Cartilage debris and osteoarthritis risk factors influence gene expression in the synovium in end stage osteoarthritis.

BACKGROUND: Gene expression in healthy synovium remains poorly characterised. Thus, synovial functional activity changes associated with osteoarthritis (OA) are difficult to define. This study sought to identify differentially expressed genes (DEG) of end-stage OA and assess the influence of OA risk factors on these DEG. METHODS: Anonymised patient clinical data and x-ray images were analysed. Osteoarthritic and non-osteoarthritic patients with soft tissue or traumatic knee injuries were matched for body mass index (BMI) and sex. Tissue samples were partitioned for immunocytochemistry (IHC) and microarray analysis. Multiple bioinformatics applications were utilised to determine changes in functional and canonical pathway activation. RESULTS: Age, disease-modifying injections and hypertension were confounding factors between patient groups. Inflammation was present in all tissues. Cartilage debris and inflammatory aggregates were noted in many osteoarthritic patient tissues. IHC and expression analyses revealed upregulation of synoviolin 1 (SYVN1) in osteoarthritic synovium. Significant differential expression was noted in 2084 genes. Osteoarthritic synovium displayed a significant upregulation of 95% of DEG coding for proteins, relative to non-osteoarthritic synovium tissues. Unfolded protein response (UPR)-related genes were upregulated in osteoarthritic synovium; gene expression of molecules within many canonical pathways including protein ubiquitination and UPR pathways was modified by BMI and sex. CONCLUSIONS: The synovium of all three pathologies exhibited elements of an inflammatory response. Cartilage debris, age, BMI and sex influence DEG of osteoarthritic synovium. UPR pathway is the top deregulated canonical pathway identified in osteoarthritic synovium regardless of BMI and sex, while typical OA-associated inflammatory and matrix gene responses were minimal.

Histopathological dataset and demographic details of synovial tissues from patients with end-stage osteoarthritis, soft tissue and traumatic injuries of the knee.

Degradation of articular cartilage is the defining feature of end-stage osteoarthritis (OA) with osteophytes, subchondral sclerosis, malalignment and joint space narrowing being additional indicators of advanced disease. Obesity, older age and female gender are OA risk factors. Differing degrees of synovitis are observed in OA, soft tissue and traumatic injuries of the knee. The synovium is also subject to systemic, enhanced lipids and inflammatory mediators characteristic of obesity. Synovial cellular composition changes specific to OA and associated with its handling of cartilage debris are unclear. Triangulation of data from three knee pathologies was used to highlight findings pertaining to OA compared to non-OA. OA patient data was compared to non-OA from knee ligament and tibial frature patients at surgery. Knee pathology, gender and BMI informed patient identification. Once consented, patient inclusion and characterisation utilised data from clinical assessments, blood tests, function scores, and radiological imaging, scores and intraoperative assessment. Intra-operative synovial tissues from the same site and processed identically underpins in-depth analyses and comparisons of histopathological images from these different knee pathologies. This supports the identification of distinct changes in the cellular composition of the knee synovium characteristic of OA. This data underpins a better understanding of OA pathogenesis and disease progression vital for the design of targeted therapeutics. The tissue and cell data include detailed results from the semi-quantitative synovitis score established by Krenn and observational data for morphological features such as cartilage debris inclusion, inflammatory cells aggregate and infiltration. This histopathological data is presented in the context of detailed clinical and functional information. This data and the holistic study design can be used as a foundation for the multifactorial collection and analysis of clinical data from OA patients, OA severity measures, tissue immuno-histology and synovial inflammation analysis to underpin the details and comparisons needed in further studies into OA and its treatment globally.

Naturally-derived endoplasmic reticulum stress inhibitors for osteoarthritis?

Osteoarthritis (OA) is a chronic degenerative disease that affects the whole synovial joint. OA causes severe pain and disability that significantly affects the livelihood of an individual and incurs a huge socioeconomic burden. Current management strategies are limited to supporting functional improvement with physiotherapy and pain reduction as there are no drugs available that can reverse the progression of OA with only joint replacement surgery for late stage OA. OA is associated with advancing age and obesity, both of which compromise the functions of key endoplasmic reticulum (ER) molecular chaperones leading to improper protein folding and ER stress. Failure to restore protein homeostasis leads to increased cellular stress and eventually apoptotic cell death. Cartilage is avascular and is dependent on its constituent cells, chondrocytes, for extracellular matrix maintenance. Chondrocytes have limited proliferative capacity and their apoptosis eventually leads to extracellular matrix loss and cartilage degeneration. Recent studies on attenuating ER stress and chondrocytes apoptosis offer a credible strategy for reducing OA progression. The established roles of ER stress responses in OA have paved the way for targeted drug discovery studies aiming to mitigate ER stress and OA progression. In this review, in vitro, pre-clinical and clinical evidence of naturally-derived ER stress inhibitors for OA, the prospect and challenges in bringing these compounds to clinics are discussed.

Modulation of the mechanical responses of synovial fibroblasts by osteoarthritis-associated inflammatory stressors.

OBJECTIVES: To compare mechanobiological response of synovial fibroblasts (SFb) from OA patient cohorts under mechanical load and inflammatory stressors for better understanding of SFb homeostatic functions. METHODS: Primary SFb isolated from knee synovium of OA obese (OA-ob:SFb), OA-pre-obese (OA-Pob:SFb), non-OA arthroscopic (scope:SFb), and non-OA arthroscopic with cartilage damage (scope-CD:SFb) were exposed to OA-conditioned media (OACM), derived from OA obese (OA-ob:CM), OA-pre-obese (OA-Pob:CM), and mechanical stretch at either 0 %, 6 % or 10 % for 24 h. Differences in the mRNA levels of genes involved in extracellular matrix production, inflammation and secretory activity were measured. RESULTS: Despite the significant BMI differences between the OA-ob and OA-Pob groups, OA-Pob has more patients with underlying dyslipidaemia, and low-grade synovitis with higher levels of secreted proteins, CXCL8, COL4A1, CCL4, SPARC and FGF2 in OA-Pob:CM. All primary SFb exhibited anti-proliferative activity with both OA-CM. Mechanical stretch stimulated lubricin production in scope:SFb, higher TGFß1 and COL1A1 expressions in scope-CD:SFb. OA-Pob:CM stimulated greater detrimental effects than the OA-ob:CM, with higher pro-inflammatory cytokines, IL1ß, IL6, COX2 and proteases such as aggrecanases, ADAMTS4 and ADAMTS5, and lower ECM matrix, COL1A1 expressions in all SFb. OA-ob:SFb were unresponsive but expressed higher pro-inflammatory cytokines under OA-Pob:CM treatment. CONCLUSION: Both mechanical and inflammatory stressors regulate SFb molecular functions with heterogeneity in responses that are dependent on their pathological tissue of origins. While mechanical stretch promotes a favorable effect with enhanced lubricin production in scope:SFb and TGFß1 and COL1A1 in scope-CD:SFb, the presence of excessively high OA-associated inflammatory mediators in OA-Pob:CM, predominantly SPARC, CXCL8 and FGF2 drive all SFb regardless of pathology, towards greater pro-inflammatory activities.

Senescent HUVECs-secreted exosomes trigger endothelial barrier dysfunction in young endothelial cells.

Accumulation of senescent endothelial cells can cause endothelium dysfunction which eventually leads to age-related vascular disorders. The senescent-associated secretory phenotype (SASP) cells secrete a plethora of soluble factors that negatively influence the surrounding tissue microenvironment. The present study sought to investigate the effects of exosomes, which are nano-sized extracellular vesicles known for intercellular communications secreted by SASP cells on young endothelial cells. Exosomes were isolated from the condition media of senescent human umbilical vein endothelial cells (HUVECs) and then confirmed by the detection of exosome specific CD63 and CD9 expressions, electron microscopy and acetylcholinesterase assay. The purified exosomes were used to treat young HUVECs. Exposure to exosomes repressed the expression of adherens junction proteins including vascular endothelial (VE)-cadherin and beta-catenin, decreased cell growth kinetics and impaired endothelial migration potential of young endothelial cells. These findings suggest that senescent HUVECs-secreted exosomes could disrupt barrier integrity that underpins endothelial barrier dysfunction in healthy young endothelial cells.

Deregulation of hsa-miR-20b expression in TNF-α-induced premature senescence of human pulmonary microvascular endothelial cells.

miRNAs are important regulators of cellular senescence yet the extent of their involvement remains to be investigated. We sought to identify miRNAs that are involved in cytokine-induced premature senescence (CIPS) in endothelial cells. CIPS was established in young human pulmonary microvascular endothelial cells (HMVEC-Ls) following treatment with a sublethal dose (20ng/ml) of tumor necrosis factor alpha (TNF-α) for 15days. In parallel, HMVEC-Ls were grown and routinely passaged until the onset of replicative senescence (RS). Differential expression analysis following miRNA microarray profiling revealed an overlapped of eight deregulated miRNAs in both the miRNA profiles of RS and TNF-α-induced premature senescence cells. Amongst the deregulated miRNAs were members of the miR 17-92 cluster which are known regulators of angiogenesis. The role of hsa-miR-20b in TNF-α-induced premature senescence, a paralog member of the miR 17-92 cluster, was further investigated. Biotin-labeled hsa-miR-20b captured the enriched transcripts of retinoblastoma-like 1 (RBL1), indicating that RBL1 is a target of hsa-miR-20b. Knockdown of hsa-miR-20b attenuated premature senescence in the TNF-α-treated HMVEC-Ls as evidenced by increased cell proliferation, increased RBL1 mRNA expression level but decreased protein expression of p16INK4a, a cellular senescence marker. These findings provide an early insight into the role of hsa-miR-20b in endothelial senescence.

Evaluation of stem-like side population cells in a recurrent nasopharyngeal carcinoma cell line.

BACKGROUND: Side population (SP) assay identifies cells with dye/drug extrusion ability, a characteristic of stem cells. Here, we determined if SP cells exist in a verified cell line originating from recurrent nasopharyngeal carcinoma (NPC) and a xenograft established from recurrent metastatic NPC. These cells were evaluated for stem-like properties via functional assays as well as for tumourigenicity. METHODS: We used Hoechst 33342 to identify the SP from non-SP (NSP) cells in HK1 NPC cell line and xeno-284 NPC xenograft. The cells were assayed for in vitro characteristics of cancer stem cells (CSC), gene expression and tumourigenicity ability. Student's t test was used to test for significance. RESULTS: Five to ten percent and less than 0.5% of HK1 and xeno-284 NPC cells, respectively, were SP cells. Fumitremorgin C (FTC), as opposed to verapamil, was effective in causing the cells to retain Hoechst 33342 dye. HK1 SP cells formed more holoclones, had more aldehyde dehydrogenase (ALDH) activity, divided asymmetrically and contained slow-proliferating cells. ABCG2, SOX2, TERT, MYC, Hedgehog, Notch, TGFß and Wnt signalling pathway genes were significantly upregulated in the SP cells. However, despite these differences in vitro, both HK1 SP and NSP cells had an overall similar tumourigenic potential in vivo. CONCLUSIONS: HK1 SP cells were ABCG2-specific as confirmed by FTC inhibition and gene expression data. Despite data from in vitro and gene expression experiments suggesting stem-like features, there was no significant difference in tumourigenic potential between SP and NSP cells. We conclude that SP assay alone is not sufficient to identify CSCs in HK1 cells. Our work also suggests the presence of a stem-cell like population among NPC cells which do not display increased tumourigenicity.

Paeonol protects against premature senescence in endothelial cells by modulating Sirtuin 1 pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Paeonol is a phenolic compound isolated mainly from Moutan cortex, root bark of Chinese Peony tree. Moutan cortex holds a significant value in traditional Chinese medicine for alleviating various oxidative stress-related diseases mainly atherosclerosis and myocardial infarction. The present study seeks to identify the protective mechanisms of paeonol in oxidative stress-induced premature senescence in endothelial cells. MATERIALS AND METHODS: HUVECs were pretreated with paeonol or DMSO control at different doses for 24h prior to an exposure of 200µM of reactive oxygen species (ROS) inducer, hydrogen peroxide (H2O2). The protective effects of paeonol against H2O2-induced senescence were evaluated and the activation of Sirtuin 1 pathway by paeonol pretreatment was investigated in HUVECs. RESULTS: Paeonol attenuated H2O2-induced cell growth arrest at G0/G1 phase, reduced the percentage of SA-ß-Gal positive cells and increased BrdU incorporation. In addition, enzymatic Sirt1 activation assay indicated that paeonol significantly increased lysyl deactylase activity of Sirt1 enzyme with a fold change of 2.4±0.195 (p<0.05). Furthermore, pretreatment with paeonol significantly decreased the levels of p53, acetyl H3K14 and H4K16 protein expression upregulated by H2O2 stimulation. The changes in the histone protein levels were accompanied with an increase in Sirt1 protein expression level. CONCLUSION: These findings suggest that paeonol protects endothelial cells against oxidative stress-induced premature senescence by modulating the expressions of Sirt1 protein and its substrates.