Increased Extracellular Matrix Deposition Research Articles

TMCO1 promotes ferroptosis and ECM deposition in glaucomatous trabecular meshwork via ERK1/2 signaling

Glaucoma, a leading cause of global blindness, is marked by irreversible retinal ganglion cells (RGCs) loss, elevated intraocular pressure (IOP), and extracellular matrix (ECM) deposition in the trabecular meshwork (TM). Transmembrane and coiled-coil domain protein 1 (TMCO1), implicated in calcium regulation, has potential links to primary open-angle glaucoma (POAG). Ferroptosis, an iron-dependent cell death mechanism driven by lipid peroxidation, is also observed in glaucoma. This study investigates the role of TMCO1 in POAG, focusing on its involvement in TM ECM deposition via ferroptosis induction and ERK1/2 phosphorylation inhibition.In both in vivo and in vitro models, we demonstrated that dexamethasone (DEX) stimulation upregulates TMCO1, leading to increased ECM deposition and ferroptosis in human trabecular meshwork cells (HTMCs). Furthermore, treatment with ferrostatin-1 (Fer-1), a ferroptosis inhibitor, significantly reduced ECM deposition and ferroptosis in HTMCs. These findings establish TMCO1 as a critical regulator of ferroptosis and ECM deposition through the ERK/MAPK pathway, positioning it as a promising therapeutic target for glaucoma.

Mammary tissue-derived extracellular matrix hydrogels reveal the role of irradiation in driving a pro-tumor and immunosuppressive microenvironment

Radiation therapy (RT) is essential for triple negative breast cancer (TNBC) treatment. However, patients with TNBC continue to experience recurrence after RT. The role of the extracellular matrix (ECM) of irradiated breast tissue in tumor recurrence is still unknown. In this study, we evaluated the structure, molecular composition, and mechanical properties of irradiated murine mammary fat pads (MFPs) and developed ECM hydrogels from decellularized tissues (dECM) to assess the effects of RT-induced ECM changes on breast cancer cell behavior. Irradiated MFPs were characterized by increased ECM deposition and fiber density compared to unirradiated controls, which may provide a platform for cell invasion and proliferation. ECM component changes in collagens I, IV, and VI, and fibronectin were observed following irradiation in both MFPs and dECM hydrogels. Encapsulated TNBC cell proliferation and invasive capacity was enhanced in irradiated dECM hydrogels. In addition, TNBC cells co-cultured with macrophages in irradiated dECM hydrogels induced M2 macrophage polarization and exhibited further increases in proliferation. Our study establishes that the ECM in radiation-damaged sites promotes TNBC invasion and proliferation as well as an immunosuppressive microenvironment. This work represents an important step toward elucidating how changes in the ECM after RT contribute to breast cancer recurrence.

A MODEL TO STUDY INTESTINAL FIBROSIS IN IPSC-DERIVED HUMAN EPITHELIAL AND MESENCHYMAL CELLS FROM CROHN’S DISEASE PATIENTS

Abstract INTRODUCTION Intestinal fibrosis is a serious complication of Crohn's disease (CD) and is caused by the excess deposition of extracellular matrix (ECM) proteins. Numerous cell types, including intestinal epithelial and mesenchymal cells, are implicated in this but research efforts are impaired by a lack of in vitro models. To overcome this, we have previously developed a human model of intestinal fibrosis using iPSC-derived epithelial and mesenchymal cells generated from both control and CD patients. Using this model, we aimed to further characterize the transcriptomic signature associated with the fibrotic response as well as other functional hallmarks of fibrosis. METHODS iPSCs from 19 CD patients with (10) and without (9) fibrotic complications were directed to form HIOs. As iPSC-derived HIOs contain epithelial and mesenchymal cells, the organoids were dissociated to generate purified cultures of epithelial only-HIOs (eHIOs) and intestinal mesenchymal cells (MSCs). eHIO and MSC cultures were treated with TNFα and TGFβ, alone or in combination. We evaluated the transcriptomic profile of both cell types via RNA-seq and carried out additional studies via ELISA, ECM staining and scratch assay. RESULTS RNAseq analysis on the effects of cytokine induction in eHIOs lead to an enrichment in pathways associated with inflammation and regulation of the immune response. Furthermore, eHIOs from patients with fibrotic complications showed a significant upregulation of ORM1 regardless of culture conditions, which was subsequently confirmed at the protein level indicating an intrinsic difference between both patient groups. RNAseq analysis on MSC cultures found differentially expressed genes clustered in pathways associated with ECM production and remodeling, and cell migration and proliferation. Cell migration capacity of MSCs was assessed by measuring the reduction in scratch area and under basal conditions, MSCs from the fibrotic cohort had a lower rate of cell migration (40.81%) compared to the non-fibrotic cohort (20.97%); notably, the addition of TNFα appeared to accelerate the migration capacity of MSCs in both fibrotic (14.54%) and non-fibrotic (6.56%) samples. Finally, compared to untreated cultures, MSCs treated with TNFα/TGFβ exhibited a significant increase in ECM deposition (2.64- and 2.29-fold increase for non-fibrotic and fibrotic, respectively). CONCLUSION We have demonstrated the functionality of our model of intestinal fibrosis generated from iPSC-derived epithelial and mesenchymal cells from CD patients. Furthermore, we have identified both generalized and intrinsic differences in our cohort of patients with fibrotic complications. Future experiments on the role of ORM1 in intestinal fibrosis and its effects on mesenchymal cells could provide a deeper understanding of the events leading the development of intestinal fibrosis in CD patients.

Matrix stiffness triggers lipid metabolic crosstalk between tumor and stromal cells to mediate bevacizumab resistance in colorectal cancer liver metastases.

Bevacizumab is an anti-vascular endothelial growth factor (VEGF) monoclonal antibody that plays an important role in the combination treatment of advanced colorectal cancer (CRC). However, resistance remains a major hurdle limiting bevacizumab efficacy, highlighting the importance of identifying mechanism of anti-angiogenic therapy resistance. Here, we investigated biophysical properties of the extracellular matrix (ECM) related to metabolic processes and acquired resistance to bevacizumab. Evaluation of paired pre- and post-treatment samples of liver metastases from 20 CRC patients treated with combination bevacizumab therapy, including 10 responders and 10 non-responders, indicated that ECM deposition in liver metastases and a highly activated fatty acid oxidation (FAO) pathway were elevated in non-responders after anti-angiogenic therapy compared to responders. In mouse models of liver metastatic CRC, anti-VEGF increased ECM deposition and FAO in CRC cells, and treatment with the FAO inhibitor etomoxir enhanced the efficacy of antiangiogenic therapy. Hepatic stellate cells (HSCs) were essential for matrix stiffness-mediated FAO in colon cancer cells. Matrix stiffness activated lipolysis in HSCs via the focal adhesion kinase (FAK)/yes-associated protein (YAP) pathway, and free fatty acids secreted by HSCs were absorbed as metabolic substrates and activated FAO in colon cancer cells. Suppressing HSC lipolysis using FAK and YAP inhibition enhanced the efficacy of anti-VEGF therapy. Together, these results indicate that bevacizumab-induced ECM remodeling triggers lipid metabolic crosstalk between colon cancer cells and HSCs. This metabolic mechanism of bevacizumab resistance mediated by the physical tumor microenvironment represents a potential therapeutic target for reversing drug resistance.

Oxidative Stress and Lung Fibrosis: Towards an Adverse Outcome Pathway.

Lung fibrosis is a progressive fatal disease in which deregulated wound healing of lung epithelial cells drives progressive fibrotic changes. Persistent lung injury due to oxidative stress and chronic inflammation are central features of lung fibrosis. Chronic cigarette smoking causes oxidative stress and is a major risk factor for lung fibrosis. The objective of this manuscript is to develop an adverse outcome pathway (AOP) that serves as a framework for investigation of the mechanisms of lung fibrosis due to lung injury caused by inhaled toxicants, including cigarette smoke. Based on the weight of evidence, oxidative stress is proposed as a molecular initiating event (MIE) which leads to increased secretion of proinflammatory and profibrotic mediators (key event 1 (KE1)). At the cellular level, these proinflammatory signals induce the recruitment of inflammatory cells (KE2), which in turn, increase fibroblast proliferation and myofibroblast differentiation (KE3). At the tissue level, an increase in extracellular matrix deposition (KE4) subsequently culminates in lung fibrosis, the adverse outcome. We have also defined a new KE relationship between the MIE and KE3. This AOP provides a mechanistic platform to understand and evaluate how persistent oxidative stress from lung injury may develop into lung fibrosis.

Extracellular matrix remodelling in obesity and metabolic disorders.

Obesity causes extracellular matrix (ECM) remodelling which can develop into serious pathology and fibrosis, having metabolic effects in insulin-sensitive tissues. The ECM components may be increased in response to overnutrition. This review will focus on specific obesity-associated molecular and pathophysiological mechanisms of ECM remodelling and the impact of specific interactions on tissue metabolism. In obesity, complex network of signalling molecules such as cytokines and growth factors have been implicated in fibrosis. Increased ECM deposition contributes to the pathogenesis of insulin resistance at least in part through activation of cell surface integrin receptors and CD44 signalling cascades. These cell surface receptors transmit signals to the cell adhesome which orchestrates an intracellular response that adapts to the extracellular environment. Matrix proteins, glycoproteins, and polysaccharides interact through ligand-specific cell surface receptors that interact with the cytosolic adhesion proteins to elicit specific actions. Cell adhesion proteins may have catalytic activity or serve as scaffolds. The vast number of cell surface receptors and the complexity of the cell adhesome have made study of their roles challenging in health and disease. Further complicating the role of ECM-cell receptor interactions is the variation between cell types. This review will focus on recent insights gained from studies of two highly conserved, ubiquitously axes and how they contribute to insulin resistance and metabolic dysfunction in obesity. These are the collagen-integrin receptor-IPP (ILK-PINCH-Parvin) axis and the hyaluronan-CD44 interaction. We speculate that targeting ECM components or their receptor-mediated cell signalling may provide novel insights into the treatment of obesity-associated cardiometabolic complications.

Inflammation and hypertension: Underlying mechanisms and emerging understandings.

Hypertension remains a major contributor to cardiovascular disease (CVD), a leading cause of global death. One of the major insults that drive increased blood pressure is inflammation. While it is the body's defensive response against some homeostatic imbalances, inflammation, when dysregulated, can be very deleterious. In this review, we highlight and discuss the causative relationship between inflammation and hypertension. We critically discuss how the interplay between inflammation and reactive oxygen species evokes endothelial damage and dysfunction, ultimately leading to narrowing and stiffness of blood vessels. This, along with phenotypic switching of the vascular smooth muscle cells and the abnormal increase in extracellular matrix deposition further exacerbates arterial stiffness and noncompliance. We also discuss how hyperhomocysteinemia and microRNA act as links between inflammation and hypertension. The premises we discuss suggest that the blue-sky scenarios for targeting the underlying mechanisms of hypertension necessitate further research.

Single nuclei transcriptomics delineates complex immune and kidney cell interactions contributing to kidney allograft fibrosis

Chronic allograft dysfunction (CAD), characterized histologically by interstitial fibrosis and tubular atrophy, is the major cause of kidney allograft loss. Here, using single nuclei RNA sequencing and transcriptome analysis, we identified the origin, functional heterogeneity, and regulation of fibrosis-forming cells in kidney allografts with CAD. A robust technique was used to isolate individual nuclei from kidney allograft biopsies and successfully profiled 23,980 nuclei from five kidney transplant recipients with CAD and 17,913 nuclei from three patients with normal allograft function. Our analysis revealed two distinct states of fibrosis in CAD; low and high extracellular matrix (ECM) with distinct kidney cell subclusters, immune cell types, and transcriptional profiles. Imaging mass cytometry analysis confirmed increased ECM deposition at the protein level. Proximal tubular cells transitioned to an injured mixed tubular (MT1) phenotype comprised of activated fibroblasts and myofibroblast markers, generated provisional ECM which recruited inflammatory cells, and served as the main driver of fibrosis. MT1 cells in the high ECM state achieved replicative repair evidenced by dedifferentiation and nephrogenic transcriptional signatures. MT1 in the low ECM state showed decreased apoptosis, decreased cycling tubular cells, and severe metabolic dysfunction, limiting the potential for repair. Activated B, T and plasma cells were increased in the high ECM state, while macrophage subtypes were increased in the low ECM state. Intercellular communication between kidney parenchymal cells and donor-derived macrophages, detected several years post-transplantation, played a key role in injury propagation. Thus, our study identified novel molecular targets for interventions aimed to ameliorate or prevent allograft fibrogenesis in kidney transplant recipients.

AMPK/Sirt1-mediated inflammation is positively correlated with myocardial fibrosis during ageing

Objectives Cardiovascular disease is the leading cause of death in the world, and it increases dramatically with ageing. The objective of this study was to elucidate age-dependent molecular changes of inflammation and its correlation with the progression of myocardial fibrosis. Methods Methods: Male SD rats aged 3, 6, 9 and 24 months were used in this study. H&E staining was used to assessed histo-morphological changes in different ages. Masson’s trichrome staining was used to evaluate myocardial fibrosis. Immunofluorescence as well as western blot was carried out to detect the expression of vimentin. Real-time PCR was used to detect the level of pro-inflammatory chemokines MCP-1, IL1β, TNFα and IL-6. Western blotting was also carried out to detect p-AMPK, Sirt1, AC-NF-κB expression. Results Myocardial pathological changes and fibrosis are positively correlated with age. Ageing rats showed an enhanced expression of inflammatory factors and the activation of cardiac fibroblasts increases. Meanwhile, the expression of p-AMPK, Sirt1 and downstream AC-NF-κB increased significantly during ageing. Furthermore, the 15–24 months of age in rats is the fastest changing stage of increased inflammation and decreased Sirt1 activity. Conclusions Ageing is an independent risk factor for the occurrence and development of myocardial fibrosis. During ageing, myocardial fibroblasts are activated, accompanied by an increase in extracellular matrix deposition. The inflammation mediated by AMPK/Sirt1/NF-κB signalling pathway is closely positively correlated with the activation of myocardial fibroblasts and the progression of myocardial fibrosis.

Cancer-Associated Fibroblasts in a 3D Engineered Tissue Model Induce Tumor-like Matrix Stiffening and EMT Transition.

Simple SummaryThe physical properties of a tumor, such as stiffness, are important drivers of tumor progression. However, current in vitro tumor models fail to recapitulate the full range of physical properties observed in solid tumors. Here, we proposed a 3D self-assembly engineered bladder model using cancer-associated fibroblasts in which stromal cells produce their extracellular matrix. We then proceeded to assess how our model recapitulates biological and mechanical features found in tumors. We confirmed that stroma assembled by cancer-associated fibroblasts have increased extracellular matrix content and display increased remodeling and higher stiffness. Moreover, normal urothelial cells seeded on the tumor model displayed a mechanotransduction response, increased cell proliferation, cell infiltration within stroma, and displayed features of the epithelial-to-mesenchymal transition. Altogether, we demonstrated that our cancer-associated fibroblast-derived tumor stroma recapitulates several biological and physical features expected from a tumor-like environment and, thus, provides the basis for more accurate cancer models.A tumor microenvironment is characterized by its altered mechanical properties. However, most models remain unable to faithfully recreate the mechanical properties of a tumor. Engineered models based on the self-assembly method have the potential to better recapitulate the stroma architecture and composition. Here, we used the self-assembly method based on a bladder tissue model to engineer a tumor-like environment. The tissue-engineered tumor models were reconstituted from stroma-derived healthy primary fibroblasts (HFs) induced into cancer-associated fibroblast cells (iCAFs) along with an urothelium overlay. The iCAFs-derived extracellular matrix (ECM) composition was found to be stiffer, with increased ECM deposition and remodeling. The urothelial cells overlaid on the iCAFs-derived ECM were more contractile, as measured by quantitative polarization microscopy, and displayed increased YAP nuclear translocation. We further showed that the proliferation and expression of epithelial-to-mesenchymal transition (EMT) marker in the urothelial cells correlate with the increased stiffness of the iCAFs-derived ECM. Our data showed an increased expression of EMT markers within the urothelium on the iCAFs-derived ECM. Together, our results demonstrate that our tissue-engineered tumor model can achieve stiffness levels comparable to that of a bladder tumor, while triggering a tumor-like response from the urothelium.

Macromolecular crowding tuned extracellular matrix deposition in a bioprinted human rhabdomyosarcoma model

The role of the extracellular matrix (ECM) in tumor recurrence and metastasis has been gaining attention. Indeed, not only cellular, but also structural proteins influence migratory and invasive capacity of tumor cells, including growth and resistance to drugs. Therefore, new in vitro tumor models that entail improved ECM formation and deposition are needed. Here, we are developed three-dimensional (3D) models of pediatric soft tissue sarcoma (Rhabdomyosarcoma [RMS]) with the two major subgroups, the embryonal (ERMS) and the alveolar (ARMS) form. We applied macromolecular crowding (MMC) technology to monolayer cultures, spheroids, and 3D bioprinted constructs. In all culture models, exposure to MMC significantly increased ECM deposition. Interestingly, bioprinted constructs showed a collagen and fibronectin matrix architecture that was comparable to that of tumor xenografts. Furthermore, the bioprinted model not only showed tumor cell growth inside the structure but also displayed cell clusters leaving the edges of the bioprinted construct, probably emulating a metastatic mechanism. ARMS and ERMS cells reacted differently in the bioprinted structure. Indeed, the characteristic metastatic behavior was much more pronounced in the more aggressive ARMS subtype. This promising approach opens new avenues for studying RMS microenvironment and creating a platform for cancer drug testing including the native tumor ECM.

Interplay between extracellular matrix components and cellular and molecular mechanisms in kidney fibrosis

Chronic kidney disease (CKD) is characterized by pathological accumulation of extracellular matrix (ECM) proteins in renal structures. Tubulointerstitial fibrosis is observed in glomerular diseases as well as in the regeneration failure of acute kidney injury (AKI). Therefore, finding antifibrotic therapies comprises an intensive research field in Nephrology. Nowadays, ECM is not only considered as a cellular scaffold, but also exerts important cellular functions. In this review, we describe the cellular and molecular mechanisms involved in kidney fibrosis, paying particular attention to ECM components, profibrotic factors and cell-matrix interactions. In response to kidney damage, activation of glomerular and/or tubular cells may induce aberrant phenotypes characterized by overproduction of proinflammatory and profibrotic factors, and thus contribute to CKD progression. Among ECM components, matricellular proteins can regulate cell-ECM interactions, as well as cellular phenotype changes. Regarding kidney fibrosis, one of the most studied matricellular proteins is cellular communication network-2 (CCN2), also called connective tissue growth factor (CTGF), currently considered as a fibrotic marker and a potential therapeutic target. Integrins connect the ECM proteins to the actin cytoskeleton and several downstream signaling pathways that enable cells to respond to external stimuli in a coordinated manner and maintain optimal tissue stiffness. In kidney fibrosis, there is an increase in ECM deposition, lower ECM degradation and ECM proteins cross-linking, leading to an alteration in the tissue mechanical properties and their responses to injurious stimuli. A better understanding of these complex cellular and molecular events could help us to improve the antifibrotic therapies for CKD.

Bi-allelic premature truncating variants in LTBP1 cause cutis laxa syndrome

Latent transforming growth factor β (TGFβ)-binding proteins (LTBPs) are microfibril-associated proteins essential for anchoring TGFβ in the extracellular matrix (ECM) as well as for correct assembly of ECM components. Variants in LTBP2, LTBP3, and LTBP4 have been identified in several autosomal recessive Mendelian disorders with skeletal abnormalities with or without impaired development of elastin-rich tissues. Thus far, the human phenotype associated with LTBP1 deficiency has remained enigmatic. In this study, we report homozygous premature truncating LTBP1 variants in eight affected individuals from four unrelated consanguineous families. Affected individuals present with connective tissue features (cutis laxa and inguinal hernia), craniofacial dysmorphology, variable heart defects, and prominent skeletal features (craniosynostosis, short stature, brachydactyly, and syndactyly). Invitro studies on proband-derived dermal fibroblasts indicate distinct molecular mechanisms depending on the position of the variant in LTBP1. C-terminal variants lead to an altered LTBP1 loosely anchored in the microfibrillar network and cause increased ECM deposition in cultured fibroblasts associated with excessive TGFβ growth factor activation and signaling. In contrast, N-terminal truncation results in a loss of LTBP1 that does not alter TGFβ levels or ECM assembly. Invivo validation with two independent zebrafish lines carrying mutations in ltbp1 induce abnormal collagen fibrillogenesis in skin and intervertebral ligaments and ectopic bone formation on the vertebrae. In addition, one of the mutant zebrafish lines shows voluminous and hypo-mineralized vertebrae. Overall, our findings in humans and zebrafish show that LTBP1 function is crucial for skin and bone ECM assembly and homeostasis.

HGF and TSG-6 Released by Mesenchymal Stem Cells Attenuate Colon Radiation-Induced Fibrosis.

Fibrosis is a leading cause of death in occidental states. The increasing number of patients with fibrosis requires innovative approaches. Despite the proven beneficial effects of mesenchymal stem cell (MSC) therapy on fibrosis, there is little evidence of their anti-fibrotic effects in colorectal fibrosis. The ability of MSCs to reduce radiation-induced colorectal fibrosis has been studied in vivo in Sprague–Dawley rats. After local radiation exposure, rats were injected with MSCs before an initiation of fibrosis. MSCs mediated a downregulation of fibrogenesis by a control of extra cellular matrix (ECM) turnover. For a better understanding of the mechanisms, we used an in vitro model of irradiated cocultured colorectal fibrosis in the presence of human MSCs. Pro-fibrotic cells in the colon are mainly intestinal fibroblasts and smooth muscle cells. Intestinal fibroblasts and smooth muscle cells were irradiated and cocultured in the presence of unirradiated MSCs. MSCs mediated a decrease in profibrotic gene expression and proteins secretion. Silencing hepatocyte growth factor (HGF) and tumor necrosis factor-stimulated gene 6 (TSG-6) in MSCs confirmed the complementary effects of these two genes. HGF and TSG-6 limited the progression of fibrosis by reducing activation of the smooth muscle cells and myofibroblast. To settle in vivo the contribution of HGF and TSG-6 in MSC-antifibrotic effects, rats were treated with MSCs silenced for HGF or TSG-6. HGF and TSG-6 silencing in transplanted MSCs resulted in a significant increase in ECM deposition in colon. These results emphasize the potential of MSCs to influence the pathophysiology of fibrosis-related diseases, which represent a challenging area for innovative treatments.

Extracellular matrix remodelling is associated with muscle force increase in overloaded mouse plantaris muscle.

Transforming growth factor-β (TGF-β) signalling is thought to contribute to the remodelling of extracellular matrix (ECM) of skeletal muscle and to functional decline in patients with muscular dystrophies. We wanted to determine the role of TGF-β-induced ECM remodelling in dystrophic muscle. We experimentally induced the pathological hallmarks of severe muscular dystrophy by mechanically overloading the plantaris muscle in mice. Furthermore, we determined the role of TGF-β signalling on dystrophic tissue modulation and on muscle function by (i) overloading myostatin knockout (Mstn-/- ) mice and (ii) by additional pharmacological TGF-β inhibition via halofuginone. Transcriptome analysis of overloaded muscles revealed upregulation predominantly of genes associated with ECM, inflammation and metalloproteinase activity. Histology revealed in wild-type mice signs of severe muscular dystrophy including myofibres with large variation in size and internalized myonuclei, as well as increased ECM deposition. At the same time, muscle weight had increased by 208% and muscle force by 234%. Myostatin deficiency blunted the effect of overload on muscle mass (59% increase) and force (76% increase), while having no effect on ECM deposition. Concomitant treatment with halofuginone blunted overload-induced muscle hypertrophy and muscle force increase, while reducing ECM deposition and increasing myofibre size. ECM remodelling is associated with an increase in muscle mass and force in overload-modelled dystrophic muscle. Lack of myostatin is not advantageous and inhibition of ECM deposition by halofuginone is disadvantageous for muscle plasticity in response to stimuli that induce dystrophic muscle.

The Thymus in Chagas Disease: Molecular Interactions Involved in Abnormal T-Cell Migration and Differentiation.

Chagas disease, caused by the protozoan parasite T. cruzi, is a prevalent parasitic disease in Latin America. Presently, it is spreading around the world by human migration, thus representing a new global health issue. Chronically infected individuals reveal a dissimilar disease progression: while nearly 60% remain without apparent disease for life, 30% develop life-threatening pathologies, such as chronic chagasic cardiomyopathy (CCC) or megaviscerae. Inflammation driven by parasite persistence seems to be involved in the pathophysiology of the disease. However, there is also evidence of the occurrence of autoimmune events, mainly caused by molecular mimicry and bystander activation. In experimental models of disease, is well-established that T. cruzi infects the thymus and causes locally profound structural and functional alterations. The hallmark is a massive loss of CD4+CD8+ double positive (DP) thymocytes, mainly triggered by increased levels of glucocorticoids, although other mechanisms seem to act simultaneously. Thymic epithelial cells (TEC) exhibited an increase in extracellular matrix deposition, which are related to thymocyte migratory alterations. Moreover, medullary TEC showed a decreased expression of AIRE and altered expression of microRNAs, which might be linked to a disrupted negative selection of the T-cell repertoire. Also, almost all stages of thymocyte development are altered, including an abnormal output of CD4−CD8− double negative (DN) and DP immature and mature cells, many of them carrying prohibited TCR-Vβ segments. Evidence has shown that DN and DP cells with an activated phenotype can be tracked in the blood of humans with chronic Chagas disease and also in the secondary lymphoid organs and heart of infected mice, raising new questions about the relevance of these populations in the pathogenesis of Chagas disease and their possible link with thymic alterations and an immunoendocrine imbalance. Here, we discuss diverse molecular mechanisms underlying thymic abnormalities occurring during T. cruzi infection and their link with CCC, which may contribute to the design of innovative strategies to control Chagas disease pathology.

Ex-vivo cultured human corneoscleral segment model to study the effects of glaucoma factors on trabecular meshwork.

Glaucoma is the second leading cause of irreversible blindness worldwide. Primary open angle glaucoma (POAG), the most common form of glaucoma, is often associated with elevation of intraocular pressure (IOP) due to the dysfunction of trabecular meshwork (TM) tissues. Currently, an ex vivo human anterior segment perfusion cultured system is widely used to study the effects of glaucoma factors and disease modifying drugs on physiological parameters like aqueous humor (AH) dynamics and IOP homeostasis. This system requires the use of freshly enucleated intact human eyes, which are sparsely available at very high cost. In this study, we explored the feasibility of using human donor corneoscleral segments for modeling morphological and biochemical changes associated with POAG. Among the number of corneas donated each year, many are deemed ineligible for transplantation due to stringent acceptance criteria. These ineligible corneoscleral segments were obtained from the Lions Eye Bank, Tampa, Florida. Each human donor anterior corneoscleral segment was dissected into four equal quadrants and cultured for 7 days by treating with the glaucoma factors dexamethasone (Dex) or recombinant transforming growth factor (TGF) β2 or transduced with lentiviral expression vectors containing wild type (WT) and mutant myocilin. Hematoxylin and Eosin (H&E) staining analysis revealed that the TM structural integrity is maintained after 7 days in culture. Increased TUNEL positive TM cells were observed in corneoscleral quadrants treated with glaucoma factors compared to their respective controls. However, these TUNEL positive cells were mainly confined to the scleral region adjacent to the TM. Treatment of corneoscleral quadrants with Dex or TGFβ2 resulted in glaucomatous changes at the TM, which included increased extracellular matrix (ECM) proteins and induction of endoplasmic reticulum (ER) stress. Western blot analysis of the conditioned medium showed an increase in ECM (fibronectin and collagen IV) levels in Dex- or TGFβ2-treated samples compared to control. Lentiviral transduction of quadrants resulted in expression of WT and mutant myocilin in TM tissues. Western blot analysis of conditioned medium revealed decreased secretion of mutant myocilin compared to WT myocilin. Moreover, increased ECM deposition and ER stress induction was observed in the TM of mutant myocilin transduced quadrants. Our findings suggest that the ex-vivo cultured human corneoscleral segment model is cost-effective and can be used as a pre-screening tool to study the effects of glaucoma factors and anti-glaucoma therapeutics on the TM.

Predifferentiated Smooth Muscle-Like Adipose-Derived Stem Cells for Bladder Engineering.

Introduction: All organs of human body are a conglomerate of various cell types with multidirectional interplay between the different cells and the surrounding microenvironment, leading to a stable tissue formation, homeostasis, and function. To develop a functional smooth muscle tissue, we need to simulate and create a multicellular microenvironment. The multilineage adipose-derived stem cells (ADSCs), which can be easily harvested in large numbers, may provide an alternative cell source for the replacement of smooth muscle cells (SMCs) in cell-based detrusor bioengineering therapeutic approaches. The aim of this study was to investigate whether predifferentiated smooth muscle-like ADSC (pADSC) can support SMCs to generate stable smooth muscle tissue through remodeling of extracellular matrix (ECM) and factor secretion. Methods: Rat SMC and pADSC were mono- and cocultured in the cell ratios 1:1, 1:2, 1:3, and 1:5 (SMC-pADSC) and grown for up to 2 weeks in vitro. The expression of the SMC-specific markers alpha-smooth muscle actin, calponin, myosin heavy chain 11 (MyH11), and smoothelin was assessed, and cell proliferation and contractility were analyzed. Proteomic analysis of the secretome (cell-cell contact was compared with a noncontact transwell 1:1 coculture) and the cell pellets was performed, with the focus on ECM deposition and remodeling, integrin expression and growth factor secretion. Results: SMC and pADSC were strongly positive for all smooth muscle markers. After 1 and 2 weeks of culture, the 1:1 cell ratio developed a significantly higher number of smooth muscle organoids and improved contractility. These organoids were highly structured, consisting of an SMC core surrounded by a pADSC layer. The deposition of various EMC proteins, such as collagens 1a1, 1a2, 2a1, 3a1, 5a2, 6a2, 12a1, and fibrillin 1, was significantly increased. A decreased matrix metalloproteinase 3 (MMP3), MMP9 and MMP13 secretion, as well as increased tissue inhibitors of metalloproteinase 1 (TIMP1) and TIMP2 secretion were found in the contact coculture compared with the monoculture controls. Conclusion: SMC-pADSC 1:1 cocultures exhibit an improved cell proliferation, contractility, and organoid formation compared with all other ratios and monoculture, while retaining a stable phenotype that is comparable with the SMC monoculture. These effects are mediated by increased ECM deposition and tight ECM remodeling by the secreted MMP and TIMP. Impact statement Harvesting smooth muscle cells (SMCs) from diseased bladders represents a significant limitation for clinical translation of bladder Tissue Engineering. Our results suggest that autologous predifferentiated smooth muscle-like adipose-derived stem cell can substitute SMCs, and may be used in combination with SMCs to generate contractile detrusor muscle tissue for patients suffering from end-stage bladder diseases. We demonstrate a beneficial effect when using these cells in a 1:1 ratio with improved deposition of extracellular matrix (ECM) molecules and superior remodeling of the ECM by matrix metalloproteinases and decreased tissue inhibitors of metalloproteinase activity.

Collagen Stiffness and Architecture Regulate Fibrotic Gene Expression in Engineered Adipose Tissue.

Adipose tissue (AT) has a dynamic extracellular matrix (ECM) surrounding adipocytes that allows for remodeling during metabolic fluctuations. During the progression of obesity, AT has increased ECM deposition, stiffening, and remodeling, resulting in a pro-fibrotic dysfunctional state. Here, the incorporation of ethylene glycol-bis-succinic acid N-hydroxysuccinimide ester (PEGDS) allows for control over 3D collagen hydrogel stiffness and architecture to investigate its influence on adipocyte metabolic and fibrotic function. Upon stiffening and altering ECM architecture, adipocytes did not alter their expression of key adipokines, leptin, and adiponectin. However, they do increase actin cytoskeletal fiber formation, pro-fibrotic gene expression, ECM deposition, and remodeling within a stiffer, 3D collagen hydrogel. For example, COL6A3 gene expression is upregulated approximately twofold, resulting in increased deposition of pericellular collagen VI alpha 3 surrounding adipocytes. Furthermore, inhibition of actin contractility results in a reversal of pro-fibrotic gene expression and ECM deposition, indicating that adipocytes are mediating mechanical cues through actin cytoskeletal networks. This study demonstrates that ECM stiffness and architecture plays a critical regulatory role in adipocyte fibrotic function and contributes to the overall pro-fibrotic dysfunctional state of AT during the progression of obesity and AT fibrosis.

Aberrant DNA methylation of miRNAs in Fuchs endothelial corneal dystrophy

Homeostatic maintenance of corneal endothelial cells is essential for maintenance of corneal deturgescence and transparency. In Fuchs endothelial corneal dystrophy (FECD), an accelerated loss and dysfunction of endothelial cells leads to progressively severe visual impairment. An abnormal accumulation of extracellular matrix (ECM) is a distinctive hallmark of the disease, however the molecular pathogenic mechanisms underlying this phenomenon are not fully understood. Here, we investigate genome-wide and sequence-specific DNA methylation changes of miRNA genes in corneal endothelial samples from FECD patients. We discover that miRNA gene promoters are frequent targets of aberrant DNA methylation in FECD. More specifically, miR-199B is extensively hypermethylated and its mature transcript miR-199b-5p was previously found to be almost completely silenced in FECD. Furthermore, we find that miR-199b-5p directly and negatively regulates Snai1 and ZEB1, two zinc finger transcription factors that lead to increased ECM deposition in FECD. Taken together, these findings suggest a novel epigenetic regulatory mechanism of matrix protein production by corneal endothelial cells in which miR-199B hypermethylation leads to miR-199b-5p downregulation and thereby the increased expression of its target genes, including Snai1 and ZEB1. Our results support miR-199b-5p as a potential therapeutic target to prevent or slow down the progression of FECD disease.