Integration of Transcriptome and MicroRNA Profile Analysis of iMSCs Defines Their Rejuvenated State and Conveys Them into a Novel Resource for Cell Therapy in Osteoarthritis.

Although MSCs grant pronounced potential for cell therapies, several factors, such as their heterogeneity restrict their use. To overcome these limitations, iMSCs (MSCs derived from induced pluripotent stem cells (iPSCs) have attracted attention. Here, we analyzed the transcriptome of MSCs, iPSCs and iMSCs derived from healthy individuals and osteoarthritis (OA) patients and explored miRNA-mRNA interactions during these transitions. We performed RNA-seq and gene expression comparisons and Protein-Protein-Interaction analysis followed by GO enrichment and KEGG pathway analyses. MicroRNAs' (miRNA) expression profile using miRarrays and differentially expressed miRNA's impact on regulating iMSCs gene expression was also explored. Our analyses revealed that iMSCs derivation from iPSCs favors the expression of genes conferring high proliferation, differentiation, and migration properties, all of which contribute to a rejuvenated state of iMSCs compared to primary MSCs. Additionally, our exploration of the involvement of miRNAs in this rejuvenated iMSCs transcriptome concluded in twenty-six miRNAs that, as our analysis showed, are implicated in pluripotency. Notably, the identified here interactions between hsa-let7b/i, hsa-miR-221/222-3p, hsa-miR-302c, hsa-miR-181a, hsa-miR-331 with target genes HMGA2, IGF2BP3, STARD4, and APOL6 could prove to be the necessary tools that will convey iMSCs into the ideal mean for cell therapy in osteoarthritis.

NEAT1-mediated miR-150-5p downregulation regulates b-catenin expression in OA chondrocytes.

We investigated the role of miR-150-5p in osteoarthritic (OA) chondrocytes, as well as the possible regulatory role of long non-coding RNAs (lncRNAs) in miR-150-5p expression. TargetScan, StarBase, DIANA-LncBase, and Open Targets databases were used to predict miR-150-5p target genes, lncRNAs/miR-150-5p interactions, and OA-related genes. Protein-protein interaction (PPI) network was constructed using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING). Gene ontology (GO) and pathway analysis were performed using Enrichr database. A publicly available RNA-seq dataset was retrieved to identify differentially expressed lncRNAs in damaged vs intact cartilage. We re-analyzed the retrieved RNA-seq data and revealed 177 differentially expressed lncRNAs in damage vs intact cartilage, including Nuclear Paraspeckle Assembly Transcript 1(NEAT1). MiR-150-5p, NEAT1, b-catenin, matrix metallopeptidase 13 (MMP-13), and ADAM metallopeptidase with thrombospondin type 1 motif 5 (ADAMTS-5) expressions were assessed by reverse transcription-quantitative PCR (RT-qPCR) and western blot assay. Knockout and transfection experiments were conducted to investigate the role of NEAT1/miR-150-5p/b-catenin in cartilage degradation. Bioinformatics analysis revealed that b-catenin was an OA-related miR-150-5p target. MiR-150-5p overexpression in OA chondrocytes resulted in decreased expression of b-catenin, as well as MMP-13 and ADAMTS-5, both being Wnt/b-catenin downstream target genes. NEAT1/miR-150-5p interaction was predicted by bioinformatics analysis, while NEAT1 knockout led to increased expression of miR-150-5p in OA chondrocytes. Moreover, inhibition of miR-150-5p reversed the repressive effects of NEAT1 silencing in b-catenin expression in OA chondrocytes. Our results support a possible catabolic role of NEAT1/miR-150-5p interaction in OA progression by regulating b-catenin expression.

DNA methylation regulates Sirtuin 1 expression in osteoarthritic chondrocytes.

PURPOSE: Sirtuin 1 (SIRT1) comprises a major anti-aging longevity factor with multiple protective effects on chondrocyte homeostasis. Previous studies have reported that downregulation of SIRT1 is linked to osteoarthritis (OA) progression. In this study, we aimed to investigate the role of DNA methylation on SIRT1 expression regulation and deacetylase activity in human OA chondrocytes. MATERIALS AND METHODS: Methylation status of SIRT1 promoter was analyzed in normal and OA chondrocytes using bisulfite sequencing analysis. CCAAT/enhancer binding protein alpha (C/EBPα) binding to SIRT1 promoter was assessed by chromatin immunoprecipitation (ChIP) assay. Subsequently, C/EBPα's interaction with SIRT1 promoter and SIRT1 expression levels were evaluated after treatment of OA chondrocytes with 5-Aza-2'-Deoxycytidine (5-AzadC). Acetylation and nuclear levels of nuclear factor kappa-B p65 subunit (NF-κΒp65) and expression levels of selected OA-related inflammatory mediators, interleukin 1ß (IL-1ß) and IL-6 and catabolic genes (metalloproteinase-1 (MMP-1) and MMP-9) were evaluated in 5-AzadC-treated OA chondrocytes with or without subsequent transfection with siRNA against SIRT1. RESULTS: Hypermethylation of specific CpG dinucleotides on SIRT1 promoter was associated with downregulation of SIRT1 expression in OA chondrocytes. Moreover, we found decreased binding affinity of C/EBPα on the hypermethylated SIRT1 promoter. 5-AzadC treatment restored C/EBPα's transcriptional activity inducing SIRT1 upregulation in OA chondrocytes. Deacetylation of NF-κΒp65 in 5-AzadC-treated OA chondrocytes was prevented by siSIRT1 transfection. Similarly, 5-AzadC-treated OA chondrocytes exhibited decreased expression of IL-1ß, IL-6, MMP-1 and MMP-9 which was reversed following 5-AzadC/siSIRT1 treatment. CONCLUSIONS: Our results suggest the impact of DNA methylation on SIRT1 suppression in OA chondrocytes contributing to OA pathogenesis.

Leptin-depended NLRP3 inflammasome activation in osteoarthritic chondrocytes is mediated by ROS.

Leptin and ROS are implicated in the regulation of inflammatory pathways including NLRP3-inflammasome. We investigated the functional link between leptin, ROS and NLRP3-inflammasome formation/activation in osteoarthritis (OA), an age-related disease. We found that inflammasome components' (NLRP3, ASC, Caspase-1 and cleaved Caspase-1) protein expression were increased in OA cartilage biopsies and chondrocytes compared to healthy cartilage and chondrocytes. Immunofluorescence showed increased co-localization of NLRP3/ASC and NLRP3/Caspase-1, ASC-specks formation and ROS levels in OA compared to normal chondrocytes. NOX4 mRNA expression and IL-1ß/IL-18 secretion levels were also elevated in OA chondrocytes. Furthermore, NLRP3-siRNA in OA chondrocytes revealed significant MMP-9/MMP-13 downregulation. To elucidate leptin/ROS/NLRP3-inflammasome interactions, OA chondrocytes were treated with ROS-inhibitor NAC, NOXs-inhibitor DPI, NOX4-inhibitor GLX351322 and leptin-siRNA, while normal chondrocytes were incubated with leptin with or without DPI or GLX351322. We observed attenuated ROS levels and NLRP3-inflammasome formation/activation in NAC-, DPI- or GLX351322-treated OA chondrocytes, while the same effect was shown after transfection with leptin-siRNA. Furthermore, incubation of normal chondrocytes with leptin enhanced ROS production and inflammasome formation/activation, while pretreatment with DPI or GLX351322 abolished leptin's stimulatory effects confirming leptin-NOX4-ROS-inflammasome regulatory axis. Overall, our findings provide novel evidence indicating that leptin-induced NLRP3-inflammasome formation/activation in OA chondrocytes is mediated by NOX4-dependent ROS production.

Chondrocyte protein co-synthesis network analysis links ECM mechanosensing to metabolic adaptation in osteoarthritis.

BACKGROUND: Knee osteoarthritis (OA) is one of the most common structural OA disorders globally. Incomplete understanding of the fundamental biological aspects of osteoarthritis underlies the current lack of effective treatment or disease modifying drugs. RESEARCH DESIGN AND METHODS: We implemented a systems approach by making use of the statistical network concepts in Weighted Gene Co-expression Analysis to reconstruct the organization of the core proteome network in chondrocytes obtained from OA patients and healthy individuals. Protein modules reflect groups of tightly co-ordinated changes in protein abundance across healthy and OA chondrocytes. RESULTS: The unbiased systems analysis identified extracellular matrix (ECM) mechanosensing and glycolysis as two modules that are most highly correlated with ΟΑ. The ECM module was enriched in the OA genetic risk factors tenascin-C (TNC) and collagen 11A1 (COL11A1), as well as in cartilage oligomeric matrix protein (COMP), a biomarker associated with cartilage integrity. Mapping proteins that are unique to OA or healthy chondrocytes onto the core interactome, which connects microenvironment sensing and regulation of glycolysis, identified differences in metabolic and anti-inflammatory adaptation. CONCLUSION: The interconnection between cartilage ECM remodeling and metabolism is indicative of the dynamic chondrocyte states and their significance in osteoarthritis.

Oxidative Stress Response Is Mediated by Overexpression and Spatiotemporal Regulation of Caveolin-1.

Oxidative stress (OS) has been linked to the aetiology of many diseases including osteoarthritis (OA). Recent studies have shown that caveolin-1-a structural protein of plasma membrane's caveolae-is upregulated in response to OS. Here, we explore the function of caveolin-1 in chondrocytes derived from healthy individuals (control) and OA patients that were subjected to exogenous OS. We showed that caveolin-1 was upregulated in response to acute OS in the control, but not in OA chondrocytes. Moreover, OS-induced DNA damage analysis revealed that control cells started repairing the DNA lesions 6 h post-oxidative treatment, while OA cells seemed unable to restore these damages. Importantly, in the control cells, we observed a translocation of caveolin-1 from the membrane/cytoplasm in and out of the nucleus, which coincided with the appearance and restoration of DNA lesions. When caveolin-1 was prevented from translocating to the nucleus, the control cells were unable to repair DNA damage. In OA cells, no such translocation of caveolin-1 was observed, which could account for their inability to repair DNA damage. Taken together, these results provide novel insights considering the role of caveolin-1 in response to OS-induced DNA damage while revealing its implication in the pathophysiology of OA.

Impact of miR-SNP rs2910164 on miR-146a expression in osteoarthritic chondrocytes.

PURPOSE: MiR-146a acts as a negative inflammatory mediator in different diseases and has been implicated in osteoarthritis (OA) pathogenesis. In our study, we investigated the association between miR-SNP rs2910164 and OA susceptibility and its role on the expression of miR-146a, inflammatory and catabolic mediators in osteoarthritic chondrocytes. MATERIALS AND METHODS: Genetic association analysis was performed in 1688 knee OA patients and healthy individuals of Greek origin. Genomic DNA was extracted from blood and genotyped for rs2910164 (G > C) using Restriction-Fragment Length Polymorphism (RFLP). Total RNA was extracted from chondrocytes of 18 OA patients and miR-146a, IL-1 Receptor-Associated Kinase 1 (IRAK-1), TNF Receptor-Associated Factor 6 (TRAF-6), A Disintegrin and Metalloproteinase with Thrombospondin Motifs 5 (ADAMTS-5), Matrix Metalloproteinase-13 (MMP-13), Interleukin-6 (IL-6), Interleukin-1 Beta (IL-1ß) and Tumor Necrosis Factor-Alpha (TNF-α) expression was evaluated using quantitative Real-Time PCR (qRT-PCR). RESULTS: OA patients carrying rs2910164-GC and CC genotypes did not have an increased risk for OA development compared to GG genotype carriers. MiR-146a expression in OA chondrocytes was significantly lower in patients with rs2910164-GC genotype than in the GG carriers. OA patients carrying the rs2910164-GC genotype in their chondrocytes exhibited increased IRAK-1, TRAF-6, MMP-13, IL-1ß and IL-6 expression levels compared with rs2910164-GG carriers. CONCLUSION: We demonstrate, for the first time, that miR-SNP rs2910164 in miR-146a gene is associated with reduced miR-146a and increased inflammatory and catabolic mediators' expression in OA chondrocytes. Our data imply that genetic variations in miRNAs linked to OA pathogenesis may regulate their expression levels, suggesting new therapeutic strategies for patients with cartilage diseases.

The synergistic function of miR-140-5p and miR-146a on TLR4-mediated cytokine secretion in osteoarthritic chondrocytes.

ΜiR-140-5p and miR-146a regulate inflammatory pathways including TLR4/NF-κB signaling and have been found to be involved in OA pathogenesis. In this study, we investigated the effect of the synergistic function of miR-140-5p and miR-146a on inflammation mediated by TLR4 in ΟΑ chondrocytes. Bioinformatics analysis revealed that TLR4 was the only common OA-related target gene of miR-140-5p and miR-146a, located in the sub-network with the highest MCODE score; it also showed that the target genes of miR-140-5p and miR-146a which located in MCODE sub-networks were enriched in OA-related biological processes and pathways. Overexpression of miR-140-5p or miR-146a and combined miR-140-5p/miR-146a overexpression in OA chondrocytes demonstrated that combined treatment had the strongest negative effect on TLR4 expression. Moreover, simultaneous overexpression of miR-140-5p and miR-146a resulted in the highest reduction of NF-κΒ phosphorylation levels, as well as IL-1b, IL-6 and TNFa expression levels in OA chondrocytes as compared to the reductions observed when either miR-140-5p or miR-146a was overexpressed. Our results, therefore, demonstrate for the first time, that the synergistic function of miR-140-5p and miR-146a have a strong protective effect against inflammatory mediators' production in OA chondrocytes through targeting the TLR4/NF-κB signaling.

DNA methylation regulates miR-140-5p and miR-146a expression in osteoarthritis.

AIMS: Previous studies have demonstrated that transcriptional silencing of miRNAs due to DNA hypermethylation is associated with different pathologies. It has also been reported that abnormal expression of miR-140-5p and miR-146a is linked to osteoarthritis (OA) progression. In this study, we investigated the role of DNA methylation on miR-140-5p and miR-146a expression in OA. MAIN METHODS: miR-140-5p and miR-146a expression was investigated by qRT-PCR. The methylation status of miR-140 and miR-146a regulatory regions was analyzed using qMSP and bisulfite sequencing analysis. SMAD-3 and NF-kB binding to miR-140 and miR-146a regulatory regions was assessed by ChIP assay and knockdown experiments. OA-related genes' expression was evaluated in 5-AzadC, miRNAs inhibitor and 5-AzadC/miRNAs inhibitor-treated cells. KEY FINDINGS: Hypermethylation of specific CpG sites in miR-140 and miR-146a regulatory regions was associated with downregulation of miR-140-5p and miR-146a in OA chondrocytes and synoviocytes, respectively. 5-AzadC-induced miR-140-5p and miR-146a upregulation was observed in OA chondrocytes and synoviocytes. Moreover, we found decreased binding affinity of SMAD-3 and NF-kB transcription factors on the hypermethylated miR-140-5p and miR-146a regulatory regions, respectively. Downregulation of MMP-13 and ADAMTS-5 in 5-AzadC-treated OA chondrocytes was prevented by miR-140-5p inhibitor transfection. Similarly, 5-AzadC-treated OA synoviocytes showed decreased expression of IRAK-1, IL1Β and IL-6, which was reversed following 5-AzadC-/miR-146a inhibitor treatment. SIGNIFICANCE: Our results strongly suggest the impact of DNA methylation on miR-140-5p and miR-146a suppression in OA chondrocytes and synoviocytes, contributing to OA pathogenesis.

Understanding the role of chondrocytes in osteoarthritis: utilizing proteomics.

INTRODUCTION: Proteomic analyses have been acknowledged to carry a significant prospective in elucidating the pathogenesis of several diseases, including osteoarthritis (OA). But it has not been an easy road: major technical issues, mainly derived from the complex and rigid nature of the cartilage tissue, had to be faced; an obstacle that led to the development of different approaches. Areas covered: In this review, we categorized the proteomic studies undertaken (proteomic analyses of the cartilage, cartilage explants, cultured chondrocytes, and chondrocytes' secretome) as part of the different strategies developed in order to overcome tissue and disease-specific challenges. Essentially these approaches aimed at identifying differences in the proteome of healthy vs diseased tissue. Our aim was to point out the novel players that have emerged from these analyses and highlight the associated mechanism(s) suggested to play a role in the pathogenesis of OA. Expert commentary: The identified factors indicate the implication of age-associated mechanisms, such as metabolic deregulation, inflammation, and redox imbalance, in OA onset and/or progression. Taken together these results outline the causal network of the disease and place chondrocytes' senescence at the center of the emerging aetiopathological atlas.

Risk Factor Assessment in a Greek Cohort of Patients With Large Abdominal Aortic Aneurysms.

Environmental and genetic risk factors contribute to the etiology of abdominal aortic aneurysms (AAAs). Matrix metalloproteinases (MMPs) have been associated with the pathophysiology of AAAs. A prospective, nonrandomized case-control study was undertaken to investigate the risk factors for large AAAs (≥5.5 cm) among 175 male Greek AAA patients and to compare the results with a cohort of 166 male controls free from any aortic dilatation, as confirmed by ultrasonography from an existing AAA screening program in the same region. We also assessed the potential association between 2 functional single nucleotide polymorphisms in the genes MMP9 (-1561C/T; rs3918242) and MMP13 (-77A/G; rs2252070), and the presence of large AAAs. Multiple logistic regression analysis revealed AAA family history ( P = .028), hypercholesterolemia ( P < .001), and current smoking ( P < .001) as AAA risk factors. Statistical difference was reached in genotype ( P = .047) and allele ( P = .037) frequencies for rs2252070, but the results did not remain significant after correction for multiple testing. No significant differences in genotype or allele frequencies for rs3918242 were detected. In summary, AAA family history, hypercholesterolemia, and current smoking were found to be risk factors for large AAAs.

Evidence of deregulated cholesterol efflux in abdominal aortic aneurysm.

Previous studies indicated that lipids may be associated with abdominal aortic aneurysm (AAA); however the molecular mechanism involved is unclear. Our study aimed to investigate the expression pattern of cholesterol efflux related proteins in AAA. Liver X receptors (LXRα and LXRß), ATP-binding-cassette transporter A1 (ABCA1), Apolipoprotein AI (ApoAI), smooth muscle α-actin (α-SM) and vimentin expression levels were evaluated in human AAA, atherosclerotic (ATH) and normal abdominal aortic tissues. We found significant differences in LXRα, LXRß and ABCA1 mRNA expression levels between AAA, ATH and normal whole aortic tissues and also within the AAA, ATH and normal "intima-media" layers. Specifically, LXRα, LXRß and ABCA1 mRNA levels were decreased in AAA compared to ATH-whole tissues, as well as in AAA "intima-media" compared to ATH and normal "intima-media" layers. Moreover, immunohistochemical evaluation revealed that LXRα and ABCA1 immunoreactivities (IR) were reduced in the AAA media compared to the normal and ATH media layers and that they were also reduced in the intima layer of AAA and ATH tissues, whereas ApoAI-IR was increased in the AAA and ATH aortic walls compared to normal pointing to possible deregulation of the cholesterol efflux mechanism in AAA. Furthermore, double staining for vimentin and α-SM showed vimentin expression in the intima and inner media layer of AAA with sparse vimentin positive SMCs designating possible SMCs phenotype switch from contractile to synthetic form. In addition, histochemical analysis showed excessive lipid accumulation in the AAA wall, while co-staining using Oil Red O with α-SM or CD68 revealed lipid accumulation in SMCs and macrophages, respectively. Our study provides novel evidence for impaired cholesterol efflux in AAA associated with lipid accumulation in SMCs and macrophages, as well as switch of SMCs phenotype from contractile to synthetic form.