Healthy Cartilage Samples Research Articles

HECTD1-Mediated Ubiquitination and Degradation of Rubicon Regulates Autophagy and Osteoarthritis Pathogenesis.

Osteoarthritis (OA) is one of the most common degenerative joint diseases and is associated with autophagy suppression. However, the molecular mechanism of autophagy regulation in the context of OA is not fully understood. In this study, we sought to determine the role that HECTD1 plays in the pathogenesis of OA. We used RNA sequencing analysis to explore the differential expression of E3 ubiquitin ligase genes in healthy human cartilage and human cartilage affected by OA. Using surgery- and aging-induced OA mouse models, we comprehensively analyzed the function of the screened gene Hectd1 in the development of OA; furthermore, we dissected the mechanism by which HECTD1 regulates autophagy and OA progression using a combination of molecular biologic, cell biologic, and biochemical approaches. HECTD1 was significantly down-regulated in human OA cartilage samples compared to healthy cartilage samples. Overexpression of HECTD1 in mouse joints alleviated OA pathogenesis, whereas conditional depletion of Hectd1 in cartilage samples aggravated surgery- and aging-induced OA pathogenesis. Mechanistically, HECTD1 bound to Rubicon and ubiquitinated Rubicon at lysine residue 534, which targets Rubicon for proteasomal degradation. More importantly, HECTD1-mediated Rubicon degradation regulated chondrocyte autophagy, leading to mitigation of stress-induced chondrocyte death and the subsequent progression of OA. HECTD1 plays a crucial role in the pathogenesis of OA, in that HECTD1 regulates chondrocyte autophagy by ubiquitinating and targeting Rubicon for proteasomal degradation.

Males and Females Have Distinct Molecular Events in the Articular Cartilage during Knee Osteoarthritis.

Osteoarthritis (OA) is a major public health challenge that imposes a remarkable burden on the affected individuals and the healthcare system. Based on the clinical observation, males and females have different prevalence rates and severity levels of OA. Thus, sex-based differences may play essential roles in OA’s prognosis and treatment outcomes. To date, the comprehensive understanding of the relationship between sex and OA is still largely lacking. In the current study, we analyzed a published transcriptome dataset of knee articular cartilage (GSE114007) from 18 healthy (five females, 13 males) and 20 OA (11 females, nine males) donors to provide a slight insight into this important but complex issue. First, comparing female healthy cartilage samples with those of males revealed 36 differential expression genes (DEGs), indicating the fundamental sex-related differences at the molecular level. Meanwhile, 923 DEGs were distinguished between OA and healthy female cartilage, which can be enriched to 15 Reactome pathways. On the other hand, when comparing OA and healthy male cartilage, there are only 419 DEGs were identified, and only six pathways were enriched against the Reactome database. The different signaling response to OA in the male and female cartilage was further enforced by recognizing 50 genes with significantly different OA-responsive expression fold changes in males and females. Particularly, 14 Reactome pathways, such as “Extracellular matrix organization”, “Collagen biosynthesis and modifying enzymes”, “Dissolution of fibrin clot”, and “Platelet Aggregation (Plug formation)”, can be noted from these 50 sex-dependent OA-responsive genes. Overall, the current study explores the Sex as a Biological Variable (SABV) at the transcriptomic level in the knee articular cartilage in both healthy status and OA event, which could help predict the differential OA prognosis and treatment outcome of males and female patients.

Intra-operative Raman spectroscopy and ex vivo Raman mapping for assessment of cartilage degradation

The development of a label-free, non-destructive and safe analytical method such as Raman spectroscopy for assessing cartilage degradation is highly desirable. Compared to non-optical imaging modalities, Raman mapping offers a more sensitive means of directly assessing the chemical composition of cartilage in three-dimensional space and the potential to monitor cartilage degeneration to inform intervention and treatment strategies. Herein, we report the application of Raman spectroscopic methods ex vivo and at arthroscopy to identify molecular alterations in cartilage specimens containing minor focal lesions characteristic of the early disease phase. Our initial ex vivo analysis, obtained by single-point Raman spectroscopy of cartilage samples, supports previous findings based on S-O stretching vibration bands associated with sulphated glycosaminoglycans (sGAGs). We extended the analyses to the high-wavenumber region where we observed that vibrational bands assigned to C-H and O-H stretching modes discriminated early cartilage alterations from healthy cartilage samples. Furthermore, we performed a proof-of-concept in-clinic study using a custom-built optical probe to acquire Raman spectral measurements for the first time in patients undergoing arthroscopy of knee joints. Spectra were obtained with adequate signal-to-noise ratios that similarly discriminated between lesion and adjacent cartilage sites and identified reductions in sGAGs in apparently healthy cartilage. Building on this, we present initial results from Raman mapping to spatially resolve the molecular constituents of cartilage through its depth and across a lesion. Mapping revealed a non-uniform and reduced sGAG distribution within the lesion and peripheral cartilage that was otherwise visually normal, similar to the in-clinic observations, showing that the degradative influence of the lesion extended beyond its border. This was accompanied by a decreased fluorescence signal intensity, which suggests that fluorescence may provide valuable information as an adjunct to the Raman signal in discriminating normal and degenerating cartilage. This work demonstrates the value of Raman mapping over single-point Raman measurements for the analysis of the anisotropy of articular cartilage and highlights the potential of the technology for in vivo articular joint arthroscopy applications.

AB0067 CHONDROCALCINOSIS IS ASSOCIATED WITH A SPECIFIC EFFECT ON THE CHONDROCYTE PHENOTYPE THAT MARKEDLY DIFFERS FROM OA

Background:Calcification of cartilage with BCP crystals is a common finding during osteoarthritis (OA) and is directly linked to the severity of the disease and hypertrophic differentiation of chondrocytes. Chondrocalcinosis (CC) is associated with CPPD crystal formation. There is only little knowledge about the effect of CPPD crystals on chondrocytes.Objectives:The aim of this study was to investigate the chondrocyte phenotype in CC cartilage and the effect of CPPD crystals on chondrocytes.Methods:Cartilage samples of patients with CC were used and compared with samples of severe OA patients without chondrocalcinosis and healthy cartilage samples served as control. Radiological presence of chondrocalcinosis was evaluated using standard X-ray pictures, as well as macroscopically inspection. The cartilage samples were stained using von Kossa/Safranin-orange staining. These stainings were used for OA severity scoring using the Chambers-Score. FTIR analyses was performed to distinguish CPPD and BCP crystals in cartilage. Chondrocyte differentiation markers were evaluated using Collagen 2 and X, as well as Sox9 and aggrecan as markers for chondrocyte hypertrophic differentiation in immunohistochemistry and qRT-PCR. TUNEL staining was performed to investigate cell death. In vivo results were validated using qRT-PCR for the expression of the respective genes after stimulation of C28 chondrocytes with CPPD and BCP crystals.Results:Radiologically detectable cartilage calcifications were evident in chondrocalcinosis patients, but absent in OA patients without CC. CPPD crystals were detected on the cartilage surface, whereas BCP crystals were detected in the pericellular matrix of hypertrophic chondrocytes. CC cartilage exhibited an increased collagen X expression compared to healthy cartilage, as well as to severe OA cartilage containing BCP calcification. Interestingly, aggrecan and collagen 2 were not reduced in CC cartilage, but markedly reduced in OA cartilage. TUNEL positive cells were significantly increased in CPPD cartilage compared to OA cartilage, although the histological OA severity was lower. qRT-PCR indicated no relevant influence of CPPD crystals on hypertrophic marker genes, whereas BCP crystals significantly induced hypertrophic differentiation.Conclusion:BCP and CPPD crystals seem to trigger differential effects on the chondrocyte phenotype. BCP crystals induce hypertrophic differentiation, which is not induced by CPPD crystals.Acknowledgments:The project was funded by theDeutsche Rheumastifung by the sponsor Dr. Sigrid Schuler.Disclosure of Interests:Franziska Meyer: None declared, Miriam Bollmann: None declared, Uwe Kornak: None declared, Jessica Bertrand Grant/research support from: Pfizer, Speakers bureau: Pfizer

Altered spontaneous calcium signaling of in situ chondrocytes in human osteoarthritic cartilage

Intracellular calcium ([Ca2+]i) signaling is an essential universal secondary messenger in articular chondrocytes. However, little is known about its spatiotemporal features in the context of osteoarthritis (OA). Herein, by examining the cartilage samples collected from patients undergoing knee arthroscopic surgery, we investigated the spatiotemporal features of spontaneous [Ca2+]i signaling in in situ chondrocytes at different OA stages. Our data showed zonal dependent spontaneous [Ca2+]i signaling in healthy cartilage samples under 4 mM calcium environment. This signal was significantly attenuated in healthy cartilage samples but increased in early-degenerated cartilage when cultured in 0 mM calcium environment. No significant difference was found in [Ca2+]i intensity oscillation in chondrocytes located in middle zones among ICRS 1–3 samples under both 4 and 0 mM calcium environments. However, the correlation was found in deep zone chondrocytes incubated in 4 mM calcium environment. In addition, increased protein abundance of Cav3.3 T-type voltage dependent calcium channel and Nfatc2 activity were observed in early-degenerated cartilage samples. The present study exhibited OA severity dependent spatiotemporal features of spontaneous [Ca2+]i oscillations of in situ chondrocytes, which might reflect the zonal specific role of chondrocytes during OA progression and provide new insight in articular cartilage degradation during OA progression.

Genome-wide DNA methylation study of hip and knee cartilage reveals embryonic organ and ‎skeletal system morphogenesis as major pathways involved in osteoarthritis

Purpose: The objectives of this study were to describe the genome wide DNA methylation changes in hip ‎and knee osteoarthritis (OA) and to identify novel genes and pathways involved in OA by ‎comparing the DNA methylome of the hip and knee osteoarthritic cartilage with those of healthy ‎individuals. ‎ Methods: OA cartilage samples were collected from the tibial plateau and femoral head articular surfaces of ‎patients with severe OA undergoing total hip/knee replacement surgery. Healthy cartilage ‎samples were obtained from individuals with femoral neck fractures, but no OA. Chondrocyte ‎DNA methylation was assayed by Illumina Infinium HumanMethylation450 BeadChip array, ‎allowing for the analysis of >480,000 CpG sites. A sub-analysis was also done to compare hip ‎and knee OA. An independent sample T-test was conducted for each CpG site and those sites ‎with at least 10% methylation difference and a P-value of <0.0001 were selected as differentially ‎methylated regions (DMR). DAVID v6.7 was used for the functional annotation clustering of ‎the DMR genes. ‎ Results: The study population comprises 5 patients with hip OA, 6 patients with knee OA and 7 hip ‎samples from healthy individuals. The comparisons of hip, knee and combined hip/knee OA ‎patients with controls resulted in 26, 72, and 103 DMRs, respectively. The comparison between ‎hip and knee OA revealed 67 DMRs. The overall number of the sites after considering the ‎overlaps was 239, among which 151 sites were annotated to 145 genes. 28 of these genes were ‎reported in previous methylation studies of OA. The functional annotation clustering of the ‎identified genes revealed clusters significantly enriched in skeletal system morphogenesis ‎‎(bonferroni corrected p=8.4e-9) and development (bonferroni corrected p=9.0e-6), embryonic ‎organ (bonferroni corrected p=1.4e-5) and skeletal system morphogenesis (bonferroni corrected ‎p=9.9e-7), as well as Homeobox genes (bonferroni corrected p=3.1e-4). A sub-analysis after the ‎removal of the genes differentially methylated between hip and knee OA highlighted the same ‎pathways excluding Homeobox genes.‎ Conclusions: We demonstrated a number of CpG sites and genes across the genome differentially methylated ‎among OA patients and controls, a remarkable portion of which seem to be involved in potential ‎etiologic mechanisms of OA. Genes involved in skeletal developmental pathways and embryonic ‎organ morphogenesis may be a potential area of concentration for future osteoarthritis ‎genetic/epigenetic research. ‎

SMAD3 is up-regulated in human osteoarthritic cartilage independent of promoter dna ‎methylation

Purpose: We previously reported that SMAD3 was associated with the total burden of radiographic ‎osteoarthritis (OA). SMAD3 is a mediator of TGF-β signalling pathway that is known to be ‎involved in the cartilage maintenance and repair. SMAD3 knocked-out mice are deficient for ‎Collagen and Aggrecan, and develop OA-like features in the joints. The aim of the present study ‎was to investigate the SMAD3 gene expression in osteoarthritic and healthy human cartilage and ‎to examine whether the gene expression is regulated by the promoter DNA methylation.‎ Methods: Osteoarthritic cartilage samples were collected from patients who underwent total hip/knee joint ‎replacement surgery due to primary OA. Healthy cartilage samples were obtained from patients ‎with hip fracture without any evidence of hip OA. DNA and RNA were extracted from the ‎cartilage samples using Qiagen’s AllPrep DNA/RNA Mini Kit. Quantitative PCR experiment ‎was done using ABI-7500 real time PCR system to measure RNA expression after cDNA ‎synthesis by ThermoScript cDNA synthesis kit. DNA Methylation was assayed by Sequonom’s ‎EpiTYPER after DNA bisulphate conversion using Qiagen’s EpiTect Bisulfite Kit. Mann-‎Whitney test was utilized to examine the association between OA cases and controls for SMAD3 ‎expression and its promoter DNA methylation levels. Spearman's rank correlation analysis was ‎performed to examine the association between the promoter methylation and gene expression. A ‎P-value less than 0.05 was considered as significant. ‎ Results: A total of 49 OA patients (38 hip OA and 11 knee OA) and 51 controls were included in the ‎study. Mean age was 64.4 in OA patients and 78.7 in controls. Four CpG sites, located ∼450bp ‎upstream of the first SMAD3 exon, were assayed, and we found no difference in methylation ‎between OA cases and controls after adjusting for age. The expression experiment was ‎performed for 38 patients with OA (32 hips and 6 knees) and 28 healthy controls, and we found ‎that the SMAD3 gene was expressed in OA cartilage on average 1.8 times higher than controls ‎‎(p=0.0005). Similar results were obtained when we looked at hip and knee OA separately ‎‎(p=0.01). We found no association between SMAD3 expression and the methylation at the ‎promoter region of the gene. ‎ Conclusions: Our study demonstrated that SMAD3 is up-regulated in OA. This up-regulation, however, cannot ‎be explained by the changes in the promoter DNA methylation. Given that TGF-β/SMAD3 ‎pathway may play a protective role in the cartilage, the up-regulation of SMAD3 is more likely ‎due to the consequence of OA, thus reflecting repairing activity stimulated by the cartilage lesion ‎in OA. ‎

Identification of an extracelular o-n-acetyl-glycosylase responsible for an increased level of O-linked n-acetylglucosamine modified proteins in the cartilage of patients with osteoarthritis

Identification of an extracelular o-n-acetyl-glycosylase responsible for an increased level of O-linked n-acetylglucosamine modified proteins in the cartilage of patients with osteoarthritis