Extracellular Matrix Production Research Articles

Fibrotic Changes in Rhegmatogenous Retinal Detachment

Rhegmatogenous retinal detachment (RRD) is a sight-threatening condition involving retinal detachment and the accumulation of fluid in the subretinal space. Proliferative vitreoretinopathy (PVR) is a pathologic complication that develops after RRD surgery, and approximately 5–10% of RRD cases develop post-operative PVR. Prolonged inflammation in the wound healing process, epithelial–mesenchymal transition (EMT), retinal pigment epithelial (RPE) cell migration and proliferation, and epiretinal, intraretinal, and subretinal fibrosis are typical in the formation of PVR. RPE cells undergo EMT and become fibroblast-like cells that migrate to the retina and vitreous, promoting PVR formation. Fibroblasts transform into myofibroblasts, which promote fibrosis by overproducing the extracellular matrix (ECM). RPE cells, fibroblasts, glial cells, macrophages, T lymphocytes, and increased ECM production form contractile epiretinal membranes. Cytokine release, complement activation, RPE cells, glial cells, and endothelial cells are all involved in retinal immune responses. Normally, wounds heal within 4 to 6 weeks, including hemostasis, inflammation, proliferation, and remodeling phases. Properly initiated inflammation, complement activation, and the function of neutrophils and glial cells heal the wound in the first stage. In a retinal wound, glial cells proliferate and fill the injured area. Gliosis tries to protect the neurons and prevent damage, but it becomes harmful when it causes scarring. If healing is complicated, prolonged inflammation leads to pathological fibrosis. Currently, there is no preventive treatment for the formation of PVR, and it is worth studying in the future.

P0196 Metabolic dysregulation in Crohn’s disease fibrosis: Targeting WISP1 to restore ECM homeostasis

Abstract Background In patients with Crohn’s disease (CD), homeostasis of the extracellular matrix (ECM) is significantly disrupted, frequently leading to severe complications such as intestinal stenosis. The maintenance of organ functionality depends on a delicate balance between inflammatory mediators, ECM production, and degradation processes. However, these mechanisms remain poorly understood. This study investigates the dynamic interplay between ECM homeostasis and metabolic pathways in CD, providing novel insights into their role in disease pathogenesis and potential therapeutic intervention. Methods Bulk RNA sequencing was performed to compare fibrotic and non-fibrotic intestinal tissues from CD patients undergoing ileocecal resection, aiming to identify key metabolic and ECM-related pathways. Primary matrix-producing fibroblasts were studied in 2D cultures and spheroids to characterize their metabolic activity, with a focus on fatty acid oxidation (FAO) and glycolysis through gene and protein analysis, metabolic assays, and fatty acid profiling (GC-FID). Chronic intestinal fibrosis was modelled in vivo using dextran sodium sulfate (DSS)-treated mice, allowing therapeutic evaluation of WISP1 neutralisation. The in vivo model was characterized using histological and clinical scoring, as well as gene and protein expression analysis. Results Gene set enrichment analysis revealed increased WNT signaling coupled with a reduction in fatty acid metabolism. Additionally, fibrotic tissue exhibited elevated levels of oleic acid and palmitate, indicative of increased lipid accumulation. WISP1, a WNT mediator, was identified as an adipokine and a key regulator of the metabolic shift from fatty acid oxidation to glycolysis. The disruption of FAO-related genes, including PPARγ, CPT1A, CPT2, and ACOX1, demonstrated reciprocal effects on fibroblast activity: pharmacological inhibition of FAO increased matrix production by fibroblasts, whereas activation of FAO via the PPARα agonist fenofibrate induced a catabolic fibroblast phenotype. This phenotype was characterized by suppressed ECM expression and enhanced ECM degradation via MMP1. Importantly, therapeutic neutralization of WISP1 significantly reduced fibrosis in a chronic DSS-induced colitis model. Conclusion This study presents a novel therapeutic strategy for managing fibrosis in Crohn’s disease. By pharmacologically neutralizing WISP1, this approach aims to correct the underlying metabolic dysfunction, thereby restoring metabolic balance and ECM homeostasis.

P0142 CD47-SIRPα Contributes to the Microenvironment of Stricturing Crohn's Disease by Mediating the Crosstalk between Macrophages and Stromal cells: “Eat Me” or “Don't Eat Me”

Abstract Background Intestinal fibrosis is a common complication of Crohn’s disease (CD), but no anti-fibrotic therapy is currently available. Collagen deposition, the pathological characteristics of intestinal fibrosis, is driven by an imbalance between extracellular matrix (ECM) production and degradation, which is controlled by the crosstalk between stromal cells and macrophage phagocytosis. The underlying mechanism of macrophage phagocytosis is the interplay between “eat me” signals and “don’t eat me”, among which CD47-SIRPa is the dominant pair. This study aims to investigate the role and therapeutic potential of CD47-SIRPa in intestinal fibrosis. Methods Thirty CD patients who underwent surgery due to fibrosis-induced intestinal obstruction were included in this study and the intestinal tissues were collected from both fibrotic and normal sites. Col1a2-CreERT2; Cd47 flox mice and Lyz2-Cre; SIRPα flox mice were used to conditionally ablate CD47/SIRPα. Mice intestinal fibrosis was induced by chronic dextrane sodium sulfate (DSS) and anti-CD47/anti-SIRPα antibody was treated intraperitoneally. Human and mice tissues were applied for single-cell RNA sequencing, flow cytometry, pathological evaluation, immunofluorescence staining, qPCR and in vitro experiment. Smart-seq were performed to detect the functional change of mice macrophage. Results Single-cell RNA sequencing and flow cytometry showed the proportion of CD47+ stromal cells and SIRPα+ macrophages significantly increased in intestinal fibrosis (Figure 1A). CD47+ stromal cells were in senescence-associated secretory phenotype (SASP), while macrophages also acquired a pro-fibrotic immature phenotype (all p<0.05). Moreover, we found the elevation of macrophage SIRPα expression was caused by CD47+ stromal cells and they extensively co-located in fibrotic lesions to form a pathogenic niche(Figure 1B). Conditionally ablation of CD47 in stromal cell or SIRPα in macrophage could significantly reduce collagen deposition(Figure 1C). Meanwhile, both anti-CD47 and anti-SIRPα treatment significantly attenuated mice intestinal fibrosis compared to the control.(Figure 1D) Mice intestinal macrophages acquired an anti-fibrotic phenotype and regained the capacity of phagocytosis by upregulating Clec7a and MMP10 after antibody treatment (all p<0.05) (Figure 1E). Furthermore, under the same total treatment dose, initiating antibody intervention at the earliest stage could achieve the best prognostic efficacy, highlighting its potential to be a therapeutic and preventive strategy against intestinal fibrosis (Figure 1F). Conclusion CD47-SIRPa mediated macrophage-stromal cell crosstalk significantly contributed to structuring CD and may be a novel therapeutic target for intestinal fibrosis.

P0007 mAb-based targeting of the eAGR2-Fibrinogen interaction as a promising new therapy in Inflammatory Bowel Disease

Abstract Background Historically, Fibrinogen (Fg) is known to be crucial in IBD by aiding fibrin deposition, which leads to scar tissue formation and tissue remodeling. High Fg levels in IBD foster chronic inflammation, activate fibroblasts, stimulate extracellular matrix production, promote fibrosis, and hinder normal mucosal repair. Recently, Anterior Gradient 2 (AGR2), an endoplasmic reticulum chaperone protein that is expressed by mucin-secreting epithelial cells, is secreted (eAGR2) under stress and contributes to a plethora of pathogenic processes (e.g. epithelial integrity disruption, monocyte attraction, fibroblast activation, and oncogenesis). AGR2 overexpression in the colon of IBD patients correlates with disease severity and activity. The present report identifies the eAGR2-Fg physical interaction, which opens new therapeutic avenues in IBD care. Methods CoIP-MS was used to identify interactors of eAGR2 in Caco2 supernatant. The binding between AGR2 and interactors was orthogonally validated using ELISA and abrogated by proprietary anti-eAGR2 mAb (TH-009). The efficacy of TH-009 in preventing the development of fibrosis was assessed using a murine chronic DSS model of colitis. Results IP-MS identified the three Fg subunits as the most abundant eAGR2 interactors in the Caco-2 supernatant. This interaction decreased fourfold with anti-eAGR2 mAbs. ELISA confirmed the specific binding of Fg to AGR2 (p<0.01), which was abrogated by TH-009 in a dose-dependent manner (p<0.001). In a murine chronic DSS model of colitis, multiple features of inflammation and fibrosis went down significantly when treated with TH-009, and anti-inflammatory molecular signature was promoted. For example, fibrosis was decreased by two-fold in TH-009 treated mice compared to that in the untreated DSS group (p<0.001). Also, collagen deposition and aSMA were decreased by 2.4 and 17-fold, respectively (p<0.001). At the mRNA level, a 2-fold decrease of TGFb, and fibronectin and a 3-fold decrease of ColA1 were observed (p=0,05; p<0.05 and p<0.01). Conclusion We identified Fg as a central eAGR2 partner and showed that this interaction was disrupted by the anti-eAGR2 mAb. Our results may suggest that eAGR2 binding to Fg impairs the fibrinogen-fibrin maturation cycle, thus maintaining the deleterious signaling observed in IBD. Considering the TH-009 efficacy in preventing fibrosis in vivo, our results support the therapeutic interest of targeting the eAGR2-Fg complex in IBD and other mucosal inflammatory diseases.

Transcriptome analysis of regenerated dermis stimulated by mechanical stretch.

Mechanical stretch is utilized in the process of tissue expansion to promote skin regeneration, which is crucial for wound healing and organ reconstruction purposes. Enlarged dermal area is one of the significant histological characteristics of the expanded skin. However, the underlying biological processes and molecular pathways associated with dermal regeneration triggered by mechanical stretch are still not well understood. Twelve male Sprague-Dawley (SD) rats were divided into the expansion group and sham group randomly. Upon creating a rat scalp expansion model, the dermis was isolated from the full-thickness skin in both experimental groups for RNA sequencing. This process led to the identification of differentially expressed genes (DEGs). Subsequently, we conducted Gene Ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and Gene Set Enrichment Analysis (GSEA) to identify the essential biological processes associated with dermal regeneration induced by mechanical stretch, leveraging data from the DEGs. A network of protein-protein interactions (PPI) was built to detect the critical modules and central genes. The expression levels of these hub genes were evaluated using quantitative real-time polymerase chain reaction (qPCR). Increased expanded skin area and dermal thinning which represent the typical changes of expanded skin were observed in the expansion group. A total of 782 DEGs were identified in the expansion group relative to the sham group. The DEGs were associated with several biological processes, including the organization of the extracellular matrix, the enhancement of macrophage activation, and the promotion of angiogenesis, among others. Cell components encompassing Toll-like receptor 2-Toll-like receptor 6 protein complex, interstitial matrix, extracellular matrix (ECM), and collagen trimer were discovered. Molecular function categories including integrin binding, insulin-like growth factor binding, and fatty acid elongase activity were involved. The KEGG pathway analysis demonstrated the significant enrichment of pathways including the PI3K-Akt signaling pathway, fatty acid metabolism, and extracellular matrix-receptor interactions. GSEA results displayed that mechanical stretch correlated with the regulation of cell activation processes, cytokine-mediated signaling pathways, and immune system processes. PPI network resulted in the identification of 598 nodes along with a total of 5,304 interaction pairs between proteins. And ten hub genes containing Ccl2, Cxcl10, Fasn, Itgad, Cd163, Mmp9, Cd36, Tlr2, Igf1, and Wnt2 were identified by bioinformatics analysis and validated by qPCR. This in vivo study for the first time revealed the DEGs related to mechanical stretch stimulated dermal regeneration and identified the involved pathways and hub genes correlated with macrophage recruitment and polarization, fibroblast proliferation and ECM production and angiogenesis, which may benefit further studies aimed at developing therapeutic strategies for facilitating expanded skin regeneration.

Current Treatment Regimens and Promising Molecular Therapies for Chronic Hepatobiliary Diseases.

Chronic hepatobiliary damage progressively leads to fibrosis, which may evolve into cirrhosis and/or hepatocellular carcinoma. The fight against the increasing incidence of liver-related morbidity and mortality is challenged by a lack of clinically validated early-stage biomarkers and the limited availability of effective anti-fibrotic therapies. Current research is focused on uncovering the pathogenetic mechanisms that drive liver fibrosis. Drugs targeting molecular pathways involved in chronic hepatobiliary diseases, such as inflammation, hepatic stellate cell activation and proliferation, and extracellular matrix production, are being developed. Etiology-specific treatments, such as those for hepatitis B and C viruses, are already in clinical use, and efforts to develop new, targeted therapies for other chronic hepatobiliary diseases are ongoing. In this review, we highlight the major molecular changes occurring in patients affected by metabolic dysfunction-associated steatotic liver disease, viral hepatitis (Delta virus), and autoimmune chronic liver diseases (autoimmune hepatitis, primary biliary cholangitis, and primary sclerosing cholangitis). Further, we describe how this knowledge is linked to current molecular therapies as well as ongoing preclinical and clinical research on novel targeting strategies, including nucleic acid-, mesenchymal stromal/stem cell-, and extracellular vesicle-based options. Much clinical development is obviously still missing, but the plethora of promising potential treatment strategies in chronic hepatobiliary diseases holds promise for a future reversal of the current increase in morbidity and mortality in this group of patients.

Microenvironment Remodeling Microgel Repairs Degenerated Intervertebral Disc via Programmed Delivery of MicroRNA-155.

The progression of intervertebral disc degeneration (IVDD) is associated with increased cell apoptosis and reduced extracellular matrix (ECM) production, both of which are driven by ongoing inflammation. Thus, alleviating the acidic inflammatory microenvironment and mitigating the apoptosis of nucleus pulposus cells (NPCs) are essential for intervertebral disc (IVD) regeneration. Regulating pH levels in the local environment can reduce inflammation and promote tissue recovery. In this study, a lactic acid-capturing microgel carrying a functionalized miRNA-155 nanocarrier was designed for IVD regeneration. microRNA-155 was loaded into the NPC-targeted nanogel via host-guest binding. The miR-155 nanocarrier (NGM) achieved lactic acid-sensitive release of miRNA-155, resulting in rapid regulation of apoptosis. Moreover, SS31, which dissociated from the nanogel network, had the ability to regulate mitochondrial metabolism. Moreover, the microgel was constructed using a matrix metalloproteinase-responsive peptide. The chitosan coating on the microgel system was ingeniously employed to capture lactic acid and enable pH-responsive dissociation, thereby alleviating the acidic microenvironment to protect cell viability and facilitate the delivery of the NGM. The microgel system effectively promoted IVD regeneration by alleviating the acidic microenvironment and preventing NPC apoptosis.

Targeting p97/Valosin-Containing Protein Promotes Hepatic Stellate Cell Senescence and Mitigates Liver Fibrosis.

Liver fibrosis, one of the main histological determinants of various chronic liver diseases, currently lacks effective treatment. Hepatic stellate cells (HSCs) are pivotal in the production of extracellular matrix and amplify the fibrogenic response. Inhibiting the activation of HSCs or promoting the senescence of activated HSCs is crucial for the regression of liver fibrosis. The ATPase p97, also known as valosin-containing protein (VCP), is a central component of the ubiquitin-proteasome system, and it regulates numerous cellular processes by influencing protein homeostasis. In this study, we observed an upregulation of p97 expression around regions exhibiting fibrosis in a diet- and chemical-induced nonalcoholic steatohepatitis and fibrosis murine model. Intervention with the p97 antagonist CB-5083 or the knockdown of p97 reduced the expression of alpha-smooth muscle actin and collagen-I in both mouse or human HSCs. The administration of CB-5083 induced HSC senescence and resulted in the upregulation of senescence markers, including p21, p53, GPX4, and senescence-associated β-galactosidase. Furthermore, CB-5083 treatment also inhibited the expression of Yes-associated protein (YAP), which is also a senescence-related regulatory protein and has a profibrotic function. We used CB-5083 to treat fibrotic mice and found that the activation of HSCs was inhibited, and the liver fibrosis was attenuated. In addition, invivo experiments confirmed that CB-5083 facilitated HSC senescence and reduced YAP expression. These findings underscore the potential of pharmacological targeting p97/VCP to induce HSC senescence and alleviate liver fibrosis.

Interpenetrating Polymer Network of Hydroxyethyl Methacrylate and Chitosan as Cryogels for Tissue Engineering Applications

ABSTRACTTissue engineering is a vast expanding field with applications in areas such as tissue/organ transplantation, drug delivery, in vitro models, and so on. Biomaterials form an essential component in tissue engineering by acting as a template for cellular activity, therefore, novel tissue‐engineered biomaterials with innovative properties are in high demand. Hence, this work proposes an interpenetrating polymer network cryogel of chitosan and hydroxyethyl methacrylate (HEMA) as a tissue‐engineered biomaterial with uniform and high cell seeding throughout the cryogel matrix. The physical analysis of the cryogels demonstrated a highly macroporous structure exhibiting uniform pore size distribution and overall porosity through field emission‐scanning electron microscopy (FE‐SEM) analysis with pore sizes lying in the range of 50–200 μm and 150–400 μm in horizontal and transverse plane, respectively. The cryogels were also found to be degradable with an average percent degradation of 17.28 ± 1.47% in 4 weeks, and their mechanical properties revealed an average compressive strength of 0.05 MPa and an elastic modulus of 3 MPa. Further, biological characterization of the cryogel through direct contact test depicted an excellent biocompatibility with L929 mouse fibroblast and MC3T3‐E1 preosteoblasts with negligible presence of dead cells in and around the cryogel. Uniform and high cell seeding with an increasing proliferation trend of MC3T3‐E1 cells was observed on these cryogels through live‐dead staining and MTT assay at day 1, 3, and 7‐time point. Cell adherence studies via FE‐SEM analysis also depicted a similar trend with uniform MC3T3‐E1 cell distribution along with highly flattened morphology and extracellular matrix production. Therefore, based on their promising physico‐chemical and biological properties, HEMA‐Chitosan cryogels exhibit a strong potential application as tissue engineered biomaterials.

The Transcriptomic and Gene Fusion Landscape of Pleomorphic Salivary Gland Adenomas.

Pleomorphic adenoma (PA) is the most common salivary gland tumor. PAs are characterized by chromosomal rearrangements of 8q12 and 12q14-15, leading to gene fusions involving the PLAG1 and HMGA2 oncogenes. Here, we performed the first comprehensive study of the transcriptomic and gene fusion landscape of 38 cytogenetically characterized PAs. RNA-seq identified PLAG1 or HMGA2 fusions in 33/38 cases (87%), of which 15 were novel fusions. Fusions were found also in tumors with normal karyotype, demonstrating that they are generated by cryptic rearrangements. PLAG1 was mainly activated by promoter swapping and HMGA2 by truncation of its 3'-part. RNA-seq revealed upregulation of genes involved in extracellular matrix production, WNT-signaling, and epithelial-mesenchymal transition in PA compared to normal salivary tissue. Principal component analysis identified two PA subclusters characterized by PLAG1- and HMGA2-activation, respectively, that differed in expression of genes involved in the immune system, cell adhesion, and microenvironment remodeling. Moreover, comparative analyses of PA and salivary carcinomas revealed that PA resembles myoepithelial carcinoma. Our study reveals new oncogenic gene fusions and expands our knowledge about the molecular underpinnings of PA.

FGF and TGF-β Growth Factor Isoform Modulation of Human Gingival and Periodontal Ligament Fibroblast Wound Healing Phenotype.

Release of growth factors in the tissue microenvironment is a critical process in the repair and regeneration of periodontal tissues, regulating fibroblast behavior and phenotype. As a result of the complex architecture of the periodontium, distinct fibroblast populations in the periodontal ligament and gingival connective tissue exist in close proximity. Growth factor therapies for periodontal regeneration have gained traction, but quantification of their effects on multiple different fibroblast populations that are required for repair has been poorly investigated. In this study, we examined the effects of TGF-β1, TGF-β3, FGF-2, and FGF-9 on human gingival fibroblasts (hGF) and human periodontal ligament cells (hPDL), as well as the combined effects of TGF-β3 and FGF-2. We show that FGF-2 enhances cell migration while TGF-β1 and TGF-β3 promotes matrix production, and TGF-β1 promotes fibroblast to myofibroblast transition. Interestingly, the combination of TGF-β3 and FGF-2, acting through both p-SMAD3 and p-ERK pathways, mitigates the inhibitory effects of TGF-β3 on migration in hPDL cells, suggesting synegystic and complimentary effects of FGF-2 and TGF-β3. Additionally, fibronectin production in hGF increased when treated with the combined TGF-β3+FGF-2 compared to FGF-2 alone, indicating that the effects of TGF-β3 in promoting extracellular matrix production are still active in the combined treatment condition. Finally, our study highlights that FGF-9 did not influence migration, α-SMA expression, or extracellular matrix production in either cell type, emphasizing the unique roles of specific growth factors in cellular responses. The synergistic effects observed with combined TGF-β3 and FGF-2 treatments present promising avenues for further research and clinical advancements in regenerative medicine.

The matrix protease ADAMTS1 is transcriptionally activated by KLF6 and contributes to cardiac fibrosis in non-ischemic cardiomyopathy

AimsAberrant cardiac fibrosis, defined as excessive production and deposition of extracellular matrix (ECM), is mediated by myofibroblasts. ECM-producing myofibroblasts are primarily derived from resident fibroblasts during cardiac fibrosis. The mechanism underlying fibroblast-myofibroblast transition is not fully understood. MethodsCardiac fibrosis was induced by transverse aortic constriction (TAC) or by angiotensin II (Ang II) infusion in C57B6/j mice. Cellular transcriptome was evaluated by RNA-seq and CUT&Tag-seq. ResultsIntegrated transcriptomic screening revealed that a disintegrin and metalloproteinase with thrombospondin motifs 1 (ADAMTS1) was a novel transcriptional target for Kruppel-like factor 6 (KLF6) in cardiac fibroblasts. Treatment with either TGF-β or Ang II up-regulated ADAMTS1 expression. KLF6 knockdown attenuated whereas KLF6 over-expression enhanced ADAMTS1 induction. ChIP assay and reporter assay showed that KLF6 was recruited to the ADAMTS1 promoter to activate its transcription. Consistently, ADAMTS1 knockdown suppressed fibroblast-myofibroblast transition in vitro. Importantly, myofibroblast-specific ADAMTS1 depletion attenuated cardiac fibrosis and normalized heart function in mice. SignificanceIn conclusion, our data demonstrate that ADAMTS1, as a downstream target of KLF6, contributes to cardiac fibrosis by regulating fibroblast-myofibroblast transition.

Fabrication and applications of biofunctional collagen biomaterials in tissue engineering.

Collagen is extensively used in tissue engineering for various organ tissue regeneration due to the main component of human organ extracellular matrix (ECM) and their inherent nature bioactivity. Collagen various types naturally exist in different organ ECMs. Collagen fabricated with natural ECM mimics architecture, composition and mechanical properties for various organ tissue regeneration. Collagen fabrication with organ-specific biofunctionality facilitated organ tissue engineering as compared to unmodified collagen biomaterials. Collagen biofunctionality improved by subjecting collagen to synthesis, fibers and surface modifications, and blending with other components. Furthermore, collagen is loaded with bioactive molecules, growth factors, drugs and cells also enhancing the biofunctionality of collagen biomaterials. In this review, we will explore the recent advancements in biofunctional collagen biomaterials fabrication with organ-specific biofunctionality in tissue engineering to resolve various organ tissue engineering issues and regeneration challenges. Biofunctional collagen biomaterials stimulate microenvironments inside and around the implants to excellently regulate cellular activities, differentiate cells into organ native cells, enhanced ECM production and remodeling to regenerate organ tissues with native structure, function and maturation. This review critically explored biofunctional collagen biomaterials fabrication in resolving various organ tissue engineering issues and regeneration challenges, and opening new directions of biofunctional collagen biomaterials fabrication, design and applications.

Direct Scaffold-Coupled Electrical Stimulation of Chondrogenic Progenitor Cells through Graphene Foam Bioscaffolds to Control Mechanical Properties of Graphene Foam - Cell Composites.

Osteoarthritis, a major global cause of pain and disability, is driven by the irreversible degradation of hyaline cartilage in joints. Cartilage tissue engineering presents a promising therapeutic avenue, but success hinges on replicating the native physiological environment to guide cellular behavior and generate tissue constructs that mimic natural cartilage. Although electrical stimulation has been shown to enhance chondrogenesis and extracellular matrix production in 2D cultures, the mechanisms underlying these effects remain poorly understood, particularly in 3D models. Here, we report that direct scaffold-coupled electrical stimulation applied to 3D graphene foam bioscaffolds significantly enhances the mechanical properties of the resulting graphene foam - cell constructs. Using custom 3D-printed electrical stimulus chambers, we applied biphasic square impulses (20, 40, 60 mVpp at 1 kHz) for 5 minutes daily over 7 days. Stimulation at 60 mVpp increased the steady-state energy dissipation and equilibrium modulus by approximately 65% and 25%, respectively, compared to unstimulated controls, while also yielding the highest cell density among stimulated samples. In addition, our custom chambers facilitated full submersion of the hydrophobic graphene foam in media, leading to enhanced cell attachment and integration across the scaffold surface and within its hollow branches. To assess this cellular integration, we employed co-localized confocal fluorescence microscopy and X-ray microCT imaging enabled by colloidal gold nanoparticle and fluorophore staining, which allowed visualization of cell distribution within the opaque scaffold's internal structure. These findings highlight the potential of direct scaffold-coupled electrical stimulus to modulate the mechanical properties of engineered tissues and offer new insights into the emergent behavior of cells within conductive 3D bioscaffolds.

Investigating the role of Wnt3a and Wnt5a as critical factors of hepatic stellate cell activation in acute toxicant-induced liver injury

Toxicant exposure can lead to acute liver injury, characterized by hepatic reprogramming and wound healing. Hepatic stellate cells (HSC) play a key role in liver regeneration during wound healing by secreting fibrogenic factors and production of extracellular matrix (ECM). However, repetitive injury to the liver can lead to extensive scarring and liver fibrosis, indicating HSCs coordinate both regeneration and disease. Because the factors contributing to HSC reprogramming during wound healing are not fully defined, we sought to further characterize morphogenic pathways of regeneration in an acute model of toxicant-induced liver injury1. Wnt/β-catenin signaling has been recently associated with progressive liver fibrosis, but its role in HSC reprogramming is not well defined. Here, we investigated the canonical role of Wnt3a/Wnt5a on β-catenin-dependent HSC transdifferentiation and find that hepatic ECM gene expression is increased and associated with Wnt3a, Wnt5a, and their transducers (Frizzled-2 and Frizzled-7) after an acute exposure of the hepatotoxin, carbon tetrachloride(CCl4). Moreover, we find exogenous Wnt3a and Wnt5a can accelerate spontaneous, culture-induced HSC activation in vitro as evidenced by increased total expression of fibrogenic factors, including Col1a1 and α-SMA. Challenge with Wnt3a induced canonical β-catenin-dependent transcription of axin2, which was attenuated by the Wnt coreceptor antagonist, Dickkopf-1 (DKK-1). These data support a role for canonical Wnt signaling as an additional mechanism by which HSCs dynamically respond to liver injury during the early wound healing response. New & noteworthy. This study elucidates novel mechanisms of fibrotic gene reprogramming in the liver. Specifically, we describe that Wnts and their transducers are increased during early liver injury which are associated with early fibrogenic responses and for the first time, causally link Wnts as direct inducers of HSC activation in the liver.Graphical abstract

Mediator kinase inhibition drives myometrial stem cell differentiation and the uterine fibroid phenotype through super-enhancer reprogramming.

Uterine fibroids (UFs) are the most common non-cutaneous tumors in women worldwide. UFs arise from genetic alterations in myometrial stem cells (MM SCs) that trigger their transformation into tumor initiating cells (UF SCs). Mutations in the RNA polymerase II Mediator subunit MED12 are dominant drivers of UFs, accounting for 70% of these clinically significant lesions. Biochemically, UF driver mutations in MED12 disrupt CDK8/19 kinase activity in Mediator, but how Mediator kinase disruption triggers MM SC transformation remains unknown. Here, we show that pharmacologic inhibition of CDK8/19 in MM SCs removes a barrier to myogenic differentiation down an altered pathway characterized by molecular phenotypes characteristic of UFs, including oncogenic growth and extracellular matrix (ECM) production. These perturbations appear to be induced by transcriptomic changes, arising in part through epigenomic alteration and super-enhancer reprogramming, that broadly recapitulate those found in MED12-mutant UFs. Altogether these findings provide new insights concerning the biological role of CDK8/19 in MM SC biology and UF formation.

New Insights into the Pathogenesis of Intestinal Fibrosis in Inflammatory Bowel Diseases: Focusing on Intestinal Smooth Muscle Cells.

Strictures in inflammatory bowel disease, especially Crohn's disease (CD), are characterized by increased intestinal wall thickness, which, according to recent accumulating data, is mainly attributed to the expansion of the intestinal smooth muscle layers and to a lesser extent to collagen deposition. In this review, we will discuss the role of intestinal smooth muscle cells (SMCs) as crucial orchestrators of stricture formation. Activated SMCs can synthesize extracellular matrix (ECM), thus contributing to intestinal fibrosis, as well as growth factors and cytokines that can further enhance ECM production, stimulate other surrounding mesenchymal and immune cells, and increase SMC proliferation via paracrine or autocrine signaling. There is also evidence that, in stricturing CD, a phenotypic modulation of SMC toward a myofibroblast-like synthetic phenotype takes place. Moreover, the molecular mechanisms and signaling pathways that regulate SMC hyperplasia/hypertrophy will be extensively reviewed. The understanding of the cellular network and the molecular background behind stricture formation is essential for the design of effective anti-fibrotic strategies, and SMCs might be a promising therapeutic target in the future.

ADGRB3-High and POSTN-High Fibroblasts Are Markers of Endotypic Traits in Chronic Rhinosinusitis

Background: Chronic rhinosinusitis (CRS) is a disease characterized by persistent sinonasal mucosal inflammation. Fibroblasts play a crucial role in extracellular matrix production and inflammation. We investigated the heterogeneity of fibroblasts in patients with CRS. Methods: Fibroblasts were isolated from nasal polyp tissues. RNA sequencing was then performed. We also analyzed the GSE136825 dataset obtained from the Gene Expression Omnibus database. Alternatively, fibroblasts were stimulated in vitro. Results: Hierarchical clustering of samples indicated ADGRB3-high and POSTN-high fibroblasts. A Gene Set Enrichment Analysis (GSEA) revealed that cytotoxic immune responses were enriched in ADGRB3-high fibroblasts, while cell cycle pathways were enriched in POSTN-high fibroblasts. Similar GSEA results were observed in the GSE136825 dataset. Additionally, type 1 and type 3 inflammation-related genes were highly expressed in ADGRB3-high samples, whereas type 2-related genes were highly expressed in POSTN-high samples. In vitro, ADGRB3 expression increased in fibroblasts stimulated with IFN-γ, while POSTN increased in those stimulated with IL-4 and IL-13. Conclusions: Our study demonstrates that type 1 inflammation induces ADGRB3-high fibroblasts, associated with the cytotoxic immune response, while type 2 inflammation induces POSTN-high fibroblasts, linked to CRS progression via an elevated cell cycle. The further characterization of fibroblasts could provide insights into the stromal networks in the CRS microenvironment.

TEAD1-Mediated Trans-Differentiation of Vascular Smooth Muscle Cells into Fibroblast-Like Cells Contributes to the Stabilization and Repair of Disrupted Atherosclerotic Plaques.

Atherosclerotic plaque rupture mainly contributes to acute coronary syndrome (ACS). Insufficient repair of these plaques leads to thrombosis and subsequent ACS. Central to this process is the modulation of vascular smooth muscle cells (VSMCs) phenotypes, emphasizing their pivotal role in atherosclerotic plaque stability and healing post-disruption. Here, an expansion of FSP1+ cells in a tandem stenosis (TS) model of atherosclerotic mice is unveiled, predominantly originating from VSMCs through single-cell RNA sequencing (scRNA-seq) analyses and VSMC lineage tracing studies. Further investigation identified TEA domain transcription factor 1 (TEAD1) as the key transcription factor driving the trans-differentiation of VSMCs into fibroblast-like cells. In vivo experiments using a TS model of plaque rupture demonstrated that TEAD1 played a crucial role in promoting plaque stability and healing post-rupture through pharmacological or TEAD1-AAV treatments. Mechanistically, it is found that TEAD1 promoted the expression of fibroblast markers through the Wnt4/β-Catenin pathway, facilitating the trans-differentiation. Thus, this study illustrated that TEAD1 played a critical role in promoting the trans-differentiation of VSMCs into fibroblast-like cells and subsequent extracellular matrix production through the Wnt4/β-Catenin pathway. Consequently, this process enhanced the healing mechanisms following plaque rupture, elucidating potential therapeutic avenues for managing atherosclerotic instability.

Evaluation of Nutritional Supplements on Periodontal Fibroblast in Systemic Disease Conditions: An In Vitro Study

ABSTRACT Background: Periodontal health is influenced by various systemic factors, including nutritional status. Nutritional supplements have been proposed to improve periodontal health, particularly in individuals with systemic diseases. Materials and Methods: This in vitro interventional study aimed to evaluate the effects of nutritional supplements on periodontal health in systemic disease conditions. Human gingival fibroblast cells were cultured under conditions mimicking systemic diseases such as diabetes mellitus and cardiovascular disease. The cells were treated with different nutritional supplements commonly used in periodontal therapy, including vitamin C, vitamin D, omega-3 fatty acids, and probiotics. The effects of these supplements on cell viability, inflammatory response, and extracellular matrix production were assessed using various assays and molecular techniques. Results: Treatment with nutritional supplements led to improvements in cell viability, reduction in inflammatory cytokine expression, and enhanced extracellular matrix synthesis in gingival fibroblast cells under systemic disease conditions. Specifically, vitamin C and omega-3 fatty acids demonstrated significant protective effects against inflammation-induced cell damage, while probiotics promoted a balanced immune response. Conclusion: Nutritional supplements, including vitamin C, vitamin D, omega-3 fatty acids, and probiotics, exhibit promising effects on periodontal health in systemic disease conditions. These supplements may serve as adjunctive therapeutic options for managing periodontal disease in individuals with systemic comorbidities.