Decorin Research Articles

Tenogenic Potential of Equine Fetal Mesenchymal Stem Cells Under The In Vitro Effect of Bone Morphogenetic Protein-12 (BMP-12)

Equine adult bone marrow-derived MSCs (BM-MSCs) may be induced into the tenogenic lineage after exposure with bone morphogenetic protein-12 (BMP-12). Despite fetal BM-MSCs have showed a greater differentiation potential compared to adults, the tenogenic differentiation capacity of equine fetal BM-MSC have not been reported. Thus, the aim of the present study was to evaluate the in vitro tenogenic differentiation potential of equine fetal BM-MSCs under the effect of BMP-12. Equine fetal BM-MSCs were exposed to three concentrations of BMP-12 (25, 50 and 100 ng/mL) during a 21-day culture period. Levels of mRNA of tenogenic markers decorin (DCN), tenomodulin (TNMD), scleraxis (SCX), collagen 1α1 (COL1α1) and protein expression of Col1α1 were evaluated. Plastic adherent cells exhibited specific MSC profile including expression of CD73 and lack of expression of CD34. Gene expression levels of DCN, TNMD, SCX and COL1α1 were increased in equine fetal BM-MSC exposed to three different concentrations of BMP-12 during a 21-day culture period. Equine fetal BM-MSCs displayed specific expression profiles suggesting features of MSCs and multipotent capacity. Furthermore, up-regulation of tenogenic markers DCN, TNMD, COL1α1 and SCX after exposure to different concentrations of BMP-12 suggests that equine fetal BM-MSCs have potential to activate selected genes that control tenogenic differentiation.

Antidiuretic hormone inhibits osteogenic differentiation of dental follicle stem cells via V1a receptors and the PLC-IP3 pathway

ObjectiveThe aim of this study was to elucidate the molecular mechanism by which antidiuretic hormone (ADH) inhibited osteogenesis in dental follicle stem cells. DesignRat dental follicle stem cells were cultured in osteogenic differentiation medium supplemented with ADH. Alkaline phosphatase enzyme activity, Alizarin Red S staining, MTT assay and RT-qPCR was used to examine ADH’s impact on cell mineralization, viability, and osteogenic gene expression. Real-time calcium imaging analysis was performed to identify the ADH receptor and its mechanism of action. ResultsADH supplementation to the osteogenic differentiation medium inhibited cell mineralization without compromising cell viability and downregulated the expression of key osteogenic genes: DCN (Decorin), RUNX2 (Runt-related transcription factor 2) and BSP (Bone sialoprotein). Real-time calcium imaging analysis revealed that ADH (1−1000 nM) increased intracellular calcium in a concentration-dependent manner. Pretreatment of cells with V2255, a V1a receptor blocker, inhibited the calcium signals, but not with the V1b (Nelivaptan) or V2 (Tolvaptan). V2255 also reversed the inhibitory effect of ADH on osteogenesis. Furthermore, U73122, a Phospholipase C (PLC) inhibitor, 2-APB, an Inositol Triphosphate (IP3) receptor blocker, and depletion of endoplasmic reticulum calcium stores abolished the calcium signals by ADH. ConclusionsOur results demonstrated that ADH activates V1a receptors and the PLC-IP3 pathway to stimulate intracellular calcium signals, which inhibits cell mineralization and osteogenic gene expression. These findings uncovered a novel function for ADH as a negative regulator of osteogenesis in dental follicle stem cells. The role of ADH in the pathogenesis of bone diseases remains to be determined.

The glucagon-like peptide 1 receptor agonist Exendin-4 induces tenogenesis in human mesenchymal stem cells

Tendon injuries are common and account for up to 50% of musculoskeletal injuries in the United States. The poor healing nature of the tendon is attributed to poor vascularization and cellular composition. In the absence of FDA-approved growth factors for tendon repair, engineering strategies using bioactive factors, donor cells, and delivery matrices to promote tendon repair and regeneration are being explored. Growth factor alternatives in the form of small molecules, donor cells, and progenitors offer several advantages and enhance the tendon healing response. Small drug molecules and peptides offer stability over growth factors that are known to suffer from relatively short biological half-lives. The primary focus of this study was to assess the ability of the exendin-4 (Ex-4) peptide, a glucagon-like peptide 1 (GLP-1) receptor agonist, to induce tenocyte differentiation in bone marrow-derived human mesenchymal stem cells (hMSCs). We treated hMSCs with varied doses of Ex-4 in culture media to evaluate proliferation and tendonogenic differentiation. A 20 nM Ex-4 concentration was optimal for promoting cell proliferation and tendonogenic differentiation. Tendonogenic differentiation of hMSCs was evaluated via gene expression profile, immunofluorescence, and biochemical analyses. Collectively, the levels of tendon-related transcription factors (Mkx and Scx) and extracellular matrix (Col-I, Dcn, Bgn, and Tnc) genes and proteins were elevated compared to media without Ex-4 and other controls including insulin and IGF-1 treatments. The tendonogenic factor Ex-4 in conjunction with hMSCs appear to enhance tendon regeneration.

The functional role of decorin in corneal neovascularization in vivo

Our earlier decorin (Dcn) gene overexpression studies found that the targeted Dcn gene transfer into the cornea inhibited corneal angiogenesis in vivo using a rabbit model. In this study, we tested the hypothesis that anti-angiogenic effects of decorin in the cornea are mediated by alterations in a normal physiologic balance of pro- and anti-angiogenic factors using decorin deficient (Dcn−/−) and wild type (Dcn+/+) mice. Corneal neovascularization (CNV) in Dcn−/− and Dcn+/+ mice was produced with a standard chemical injury technique. The clinical progression of CNV in mice was monitored with stereo- and slit-lamp microscopes, and histopathological hematoxylin and eosin (H&E) staining. Protein and mRNA expression of pro- and anti-angiogenic factors in the cornea were evaluated using immunofluorescence and quantitative real-time PCR, respectively. Slit-lamp clinical eye examinations revealed significantly more CNV in Dcn−/- mice than the Dcn+/+ mice post-injury (p < 0.05) and AAV5-Dcn gene therapy significantly reduced CNV in Dcn−/- mice compered to no AAV5-Dcn gene therapy controls (p < 0.001). H&E-stained corneal sections exhibited morphology with several neovessels in injured corneas of the Dcn−/− mice than the Dcn+/+ mice. Immunofluorescence of corneal sections displayed significantly higher expression of α-smooth muscle actin (α-SMA) and endoglin proteins in Dcn−/- mice than Dcn+/+ mice (p < 0.05). Quantitative real-time PCR found significantly increased mRNA levels of pro-angiogenic factors endoglin (2.53-fold; p < 0.05), Vegf (2.47-fold; p < 0.05), and Pecam (2.14-fold; p < 0.05) and anti-angiogenic factor Vegfr2 (1.56-fold; p < 0.05) in the normal cornea of the Dcn−/− mice than the Dcn+/+ mice. Furthermore, neovascularized Dcn−/− mice corneas showed greater increase in mRNA expression of pro-angiogenic factors endoglin (4.58-fold; p < 0.0001), Vegf (4.16-fold; p < 0.0001), and Pdgf (2.15-fold; p < 0.0001) and reduced expression of anti-angiogenic factors Ang2 (0.12-fold; p < 0.05), Timp1 (0.22-fold; p < 0.05), and Vegfr2 (0.67-fold; p > 0.05) compared to neovascularized Dcn+/+ mice corneas. These gene deficience studies carried with transgenic Dcn−/− mice revealed decorin's role in influencing a physiologic balance between pro-and anti-angiogenic factors in the normal and injured cornea. We infer that the functional deletion of Dcn promotes irregular corneal repair and aggravates CNV.

Role of Decorin in Posterior Capsule Opacification and Eye Lens Development.

Decorin (DCN) is involved in a variety of physiological and pathological processes. Epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) has been proposed as a major cause for the development of posterior capsule opacification (PCO) after cataract surgery. We investigated the plausible target gene(s) that suppress PCO. The expression of Dcn was significantly upregulated in rat PCO tissues compared to that observed in the control using a microarray-based approach. LECs treated with fibroblast growth factor (FGF) 2 displayed an enhanced level of DCN expression, while LECs treated with transforming growth factor (TGF)β-2 showed a decrease in DCN expression. The expression of tropomyosin 1 (Tpm1), a marker of lens EMT increased after the addition of TGFβ-2 in human LEC; however, upregulation of Tpm1 mRNA or protein expression was reduced in human LECs overexpressing human DCN (hDCN). No phenotypic changes were observed in the lenses of 8- and 48-week-old transgenic mice for lens-specific hDCN (hDCN-Tg). Injury-induced EMT of the mouse lens, and the expression patterns of α smooth muscle actin, were attenuated in hDCN-Tg mice lenses. Overexpression of DCN inhibited the TGFβ-2-induced upregulation of Tpm1 and EMT observed during wound healing of the lens, but it did not affect mouse lens morphology until 48 weeks of age. Our findings demonstrate that DCN plays a significant role in regulating EMT formation of LECs and PCO, and suggest that for therapeutic intervention, maintenance of physiological expression of DCN is essential to attenuate EMT progression and PCO formation.

Development and Validation of a Hypoxia-Associated Prognostic Signature Related to Osteosarcoma Metastasis and Immune Infiltration.

BackgroundIncreasing evidence has shown that hypoxia microenvironment relates to tumor initiation and progression. However, no studies focus on the application of hypoxia-associated genes in predicting osteosarcoma patients’ prognosis. This research aims to identify the hypoxia-associated genes related to osteosarcoma metastasis and construct a gene signature to predict osteosarcoma prognosis.MethodsThe differentially expressed messenger RNAs (DEmRNAs) related to osteosarcoma metastasis were identified from Therapeutically Applicable Research to Generate Effective Treatments (Target) database. Univariate and multivariate cox regression analyses were performed to develop the hypoxia-associated prognostic signature. The Kaplan–Meier (KM) survival analyses of patients with high and low hypoxia risk scores were conducted. The nomogram was constructed and the gene signature was validated in the external Gene Expression Omnibus (GEO) cohort. Single-sample gene set enrichment analysis (ssGSEA) was conducted to investigate the relationships between immune infiltration and gene signature.ResultsTwo genes, including decorin (DCN) and prolyl 4-hydroxylase subunit alpha 1 (P4HA1), were involved in the hypoxia-associated gene signature. In training and testing datasets, patients with high-risk scores showed lower survival rates and the gene signature was identified as the independent prognostic factor. Receiver operating characteristic (ROC) curves demonstrated the robustness of signature. Functional analyses of DEmRNAs among high- and low-risk groups revealed that immune-associated functions and pathways were significantly enriched. Furthermore, ssGSEA showed that five immune cells (DCs, macrophages, neutrophils, pDCs, and TIL) and three immune features (CCR, APC co inhibition, and Check-point) were down-regulated in the high-risk group.ConclusionThe current study established and validated a novel hypoxia-associated gene signature in osteosarcoma. It could act as a prognostic biomarker and serve as therapeutic guidance in clinical applications.

CHPF Regulates the Aggressive Phenotypes of Hepatocellular Carcinoma Cells via the Modulation of the Decorin and TGF-β Pathways.

Simple SummaryAltered extracellular chondroitin sulfate (CS) contributes to tumor progression in many cancers. CHPF is a key enzyme supporting the elongation of CS. Here we showed that CHPF was frequently downregulated in hepatocellular carcinoma (HCC) tumors compared with adjacent non-tumor tissues, and its downregulation was associated with poor overall survival. CHPF regulated aggressive phenotypes of HCC cells in vitro and in vivo, and the TGF-β pathway involved in the phenotypical changes. Mechanistically, CHPF modified CS on decorin (DCN), which could facilitate DCN accumulation surrounding HCC cells, and modulate activation of TGF-β pathway. Indeed, the expression of DCN were positively associated with CHPF levels in primary HCC tissue. The research proposed novel insights into the significance of CHPF, which modified DCN and modulated TGF-β signaling.Aberrant composition of glycans in the tumor microenvironment (TME) and abnormal expression of extracellular matrix proteins are hallmarks of hepatocellular carcinoma (HCC); however, the mechanisms responsible for establishing the TME remain unclear. We demonstrate that the chondroitin polymerizing factor (CHPF), an enzyme that mediates the elongation of chondroitin sulfate (CS), is a critical elicitor of the malignant characteristics of HCC as it modifies the potent tumor suppressor, decorin (DCN). CHPF expression is frequently downregulated in HCC tumors, which is associated with the poor overall survival of HCC patients. We observed that restoring CHPF expression suppressed HCC cell growth, migration, and invasion in vitro and in vivo. Mechanistic investigations revealed that TGF-β signaling is associated with CHPF-induced phenotype changes. We found that DCN, as a TGF-β regulator, is modified by CHPF, and that it affects the distribution of DCN on the surface of HCC cells. Importantly, our results confirm that CHPF and DCN expression levels are positively correlated in primary HCC tissues. Taken together, our results suggest that CHPF dysregulation contributes to the malignancy of HCC cells, and our study provides novel insights into the significance of CS, which affects DCN expression in the TME.

Is Decorin a Promising New Agent for Facial Nerve Regeneration? An Experimental Study

Objective: The aim of this study was to investigate the effects of systemic administration of decorin (DC) on facial nerve (FN) regeneration. Methods: A total of 32 female albino Wistar rats were divided into 4 groups: control (C) group: no bilateral FN neurorrhaphy (B-FNN), no DC application, sham-operated group: B-FNN without DC application, DC group: DC application without B-FNN, and B-FNN + DC group: B-FNN and DC application. Nerve conduction studies were performed before and after skin incisions at 1st, 3rd, 5th, and 7th weeks in all groups. The amplitude and latency of compound muscle action potentials were recorded. FN samples were obtained and were investigated under light microscopy and immunohistochemical staining. The nerve and axon diameter, number of axons, H score, Schwann cell proliferation, and myelin and axonal degeneration were recorded quantitatively. Results: In the sham group, the 3rd and 5th postoperative week, amplitude values were significantly lower than those of the B-FNN + DC group (p < 0.05). Nerve diameters were found to be significantly larger in the sham, DC, and B-FNN + DC groups than in the C group (p < 0.05). The number of axons, the axon diameter, and the H scores were found to be significantly higher in the B-FNN + DC group than in the sham group (p < 0.05). The Schwann cell proliferation, myelin degeneration, and axonal degeneration scores were significantly lower in the B-FNN + DC group than in the sham group (p < 0.05). Conclusion: Electrophysiological and histopathological evaluation revealed the potential benefits provided by DC. This agent may increase FN regeneration.

Expression of cornulin in tongue squamous cell carcinoma.

The aim of the study is to identify cornulin (CRNN) protein expression associated with advancement of tongue squamous cell carcinoma (TSCC). A comparison of addictive (containing potential carcinogens) versus non-addiction causative agents was expected to allow detection of differences in CRNN expression associated with TSCC. Bespoke tissue microarrays (TMAs) were prepared and immunohistochemistry (IHC) performed to determine the changes in CRNN expression in epithelial cells of node-negative (pN-), node-positive (pN+) TSCC and non-cancer patients’ oral tissues. TMAs were validated by performing IHC on whole diagnostic tissues. Chi-square test or Fisher’s-exact tests were used to establish significant expression differences. Analogous analyses were performed for biomarkers previously associated with TSCC, namely collagen I alpha 2 (COL1A2) and decorin (DCN) to compare the significance of CRNN. Keratinisation and its level (low, extensive) were studied in relation to CRNN so that the extent of squamous differentiation could better be assessed.IHC immunoreactive score (IRS) clustered the patients based on weak/moderate (Low (IRS ≤ +3)) or strong (High (IRS ≥ +4)) expression groups. A low expression was observed in a larger number of patients in control proteins COL1A2 (77.3%), DCN (87.5%) and target protein CRNN (52.3%), respectively. Low CRNN expression was observed in TSCC where nodes were involved (pN+: mean 1.4 ± 2.1) (p = 0.248). Keratinisation (%) was low (0% ≤ 50%) in 42.2% and extensive (1% ≥ 50.0%) in 57.8% patients. In conclusion, our study suggested that Low CRNN expression was associated with grade and lymph node metastasis in TSCC. CRNN expression is independent of addiction, however potentially carcinogenic addictive substances might be aiding in the disease progression.

LncRNA FDNCR promotes apoptosis of granulosa cells by targeting the miR-543-3p/DCN/TGF-β signaling pathway in Hu sheep

Long non-coding RNAs (lncRNAs) regulate the development of follicles and reproductive diseases, but the mechanisms by which lncRNAs regulate ovarian functions and fertility remain elusive. We profiled the expression of lncRNAs in ovarian tissues of Hu sheep with different prolificacy and identified 21,327 lncRNAs. Many of the lncRNAs were differentially expressed in different groups. We further characterized an lncRNA that was predominantly expressed in the ovaries of the low prolificacy FecB+ (LPB+) group and mainly present in granulosa cells (GCs), and the expression of this lncRNA decreased during follicular development, which we named follicular development-associated lncRNA (FDNCR). Next, we found that FDNCR directly binds miR-543-3p, and decorin (DCN) was identified as a target of miR-543-3p. FDNCR overexpression promoted GC apoptosis through increased expression of DCN, which could be attenuated by miR-543-3p. Furthermore, miR-543-3p increased and FDNCR reduced the expression of transforming growth factor-β (TGF-β) pathway-related genes, including TGF-β1 and inhibin beta A (INHBA), which were upregulated upon DCN silencing. Our results demonstrated that FDNCR sponges miR-543-3p in GCs and prevents miR-543-3p from binding to the DCN 3′ UTR, resulting in DCN transactivation and TGF-β pathway inhibition and promotion of GC apoptosis in Hu sheep. These findings provide insights into the mechanisms underlying prolificacy in sheep.

Abstract LT002: The therapeutically inflamed tumor microenvironment drives melanoma progression

Abstract How microenvironmental inflammation impacts melanoma progression remains controversial. Inflammation appears beneficial during interferon stimulated expression of checkpoint targets. However, when tipped too far, therapy-driven inflammation has been linked to debilitating autoimmune disease in cancer patients. One unanswered question is whether this inflammation also promotes melanoma dissemination. Here, drug-stressed melanomas are shown to promote progression via a pro-inflammatory secretome that activates the tumor microenvironment. Analyses showed the drug specific activation of immune recruiting cytokines and the transforming growth factor beta (TGFβ) superfamily. In coculture, TGFβ from drug-stressed melanomas resulted in the paracrine hyperactivation of cancer associated fibroblasts (CAFs) as noted by the inflammatory marker alpha smooth muscle actin (αSMA). Pharmacologic inhibition of TGFβ using SB431542 or clinically relevant EW-7197, blunted the αSMA phenotype as well as downstream phospho-SMAD activation. Invasion of melanoma spheroids in CAF cocultures resulted in enhanced invasion which could suppressed by targeted MEK inhibition (AZD6244) and TGFβ receptor blockade. Engrafted melanomas in vivo presented with the rapid expansion of peri-tumoral αSMA+ CAFs surrounding invasive micrometastatic nodules in mouse livers. We have previously reported that extracellular matrix (ECM) protein tenascin-C (TNC) is enriched at the invasive fronts of melanoma, which was recapitulated in vivo and correlated to human melanomas. Human tissue microarrays were assayed for pro-inflammatory TNC, healthy ECM marker Decorin (DCN), αSMA CAFs, and fibrotic Collagen (COL1A1) expression. We detected statistically significant increases in TNC, αSMA and COL1A1 correlating to progression and a concomitant decrease in DCN. Evidence from drug treated spheroid cocultures did not show complete suppression single cell invasion. Prompted by this, ex vivo organotypic skin organ cultures containing TNC and DCN were developed to show that the ECM provides physiological pro-and-anti invasive signals. Expression of DCN therapeutically inhibited collective and single cell invasion in this model. We also observed transcriptomic suppression of TNC and key inflamed matrisome proteins in anti-TGFβ treated CAFs, even when challenged with TGFβ conditioned media. Informed by this, we used a TNC+ vemurafenib resistant patient-derived metastatic melanoma in our xenograft model. Cohorts treated with MEK inhibitor and combination therapy with EW-7197 showed drug efficacy and tolerance with detectable decreases in αSMA and tumor burden of the spleen. Treated animals also showed reduced liver metastases compared to control. Taken together, combination therapy that blocks off-target inflammation and cancer proliferation represent an underappreciated but potentially successful strategy to halt melanoma progression. Citation Format: Andrew Bradshaw, Erica Kuo, Jelena Grahovac, Cindy Sander, Howard Edington, John M. Kirkwood, Alan Wells. The therapeutically inflamed tumor microenvironment drives melanoma progression [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr LT002.

Decorin-mediated suppression of tumorigenesis, invasion, and metastasis in inflammatory breast cancer

Inflammatory breast cancer (IBC) is a clinically distinct and highly aggressive form of breast cancer with rapid onset and a strong propensity to metastasize. The molecular mechanisms underlying the aggressiveness and metastatic propensity of IBC are largely unknown. Herein, we report that decorin (DCN), a small leucine-rich extracellular matrix proteoglycan, is downregulated in tumors from patients with IBC. Overexpression of DCN in IBC cells markedly decreased migration, invasion, and cancer stem cells in vitro and inhibited tumor growth and metastasis in IBC xenograft mouse models. Mechanistically, DCN functioned as a suppressor of invasion and tumor growth in IBC by destabilizing E-cadherin and inhibiting EGFR/ERK signaling. DCN physically binds E-cadherin in IBC cells and accelerates its degradation through an autophagy-linked lysosomal pathway. We established that DCN inhibits tumorigenesis and metastasis in IBC cells by negatively regulating the E-cadherin/EGFR/ERK axis. Our findings offer a potential therapeutic strategy for IBC, and provide a novel mechanism for IBC pathobiology.

Effect of Decorin and Bevacizumab on oxygen-induced retinopathy in rat models: A comparative study.

Purpose:The aims of this study were to evaluate the effects of decorin (DCN) in rat oxygen-induced retinopathy (OIR) model and to compare the results with those of bevacizumab.Methods:Twenty-eight newborn Sprague–Dawley rats were randomly divided into four groups. Group I (control): normoxia plus intraperitoneal (ip) normal saline (NS), Group II (sham): OIR plus ip NS, Group III (DCN): OIR plus ip 0.1 mg/kg DCN, and Group IV (bevacizumab): OIR plus ip 2.5 mg/kg bevacizumab. The OIR model was induced by cycling the oxygen concentration between 50% and 10% every 24 h for 14 days following their birth. In all groups, injections were administered on postnatal day (PD) 15. All animals were sacrificed and their right eyes were enucleated on PD 18. The nuclei of neovascular endothelial cells on the vitreal side of the inner limiting membrane were counted, and vascular endothelial growth factor (VEGF) and tumor necrosis factor-alpha (TNF)-α immunoreactivity were detected in histopathological and immunohistochemical examinations. One-way analysis of variance and post hoc Tukey tests were used for statistical analyses of the data.Results:In Groups II, III, and IV, the mean neovascular cell nuclei counts were 13.14 ± 1.34, 6.57 ± 1.51, and 6.71 ± 1.49, respectively. The mean neovascular cell nuclei count was significantly reduced in treatment groups compared with sham group (P < 0.001). In immunohistochemical staining, the immunoreactivity of VEGF was 0.07 ± 0.02, 0.97 ± 0.21, 0.37 ± 0.12, and 0.23 ± 0.17, respectively. Likewise, immunoreactivity of TNF-α was 0.02 ± 0.02, 1.11 ± 0.36, 0.37 ± 0.13, and 0.62 ± 0.21, respectively. VEGF and TNF-α immunoreactivity increased markedly in the sham group compared with those in the control group (P < 0.001). VEGF and TNF-α immunoreactivity of treatment groups decreased significantly compared to sham group (P < 0.001).Conclusion:The beneficial effects obtained by DCN administration in OIR model were comparable to the effects of bevacizumab.

The Effect of Lecithins Coupled Decorin Nanoliposomes on Treatment of Carbon Tetrachloride-Induced Liver Fibrosis.

This study aimed to investigate the effect of bile duct-targeting lecithins- (PC-) coupled decorin (DCN) (PC-DCN) nanoliposomes against liver fibrosis in vitro and in vivo. We prepared PC-DCN nanoliposomes by using rat astrocytes, HSC-T6, to verify the antifibrosis effect of PC-DCN in vitro. First, we established a rat model of carbon tetrachloride-induced fibrosis. PC-DCN nanoliposomes were then injected into fibrotic rats via the portal vein or bile duct. The EdU assay was performed to analyze cell proliferation. Immunofluorescence staining was used to detect α-smooth muscle actin (α-SMA) expression. Western blot was performed to examine the expression of α-SMA, collagen type I alpha 1 (COL1A1), and transforming growth factor-β (TGF-β) protein. The levels of aspartate transaminase (AST), alanine transaminase (ALT), and total bilirubin (TBIL) were examined by enzyme-linked immunosorbent assay (ELISA) analysis. Hematoxylin and eosin (H&E) staining and Masson trichrome staining were used to determine liver tissue lesions and liver fibrosis. Compared with TGF-β group, PC-DCN treatment could significantly reduce cell proliferation. Western blot analysis indicated that the expression of α-SMA, COL1A1, and TGF-β was downregulated after treatment with PC-DCN in vitro and in vivo. Immunofluorescence staining confirmed that α-SMA expression was reduced by PC-DCN. Furthermore, H&E staining and Masson trichrome staining showed that the administration of PC-DCN nanoliposomes via the bile duct could reduce the extent of liver fibrosis. PCR analysis showed that PC-DCN administration could reduce proinflammatory cytokines IL-6, TNF-α, and IL-1β expression via the bile duct. The administration of PC-DCN nanoliposomes also significantly downregulated liver function indicators ALT, AST, and TBIL. The results of our study indicated that PC-DCN could effectively reduce the extent of liver fibrosis.

Glycosaminoglycan Modification of Decorin Depends on MMP14 Activity and Regulates Collagen Assembly.

Proper processing of collagens COL1 and COL6 is required for normal function of adipose tissue and skeletal muscle. Proteoglycan decorin (DCN) regulates collagen fiber formation. The amino-terminus of DCN is modified with an O-linked glycosaminoglycan (GAG), the function of which has remained unclear. Previously, non-glycanated DCN (ngDCN) was identified as a marker of adipose stromal cells. Here, we identify MMP14 as the metalloprotease that cleaves DCN to generate ngDCN. We demonstrate that mice ubiquitously lacking DCN GAG (ngDCN mice) have reduced matrix rigidity, enlarged adipocytes, fragile skin, as well as skeletal muscle hypotrophy, fibrosis, and dysfunction. Our results indicate that DCN deglycanation results in reduced intracellular DCN—collagen binding and increased production of truncated COL6 chains, leading to aberrant procollagen processing and extracellular localization. This study reveals that the GAG of DCN functions to regulate collagen assembly in adipose tissue and skeletal muscle and uncovers a new mechanism of matrix dysfunction in obesity and aging.

Metabolic Disorder of Extracellular Matrix Mediated by Decorin Upregulation Is Associated With Brain Arteriovenous Malformation Diffuseness.

Background and ObjectiveDiffuse brain arteriovenous malformations (BAVMs) are mixed up with normal brain parenchyma and therefore increase the difficulty of surgical resection, leading to poor surgical prognosis. Since the mechanism underlying BAVM diffuseness remains unknown, a quantitative proteomic analysis was performed to investigate the altered expression of proteins in diffuse BAVMs compared to compact ones.MethodsWe performed proteomic analysis on five diffuse BAVMs and five compact BAVMs. Bioinformatics analysis was conducted to identify potential signals related to BAVM diffuseness. Candidate proteins were then investigated in BAVM specimens using immunofluorescence and Western blot analysis. Tube formation assays were used to investigate the effects of candidate proteins on the angiogenesis of human umbilical endothelial cells (HUVECs). Finally, Masson, Sirius red staining, and immunofluorescence were used to evaluate the characteristics of extracellular matrix (ECM) in BAVM tissues.ResultsA total of 58 proteins were found to be differentially expressed between diffuse and compact BAVMs via proteomic analysis. TGF-β (transforming growth factor-beta) signaling pathway, ECM–receptor pathway, relaxin signaling pathway, and several other pathways were associated with BAVM diffuseness. The TGF-β signaling pathway is associated with angiogenesis; the role of this pathway in the formation of diffuse BAVMs was investigated, and the decorin (DCN) upregulation played an important role in this process. Immunofluorescence showed that DCN was significantly upregulated within and around the malformed vessels of diffuse BAVMs. Functional assays showed that exogenous DCN could promote the tube formation ability of HUVECs through inhibiting the TGF-β signaling pathway and overproducing ECM. Histological staining demonstrated the overproduction of ECM in diffuse BAVMs.ConclusionTGF-β signaling pathway inhibited by DCN in vascular endothelial cells is related to BAVM diffuseness. The metabolic disorder of ECM caused by DCN upregulation may significantly contribute to the formation of diffuse BAVMs.

Cardioprotective Effect of Decorin in Type 2 Diabetes.

Cardiomyopathy is the leading cause of increased mortality in diabetes. In the present study, we investigated the effects of decorin (DCN) gene therapy on left ventricular function, cardiac inflammation and fibrosis in type 2 diabetes. Type 2 diabetes was induced in male Wistar rats by high fat diet (HFD, 60% of calories as fat) and STZ (20 mg/kg, intraperitoneal). Diabetic rats were divided into (n=6 for each group) the control group, the GFP-treated group and the DCN-treated group, received intravenous injection of saline solution, recombinant adeno-associated viral (rAAV)-GFP, and rAAV-DCN, respectively. We evaluated cardiac inflammation, fibrosis, left ventricular function at 6 months after gene delivery. Results turned out that rAAV-DCN treatment attenuated diabetic cardiomyopathy with improved LV function compared with control animals, which might be related to the reduced cardiac inflammation and fibrosis. These protective effects were associated with TGFβ1 pathway (ERK1/2 and smad-2) and NF-κB pathway, which may due to the decreased activation level of IGF-IR, increased expression of PKC-α and Hsp70. In conclusion, our results show that rAAV-mediated DCN therapy may be beneficial in the treatment of Diabetic Cardiomyopathy.

Decorin inhibits the insulin-like growth factor I signaling in bone marrow mesenchymal stem cells of aged humans.

Aging impairs the IGF-I signaling of bone marrow mesenchymal stem cells (bmMSCs), but the mechanism is unclear. Here, we found that the ability to auto-phosphorylate IGF-I receptor (IGF-IR) in response to IGF-I was decreased in the bmMSCs of aged donors. Conversely, data showed that decorin (DCN) expression was prominently increased in aged bmMSCs, and that under IGF-I treatment, DCN knockdown in serum-starved aged bmMSCs potentiated their mitogenic activity and IGF-IR auto-phosphorylation, whereas DCN overexpression in serum-starved adult bmMSCs decreased both activities. Co-immunoprecipitation assays suggested that IGF-I and DCN bound to IGF-IR in a competitive manner. Online MethPrimer predicted 4 CpG islands (CGIs) in the introns of DCN gene. RT-qPCR and bisulfite sequencing showed that dimethyloxalylglycine, an inhibitor of DNA demethylation, increased DCN mRNA expression and CGI-I methylation in adult bmMSCs, whereas 5-aza-2’-deoxycytidine, a DNA methylation inhibitor, decreased DCN mRNA expression and CGI-I methylation in aged bmMSCs, and ultimately enhanced the proliferation of serum-starved aged bmMSCs under IGF-I stimulation. Thus, IGF-IR could be the prime target of aging in down-regulating the IGF-I signaling of bmMSCs, where DCN could be a critical mediator.

Comparative proteomic analysis of dental cementum from deciduous and permanent teeth.

Dental cementum (DC) is a mineralized tissue covering tooth roots that plays a critical role in dental attachment. Differences in deciduous vs. permanent tooth DC have not been explored. We hypothesized that proteomic analysis of DC matrix would identify compositional differences in deciduous (DecDC) vs. permanent (PermDC) cementum that might reflect physiological or pathological differences, such as root resorption that is physiological in deciduous teeth but can be pathological in the permanent dentition. Protein extracts from deciduous (n=25) and permanent (n=12) teeth were pooled (five pools of DecDC, five teeth each; four pools of PermDC, three teeth each). Samples were denatured, and proteins were extracted, reduced, alkylated, digested, and analyzed by liquid chromatography-mass spectrometry (LC-MS/MS). The beta-binomial statistical test was applied to normalized spectrum counts with 5% significance level to determine differentially expressed proteins. Immunohistochemistry was used to validate selected proteins. A total of 510 proteins were identified: 123 (24.1%) exclusive to DecDC; 128 (25.1%) exclusive to PermDC; 259 (50.8%) commonly expressed in both DecDC and PermDC. Out of 60 differentially expressed proteins, 17 (28.3%) were detected in DecDC, including myeloperoxidase (MPO), whereas 43 (71.7%) were detected in PermDC, including decorin (DCN) and osteocalcin (BGLAP). Overall, Gene Ontology (GO) analysis indicated that all expressed proteins were related to GO biological processes that included localization and response to stress, and the GO molecular function of differentially expressed proteins was enriched in cell adhesion, molecular binding, cytoskeletal protein binding, structural molecular activity, and macromolecular complex binding. Immunohistochemistry confirmed the trends for selected differentially expressed proteins in human teeth. Clear differences were found between the proteomes of DecDC and PermDC. These findings may lead to new insights into developmental differences between DecDC and PermDC, as well as to a better understanding of physiological/pathological events such as root resorption.

Novel potential diagnostic targets revealed by plasma proteomic analysis in chronic thromboembolic pulmonary hypertension

Abstract Background Chronic thromboembolic pulmonary hypertension (CTEPH) is associated with poor outcome if untreated, although it is a curable form of pulmonary hypertension (PH). Successful treatment requires an optimized diagnostic work-up. Purpose The aim of this study was to identify non-invasive biomarkers that might serve as new diagnostic parameters in the multifaceted pathophysiology of CTEPH. Methods The biomarker profile of 64 CTEPH patients who underwent balloon pulmonary angioplasty (BPA) was analyzed prior to and after therapy and compared with that of a healthy control group (CG1, n=25) at baseline. Proteomes were analyzed by semiquantitative screening based on a proximity extension assay of three high-throughput, multiplex immunoassay panels. Serum levels of a subset of biomarkers identified in the screening were additionally measured by immunochemical methods. Results Fifty protein biomarkers were found to differ between CTEPH patients and CG1. Eight biomarkers changed significantly after therapy. The overlap of these two groups revealed six targets that were all upregulated in CTEPH at baseline and modifiable by treatment. In this group of biomarkers, the levels of DCN (decorin), HGF (hepatocyte growth factor), BNP (B-type natriuretic peptide), and PAPP-A (papalysin-1) decreased after therapy, whereas SPON-1 (spondin-1) and MEPE (matrix extracellular phosphoglycoprotein) further increased at follow-up. The differences in these biomarkers in CTEPH as well as the dynamics after therapy were confirmed and quantified in enzyme-linked immunosorbent assays. Conclusions This study identified 6 biomarkers that might serve as new diagnostic parameters or constitute new therapeutic targets in CTEPH. Further prospective studies will be necessary to determine the specific pathophysiological role of each marker. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): SFB 1213 area CP01