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Objectives: Periodontal ligament stem cells (PDLSCs) are ideal seed cells for periodontal tissue regeneration. Our previous studies have indicated that the histone methyltransferase PRDM9 plays an important role in human periodontal ligament stem cells (hPDLSCs). Whether FBLN5, which is a downstream gene of PRDM9, also has a potential impact on hPDLSCs is still unclear., Methods: Senescence was assessed using β-galactosidase and Enzyme-linked immunosorbent assay (ELISA). Osteogenic differentiation potential of hPDLSCs was measured through Alkaline phosphatase (ALP) activity assay and Alizarin red detection, while gene expression levels were evaluated using western blot and RT-qPCR analysis., Results: FBLN5 overexpression promoted the osteogenic differentiation and senescence of hPDLSCs. FBLN5 knockdown inhibited the osteogenic differentiation and senescence of hPDLSCs. Knockdown of PRDM9 decreased the expression of FBLN5 in hPDLSCs and inhibited senescence of hPDLSCs. Additionally, both FBLN5 and PRDM9 promoted the expression of phosphorylated p38 MAPK, Erk1/2 and JNK. The p38 MAPK pathway inhibitor SB203580 and the Erk1/2 pathway inhibitor PD98059 have the same effects on inhibiting the osteogenic differentiation and senescence of hPDLSCs. The JNK pathway inhibitor SP600125 reduced the senescence of hPDLSCs., Conclusion: FBLN5 promoted senescence and osteogenic differentiation of hPDLSCs via activation of the MAPK signaling pathway. FBLN5 was positively targeted by PRDM9, which also activated the MAPK signaling pathway., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Introduction: Osteolectin is a secreted glycoprotein of the C-type lectin domain superfamily, expressed in bone tissues and is reported as a novel osteogenic factor that promotes bone regeneration. However, the effect of osteolectin on human dental pulp cells (hDPCs) has not been reported. Therefore, we aimed to investigate the odontoblastic differentiation of osteolectin in hDPCs and further attempt to reveal its underlying mechanism., Methods: Cytotoxicity assays were used to detect the cytotoxicity of osteolectin. The odontoblastic differentiation of hDPCs and its underlying mechanisms were measured by the alkaline phosphatase (ALP) activity, mineralized spots formation, and the gene and protein expression of odontoblastic differentiation through ALP staining, Alizarin red S staining, quantitative real-time polymerase chain reaction, and Western blot analysis, respectively., Results: WST-1 assay showed osteolectin at concentrations below 300 ng/ml was noncytotoxic and safe for hDPCs. The following experiment demonstrated that osteolectin could increase ALP activity, accelerate the mineralization process, and up-regulate the odontogenic differentiation markers in both gene and protein levels (P < .05). Osteolectin stimulated the phosphorylation of ERK, JNK, and Protein kinase B (AKT) in hDPCs. Extracellular signal-regulated kinase (ERK), Jun N-terminal kinase (JNK), and AKT inhibitors decreased ALP activity and mineralization capacity and suppressed the expression of dentin sialophosphoprotein and dentin matrix protein-1., Conclusion: Osteolectin can promote odontoblastic differentiation of hDPCs, and the whole process may stimulate ERK, JNK, and AKT signaling pathways by increasing p-ERK, p-JNK, and p-AKT signals., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Diabetic nephropathy (DN), a severe diabetes complication, causes kidney morphological and structural changes due to extracellular matrix accumulation. This accumulation is caused mainly by oxidative stress. Semi-essential amino acid derivative taurine has powerful antioxidant and antifibrotic effects. The aim of this study was to investigate the renoprotective effects of taurine through its possible roles in oxidative stress, extracellular matrix proteins, and the signaling pathways associated with the accumulation of extracellular matrix proteins in DN rats. 29 Wistar albino rats were randomly separated into control, taurine, diabetes, and diabetes + taurine groups. Diabetes animals were injected 45 mg/kg streptozosine. Taurine is given by adding to drinking water as 1% (w/v). Urine, serum, and kidney tissue were collected from rats for biochemical and histological analysis after 12 weeks. According to the studies, taurine significantly reduces the levels of malondialdehyde (MDA), total oxidant status (TOS), and protein expression of NADPH oxidase 4 (NOX4) that increase in diabetic kidney tissue. Also, decreased superoxide dismutase (SOD) activity levels significantly increased with taurine in diabetic rats. Moreover, increased mRNA and protein levels of fibronectin decreased with taurine. The matrix metalloproteinase (MMP)-2 and MMP-9 activities and their mRNA levels increased significantly, and this increase was significantly summed with taurine. There was a decrease in mRNA expression of Extracellular matrix metalloproteinase inducer (EMMPRIN). Taurine significantly increased this decrease. Diabetes increased mRNA expressions of transforming growth factor (TGF)-β and Smad2/3. Taurine significantly reduced this induction. TGF-β protein expression, p38, and Smad2/3 activations were also inhibited, but taurine was suppressed significantly. All these findings indicate that taurine may be an effective practical strategy to prevent renal diabetic injury., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

A keloid is a classic fibrotic skin disease characterized by excessive deposition of extracellular matrix (ECM). Osteomodulin (OMD) is a heterologous protein that is a part of osteoadherin and plays a role in modulating ECM deposition. In this study, we investigated the effects of OMD on ECM synthesis and the tumor-like phenotype of keloid fibroblasts. We enrolled 10 patients with keloids and 10 age- and sex-matched healthy individuals, whose keloid or normal skin tissues were collected during surgery. Real-time quantitative polymerase chain reaction (qRT-PCR), western blotting, and immunohistochemical staining were performed to analyze OMD expression in skin tissues. Cell transfection, CCK-8 assay, EdU staining, Transwell assay, qRT-PCR, western blotting, and immunofluorescence were performed to study the effects of OMD on primary keloid-derived fibroblasts (KFs). OMD exhibited greater expression in human keloid specimens than in normal skin tissues. Consistently, higher expression of OMD was observed in KFs, compared to that in normal fibroblasts. Silencing OMD expression in transforming growth factor (TGF)-β1-treated KFs inhibited cell proliferation and migration, as well as collagen and fibronectin expression; however, overexpression of OMD had the opposite effect. p38 mitogen-activated protein kinase (MAPK) was activated in keloid tissues but not in normal skin. OMD was positively correlated with p38 MAPK activation. Adding SB203580, p38 MAPK inhibitor, significantly reversed the effects of OMD on the regulation of KF phenotype. The high expression of OMD may contribute to hyperproliferation of KFs, their migration, and excess ECM synthesis in KFs via regulation of the p38 MAPK signaling pathway., (© 2023 Japanese Dermatological Association.)

Introduction: Substance P (SP) is a neuropeptide released from the nervous fibers in response to injury. In addition to its association with pain and reactions to anxiety and stress, SP exerts various physiological functions by binding to the neurokinin-1 receptor (NK1R). However, the expression and role of SP in reparative dentinogenesis remain elusive. Here, we explored whether SP is involved in odontoblastic differentiation during reparative dentinogenesis., Methods: Dental pulp stem cells (DPSCs) were isolated from healthy human dental pulp tissues and subjected to odontoblastic differentiation. The expression of SP and NK1R during odontoblastic differentiation was investigated in vitro. The effects of SP on odontoblastic differentiation of DPSCs were evaluated using alizarin red staining, alkaline phosphatase staining, and real-time polymerase chain reaction. After direct pulp capping with mineral trioxide aggregate, the expression of SP and NK1R during reparative dentin formation in rats were identified using histological and immunohistochemical staining., Results: SP and NK1R expression increased during the odontoblastic differentiation of DPSCs. SP translocated to the nucleus when DPSCs were exposed to differentiation medium. NK1R was always present in the nuclei of DPSCs and odontoblast-like cells. Additionally, we discovered that 10 -8  M SP marginally enhanced the odontoblastic differentiation of DPSCs, and that these effects could be impaired by the NK1R antagonist. Furthermore, SP and NK1R were expressed in odontoblast-like and dental pulp cells during reparative dentin formation in vivo., Conclusions: SP contributes to odontoblastic differentiation during reparative dentin formation by binding to the NK1R., (Copyright © 2023. Published by Elsevier Inc.)

Background: Drosophila melanogaster lipophorin receptors (LpRs), LpR1 and LpR2, are members of the LDLR family known to mediate lipid uptake in a range of organisms from Drosophila to humans. The vertebrate orthologs of LpRs, ApoER2 and VLDL-R, function as receptors of a glycoprotein involved in development of the central nervous system, Reelin, which is not present in flies. ApoER2 and VLDL-R are associated with the development and function of the hippocampus and cerebral cortex, important association areas in the mammalian brain, as well as with neurodevelopmental and neurodegenerative disorders linked to those regions. It is currently unknown whether LpRs play similar roles in the Drosophila brain., Results: We report that LpR-deficient flies exhibit impaired olfactory memory and sleep patterns, which seem to reflect anatomical defects found in a critical brain association area, the mushroom bodies (MB). Moreover, cultured MB neurons respond to mammalian Reelin by increasing the complexity of their neurite arborization. This effect depends on LpRs and Dab, the Drosophila ortholog of the Reelin signaling adaptor protein Dab1. In vitro, two of the long isoforms of LpRs allow the internalization of Reelin, suggesting that Drosophila LpRs interact with human Reelin to induce downstream cellular events., Conclusions: These findings demonstrate that LpRs contribute to MB development and function, supporting the existence of a LpR-dependent signaling in Drosophila, and advance our understanding of the molecular factors functioning in neural systems to generate complex behaviors in this model. Our results further emphasize the importance of Drosophila as a model to investigate the alterations in specific genes contributing to neural disorders., (© 2022. The Author(s).)

Pulmonary fibrosis, a severe condition that can progress to respiratory failure and death, is characterized by aberrant activation/proliferation of fibroblasts and excessive extracellular matrix (ECM) deposition and has limited therapeutic options. Identifying novel mediators of pulmonary fibrosis is currently needed to facilitate the development of more effective therapeutic strategies targeting pulmonary fibrosis. The present study was designed to investigate whether transforming growth factor-β (TGF-β) induced protein (TGFBI), an extracellular matrix protein, regulates pulmonary fibrosis in vitro and in vivo and the possible mechanism of actions. It was found that protein expressions of TGFBI were significantly upregulated and G-protein signaling modulator 2 (GPSM2) expression downregulated in fibrotic lung tissues from bleomycin (BLM)-induced rats and TGF-β1-stimulated human lung IMR-90 fibroblasts. Either silencing TGFBI with specific siRNA or treatment with the TGF-β signaling inhibitor SB431542 significantly inhibited TGF-β1-induced fibrotic effects and dysregulation of GPSM2 and Snail expressions in IMR-90 fibroblasts. Moreover, GPSM2 overexpression also inhibited TGF-β1-induced fibrotic effects and Snail upregulation in IMR-90 fibroblasts. Silencing Snail with specific siRNA attenuated TGF-β1-induced fibrotic effects. Therefore, our findings suggest that the extracellular matrix protein TGFBI mediates pulmonary fibrosis through regulation of the GPSM2/Snail axis, which identifies TGFBI as a novel mediator of pulmonary fibrosis and may be a potential therapeutic target for the treatment of pulmonary fibrosis., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Cutaneous wound healing is an intricate orchestration of three overlapping phases of repair that encompass numerous cell types, signalling cascades, and microenvironment modifications to reach a successful resolution. Disruption of any of these steps will create an abnormal healing response resulting in either ulceration or excessive scarring. It has become evident that the extracellular matrix and its associated components are key orchestrators during this process. One of these essential matrix proteins is decorin, a small leucine-rich proteoglycan (SLRP) that acts as a regulator of collagen fibrillogenesis and a non-competitive inhibitor of multiple growth factors signalling cascades. Decorin is a necessary shut-off switch for the pro-reparative mechanism of the tissue replacement phase and limits the occurrence of hypertrophic scarring by preventing excessive repair. We investigated the use of decorin as a therapeutic by administering the matrix protein anchored in a slow-release coacervate in a hypertrophic scarring mouse model. The results show that early wound healing phase measurements exhibit little difference in performance compared to our coacervate-only baseline or HB-EGF-treated control mice. However, during the resolution phase of wound healing, the decorin-treatment significantly reduces cutaneous thickness, enhances collagen alignment, and improves overall wound scoring in the mice. Thus, mice treated with decorin display better healing outcomes and could limit the hypertrophic scarring phenotype in the coacervate only, and HB-EGF controls. These results suggest that decorin may be a promising tool and alternative therapy for patients who suffer from over-exuberant matrix deposition during wound healing., (© 2022 The Wound Healing Society.)

Gallbladder carcinoma (GBC), with early metastasis and high recurrence rates, is an enormous threat to health. As an anthraquinones monomer of traditional Chinese medicine Hedyotis diffusa, 2-hydroxy-3-methylanthraquinone (HMA) has been reported to inhibit the growth of several cancers. But in our preliminary study, HMA could only weakly induce GBC cell apoptosis. To explore other possible mechanism underlying the inhibition effect of HMA on GBC, this proteomics analysis was performed. A proteomics analysis was performed on one GBC cell line bought from the China Life Science Cell Bank. Several computational techniques were merged to develop analysis for those differently expressed proteins. A comparative protein-protein interaction network analysis was carried out among the differently expressed proteins to identify the proteins potentially inhibiting GBC. Thus, a GO and KEGG analysis was performed to identify the signaling pathways underlying a potential therapeutic role for HMA. A total of 285 proteins were affected by HMA, including 187 upregulated and 98 downregulated. The subcellular localization of differently expressed proteins were identified, including 142 in nuclear, 67 in cytoplasm, 67 in extracellular matrix, 46 in plasma membrane, 13 in mitochondrion, 3 in lysosome, and 1 in cytoskeleton. HMA could regulate EGFR, FN1, PLG, PLAUR, LAMA3, HRG, THBS1, PLAT, KNG1, ENAM, SERPINE1, ECM1, interleukin-8, and trypsin in GBC. Most of the regulated proteins involve in cell migration. Pathways including PI3K-Akt, Wnt, HIF-1, focal adhesion, microRNAs were regulated by HMA. HMA was shown to be an inhibition agent for GBC development, and this analysis would contribute to the development of new anti-GBC drugs., (© 2021 Société Française de Pharmacologie et de Thérapeutique.)

Rationale: Vascular calcification is a prominent feature of late-stage diabetes, renal and cardiovascular disease (CVD), and has been linked to adverse events. Recent studies in patients reported that plasma levels of osteomodulin (OMD), a proteoglycan involved in bone mineralisation, associate with diabetes and CVD. We hypothesised that OMD could be implicated in these diseases via vascular calcification as a common underlying factor and aimed to investigate its role in this context., Methods and Results: In patients with chronic kidney disease, plasma OMD levels correlated with markers of inflammation and bone turnover, with the protein present in calcified arterial media. Plasma OMD also associated with cardiac calcification and the protein was detected in calcified valve leaflets by immunohistochemistry. In patients with carotid atherosclerosis, circulating OMD was increased in association with plaque calcification as assessed by computed tomography. Transcriptomic and proteomic data showed that OMD was upregulated in atherosclerotic compared to control arteries, particularly in calcified plaques, where OMD expression correlated positively with markers of smooth muscle cells (SMCs), osteoblasts and glycoproteins. Immunostaining confirmed that OMD was abundantly present in calcified plaques, localised to extracellular matrix and regions rich in α-SMA + cells. In vivo, OMD was enriched in SMCs around calcified nodules in aortic media of nephrectomised rats and in plaques from ApoE -/- mice on warfarin. In vitro experiments revealed that OMD mRNA was upregulated in SMCs stimulated with IFNγ, BMP2, TGFβ1, phosphate and β-glycerophosphate, and by administration of recombinant human OMD protein (rhOMD). Mechanistically, addition of rhOMD repressed the calcification process of SMCs treated with phosphate by maintaining their contractile phenotype along with enriched matrix organisation, thereby attenuating SMC osteoblastic transformation. Mechanistically, the role of OMD is exerted likely through its link with SMAD3 and TGFB1 signalling, and interplay with BMP2 in vascular tissues., Conclusion: We report a consistent association of both circulating and tissue OMD levels with cardiovascular calcification, highlighting the potential of OMD as a clinical biomarker. OMD was localised in medial and intimal α-SMA + regions of calcified cardiovascular tissues, induced by pro-inflammatory and pro-osteogenic stimuli, while the presence of OMD in extracellular environment attenuated SMC calcification., (© 2022 The Authors. Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.)

Dentin matrix protein 1 (DMP1) contains a large number of acidic domains, multiple phosphorylation sites, a functional arginine-glycine-aspartate (RGD) motif, and a DNA binding domain, and has been shown to play essential regulatory function in dentin and bone mineralization. DMP1 could also orchestrate bone matrix formation, but the ability of DMP1 on Ti to human mesenchymal stem cell (hMSC) conversion to osteoblasts has not been studied. There is importance to test if the DMP1 coated Ti surface would promote cell migration and attachment to the metal surface and promote the differentiation of the attached stem cells to an osteogenic lineage. This study aimed to study the human mesenchymal stem cells (hMSCs) attachment and proliferation on DMP1 coated titanium (Ti) disks compared to non-coated disks, and to assess possible osteoblastic differentiation of attached hMSCs. Sixty-eight Ti disks were divided into two groups. Group 1 disks were coated with dentin matrix protein 1 and group 2 disks served as control. Assessment with light microscopy was used to verify hMSC attachment and proliferation. Cell viability was confirmed through fluorescence microscopy and mitochondrial dehydrogenase activity. Real-time polymerase chain reaction analysis was done to study the gene expression. The proliferation assay showed significantly greater cell proliferation with DMP1 coated disks compared to the control group ( p -value < 0.001). Cell vitality analysis showed a greater density of live cells on DMP1 coated disks compared to the control group. Alkaline phosphatase staining revealed higher enzyme activity on DMP1 coated disks and showed itself to be significantly higher than the control group ( p -value < 0.001). von Kossa staining revealed higher positive areas for mineralized deposits on DMP1 coated disks than the control group ( p -value < 0.05). Gene expression analysis confirmed upregulation of runt-related transcription factor 2, osteoprotegerin, osteocalcin, osteopontin, and alkaline phosphatase on DMP1 coated disks ( p -value < 0.001). The dentin matrix protein promoted the adhesion, proliferation, facilitation differentiation of hMSC, and mineralized matrix formation.

Reelin is a large secreted glycoprotein that regulates neuronal migration, lamination and establishment of dendritic architecture in the embryonic brain. Reelin expression switches postnatally from Cajal-Retzius cells to interneurons. However, reelin function in interneuron development is still poorly understood. Here, we have investigated the role of reelin in interneuron development in the postnatal neocortex. To preclude early cortical migration defects caused by reelin deficiency, we employed a conditional reelin knockout (RelncKO) mouse to induce postnatal reelin deficiency. Induced reelin deficiency caused dendritic hypertrophy in distal dendritic segments of neuropeptide Y-positive (NPY+) and calretinin-positive (Calr+) interneurons, and in proximal dendritic segments of parvalbumin-positive (Parv+) interneurons. Chronic recombinant Reelin treatment rescued dendritic hypertrophy in Relncko interneurons. Moreover, we provide evidence that RelncKO interneuron hypertrophy is due to presynaptic GABABR dysfunction. Thus, GABABRs in RelncKO interneurons were unable to block N-type (Cav2.2) Ca2+ channels that control neurotransmitter release. Consequently, the excessive Ca2+ influx through AMPA receptors, but not NMDA receptors, caused interneuron dendritic hypertrophy. These findings suggest that reelin acts as a 'stop-growth-signal' for postnatal interneuron maturation., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Extracellular matrix (ECM) products have the potential to improve cellular attachment and promote tissue-specific development by mimicking the native cellular niche. In this study, the therapeutic efficacy of an ECM substratum produced by bone marrow stem cells (BM-MSCs) to promote bone regeneration in vitro and in vivo were evaluated. Fluorescence-activated cell sorting analysis and phenotypic expression were employed to characterize the in vitro BM-MSC response to bone marrow specific ECM (BM-ECM). BM-ECM encouraged cell proliferation and stemness maintenance. The efficacy of BM-ECM as an adjuvant in promoting bone regeneration was evaluated in an orthotopic, segmental critical-sized bone defect in the rat femur over 8 weeks. The groups evaluated were either untreated (negative control); packed with calcium phosphate granules or granules+BM-ECM free protein and stabilized by collagenous membrane. Bone regeneration in vivo was analyzed using microcomputed tomography and histology. in vivo results demonstrated improvements in mineralization, osteogenesis, and tissue infiltration (114 ± 15% increase) in the BM-ECM complex group from 4 to 8 weeks compared to mineral granules only (45 ± 21% increase). Histological observations suggested direct apposition of early bone after 4 weeks and mineral consolidation after 8 weeks implantation for the group supplemented with BM-ECM. Significant osteoid formation and greater functional bone formation (polar moment of inertia was 71 ± 0.2 mm 4 with BM-ECM supplementation compared to 48 ± 0.2 mm 4 in untreated defects) validated in vivo indicated support of osteoconductivity and increased defect site cellularity. In conclusion, these results suggest that BM-ECM free protein is potentially a therapeutic supplement for stemness maintenance and sustaining osteogenesis., (© 2020 Wiley Periodicals LLC.)

Despite the poor regenerative capacity of the adult central nervous system (CNS) in mammals, two distinct regions, subventricular zone (SVZ) and the subgranular zone (SGZ), continue to generate new functional neurons throughout life which integrate into the pre-existing neuronal circuitry. This process is not fixed but highly modulated, revealing many intrinsic and extrinsic mechanisms by which this performance can be optimized for a given environment. The capacity for self-renewal, proliferation, migration, and multi-lineage potency of neural stem cells (NSCs) underlines the necessity of controlling stem cell fate. In this context, the native and local microenvironment plays a critical role, and the application of this highly organized architecture in the CNS has been considered as a fundamental concept in the generation of new effective therapeutic strategies in tissue engineering approaches. The brain extracellular matrix (ECM) is composed of biomacromolecules, including glycosaminoglycans, proteoglycans, and glycoproteins that provide various biological actions through biophysical and biochemical signaling pathways. Herein, we review predominantly the structure and function of the mentioned ECM composition and their regulatory impact on multiple and diversity of biological functions, including neural regeneration, survival, migration, differentiation, and final destiny of NSCs., Competing Interests: Declaration of competing interest Authors declare there is no conflict of interest., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The immature phenotype of human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) is a major limitation to the use of these valuable cells for pre-clinical toxicity testing and for disease modeling. Here we tested the hypothesis that human perinatal stem cell derived extracellular matrix (ECM) promotes hiPSC-CM maturation to a greater extent than mouse cell derived ECM. We refer to the human ECM as Matrix Plus (Matrix Plus) and compare effects to commercially available mouse ECM (Matrigel). hiPSC-CMs cultured on Matrix Plus mature functionally and structurally seven days after thaw from cryopreservation. Mature hiPSC-CMs showed rod-shaped morphology, highly organized sarcomeres, elevated cTnI expression and mitochondrial distribution and function like adult cardiomyocytes. Matrix Plus also promoted mature hiPSC-CM electrophysiological function and monolayers' response to hERG ion channel specific blocker was Torsades de Pointes (TdP) reentrant arrhythmia activations in 100% of tested monolayers. Importantly, Matrix Plus enabled high throughput cardiotoxicity screening using mature human cardiomyocytes with validation utilizing reference compounds recommended for the evolving Comprehensive In Vitro Proarrhythmia Assay (CiPA) coordinated by the Health and Environmental Sciences Institute (HESI). Matrix Plus offers a solution to the commonly encountered problem of hiPSC-CM immaturity that has hindered implementation of these human based cell assays for pre-clinical drug discovery.

Tenascin-C (TNC) is strongly expressed by fibroblasts and cancer cells in breast cancer. To assess the effects of TNC on stromal formation, we examined phenotypic changes in human mammary fibroblasts treated with TNC. The addition of TNC significantly up-regulated α-smooth muscle actin (α-SMA) and calponin. TNC increased the number of α-SMA- and/or calponin-positive cells with well-developed stress fibers in immunofluorescence, which enhanced contractile ability in collagen gel contraction. The treatment with TNC also significantly up-regulated its own synthesis. Double immunofluorescence of human breast cancer tissues showed α-SMA- and/or calponin-positive myofibroblasts in the TNC-deposited stroma. Among several receptors for TNC, the protein levels of the αv and β1 integrin subunits were significantly increased after the treatment. Immunofluorescence showed the augmented colocalization of αv and β1 at focal adhesions. Immunoprecipitation using an anti-αv antibody revealed a significant increase in coprecipitated β1 with TNC in lysates. The knockdown of αv and β1 suppressed the up-regulation of α-SMA and calponin. The addition of TNC induced the phosphorylation of SMAD2/3, whereas SB-505124 and SIS3 blocked myofibroblast differentiation. Therefore, TNC enhances its own synthesis by forming a positive feedback loop and increases integrin αvβ1 heterodimer levels to activate transforming growth factor-β signaling, which is followed by a change to highly contractile myofibroblasts. TNC may essentially contribute to the stiffer stromal formation characteristic of breast cancer tissues., (Copyright © 2020 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Recent reports highlight the potential tumorigenic role of Dentin Sialophosphoprotein (DSPP) and its cognate partner Matrix Metalloproteinase 20 (MMP-20) in Oral Squamous Cell Carcinomas (OSCCs). However, the function/mechanism of these roles is yet to be fully established. The present study aimed to investigate the effects of DSPP and MMP20 silencing on specific proteins involved in oral cancer cell adhesion, angiogenesis, metastasis, and epithelial-mesenchymal transition (EMT). Stable lines of DSPP/MMP20 silenced OSCC cell line (OSC2), previously established via lentiviral-mediated shRNA transduction, were analyzed for the effects of DSPP, MMP20, and combined DSPP-MMP20 silencing on MMP2, MMP9, integrins αvβ3 and αvβ6, VEGF, Kallikerin- 4,-5,-8,-10, E-cadherin, N-cadherin, Vimentin, met, src, snail, and Twist by Western blot. Results show a significant decrease ( p < 0.05) in the expression of MMP2, MMP9, integrin αvβ3, αvβ6, VEGF, Kallikerins -4, -5, -8, -10, N-cadherin, vimentin met, src, snail and twist following DSPP and MMP20 silencing, individually and in combination. On the other hand, the expression of E-cadherin was found to be significantly increased ( p < 0.05). These results suggest that the tumorigenic effect of DSPP and MMP20 on OSC2 cells is mediated via the upregulation of the genes involved in invasion, metastasis, angiogenesis, and epithelial-mesenchymal transition (EMT).

N16 is an active protein existing in Pinctada martensi. Our previous studies have demonstrated that N16 inhibited osteoclast differentiation in vitro. To better understand how N16 regulates osteoclast differentiation, RAW264.7 cells, a murine monocytic cell line and murine bone marrow-derived macrophages (BMMs) were adopted. Treatment of RAW264.7 cells with RANKL activated osteoclastogenesis and N16 inhibited the formation of multinucleated osteoclasts and TRAP activity. The suppression occurred at the early stage of osteoclastogenesis. Moreover, we found that N16 inhibited PU.1 and MITF expressions, mirroring the inhibition of RANK expressions, indicating that N16 inhibited RANK expression by down-regulating the expressions of MITF and PU.1, thus preventing osteoclastogenesis., Competing Interests: Declaraton of Competing Interest The authors declare no competing financial interest., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Transforming growth factor β-induced protein (TGFBIp) is an extracellular matrix protein; its expression by several cell types is greatly increased by TGF-β. TGFBIp is released by primary human umbilical vein endothelial cells (HUVECs) and functions as a mediator of experimental sepsis. 2,2'-Bipyridine-containing natural products are generally accepted to have antimicrobial, cytotoxic and anti-inflammatory properties. We hypothesized that a 2,2'-bipyridine containing natural product, collismycin C, could reduce TGFBIp-mediated severe inflammatory responses in human endothelial cells and mice. Here we investigated the effects and underlying mechanisms of collismycin C against TGFBIp-mediated septic responses. Collismycin C effectively inhibited lipopolysaccharide-induced release of TGFBIp and suppressed TGFBIp-mediated septic responses. In addition, collismycin C suppressed TGFBIp-induced sepsis lethality and pulmonary injury. This suppression of TGFBIp-mediated and CLP-induced septic responses indicates that collismycin C is a potential therapeutic agent for various severe vascular inflammatory diseases, with inhibition of the TGFBIp signaling pathway as the mechanism of action.

Cellular therapies play an important role in tendon tissue engineering, with tenocytes being the most prominent and potent cell population available. However, for the development of a rich extracellular matrix tenocyte-assembled tendon equivalent, prolonged in vitro culture is required, which is associated with phenotypic drift. Recapitulation of tendon tissue microenvironment in vitro with cues that enhance and accelerate extracellular matrix synthesis and deposition, whilst maintaining tenocyte phenotype, may lead to functional cell therapies. Herein, we assessed the synergistic effect of low oxygen tension (enhances extracellular matrix synthesis) and macromolecular crowding (enhances extracellular matrix deposition) in human tenocyte culture. Protein analysis demonstrated that human tenocytes at 2% oxygen tension and with 50 μg ml -1 carrageenan (macromolecular crowder used) significantly increased synthesis and deposition of collagen types I, III, V and VI. Gene analysis at day 7 illustrated that human tenocytes at 2% oxygen tension and with 50 μg ml -1 carrageenan significantly increased the expression of prolyl 4-hydroxylase subunit alpha 1, procollagen-lysine 2- oxoglutarate 5-dioxygenase 2, scleraxis, tenomodulin and elastin, whilst chondrogenic (e.g. runt-related transcription factor 2, cartilage oligomeric matrix protein, aggrecan) and osteogenic (e.g. secreted phosphoprotein 1, bone gamma-carboxyglutamate protein) trans-differentiation markers were significantly down-regulated or remained unchanged. Collectively, our data clearly illustrates the beneficial synergistic effect of low oxygen tension and macromolecular crowding in the accelerated development of tissue equivalents.

Objective: The objective of this study was to investigate the effects of dentin phosphoprotein (DPP) on lipopolysaccharide-induced inflammatory responses of macrophages in vitro., Design: Wildtype and mutant recombinant dentin phosphoprotein (rDPP) proteins were generated using a mammalian expression system. Macrophages, phorbol 12-myristate 13-acetate-differentiated THP-1 cells, were stimulated with lipopolysaccharide in the absence or presence of rDPP proteins. After the 24-hr incubation, the inflammatory gene expression levels were examined by quantitative reverse-transcription polymerase chain reaction and the amount of secreted TNF-α protein was evaluated by enzyme-linked immunosorbent assay. Furthermore, the subcellular localization of exogenously added rDPP was examined by immunocytochemistry, and the direct binding of rDPP to lipopolysaccharide was quantified by solid-phase binding assay., Results: rDPP dose-dependently reduced the expression of lipopolysaccharide-induced inflammatory genes, such as TNFα, IL-1β, and IL-8, and TNF-α protein secretion from the macrophages. Furthermore, mutant rDPP having a shortened serine/aspartic acid-rich repeats (SDrr) was also able to inhibit lipopolysaccharide-induced inflammatory responses of macrophages. rDPP was localized adjacent to the cellular membrane rather than in the cytoplasm, and rDPP was able to bind to lipopolysaccharide. These results suggested that rDPP inhibited lipopolysaccharide-induced inflammatory responses by binding to lipopolysaccharide., Conclusions: In addition to the well-known functions of DPP for dentin mineralization that depend on the SDrr, we demonstrated that DPP possesses anti-inflammatory effects on lipopolysaccharide-stimulated macrophages that are independent of the SDrr., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Recent reports suggest the utility of extracellular matrix (ECM) molecules as raw components in scaffolding of engineered materials. However, rapid and tunable manufacturing of ECM molecules into fibrous structures remains poorly developed. Here we report on an immersion rotary jet-spinning (iRJS) method to show high-throughput manufacturing (up to ∼1 g/min) of hyaluronic acid (HA) and other ECM fiber scaffolds using different spinning conditions and postprocessing modifications. This system allowed control over a variety of scaffold material properties, which enabled the fabrication of highly porous (70-95%) and water-absorbent (swelling ratio ∼2000-6000%) HA scaffolds with soft-tissue mimetic mechanical properties (∼0.5-1.5 kPa). Tuning these scaffolds' properties enabled the identification of porosity (∼95%) as a key facilitator for rapid and in-depth cellular ingress in vitro. We then demonstrated that porous HA scaffolds accelerated granulation tissue formation, neovascularization, and reepithelialization in vivo, altogether potentiating faster wound closure and tissue repair. Collectively, this scalable and versatile manufacturing approach enabled the fabrication of tunable ECM-mimetic nanofiber scaffolds that may provide an ideal first building block for the design of all-in-one healing materials.

Background and Objective: Amelogenins are major components of extracellular matrix proteins in developing teeth, and regulate the growth of enamel crystals. They also function as signaling molecules in cell differentiation. This study aimed to determine the biological effects of amelogenins on the differentiation of HAT-7 dental epithelial cells and MC3T3-E1 pre-osteoblastic cells using full-length recombinant human amelogenin (rh-AMEL)., Design: rh-AMEL was expressed in a mammalian cell line (Expi293F™) and was purified by DDK agarose beads. Effects of rh-AMEL on differentiation were evaluated by Mineralization and Alkaline phosphatase (ALP) activity using Alizarin Red S staining and colorimetric substrate p-nitrophenol, respectively., Results: Western blotting and silver staining confirmed the successful purification of rh-AMEL. Mineralization and ALP activity in HAT-7 cells were significantly higher after treatment with 4 μg/mL rh-AMEL, but not after treatment with Emdogain ® (EMD). In MC3T3-E1 cells, on the other hand, rh-AMEL showed biphasic effects on differentiation. Treatment with low concentrations of rh-AMEL (0.001-0.1 μg/mL) and EMD (0.01-1 μg/mL) increased mineralization and ALP activity in MC3T3-E1 cells, whereas treatment with high concentrations of rh-AMEL (4 μg/mL) and EMD (100 μg/mL) had the opposite effect., Conclusion: High concentrations of rh-AMEL and EMD decreased the differentiation of MC3T3-E1 cells. By contrast, a high concentration of rh-AMEL, but not that of EMD, promoted the differentiation of HAT-7 cells. This study demonstrates that the effects of rh-AMEL on cell differentiation differ between HAT-7 and MC3T3-E1 cells, and suggests that different regions on AMEL may induce the differentiation of these cell types., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Corneal lymphangiogenesis plays a key role in diverse pathological conditions of the eye. Here, we demonstrate that a versatile extracellular matrix protein, transforming growth factor-β induced protein (TGFBIp), promotes lymphatic sprouting in corneal lymphangiogenesis. TGFBIp is highly up-regulated in inflamed mouse corneas. Immunolocalization of TGFBIp is detected in infiltrating macrophages in inflamed mouse corneas. Subconjunctival injection of liposomal clodronate can significantly reduce macrophage infiltration in inflamed mouse cornea, and decrease the expression of TGFBIp and areas of corneal lymphangiogenesis and angiogenesis after corneal suture placement. In brief, these results indicate that the up-regulation of TGFBIp in sutured cornea correlates with macrophage infiltration. Although TGFBIp alone cannot significantly stimulate corneal lymph vessel ingrowth in vivo, it can enhance the effect of vascular endothelial growth factor-C in promoting corneal lymphangiogenesis. The in vitro results show that TGFBIp promotes migration, tube formation and adhesion of human lymphatic endothelial cells (HLECs), but it has no effect on HLECs' proliferation. We also find that the in vitro effect of TGFBIp is mediated by the integrin α5β1-FAK pathway. Additionally, integrin α5β1 blockade can significantly inhibit lymphatic sprouting induced by TGFBIp. Taken together, these findings reveal a new molecular mechanism of lymphangiogenesis in which the TGFBIp-integrin pathways plays a pivotal role in lymphatic sprouting., (© 2019 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

This report addresses the issue of optimizing extracellular matrix protein density required to support osteogenic lineage differentiation of mesenchymal stem cells (MSCs) by culturing MSCs on surface-bound density gradients of immobilized collagen type I (COL1) and osteopontin (OPN). A chemical surface gradient is prepared by tailoring the surface chemical composition from high hydroxyl groups to aldehyde groups using a diffusion-controlled plasma polymerization technique. Osteogenesis on the gradient surface is determined by immunofluorescence staining against Runx2 as an early marker and by staining of calcium phosphate deposits as a late stage differentiation marker. The Runx2 intensity and calcified area increase with increasing COL1 density up to a critical value corresponding to 124.2 ng cm -2 , above which cell attachment and differentiation do not rise further, while this critical value for OPN is 19.0 ng cm -2 . This gradient approach may facilitate the screening of an optimal biomolecule surface density on tissue-engineered scaffolds, implants, or tissue culture ware to obtain the desired cell response, and may generate opportunities for more cost-effective regenerative medicine., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Impaired elastogenesis and increased degradation of elastic fibers has been implicated in the pathogenesis of pelvic organ prolapse. Loss of the elastogenic organizer, fibulin-5 (FBLN5), leads to pelvic organ prolapse in mice. The objective of this study was to investigate the regulation of FBLN5 after surgical injury of the vaginal wall using the rat as a preclinical animal model. Both endogenous and recombinant FBLN5 were degraded after surgical injury. Estrogen did not alter the dramatic loss of vaginal FBLN5 in the acute phase after injury (12-48 h), but resulted in rescue of the poor recovery of FBLN5 levels in the late phase (7 d) of healing in ovariectomized animals. In contrast with estrogen, the general MMP inhibitor, actinonin, abrogated injury-induced degradation of FBLN5 significantly. Further, actinonin rescued the negative effects of injury on biomechanics, histomorphology, and elastic fibers. Control of excessive matrix degradation by local application of actinonin at the time of surgery may lead to improved elastic fiber regeneration and wound healing, thereby potentially enhancing pelvic floor recovery after reconstructive surgery for prolapse.

Hantaviruses are the major causative agents of hemorrhagic fever with renal syndrome (HFRS) in humans, which is characterized by increased capillary permeability. Dentin matrix protein 1 (DMP1) has been shown to degrade components of the basal membrane and interendothelial junctions via matrix metalloproteinase-9. To study the changes of serum DMP1 in HFRS, we determined the concentration of DMP1 using sandwich enzyme-linked immunosorbent assay. We found that serum DMP1 concentrations increased significantly, and reached peak value during the oliguric phase and in the critical group in HFRS patients. Moreover, serum DMP1 concentrations were closely related to blood urea nitrogen, creatinine, cystatin C, and vascular endothelial growth factor (VEGF). We further explored the role of DMP1 in HTNV-infected human umbilical vein endothelial cells (HUVECs) model. Data from immunocytochemistry showed that VEGF and tumor necrosis factor-α (TNF-α) promoted the expression of DMP1 on HTNV-infected HUVECs. Results from transwell assays demonstrated that the permeability of HUVECs increased significantly after HTNV infection with the addition of DMP1, VEGF, and TNF-α. This study suggests that elevated DMP1 concentrations may be associated with disease stage, severity, and the degree of acute kidney injury. DMP1 is involved in the regulation of capillary permeability in HFRS caused by hantavirus infection., (Copyright © 2019 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Acinetobacter baumannii has been well recognized as a problematic human pathogen and several reports has shown the incidence of multidrug and pandrug-resistant A. baumannii strains in infirmary infections. A. baumannii grows only on an air-liquid interface and does not form a contiguous biofilm. Extracellular matrices (ECM) and slanted glass coverslips are (SGC) used as biofilm substrates and biofilms have been investigated by SEM, confocal and crystal violet staining. ECM has shown enhanced biofilm formation under dynamic conditions rather than static conditions. SGC biofilm yield assay has shown higher levels of continuous layers and packed thicker biofilm formation with glass coverslip inserts, up to 1.7 to 3 times higher biofilm formation, than when compared with no glass coverslip inserts. A media immersed ECM study revealed that biofilm grown on extracellular matrixes formed thread-like pili structures, and that these structures had contact with the ECM and also showed excellent cell-to-cell interaction. In summary, A. baumannii showed higher biofilm formation capacities with ECM, while the prominent results were directly related with the biofilm formation capacity of A. baumannii. For the initial step of biofilm formation, adherence is an important factor and, consequently, strains with a comparatively high capability to adhere to extracellular matrices and slanted glass coverslips provide a new method of enhanced biofilm growth for in vitro assays. ECM can be used as a substrate for immersed biofilm formation studies and the SGC method for air-liquid interface exposed biofilm formation studies, and these substrates can provide better biofilm growth and easy handling for in vitro adherence and biofilm assays.

Osteoarthritis (OA) is a degenerative joint disease characterised by a progressive degradation of articular cartilage and underlaying bone and is associated with pain and disability. Currently, there is no medical treatment to reverse or even retard OA. Based on our previous reports, where we establish the repair potential of short Link N (sLN) in the intervertebral disc, a cartilage-like tissue, we hypothesise that sLN may hold similar promises in the repair of articular cartilage. This study aimed to determine if sLN, could prevent OA disease progression. Skeletally mature New Zealand white rabbits underwent unilateral anterior cruciate ligament transection (ACLT) of their left femorotibial joints to induce joint degeneration typical of OA. Beginning 3 weeks post-operatively, and every three weeks thereafter for 12 weeks, either saline (1 mL) or sLN (100 μg in 1 mL saline) was injected intraarticularly into the operated knee. Six additional rabbits underwent sham surgery but without ACLT or post-operative injections. The effects on gross joint morphology and cartilage histologic changes were evaluated. In the Saline group, prominent erosion of articular cartilage occurred in both femoral condyle compartments and the lateral compartment of the tibial plateau while, sLN treatment reduced the severity of the cartilage damage in these compartments of the knee showing erosion. Furthermore, statistically significant differences were detected between the joint OA score of the saline and sLN treated groups (p = 0.0118). Therefore, periodic intraarticular injection of sLN is a promising nonsurgical treatment for preventing or retarding OA progression, by reducing cartilage degradation.

The healing of acute wounds is vital to humans and is a well-orchestrated process that involves systemic and local factors. However, there is a lack of effective and safe clinical therapies. The collagen triple helix repeat containing 1 (CTHRC1) protein is a type of exocrine protein that has been recently reported to contribute to tissue repair. Our aim is to validate the promoting effects of CTHRC1 on the healing of acute wounds and to elucidate the underlying molecular mechanism. Therefore, we first established acute wound healing mouse models and confirmed that CTHRC1 accelerates the healing process of acute wounds. Then, we characterized wound macrophages using a polyvinylalcohol (PVA) sponge model and used Western blotting to investigate the molecular mechanism. We found that CTHRC1 increased the M2 macrophage population and the TGF-β expression level as a result of the activation of the TGF-β and Notch pathways, which eventually contributed to the promotion of wound healing. Inhibition of the Notch pathway showed attenuated M2 macrophage recruitment, and it decreased the TGF-β expression level. These results substantiate our hypothesis that CTHRC1 promotes wound healing by recruiting M2 macrophages and regulating the TGF-β and Notch pathways., (Copyright © 2019 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Tissue engineers rely on expensive, time-consuming, and destructive techniques to monitor the composition, microstructure, and function of engineered tissue equivalents. A non-destructive solution to monitor tissue quality and maturation would greatly reduce costs and accelerate the development of tissue-engineered products. The objectives of this study were to (a) determine whether matrix stabilization with exogenous lysyl oxidase-like protein-2 (LOXL2) with recombinant hyaluronan and proteoglycan link protein-1 (LINK) would result in increased compressive and tensile properties in self-assembled articular cartilage constructs, (b) evaluate whether label-free, non-destructive fluorescence lifetime imaging (FLIm) could be used to infer changes in both biochemical composition and biomechanical properties, (c) form quantitative relationships between destructive and non-destructive measurements to determine whether the strength of these correlations is sufficient to replace destructive testing methods, and (d) determine whether support vector machine (SVM) learning can predict LOXL2-induced collagen crosslinking. The combination of exogenous LOXL2 and LINK proteins created a synergistic 4.9-fold increase in collagen crosslinking density and an 8.3-fold increase in tensile strength as compared with control (CTL). Compressive relaxation modulus was increased 5.9-fold with addition of LOXL2 and 3.4-fold with combined treatments over CTL. FLIm parameters had strong and significant correlations with tensile properties (R 2  = 0.82; p < 0.001) and compressive properties (R 2  = 0.59; p < 0.001). SVM learning based on FLIm-derived parameters was capable of automating tissue maturation assessment with a discriminant ability of 98.4%. These results showed marked improvements in mechanical properties with matrix stabilization and suggest that FLIm-based tools have great potential for the non-destructive assessment of tissue-engineered cartilage., (© 2019 John Wiley & Sons, Ltd.)

We challenge the conventional designation of structural matrix proteins primarily as supporting scaffolds for resident cells. The extracellular matrix protein tropoelastin is classically regarded as a structural component that confers mechanical strength and resilience to tissues subject to repetitive elastic deformation. Here we describe how tropoelastin inherently induces a range of biological responses, even in cells not typically associated with elastic tissues and in a manner unexpected of typical substrate-dependent matrix proteins. We show that tropoelastin alone drives mesenchymal stem cell (MSC) proliferation and phenotypic maintenance, akin to the synergistic effects of potent growth factors such as insulin-like growth factor 1 and basic fibroblast growth factor. In addition, tropoelastin functionally surpasses these growth factors, as well as fibronectin, in allowing substantial media serum reduction without loss of proliferative potential. We further demonstrate that tropoelastin elicits strong mitogenic and cell-attractive responses, both as an immobilized substrate and as a soluble additive, via direct interactions with cell surface integrins αvβ3 and αvβ5. This duality of action converges the long-held mechanistic dichotomy between adhesive matrix proteins and soluble growth factors and uncovers the powerful, untapped potential of tropoelastin for clinical MSC expansion and therapeutic MSC recruitment. We propose that the potent, growth factor-like mitogenic and motogenic abilities of tropoelastin are biologically rooted in the need for rapid stem cell homing and proliferation during early development and/or wound repair., Competing Interests: Conflict of interest statement: A.S.W. is the founding scientist of Elastagen Pty. Ltd., now sold to Allergan, Inc.

Background: Carcinogenesis occurs in elastin-rich tissues and leads to local inflammation and elastolytic proteinase release. This contributes to bioactive matrix fragment (Matrikine) accumulation like elastin degradation products (EDP) stimulating tumour cell invasive and metastatic properties. We previously demonstrate that EDPs exert protumoural activities through Hsp90 secretion to stabilised extracellular proteinases., Methods: EDP influence on cancer cell blebbing and extracellular vesicle shedding were examined with a videomicroscope coupled with confocal Yokogawa spinning disk, by transmission electron microscopy, scanning electron microscopy and confocal microscopy. The ribosomal protein SA (RPSA) elastin receptor was identified after affinity chromatography by western blotting and cell immunolocalisation. mRNA expression was studied using real-time PCR. SiRNA were used to confirm the essential role of RPSA., Results: We demonstrate that extracellular matrix degradation products like EDPs induce tumour amoeboid phenotype with cell membrane blebbing and shedding of extracellular vesicle containing Hsp90 and proteinases in the extracellular space. EDPs influence intracellular calcium influx and cytoskeleton reorganisation. Among matrikines, VGVAPG and AGVPGLGVG peptides reproduced EDP effects through RPSA binding., Conclusions: Our data suggests that matrikines induce cancer cell blebbing and extracellular vesicle release through RPSA binding, favouring dissemination, cell-to-cell communication and growth of cancer cells in metastatic sites.

Objective: Upregulation of calcium/calmodulin-dependent kinase II (CaMKII) is implicated in the pathogenesis of osteoarthritis (OA) and reactivation of articular cartilage hypertrophy. However, direct inhibition of CaMKII unexpectedly augmented symptoms of OA in animal models. The role of CaMKII in OA remains unclear and requires further investigation., Methods: Analysis of CaMKII expression was performed in normal human and OA articular chondrocytes, and signaling mechanisms were assessed in articular, fetal and Pluripotent Stem Cell (PSC)-derived human chondrocytes using pharmacological (KN93), peptide (AC3-I) and small interfering RNA (siRNA) inhibitors of CaMKII., Results: Expression levels of phospho-CaMKII (pCaMKII) were significantly and consistently increased in human OA specimens. BMP2/4 activated expression of pCaMKII as well as COLII and COLX in human adult articular chondrocytes, and also increased the levels and nuclear localization of SMADs1/5/8, while TGFβ1 showed minimal or no activation of the chondrogenic program in adult chondrocytes. Targeted blockade of CaMKII with specific siRNAs decreased levels of pSMADs, COLII, COLX and proteoglycans in normal and OA adult articular chondrocytes in the presence of both BMP4 and TGFβ1. Both human fetal and PSC-derived chondrocytes also demonstrated a decrease of chondrogenic differentiation in the presence of small molecule and peptide inhibitors of CaMKII. Furthermore, immunoprecipitation for SMADs1/5/8 or 2/3 followed by western blotting for pCaMKII showed direct interaction between SMADs and pCaMKII in primary chondrocytes., Conclusion: Current study demonstrates a direct role for CaMKII in TGF-β and BMP-mediated responses in primary and PSC-derived chondrocytes. These findings have direct implications for tissue engineering of cartilage tissue from stem cells and therapeutic management of OA., (Copyright © 2018 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Background/aims: Different components of the tumor microenvironment can be either tumor-promoting or tumor-suppressive agents depending on factors which are not fully understood. Fibulins are components of the extracellular matrix from different tissues and constitute a clear example of this dual function. In fact, fibulins may either support tumor growth or abolish progression of malignant cells depending on the crosstalk between tumor cells and their surrounding stroma through mechanisms that remain to be elucidated. Among all fibulins, fibulin-5 contains a particular structural hallmark which consists in the presence of a RGD motif within its architecture. Previous reports have highlighted the importance of the interaction of this motif with integrins, and not only in normal functions but also in a tumor context., Methods: Site-Directed Mutagenesis technique was employed to introduce the change RGD to RGE (RGD-to-RGE) within Fbln5 cDNA sequence. Cell proliferation was measured using the MTT assay or by counting Ki-67 positive cell nuclei. Cell adhesion was analysed using culture plates coated with different extracellular matrix components. Cell invasion was evaluated using 24-well Matrigel-coated invasion chambers, and mammosphere formation was monitored using ultralow attachment culture plates. BALB/c mice were employed to induce subcutaneous tumors., Results: The RGD-to-RGE change alters the capacity of breast cancer cells to adhere to different extracellular matrix proteins as well as to α v β 3 and α 5 β 1 integrins, and promotes protumor effects using different cell-based assays. Moreover, 4T1 cells, a mouse breast cancer cell line model, shows an increased capacity to generate tumors when exogenously expresses fibulin-5 with a RGD-to-RGE change, and such capacity is similar to that shown for 4T1 cells with an interfered Fbln5 gene., Conclusion: These data highlight the importance of the RGD motif of fibulin-5 to induce antitumor effects and provide new insights into the involvement of fibulins in tumor processes., Competing Interests: The authors declare no conflicts of interest., (© Copyright by the Author(s). Published by Cell Physiol Biochem Press.)

Dentin phosphoprotein (DPP) is a major component of the dentin matrix playing crucial role in hydroxyapatite deposition during bone mineralization, making it a prime candidate for the design of novel materials for bone and tooth regeneration. The bioactivity of DPP-derived proteins is controlled by the phosphorylation and dephosphorylation of the serine residues. Here an enzyme-responsive peptide nanofiber system inducing biomineralization is demonstrated. It closely emulates the structural and functional properties of DPP and facilitates apatite-like mineral deposition. The DPP-mimetic peptide molecules self-assemble through dephosphorylation by alkaline phosphatase (ALP), an enzyme participating in tooth and bone matrix mineralization. Nanofiber network formation is also induced through addition of calcium ions. The gelation process following nanofiber formation produces a mineralized extracellular matrix like material, where scaffold properties and phosphate groups promote mineralization. It is demonstrated that the DPP-mimetic peptide nanofiber networks can be used for apatite-like mineral deposition for bone regeneration., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Angiogenesis is crucial for tissue development and homeostasis; however, excessive angiogenesis can lead to diseases, including arthritis and cancer metastasis. Some antiangiogenic drugs are available, but side effects remain problematic. Thus, alternative angiogenesis inhibition strategies are needed. Fibulin-7 (Fbln7) is a newly discovered member of the fibulin protein family, a group of cell-secreted glycoproteins, that functions as a cell adhesion molecule and interacts with other extracellular matrix (ECM) proteins as well as cell receptors. We previously showed that a recombinant C-terminal Fbln7 fragment (Fbln7-C) inhibits tube formation by human umbilical vein endothelial cells (HUVECs) in vitro. In the present study, we examined the in vivo antiangiogenic activity of recombinant full-length Fbln7 (Fbln7-FL) and Fbln7-C proteins using a rat corneal angiogenesis model. We found that both Fbln7-FL and Fbln7-C inhibited neovascularization. Fbln7-C bound to vascular endothelial growth factor receptor 2 (VEGFR2), inhibiting VEGFR2 and ERK phosphorylation and resulting in reduced HUVEC motility. HUVEC attachment to Fbln7-C occurred through an interaction with integrin α5β1 and regulated changes in cellular morphology. These results suggest that Fbln7-C action may target neovascularization by altering cell/ECM associations. Therefore, Fbln7-C could have potential as a therapeutic agent for diseases associated with angiogenesis.

Matrikines, peptides originating from the fragmentation of extracellular matrix proteins are identified to play important role in both health and disease. They possess biological activities, much different from their parent protein. Identification of such bioactive cryptic regions in the extracellular matrix proteins has attracted the researchers all over the world in the recent decade. These bioactive peptides could find use in preparation of biomaterials and tissue engineering applications. Matrikines identified in major extracellular matrix (ECM) proteins like collagen, elastin, fibronectin, and laminin are being extensively studied for use in tissue engineering and regenerative medicine. They are identified to modulate cellular activity like cell growth, proliferation, migration and may induce apoptosis. RGD, a well-known peptide identified in fibronectin with cell adhesive property is being investigated in designing biomaterials. Collagen hexapeptide GFOGER was found to promote cell adhesion and differentiation. Laminin also possesses regions with strong cell adhesion property. Recently, cell-penetrating peptides from elastin are used as a targeted delivery system for therapeutic drugs. The continued search for cryptic sequences in the extracellular matrix proteins along with advanced peptide coupling chemistries would lead to biomaterials with improved surface properties. This review article outlines the peptides derived from extracellular matrix and some of the possible applications of these peptides in therapeutics and tissue engineering applications., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Collagen triple helix repeat containing (CTHRC1), which was identified as a cancer-related factor, is a promigratory protein involved in multiple processes, including vascular remodeling, antifibrosis, metabolism, bone formation, and cancer. In this study, we aimed to investigate the clinical significance and possible role of CTHRC1 in the process of epithelial-mesenchymal transition (EMT) in colorectal cancer (CRC). Here, we revealed that CTHRC1 mRNA and protein levels are both upregulated in CRC tissues compared with those of paired noncancerous tissues. Moreover, the overexpression of CTHRC1 correlated with poor prognosis in patients with CRC (especially colon cancer). Furthermore, we showed that CTHRC1 induced EMT and promoted cell motility in CRC cells. Importantly, we demonstrated that CTHRC1 promoted EMT by activating transforming growth factor-β (TGF-β) signaling, revealing a possible effective therapeutic treatment for patients with CRC., (© 2018 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Water stress proteins (WSP1) from Nostoc commune Vauch. had been proven to selectively induce colon cancer cells apoptosis. In this study, the effect of WSP1 on migration of human colon cancer cells was investigated. It showed that WSP1 inhibited DLD-1 cell migration, but with an insignificant effect on normal human colon epithelial cells. The data further indicated that WSP1 activated autophagy through down regulation of PI3K/AKT/mTOR pathway. Meanwhile, β‑catenin was degraded by autophagy, which then restrained epithelial-mesenchymal transition (EMT) of DLD-1 cell and its migration was subsequently suppressed significantly. The same changes occurred in xenografted nude mice according to the obtained immunohistochemical results. Consistently, the application of autophagy inhibitor largely reversed the inhibited migration by WSP1 treatment. Taken together, WSP1 could suppress migration of DLD-1 cells by autophagy inhibited EMT. The results suggested that WSP1 possessed the potential as a selective therapeutic agent against metastatic colon cancer., (Copyright © 2018. Published by Elsevier B.V.)

Congenital scoliosis is a lateral curvature of the spine that is due to the presence of vertebral anomalies. Although genetic and environmental factors are involved in the pathogenesis of congenital scoliosis, the specific cause of only a small number of individuals has been identified to date. We identified a de novo missense mutation in the olfactomedin-like 1 (OLFML1) gene by whole-exome sequencing of a patient with congenital scoliosis. Then, we carried out further functional investigation in mice. An assessment of the tissue distribution of Olfml1 revealed it to be prominently expressed in developing skeletal tissues, specifically osteoblasts. Short hairpin RNA-mediated knockdown of Olfml1 in osteoblasts induced the translocation of Yes-associated protein (YAP) transcriptional coactivator from the cytoplasm to the nucleus, which accelerated the Hippo signaling pathway to promote osteoblast mineralization. In contrast, experimentally induced gain of function of Olfml1 retained YAP in the cytoplasm. There appears to exist a novel cell-autonomous mechanism by which osteoblasts avoid excess mineralization through Olfml1. Our results also indicate that mutation of OLFML1 leads to impaired osteoblast differentiation and abnormal development of bone tissue., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: iMatrix-511 is a novel integrin-binding fragment derived from laminin-511. Previous studies showed its superiority as a culture substrate for xeno-free culture and maintenance of pluripotency in stem cells. However, its effects in the dental field remain largely unknown. The aim of the present study was to unravel the in vitro effects of iMatrix-511 in comparison with vitronectin (VN)., Methods: Biochemical assays were performed in vitro in MDPC-23 cells. The optimal coating density for 2 proteins was determined using the cell counting kit-8. To evaluate cell proliferation to both proteins, MDPC-23 cells were directly seeded onto the iMatrix-511 or VN-modified polystyrene and analyzed by the cell counting kit-8. The phenotype of cells seeded on iMatrix-511 and VN was characterized. Phenotypic characterization included real-time reverse-transcription polymerase chain reaction and alizarin red staining., Results: The optimal coating density for iMatrix-511 and VN was determined to be 1 μg/cm 2 and 0.25 μg/cm 2 , respectively. Cells cultured on iMatrix-511 showed higher cell proliferative activity than the noncoated control and VN on days 1, 2, and 4. Cell morphology observation revealed MDPC-23 cells attach preferentially to iMatrix-511 and start to spread as early as 1 hour after inoculation. MDPC-23 cells exhibited more potent odontogenic differentiation on iMatrix-511 than the control and VN as shown by the marked enhancement of dentin matrix protein 1 and dentin sialophosphoprotein messenger RNA expression. Although both proteins showed more mineralized nodule formation than the control, iMatrix-511 remained to be the one that elicited stronger calcific deposition., Conclusions: iMatrix-511 supported the proliferation and acquisition of odontogenic cell phenotype in vitro, rendering this novel material a potential candidate for dentin regeneration., (Copyright © 2018 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Background: The degeneration of the intervertebral disc (IVD) is characterized by proteolytic degradation of the extracellular matrix, and its repair requires the production of an extracellular matrix with a high proteoglycan-to-collagen ratio characteristic of a nucleus pulposus (NP)-like phenotype in vivo. At the moment, there is no medical treatment to reverse or even retard disc degeneration. The purpose of the present study was to determine if a low dose of short link N (sLN), a recently discovered fragment of the link N peptide, could behave in a manner similar to that of link N in restoring the proteoglycan content and proteoglycan-to-collagen ratio of the disc in a rabbit model of IVD degeneration, as an indication of its potential therapeutic benefit in reversing disc degeneration., Methods: Adolescent New Zealand white rabbits received an annular puncture with an 18-gauge needle into two noncontiguous discs to induce disc degeneration. Two weeks later, either saline (10 μL) or sLN (25 μg in 10 μL saline) was injected into the center of the NP. The sLN concentration was empirically chosen at a lower molar concentration equivalent to half that of link N (100 μg in 10 μL). The effect on radiographic, biochemical and histologic changes were evaluated., Results: Following needle puncture, disc height decreased by about 25-30% within 2 weeks and maintained this loss for the duration of the 12-week study; a single 25-μg sLN injection at 2 weeks partially restored this loss in disc height. sLN injection led to an increase in glycosaminoglycans (GAG) content 12 weeks post-injection in both the NP and annulus fibrosus (AF). There was a trend towards maintaining control disc collagen-content with sLN supplementation and the GAG-to-collagen ratio in the NP was increased when compared to the saline group., Conclusions: When administered to the degenerative disc in vivo, sLN injection leads to an increase in proteoglycan content and a trend towards maintaining control disc collagen content in both the NP and AF. This is similar to link N when it is administered to the degenerative disc. Thus, pharmacologically, sLN supplementation could be a novel therapeutic approach for treating disc degeneration.

Crohn's disease (CD) is a chronic inflammatory bowel disease that is difficult to treat. However, previous preclinical and clinical studies have shown that mesenchymal stromal cells (MSCs) are a promising therapeutic approach, whereas the exact underlying molecular mechanisms of MSCs in treating CD remain unclear. Furthermore, the heterogeneity of MSCs, as well as the in vivo microenvironments may influence the therapeutic efficacy. In our previous study, we found that a subpopulation of mouse MSCs with a high expression of matrix Gla protein (MGP), one of the members of vitamin K-dependent protein family, possessed better immunoregulatory properties. Therefore, in this study we investigate whether the abundant MSCs-derived MGP participate in the therapeutic mechanisms for MSCs treating CD. Obvious suppression of cell proliferation and cytokine production in T cells were observed in vitro through MSCs-derived MGP. Moreover, MGP alleviated the clinical and histopathological severity of colonic inflammation in mouse experimental colitis models to a remarkable degree. Our results indicate that MGP might be a novel important mediator of MSCs-mediated immunomodulation in treating CD.

Innate immune recognition is classically mediated by the interaction of host pattern-recognition receptors and pathogen-associated molecular patterns; this triggers a series of downstream signaling events that facilitate killing and elimination of invading pathogens. In this report, we provide the first evidence that peroxidasin (PXDN; also known as vascular peroxidase-1) directly binds to gram-negative bacteria and mediates bactericidal activity, thus, contributing to lung host defense. PXDN contains five leucine-rich repeats and four immunoglobulin domains, which allows for its interaction with lipopolysaccharide, a membrane component of gram-negative bacteria. Bactericidal activity of PXDN is mediated via its capacity to generate hypohalous acids. Deficiency of PXDN results in a failure to eradicate Pseudomonas aeruginosa and increased mortality in a murine model of Pseudomonas lung infection. These observations indicate that PXDN mediates previously unrecognized host defense functions against gram-negative bacterial pathogens., Competing Interests: The authors have declared that no competing interests exist.

Lumbar disc disease (LDD) is common in aged populations, and it is primarily caused by intervertebral disc degeneration (IDD). Cartilage intermediate layer protein (CILP), which is specifically expressed in intervertebral discs (IVDs), is suspected to be associated with IDD. However, it remains unclear whether CILP contributes to IDD in humans. Furthermore, the regulation of CILP in human IVDs is poorly understood, especially by mechanical stimuli, which are regarded as primary factors promoting IDD. To address these issues, the present study collected nucleus pulposus (NP) cells from patients undergoing lumbar spinal surgery for degenerative disc disease (DDD). Subsequently, CILP expression was measured in human NP cells in response to mechanical stimuli, including cyclic compressive stress and cyclic tensile strain (CTS), by reverse transcription‑quantitative polymerase chain reaction and western blotting. Aggrecan and collagen II, which are the main components of the extracellular matrix (ECM) and traditional degenerative markers for IDD, were detected following the treatment with CILP small interfering (si)RNA or recombinant human CILP (rhCILP) at various concentrations to determine whether CILP contributes to IDD by negatively regulating expression of the ECM. The results revealed that CILP expression in loaded NP cells was significantly increased compared with that in non‑loaded cells under compressive loading, and that it was markedly decreased in cells under tensile loading, in contrast with the expression of aggrecan and collagen II in response to the same stimuli. Furthermore, CILP siRNA effectively inhibited CILP expression and significantly increased the expression of aggrecan and collagen II. In addition, treatment of NP cells with a high concentration of rhCILP resulted in significantly decreased expression of aggrecan and collagen II. In conclusion, these results demonstrated for the first time, to the best of our knowledge, that in human NP cells, CILP is regulated by mechanical stress and that its expression affects ECM synthesis. Therefore, CILP represents a promising therapeutic target for preventing loss of the matrix during IDD as a novel treatment strategy.

The rising prevalence of chronic liver disease, coupled with a permanent shortage of organs for liver transplantation, has sparked enormous interest in alternative treatment strategies. Previous protocols to generate hepatocyte-like cells (HLCs) via pancreas-to-liver transdifferentiation have utilised fetal bovine serum, introducing unknown variables and severely limiting study reproducibility. Therefore, the main goal of this study was to develop a protocol for transdifferentiation of pancreatic progenitor cells to HLCs in a chemically defined, serum-free culture medium. The clonal pancreatic progenitor cell line AR42J-B13 was cultured in basal growth medium on uncoated plastic culture dishes in the absence or presence of Dexamethasone on uncoated, laminin- or fibronectin-coated culture substrata, with or without serum supplementation. The hepatocytic differentiation potential was evaluated: (i) morphologically through bright-field and scanning electron microscopy, (ii) by assessing pancreatic and hepatic marker expression and (iii) by determining the function of HLCs through their ability to synthesise glycogen or take up and release indocyanine green. Here we demonstrate for the first time that transdifferentiation of pancreatic cells to HLCs is not dependent on serum. These results will assist in converting current differentiation protocols into procedures that are compliant with clinical use in future cell-based therapies to treat liver-related metabolic disorders.

Purpose: Intervertebral disc degeneration is a major cause of back pain. Novel therapies for prevention or reversal of disc degeneration are needed. It is desirable for potential therapies to target both inflammation and matrix degeneration., Materials and Methods: The combined regenerative potential of link protein N-terminal peptide (LN) and fullerol on annulus fibrosus (AF) cells was evaluated in a 3D culture model., Results: Interleukin-1α (IL-1α)-induced AF cell degeneration was counteracted by fullerol, LN, and fullerol + LN, with the latter having the greatest effect on matrix production as evaluated by real-time polymerase chain reaction and glycosaminoglycan assay. IL-1α-induced increases in pro-inflammatory mediators (interleukin-6 and cyclooxygenase-2) and matrix metalloproteinases (MMP-1, -2, -9, and -13) were also counteracted by fullerol and LN., Conclusion: Our data demonstrate that LN and fullerol individually, and in combination, promote matrix production and have anti-inflammatory and anti-catabolic effects on AF cells.

Background/aims: Fibulin-3, an extracellular matrix glycoprotein, inhibits vascular oxidative stress and remodeling in hypertension. Oxidative stress is prevalent in chronic kidney disease (CKD) patients and is an important mediator of osteo-/chondrogenic transdifferentiation and calcification of vascular smooth muscle cells (VSMCs) during hyperphosphatemia. Therefore, the present study explored the effects of Fibulin-3 on phosphate-induced vascular calcification., Methods: Experiments were performed in primary human aortic smooth muscle cells (HAoSMCs) treated with control or with phosphate without or with additional treatment with recombinant human Fibulin-3 protein or with hydrogen peroxide as an exogenous source of oxidative stress., Results: Treatment with calcification medium significantly increased calcium deposition in HAoSMCs, an effect significantly blunted by additional treatment with Fibulin-3. Moreover, phosphate-induced alkaline phosphatase activity and mRNA expression of osteogenic and chondrogenic markers MSX2, CBFA1, SOX9 and ALPL were all significantly reduced by addition of Fibulin-3. These effects were paralleled by similar regulation of oxidative stress in HAoSMCs. Phosphate treatment significantly up-regulated mRNA expression of the oxidative stress markers NOX4 and CYBA, down-regulated total antioxidant capacity and increased the expression of downstream effectors of oxidative stress PAI-1, MMP2 and MMP9 as well as BAX/BLC2 ratio in HAoSMCs, all effects blocked by additional treatment with Fibulin-3. Furthermore, the protective effects of Fibulin-3 on phosphate-induced osteogenic and chondrogenic markers expression in HAoSMCs were reversed by additional treatment with hydrogen peroxide., Conclusions: Fibulin-3 attenuates phosphate-induced osteo-/ chondrogenic transdifferentiation and calcification of VSMCs, effects involving inhibition of oxidative stress. Up-regulation or supplementation of Fibulin-3 may be beneficial in reducing the progression of vascular calcification during hyperphosphatemic conditions such as CKD., (© 2018 The Author(s). Published by S. Karger AG, Basel.)

Transforming growth factor-β-induced protein (TGFBIp), an extracellular protein, is expressed on several cell types in response to TGF-β stimulation. Human umbilical vein endothelial cell (HUVEC)-derived TGFBIp functions as a mediator of sepsis. Screening of bioactive compound libraries is an effective approach for repositioning FDA-approved drugs or discovering new treatments for human diseases (drug repositioning). Dabrafenib (DAB), a B-Raf inhibitor, was initially used for treating metastatic melanoma. The present study determined whether DAB modulated TGFBIp-mediated septic responses in HUVECs and in mice. Antiseptic functions of DAB were examined by measuring permeability, leukocyte adhesion and migration, and proinflammatory protein activation in TGFBIp-stimulated HUVECs and mice. In addition, beneficial effects of DAB on survival rate were examined using a mouse model of sepsis. We found that DAB inhibited TGFBIp-induced vascular barrier disruption, cell adhesion molecule (CAM) expression, and neutrophil adhesion/transendothelial migration toward human endothelial cells. DAB also suppressed TGFBIp-induced hyperpermeability and leukocyte migration in vivo. These results suggest that DAB exerts anti-inflammatory effects by inhibiting hyperpermeability, CAM expression, and leukocyte adhesion and migration, indicating its utility for treating vascular inflammatory diseases., (Copyright © 2017 Elsevier B.V. All rights reserved.)