Extracellular Matrix Production Research Articles

Alveolar epithelial cells are competent producers of interstitial extracellular matrix with disease relevant plasticity in a human in vitro 3D model

Alveolar epithelial cells (AEC) have been implicated in pathological remodelling. We examined the capacity of AEC to produce extracellular matrix (ECM) and thereby directly contribute towards remodelling in chronic lung diseases. Cryopreserved type 2 AEC (AEC2) from healthy lungs and chronic obstructive pulmonary disease (COPD) afflicted lungs were cultured in decellularized healthy human lung slices for 13 days. Healthy-derived AEC2 were treated with transforming growth factor ß1 (TGF-β1) to evaluate the plasticity of their ECM production. Evaluation of phenotypic markers and expression of matrisome genes and proteins were evaluated by RNA-sequencing, mass spectrometry and immunohistochemistry. The AEC2 displayed an AEC marker profile similar to freshly isolated AEC2 throughout the 13-day culture period. COPD-derived AECs proliferated as healthy AECs with few differences in gene and protein expression while retaining increased expression of disease marker HLA-A. The AEC2 expressed basement membrane components and a complex set of interstitial ECM proteins. TGF-β1 stimuli induced a significant change in interstitial ECM production from AEC2 without loss of specific AEC marker expression. This study reveals a previously unexplored potential of AEC to directly contribute to ECM turnover by producing interstitial ECM proteins, motivating a re-evaluation of the role of AEC2 in pathological lung remodelling.

UCHL1 aggravates skin fibrosis through an IGF-1-induced Akt/mTOR/HIF-1α pathway in keloid.

Keloid is a heterogeneous disease featured by the excessive production of extracellular matrix. It is a great challenge for both clinicians and patients regarding the exaggerated and uncontrolled outgrowth and the therapeutic resistance of the disease. In this study, we verified that UCHL1 was drastically upregulated in keloid fibroblasts. UCHL1 had no effects on cell proliferation and migration, but instead promoted collagen I and α-SMA expression that was inhibited by silencing UCHL1 gene and by adding in LDN-57444, a pharmacological inhibitor for UCHL1 activity as well. The pathological process was mediated by IGF-1 promoted Akt/mTOR/HIF-1α signaling pathway because inhibition of any of them could reduce the expression of collagen I and α-SMA driven by UCHL1 in fibroblasts. Also, we found that UCHL1 expression in keloid fibroblasts was promoted by M2 macrophages via TGF-β1. These findings extend our understanding of the pathogenesis of keloid and provide potential therapeutic targets for the disease.

The Extracellular Matrix of Yeasts: A Key Player in the Microbial Biology Change of Paradigm.

Microbes are traditionally regarded as planktonic organisms, individual cells that live independently from each other. Although this is true, microbes in nature mostly live within large multi-species communities forming complex ecosystems. In these communities, microbial cells are held together and organised spatially by an extracellular matrix (ECM). Unlike the ECM from the tissues of higher eukaryotes, microbial ECM, mostly that of yeasts, is still poorly studied. However, microbial biofilms are a serious cause for concern, for being responsible for the development of nosocomial infections by pharmacological drugs-resistant strains of pathogens, or for critically threatening plant health and food security under climate change. Understanding the organization and behaviour of cells in biofilms or other communities is therefore of extreme importance. Within colonies or biofilms, extremely large numbers of individual microbial cells adhere to inert surfaces or living tissues, differentiate, die or multiply and invade adjacent space, often following a 3D architectural programme genetically determined. For all this, cells depend on the production and secretion of ECM, which might, as in higher eukaryotes, actively participate in the regulation of the group behaviour. This work presents an overview of the state-of-the-art on the composition and structure of the ECM produced by yeasts, and the inherent physicochemical properties so often undermined, as well as the available information on its production and delivery pathways.

BIOSCAFFOLD FROM MOUSE EMBRYONIC FIBROBLAST MAINTAINS THE PLURIPOTENCY OF MOUSE EMBRYONIC STEM CELLS

Cell culture using a 3D method provides Various cell culture strategies have been developed using synthetic or biological materials; most existing publications use many reagents. Bioscaffold from mouse embryonic fibroblast (MEF) enhances cell attachment, interaction, and production of growth factors. Since bioscaffolds could maintain and stimulate pluripotency of stem cells, we conducted this study to prove bioscaffold function. Bioscaffold was prepared from MEF cultured in DMEM complete medium supplemented with dextran sulfate and L-ascorbic acid to increase extracellular matrix production. This medium acts as an embryo stem cell (ESC) culture medium. We used a Tali-cytometer to identify and quantify stem cells based on Sox2 and Oct4 proteins, markers of stemness. ESC culture using bioscaffold maintained the pluripotency of ESC as indicated by the presence of Oct 4 and Sox2 as ESC markers compared to MEF culture. From this research, the bioscaffold from MEF can be developed as media for ESC to improve propagation. Furthermore, it is a model for tissue engineering and in vitro organ development.

C–C motif chemokine CCL11 is a novel regulator and a potential therapeutic target in non-alcoholic fatty liver disease

Background & AimsNon-alcoholic fatty liver disease (NAFLD) is characterised by accelerated lipid deposition, aberrant inflammation, and excessive extracellular matrix production in the liver. Short of effective intervention, NAFLD can progress to cirrhosis and hepatocellular carcinoma. In the present study we investigated the involvement of the C–C motif ligand 11 (CCL11) in NAFLD pathogenesis. MethodsNAFLD was induced by feeding mice with a high-fat high-carbohydrate diet. CCL11 targeting was achieved by genetic deletion or pharmaceutical inhibition. The transcriptome was analysed using RNA-seq. ResultsWe report that CCL11 expression was activated at the transcription level by free fatty acids (palmitate) in hepatocytes. CCL11 knockdown attenuated whereas CCL11 treatment directly promoted production of pro-inflammatory/pro-lipogenic mediators in hepatocytes. Compared with wild-type littermates, CCL11 knockout mice displayed an ameliorated phenotype of NAFLD when fed a high-fat high-carbohydrate diet as evidenced by decelerated body weight gain, improved insulin sensitivity, dampened lipid accumulation, reduced immune cell infiltration, and weakened liver fibrosis. RNA-seq revealed that interferon regulatory factor 1 as a mediator of CCL11 induced changes in hepatocytes. Importantly, CCL11 neutralisation or antagonism mitigated NAFLD pathogenesis in mice. Finally, a positive correlation between CCL11 expression and NAFLD parameters was identified in human patients. ConclusionsOur data suggest that CCL11 is a novel regulator of NAFLD and can be effectively targeted for NAFLD intervention. Impact and implicationsNon-alcoholic fatty liver disease (NAFLD) precedes cirrhosis and hepatocellular carcinoma. In this paper we describe the regulatory role of CCL11, a C–C motif ligand chemokine, in NAFLD pathogenesis. Our data provide novel insights and translational potential for NAFLD intervention.

Modeling fibrotic alveolar transitional cells with pluripotent stem cell-derived alveolar organoids.

Repeated injury of the lung epithelium is proposed to be the main driver of idiopathic pulmonary fibrosis (IPF). However, available therapies do not specifically target the epithelium and human models of fibrotic epithelial damage with suitability for drug discovery are lacking. We developed a model of the aberrant epithelial reprogramming observed in IPF using alveolar organoids derived from human-induced pluripotent stem cells stimulated with a cocktail of pro-fibrotic and inflammatory cytokines. Deconvolution of RNA-seq data of alveolar organoids indicated that the fibrosis cocktail rapidly increased the proportion of transitional cell types including the KRT5 - /KRT17 + aberrant basaloid phenotype recently identified in the lungs of IPF patients. We found that epithelial reprogramming and extracellular matrix (ECM) production persisted after removal of the fibrosis cocktail. We evaluated the effect of the two clinically approved compounds for IPF, nintedanib and pirfenidone, and found that they reduced the expression of ECM and pro-fibrotic mediators but did not completely reverse epithelial reprogramming. Thus, our system recapitulates key aspects of IPF and is a promising system for drug discovery.

Crosstalk between Interleukin-1 Receptor-Like 1 and Transforming Growth Factor-β Receptor Signaling Promotes Renal Fibrosis

IL-33, a member of the IL-1 family, acts as an alarmin in immune response. Epithelial-mesenchymal transition and transforming growth factor-β (TGF-β)-induced fibroblast activation are key events in the development of renal interstitial fibrosis. The current study found increased expression of IL-33 and suppression of tumorigenicity 2 (ST2), the receptor for IL-33, in human fibrotic renal tissues. In addition, IL-33- or ST2-deficient mice showed significantly reduced levels of fibronectin, α-smooth muscle actin, and vimentin, and increased E-cadherin levels. In HK-2 cells, IL-33 promotes the phosphorylation of the TGF-β receptor (TGF-βR), Smad2, and Smad3, and the production of extracellular matrix (ECM), with reduced expression of E-cadherin. Blocking TGF-βR signaling or suppressing ST2 expression impeded Smad2 and Smad3 phosphorylation, thereby reducing ECM production, suggesting that IL-33-induced ECM synthesis requires cooperation between the two pathways. Mechanistically, IL-33 treatment induced a proximate interaction between ST2 and TGF-βRs, activating downstream Smad2 and Smad3 for ECM production in renal epithelial cells. Collectively, this study identified a novel and essential role for IL-33 in promoting TGF-β signaling and ECM production in the development of renal fibrosis. Therefore, targeting IL-33/ST2 signaling may be an effective therapeutic strategy for renal fibrosis.

Rev-erbα agonists suppresses TGFβ1-induced fibroblast-to-myofibroblast transition and pro-fibrotic phenotype in human lung fibroblasts

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease characterized by excessive scarring of the lungs that can lead to respiratory failure and death. Lungs of patients with IPF demonstrate excessive deposition of extracellular matrix (ECM) and an increased presence of pro-fibrotic mediators such as transforming growth factor-beta 1 (TGFβ1), which is a major driver of fibroblast-to-myofibroblast transition (FMT). Current literature supports that circadian clock dysfunction plays an essential role in the pathophysiology of various chronic inflammatory lung diseases such as asthma, chronic obstructive pulmonary disease, and IPF. The circadian clock transcription factor Rev-erbα is encoded by Nr1d1 that regulates daily rhythms of gene expression linked to immunity, inflammation, and metabolism. However, investigations into the potential roles of Rev-erbα in TGFβ-induced FMT and ECM accumulation are limited. In this study, we utilized several novel small molecule Rev-erbα agonists (GSK41122, SR9009, and SR9011) and a Rev-erbα antagonist (SR8278) to determine the roles of Rev-erbα in regulating TGFβ1-induced FMT and pro-fibrotic phenotypes in human lung fibroblasts. WI-38 cells were either pre-treated/co-treated with or without Rev-erbα agonist/antagonist along with TGFβ1. After 48 h, the following parameters were evaluated: secretion of COL1A1 (Slot-Blot analysis) and IL-6 (ELISA) into condition media, expressions of α-smooth muscle actin (αSMA: immunostaining and confocal microscopy), and pro-fibrotic proteins (αSMA and COL1A1 by immunoblotting), as well as gene expression of pro-fibrotic targets (qRT-PCR: Acta2, Fn1, and Col1a1). Results revealed that Rev-erbα agonists inhibited TGFβ1-induced FMT (αSMA and COL1A1), and ECM production (reduced gene expression of Acta2, Fn1, and Col1a1), and decreased pro-inflammatory cytokine IL-6 release. The Rev-erbα antagonist promoted TGFβ1-induced pro-fibrotic phenotypes. These findings support the potential of novel circadian clock-based therapeutics, such as Rev-erbα agonist, for the treatment and management of fibrotic lung diseases and disorders.

Identifying fibrogenic cells following salivary gland obstructive injury.

Fibrosis results from excess extracellular matrix accumulation, which alters normal tissue architecture and impedes function. In the salivary gland, fibrosis can be induced by irradiation treatment for cancer therapy, Sjögren's Disease, and other causes; however, it is unclear which stromal cells and signals participate in injury responses and disease progression. As hedgehog signaling has been implicated in fibrosis of the salivary gland and other organs, we examined contributions of the hedgehog effector, Gli1, to fibrotic responses in salivary glands. To experimentally induce a fibrotic response in female murine submandibular salivary glands, we performed ductal ligation surgery. We detected a progressive fibrotic response where both extracellular matrix accumulation and actively remodeled collagen significantly increased at 14days post-ligation. Macrophages, which participate in extracellular matrix remodeling, and Gli1+ and PDGFRα+ stromal cells, which may deposit extracellular matrix, both increased with injury. Using single-cell RNA-sequencing, Gli1 + cells were not found in discrete clusters at embryonic day 16 but were found in clusters expressing the stromal genes Pdgfra and/or Pdgfrb. In adult mice, Gli1+ cells were similarly heterogeneous but more cells co-expressed PDGFRα and PDGFRβ. Using Gli1-CreERT2; ROSA26tdTomato lineage-tracing mice, we found that Gli1-derived cells expand with ductal ligation injury. Although some of the Gli1 lineage-traced tdTomato+ cells expressed vimentin and PDGFRβ following injury, there was no increase in the classic myofibroblast marker, smooth muscle alpha-actin. Additionally, there was little change in extracellular matrix area, remodeled collagen area, PDGFRα, PDGFRβ, endothelial cells, neurons, or macrophages in Gli1 null salivary glands following injury when compared with controls, suggesting that Gli1 signaling and Gli1+ cells have only a minor contribution to mechanical injury-induced fibrotic changes in the salivary gland. We used scRNA-seq to examine cell populations that expand with ligation and/or showed increased expression of matrisome genes. Some Pdgfra + /Pdgfrb + stromal cell subpopulations expanded in response to ligation, with two stromal cell subpopulations showing increased expression of Col1a1 and a greater diversity of matrisome genes, consistent with these cells being fibrogenic. However, only a few cells in these subpopulations expressed Gli1, consistent with a minor contribution of these cells to extracellular matrix production. Defining the signaling pathways driving fibrotic responses in stromal cell sub-types could reveal future therapeutic targets.

(375) Can Tamoxifen and a PDE5 inhibitor slow the progression of Peyronie’s Disease?

Abstract Introduction The available treatments for Peyronie’s Disease as recommended by international guidelines are either surgery or intralesional injection therapy. In recently cell and rat studies it has been shown that using a phosophdiesterase type 5 inhibitor (PDE5i) and a selective oestrogen receptor modulator (Tamoxifen) inhibited myofibroblast transformation and extracellular matrix production. Objective The aim of this study is to compare the combination of tamoxifen and a PDE5i versus no treatment or other oral medications in the progression of early Peyronie’s Disease. Methods This is a retrospective study of all patients with early Peyronie’s Disease, defined as less than 6 months duration or persistent pain presenting over a 3-year period. Patients were given Tamoxifen 20mg twice daily + Tadalafil 5mg daily for 2 months. The control group either received no treatment or simple oral medication such as Vitamin E. Data retrieved included demographics, duration of disease, change in curvature and pain, as well as further treatment modalities. Assessment of curvature was with photos or an artificial erection test. Data was analysed using the Mann-Whitney Test. Results A total of 147 patients were recruited with early Peyronie’s Disease, 102 patients given the active drug combination (median age 49, range 24-71) and 45 controls (median age 55, range 27-76). The results in each group are presented in Table 1. Overall, the patients on Tamoxifen and Tadalafil showed more improvement and less deterioration which was statistically significant (p=0.009) Conclusions The combination of Tamoxifen and Tadalafil has been shown to prevent the progression of Peyronie’s Disease in vitro and in this study in patients with early disease. Future prospective studies are warranted to confirm this finding. Disclosure No

Chondroitinase ABC Treatment Improves the Organization and Mechanics of 3D Bioprinted Meniscal Tissue.

The meniscus is a fibrocartilage tissue that is integral to the correct functioning of the knee joint. The tissue possesses a unique collagen fiber architecture that is integral to its biomechanical functionality. In particular, a network of circumferentially aligned collagen fibers function to bear the high tensile forces generated in the tissue during normal daily activities. The limited regenerative capacity of the meniscus has motivated increased interest in meniscus tissue engineering; however, the in vitro generation of structurally organized meniscal grafts with a collagen architecture mimetic of the native meniscus remains a significant challenge. Here we used melt electrowriting (MEW) to produce scaffolds with defined pore architectures to impose physical boundaries upon cell growth and extracellular matrix production. This enabled the bioprinting of anisotropic tissues with collagen fibers preferentially oriented parallel to the long axis of the scaffold pores. Furthermore, temporally removing glycosaminoglycans (sGAGs) during the early stages of in vitro tissue development using chondroitinase ABC (cABC) was found to positively impact collagen network maturation. Specially we found that temporal depletion of sGAGs is associated with an increase in collagen fiber diameter without any detrimental effect on the development of a meniscal tissue phenotype or subsequent extracellular matrix production. Moreover, temporal cABC treatment supported the development of engineered tissues with superior tensile mechanical properties compared to empty MEW scaffolds. These findings demonstrate the benefit of temporal enzymatic treatments when engineering structurally anisotropic tissues using emerging biofabrication technologies such as MEW and inkjet bioprinting.

Cardiac pericytes mediate the remodeling response to myocardial infarction.

Despite the prevalence of pericytes in the microvasculature of the heart, their role during ischemia-induced remodeling remains unclear. We used multiple lineage-tracing mouse models and found that pericytes migrated to the injury site and expressed profibrotic genes, coinciding with increased vessel leakage after myocardial infarction (MI). Single-cell RNA-Seq of cardiac pericytes at various time points after MI revealed the temporally regulated induction of genes related to vascular permeability, extracellular matrix production, basement membrane degradation, and TGF-β signaling. Deleting TGF-β receptor 1 in chondroitin sulfate proteoglycan 4-expressing (Cspg4-expressing) cells reduced fibrosis following MI, leading to a transient improvement in the cardiac ejection fraction. Furthermore, genetic ablation of Cspg4-expressing cells resulted in excessive vascular permeability, a decline in cardiac function, and increased mortality in the second week after MI. These data reveal an essential role for cardiac pericytes in the control of vascular homeostasis and the fibrotic response after acute ischemic injury, information that will help guide the development of novel strategies to preserve vascular integrity and attenuate pathological cardiac remodeling.

Oxymatrine ameliorates osteoarthritis via the Nrf2/NF-κB axis in vitro and in vivo

PurposeOsteoarthritis (OA) is a common degenerative joint disorder. Currently, the underlying etiology of OA is still far from fully elucidated and there is no cure for OA progression. Previous studies have demonstrated that oxymatrine (OMT) could inhibit inflammation and oxidative stress in several animal models. However, the potential effects of OMT on OA remain largely elusive. The aim of the study is to investigate the anti-inflammatory and chondrocyte protective effect of OMT, and delineate the potential mechanism in vitro and in vivo. MethodsWestern blotting, RT-PCR, ELISA and tissue staining were employed to explore the mechanisms by which OMT exerted a protective effect on IL-1β-induced production of pro-inflammation cytokines and extracellular matrix (ECM) degradation in primary murine chondrocytes and DMM mouse models. ResultsThe results showed that OMT reduced the IL-1β-induced over-production of pro-inflammation cytokines and ECM degradation. Mechanistically, OMT inhibited the NF-κB pathway via activating Nrf2. In vivo studies also demonstrated that OMT ameliorated OA progression. ConclusionsOMT reduced pro-inflammation cytokines, ECM degradation and OA progression via activating Nrf2 and inhibiting NF-κB pathway.

Nanovaccine Displaying Immunodominant T Cell Epitopes of Fibroblast Activation Protein Is Effective Against Desmoplastic Tumors.

Cancer-associated fibroblasts (CAFs), which are dominant cell types in the tumor microenvironment (TME), support tumor growth by secreting cytokines and forming an extracellular matrix (ECM) that hampers the penetration of chemical and biological therapeutics within the tumor and thereby limits their therapeutic efficacy. Here, we report a cancer nanovaccine targeting fibroblast activation protein α (FAP)-expressing CAFs as a potential pan-tumor vaccine. We predicted immunodominant FAP-specific epitope peptides in silico and selected two candidate peptides after in vitro and in vivo screening for immunogenicity and antitumor efficacy. Next, we developed a nanoparticle-based vaccine that displays the two selected epitope peptides on the surface of lipid nanoparticles encapsulating CpG adjuvant (FAPPEP-SLNPs). Immunization with one of two FAPPEP-SLNP nanovaccines led to considerable growth inhibition of various tumors, including desmoplastic tumors, by depleting FAP+ CAFs and thereby reducing ECM production in the TME while causing little appreciable adverse effects. Furthermore, when combined with a chemotherapeutic drug, the FAPPEP-SLNP nanovaccine increased drug accumulation and resulted in a synergistic antitumor efficacy far better than that of each corresponding monotherapy. These findings suggest that our FAPPEP-SLNP nanovaccine has potential for use as an "off-the-shelf" pan-tumor vaccine applicable to a variety of tumors and may be a suitable platform for use in various combination therapies.

Insights into Overlappings of Fibrosis and Cancer: Exploring the Tumor-related Cardinal Genes in Idiopathic Pulmonary Fibrosis.

The pathogenesis of idiopathic pulmonary fibrosis (IPF) is quite similar to that of cancer pathogenesis, and several pathways appear to be involved in both disorders. The mammalian target of the rapamycin (mTOR) pathway harbors several established oncogenes and tumor suppressors. The same signaling molecules and growth factors, such as vascular endothelial growth factor (VEGF), contributing to cancer development and progression play a part in fibroblast proliferation, myofibroblast differentiation, and the production of extracellular matrix in IPF development as well. The expression of candidate genes acting upstream and downstream of mTORC1, as well as Vegf and low-density lipoprotein receptor related protein 1(Lrp1), was assessed using specific primers and quantitative polymerase chain reaction (qPCR) within the lung tissues of bleomycin (BLM)-induced IPF mouse models. Lung fibrosis was evaluated by histological examinations and hydroxyproline colorimetric assay. BLM-exposed mice developed lung injuries characterized by inflammatory manifestations and fibrotic features, along with higher levels of collagen and hydroxyproline. Gene expression analyses indicated a significant elevation of regulatory associated protein of mTOR (Raptor), Ras homolog enriched in brain (Rheb), S6 kinase 1, and Eukaryotic translation initiation factor 4E-binding protein 1 (4Ebp1), as well as a significant reduction of Vegfa, Tuberous sclerosis complex (Tsc2), and Lrp1; no changes were observed in the Tsc1 mRNA level. Our findings support the elevation of S6K1 and 4EBP1 in response to the TSC/RHEB/mTORC1 axis, which profoundly encourages the development and establishment of IPF and cancer. In addition, this study suggests a possible preventive role for VEGF-A and LRP1 in the development of IPF.

Pancreatic cancer-derived small extracellular vesical ezrin activates fibroblasts to exacerbate cancer metastasis through STAT3 and YAP-1 signaling pathways.

Cancer-associated fibroblasts (CAFs), a major component of the tumor microenvironment (TME) in pancreatic ductal adenocarcinoma (PDAC), play an important role in tumorigenesis, metastasis, and chemoresistance. Tumor-derived small extracellular vesicles (sEVs), which mediate cell-to-cell communication between cancer cells and fibroblasts, are also critical for cancer progression and metastasis. However, it remains unclear how PDAC cell-derived sEVs activate fibroblasts, which contributes to tumor progression. Here, we report that ezrin (EZR) expression in PDAC cell-derived sEVs (sEV-EZR) can activate fibroblasts, resulting in increased migration ability and high expression of α-SMA, PDGFRB, and high production of extracellular matrix in fibroblasts. Reciprocally, sEV-EZR-activated fibroblasts enhanced PDAC cell proliferation, invasion, and metastasis to the liver in animal models. Conversely, fibroblasts treated with PDAC cell-derived sEVs with EZR knockdown resulted in the reduced metastatic ability of PDAC. Mechanistically, we demonstrated that PDAC cell-derived sEV-EZR increases the STAT3 and YAP-1 signaling pathways to induce fibroblast activation, and the activated fibroblasts promote PDAC cell proliferation, invasion, and liver metastasis. Inhibition of the STAT3 and YAP-1 signaling pathways by gene knockdown can abrogate sEV-EZR-induced effects. These findings suggest that targeting the interaction between PDAC cell-derived sEV-EZR and fibroblasts is a potential therapeutic strategy for PDAC.

Expression and biological function of the cellular communication network factor 5 (CCN5) in primary liver cells

The cellular (centralized) communication network (CCN) factor protein family contains six small secreted cysteine-rich proteins sharing high structural similarity. These matricellular proteins have vital biological functions in cell adhesion, migration, cell cycle progression, and control of production and degradation of extracellular matrix. However, in liver the biological functions of CCN proteins become most visible during hepatic injury, disease, and remodeling. In particular, most of the hepatic functions of CCN proteins were derived from CCN2/CTGF, which becomes highly expressed in damaged hepatocytes and acts as a profibrogenic molecule. On the contrary, CCN1/CYR61 seems to have opposite effects, while the biological activity during hepatic fibrosis is somewhat controversially discussed for other CCN family members. In the present study, we analyzed the expression of CCN5/WISP2 in cultures of different types of primary liver cells and in an experimental model of hepatic fibrosis. We found that CCN5 is expressed in hepatic stellate cells, myofibroblasts and portal myofibroblasts, while CCN5 expression is virtually absent in hepatocytes. During hepatic fibrogenesis, CCN5 is significantly upregulated. Overexpression of CCN5 in portal myofibroblasts reduced expression of transforming growth factor-β receptor I (ALK5) and concomitant Smad2 activation, whereas JunB expression is upregulated. Moreover, elevated expression of CCN5 induces endoplasmic reticulum stress, unfolded protein response and apoptosis in portal myofibroblasts. We suggest that upregulated expression of CCN5 might be an intrinsic control mechanism that counteracts overshooting fibrotic responses in profibrogenic liver cells.Graphical abstract

Enhanced chondrogenic differentiation of iPS cell-derived mesenchymal stem/stromal cells via neural crest cell induction for hyaline cartilage repair.

Background: To date, there is no effective long-lasting treatment for cartilage tissue repair. Primary chondrocytes and mesenchymal stem/stromal cells are the most commonly used cell sources in regenerative medicine. However, both cell types have limitations, such as dedifferentiation, donor morbidity, and limited expansion. Here, we report a stepwise differentiation method to generate matrix-rich cartilage spheroids from induced pluripotent stem cell-derived mesenchymal stem/stromal cells (iMSCs) via the induction of neural crest cells under xeno-free conditions. Methods: The genes and signaling pathways regulating the chondrogenic susceptibility of iMSCs generated under different conditions were studied. Enhanced chondrogenic differentiation was achieved using a combination of growth factors and small-molecule inducers. Results: We demonstrated that the use of a thienoindazole derivative, TD-198946, synergistically improves chondrogenesis in iMSCs. The proposed strategy produced controlled-size spheroids and increased cartilage extracellular matrix production with no signs of dedifferentiation, fibrotic cartilage formation, or hypertrophy in vivo. Conclusion: These findings provide a novel cell source for stem cell-based cartilage repair. Furthermore, since chondrogenic spheroids have the potential to fuse within a few days, they can be used as building blocks for biofabrication of larger cartilage tissues using technologies such as the Kenzan Bioprinting method.

Exploring the Potential of Ultrasound Therapy to Reduce Skin Scars: An In Vitro Study Using a Multi-Well Device Based on Printable Piezoelectric Transducers.

Excessive skin scarring affects over 100 million patients worldwide, with effects ranging from cosmetic to systemic problems, and an effective treatment is yet to be found. Ultrasound-based therapies have been used to treat a variety of skin disorders, but the exact mechanisms behind the observed effects are still unclear. The aim of this work was to demonstrate the potential of ultrasound for the treatment of abnormal scarring by developing a multi-well device based on printable piezoelectric material (PiezoPaint™). First, compatibility with cell cultures was evaluated using measurements of heat shock response and cell viability. Second, the multi-well device was used to treat human fibroblasts with ultrasound and quantify their proliferation, focal adhesions, and extracellular matrix (ECM) production. Ultrasound caused a significant reduction in fibroblast growth and ECM deposition without changes in cell viability or adhesion. The data suggest that these effects were mediated by nonthermal mechanisms. Interestingly, the overall results suggest that ultrasound treatment would a be beneficial therapy for scar reduction. In addition, it is expected that this device will be a useful tool for mapping the effects of ultrasound treatment on cultured cells.

Iron overload triggering ECM-mediated Hippo/YAP pathway in follicle development: a hypothetical model endowed with therapeutic implications.

Disruption of iron homeostasis plays a negative role in follicle development. The dynamic changes in follicle growth are dependent on Hippo/YAP signaling and mechanical forces. However, little is known about the liaison between iron overload and the Hippo/YAP signalling pathway in term of folliculogenesis. Here, based on the available evidence, we established a hypothesized model linking excessive iron, extracellular matrix (ECM), transforming growth factor-β (TGF-β) and Hippo/Yes-associated protein (YAP) signal regarding follicle development. Hypothetically, the TGF-β signal and iron overload may play a synergistic role in ECM production via YAP. We speculate that the dynamic homeostasis of follicular iron interacts with YAP, increasing the risk of ovarian reserve loss and may enhance the sensitivity of follicles to accumulated iron. Hence, therapeutic interventions targeting iron metabolism disorders, and Hippo/YAP signal may alter the consequences of the impaired developmental process based on our hypothesis, which provides potential targets and inspiration for further drug discovery and development applied to clinical treatment.