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Dysregulation and fibrotic transformation of the skeletal muscle extracellular matrix (ECM) is a common pathology associated with injury and aging Studies of the muscle ECM in humans have necessitated the use of biopsies, which are impractical in many settings Non-invasive MRI T1ρ relaxation time was validated to predict ECM collagen composition and organization with aligned T1ρ imaging and biopsies of the vastus lateralis in the heathy limb and Anterior Cruciate Ligament (ACL)-deficient limb of 27 subjects T1ρ relaxation time was strongly associated with collagen abundance and unfolding in the ACL deficient limb, and T1ρ relaxation time was strongly associated with total collagen abundance in the healthy limb T1ρ relaxation time was significantly longer in the ACL deficient limb, coinciding with significant increases in several indices of muscle collagen content and remodeling supporting the use of T1ρ to non-invasively evaluate ECM composition and pathology in skeletal muscle ABSTRACT: Dysregulation and fibrosis of the extracellular matrix (ECM) in skeletal muscle is a consequence of injury. Current ECM assessment necessitates muscle biopsies to evaluate alterations to the muscle ECM, which is often not practical in humans. The goal of this study was to evaluate the potential of a magnetic resonance imaging sequence that quantifies T1ρ relaxation time to predict ECM collagen composition and organization. T1ρ imaging was performed and muscle biopsies obtained from the involved and non-involved vastus lateralis muscle on 27 subjects who had an anterior cruciate ligament (ACL) tear. T1ρ times were quantified via mono exponential decay curve fitted to a series of T1ρ-weighted images. Several ECM indices, including collagen content and organization, were obtained using immunohistochemistry and histochemistry in addition to hydroxyproline. Model selection with multiple linear regression was used to evaluate the relationships between T1ρ times and ECM composition. Additionally, the ACL-deficient and healthy limb were compared to determine sensitivity of T1ρ to detect early adaptations in the muscle ECM following injury. We show that T1ρ relaxation time was strongly associated with collagen unfolding (t = 4.093, p = 0.0007) in the ACL-deficient limb, and collagen 1 abundance in the healthy limb (t = 2.75, p = 0.014). In addition, we show that T1ρ relaxation time is significantly longer in the injured limb, coinciding with significant differences in several indices of collagen content and remodeling in the ACL-deficient limb. These results support the use of T1ρ to evaluate ECM composition in skeletal muscle in a non-invasive manner. This article is protected by copyright. All rights reserved.

Objective To screen 14 different plant extracts for their antifibrotic effect on human primary leiomyoma and healthy myometrial cells. Design Preclinical study. Setting University research laboratory. Patient(s) Human uterine leiomyoma and matched myometrial tissues were obtained from Caucasian premenopausal women with symptomatic uterine fibroids at the time of hysterectomy. Intervention(s) Primary human leiomyoma and myometrial cells were cultured in the absence or presence of the plant extracts. Main Outcome Measure(s) Quantification of the expression of extracellular matrix components, such as fibronectin 1 (FN1), collagen type I alpha 1 (COL1A1), and versican (VCAN), and the profibrotic growth factor activin A or inhibin beta A subunit (INHBA). Result(s) The cells were treated with the 14 extracts for 48 hours, and we measured FN1 messenger RNA (mRNA) expression. Of the 14 extracts, about (ABO) ABO-2 (hop) and ABO-9 (artichoke) significantly reduced FN1 expression in both the cell types. Next, we evaluated the effect of fractions of these 2 extracts on the mRNA expression of FN1 and other extracellular matrix components, such as COL1A1, VCAN, and INHBA, in leiomyoma and myometrial cells. We found that ABO-2 (hop) and ABO-9 (artichoke) as well as their fractions, ABO-AR-2016-015 (fraction of ABO-2) and ABO-AR-2014-168 (fraction of ABO-9), reduced the mRNA expression of FN1, COL1A1, VCAN, and INHBA in primary leiomyoma cells. In primary myometrial cells, the mRNA expression of FN1, COL1A1, VCAN, and INHBA was not greatly affected. Conclusion(s) These results suggest that the hop and artichoke extracts possess antifibrotic properties and support additional evaluation using in vivo models.

PURPOSE Graves' disease (GD) is an autoimmune disorder affecting primarily the thyroid gland. The most common extrathyroidal manifestation of GD is known as Graves' orbitopathy (GO). Bone marrow-derived fibrocytes represent a subset of monocytes in peripheral blood mononuclear cells (PBMCs), infiltrate the orbital tissues, and contribute to the pathogenesis of GO. Hence objectives of the study included whether the concentration of fibrocytes in peripheral blood was higher in GO, whether TSHR m RNA expression and TSHR surface expression in peripheral blood were higher in GO in comparison to Graves' Disease (GD) and Control subjects. METHODS The percentage of circulating fibrocytes (FC) along with TSHR on its cell surface (CD 34+, CD 45+, CXCR4+, Collagen 1+, TSHR+) were assessed by flow cytometry of 50 patients with GD and GO and 15 healthy donors (Control). TSHR mRNA expression was measured by q RT PCR. RESULT The concentration of circulating fibrocytes was significantly higher in GO compared to GD and control [GO 17% vs GD 3% vs control 0.7% (p

MicroRNAs (miRNAs) play an important role in the pathogenesis of atrial fibrillation (AF). Exosomal miRNAs may develop as promising biomarkers for AF. To explore significant exosomal miRNAs in AF, plasma exosomes were extracted from 3 patients with AF and 3 patients with sinus rhythm (SR), respectively. Differential expression of exosomal miRNAs were screened by high-throughput sequencing analysis and verified by qRT-PCR from 40 patients with AF and 40 patients with SR. The target genes prediction, biological function, and signaling pathways analysis were conducted by miRanda software, gene ontology (GO), and KEGG analysis. The results showed that there were 40 differently expressed exosomal miRNAs from AF patients compared with SR patients, of which 13 miRNAs were upregulated and 27 miRNAs were downregulated. qRT-PCR validation demonstrated that miR-124-3p, miR-378d, miR-2110, and miR-3180-3p were remarkably upregulated, while miR-223-5p, miR-574-3p, miR-125a-3p, and miR-1299 were downregulated. To explore the function of miR-124-3p associated with AF, plasma exosomes derived from AF patients were co-incubated with rat myocardial fibroblasts. The expression of miR-124-3p was upregulated in myocardial fibroblasts. The viability and proliferation of myocardial fibroblasts were elevated by transfecting with miR-124-3p overexpression plasmids using CCK8 and immunofluorescence-staining methods. AXIN1 was verified to be the target of miR-124-3p by luciferase assay in vitro. Expression of AXIN1 was reduced, while β-catenin, Collagen 1, and α-SMA were increased in myocardial fibroblasts with miR-124-3p overexpression. In conclusion, these findings suggested that circulating exosomal miRNAs may serve as novel biomarkers for AF, and miR-124-3p promotes fibroblast activation and proliferation through regulating WNT/β-catenin signaling pathway via AXIN1.

Epidemiological studies have suggested that fine particulate matter (PM2.5) and asthma have been independently associated with pulmonary fibrosis but rarely studied together. Furthermore, it is unknown whether airway fibrosis in asthma is more attributable to water-soluble ions of PM2.5. Our current study was to explore the potential mechanism of PM2.5 and water-soluble components on airway fibrosis in ovalbumin (OVA)-sensitized asthmatic rats. Rats were intratracheally instilled with PM2.5 and water-soluble components every 3 days for 4 times or 8 times. Histopathological examination demonstrated that lung inflammatory and airway fibrosis were induced after PM2.5 and water-soluble components exposure. Meanwhile, PM2.5, in particular water-soluble extracts, increased expression of collagen 1 (COL-1), connective tissue growth factor (CTGF), interleukin-6 (IL-6), transforming growth factor-β1 (TGF-β1), Smad family member 3 (Smad3), and p-Smad3, whereas decreased secretion of heme oxygenase-1 (HO-1). However, pretreating asthmatic rats with SB432542, the inhibitor of TGF-β1, and SIS3 HCl, the antagonist of Smad3, both reversed the activation of airway fibrosis induced by water-soluble extracts. Therefore, TGF-β1/Smad3 signaling pathway may be responsible for the pathological process of airway fibrosis in asthmatic rats following PM2.5 and water-soluble components exposure.

Background Fibrocytes are hematopoietic cells with features of mesenchymal cells found in the circulation and inflammatory sites implicated in promoting fibrosis in many fibroinflammatory diseases. However, their role(s) in the development of intestinal fibrosis is poorly understood. Here, we investigated a potential role of fibrocytes in the development of fibrosis in Crohn’s disease (CD) and sought factors that may impact their development and function. Methods Plasma and mononuclear cells were collected from patients with and without fibrostenotic CD. Fibrocytes defined as CD11b+, CD34+, and Collagen 1+ were correlated with clinical assessments of fibrosis, including evaluation using intestinal ultrasound. We measured the levels of relevant circulating molecules via Luminex and studied the effect of patient plasma proteins on fibrocyte differentiation. Results Fibrocyte numbers were increased in CD patients with stricturing Crohn’s disease compared with patients with an inflammatory phenotype (P = .0013), with strong correlation between fibrocyte numbers and acoustic radiation force impulse (ARFI), a measure of bowel elasticity on intestinal ultrasound (R = .8383, P = .0127). Fibrostenotic plasma was a more potent inducer of fibrocyte differentiation in both primary human monocytes and cell line and contained increased levels of cytokines implicated in fibrocyte differentiation compared with plasma from inflammatory patients. Interestingly, increased fibrocyte numbers at time of ultrasound were associated with escalation of medical therapy and endoscopic/surgical management of small bowel strictures at 30 months follow-up. Conclusions Circulating fibrocytes strongly correlate with fibrostenotic disease in CD, and they may serve as predictors for escalation of medical +/- surgical therapy.

The histological distinction between acute and chronic liver injury is a challenging aspect of liver histopathology. It is traditionally based on the interpretation of morphological changes to the extracellular matrix (ECM) at sites of hepatocyte loss using histochemical stains. Our aim was to investigate whether immunohistochemistry and multiplexing for collagen type (I & III) and elastic fibres and a modified Victoria blue method could be helpful. We studied 43 livers removed at transplantation for acute liver failure (ALF, 20 cases) or cirrhosis (23) plus 8 normal controls. In ALF the periportal ECM was normal in 2 cases, contained mainly collagen I associated with a ductular reaction in 6 cases, and delicate elastic strands in 11 cases. Periportal deposition of mainly collagen I and mature elastic fibres was observed in cirrhosis. In ALF the perisinusoidal ECM was intact in 4 cases, collapsed or condensed but of normal composition (predominantly collagen III) in 2 cases, and collapsed and condensed containing mostly collagen I in 17 cases (7 including delicate immature elastic strands). In contrast, bridging fibrous septa of cirrhosis contained abundant collagen 1 and bundles of mature elastin. We propose the use of a scale and the use of immunohistochemistry and multiplexing in additional to histochemical stains to characterise the ECM changes in acute and chronic liver injury.

BACKGROUND AND AIMS Myofibroblasts play a pivotal role in the development and progression of HCC. Here, we aimed to explore the role and mechanism of myofibroblast Musashi RNA binding protein 2 (MSI2) in HCC progression. APPROACH AND RESULTS Myofibroblast infiltration and collagen deposition were detected and assessed in the tissues from 117 patients with HCC. Transgenic mice (Msi2ΔCol1a1 ) with floxed Msi2 allele and collagen type I alpha 1 chain (Col1a1)-ligand inducible Cre recombinases (CreER) were constructed to generate a myofibroblast-specific Msi2 knockout model. Mouse HCC cells were orthotopically transplanted into the Msi2ΔCol1a1 or the control mice (Msi2F/F ). We found that the deposition of collagen fibers, the main product of myofibroblasts, predicted a poor prognosis for HCC; meanwhile, we detected high MSI2 expression in the peritumoral infiltrated myofibroblasts. Conditional deletion of Msi2 in myofibroblasts significantly inhibited the growth of orthotopically implanted HCC, reduced both intrahepatic and lung metastasis, and prolonged the overall survival of tumor-bearing mice (P = 0.002). In vitro analysis demonstrated that myofibroblasts promoted cell proliferation, invasion, and epithelial-mesenchymal transformation of HCC cells, whereas Msi2 deletion in myofibroblasts reversed these effects. Mechanically, Msi2 knockout decreased myofibroblast-derived IL-6 and IL-11 secretion by inhibiting the extracellular signal-regulated kinase 1/2 pathway, and thus attenuated the cancer stem cell-promoting effect of myofibroblasts. Interestingly, we found that the simultaneous knockout of Msi2 in myofibroblasts and knockdown of Msi2 in HCC cells could not further attenuate the implanted HCC progression. CONCLUSIONS Myofibroblast-specific Msi2 knockout abrogated the tumor-promoting function of myofibroblasts and inhibited HCC progression in mouse models. Targeting myofibroblast MSI2 expression may therefore prove to be a therapeutic strategy for HCC treatment in the future.

Summary Our study reports a novel dominant COL1A1 mutation in OI. Using a zebrafish model, we confirmed that the glycine to serine substitution at position 608 of the COL1A1 protein has deleterious effects on bone development. Purpose Osteogenesis imperfecta (OI), also known as brittle bone disease, is a group of genetic disorders. Mutations in two genes, collagen type I alpha 1 chain (COL1A1) and collagen type I alpha 1 chain (COL1A2), which encode the pro-a1 (I) and pro-a2 (I) chains of type I collagen, respectively, the most abundant form of collagen in the human body, cause most cases of OI. Methods In this study, we used panel-based next-generation sequencing for prenatal diagnosis of a fetus whose ultrasound images suggested OI. A de novo mutation in COL1A1 gene was suspected to cause the phenotype. To validate the effect of this mutation in a zebrafish model, we constructed a plasmid containing the corresponding mutation in the zebrafish gene col1a1a (c.1744 G > A), and overexpressed the mutant protein in zebrafish larvae. Results We identified a novel COL1A1 mutation (c.1822 G > A; p.Gly608Ser) in the fetus but not in her parents by an skeletal dysplasias panel. Bioinformatic analysis showed that the affected residue (p.Gly608) is highly conserved from zebrafish to humans. In contrast to larvae expressing wild-type (WT) col1a1a and enhanced green fluorescent protein (EGFP), col1a1a mutation-expressing larvae showed significant spinal curvature and embryonic lethality, mimicking the phenotype of human OI. Conclusions Our study revealed the pathogenicity of a de novo mutation, c.1822 G > A, in human COL1A1, which expands the mutation spectrum of OI.

AimCruciate ligaments (CLs) of the knee joint are commonly injured following trauma or ageing. MicroRNAs (miRs) are potential therapeutic targets in musculoskeletal disorders. This study aimed to 1) identify if wild-stock house (WSH) mice are an appropriate model to study age-related changes of the knee joint and 2) investigate expression of miRs in ageing murine CLs.MethodsKnee joints were collected from 6 and 24 months old C57BL/6 and WSH mice (Mus musculus domesticus) for histological analysis. RNA extraction and qPCR gene expression were performed on CLs in 6, 12, 24, and 30 month WSH old mice. Expression of miR targets in CLs was determined, followed by analysis of predicted mRNA target genes and Ingenuity Pathway Analysis.ResultsHigher CL and knee OARSI histological scores were found in 24 month old WSH mice compared to 6 and 12 month old C57BL/6 and 6 month old WSH mice (p< 0.05). miR-29a and miR-34a were upregulated in 30 month-old WSH mice in comparison to 6, 12 and 24-month-old WSH mice (pConclusionThe findings of this study support WSH house mice as an accelerated ageing model of the murine knee joint. This study also indicated that miR-29a and 34a may be important regulators of COL1A1 gene expression in murine CLs.

Engineering osteoinductive, self-fitting scaffolds offers a potential treatment modality to repair irregularly shaped craniomaxillofacial bone defects. Recently, we innovated on osteoinductive poly(ε-caprolactone)-diacrylate (PCL-DA) shape memory polymers (SMPs) to incorporate poly-L-lactic acid (PLLA) into the PCL-DA network, forming a semi-interpenetrating network (semi-IPN). Scaffolds formed from these PCL-DA/PLLA semi-IPNs display stiffnesses within the range of trabecular bone and accelerated degradation relative to scaffolds formed from slowly degrading PCL-DA SMPs. Herein, we demonstrate for the first time that PCL-DA/PLLA semi-IPN SMP scaffolds show increased intrinsic osteoinductivity relative to PCL-DA. We also confirm that application of a bioinspired polydopamine (PD) coating further improves the osteoinductive capacity of these PCL-DA/PLLA semi-IPN SMPs. In the absence of osteogenic supplements, protein level assessment of human mesenchymal stem cells (h-MSCs) cultured in PCL-DA/PLLA scaffolds revealed an increase in expression of osteogenic markers osterix, bone morphogenetic protein-4 (BMP-4), and collagen 1 alpha 1 (COL1A1), relative to PCL-DA scaffolds and osteogenic medium controls. Likewise, the expression of runt-related transcription factor 2 (RUNX2) and BMP-4 was elevated in the presence of PD-coating. In contrast, the chondrogenic and adipogenic responses associated with the scaffolds matched or were reduced relative to osteogenic medium controls, indicating that the scaffolds display intrinsic osteoinductivity.

BACKGROUND: Runt-related transcription factor 2 (RUNX2) is an important gene that has been implicated in the progression of human cancer. Aberrant expression of RUNX2 predicts gastric cancer (GC) metastasis. However, the molecular mechanism of RUNX2 remains unknown. OBJECTIVE: We hypothesize that RUNX2 promotes GC metastasis by regulating the extracellular matrix component collagen type I alpha 1 (COL1A1). METHODS: The GEPIA database and immunohistochemical staining of 60 GC tissues were used to analyse the correlations between RUNX2 or COL1A1 expression and clinicopathological features, and the Kaplan-Meier method was used to evaluate survival. RT-PCR, western blotting and immunofluorescence were used to detect RUNX2 and COL1A1 expression in GC cells. Migration and invasion assays were performed to assess the influence of RUNX2 and COL1A1 on metastasis. RESULTS: RUNX2 and COL1A1 were highly expressed at both the gene and protein levels in GC, and patients who were positive for RUNX2 and COL1A1 had shorter survival. RUNX2 and COL1A1 expression linearly correlated with each other (r= 0.15, p< 0.01) and with clinical stage and lymph node metastasis (p< 0.05). Overexpressing RUNX2in vitro enhanced COL1A1 expression and promoted GC cell invasion and migration, whereas COL1A1 knockdown inhibited the increase in cell metastatic capacity promoted by RUNX2. In vivo, GC cells overexpressing RUNX2 promoted lung metastasis, and the downregulation of COL1A1 reduced the metastasis promoted by RUNX2. CONCLUSIONS: RUNX2 may promote GC metastasis by regulating COL1A1. RUNX2/COL1A1 can be employed as a novel target for therapy in GC.

Angelicae Sinensis Radix (Danggui) and Ligusticum Chuanxiong Rhizoma (Chuan Xiong) herb-pair (DC) have been frequently used in Traditional Chinese medicine (TCM) prescriptions for hundreds of years to prevent vascular diseases and alleviate pain. However, the mechanism of DC herb-pair in the prevention of liver fibrosis development was still unclear. In the present study, the effects and mechanisms of DC herb-pair on liver fibrosis were examined using network pharmacology and mouse fibrotic model. Based on the network pharmacological analysis of 13 bioactive ingredients found in DC, a total of 46 targets and 71 pathways related to anti-fibrosis effects were obtained, which was associated with mitogen-activated protein kinase (MAPK) signal pathway, hepatic inflammation and fibrotic response. Furthermore, this hypothesis was verified using carbon tetrachloride (CCl4)-induced fibrosis model. Measurement of liver functional enzyme activities and histopathological examination showed that DC dramatically reduced bile acid levels, inflammatory cell infiltration and collagen deposition caused by CCl4. The increased expression of liver fibrosis markers, such as collagen 1, fibronectin, α-smooth muscle actin (α-SMA) and transforming growth factor-β (TGF-β), and inflammatory factors, such as chemokine (C-C motif) ligand 2 (MCP-1), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and IL-6 in fibrotic mice were significantly downregulated by DC herb-pair through regulation of extracellular signal-regulated kinase 1/2 (ERK1/2)-protein kinase B (AKT) signaling pathways. Collectively, these results suggest that DC prevents the development of liver fibrosis by inhibiting collagen deposition, decreasing inflammatory reactions and bile acid accumulation, which provides insights into the mechanisms of herb-pair in improving liver fibrosis.

ObjectivesIncreasing evidence suggests that inflammatory mechanisms play a key role in chronic tendon disease. After observing T cell signatures in human tendinopathy, we explored the interaction between T cells and tendon stromal cells or tenocytes to define their functional contribution to tissue remodelling and inflammation amplification and hence disease perpetuation.MethodsT cells were quantified and characterised in healthy and tendinopathic tissues by flow cytometry (FACS), imaging mass cytometry (IMC) and single cell RNA-seq. Tenocyte activation induced by conditioned media from primary damaged tendon or interleukin-1β was evaluated by qPCR. The role of tenocytes in regulating T cell migration was interrogated in a standard transwell membrane system. T cell activation (cell surface markers by FACS and cytokine release by ELISA) and changes in gene expression in tenocytes (qPCR) were assessed in cocultures of T cells and explanted tenocytes.ResultsSignificant quantitative differences were observed in healthy compared with tendinopathic tissues. IMC showed T cells in close proximity to tenocytes, suggesting tenocyte–T cell interactions. On activation, tenocytes upregulated inflammatory cytokines, chemokines and adhesion molecules implicated in T cell recruitment and activation. Conditioned media from activated tenocytes induced T cell migration and coculture of tenocytes with T cells resulted in reciprocal activation of T cells. In turn, these activated T cells upregulated production of inflammatory mediators in tenocytes, while increasing the pathogenic collagen 3/collagen 1 ratio.ConclusionsInteraction between T cells and tenocytes induces the expression of inflammatory cytokines/chemokines in tenocytes, alters collagen composition favouring collagen 3 and self-amplifies T cell activation via an auto-regulatory feedback loop. Selectively targeting this adaptive/stromal interface may provide novel translational strategies in the management of human tendon disorders.

Objective Pelvic organ prolapse (POP) is a common condition in older women. A decrease in collagen 1 (Col-1) expression is one of the main causes of POP. Many microRNAs play an important role in regulating target genes. The relationship between miR-19-3p and POP is investigated in this study, and the molecular mechanism was also explored to find whether miR-19-3p may be a potential target for early diagnosis and prevention of POP. Methods A total of 60 patients with POP and 60 patients without POP were included in this study. Reverse transcription-polymerase chain reaction and Western blot were used to detect the expression of miR-19-3p, insulin-like growth factor 1 (IGF-1), and the Akt/mTOR/p70S6K pathway. Cell cycle was defined by flow cytometric analysis. The combination of miR-19-3p and IGF-1 was revealed by luciferase assays. Results The results of this study show that miR-19-3p was upregulated in the tissue of patients with POP, whereas COL-1 and IGF-1 expressions were lower in the POP group. miR-19-3p promoted excessive fibroblast autophagy and apoptosis. miR-19-3p negatively regulated the Akt/mTOR/p70S6K pathway and inhibited COL-1 secretion. Luciferase reporter assay showed that miR-19-3p regulated IGF-1 expression by direct target binding. Conclusions miR-19-3p has negative associations with the expression of Col-1. Our study highlights that miR-19-3p may affect the synthesis of Col-1 by targeting IGF-1 and that it may play an vital role in POP.

Objectives To investigate the association between selected single nucleotide polymorphisms (SNPs) with cervical insufficiency and its relationship with obstetric history. Methods Twenty-eight women with cervical insufficiency (case group) and 29 non-pregnant women (control group) were included. The SNPs sequenced included rs2586490 in collagen type I alpha 1 chain (COL1A1), rs1882435 in collagen type IV alpha 3 chain (COL4A3), rs2277698 in metallopeptidase inhibitor 2 (TIMP2), and rs1800468 in transforming growth factor beta 1 (TGFB1). Results We found a higher frequency of the normal allele in the control group (65.5%) and the homozygous mutated genotype in the case group (64.3%) for rs2586490 in COL1A1 (p=0.023). An unplanned finding in the cervical insufficiency group was a higher gestational age of delivery (median≥38 weeks) in the mutated allele than in the wild-type genotype (median of 28.2 weeks) for rs2857396, which is also in the COL1A1 gene (p=0.011). Conclusions The findings of the present study corroborate the hypothesis that cervical insufficiency has a genetic component and probably involves genes encoding proteins in the extracellular matrix, in addition to inflammatory processes.

Background and aim Studies on the resolution of liver fibrosis are becoming more important in this era of etiologic eradication. In contrast to the extensive research on the recovery of liver fibrosis induced by hepatotoxic injuries, regression of cholestatic liver fibrosis has been insufficiently examined owing to the limited availability of animal models. Methods We examined our novel recanalization mice model of biliary obstruction, involving anastomosis between the gallbladder and jejunum (G-J anastomosis) by invagination. Transgenic mice expressing green fluorescent protein (GFP) under the collagen 1(α)1 promoter underwent G-J anastomosis 14 days after bile duct ligation (BDL) and were sacrificed 14 days later. Results Transaminase and total bilirubin levels decreased to almost normal values on day 14 after G-J anastomosis. G-J anastomosis resulted in dramatic reversal of liver fibrosis induced by BDL. Activated portal fibroblasts (PFs) double-positive for GFP and Thy-1 on immunofluorescence in the liver of BDL-injured mice became less noticeable following G-J anastomosis. Messenger RNA expression of markers for activated PFs in the liver was downregulated after anastomosis. Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) were induced by BDL. After anastomosis, expressions of MMP-3, 8 as well as hepatocyte growth factor were further upregulated, whereas those of TIMP-1 and -3 were markedly downregulated. Conclusions Our established G-J anastomosis model is associated with fibrosis resolution and reduced PF activation through reopening of bile duct obstruction and will be valuable for studying the recovery process of cholestatic liver fibrosis.

Sorbaria kirilowii (Regel) Maxim, a plant found in China, Korea, Japan, and east of Europe, is a common herb used for traditional medicinal purposes. However, its ability to prevent photoaging has not been studied. In this study, we investigated the anti-photoaging functions of an ethanol extract (Sk-EE) of S. kirilowii (Regel) Maxim using human keratinocytes exposed to UVB. First, we analyzed the cytotoxicity of Sk-EE. Then, we determine the expression of genes related to inflammation, collagen degradation, and moisture retention. We also explored the anti-photoaging mechanism of Sk-EE by determining correlated signaling pathways and target molecules using reporter gene assays and immunoblotting analyses. Sk-EE treatment of cells increased hyaluronic acid synthase (HAS), filaggrin (FLG), and collagen type I alpha 1 (COL1A1) expression. Sk-EE dose-dependently inhibited the UVB-induced expression of matrix metalloproteinases (MMPs) 1, 2, 9 and cyclooxygenase (COX)-2 by blocking the activator protein (AP)-1 signaling pathway, in particular the phosphorylation of c-Jun N-terminal kinase (JNK), p38, and extracellular response kinase (ERK). In addition, c-Fos and c-Jun were targeted by Sk-EE. Our results indicate that Sk-EE has anti-inflammatory and skin-protective properties, and could be a candidate to treat signs of photoaging.

Purpose To analyze the effect of riboflavin UV-A illumination on mRNA and protein expression of healthy (HCFs) and keratoconus human corneal fibroblasts (KC-HCFs), concerning the inflammatory markers NF-κB, iNOS, IL-6, and collagen 1 and 5 (Col 1/Col 5). Methods Keratocytes were isolated from healthy (n = 3) and keratoconus (KC) corneas (n = 3) and were cultivated in basal medium with 5% fetal calf serum, which resulted in their transformation into human corneal fibroblasts (HCFs/KC-HCFs). Cells underwent 0.1% riboflavin UV-A illumination for 250 s (CXL). NF-κB, iNOS, IL-6, Col 1, and Col 5 expression was investigated by qPCR and Western blot analysis. IL-6 concentration of the cell culture supernatant and cell lysate was determined by ELISA. Results In untreated KC-HCFs, NF-κB (p = 0.0002), iNOS (p = 0.0019), Col 1 (p = 0.0286), and Col 5 (p = 0.0054) mRNA expression was higher and IL-6 expression was lower (p = 0.0057), than in healthy controls. In HCFs, CXL led to an increased NF-κB (p = 0.0286) and IL-6 (p = 0.0057) mRNA expression. The IL-6 concentration in the cell culture supernatant was increased in HCFs (p = 0.0485) and KC-HCFs (p = 0.0485) after CXL. CXL increased intracellular IL-6 concentration only in KC-HCFs (p = 0.0357). In the HCF group (p = 0.0286), an increased Col 1 mRNA expression after CXL could be observed. Conclusion Our study confirmed altered gene expression in untreated KC-HCFs compared to untreated HCFs. Riboflavin UV-A illumination affected gene expression only in HCFs. Increased IL-6 concentration in the cell culture supernatant and cell lysate indicate a secondary inflammatory response of HCFs and KC-HCFs to riboflavin UV-A illumination.

Background Hypospadias, the most frequent congenital male external genitalia abnormality, is usually associated with curvature of the ventral penis, i.e. chordee. Abnormality of darto tissue has been suggested as the pathophysiology of chordees. Collagen is one of the most abundant fibrous proteins within the extracellular matrix. In this study, we determined the expression of collagen 1 (COL1A1) and COL6A1 in patients with hypospadias and associated them with the severity of penile curvature. Methods We included 60 children COL1A1 and COL6A1 in darto tissue was determined by reverse-transcriptase polymerase chain reaction (qPCR). The penile curvature severity was classified as mild ( 60 degrees). Results qPCR showed that COL1A1 and COL6A1 expression was significantly downregulated in the distal (0.88 (0.38–2.53) and 0.54 (0.16–4.35), respectively) and proximal 0.76 (0.33–2.57) and 0.57 (0.18–1.38), respectively) hypospadias groups compared to controls (1.85 (0.24–4.61) and 0.93 (0.17–4.06), respectively) with p-values of 0.024 and 0.018, respectively. Furthermore, there was a moderate correlation between COL1A1 and COL6A1 expression (r = 0.458, p COL1A1 and COL6A1 were also significantly downregulated in the moderate and severe chordee groups compared to the mild chordee groups, with p-values of 0.003 and 0.037, respectively. Conclusions Aberrant COL1A1 and COL6A1 expression might affect abnormalities in darto tissue and penile curvature severity in hypospadias patients.

BACKGROUND: This study investigates the effects of a neuropeptide, secretoneurin (SN), on bone regeneration in an experimental mouse model. METHODS: The effects of SN on cell proliferation, osteoblast marker genes expression, and mineralization were evaluated using the CCK-8 assay, quantitative reverse transcriptase polymerase chain reaction (RT-PCR), and alizarin red S staining, respectively. To examine the effects of SN on bone regeneration in vivo, bone defects were created in the calvaria of ICR mice, and 0.5 or 1 µg/ml SN was applied. New bone formation was analyzed by micro-computed tomography (micro-CT) and histology. New blood vessel formation was assessed by CD34 immunohistochemistry. RESULTS: SN had no significant effect on proliferation and mineralization of MC3T3-E1 cells. However, SN partially induced the gene expression of osteoblast differentiation markers such as runt-related transcription factor 2, alkaline phosphatase, collagen type I alpha 1, and osteopontin. A significant increase of bone regeneration was observed in SN treated calvarial defects. The bone volume (BV), BV/tissue volume, trabecular thickness and trabecular number values were significantly increased in the collagen sponge plus 0.5 or 1 µg/ml SN group (p

The use of bone morphogenic protein-2 (BMP-2) to accelerate the healing of bone defects has resulted in side effects such as ectopic bone formation, soft tissue swelling, and cyst-like bone void formation. An equipotential and a cost-effective replacement for BMP-2 is therefore required. In this study, different combinations of small molecules like FK506 (F), an immunosuppressant; purmorphamine (P), a hedgehog agonist; and Ku-0063794 (K), an mTOR inhibitor, were evaluated on human mesenchymal stem cells (hMSCs) to assess the osteoinduction behavior. Dose optimization and various combinations of small molecules were evaluated using MTT and ALP assay. The gene and protein expression of osteogenic markers RunX2, Osteopontin (OPN), and collagen type 1 on treatment with small molecules was evaluated and compared with BMP-2 expression. The confirmation of the osteogenic potential of small molecules was calcium deposits formed in hMSCs after staining with Alizarin Red S. Optimum concentration of individual small molecule (F 5 nM, K 10 nM, and P 2 μM) was determined based on the MTT and ALP activity. Small molecules (F + K + P) with osteogenic media (OS) showed 1.5-fold higher ALP activity with respect to OS. The synergistic effect of F + K + P and BMP-2/OS demonstrated an upregulation of RUNX2, OPN, and collagen 1 proteins and genes. Small molecules alone (F- 5 nM, K- 10 nM, and P- 2 μM) expressed the formation of calcium ions stained by Alizarin Red S which confirmed their osteoinductive effect. Thus, small molecules may have the potential to substitute or enable the use of reduced dose of BMP-2 for enhanced osteogenesis of progenitor cells. The use of Bone Morphogenic Protein-2 (BMP-2) to accelerate the healing of bone defects has resulted in side effects such as ectopic bone formation, soft tissue swelling, and cyst-like bone void formation. We show that cost-effective small molecules, in combination with osteogenic differentiation cocktail, have the potential to substitute BMP-2 for enhanced osteogenesis of progenitor cells. Graphical abstract

Background Osteoporosis and neurological complications are consequences of acute lymphoblastic leukemia (ALL). Collagen type I alpha 1 gene (COL1A1) polymorphism is associated with osteoporosis. This study aimed to detect the COL1A1 polymorphism and the neurological complications in ALL patients and their association with decreased lumbar spine bone mineral density (BMDLS). This study included 100 pediatric ALL patients and 100 controls. All participants were subjected to laboratory assessment and assessment of BMDLS at the start of the study and 3 years later. COLIA1 genotyping was done once for all participants. Results At the start of the study, there was a significant decrease in osteocalcin (OC), alkaline phosphatase (ALP), and BMDLS levels in the patients. G/T variants and “T” alleles were significantly more detected in the patients (34% and 35% respectively); also, significant differences were detected between patients with polymorphism (G/T and T/T) and those without polymorphism (G/G) regarding OC, ALP, and BMDLS. After 3 years, significant decrement in BMDLS, OC, and ALP was detected in the patients. Twenty-four patients had neurological complications and seven patients had bone fractures. Those patients had significant decrement in BMDLS, OC, and ALP levels. As regards COL1A1 gene polymorphism, the GT and TT variants were significantly detected in fractured patients, while there was no significant difference regarding GT and TT variants in the patients with neurological complications. T allele, neurological complications, high-risk stratification, and age were significantly associated with decreased BMDLS. T allele was the most significant risk factor. Conclusion COLIA1 gene polymorphism, decreased BMDLS, and neurological complications were significantly detected in pediatric ALL patients. COLIA1 gene polymorphism is a significant risk factor for decreased BMDLS in pediatric ALL patients. There is no significant relation between COLIA1 gene polymorphism and the development of neurologic complications.

Morphine is routinely used for pain management in heart failure patients. However, extended morphine exposure associates with major adverse cardiovascular events. Reports link the dopamine receptor D2-family with morphine-induced nociception modulation. This study first assessed whether morphine induces cardiac remodeling in healthy mice, then whether DRD3 agonist (DRD3ag, D2-family member) adjunct therapy prevents morphine-induced cardiac remodeling. Mice received morphine (2 mg/kg/day i. p.) for 7 days (D7) and were either euthanized at D7 or kept 7 more days without morphine (i.e. withdrawal period, D8-D14): G1, morphine; G2, morphine/DRD3ag; G3, morphine + withdrawal; G4, morphine/DRD3ag + withdrawal; G5, morphine + withdrawal/DRD3ag. A separate cohort of animals were used as naïve tissues. We evaluated functional and molecular parameters of cardiac remodeling. Although we did not observe significant differences in systolic function, morphine induced both interstitial fibrosis and cardiomyocyte hypertrophy. Interestingly, DRD3ag abolished these effects. Compared to naïve tissues, collagen 1 increased after withdrawal in G3 and G4 and collagen 3 increased in G1-G4 but at higher levels in G1 and G2. Only G5 did not show collagen differences compared to naïve, suggesting DRD3ag treatment during withdrawal may be beneficial and prevent morphine-induced fibrosis. Smad2/3 phosphorylation increased during withdrawal, indicating a likely upstream pathway for the observed morphine-induced fibrosis. Overall, our data suggest that DRD3ag adjunct therapy decreases morphine-induced adverse cardiac remodeling.

OBJECTIVES Iatrogenic laryngotracheal stenosis (iLTS) is the pathological narrowing of the glottis, subglottis, and/or trachea due to scar tissue. Patients with type 2 diabetes mellitus (T2DM) are over 8 times more likely to develop iLTS and represent 26% to 53% of all iLTS patients. In this investigation, we compared iLTS scar-derived fibroblasts in patients with and without T2DM. STUDY DESIGN Controlled ex vivo study. METHODS iLTS scar fibroblasts were isolated and cultured from subglottic scar biopsies in iLTS patients diagnosed with or without type 2 diabetes (non-T2DM). Fibroblast proliferation, fibrosis-related gene expression, and metabolic utilization of oxidative phosphorylation (OXPHOS) and glycolysis were assessed. Contractility was measured using a collagen-based assay. Metabolically targeted drugs (metformin, phenformin, amobarbital) were tested, and changes in fibrosis-related gene expression, collagen protein, and contractility were evaluated. RESULTS Compared to non-T2DM, T2DM iLTS scar fibroblasts had increased α-smooth muscle actin (αSMA) expression (8.2× increased, P = .020), increased contractility (mean 71.4 ± 4.3% vs. 51.7 ± 16% Δ area × 90 minute-1 , P = .016), and reduced proliferation (1.9× reduction at 5 days, P

Background Although immune checkpoint inhibitors (ICIs) have made an immense breakthrough in cancer therapeutics, they can exert unique, immune-related adverse events. Among them, myocarditis is less frequent, but it is serious and often follows a lethal course. Methods To examine the changes in cardiac autoimmunity after ICI administration, we developed a mouse experimental autoimmune myocarditis (EAM) model via intraperitoneal administration of murine α-cardiac myosin heavy chain (MyHC-α) fragment. Thereafter, the mouse anti-PD-1 antibody (mPD1ab) was administered at two time points, subsequent to and concurrent with MyHC-α fragment administration. Results Severe EAM developed in 3 weeks; wide inflammatory lesions were observed in the cardiac sections. Furthermore, inflammatory/fibrotic genes, such as interleukin 1β, interleukin 6, and collagen 1, were upregulated, although the cardiac function was not significantly affected. The subsequent administration of mPD1ab at 2 weeks post administration of the first MyHC-α fragment exacerbated EAM, whereas the administration of mPD1ab concurrent with MyHC-α fragment administration did not exacerbate EAM. The subsequent administration of mPD1ab significantly increased the infiltration of cluster of differentiation (CD)4- and F4/80-positive cells, whereas the concurrent administration of mPD1ab significantly decreased the infiltration of CD4-positive cells, indicating that the concurrent and subsequent administration of mPD1ab had opposite effects on immune/inflammatory cell infiltration. Conclusions These data suggest that the appearance of ICI-induced autoimmune myocarditis might be related to autoimmune system activity before ICI administration. Although ICIs do not adversely affect patients with normal immune systems, we propose that ICI administration should be avoided in patients with autoimmune disorders.

Introduction and Objective: Alveolar osteoblasts have critical functions during alveolar bone regeneration. Berberine (BBR) and microRNAs (miRNAs) are considered to play important roles in regulating osteoblast differentiation. The study aimed to investigate the role and mechanisms of BBR in osteogenic differentiation of human alveolar osteoblasts (HAOBs) and determine miR-214 expression in the process. Methods: Healthy human alveolar bones were cultured in vitro and prepared for morphological observation and alkaline phosphatase (ALP) staining. The third generation of HAOBs was used for cell transfection and treated by different concentrations of BBR. Cell Counting Kit-8 was used to detect the effect of BBR and increased miR-214 on the proliferation of HAOBs. qRT-PCR and Western blot were used to detect the expression of osteogenic differentiation-related genes and miR-214 level, respectively. Results: The ALP staining results were positive, indicating that cultured cells were HAOBs. Different concentrations of BBR significantly promoted the proliferation of HAOBs and increased the expression levels of ALP, osteocalcin (OCN), collagen type I alpha 1 (COL1A1), runt related transcription factor 2 (RUNX2), and osterix (OSX). Moreover, the expression of miR-214 was reduced as BBR concentrations increased, and the increase of miR-214 reversed the BBR-induced proliferation and osteogenic differentiation of HAOBs. Conclusion: BBR could promote the proliferation and osteogenic differentiation of HAOBs through downregulating the expression of miR-214.

In this study, we examined the effects of injectable platelet-rich fibrin (iPRF) on proliferation and osteodifferentiation in mesenchymal stem cells (MSCs) isolated from human gingiva. Gingival MSCs (gMSCs) were grown in experimental culture media with different concentrations of iPRF [5%, 10%, and replacement of fetal calf serum (FCS) in the standard media with 10% iPRF-10% iPRF-FCS]. Immunophenotyping of gMSCs was performed after seven days by flow cytometry, and their proliferation was examined after three and seven days using the Cell Counting Kit-8 method. After 14 days in culture, spontaneous osteogenic differentiation of gMSCs was evaluated via real-time polymerase chain reaction. All gMSCs were positive for cluster of differentiation (CD) 105, CD73, CD90, and CD44, and negative for CD34∕45, CD14, CD79a, and human leukocyte antigen, DR isotype (HLA-DR). Reduced expression of some surface antigens was observed in the gMSCs grown in 10% iPRF-FCS medium compared to the other groups. After three days, gMSCs grown in 10% iPRF had proliferated significantly less than the other groups. After seven days, proliferation was significantly higher in the 5% iPRF cells compared to the control, while proliferation in the 10% iPRF and 10% iPRF-FCS groups was significantly lower. No spontaneous osteogenic differentiation was observed in the presence of iPRF, as observed by low runt-related transcription factor 2 (RUNX2) expression. Some expression of secreted protein acidic and cysteine rich (SPARC) and collagen 1 alpha (COL1A) was observed for all the gMSCs regardless of the culture medium composition. gMSCs grown in 10% iPRF had significantly lower SPARC expression. In conclusion, 5% iPRF stimulated gMSC proliferation, and an excessively high concentration of iPRF can impair osteogenic induction.

Background TGF-β2-induced epithelial-mesenchymal transition (EMT) is an important mechanism for posterior capsule opacity (PCO) in lens epithelial cells (LECs). This study aimed to investigate if MicroRNA-184 (miR-184) plays a role in the TGF-β2-induced EMT in LECs. Methods Human LECs (HLE-B3 cells) were used in this study. Quantitative real-time polymerase chain reaction (PCR) (qRT-PCR) was performed to analyse miR-184 expressions in HLE-B3 treated with TGF-β2 at different concentrations (0-15 ng/mL) and different time (10 ng/mL, 0-48 hours). After transfection of miR-184 mimics or miR-184 inhibitor, cells were treated with 10 ng/mL TGF-β2 for 24 hours, and the expression levels of miR-184, E-cadherin, vimentin, zinc finger E-box binding homeobox 2 (ZEB2), α-Smooth muscle actin (α-SMA), Collagen 1 and bin3 were determined by qRT-PCR and Western blot, respectively. Results TGF-β2 treatment significantly downregulated E-cadherin and upregulated vimentin generally in a dose-dependent and time-dependent manner. TGF-β2 treatment significantly elevated the level of miR-184 in both dose- and time-dependent manners. In addition, transfection of miR-184 inhibitor RNA significantly attenuated TGF-β2-induced downregulation of E-cadherin as well as upregulation of vimentin, ZEB2, α-SMA and Collagen 1, whereas transfection of miR-184 mimic further enhanced the effects of TGF-β2 on the expressions of these markers. Furthermore, TGF-β2 treatment significantly downregulated bin3, and transfection of miR-184 mimic and miR-184 inhibitor significantly enhanced and attenuated the inhibition effect of TGF-β2 on bin3, respectively. Conclusions miR-184 plays a key role in the TGF-β2-induced EMT in LECs, and bin3 may be a downstream protein.

Background: Electrospinning is a widely used technology that can produce scaffolds with high porosity and surface area for bone regeneration. However, the small pore sizes in electrospun scaffolds constrain cell growth and tissue-ingrowth. In this study, novel drug-loading core-shell scaffolds were fabricated via electrospinning and freeze drying to facilitate the repair of tibia bone defects in rabbit models. Materials and Methods: The collagen core scaffolds were freeze-dried containing icariin (ICA)-loaded chitosan microspheres. The shell scaffolds were electrospun using collagen, polycaprolactone and hydroxyapatite materials to form CPH composite scaffolds with the ones containing ICA microspheres named CPHI. The core-shell scaffolds were then cross-linked by genipin. The morphology, microstructure, physical and mechanical properties of the scaffolds were assessed. Rat marrow mesenchymal stem cells from the wistar rat were cultured with the scaffolds. The cell adhesion and proliferation were analysed. Adult rabbit models with tibial plateau defects were used to evaluate the performance of these scaffolds in repairing the bone defects over 4 to 12 weeks. Results: The results reveal that the novel drug-loading core-shell scaffolds were successfully fabricated, which showed good physical and chemical properties and appropriate mechanical properties. Furthermore, excellent cells attachment was observed on the CPHI scaffolds. The results from radiography, micro-computed tomography, histological and immunohistochemical analysis demonstrated that abundant new bones were formed on the CPHI scaffolds. Conclusion: These new core-shell composite scaffolds have great potential for bone tissue engineering applications and may lead to effective bone regeneration and repair.

Background and purpose Recruitment and involvement of bone-/blood-derived circulating fibrocytes (CF) in the promotion of fibrotic tissue remodelling processes have been shown. However, their direct contribution to pathological changes is not clear. The present study investigates the causal role of CF in the pathogenesis of pulmonary hypertension (PH). Experimental approach For selective ablation of CF, we applied the suicidal gene strategy with herpes simplex virus thymidine kinase (HSV-TK) and ganciclovir. The transgenic mice were generated, having HSV-TK-GFP transgene under the collagen 1 promoter. To selectively target CF, HSV-TK-GFP+ bone marrow transplanted into irradiated wild type mice. These chimera mice were subjected to hypoxia for PH induction and ganciclovir for CF ablation. Key results In vivo CF ablation reduced right ventricular hypertrophy and vascular remodelling with reduced total collagen content. We quantified the CF recruited in the perivascular area and arterial wall of small pulmonary arteries. There was significant recruitment of CF in the lung in response to hypoxia. The characterization of CF showed the expression of CD45 and collagen1 (GFP) along with α-smooth muscle actin (αSMA). Conclusion and implications Our data demonstrated that CF ablation has a potential impact on right ventricular hypertrophy and vascular remodelling in the setting of experimental pulmonary hypertension induced by hypoxia. The beneficial effects may be related to the direct contribution of fibrocytes or its paracrine effect on other resident cell types. Thus, clinical manipulation of CF may represent a novel therapeutic approach to ameliorate the disease state in pulmonary hypertension.

Summary: Background: Non-alcoholic steatohepatitis (NASH), a subtype of non-alcoholic fatty liver disease (NAFLD), is a potentially progressive liver disease that can lead to cirrhosis. Obesity increases the risk of NAFLD/NASH, but this disease can also be observed in non-obese individuals. Methods: We investigated the metabolic and histopathological changes in 13 obesity-resistant Slc:Wistar/ST rats fed a high-fat and high-cholesterol (HFC) diet for 9 weeks, and also retrospectively compared the results of 41 Sprague-Dawley (SD) rats that were previously fed with the same protocol to the results of the Slc:Wistar/ST rats. Results: Of the 13 Slc:Wistar/ST rats fed an HFC diet containing 1.25% or 2.5% cholesterol, 11 (84.6%) developed histologically proven NASH without obesity, an increased visceral fat volume, insulin resistance, histopatological severe lobular inflammation and severe hepatic fibrosis. The HFC diets significantly increased the levels of mRNA encoding collagen type I alpha 1 (COL1A1), transforming growth factor-β1 (TGF-β1), tumor necrosis factor-α (TNF-α) and monocyte chemoattractant protein-1 (MCP-1). The SD rats also developed NASH without obesity, an increased visceral fat volume and insulin resistance, but the metabolic and histopathological effects, such as lower serum adiponectin levels, higher serum leptin levels, histopatological severe lobular inflammation and hepatic fibrosis, seemed to be more pronounced in the SD rats than in the Slc:Wistar/ST rats. Conclusions: These two rat models may reflect the human etiology of NASH that is influenced by dietary factors, and the obesity-resistant Slc:Wistar/ST rat model may be particularly useful for elucidating the pathophysiological mechanism of the so-called “lean NASH”. Keywords: Non-alcoholic fatty liver disease, Obesity, Wistar rat, Sprague-Dawley rat, High-fat diet, Cholesterol

Non-coding microRNAs (miRNAs) are powerful regulators of gene expression and critically involved in cardiovascular pathophysiology. The aim of the current study was to identify miRNAs regulating cardiac fibrosis. Cardiac samples of age-matched control subjects and sudden cardiac death (SCD) victims with primary myocardial fibrosis (PMF) were subjected to miRNA profiling. Old SCD victims with PMF and healthy aged human hearts showed increased expression of miR-1468-3p. In vitro studies in human cardiac fibroblasts showed that augmenting miR-1468-3p levels induces collagen deposition and cell metabolic activity and enhances collagen 1, connective tissue growth factor, and periostin expression. In addition, miR-1468-3p promotes cellular senescence with increased senescence-associated β-galactosidase activity and increased expression of p53 and p16. AntimiR-1468-3p antagonized transforming growth factor β1 (TGF-β1)-induced collagen deposition and metabolic activity. Mechanistically, mimic-1468-3p enhanced p38 phosphorylation, while antimiR-1468-3p decreased TGF-β1-induced p38 activation and abolished p38-induced collagen deposition. RNA sequencing analysis, a computational prediction model, and qPCR analysis identified dual-specificity phosphatases (DUSPs) as miR-1468-3p target genes, and regulation of DUSP1 by miR-1468-3p was confirmed with a dual-luciferase reporter assay. In conclusion, miR-1468-3p promotes cardiac fibrosis by enhancing TGF-β1-p38 signaling. Targeting miR-1468-3p in the older population may be of therapeutic interest to reduce cardiac fibrosis., Graphical Abstract, Cardiac expression of miR-1468-3p is increased in healthy aging subjects and in sudden cardiac death victims with primary myocardial fibrosis. miR-1468-3p promotes cell senescence and induces collagen production in fibroblasts by enhancing p38 signaling, and DUSP1 is a target gene of miR-1468-3p.

Alcoholic liver disease (ALD) is a leading cause of cirrhosis in the United States, which is characterized by extensive deposition of extracellular matrix proteins and formation of a fibrous scar. Hepatic stellate cells (HSCs) are the major source of collagen type 1 producing myofibroblasts in ALD fibrosis. However, the mechanism of alcohol‐induced activation of human and mouse HSCs is not fully understood. We compared the gene‐expression profiles of primary cultured human HSCs (hHSCs) isolated from patients with ALD (n = 3) or without underlying liver disease (n = 4) using RNA‐sequencing analysis. Furthermore, the gene‐expression profile of ALD hHSCs was compared with that of alcohol‐activated mHSCs (isolated from intragastric alcohol‐fed mice) or CCl4‐activated mouse HSCs (mHSCs). Comparative transcriptome analysis revealed that ALD hHSCs, in addition to alcohol‐activated and CCl4‐activated mHSCs, share the expression of common HSC activation (Col1a1 [collagen type I alpha 1 chain], Acta1 [actin alpha 1, skeletal muscle], PAI1 [plasminogen activator inhibitor‐1], TIMP1 [tissue inhibitor of metalloproteinase 1], and LOXL2 [lysyl oxidase homolog 2]), indicating that a common mechanism underlies the activation of human and mouse HSCs. Furthermore, alcohol‐activated mHSCs most closely recapitulate the gene‐expression profile of ALD hHSCs. We identified the genes that are similarly and uniquely up‐regulated in primary cultured alcohol‐activated hHSCs and freshly isolated mHSCs, which include CSF1R (macrophage colony‐stimulating factor 1 receptor), PLEK (pleckstrin), LAPTM5 (lysosmal‐associated transmembrane protein 5), CD74 (class I transactivator, the invariant chain), CD53, MMP9 (matrix metallopeptidase 9), CD14, CTSS (cathepsin S), TYROBP (TYRO protein tyrosine kinase‐binding protein), and ITGB2 (integrin beta‐2), and other genes (compared with CCl4‐activated mHSCs). Conclusion: We identified genes in alcohol‐activated mHSCs from intragastric alcohol‐fed mice that are largely consistent with the gene‐expression profile of primary cultured hHSCs from patients with ALD. These genes are unique to alcohol‐induced HSC activation in two species, and therefore may become targets or readout for antifibrotic therapy in experimental models of ALD., We identified genes in alcohol‐activated mHSCs from IG alcohol‐fed mice that are largely consistent with the gene‐expression profile of primary cultured hHSCs from patients with ALD. These genes are unique to alcohol‐induced HSC activation in two species, and therefore may become targets or readout for antifibrotic therapy in experimental models of ALD.

The in vivo antifibrotic effect of a fucoidan extract (FE) from Sargassum fluitans Borgesen was evaluated in a carbon tetrachloride-induced liver damage model in rats over twelve weeks. Chemical analysis showed the FE to contain carbohydrates, sulfates, uronic acids, protein, phenols, and to have a molecular weight of ~60 kDa. Physiological, biochemical, histological and genetic assays were done. Daily oral administration of FE (50 mg/kg) reduced liver enzymatic activity, liver infiltration of inflammatory cells, collagen fiber deposition and gene expression cytokines such as interleukin beta 1 (IL-β1), tumor necrosis factor alpha (TNF-α), transforming growth factor beta 1 (TGF-β1), Smad-3, Smad-2, collagen 1 alpha 1 (col1α1) and tissue inhibitor of metalloproteinase 1 (TIMP-1). It also increased RNA expression of Smad-7 and metalloproteinase 2 and 9 (MMP2 and MMP9). The fucoidan extract exhibited an antifibrotic effect mediated by the inhibiting TGF-β1/Smad pathway, as well as anti-inflammatory effects.

To study whether TGF-β1/IL-11/MEK/ERK (TIME) signaling mediates senescence-associated pulmonary fibrosis (SAPF) in Bmi-1-deficient (Bmi-1−/−) mice and determines the major downstream mediator of Bmi-1 and crosstalk between p16INK4a and reactive oxygen species that regulates SAPF, phenotypes were compared among 7-week-old p16INK4a and Bmi-1 double-knockout, N-acetylcysteine (NAC)-treated Bmi-1−/−, Bmi-1−/−, and wild-type mice. Pulmonary fibroblasts and alveolar type II epithelial (AT2) cells were used for experiments. Human pulmonary tissues were tested for type Ι collagen, α-smooth muscle actin (α-SMA), p16INK4a, p53, p21, and TIME signaling by using enzyme-linked immunosorbent assay (ELISA). Our results demonstrated that Bmi-1 deficiency resulted in a shortened lifespan, ventilatory resistance, poor ventilatory compliance, and SAPF, including cell senescence, DNA damage, a senescence-associated secretory phenotype and collagen overdeposition that was mediated by the upregulation of TIME signaling. The signaling stimulated cell senescence, senescence-related secretion of TGF-β1 and IL-11 and production of collagen 1 by pulmonary fibroblasts and the epithelial-to-mesenchymal transition of AT2 cells. These processes were inhibited by anti-IL-11 or the MEK inhibitor PD98059. NAC treatment prolonged the lifespan and ameliorated pulmonary dysfunction and SAPF by downregulating TIME signaling more than p16INK4a deletion by inhibiting oxidative stress and DNA damage and promoting ubiquitin-proteasome degradation of p16INK4a and p53. Cytoplasmic p16INK4a accumulation upregulated MEK/ERK signaling by inhibiting the translocation of pERK1/2 (Thr202/Tyr204) from the cytoplasm to the nucleus in senescent fibroblasts. The accumulation of collagen 1 and α-SMA in human lungs accompanied by cell senescence may be mediated by TIME signaling. Thus, this signaling in aging fibroblasts or AT2 cells could be a therapeutic target for preventing SAPF., Lung fibrosis: calling TIME on disease progression Targeting cellular signals that are increased in lung fibrosis may help halt disease progression. The build-up of scarred and thickened tissues in the lungs associated with aging and cellular deterioration is known as senescence-associated pulmonary fibrosis (SAPF). There are limited treatment options, and Jianliang Jin at Nanjing Medical University, China, and co-workers believe that targeting a complex of cellular signaling pathways called TGF-β1/IL-11/MEK/ERK (TIME) signals, the disruption of which affects tissue homeostasis, may hold the key to tackling the disease. The team conducted experiments on mouse models of SAPF, deficient in a gene called Bmi-1. The protein encoded by Bmi-1 is critical for cell division and DNA damage repair. In the Bmi-1-deficient mice, the researchers found that TIME signals were overexpressed, resulting in premature cellular deterioration, increased inflammation and accelerated collagen production.

Background Accumulating evidence shows that mesenchymal stem cells (MSCs) have the potential as a cellular therapy avenue for schistosome-induced liver injury. Extracellular vesicles (EVs) are membranous vesicles released by almost all cell types, and EVs produced by MSCs (MSC-EVs) exert therapeutic effects in several disease models. However, the potential of MSC-EVs in schistosomiasis treatment remains unclear. Methods Using survival analysis, HE and Masson’s trichrome staining, immunohistochemical, western blot analysis, real-time PCR, and EdU proliferation, we investigated the effects of human umbilical cord MSC-derived EVs (hUCMSC-EVs) on the survival and liver injury in the S. japonicum-infected mice and explored the underlying mechanism. Results Here, we found that like hUCMSCs, hUCMSC-EVs significantly ameliorated liver injury and improved the survival of schistosome-infected mice. Indeed, the hUCMSC-EV-mediated alleviation of liver injury is associated with decreased expression of α-smooth muscle actin (α-SMA), collagen 1, and collagen 3. More importantly, we showed that hUCMSC-EVs directly suppressed the proliferation of LX2 (human hepatic stellate cell) in vitro. In addition, hUCMSC-EVs significantly downregulated the activation of LX2 after transforming growth factor-β1 (TGF-β1) treatment. Conclusion Our results provided the first evidence that hUCMSC-EVs reduced liver injury in S. japonicum-infected mice, potentially creating new avenues for the treatment of liver damage in schistosomiasis.

Genomic features have been gradually regarded as part of the fundamentals to the clinical diagnosis and treatment for gastric cancer. However, the molecular alterations taking place during the progression of gastric cancer remain unclear. Therefore, identification of potential key genes and pathways in the gastric cancer progression is crucial to clinical practices. The gene expression profile, GSE103236, was retrieved for the identification of the differentially expressed genes (DEGs), followed by gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichments, gene set enrichment analysis (GSEA) and the protein-protein interaction (PPI) networks. Multiple bioinformatics platforms were employed for expression and prognostic analysis. Fresh frozen gastric cancer tissues were used for external validation. A total of 161 DEGs were identified from GSE103236. The PPI network-derived hub genes included collagen type I alpha 1 chain (COL1A1), tissue inhibitor of the metalloproteinases (TIMP1), Secreted Phosphoprotein 1 (SPP1), somatostatin (SST), neuropeptide Y (NPY), biglycan (BGN), matrix metallopeptidase 3 (MMP3), apolipoprotein E (APOE), ATPase H+/K+ transporting alpha subunit (ATP4A), lysyl oxidase (LOX). SPP1 (log rank p = 0.0048, HR = 1.39 [1.1–1.75]) and MMP3 (log rank p

Background Scars affects the appearance and results in tissue damage. In this research, we preliminarily studied the function and mechanism of curcumin (CUR) on cell proliferation and soluble collagen synthesis in NIH-3T3 cells. Methods CCK-8 was used to detect the IC50 of CUR. Moreover, Western blot was used to measure the expression of cell proliferation-related, soluble collagen synthesis and pathway-related proteins. Sircol assay was determined the expression of soluble collagen. Furthermore, reverse transcription quantitative PCR (RT-qPCR) was used to determined miR-29a, α-smooth muscle aorta (α-SMA), soluble collagen 1 (Col 1) and Col 3 expression. Results CUR inhibited cell viability and proliferation-related proteins expression. Transforming growth factor-β (TGFβ1)-induced heightened the expression of proliferation-related proteins and soluble collagen synthesis-related proteins. CUR inhibited TGFβ1-induced proliferation and soluble collagen synthesis. Furthermore, CUR positively related miR-29a and miR-29a mimic inhibited TGFβ1-induced proliferation and soluble collagen synthesis. Besides, transfection with miR-29a inhibitor could partly reverse the effects of CUR. CUR inhibited the ERK1/2 and β-catenin pathways and the miR-29a inhibitor reversed the above results. Otherwise, soluble collagen 1 (Col 1) partly reversed the effects of CUR on proliferation and soluble collagen synthesis and silenced Col 1/3 could inhibit ERK1/2 and β-catenin signaling pathways. Conclusion CUR restrained TGFβ1-induced proliferation and soluble collagen synthesis in NIH-3T3 cells by up-regulation of miR-29a via ERK1/2 and β-catenin signaling pathways.

Osteoporosis significantly impacts the normal life of the elderly and is reported to be closely related to dysfunction of osteoblastic differentiation. Runt-related transcription factor-2 (Runx2) is a critical transcriptional factor involved in the regulation of osteoblast differentiation. Omarigliptin is a novel dipeptidyl peptidase-4 (DDP-4) inhibitor and this study proposes to probe into its possible therapeutic function against Osteoporosis by investigating its impacts on osteoblastic differentiation. Osteogenic medium was used to induce osteoblastic differentiation in MC3T3‑E1 cells, and was verified by the increased alkaline phosphatase (ALP) activity, enhanced mineralization, and promoted expression level of osteoblastic differentiation-related factors, including bone morphogenetic protein-2 (BMP-2), ALP, osteocalcin (Ocn), collagen type I alpha 1 (Col1a1), Collagen Type I alpha 2 (Col1a2), Runx2, osterix (Sp7), fibroblast growth factor receptor 2 (Fgfr2), and fibroblast growth factor receptor 3 (Fgfr3), accompanied by the activation of the p38 and Akt pathways. After treatment with Omarigliptin, the ALP activity and mineralization were further promoted, accompanied by the further upregulation of osteoblastic differentiation-related factors, and activation of the p38 and Akt pathways. Lastly, Omarigliptin-induced osteoblastic differentiation, promoted ALP activity, and increased expression levels of Sp7, Fgfr2, Fgfr3, BMP-2, Ocn, ALP, Col1a1, and Col1a2, in the osteogenic medium- cultured MC3T3‑E1 cells were dramatically abolished by the knockdown of Runx2. Taken together, our data reveal that Omarigliptin promoted osteoblastic differentiation by regulating Runx2.

Glomerular mesangial cell (MC) proliferation and extracellular matrix deposition are the main pathological changes in diabetic nephropathy. Hydrogen sulfide (H2S) inhibits the proliferation of MCs. Dopamine 1 receptors (DR1) are expressed in MCs and serve important physiological roles. However, it is unclear whether DR1 activation inhibits MC proliferation by increasing endogenous H2S. The present study found that the production of H2S and the expression of DR1 and cystathionine‑γ‑lyase (CSE) were decreased in the renal tissues of diabetic mice and high glucose (HG)‑induced MCs. SKF38393 (a DR1 agonist) increased the production of H2S and the expression of DR1 and CSE and NaHS (an exogenous H2S donor) only increased H2S production and CSE expression but not DR1 expression. HG increased the thickness of the glomerular basement membrane, cell viability and proliferation, the expression of cyclin D1, PCNA, collagen 1 and α‑smooth muscle actin and the activity of phosphorylated ERK1/2 and decreased the expression of P21 and MMP9. SKF38393 and NaHS reversed the effects of HG. PPG (a CSE inhibitor) abolished the beneficial effects of SKF38393. The beneficial effects of SKF38393 were similar to those of PD98059 (an ERK1/2 inhibitor). Taken together, the findings suggested that the DR1‑CSE/H2S pathway activation attenuated diabetic MC proliferation and extracellular matrix deposition by downregulating the ERK1/2 signaling pathway.

Chewing areca nut (betel quid) is strongly associated with oral submucous fibrosis (OSF), a pre-cancerous lesion. Among the areca alkaloids, arecoline is the main agent responsible for fibroblast proliferation; however, the specific molecular mechanism of arecoline affecting the OSF remains unclear. The present study revealed that arecoline treatment significantly enhanced Transforming growth factor-β (TGF-β)-induced buccal mucosal fibroblast (BMF) activation and fibrotic changes. Arecoline interacts with phosphodiesterase 4A (PDE4A) to exert its effects through modulating PDE4A activity but not PDE4A expression. PDE4A silence reversed the effects of arecoline on TGF-β-induced BMFs activation and fibrotic changes. Moreover, the exchange protein directly activated by cAMP 1 (Epac1)-selective Cyclic adenosine 3′,5′-monophosphate (cAMP) analog (8-Me-cAMP) but not the protein kinase A (PKA)-selective cAMP analog (N6-cAMP) remarkably suppressed α-smooth muscle actin(α-SMA) and Collagen Type I Alpha 1 Chain (Col1A1) protein levels in response to TGF-β1 and arecoline co-treatment, indicating that cAMP-Epac1 but not cAMP-PKA signaling is involved in arecoline functions on TGF-β1-induced BMFs activation. In conclusion, arecoline promotes TGF-β1-induced BMFs activation through enhancing PDE4A activity and the cAMP-Epac1 signaling pathway during OSF. This novel mechanism might provide more powerful strategies for OSF treatment, requiring further in vivo and clinical investigation.

Background MicroRNA-101 (miR-101) is a tumor suppressor microRNA (miRNA) and its loss is associated with the occurrence and progression of various diseases. However, the biological function and target of miR-101 in the pathogenesis of hypertrophic scars (HS) remains unknown. Methods We harvested HS and paired normal skin (NS) tissue samples from patients and cultured their fibroblasts (HSF and NSF, respectively). We used quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), fluorescence in situ hybridization (FISH), enzyme-linked immunosorbent assays (ELISA) and Western blot analyses to measure mRNA levels and protein expression of miR-101, enhancer of zeste homolog 2 (EZH2), collagen 1 and 3 (Col1 and Col3) and α-smooth muscle actin (α-SMA) in different in vitro conditions. We also used RNA sequencing to evaluate the relevant signaling pathways and bioinformatics analysis and dual-luciferase reporter assays to predict miR-101 targets. We utilized a bleomycin-induced fibrosis mouse model in which we injected miR-101 mimics to evaluate collagen deposition in vivo. Results We found low expression of miR-101 in HS and HSF compared to NS and NSF. Overexpressing miR-101 decreased Col1, Col3 and α-SMA expression in HSF. We detected high expression of EZH2 in HS and HSF. Knockdown of EZH2 decreased Col1, Col3 and α-SMA in HSF. Mechanistically, miR-101 targeted the 3′-untranslated region (3′UTR) of EZH2, as indicated by the decreased expression of EZH2. Overexpressing EZH2 rescued miR-101-induced collagen repression. MiR-101 mimics effectively suppressed collagen deposition in the bleomycin-induced fibrosis mouse model. Conclusions Our data reveal that miR-101 targets EZH2 in HS collagen production, providing new insight into the pathological mechanisms underlying HS formation.

Mesothelial to mesenchymal transition (MesoMT) is one of the crucial mechanisms underlying pleural fibrosis, which results in restrictive lung disease. DOCK2 (dedicator of cytokinesis 2) plays important roles in immune functions; however, its role in pleural fibrosis, particularly MesoMT, remains unknown. We found that amounts of DOCK2 and the MesoMT marker α-SMA (α-smooth muscle actin) were significantly elevated and colocalized in the thickened pleura of patients with nonspecific pleuritis, suggesting the involvement of DOCK2 in the pathogenesis of MesoMT and pleural fibrosis. Likewise, data from three different pleural fibrosis models (TGF-β [transforming growth factor-β], carbon black/bleomycin, and streptococcal empyema) consistently demonstrated DOCK2 upregulation and its colocalization with α-SMA in the pleura. In addition, induced DOCK2 colocalized with the mesothelial marker calretinin, implicating DOCK2 in the regulation of MesoMT. Our in vivo data also showed that DOCK2-knockout mice were protected from Streptococcus pneumoniae–induced pleural fibrosis, impaired lung compliance, and collagen deposition. To determine the involvement of DOCK2 in MesoMT, we treated primary human pleural mesothelial cells with the potent MesoMT inducer TGF-β. TGF-β significantly induced DOCK2 expression in a time-dependent manner, together with α-SMA, collagen 1, and fibronectin. Furthermore, DOCK2 knockdown significantly attenuated TGF-β–induced α-SMA, collagen 1, and fibronectin expression, suggesting the importance of DOCK2 in TGF-β–induced MesoMT. DOCK2 knockdown also inhibited TGF-β–induced Snail upregulation, which may account for its role in regulating MesoMT. Taken together, the current study provides evidence that DOCK2 contributes to the pathogenesis of pleural fibrosis by mediating MesoMT and deposition of neomatrix and may represent a novel target for its prevention or treatment.

Human dental pulp cells (hDPCs) have shown their plasticity when treated with the hydroxamate-based histone deacetylase (HDAC) inhibitor members, Trichostatin A (TSA), and suberoylanilide hydroxamic acid (SAHA). However, a comparison of their potency to stimulate odontoblast-like differentiation and mineralization has not been reported. The aim of our study was to confirm and compare these TSA and SAHA effects. Primary hDPCs cultured with/without various TSA or SAHA concentrations were evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), ALP activity, alizarin red staining, and scratch wound healing assays. The inhibitory effect of TSA and SAHA on inhibiting the activity of HDAC was evaluated by HDAC activity assay. Odontoblast-related gene expression was determined using RT-qPCR. The MTT assay indicated that TSA or SAHA did not affect hDPC viability. TSA or SAHA treatment-induced odontoblast-like differentiation as evidenced by a significant increase in alkaline phosphatase activity and mineral deposition after 400 nM TSA or 1 μM SAHA treatment. A significant increase in nuclear factor I C, kruppel like factor 4, dentin matrix acidic phosphoprotein 1, dentin sialophosphoprotein, collagen type I alpha 1 chain, alkaline phosphatase (ALPL), integrin-binding sialoprotein, bone gamma-carboxyglutamate protein, vascular endothelial growth factor A, and cyclin-dependent kinase inhibitor 1A gene expression analyzed by RT-qPCR, at 24, 72 h, 7, and 10 days of treatment. The activity of HDAC in hDPCs culture was significantly inhibited after 72 h TSA and SAHA treatment. The scratch wound healing assay displayed enhanced cell migration at 72 h after TSA or SAHA treatment. Our findings demonstrated that TSA and SAHA have similar stimulatory effects in inducing HDPC odontogenic differentiation and mineralization and propose another potential use of TSA and SAHA to promote dentin regeneration.

We generated and characterized a transgenic mouse line with the tendon-specific expression of a double fluorescent reporter system, which will fulfill an unmet need for animal models to support real-time monitoring cell behaviors during tendon development, growth, and repair in vitro and in vivo. The mScarlet red fluorescent protein is driven by the Scleraxis (Scx) promoter to report the cell lineage alteration. The blue fluorescent protein reporter is expressed under the control of the 3.6kb Collagen Type I Alpha 1 Chain (Col1a1) proximal promoter. In this promoter, the existence of two promoter regions named tendon-specific cis-acting elements (TSE1, TSE2) ensure the specific expression of blue fluorescent protein (BFP) in tendon tissue. Collagen I is a crucial marker for tendon regeneration that is a major component of healthy tendons. Thus, the alteration of function during tendon repair can be estimated by BFP expression. After mechanical stimulation, the expression of mScarlet and BFP increased in adipose-derived mesenchymal stem cells (ADMSCs) from our transgenic mouse line, and there was a rising trend on tendon key markers. These results suggest that our tendon-specific double reporter system is a novel model used to study cell re-differentiation and extracellular matrix alteration in vitro and in vivo.

Liver fibrosis, which is the outcome of wound-healing response to chronic liver damage, represents an unmet clinical need. This study evaluated the anti-fibrotic and anti-inflammatory effects of the polyphenol oleocanthal (OC) extracted from extra virgin olive oil (EVOO) by an in vitro/in vivo approach. The hepatic cell lines LX2 and HepG2 were used as in vitro models. The mRNA expression of pro-fibrogenic markers, namely alpha-smooth muscle actin (α-SMA), collagen type I alpha 1 chain (COL1A1), a panel of metalloproteinases (MMP1, MMP2, MMP3, MMP7, MMP9) and vascular endothelial growth factor A (VEGFA) as well as the pro-oxidant genes NADPH oxidases (NOXs) 1 and 4 were evaluated in TGF-β activated LX2 cells by qRT-PCR. α-SMA and COL1A1 protein expression was assessed by immunofluorescence coupled to confocal microscopy. VEGFA release from LX2 was measured by ELISA. We also evaluated the amount of reactive oxygen species (ROS) produced by H2O2 activated- HepG2 cells. In vivo, OC was administered daily by oral gavage to Balb/C mice with CCl4-induced liver fibrosis. In this model, we measured the mRNA hepatic expression of the three pro-inflammatory interleukins (IL) IL6, IL17, IL23, chemokines such as C-C Motif Chemokine Ligand 2 (CCL2) and C-X-C Motif Chemokine Ligand 12 (CXCL12), and selected miRNAs (miR-181-5p, miR-221-3p, miR-29b-3p and miR-101b-3p) by qRT-PCR. We demonstrated that OC significantly downregulated the gene/protein expression of α-SMA, COL1A1, MMP2, MMP3, MMP7 and VEGF as well as the oxidative enzymes NOX1 and 4 in TGFβ1-activated LX2 cells, and reduced the production of ROS by HepG2. In vivo OC, beside causing a significant reduction of fibrosis at histological assessment, counteracted the CCl4-induced upregulation of pro-fibrotic and inflammatory genes. Moreover, OC upregulated the anti-fibrotic miRNAs (miR-29b-3p and miR-101b-3p) reduced in fibrotic mice, while downregulated the pro-fibrotic miRNAs (miR-221-3p and miR-181-5p), which were dramatically upregulated in fibrotic mice. In conclusion, OC exerts a promising antifibrotic effect via a combined reduction of oxidative stress and inflammation involving putative miRNAs, which in turn reduces hepatic stellate cells activation and liver fibrosis.

OBJECTIVE Lymphatic vessels (LVs) maintain fluid homeostasis by draining interstitial fluid. A failure in lymphatic drainage triggers lymphatic diseases such as lymphedema. Since lymphatic drainage is regulated by lymphatic barrier function, developing experimental models that assess lymphatic barrier function is critical for better understanding of lymphatic physiology and disease. METHODS We built a lymphatic vessel-on-chip (LV-on-chip) by fabricating a microfluidic device that includes a hollow microchannel embedded in three-dimensional (3D) hydrogel. Employing luminal flow in the microchannel, human lymphatic endothelial cells (LECs) seeded in the microchannel formed an engineered LV exhibiting 3D conduit structure. RESULTS Lymphatic endothelial cells formed relatively permeable junctions in 3D collagen 1. However, adding fibronectin to the collagen 1 apparently tightened LEC junctions. We tested lymphatic barrier function by introducing dextran into LV lumens. While LECs in collagen 1 showed permeable barriers, LECs in fibronectin/collagen 1 showed reduced permeability, which was reversed by integrin α5 inhibition. Mechanistically, LECs expressed inactivated integrin α5 in collagen 1. However, integrin α5 is activated in fibronectin and enhances barrier function. Integrin α5 activation itself also tightened LEC junctions in the absence of fibronectin. CONCLUSIONS Lymphatic vessel-on-chip reveals integrin α5 as a regulator of lymphatic barrier function and provides a platform for studying lymphatic barrier function in various conditions.

Creating a pro-regenerative immune microenvironment around implant biomaterial surfaces is significant to osseointegration. Immune cells, especially macrophages that participate in the osseointegration, including osteogenesis, osteoclastogenesis, and angiogenesis, should be considered when testing biomaterials. In this study, we immobilized an antimicrobial peptide GL13K with immunomodulatory properties onto a titanium surface via silanization. The modified surfaces show good biocompatibility with bone mesenchymal stromal cells (BMSCs), human umbilical vein endothelial cells (HUVECs), and RAW264.7. By co-culturing BMSCs with RAW264.7, we found that the GL13K-coated titanium surfaces could promote late-stage osteogenesis as demonstrated by the upregulated expression of recombinant collagen type I alpha 1 (COL-1α1) and more extracellular matrix mineralization, while the early phase remained unchanged. The surfaces inhibited the osteoclastogenic differentiation of RAW264.7 cells by restraining nuclear factor-activated T cells, cytoplasmic 1 (NFATc1), the main factor of the receptor activator of nuclear factor-κ B, and the receptor activator of the nuclear factor-κ B ligand signaling pathway, from entering the nucleus and further reduced the expression of the activating osteoclastogenic tartrate-resistant acid phosphatase gene. Moreover, the GL13K-coated titanium surface demonstrated significant promotion of angiogenesis differentiation of HUVECs as indicated by the upregulated expression of essential angiogenesis function genes, including hypoxia-inducible factor-1α, endothelial nitric oxide synthase, kinase insert domain receptor, and vascular endothelial growth factor A (HIF-1α, eNOS, KDR, and VEGF-A). Taken together, these results demonstrated that the GL13K coating had properties of osteogenesis, angiogenesis, and anti-osteoclastogenesis via its immunomodulatory potential.

Background Carpal tunnel steroid injection is a nonoperative intervention for the treatment for idiopathic carpal tunnel syndrome (CTS). The antifibrotic, anti-inflammatory, and antiedematous properties of steroids account for their therapeutic effects in the context of CTS; however, their relative contribution has not been clarified. Methods Fibroblasts from subsynovial connective tissues (SSCT) were intraoperatively collected from patients with idiopathic CTS and were incubated with or without the steroid triamcinolone acetonide (TA) for 1, 3, and 7 days; the expression of fibrosis-related genes and inflammatory cytokines was evaluated using quantitative reverse transcription-polymerase chain reaction. A clinical prospective study was conducted with patients who received carpal tunnel TA injections. We performed clinical and electrophysiological evaluations before and 1, 3, and 5 months after TA injection; and we compared the median nerve, flexor tendon, and SSCT areas and the median nerve flattening ratio before and 1 month after TA injection using 3-T magnetic resonance imaging (MRI). Results TA induced downregulation of the fibrosis-related genes Col1A1 (collagen type I alpha 1 chain), Col1A2, and Col3A1 but not the inflammation-related genes. The nerve flattening ratio did not change after TA injection according to the MRI-based observation of the median nerve, flexor tendon, and SSCT areas. Conclusions The therapeutic effects of injected TA are apparently mediated by its antifibrotic rather than its anti-inflammatory and antiedematous properties. TA probably alters the properties but not the morphology of SSCT. Level of evidence Therapeutic Level II. See Instructions for Authors for a complete description of levels of evidence.