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Collagen triple helix repeat containing 1 (CTHRC1), a secreted glycoprotein, is widely expressed in many tissues. It has been recently defined as a novel marker for rheumatoid arthritis (RA), a systemic inflammatory disorder. However, the precise role of CTHRC1 in other chronic inflammatory diseases, like periodontal disease, remains unclear. This research aimed to explore the presence of CTHRC1 in periodontal inflammation, determine the precise role in inflammatory response modulation in periodontal ligament cells (PDLCs), and explore its underlying mechanisms. In vivo gingival crevicular fluid (GCF) and gingivae were obtained from healthy people and chronic periodontitis patients. Maxillary tissues of mice with or without ligature-induced periodontitis were immunostained for CTHRC1. In vitro human PDLCs were treated with tumor necrosis factor alpha (TNF-α) to mimic the inflammatory environment. Small interfering RNA (siRNA) was used to silence CTHRC1. SB203580 was used to inhibit the p38 mitogen-activated protein kinase (MAPK) pathway. CTHRC1 was highly expressed in GCF and gingival tissues of periodontitis patients. Animal models also revealed the same tendency. CTHRC1 knockdown promoted inflammatory cytokine production and activated the p38 MAPK signaling pathway in PDLCs. Inhibiting the p38 MAPK signaling pathway partially attenuated the inflammatory responses. This study revealed that CTHRC1 was highly expressed in periodontitis and suggested that CTHRC1 might play an important role in modulating periodontal inflammation., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Matrix Gla protein (MGP), an extracellular matrix protein, has been widely reported to participate in the tumorigenic process and is abnormally expressed in several tumors. However, the role of MGP in colorectal cancer (CRC) remains unknown. Chemotherapy resistance represents a significant limitation in the treatment of CRC. Here, a comprehensive bioinformatics analysis revealed that MGP, which is overexpressed in CRC, might act as one of the critical genes conferring resistance to oxaliplatin (OXA). Furthermore, we found that MGP overexpression in tumor tissue might be correlated with cancer stage and patient prognosis, consistent with the bioinformatics analysis. The upregulation of MGP may act as an independent risk factor for CRC. The knockdown of MGP or inhibition of MGP expression significantly increased the sensitivity of the CRC cell lines to OXA. Suppression of MGP may reverse OXA resistance by upregulating copper transporter 1 (CTR1) and downregulating ATP7A and ATP7B. When used in combination with OXA, the inhibition of MGP reduced cancer cell proliferation, invasion, and migration and increased cell apoptosis in vitro. Suppression of MGP or OXA treatment alone significantly inhibited tumor growth in the CRC mouse model. Additionally, we found that OXA might promote the antitumor immune response in vivo. In summary, our study is the first to provide evidence that MGP expression confers OXA chemotherapy resistance in CRC and provides novel strategies to overcome chemotherapy resistance in CRC., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Calcineurin inhibitors, such as Cyclosporin (CsA), are the mainstay of anti-rejection therapy in solid organ transplants but can paradoxically induce progressive nephropathy characterised by renal dysfunction and interstitial fibrosis. Lysyl oxidases (LOXs), a group of enzymes that catalyse extracellular matrix (ECM) crosslinking, were shown to implicate in tissue scarring. It is hypothesized that inhibition of these enzymes may render therapeutic effects against CsA-induced nephropathy. In this study, 6-to-8 weeks old C57BL/6 J mice were administered saline or CsA (30 mg/kg/day s.c) for 16 weeks. At 8 weeks, CsA-treated animals were divided into 5 groups respectively treated with: (1) vehicle, (2) PXS-5505 (Pan-LOX inhibitor), (3) PXS-5382 (LOX-like 2 inhibitor), (4) PXS-5505 for 4 weeks then PXS-5382 for 4 weeks (sequential therapy), and (5) Telmisartan (standard therapy). Our results indicate that CsA administration significantly increased the levels of blood urea nitrogen, glomerular and tubular injury, tubulointerstitial fibrosis, inflammation and oxidative stress in mouse kidney. These changes were associated with upregulated mRNA expression of LOX and LOXL2. Administration of Pan-LOX or LOXL2 inhibitors or the sequential therapy suppressed the expression of ECM proteins (α-SMA, FN and COL1A), matrix metalloproteases (MMP)2 and 9, inflammatory markers (TNFα and MCP-1) and TGF-β1-Smad3 signalling. Among all regimens including telmisartan, only Pan-LOX inhibitor PXS-5505 was able to attenuate uraemia. Collectively, our study suggests that Pan-LOX and LOXL2 inhibition can attenuate progressive nephropathy due to CsA administration.

Glaucoma is a progressive optic neuropathy in more than 25 % of cases in patients with permanent blindness. The microRNA is implicated in modulating the cellular function of the trabecular meshwork (TM). The aim of this study is to investigate the role of miR-137 in glaucoma and illustrate the potential molecular mechanisms. We show that miR-137 was down-regulated in H 2 O 2 -induced human trabecular meshwork cells (HTMCs), and overexpression of miR-137 attenuated H 2 O 2 -induced cell growth inhibition, apoptosis and elevated extracellular matrix (ECM) protein expression. In addition, miR-137 blocked the activation of YAP/TAZ by directly targeting src. Overexpression of src or activation of the YAP/TAZ pathway partly abrogated the effects of miR-137 on H 2 O 2 -induced cell viability and apoptosis and dampened the inhibition effect on ECM protein expression. In conclusion, miR-137 promotes cell growth and inhibits extracellular matrix protein expression in H 2 O 2 -induced human trabecular meshwork cells via the YAP/TAZ pathway by targeting src. Hence, miR-137 might be used as a novel therapeutic target to treat glaucoma., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Systemic sclerosis a chronic, fibrotic disorder associated with high disease-specific mortality and morbidity. Cutaneous manifestations include dermal thickening and obliteration of dermal adipose tissue. Accumulation of low-molecular-weight hyaluronan, which signals through the receptor for hyaluronan-mediated motility, RHAMM, leads to progressive fibrosis and is correlated with increased severity of systemic sclerosis. The purpose of this study is to test the efficacy of two function-blocking RHAMM peptides, NPI-110 and NPI-106, in reducing skin fibrosis in a bleomycin-induced mouse model of systemic sclerosis. NPI-110 reduced visible measures of fibrosis (dermal thickness and collagen production, deposition, and organization) and profibrotic gene expression (Tgfb1, c-Myc, Col1a1, Col3a1). NPI-110 treatment also increased the expression of the antifibrotic adipokines perilipin and adiponectin. Both RHAMM peptides strongly reduced dermal RHAMM expression, predicting that dermal fibroblasts are peptide targets. Transcriptome and cell culture analyses using Rhamm-/- and Rhamm-rescued dermal fibroblasts reveal a TGFβ1/RHAMM/MYC signaling axis that promotes fibrogenic gene expression and myofibroblast differentiation. RHAMM function‒blocking peptides suppress this signaling and prevent TGFβ1-induced myofibroblast differentiation. These results suggest that inhibiting RHAMM signaling will offer a treatment method for cutaneous fibrosis in systemic sclerosis., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Metastasis is a major cause of cancer-related deaths. Tumor-intrinsic properties can determine whether tumor metastasis occurs or not. Here, by comparing the gene expression patterns in primary colorectal cancer (CRC) patients with or without metastasis, we found that Collagen Triple Helix Repeat Containing 1 (CTHRC1) in primary CRC served as a metastasis-associated gene. Animal experiments verified that CTHRC1 secreted by CRC cells promoted hepatic metastasis, which was closely correlated with macrophage infiltration. Depletion of macrophages by liposomal clodronate largely abolished the promoting effect of CTHRC1 on CRC liver metastasis. Furthermore, we demonstrated that CTHRC1 modulated macrophage polarization to M2 phenotypes through TGF-β signaling. A mechanistic study revealed that CTHRC1 bound directly to TGF-β receptor II and TGF-β receptor III, stabilized the TGF-β receptor complex, and activated TGF-β signaling. The combination treatment of CTHRC1 monoclonal antibody and anti-PD-1 blocking antibody effectively suppressed CRC hepatic metastasis. Taken together, our data demonstrated that CTHRC1 is an intrinsic marker of CRC metastasis and further revealed that CTHRC1 promoted CRC liver metastasis by reshaping infiltrated macrophages through TGF-β signaling, suggesting that CTHRC1 could be a potential biomarker for the early prediction of and a therapeutic target of CRC hepatic metastasis.

Ethnopharmacological Relevance: Peel of Citrus reticulata, a Chinese herbal drug with functions of regulating Qi and expelling phlegm, has been used for the treatment of lung related diseases in Chinese medicine for a long time. Its detailed effects on collagen in anti-idiopathic pulmonary fibrosis (IPF) is still unclear., Aim of the Study: To explore the effects of citrus alkaline extract (CAE) on collagen synthesis, crosslinking and deposition in pulmonary fibrosis and understand the possible signal pathways involved in the activity., Materials and Methods: CAE was prepared from C. reticulata. Bleomycin-induced pulmonary fibrosis mouse model was applied. Pulmonary fibrosis of lung was estimated with histopathology analysis, and collagen deposition was evaluated with immunohistochemistry. Collagen crosslinking related biomarkers and enzymes were analyzed with chemical methods, immunohistochemical and western blot analyses., Results: CAE oral administration lowered hydroxyproline content, inhibited the collagen deposition including expressions of collagen I and III, and relieved bleomycin-induced pulmonary fibrosis in mice model. The productions of a collagen crosslink pyridinoline and crosslinking related enzymes including lysyl oxidase (LOX), lysyl oxidase-like protein 1 (LOXL1) in lung were suppressed by CAE treatment. Furthermore, the protein expressions of TGF-β1 and Smad3 levels in lungs were also downregulated by CAE., Conclusions: This study demonstrated that CAE inhibited collagen synthesis, crosslinking and deposition, and ameliorated bleomycin-induced pulmonary fibrosis. Preliminary mechanism study revealed that CAE exerted its bioactivity at least via downregulation of TGF-β1/Smad3 pathway. Our findings provided a great potential in fighting IPF based on CAE., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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Neuronal synapse formation is critical for brain development and depends on secreted factors from astrocytes. Here, we report that small extracellular vesicles (EVs) secreted from primary astrocytes, but not from neurons or C6 glioma cells, greatly enhance spine and synapse formation by primary cortical neurons. A comparative proteomics analysis of small EVs from astrocytes, neurons, and C6 glioma cells identified fibulin-2 as a promising EV cargo to regulate synaptogenesis. Treatment of cortical neurons with recombinant fibulin-2 increased the formation of spines and synapses, similar to the effect of small EVs. In addition, treatment of neurons with fibulin-2 or astrocyte-derived small EVs led to increased phosphorylation of Smad2, an indicator of TGF-β signaling. Finally, the effects of fibulin-2 and astrocyte-derived small EVs on synapse formation were reversed by inhibiting transforming growth factor β (TGF-β) signaling. These data suggest a model in which astrocyte EVs promote synapse formation via fibulin-2-mediated activation of TGF-β signaling., Competing Interests: Declaration of interests A.M.W. is a member of the advisory board for Cell Reports. Otherwise, there are no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The family with sequence similarity 20, member C (Fam20C), a Golgi casein kinase, has been recently regarded as a potential therapeutic target for the treatment of triple negative breast cancer (TNBC). Lacking enzyme activity center has been becoming an obstacle to the development of small-molecule inhibitors of Fam20C. Herein, we combined in silico high-throughput screening with chemical synthesis methods to obtain a new small-molecule Fam20C inhibitor 3r, which exhibited desired anti-proliferative activities against MDA-MB-231 cells and also inhibited migration. Subsequently, the enzymatic assay, molecular docking, and molecular dynamics (MD) simulations were carried out for validating that 3r could bind to Fam20C. In addition, 3r was found to induce apoptosis via the mitochondrial pathway in MDA-MB-231 cells as well as to inhibit cell migration. Moreover, we demonstrated that 3r inhibited tumor growth in vivo and thereby having a good therapeutic potential on TNBC. Taken together, these results suggest that 3r may be a novel Fam20C inhibitor with anti-proliferative and apoptosis-inducing activities, which would shed light on discovering more small-molecule drugs for the future TNBC therapy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

Background: Dentin matrix protein 1 (DMP1) is highly expressed in mineralized tooth and bone, playing a critical role in mineralization and phosphate metabolism. One important role for the expression of DMP1 in the nucleus of preosteoblasts is the up-regulation of osteoblast-specific genes such as osteocalcin and alkaline phosphatase 1 . The present study aimed to investigate the potential application of human DMP1 promoter as an indicator marker of osteoblastic differentiation., Methods: In the present study, we developed DMP1 promoter-DsRed-GFP knock-in mesenchymal stem cell (MSCs) via the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system that enabled automatic detection of osteoblast differentiation. With the application of a homology-directed knock-in strategy, a 2-kb fragment of DMP1 promoter, which was inserted upstream of the GFP and DsRed reporter cassette, was integrated into the human ROSA locus to generate double fluorescent cells. We further differentiated MSCs under osteogenic media to monitor the fate of MSCs. First, cells were transfected using CRISPR/Cas9 plasmids, which culminated in MSCs with a green fluorescence intensity, then GFP-positive cells were selected using puromycin. Second, the GFP-positive MSCs were differentiated toward osteoblasts, which demonstrated an increased red fluorescence intensity. The osteoblast differentiation of MSCs was also verified by performing alkaline phosphatase and Alizarin Red assays., Results: We have exploited the DMP1 promoter as a predictive marker of MSC differentiation toward osteoblasts. Using the CRISPR/Cas9 technology, we have identified a distinctive change in the fluorescence intensities of GFP knock-in (green) and osteoblast differentiated MSCs 2 ., Conclusions: The data show that DMP1-DsRed-GFP knock-in MSCs through CRISPR/Cas9 technology provide a valuable indicator for osteoblast differentiation. Moreover, The DMP1 promoter might be used as a predictive marker of MSCs differentiated toward osteoblasts., (© 2020 John Wiley & Sons, Ltd.)

Vascular calcification (VC) is a major risk factor for increasing cardiovascular morbidity and mortality in patients with chronic kidney disease (CKD). Indoxyl sulfate (IS), a representative uremic toxin, is closely associated with VC in CKD patients. Matrix Gla protein (MGP) plays pivotal role in VC as a calcification inhibitor. The aim of this work was to explore whether MGP was involved in IS-induced VC. Here, we demonstrated the role of MGP in the IS-induced osteogenic differentiation of human aortic smooth muscle cells (HASMCs). The methods included Von Kossa staining, immunohistochemistry, Alizarin Red staining, quantitative real-time PCR and western blotting. MGP was decreased in calcified arteries both in CKD patients and rats. In vitro, IS suppressed MGP expression in HASMCs by activating ROS/NF-κB signaling in parallel with osteogenic differentiation, which was mitigated by inhibiting ROS and NF-κB with diphenyleneiodonium and Bay11-7082. Further investigation showed that IS induced NF-κB-responsive microRNA (miR)-155-5p mediating MGP downregulation. Overexpression of miR-155-5p with mimics aggravated IS-induced MGP reduction and osteogenic differentiation. In contrast, these conditions were diminished by silencing miR-155-5p. We demonstrate that IS promotes the HASMCs phenotype switch by suppressing MGP expression via ROS/NF-κB/miR-155-5p signaling and provide a new insight for the pathogenesis of IS-induced VC., (Copyright © 2020. Published by Elsevier Inc.)

High-grade serous ovarian carcinoma (HGSOC) is one of the most lethal gynecological malignancies; however, the precise molecular mechanisms have not been fully characterized. Fibulin‑5 (FBLN‑5) is an extracellular matrix (ECM) glycoprotein, and plays a crucial role in maintaining the stability of ECM structures, regulating cell proliferation and tumorigenesis. In the present study, the expression of FBLN‑5, as determined by western blot analysis and immunohistochemistry, was significantly increased in normal fallopian tube (FT) samples compared with that in HGSOC samples, and decreased FBLN5 expression was associated with unfavorable prognosis of HGSOC. Functional characterization revealed that FBLN5 overexpression significantly inhibited migration, invasion and proliferation abilities of ovarian cancer cells in vitro. Furthermore, micro (mi)RNA‑27a‑3p (miR‑27a‑3p) was revealed to be increased in HGSOC, and dual‑luciferase reporter assay indicated that miR‑27a‑3p was functioned as a negative regulator of FBLN5 by directly binding with its 3'‑untranslated region. Collectively, FBLN5 expression was associated with prognosis, proliferation, and metastasis in HGSOC. We hypothesized that FBLN5 was targeted by miR‑27a‑3p and may serve as a biomarker and provide a new therapeutic approach for the treatment of HGSOC.

Tumor growth and invasion occurs through a dynamic interaction between cancer and stromal cells, which support an aggressive niche. MicroRNAs are thought to act as tumor messengers to "corrupt" stromal cells. We previously demonstrated that miR-9, a known metastamiR, is released by triple negative breast cancer (TNBC) cells to enhance the transition of normal fibroblasts (NFs) into cancer-associated fibroblast (CAF)-like cells. EGF containing fibulin extracellular matrix protein 1 ( EFEMP1 ), which encodes for the ECM glycoprotein fibulin-3, emerged as a miR-9 putative target upon miRNA's exogenous upmodulation in NFs. Here we explored the impact of EFEMP1 downmodulation on fibroblast's acquisition of CAF-like features, and how this phenotype influences neoplastic cells to gain chemoresistance. Indeed, upon miR-9 overexpression in NFs, EFEMP1 resulted downmodulated, both at RNA and protein levels. The luciferase reporter assay showed that miR-9 directly targets EFEMP1 and its silencing recapitulates miR-9-induced pro-tumoral phenotype in fibroblasts. In particular, EFEMP1 siRNA-transfected (si- EFEMP1 ) fibroblasts have an increased ability to migrate and invade. Moreover, TNBC cells conditioned with the supernatant of NFs transfected with miR-9 or si- EFEMP1 became more resistant to cisplatin. Overall, our results demonstrate that miR-9/ EFEMP1 axis is crucial for the conversion of NFs to CAF-like cells under TNBC signaling.

Background: As a substrate of apoER2, Reelin has been verified to exert neuroprotection by preventing memory impairment. Pinocembrin is the most abundant natural flavonoid found in propolis, and it has been used to exert neuroprotection, blood-brain barrier protection, anti-oxidation, and inflammation diminishing, both in vitro and in vivo. However, the roles and molecular mechanisms of pinocembrin in neurobehavioral outcomes and neuronal repair after vascular dementia are still under investigation., Purpose: To explore the role of pinocembrin in the involvement of the Reelin-dab1 signaling pathway in improving memory impairment, both in cell culture and animals experiments., Material and Methods: Behavioral tests were conducted on day 48 to confirm the protection of pinocembrin against cognitive impairment. Cell and molecular biology experiments demonstrated that the Reelin-dab1 pathway mediates the underlying mechanism of cognitive improvement by pinocembrin., Results: It was showed that pinocembrin alleviated learning and memory deficits induced by vascular dementia, by inducing the expression of Reelin, apoER2, and p-dab1 in the hippocampus. The expression of Reelin and p-dab1 was both inhibited following Reelin RNA interference in SH-SY5Y prior to oxygen glucose deprivation (OGD) injury, suggesting that Reelin played a core role in pinocembrin's effect on OGD in vitro., Conclusion: Pinocembrin improves the cognition via the Reelin-dab1 signaling pathway., Competing Interests: The authors report no conflicts of interest in this work., (© 2020 Kang et al.)

The outbreak of the highly contagious and deadly severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), also known as coronavirus disease 2019 (COVID-19), has posed a serious threat to public health across the globe, calling for the development of effective diagnostic markers and therapeutics. Here, we report a highly reliable severity diagnostic biomarker, acetylated 676th lysine transforming growth factor-beta-induced protein (TGFBIp K676Ac). TGFBIp K676Ac was consistently elevated in the blood of patients with SARS-CoV-2 pneumonia ( n = 113), especially in patients in the intensive care unit (ICU) compared to non-ICU patients. Patients' blood samples showed increased cytokines and lymphopenia, which are exemplary indicators of SARS-CoV-2 pneumonia. Treatment with TGFBIp neutralizing antibodies suppressed the cytokine storm. The increased level of TGFBIp K676Ac in ICU patients suggests the promise of this protein as a reliable severity diagnostic biomarker for severe SARS-CoV-2 disease., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Cyclovirobuxine D (CVB‑D) is an alkaloid, which is mainly derived from Buxus microphylla. It has been reported that CVB‑D has positive effects on breast cancer, gastric cancer and other malignant tumors. However, to the best of our knowledge, there are no reports regarding the effects of CVB‑D on colorectal cancer (CRC). The purpose of the present study was to determine the anticancer effects of CVB‑D and further elucidate its molecular mechanism(s). DLD‑1 and LoVo cell lines were selected to evaluate the antitumor effect of CVB‑D. Cytotoxicity, viability and proliferation were evaluated by the MTT and colony formation assays. Flow cytometry was used to detect the effects on apoptosis and the cell cycle in CVB‑D‑treated CRC cells. The migration and invasion abilities of CRC cells were examined by wound healing and Transwell assays. In addition, RNA sequencing, bioinformatics analysis and western blotting were performed to investigate the target of drug action and clarify the molecular mechanisms. A xenograft model was established using nude mice, and ultrasound was employed to assess the preclinical therapeutic effects of CVB‑D in vivo. It was identified that CVB‑D inhibited the proliferation, migration, stemness, angiogenesis and epithelial‑mesenchymal transition of CRC cells, and induced apoptosis and S‑phase arrest. In addition, CVB‑D significantly inhibited the growth of xenografts. It is notable that CVB‑D exerted anticancer effects in CRC cells partly by targeting collagen triple helix repeat containing 1 (CTHRC1), which may be upstream of the AKT and ERK pathways. CVB‑D exerted anticancer effects through the CTHRC1‑AKT/ERK‑Snail signaling pathway. Targeted therapy combining CTHRC1 with CVB‑D may offer a promising novel therapeutic approach for CRC treatment.

Lysyl oxidase (LOX) is involved in fibrosis by catalyzing collagen cross-linking. Previous work observed that Triptolide (TPL) alleviated radiation-induced pulmonary fibrosis (RIPF), but it is unknown whether the anti-RIPF effect of TPL is related to LOX. In a mouse model of RIPF, we found that LOX persistently increased in RIPF which was significantly lowered by TPL. Excessive LOX aggravated fibrotic lesions in RIPF, while LOX inhibition mitigated RIPF. Irradiation enhanced the transcription and synthesis of LOX by lung fibroblasts through IKKβ/NFκB activation, and siRNA knockdown IKKβ largely abolished LOX production. By interfering radiation induced IKKβ activation, TPL prevented NFκB nuclear translocation and DNA binding, and potently decreased LOX synthesis. Our results demonstrate that the anti-RIPF effect of TPL is associated with reduction of LOX production which mediated by inhibition of IKKβ/NFκB pathway., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

A central and unique aspect of high-grade serous ovarian carcinoma (HGSC) is the extensive transcoelomic spreading of tumor cell via the peritoneal fluid or malignant ascites. We and others identified tumor-associated macrophages (TAM) in the ascites as promoters of metastasis-associated processes like extracellular matrix (ECM) remodeling, tumor cell migration, adhesion, and invasion. The precise mechanisms and mediators involved in these functions of TAM are, however, largely unknown. We observed that HGSC migration is promoted by soluble mediators from ascites-derived TAM, which can be emulated by conditioned medium from monocyte-derived macrophages (MDM) differentiated in ascites to TAM-like asc-MDM. A similar effect was observed with IL-10-induced alternatively activated m2c-MDM but not with LPS/IFNγ-induced inflammatory m1-MDM. These observations provided the basis for deconvolution of the complex TAM secretome by performing comparative secretome analysis of matched triplets of different MDM phenotypes with different pro-migratory properties (asc-MDM, m2c-MDM, m1-MDM). Mass spectrometric analysis identified an overlapping set of nine proteins secreted by both asc-MDM and m2c-MDM, but not by m1-MDM. Of these, three proteins, i.e., transforming growth factor beta-induced (TGFBI) protein, tenascin C (TNC), and fibronectin (FN1), have been associated with migration-related functions. Intriguingly, increased ascites concentrations of TGFBI, TNC, and fibronectin were associated with short progression-free survival. Furthermore, transcriptome and secretome analyses point to TAM as major producers of these proteins, further supporting an essential role for TAM in promoting HGSC progression. Consistent with this hypothesis, we were able to demonstrate that the migration-inducing potential of asc-MDM and m2c-MDM secretomes is inhibited, at least partially, by neutralizing antibodies against TGFBI and TNC or siRNA-mediated silencing of TGFBI expression. In conclusion, the present study provides the first experimental evidence that TAM-derived TGFBI and TNC in ascites promote HGSC progression.

Vaginal delivery with obstetrical trauma is a risk factor for pelvic organ prolapse later in life. Loss of fibulin-5 (FBLN5), an elastogenesis-promoting cellular matrix protein, results in prolapse in mice. Here, we evaluated effects of pregnancy, parturition, and obstetrical injury on FBLN5 content, elastic fibers, biomechanics, and histomorphology of the vaginal wall in rats. Further, we analyzed the effects of actinonin, a protease inhibitor, on obstetrical injury of the vaginal wall. Vaginal FBLN5 decreased significantly in pregnancy, and injury resulted in further downregulation. Stiffness of the vaginal wall decreased 82% in pregnant rats and 74% (p = 0.019) with injury relative to uninjured vaginal delivery controls at 3d. Actinonin ameliorated loss of FBLN5, rescued injury-induced loss of elastic fibers and biomechanical properties after parturition, and reduced the area of injury 10-fold. We conclude that pregnancy and parturition have a profound impact on vaginal FBLN5 and biomechanics of the vaginal wall. Further, obstetrical injury has significant deleterious impact on recovery of the vaginal wall from pregnancy. Actinonin, a non-specific matrix metalloprotease inhibitor, improved recovery of the parturient vaginal wall after obstetrical injury.

The extracellular matrix protein nephronectin (Npnt) is known to be critical for kidney development, but its function in inflammatory diseases is unknown. Here, we developed a new enzyme-linked immunosorbent assay system to detect Npnt in various autoimmune diseases, which revealed that plasma Npnt levels are increased in various mouse autoimmune models. We also report that antibodies against the α8β1 integrin-binding region of Npnt protect mice from anti-type II collagen-induced arthritis, suggesting that Npnt may be a potential therapeutic target molecule for the prevention of autoimmune arthritis., (© 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

MicroRNAs (miRNAs) serve as key regulators in human disorders. Previous research reported that miR-211-5p is down-regulated in osteoarthritis (OA) and that Fibulin-4 inhibits chondrocyte differentiation. However, the role of miR-211-5p in the development of OA has not been clarified, and its downstream target has not been studied. This study aimed to explore the effect of miR-211-5p on chondrocyte differentiation and its influence on OA pathogenesis, as well as the interaction between miR-211-5p and Fibulin-4. In this study, we found that miR-211-5p is significantly down-regulated in articular cartilage tissues in an OA rat model, whereas it is clearly up-regulated during chondrocyte differentiation of ATDC5 cells. Silencing miR-211-5p in ATDC5 cells had an adverse effect on chondrocyte differentiation. Fibulin-4 was identified as a target of miR-211-5p, and miR-211-5p participated in chondrocyte differentiation by negatively regulating Fibulin-4 expression. In the OA rat model, miR-211-5p overexpression facilitated chondrocyte differentiation, along with the reduced pro-inflammatory cytokines level and the level of proteinases responsible for cartilage matrix degradation. In summary, miR-211-5p promotes chondrocyte differentiation by negatively regulating Fibulin-4 expression, and represses the expression of pro-inflammatory cytokines and proteinases responsible for cartilage matrix degradation in OA. miR-211-5p may serve as a promising target for OA treatment., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

A C 2 -symmetric bicyclic peptide bearing two RGD motifs was developed as a dimeric ligand, and it displayed enhanced inhibition of ECM protein binding to purified integrin receptors as compared to monomeric RGD analogues. Moreover, the dimeric bicyclic ligand induced cell detachment and inhibited FAK phosphorylation in U-373 MG glioblastoma cells.

Although combination of surgery and chemo-radiotherapy could cure 80-95% of patients with early cervical cancer, there is still no satisfactory therapeutic strategies for locally advanced and metastatic cervical cancer patients. Our previous study has already investigated that CTHRC-1 is highly expressed not only in the local tissue but also in circulating serum of patients with cervical cancer and played important function on metastasis of cervical cancer cells. In present study, we aimed to see whether circulating specific monoclonal antibody (mAb) to CTHRC-1could inhibit the metastasis of advanced cervical cancer. Therefore, we innovatively generated one specific and sensitive mAb against CTHRC1 and found the CTHRC1 mAb could attenuate the promoting function of rCTHRC-1 on wound healing and invasion of SiHa cell in vitro. In addition, administration of mAb on the lung metastasis mouse model of cervical cancer strongly inhibited the level of metastasis. Taken together, targeting on CTHRC-1 is greatly beneficial not only for diagnosis but also for treatment of cervical cancer, which providing experimental and theoretical basis for developing a novel precise treatment of cervical cancer and improving patient survival rate., (Copyright © 2018 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Background: The obesity paradox in COPD describes protective effects of obesity on lung pathology and inflammation. However, the underlying relationships between obesity, diet and disease outcomes in COPD are not fully understood. In this study we measured the response to dietary fatty acids upon markers of inflammation and remodelling in human lung cells from people with and without COPD., Methods: Pulmonary fibroblasts were challenged with ω-3 polyunsaturated fatty acids (PUFAs), ω-6 PUFAs, saturated fatty acids (SFAs) or the obesity-associated cytokine TNFα. After 48-72 h release of the pro-inflammatory cytokines interleukin (IL)-6 and CXCL8 was measured using ELISA and mRNA expression and deposition of the extracellular matrix (ECM) proteins fibronectin, type I collagen, tenascin and perlecan were measured using qPCR or ECM ELISA, respectively., Results: Challenge with the ω-6 PUFA arachidonic acid (AA), but not ω-3 PUFAs or SFAs, resulted in increased IL-6 and CXCL8 release from fibroblasts, however IL-6 and CXCL8 release was reduced in COPD (n = 19) compared to non-COPD (n = 36). AA-induced cytokine release was partially mediated by downstream mediators of cyclooxygenase (COX)-2 in both COPD and non-COPD. In comparison, TNFα-induced IL-6 and CXCL8 release was similar in COPD and non-COPD, indicating a specific interaction of AA in COPD. In patients with or without COPD, regression analysis revealed no relationship between BMI and cytokine release. In addition, AA, but not SFAs or ω-3 PUFAs reduced the basal deposition of fibronectin, type I collagen, tenascin and perlecan into the ECM in COPD fibroblasts. In non-COPD fibroblasts, AA-challenge decreased basal deposition of type I collagen and perlecan, but not fibronectin and tenascin., Conclusions: This study shows that AA has disease-specific effects on inflammation and ECM protein deposition. The impaired response to AA in COPD might in part explain why obesity appears to have less detrimental effects in COPD, compared to other lung diseases.

Background: Goodpasture syndrome (GP) is a pulmonary-renal syndrome characterized by autoantibodies directed against the NC1 domains of collagen IV in the glomerular and alveolar basement membranes. Exposure of the cryptic epitope is thought to occur via disruption of sulfilimine crosslinks in the NC1 domain that are formed by peroxidasin-dependent production of hypobromous acid. Peroxidasin, a heme peroxidase, has significant structural overlap with myeloperoxidase (MPO), and MPO-ANCA is present both before and at GP diagnosis in some patients. We determined whether autoantibodies directed against peroxidasin are also detected in GP., Methods: We used ELISA and competitive binding assays to assess the presence and specificity of autoantibodies in serum from patients with GP and healthy controls. Peroxidasin activity was fluorometrically measured in the presence of partially purified IgG from patients or controls. Clinical disease severity was gauged by Birmingham Vasculitis Activity Score., Results: We detected anti-peroxidasin autoantibodies in the serum of patients with GP before and at clinical presentation. Enriched anti-peroxidasin antibodies inhibited peroxidasin-mediated hypobromous acid production in vitro . The anti-peroxidasin antibodies recognized peroxidasin but not soluble MPO. However, these antibodies did crossreact with MPO coated on the polystyrene plates used for ELISAs. Finally, peroxidasin-specific antibodies were also found in serum from patients with anti-MPO vasculitis and were associated with significantly more active clinical disease., Conclusions: Anti-peroxidasin antibodies, which would previously have been mischaracterized, are associated with pulmonary-renal syndromes, both before and during active disease, and may be involved in disease activity and pathogenesis in some patients., (Copyright © 2018 by the American Society of Nephrology.)

The receptor for hyaluronan mediated motility (RHAMM, gene name HMMR) belongs to a group of proteins that bind to hyaluronan (HA), a high-molecular weight anionic polysaccharide that has pro-angiogenic and inflammatory properties when fragmented. We propose to use a chemically synthesized, truncated version of the protein (706-767), 7 kDa RHAMM, as a target receptor in the screening of novel peptide-based therapeutic agents. Chemical synthesis by Fmoc-based solid-phase peptide synthesis, and optimization using pseudoprolines, results in RHAMM protein of higher purity and yield than synthesis by recombinant protein production. 7 kDa RHAMM was evaluated for its secondary structure, ability to bind the native ligand, HA, and its bioactivity. This 62-amino acid polypeptide replicates the HA binding properties of both native and recombinant RHAMM protein. Furthermore, tubulin-derived HA peptide analogues that bind to recombinant RHAMM and were previously reported to compete with HA for interactions with RHAMM, bind with a similar affinity and specificity to the 7 kDa RHAMM. Therefore, in terms of its key binding properties, the 7 kDa RHAMM mini-protein is a suitable replacement for the full-length recombinant protein., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

BACKGROUND Let-7c-5p is down-regulated in dental pulp tissues in inflammatory disorders. The microRNA (miR) molecule shows an anti-inflammation potential due to its direct regulation of dentin matrix protein-1 (DMP1), which promotes inflammation changes in dental pulp tissues. In the present study, the effect of let-7c-5p on lipopolysaccharide (LPS)-induced pulpitis was detected and the associated mechanism was explored. MATERIAL AND METHODS Dental pulp stem cells (DPSCs) were isolated from rat dental tissues, infected with let-7c-5p lentivirus particles, and subjected to LPS administration to induce inflammation. Then, the effect of let-7c-5p overexpression on LPS-induced impairments on DPSCs were detected and the mechanism was explained by focusing on the DMP1 expression and NF-κB pathway. The role of DMP1 in the anti-inflammation effect of let-7c-5p was assessed by incubating let-7c-5p-expressed DPSCs with DMP1 protein. The results of in vitro assays were verified in LPS-induced rat pulpitis models. RESULTS LPS administration increased the production of IL-1β and TNF-α and decreased DPSCs viability by increasing the expression of DMP1 and activating NF-κB pathway. However, the induced expression of let-7c-5p relieved DPSCs from LPS-induced inflammation and suppressed DMP1 as well as NF-κB pathway. The incubation of let-7c-5p-expressed DPSCs with DMP1 protein blocked the effect of let-7c-5p. In in vivo experiments, the injection of let-7c-5p attenuated LPS-induced pulpitis by inhibiting DMP1-mediated NF-κB pathway. CONCLUSIONS Findings outlined in the current study demonstrated the dental pulp protecting function of let-7c-5p during LPS-induced inflammation, which was exerted by inhibiting the DMP1-mediated NF-κB pathway.

Objective: To address whether matrix Gla protein (MGP) can inhibit mineralization in normal rat kidney tubular cells (NRK-52E) under high concentration of calcium., Materials and Methods: NRK-52E cells were treated with high concentration of calcium. The viability and apoptosis of cells were detected by cell counting kit-8 and flow cytology, respectively. Real-time-polymerase chain, Western blotting, and immunofluorescence analysis were conducted to detect the expression of MGP. Cells were transfected with plasmid-MGP or siRNA-MGP for up- or down-regulation of the expression of MGP, respectively. Rat recombinant MGP was also used as supplementation of exogenous MGP. Alizarin red staining was conducted to detect the adherent and deposition of calcium salt., Results: High concentration of calcium suppressed MGP expression in NRK-52E cells. There was significant mineralization when NRK-52E cells were treated with high concentration of calcium. Supplementation with exogenous rat recombinant MGP and overexpression of endogenous MGP both decreased the adherent and deposition of calcium salt to NRK-52E cells, while silence of MGP showed reverse results., Conclusion: MGP plays an inhibitory role in the stone formation. However, high concentration of calcium significantly inhibits the expression of MGP and then promotes mineralization in NRK-52E cells., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Recent findings indicate that dentin sialophosphoprotein (DSPP) and matrix metalloproteinase (MMP) 20 interact in oral squamous cell carcinoma (OSCC). The objective of this study was to determine the effects of DSPP/MMP20 gene silencing on oral cancer stem cell (OCSC) markers., Methods: The expression of well-established OCSC markers: ABCG2; ALDH1; CD133; CD44; BMI1; LGR4, and Podoplanin in DSPP/MMP20-silenced OSCC cell line, OSC2, and controls were assayed by western blot (WB), and flow cytometry techniques. The sensitivity of OSC2 cells to cisplatin following DSPP/MMP20 silencing was also determined., Results: DSPP/MMP20 silencing resulted in downregulation of OCSC markers, more profoundly ABCG2 (84%) and CD44 (81%), following double silencing. Furthermore, while treatment of parent (pre-silenced) OSC2 cells with cisplatin resulted in upregulation of OCSC markers, DSPP/MMP20-silenced OSC2 cells similarly treated resulted in profound downregulation of OCSC markers (72 to 94% at 50 μM of cisplatin), and a marked reduction in the proportion of ABCG2 and ALDH1 positive cells (~ 1%)., Conclusions: We conclude that the downregulation of OCSC markers may signal a reduction in OCSC population following MMP20/DSPP silencing in OSCC cells, while also increasing their sensitivity to cisplatin. Thus, our findings suggest a potential role for DSPP and MMP20 in sustaining OCSC population in OSCCs, possibly, through mechanism(s) that alter OCSC sensitivity to treatment with chemotherapeutic agents such as cisplatin., Competing Interests: The study was approved by the University of Texas Health Science Center-Houston’s Institutional Review Board for all experimental procedures including human tissue samples and cell lines.Not applicable.The authors declare that they have no competing interests.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Peroxidasin is a heme peroxidase that catalyses the oxidation of bromide by hydrogen peroxide to form an essential sulfilimine cross-link between methionine and hydroxylysine residues in collagen IV. We investigated cross-linking by peroxidasin embedded in extracellular matrix isolated from cultured epithelial cells and its sensitivity to alternative substrates and peroxidase inhibitors. Peroxidasin showed peroxidase activity as measured with hydrogen peroxide and Amplex red. Using a specific mass spectrometry assay that measures NADH bromohydrin, we showed definitively that the enzyme releases hypobromous acid (HOBr). Less than 1 μM of the added hydrogen peroxide was used by peroxidasin. The remainder was consumed by catalase activity that was associated with the matrix. Results from NADH bromohydrin measurements indicates that low micromolar HOBr generated by peroxidasin was sufficient for maximum sulfilimine cross-linking, whereas 100 μM reagent HOBr or taurine bromamine was less efficient. This implies selectivity for the enzymatic process. Physiological concentrations of thiocyanate and urate partially inhibited cross-link formation. 4-Aminobenzoic acid hydrazide, a commonly used myeloperoxidase inhibitor, also inhibited peroxidasin, whereas acetaminophen and a 2-thioxanthine were much less effective. In conclusion, HOBr is produced by peroxidasin in the extracellular matrix. It appears to be directed at the site of collagen IV sulfilimine formation but the released HOBr may also undergo other reactions., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Purpose: We sought a novel approach against glioblastomas (GBM) focused on targeting signaling molecules localized in the tumor extracellular matrix (ECM). We investigated fibulin-3, a glycoprotein that forms the ECM scaffold of GBMs and promotes tumor progression by driving Notch and NFκB signaling. Experimental Design: We used deletion constructs to identify a key signaling motif of fibulin-3. An mAb (mAb428.2) was generated against this epitope and extensively validated for specific detection of human fibulin-3. mAb428.2 was tested in cultures to measure its inhibitory effect on fibulin-3 signaling. Nude mice carrying subcutaneous and intracranial GBM xenografts were treated with the maximum achievable dose of mAb428.2 to measure target engagement and antitumor efficacy. Results: We identified a critical 23-amino acid sequence of fibulin-3 that activates its signaling mechanisms. mAb428.2 binds to that epitope with nanomolar affinity and blocks the ability of fibulin-3 to activate ADAM17, Notch, and NFκB signaling in GBM cells. mAb428.2 treatment of subcutaneous GBM xenografts inhibited fibulin-3, increased tumor cell apoptosis, and enhanced the infiltration of inflammatory macrophages. The antibody reduced tumor growth and extended survival of mice carrying GBMs as well as other fibulin-3-expressing tumors. Locally infused mAb428.2 showed efficacy against intracranial GBMs, increasing tumor apoptosis and reducing tumor invasion and vascularization, which are enhanced by fibulin-3. Conclusions: To our knowledge, this is the first rationally developed, function-blocking antibody against an ECM target in GBM. Our results offer a proof of principle for using "anti-ECM" strategies toward more efficient targeted therapies for malignant glioma. Clin Cancer Res; 24(4); 821-33. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Studies that have focused on the role TGFβ signaling plays in osteoclast activity are gradually increasing; however, literature is rare in terms of fluorosis. The aim of this study is to observe the role the TβR1/Smad3 pathway plays in fluoride regulating cellsosteoclast-like cells that are under the treatment of TGFβ receptor 1 kinase. The RANKL-mediated osteoclast-like cells from RAW264.7 cells were used as osteoclast precursor model. The profile of miRNA expression in fluoride-treated osteoclast-like cells exhibited 303 upregulated miRNAs, 61 downregulated miRNAs, and further drew 37 signaling pathway maps by KEGG and Biocarta pathway enrichment analysis. TGFβ and its downstream effectors were included among them. Osteoclast viability, formation and function were detected via MTT method, bone resorption pit and tartrate-resistant acid phosphatase (TRACP) staining, respectively. Results demonstrated that different doses of fluoride exhibited a biphasic effect on osteoclast cell viability, differentiation, formation and function. It indicated that a low dose of fluoride treatment stimulated them, but high dose inhibited them. SB431542 acted as TβR1 kinase inhibitor and blocked viability, formation and function of osteoclast-like cells regulated by fluoride. The expression of the osteoclast marker, RANK, and TβR1/Smad3 at gene and protein level was analyzed under fluoride with and without SB431542 treatment. Fluoride treatment indicated little effect on the RANK protein expression; however it significantly influenced TRACP expression in osteoclast-like cells. The stimulation of fluoride on the expression of Smad3 gene and phosphorylated Smad3 protein exhibited dose-dependent manner. SB431542 significantly impeded phosphorylation of Smad3 protein and TRACP expression in osteoclast-like cells that were exposed to fluoride. Our work demonstrated that TGFβ signaling played a key role in fluoride regulating osteoclast differentiation, formation and function. It elucidated that TβR1/Smad3 pathway participated in the mechanism of biphasic modulation of osteoclast mode regulated by fluoride., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Epithelia are bound by both basal and apical extracellular matrices (ECM). Although the composition and function of the former have been intensively investigated, less is known about the latter. The embryonic sheath, the ECM apical to the Caenorhabditis elegans embryonic epidermis, has been suggested to promote elongation of the embryo. In an RNAi screen for the components of the sheath, we identified the zona pellucida domain proteins NOAH-1 and NOAH-2. We found that these proteins act in the same pathway, and in parallel to three other putative sheath proteins, the leucine-rich repeat proteins SYM-1, LET-4 and FBN-1/Fibrillin, to ensure embryonic integrity and promote elongation. Laser nano-ablation experiments to map the stress field show that NOAH-1 and NOAH-2, together with PAK-1/p21-activated kinase, maintain and relay the actomyosin-dependent stress generated within the lateral epidermis before muscles become active. Subsequently, loss-of-function experiments show that apical ECM proteins are essential for muscle anchoring and for relaying the mechanical input from muscle contractions, which are essential for elongation. Hence, the apical ECM contributes to morphogenesis by maintaining embryonic integrity and relaying mechanical stress., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

Spon2 is a proto-oncogene matrix protein that plays an essential role in the tumorigenesis and metastasis of gastric cancer. The protein has recently been found to function as a guanine nucleotide exchange factor through the activation of RhoGTPase. Here, computational modeling and bioinformatics analysis were employed to investigate the molecular mechanism and biological implication underlying Spon2 autoinhibition. It is revealed that the binding of PxxP motif to SH domain can stabilize the intramolecular interaction between the N-terminal helix and DH domain of Spon2, thus shifting the protein into an autoinhibitory state. Here, we proposed releasing Spon2 autoinhibition by targeting SH domain with competitive peptide ligands. To verify this notion, the PxxP sequence was adopted as the start to derive an array of efficient SH binders by using a structure-based rational design strategy, which were then substantiated with fluorescence spectroscopy analysis and guanine nucleotide exchange test. Consequently, the obtained peptide ligands were determined to have a moderate or high affinity for SH domain; they can also enhance Spon2 exchange activity by 1.2-6.1 folds, exhibiting a significant correlation with their SH-binding affinity (Pearson's coefficient=0.92). In addition, neutral substitution of conserved residues in a high-affinity peptide ligand can largely reduce its Spon2-activating potency, confirming that the designed peptide activates Spon2 by competitively disrupting SH-PxxP interaction., (Copyright © 2017 Elsevier Inc. All rights reserved.)

During postnatal development, neural circuits are extremely dynamic and develop precise connection patterns that emerge as a result of the elimination of synaptic terminals, a process instructed by molecular cues and patterns of electrical activity. In the rodent visual system, this process begins during the first postnatal week and proceeds during the second and third postnatal weeks as spontaneous retinal activity and finally use-dependent fine tuning takes place. Reelin is a large extracellular matrix glycoprotein able to affect several steps of brain development, from neuronal migration to the maturation of dendritic spines and use-dependent synaptic development. In the present study, we investigated the role of reelin on the topographical refinement of primary sensory connections studying the development of retinal ganglion cell axon terminals in the rat superior colliculus. We found that reelin levels in the visual layers of the superior colliculus are the highest between the second and third postnatal weeks. Blocking reelin signaling with a neutralizing antibody (CR-50) from PND 7 to PND 14 induced a non-specific sprouting of ipsilateral retinocollicular axons outside their typical distribution of discrete patches of axon terminals. Also we found that reelin blockade resulted in reduced levels of phospho-GAP43, increased GluN1 and GluN2B-NMDA subunits and decreased levels of GAD65 content in the visual layers of the superior colliculus. The results suggest that reelin signaling is associated with the maturation of excitatory and inhibitory synaptic machinery influencing the development and fine tuning of topographically organized neural circuits during postnatal development., (Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.)

Background: Abdominal surgery and disease cause persistent abdominal adhesions, pelvic pain, infertility and occasionally, bowel obstruction. Current treatments are ineffective and the aetiology is unclear, although excessive collagen deposition is a consistent feature. Lysyl oxidase (Lox) is a key enzyme required for crosslinking and deposition of insoluble collagen, so we investigated whether targeting Lox might be an approach to reduce abdominal adhesions., Methods: Female C57Bl/6 mice were treated intraperitoneally with multiwalled carbon nanotubes (NT) to induce fibrosis, together with chemical (ß-aminoproprionitrile-BAPN) or miRNA Lox inhibitors, progesterone or dexamethasone. Fibrotic lesions on the diaphragm, and expression of fibrosis-related genes in abdominal wall peritoneal mesothelial cells (PMC) were measured. Effects of BAPN and dexamethasone on collagen fibre alignment were observed by TEM. Isolated PMC were cultured with interleukin-1 alpha (IL-1α) and progesterone to determine effects on Lox mRNA in vitro., Results: NT-induced fibrosis and collagen deposition on the diaphragm was ameliorated by BAPN, Lox miRNA, or steroids. BAPN and dexamethasone disrupted collagen fibres. NT increased PMC Lox, Col1a1, Col3a1 and Bmp1 mRNA, which was inhibited by steroids. Progesterone significantly inhibited IL-1α induced Lox expression by PMC in vitro., Conclusion: Our results provide proof-of-concept that targeting peritoneal Lox could be an effective approach in ameliorating fibrosis and adhesion development.

This study explored the role of fibulin-3 in osteosarcoma progression and the possible signaling pathway involved. Fibulin-3 mRNA and protein expression in normal tissue, benign fibrous dysplasia, osteosarcoma, osteosarcoma cell lines (HOS and U-2OS), the normal osteoblastic cell line hFOB, and different invasive subclones was evaluated by immunohistochemistry (IHC) or immunocytochemistry (ICC) and real time reverse transcriptase-polymerase chain reaction (real time qRT-PCR). To assess the role of fibulin-3 in the invasion and metastasis of osteosarcoma cells, lentiviral vectors with fibulin-3 small hairpin RNA (shRNA) and pLVX-fibulin-3 were constructed and used to infect the highly invasive and low invasive subclones. The effects of fibulin-3 knockdown and upregulation on the biological behavior of osteosarcoma cells were investigated by functional in vitro and in vivo assays. The results revealed that fibulin-3 expression was upregulated in osteosarcoma, and was positively correlated with low differentiation, lymph node metastasis, and poor prognosis. Fibulin-3 could promote osteosarcoma cell invasion and metastasis by inducing EMT and activating the Wnt/β-catenin signaling pathway. Collectively, our findings demonstrate that fibulin-3 is a promoter of osteosarcoma development and progression, and suggest a novel therapeutic target for future studies.

Tissue microenvironments formed by extracellular matrix networks play an important role in regulating tissue structure and function. Extracellular microfibrillar networks composed of fibrillins and their associated ligands such as LTBPs, fibulins, and EMILINs are of particular interest in this regard since they provide a specialized cellular microenvironment guiding proper morphology and functional behavior of specialized cell types. To understand how cellular microenvironments composed of intricate microfibrillar networks influence cell fate decisions in a contextual manner, more information about the spatiotemporal localization, deposition, and function of their components is required. By employing confocal immunofluorescence and electron microscopy we investigated the localization and extracellular matrix deposition of EMILIN-1 and -2 in tissues of the skeletal system such as cartilage and bone as well as in in vitro cultures of osteoblasts. We found that upon RNAi mediated depletion of EMILIN-1 in primary calvarial osteoblasts and MC3T3-E1 cells only fibulin-4 matrix deposition was lost while other fibulin family members or LTBPs remained unaffected. Immunoprecipitation and ELISA-style binding assays confirmed a direct interaction between EMILIN-1 and fibulin-4. Our data suggest a new function for EMILIN-1 which implies the guidance of linear fibulin-4 matrix deposition and thereby fibulin-4 fiber formation.

Objective: Soluble Aβ 1-42 oligomers play a vital role in the development and pathogenesis of Alzheimer's disease (AD). Melatonin could delay the progress of AD through multiple mechanisms. Reelin-Dab1 signaling plays an important role in AD, including neuronal function and synaptic plasticity. However, whether melatonin could exert its neuroprotective function against soluble Aβ 1-42 -induced neurotoxicity during AD development through regulating Reelin-Dab1 signaling remains poorly understood., Methods: AD rat model was established by soluble Aβ 1-42 repeated intracerebroventricular injection. Using immunohistochemistry and Western blot analyses, the effect of melatonin on synaptic plasticity, neuritic degeneration, and astrocyte activation was investigated in cerebral cortex. Meanwhile, the expression of Reelin and Dab1 was also examined in cerebral cortex. In our in vitro study, Reelin-Dab1 signaling was inhibited by Reelin antibody, and neuroprotective effect of melatonin against Aβ 1-42 was further determined., Results: Melatonin ameliorated the neurotoxiciy and astrocyte activation induced by Aβ 1-42 in the cerebral cortex. Melatonin also blocked the reduction in Reelin and Dab1 expression induced by Aβ 1-42 . Using in vitro study, Reelin inactivation completely abolished the protective effect of melatonin against Aβ 1-42 -induced neurotoxicity., Discussion: Melatonin might play its neuroprotective role against Aβ 1-42 through mediating Reelin-Dab1 signaling pathway. Melatonin could be a safe and remarkable therapeutic candidate for AD and other aged-associated neurodegenerative diseases.

RB loss occurs commonly in neoplasia but its contributions to advanced cancer have not been assessed directly. Here we show that RB loss in multiple murine models of cancer produces a prometastatic phenotype. Gene expression analyses showed that regulation of the cell motility receptor RHAMM by the RB/E2F pathway was critical for epithelial-mesenchymal transition, motility, and invasion by cancer cells. Genetic modulation or pharmacologic inhibition of RHAMM activity was sufficient and necessary for metastatic phenotypes induced by RB loss in prostate cancer. Mechanistic studies in this setting established that RHAMM stabilized F-actin polymerization by controlling ROCK signaling. Collectively, our findings show how RB loss drives metastatic capacity and highlight RHAMM as a candidate therapeutic target for treating advanced prostate cancer. Cancer Res; 77(4); 982-95. ©2016 AACR ., (©2016 American Association for Cancer Research.)

Alterations in the extracellular matrix (ECM) production and remodeling of smooth muscle cells (SMCs) have been implicated in processes related to the differentiation in atherosclerosis. Due to the anti-atherosclerotic properties of the tetracyclines, we aimed to investigate whether cholesterol supplementation changes the effect of doxycycline over the ECM proteins synthesis and whether isoprenylated proteins and Rho A protein activation are affected. SMC primary culture isolated from chicks exposed to atherogenic factors in vivo (a cholesterol-rich diet, SMC-Ch), comparing it with control cultures isolated after a standard diet (SMC-C). After treatment with 20 nM doxycycline, [H 3 ]-proline and [H 3 ]-mevalonate incorporation were used to measure the synthesis of collagen and isoprenylated proteins, respectively. Real-time PCR was assessed to determine col1a2, col2a1, col3a1, fibronectin, and mmp2 gene expression and the pull-down technique was applied to determine the Rho A activation state. A higher synthesis of collagens and isoprenylated proteins in SMC-Ch than in SMC-C was determined showing that doxycycline inhibits ECM production and remodeling in both SMC types of cultures. Moreover, preliminary results about the effect of doxycycline on protein isoprenylation and Rho A protein activation led us to discuss the possibility that membrane G-protein activation pathways could mediate the molecular mechanism., (Copyright © 2016 Japanese Pharmacological Society. Production and hosting by Elsevier B.V. All rights reserved.)

Reelin is an extracellular matrix glycoprotein involved in the modulation of synaptic plasticity and essential for the proper radial migration of cortical neurons during development and for the integration and positioning of dentate granular cell progenitors; its expression is down-regulated as brain maturation is completed. Trimethyltin (TMT) is a potent neurotoxicant which causes selective neuronal death mainly localised in the CA1-CA3/hilus hippocampal regions. In the present study we analysed the expression of reelin and the modulation of endogenous neurogenesis in the postnatal rat hippocampus during TMT-induced neurodegeneration (TMT 6 mg/kg). Our results show that TMT administration induces changes in the physiological postnatal decrease of reelin expression in the hippocampus of developing rats. In particular, quantitative analysis of reelin-positive cells evidenced, in TMT-treated animals, a persistent reelin expression in the stratum lacunosum moleculare of Cornu Ammonis and in the molecular layer of Dentate Gyrus. In addition, a significant decrease in the number of bromodeoxyuridine (BrdU)-labeled newly-generated cells was also detectable in the subgranular zone of P21 TMT-treated rats compared with P21 control animals; no differences between P28 TMT-treated rats and age-matched control group were observed. In addition the neuronal commitment of BrdU-positive cells appeared reduced in P21 TMT-treated rats compared with P28 TMT-treated animals. Thus TMT treatment, administrated during development, induces an early reduction of endogenous neurogenesis and influences the hippocampal pattern of reelin expression in a temporally and regionally specific manner, altering the physiological decrease of this protein.

Family with sequence similarity 20, member C (Fam20C) is a physiological Golgi casein kinase that phosphorylates multiple secreted proteins. Recently, it has been reported that Fam20C can be identified as a novel kinase target for therapeutic development. Thus, inhibition of Fam20C will be a potential therapeutic strategy to prevent tumor cell progression and metastasis. In our study, based upon the systems-biology network, molecular modeling and molecular dynamics (MD) simulations, we discovered a novel Fam20C inhibitor (FL-1607) with potent anti-proliferative effects on triple-negative breast cancer (TNBC) cells. Subsequently, we found that this Fam20C inhibitor could induce apoptosis and inhibit cell migration in MDA-MB-468 cells. Together, these findings would provide a new clue to the exploration of more novel Fam20C inhibitors for future TNBC therapy.

Diketopiperazine is a naturally occurring cyclic dipeptide found from diverse living organisms. The compounds in this structure class have been known with a broad spectrum of bioactivities including anti-inflammatory activities. Transforming growth factor β-induced protein (TGFBIp) is an extracellular matrix protein whose expression in several cell types is greatly increased by TGF-β. TGFBIp is released by human umbilical vein endothelial cells and functions as a mediator of experimental sepsis. Here, three (1-3) of diketopiperazines were isolated from two strains of marine-derived bacteria and we hypothesized that 1-3 could reduce TGFBIp-mediated severe inflammatory responses in human endothelial cells and mice. Here, we investigated the anti-septic effects and underlying mechanisms of 1-3 against TGFBIp-mediated septic responses. 1-3 effectively inhibited lipopolysaccharide-induced release of TGFBIp and suppressed TGFBIp-mediated septic responses. In addition, 1-3 suppressed cecal ligation and puncture (CLP)-induced sepsis lethality and pulmonary injury. In conclusion, 1-3 suppressed TGFBIp-mediated and CLP-induced septic responses. Therefore, 1-3 could be a potential therapeutic agent for treatment of various severe vascular inflammatory diseases via inhibition of the TGFBIp signaling pathway.

Polyozellus multiplex (Thelephoraceae) is a wild mushroom in Korea and Japan and is usually harvested in early autumn for food. Polyozellin, a major constituent of the edible mushroom P multiplex, has been known to exhibit biological activities such as antioxidative and anti-inflammatory effects. Transforming growth factor β-induced protein (TGFBIp) is an extracellular matrix protein whose expression in several cell types is greatly increased by TGF-β. TGFBIp is released by human umbilical vein endothelial cells and functions as a mediator of experimental sepsis. We hypothesized that polyozellin could reduce TGFBIp-mediated severe inflammatory responses in human endothelial cells and mice. Here, we investigated the antiseptic effects and underlying mechanisms of polyozellin against TGFBIp-mediated septic responses. Polyozellin effectively inhibited lipopolysaccharide-induced release of TGFBIp and suppressed TGFBIp-mediated septic responses. In addition, polyozellin suppressed cecal ligation and puncture-induced sepsis lethality and pulmonary injury. In conclusion, polyozellin suppressed TGFBIp-mediated and cecal ligation and puncture-induced septic responses. Therefore, polyozellin could be a potential therapeutic agent for treatment of various severe vascular inflammatory diseases via inhibition of the TGFBIp signaling pathway., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Collagen triple helix repeat containing-1 (CTHRC1), a secreted protein, is transiently expressed in the arterial wall in response to injury, indicating that it may contribute to vascular remodeling by limiting collagen matrix deposition and promoting cell migration. Recent studies showed that it is aberrantly upregulated in most human solid tumors, yet its role in osteosarcoma remains unclear. In this study, the authors investigated the role of CTHRC1 in human osteosarcoma tumorigenesis. The authors used lentivirus-mediated short hairpin RNA (shRNA) against CTHRC1 to limit its endogenous expression in U2OS and SW1353 cells. Interestingly, they found that depletion of CTHRC1 significantly inhibited cell proliferation and colony formation in U2OS and SW1353 cells. Flow cytometry assay showed that knockdown of CTHRC1 increased the cell percentage of G0/G1 phase, resulting in cell cycle arrest in U2OS cells. Moreover, CTHRC1 silencing induced the cell cycle arrest by a decrease in the cell percentage in G0/G1 phase and increased in G2/M phase in SW1353 cells. In addition, crystal violet staining suggested CTHRC1 silencing inhibited migration of U2OS and SW1353 cells. These results demonstrated that CTHRC1 might play an important role in osteosarcoma progression.

Background: Esophageal squamous cell carcinoma (ESCC) is an important cause of cancer-related death worldwide. To improve prognoses in patients with ESCC, we evaluated the potential of transforming growth factor-beta-induced protein (TGFBI), which is overexpressed in ESCC, as a therapeutic candidate., Methods: We examined the clinical significance of TBFBI in 102 ESCC samples using real-time RT-PCR. Immunohistochemical studies were conducted to examine the localization of TGFBI. Knockdown of TGFBI in cocultured fibroblasts was performed to determine the roles of TGFBI in migration and invasion., Results: The level of TGFBI in ESCC tissues was higher than that in normal tissues. The high TGFBI expression group (n = 16) had higher TGFB1 expression and more frequent hematogenous recurrence than the low-expression group (n = 86). High TGFBI expression was an independent prognostic factor in patients with ESCC. TGFBI was mainly localized in stromal cells of ESCC. Moreover, suppression of TGFBI in fibroblasts inhibited the migration and invasion capacity of TE8 ESCC cells., Conclusions: High TGFBI expression in ESCC tissues could be a powerful biomarker of poor prognosis and hematogenous recurrence. TGFBI in stromal cells might be a promising molecular target for ESCC treatment.

Liver fibrosis is the pathological consequence of chronic liver diseases, where an excessive deposition of extracellular matrix (ECM) proteins occurs, concomitantly with the processes of repair and regeneration. It is characterized by increased production of matrix proteins, in particular collagens, and decreased matrix remodelling. The principal source of ECM accumulation is myofibroblasts (MFB). Most fibrogenic MFB are endogenous to the liver, coming from hepatic stellate cells (HSC) and portal fibroblasts. Dysregulated inflammatory responses have been associated with most (if not all) hepatotoxic insults and chronic oxidative stress play a role during the initial liver inflammatory phase and its progression to fibrosis. Redox-regulated processes are responsible for activation of HSC to MFB, as well as maintenance of the MFB function. Increased oxidative stress also induces hepatocyte apoptosis, which contributes to increase the liver injury and to transdifferentiate HSC to MFB, favouring the fibrogenic process. Mitochondria and other redox-active enzymes can generate superoxide and hydrogen peroxide as a by-product in liver cells. Moreover, accumulating evidence indicates that NADPH oxidases (NOXs), which play a critical role in the inflammatory response, may contribute to reactive oxygen species (ROS) production during liver fibrosis, being important players in HSC activation and hepatocyte apoptosis. Based on the knowledge of the pathogenic role of ROS, different strategies to prevent or reverse the oxidative damage have been developed to be used as therapeutic tools in liver fibrosis. This review will update all these concepts, highlighting the relevance of redox biology in chronic fibrogenic liver pathologies., (Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.)