.al. Animals. 2022; 12:779). In this context, their inability to recover from endometritis can lead to degenerative changes in the endometrium (e.g., glandular degenerative changes and associated peri-glandular and/or diffuse stromal fibrosis) (Canisso et.al. International Journal of Molecular Sciences 2020; 21:1432). This study aimed to evaluate possible variations in endometrial gene expression of the genes Defensin Beta 4B (DEFB4B), Lysozyme (LYZ), and Secretory Leukocyte Peptidase Inhibitor (SLPI) between resistant and susceptible mares. These three genes have already been identified as involved in the equine innate immune response and in the chronic uterine inflammatory response (Marth et.al. Reproduction, Fertility, Development 2018; 30:533-545). Endometrial biopsy samples were collected from 19 mares. Mares were divided into two groups concerning their age and reproductive history: the "resistant group" (RES) (N=4) consisted of maiden mares 3.00±0.00 years old with endometrial biopsy grade I according to Kenney and Doig classification (1986); the "susceptible group" (SUS) (N=15)was composed of multiparous mares 17.33±3.28 years old, with endometrial biopsy of grade IIA, IIB or III. Total RNA was extracted from each sample and the corresponding cDNAs were prepared by retro-transcription using dedicated commercial kits. The samples were then amplified by qRT-PCR procedure as described by Marth and colleagues (Marth et.al. Reproduction, Fertility, Development 2018; 30:533-545). The housekeeping gene GADPH was amplified in each sample as internal control and for data normalization. To minimize possible experimental errors, for each sample three technical replicates were performed and the quality of the extracted RNA was evaluated by spectrophotometry and gel electrophoresis. The results showed a statistically significant difference in the expression of the genes LYZ (p=0.0003) and DEFB4B (p=0.0273) which were upregulated in SUS versus RES mares. This suggests that lower gene expression found in young mares are attributable to a more balanced local immunomodulation, with consequent reduction of the inflammatory pathways and slower progression towards the deposition of fibrotic tissue compared to older mares. No differences in the expression of the SLPI gene were found between the two groups (p=0.8854). In conclusion, despite these promising results, larger studies are needed before LYZ and DEFB4B genes could be used as biomarkers to identify the mares susceptible to endometritis.

Combined application of biosponges and an antifibrotic agent for the treatment of volumetric muscle loss.

Volumetric muscle loss (VML) causes irrecoverable loss of muscle mass and strength and results in permanent disability. VML injury shows extensive fibrosis, which impedes functional tissue regeneration. Our lab has created a biosponge scaffold composed of extracellular matrix (ECM) proteins (i.e., biosponge) that can enhance muscle regeneration and function following VML. In this work, a potent small molecule inhibitor of alpha v-subunit containing integrins known as IDL-2965 was incorporated into the biosponges for localized suppression of fibrosis post-VML. Our results demonstrate that local delivery of IDL-2965 via the biosponges attenuated the deposition of fibrotic tissue preceded by a downregulation of profibrotic genes in VML-injured muscles. The reduction in fibrotic tissue had no detrimental effects on muscle mass, function, size, or vascularity. Overall, these findings suggest that the codelivery of biosponges and IDL-2965 is a safe and effective strategy for the mitigation of fibrotic tissue deposition in VML-injured muscles.

Prediction and Demonstration of Retinoic Acid Receptor Agonist Ch55 as an Antifibrotic Agent in the Dermis

The prevalence of fibrotic diseases and the lack of pharmacologic modalities to effectively treat them impart particular importance to the discovery of novel antifibrotic therapies. The repurposing of drugs with existing mechanisms of action and/or clinical data is a promising approach for the treatment of fibrotic diseases. One paradigm that pervades all fibrotic diseases is the pathological myofibroblast, a collagen-secreting, contractile mesenchymal cell that is responsible for the deposition of fibrotic tissue. In this study, we use a gene expression paradigm characteristic of activated myofibroblasts in combination with the Connectivity Map to select compounds that are predicted to reverse the pathological gene expression signature associated with the myofibroblast and thus contain the potential for use as antifibrotic compounds. We tested a small list of these compounds in a first-pass screen, applying them to fibroblasts, and identified the retinoic acid receptor agonist Ch55 as a potential hit. Further investigation exhibited and elucidated the antifibrotic effects of Ch55 invitro as well as showing antiscarring activity upon intradermal application in a preclinical rabbit ear hypertrophic scar model. We hope that similar predictions to uncover antiscarring compounds may yield further preclinical and ultimately clinical success.

Pulmonary Fibrosis as a Result of Acute Lung Inflammation: Molecular Mechanisms, Relevant In Vivo Models, Prognostic and Therapeutic Approaches

Pulmonary fibrosis is a chronic progressive lung disease that steadily leads to lung architecture disruption and respiratory failure. The development of pulmonary fibrosis is mostly the result of previous acute lung inflammation, caused by a wide variety of etiological factors, not resolved over time and causing the deposition of fibrotic tissue in the lungs. Despite a long history of study and good coverage of the problem in the scientific literature, the effective therapeutic approaches for pulmonary fibrosis treatment are currently lacking. Thus, the study of the molecular mechanisms underlying the transition from acute lung inflammation to pulmonary fibrosis, and the search for new molecular markers and promising therapeutic targets to prevent pulmonary fibrosis development, remain highly relevant tasks. This review focuses on the etiology, pathogenesis, morphological characteristics and outcomes of acute lung inflammation as a precursor of pulmonary fibrosis; the pathomorphological changes in the lungs during fibrosis development; the known molecular mechanisms and key players of the signaling pathways mediating acute lung inflammation and pulmonary fibrosis, as well as the characteristics of the most common in vivo models of these processes. Moreover, the prognostic markers of acute lung injury severity and pulmonary fibrosis development as well as approved and potential therapeutic approaches suppressing the transition from acute lung inflammation to fibrosis are discussed.

A new mouse model of post-traumatic joint injury allows to identify the contribution of Gli1+ mesenchymal progenitors in arthrofibrosis and acquired heterotopic endochondral ossification.

Complex injury and open reconstructive surgeries of the knee often lead to joint dysfunction that may alter the normal biomechanics of the joint. Two major complications that often arise are excessive deposition of fibrotic tissue and acquired heterotopic endochondral ossification. Knee arthrofibrosis is a fibrotic joint disorder where aberrant buildup of scar tissue and adhesions develop around the joint. Heterotopic ossification is ectopic bone formation around the periarticular tissues. Even though arthrofibrosis and heterotopic ossification pose an immense clinical problem, limited studies focus on their cellular and molecular mechanisms. Effective cell-targeted therapeutics are needed, but the cellular origin of both knee disorders remains elusive. Moreover, all the current animal models of knee arthrofibrosis and stiffness are developed in rats and rabbits, limiting genetic experiments that would allow us to explore the contribution of specific cellular targets to these knee pathologies. Here, we present a novel mouse model where surgically induced injury and hyperextension of the knee lead to excessive deposition of disorganized collagen in the meniscus, synovium, and joint capsule in addition to formation of extra-skeletal bone in muscle and soft tissues within the joint capsule. As a functional outcome, arthrofibrosis and acquired heterotopic endochondral ossification coupled with a significant increase in total joint stiffness were observed. By employing this injury model and genetic lineage tracing, we also demonstrate that Gli1+ mesenchymal progenitors proliferate after joint injury and contribute to the pool of fibrotic cells in the synovium and ectopic osteoblasts within the joint capsule. These findings demonstrate that Gli1+ cells are a major cellular contributor to knee arthrofibrosis and acquired heterotopic ossification that manifest after knee injury. Our data demonstrate that genetic manipulation of Gli1+ cells in mice may offer a platform for identification of novel therapeutic targets to prevent knee joint dysfunction after chronic injury.

Force-Bioreactor for Assessing Pharmacological Therapies for Mechanobiological Targets.

Tissue fibrosis is a major health issue that impacts millions of people and is costly to treat. However, few effective anti-fibrotic treatments are available. Due to their central role in fibrotic tissue deposition, fibroblasts and myofibroblasts are the target of many therapeutic strategies centered primarily on either inducing apoptosis or blocking mechanical or biochemical stimulation that leads to excessive collagen production. Part of the development of these drugs for clinical use involves in vitro prescreening. 2D screens, however, are not ideal for discovering mechanobiologically significant compounds that impact functions like force generation and other cell activities related to tissue remodeling that are highly dependent on the conditions of the microenvironment. Thus, higher fidelity models are needed to better simulate in vivo conditions and relate drug activity to quantifiable functional outcomes. To provide guidance on effective drug dosing strategies for mechanoresponsive drugs, we describe a custom force-bioreactor that uses a fibroblast-seeded fibrin gels as a relatively simple mimic of the provisional matrix of a healing wound. As cells generate traction forces, the volume of the gel reduces, and a calibrated and embedded Nitinol wire deflects in proportion to the generated forces over the course of 6 days while overhead images of the gel are acquired hourly. This system is a useful in vitro tool for quantifying myofibroblast dose-dependent responses to candidate biomolecules, such as blebbistatin. Administration of 50 μM blebbistatin reliably reduced fibroblast force generation approximately 40% and lasted at least 40 h, which in turn resulted in qualitatively less collagen production as determined via fluorescent labeling of collagen.

Role of caveolin-1 in heart extracellular matrix deposition and remodelling upon myocardial infarction

Introdution: Changes in tissue stiffness or forces transmitted across tissues drive cell functions and behaviour. Cardiovascular system is inherently mechanical, and how its cells and tissues interplay with mechanical forces are an important aspect of its physiology. For example, upon heart damage, the non-injured myocardium is subjected to mechanical overload to preserve heart functionality. Caveolin-1 (Cav1) is an essential constituent of caveolae, flask- shaped invaginated nanodomains of the plasma membrane that act as mechanosensing and mechanoadapting structures through tension-driven flattening and disassembly and membrane trafficking. Cav1 phospho-Tyr14 is an important regulatory node that regulates caveolae internalization and specific downstream functions, but its relevance to cardiac remodelling and function has not been explored. In this study, we have assessed the role of Cav1 on mechanical adaptation to myocardial infarction, specifically on scar establishment and remodelling. Matherial and methods: To study the role of Cav1 on cardiac regeneration potential and extracellular matrix (ECM) remodelling ability upon myocardial infarction (MI), mice that express a non-phosphorylatable form of Cav1 on Tyr14 (Cav1Y14F/Y14F) were used. As a model of MI, postnatal day 1 mice were subjected to heart cryoinjury. Mice were anesthetized by hypothermia, lateral thoracotomy was performed to expose the heart and a metal probe cooled in liquid nitrogen was applied to the left ventricle. The deposition of fibrotic tissue at 7 days postinjury was assessed by H&amp;E and Picrosirius Red histological stainings. Proteomics analysis of cryoinjured hearts was performed. Cardiac lesions were further characterized by immunofluorescence for several markers (CD68, αSMA, Cav1, pHis3, etc.). Results: We previously found that Cav1 is a pivotal regulator of physical ECM remodelling, determining cell migration and tissue fibrosis, and also that specific non-collagen ECM components, are sorted into exosomes through a Cav1- dependent mechanisms. Proteomics analysis of infarcted hearts suggest that Cav1 mutant mice present an altered deposition of ECM proteins, which might impair the recovery of cardiac function. In agreement with this, aberrant histopathological features were observed in scars formed in mutant hearts. Increased macrophage density was observed in Cav1Y14F/Y14F mice, but no significant differences on cell proliferation were found. Conclusions: Here, we provide several evidences suggesting that Cav1 is a key player on mechanoadaptation and machanoresponse in challenged hearts and that Cav1 phospho-Tyr14 is involved in ECM deposition and remodelling and immune infiltration in cryoinjured hearts.

Therapeutic targets for cardiac fibrosis: from old school to next-gen

Cardiovascular diseases remain the leading cause of death worldwide, with pathological fibrotic remodeling mediated by activated cardiac myofibroblasts representing a unifying theme across etiologies. Despite the profound contributions of myocardial fibrosis to cardiac dysfunction and heart failure, there currently exist limited clinical interventions that effectively target the cardiac fibroblast and its role in fibrotic tissue deposition. Exploration of novel strategies designed to mitigate or reverse myofibroblast activation and cardiac fibrosis will likely yield powerful therapeutic approaches for the treatment of multiple diseases of the heart, including heart failure with preserved or reduced ejection fraction, acute coronary syndrome, and cardiovascular disease linked to type 2 diabetes. In this Review, we provide an overview of classical regulators of cardiac fibrosis and highlight emerging, next-generation epigenetic regulatory targets that have the potential to revolutionize treatment of the expanding cardiovascular disease patient population.

Inhibition of PKCθ Improves Dystrophic Heart Phenotype and Function in a Novel Model of DMD Cardiomyopathy.

Chronic cardiac muscle inflammation and subsequent fibrotic tissue deposition are key features in Duchenne Muscular Dystrophy (DMD). The treatment of choice for delaying DMD progression both in skeletal and cardiac muscle are corticosteroids, supporting the notion that chronic inflammation in the heart plays a pivotal role in fibrosis deposition and subsequent cardiac dysfunction. Nevertheless, considering the adverse effects associated with long-term corticosteroid treatments, there is a need for novel anti-inflammatory therapies. In this study, we used our recently described exercised mdx (ex mdx) mouse model characterised by accelerated heart pathology, and the specific PKCθ inhibitor Compound 20 (C20), to show that inhibition of this kinase leads to a significant reduction in the number of immune cells infiltrating the heart, as well as necrosis and fibrosis. Functionally, C20 treatment also prevented the reduction in left ventricle fractional shortening, which was typically observed in the vehicle-treated ex mdx mice. Based on these findings, we propose that PKCθ pharmacological inhibition could be an attractive therapeutic approach to treating dystrophic cardiomyopathy

Sunitinib inhibits STAT3 phosphorylation in cardiac muscle and prevents cardiomyopathy in the mdx mouse model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a fatal X-linked genetic disorder affecting approximately 1 in 5000 male births worldwide. DMD is caused by mutations in the dystrophin gene. Dystrophin is essential for maintaining muscle cell membrane integrity and stability by linking the cytoskeleton to the extracellular matrix, which protects myofibers from contraction-induced damage. Loss of dystrophin leads to mechanically induced skeletal and cardiac muscle damage. Although the disease is not evident in DMD patients at birth, muscular dystrophy rapidly progresses and results in respiratory and cardiac muscle failure as early as the teenage years. Premature death in DMD patients is due to cardiac arrhythmias and left ventricular dysfunction. Currently, there is no effective treatment for DMD-related cardiac failure. Recently, we have shown that a Food and Drug Administration-approved small molecule, sunitinib, a multi-targeted tyrosine kinase inhibitor can mitigate skeletal muscle disease through an increase in myogenic capacity, cell membrane integrity, and improvement of skeletal muscle function via regulation of STAT3-related signaling pathway. Chronic activation of STAT3 has been shown to promote cardiac hypertrophy and failure. In this study, we examined the effects of long-term sunitinib treatment on cardiac pathology and function. Our results showed sunitinib treatment reduced STAT3 phosphorylation in the heart muscle of mdx mice, improved cardiac electrical function, increased cardiac output and stroke volume, decreased ventricular hypertrophy, reduced cardiomyocytes membrane damage, fibrotic tissue deposition and slightly decreased cardiac inflammation. Together, our studies support the idea that sunitinib could serve as a novel treatment to slow cardiomyopathy progression in DMD. One Sentence Summary In this study, we determined if sunitinib, a Food and Drug Administration-approved drug, could reduce the pathology and improve cardiac function in an animal model for DMD.

A Safe, Fibrosis-Mitigating, and Scalable Encapsulation Device Supports Long-Term Function of Insulin-Producing Cells.

Encapsulation and transplantation of insulin-producing cells offer a promising curative treatment for type 1 diabetes (T1D) without immunosuppression. However, biomaterials used to encapsulate cells often elicit foreign body responses, leading to cellular overgrowth and deposition of fibrotic tissue, which in turn diminishes mass transfer to and from transplanted cells. Meanwhile, the encapsulation device must be safe, scalable, and ideally retrievable to meet clinical requirements. Here, a durable and safe nanofibrous device coated with a thin and uniform, fibrosis-mitigating, zwitterionically modified alginate hydrogel for encapsulation of islets and stem cell-derived beta (SC-β) cells is reported. The device with a configuration that has cells encapsulated within the cylindrical wall, allowing scale-up in both radial and longitudinal directions without sacrificing mass transfer, is designed. Due to its facile mass transfer and low level of fibrotic reactions, the device supports long-term cell engraftment, correcting diabetes in C57BL6/J mice with rat islets for up to 399 days and SCID-beige mice with human SC-β cells for up to 238 days. The scalability and retrievability in dogs are further demonstrated. These results suggest the potential of this new device for cell therapies to treat T1D and other diseases.

Evaluating the potential use of functional fibrosis to facilitate improved outcomes following volumetric muscle loss injury

Volumetric muscle loss (VML) was defined as the frank loss of skeletal muscle tissue with associated chronic functional deficits. Significant effort has been dedicated to developing approaches for treating VML injuries, most of which have focused on stimulating regeneration of the affected musculature via a variety of approaches (e.g., biomaterials). VML injury induces a prolonged inflammatory response which causes fibrotic tissue deposition and is thought to inhibit de novo myofiber regeneration despite observed improvements in functional outcomes (i.e., functional fibrosis; FF). Recent approaches have sought to attenuate inflammation and/or fibrosis as a means to create a permissive environment for regenerative therapies. However, there are currently no clinically available interventions capable of facilitating full restoration of form and function following VML injury; thus, an unmet clinical need exists for a near-term interventional strategy to treat affected patients. FF could serve as an alternative approach to facilitate improved functional outcomes following VML injuries. We sought to investigate whether intentionally exploiting the concept of FF (i.e., induction of a supraphysiological fibrotic response via the delivery of a polypropylene mesh combined with TGFβ) would enhance the function of the VML affected musculature. We found that FF treatment induces enhanced fibrotic tissue deposition within the VML defect as evidenced by histological and molecular analysis. FF-treated animals exhibit improved in vivo muscle function compared to untreated control animals at 8 weeks post-injury, thus substantiating the concept that FF could serve as an efficacious approach for facilitating improved functional outcomes following VML injury. Statement of SignificanceVML injuries result in long-term functional impairments and reduced quality of life for affected individuals, namely combat injured US Service members, and no clinical interventions can restore the form and function of the injured limb. Extensive efforts have been aimed at developing therapeutics to address this critical gap; unfortunately, most interventions facilitate only modest regeneration. Interestingly, improved muscle function has been observed in VML studies following treatment with a therapeutic, despite a lack of myogenic tissue formation; a phenomenon termed Functional Fibrosis (FF). Herein we exploited the concept of FF to enhance the function of VML affected musculature. This finding is significant in that the commercially available interventions used to induce FF can be translated into the clinic near-term, thus improving the standard of care for VML injuries.

Swertia purpurascens Wall ethanolic extract mitigates hepatic fibrosis and restores hepatic hepcidin levels via inhibition of TGFβ/SMAD/NFκB signaling in rats

Ethnopharmacological relevanceSwertia purpurascens Wall belongs to a well-known genus in traditional systems of medicine worldwide. In folklore, it is used to treat various ailments, including hepatic disorders, as an alternative to the endangered species Swertia chirayita. However, the therapeutic potential of Swertia purpurascens Wall against hepatic fibrosis has not been validated yet. Aim of the studyThe present study was planned to evaluate the efficacy of the Swertia purpurascens Wall extract (SPE) against hepatic fibrosis and elucidate the underlying mechanism of action. Materials and methodsThe metabolite profiling of the SPE was done using UHPLC-QTOF-MS/MS. The acute oral toxicity study of SPE at 2 g/kg BW dose was done in rats. Further, the liver fibrosis was induced by the CCl4 intoxication, and the efficacy of SPE at three doses (100, 200 and 400 mg/kg BW) was evaluated by studying biochemical parameters, histopathology, immunohistochemistry, qRT-PCR, western blotting and in silico analysis. ResultsUHPLC-QTOF-MS/MS analysis revealed the presence of a total of 23 compounds in SPE. Acute oral toxicity study of SPE at 2 g/kg BW showed no harmful effects in rats. Further, the liver fibrosis was induced by the CCl4 administration, and the efficacy of SPE was evaluated at three doses (100, 200 and 400 mg/kg BW). SPE treatment significantly improved the body weight gain, the relative liver weight, serum liver injury markers and endogenous antioxidant enzyme levels in the CCl4-treated rats. SPE also recovered the altered liver histology and effectively reduced the fibrotic tissue deposition in the hepatic parenchyma. Further, SPE significantly inhibited the fibrotic (TGFβ, αSMA, SMADs and Col1A), proinflammatory markers (NFκB, TNFα and IL1β) and apoptosis in the liver tissue. Interestingly, SPE treatment also restored the altered hepcidin levels in the liver tissue. In silico study revealed the potential of various metabolites as drug candidates and their interaction with target proteins. ConclusionAltogether, SPE showed its therapeutic potential against CCl4-induced hepatic fibrosis by restoring the hepatic hepcidin levels and inhibiting TGFβ/SMAD/NFκB signaling in rats.