Post-traumatic osteoarthritis: the role of accelerated chondrocyte senescence

Joint injuries frequently lead to progressive joint degeneration that causes the clinical syndrome of post-traumatic osteoarthritis. The pathogenesis of osteoarthritis remains poorly understood, but patient age is a significant risk factor for progressive joint degeneration. We have found that articular cartilage chondrocytes show strong evidence of senescence with increasing age, including synthesis of smaller more irregular aggrecans; increased expression of lysosomal beta-galactosidase and telomere erosion; and decreased proteoglycan synthesis, response to the anabolic cytokine IGF-I, proliferative capacity, and mitochondrial function. These observations help explain the strong association between age and joint degeneration, but they do not explain how joint injury increases the risk of joint degeneration in younger individuals. We hypothesized that excessive loading of articular surfaces due to acute joint trauma or post-traumatic joint instability, incongruity or mal-alignment increases release of reactive oxygen species, and that the increased oxidative stress on chondrocytes accelerates chondrocyte senescence thereby decreasing the ability of the cells to maintain or restore the tissue. To test this hypothesis, we exposed human articular cartilage chondrocytes from young adults to mechanical and oxidative stress. We found that shear stress applied to cartilage explants in a triaxial pressure vessel increased release of reactive oxygen species and oxidative stress induced chondrocyte senescence (as measured by expression of lysosomal beta-galactosidase, nuclear and mitochondrial DNA damage and decreased mitochondrial function). These observations support the hypothesis that joint injury accelerates chondrocyte senescence and that this acceleration plays a role in the joint degeneration responsible for post-traumatic osteoarthritis.     

Human chondrocyte senescence and osteoarthritis

Although osteoarthritis (OA) is not an inevitable consequence of aging, a strong association exists between age and increasing incidence of OA. We hypothesized that this association is due to in vivo articular cartilage chondrocyte senescence which causes an age-related decline in the ability of the cells to maintain articular cartilage, that is, increasing age increases the risk of OA because chondrocytes lose their ability to replace their extracellular matrix. To test this hypothesis, we measured senescence markers in human articular cartilage chondrocytes from 27 donors ranging in age from one to 87 years. The markers included expression of the senescence-associated enzyme beta-galactosidase, mitotic activity measured by 3H-thymidine incorporation, and telomere length. beta-galactosidase expression increased with age (r=0.84, p=0.0001) while mitotic activity and mean telomere length declined (r=-0.774, p=0.001 and r=-0.71, p=0.0004, respectively). Decreasing telomere length was strongly correlated with increasing expression of beta-galactosidase and decreasing mitotic activity. These findings help explain the previously reported age related declines in chondrocyte synthetic activity and responsiveness to anabolic growth factors and indicate that in vivo articular cartilage chondrocyte senescence is responsible, at least in part, for the age related increased incidence of OA. The data also imply that people vary in their risk of developing OA because of differences in onset of chondrocyte senescence; and, the success of chondrocyte transplantation procedures performed to restore damaged articular surfaces in older patients could be limited by the inability of older chondrocytes to form new cartilage. New efforts to prevent the development or progression of OA might include strategies that delay the onset of chondrocyte senescence or replace senescent cells.     

长非编码RNA SNAI3-AS1调控人软骨细胞C28/I2增殖能力及退变的机制研究

摘要 目的：为了探究长非编码RNA SNAI3-AS1（LncRNA SNAI3-AS1,即SNAI3-AS1）在骨性关节炎（osteoarthritis,OA）进展中的作用与机制。方法：通过全转录组测序筛选出在OA中差异表达的lncRNA SNAI3-AS1,并通过实时荧光定量PCR（qRT-PCR）检测SNAI3-AS1在软骨细胞退变模型中的表达情况。在软骨细胞C28/I2中分别转染SNAI3-AS1特异性siRNA或真核过表达质粒,分别敲低或过表达SNAI3-AS1,通过MTT、平板克隆形成和EdU掺入实验检测细胞增殖活力,Western Blot检测炎症和细胞外基质蛋白的表达情况。通过生物信息学网站预测SNAI3-AS1相互作用的miRNA和下游靶基因,并通过双荧光素酶报告基因和RIP实验进行验证。结果：相较于正常软骨细胞, SNAI3-AS1的表达水平在OA中显著下调。敲低正常软骨细胞中SNAI3-AS1的表达后,软骨细胞的增殖能力减弱并促进了软骨细胞的退变,而在OA模型的软骨细胞中过表达SNAI3-AS1后,软骨细胞的增殖活力加强并抑制了软骨细胞的退变。在机制上,SNAI3-AS1可充当竞争性内源性RNA（ceRNA）,经海绵吸附miR-2278间接上调PRELP,发挥促进软骨细胞增殖和抑制其退变的作用。结论：LncRNA SNAI3-AS1通过LncRNA SNAI3-AS1/ miR-2278/PRELP轴参与骨性关节炎的发生发展过程。     

Potential involvement of oxidative stress in cartilage senescence and development of osteoarthritis: oxidative stress induces chondrocyte telomere instability and downregulation of chondrocyte function

Oxidative stress leads to increased risk for osteoarthritis (OA) but the precise mechanism remains unclear. We undertook this study to clarify the impact of oxidative stress on the progression of OA from the viewpoint of oxygen free radical induced genomic instability, including telomere instability and resulting replicative senescence and dysfunction in human chondrocytes. Human chondrocytes and articular cartilage explants were isolated from knee joints of patients undergoing arthroplastic knee surgery for OA. Oxidative damage and antioxidative capacity in OA cartilage were investigated in donor-matched pairs of intact and degenerated regions of tissue isolated from the same cartilage explants. The results were histologically confirmed by immunohistochemistry for nitrotyrosine, which is considered to be a maker of oxidative damage. Under treatment with reactive oxygen species (ROS; 0.1 μmol/l H₂O₂) or an antioxidative agent (ascorbic acid: 100.0 μmol/l), cellular replicative potential, telomere instability and production of glycosaminoglycan (GAG) were assessed in cultured chondrocytes. In tissue cultures of articular cartilage explants, the presence of oxidative damage, chondrocyte telomere length and loss of GAG to the medium were analyzed in the presence or absence of ROS or ascorbic acid. Lower antioxidative capacity and stronger staining of nitrotyrosine were observed in the degenerating regions of OA cartilages as compared with the intact regions from same explants. Immunostaining for nitrotyrosine correlated with the severity of histological changes to OA cartilage, suggesting a correlation between oxidative damage and articular cartilage degeneration. During continuous culture of chondrocytes, telomere length, replicative capacity and GAG production were decreased by treatment with ROS. In contrast, treatment with an antioxidative agent resulted in a tendency to elongate telomere length and replicative lifespan in cultured chondrocytes. In tissue cultures of cartilage explants, nitrotyrosine staining, chondrocyte telomere length and GAG remaining in the cartilage tissue were lower in ROS-treated cartilages than in control groups, whereas the antioxidative agent treated group exhibited a tendency to maintain the chondrocyte telomere length and proteoglycan remaining in the cartilage explants, suggesting that oxidative stress induces chondrocyte telomere instability and catabolic changes in cartilage matrix structure and composition. Our findings clearly show that the presence of oxidative stress induces telomere genomic instability, replicative senescence and dysfunction of chondrocytes in OA cartilage, suggesting that oxidative stress, leading to chondrocyte senescence and cartilage ageing, might be responsible for the development of OA. New efforts to prevent the development and progression of OA may include strategies and interventions aimed at reducing oxidative damage in articular cartilage.     

荷叶碱对脂多糖诱导的大鼠软骨细胞炎症的抗炎作用研究

目的探讨荷叶碱对软骨细胞的保护作用及对软骨细胞炎症的抗炎作用。方法取体外分离培养3~5天的SD大鼠膝关节软骨细胞,应用10μg/ml LPS诱导软骨细胞建立体外骨关节炎模型。将实验分为3组,即空白组、模型组(LPS)和实验组(LPS+荷叶碱)。通过活/死细胞(Calcein-AM/EthD-I)双染色,实时荧光定量聚合酶链反应(qRT-PCR),HE染色(苏木素-伊红),番红O染色,免疫荧光染色,检测荷叶碱抑制软骨细胞外基质降解作用及缓解软骨细胞炎症的作用。结果荷叶碱浓度为10μM时,无明显毒性,Calcein-AM/EthD-I染色表明该浓度对炎症诱导的软骨细胞有保护作用。qRT-PCR表明,与模型组相比,实验组的Col2al表达升高,MMP-13和IL-6表达下降。HE染色(苏木素-伊红)、番红O染色、免疫荧光染色结果表明荷叶碱能够维持软骨细胞的形态,促进软骨细胞蛋白多糖的分泌,抑制MMP-13的表达。结论浓度为10μM的荷叶碱对LPS诱导的软骨细胞有较好保护作用及抗炎作用,无毒副作用。     

Curcumin ameliorates IL-1β-induced apoptosis by activating autophagy and inhibiting the NF-κB signaling pathway in rat primary articular chondrocytes

Articular cartilage damage and chondrocyte apoptosis are common features of rheumatoid arthritis and osteoarthritis. Recently, curcumin has been reported to exhibit protective effects on degeneration in articular cartilage diseases. However, the effects and mechanisms of curcumin on articular chondrocyte injury remain to be elucidated. The aim of the present study is to investigate the chondroprotective mechanisms of curcumin on interleukin-1β (IL-1β)-induced chondrocyte apoptosis in vitro. The results revealed that IL-1β decreased cell viability and induced apoptosis in primary articular chondrocytes. Curcumin pretreatment reduced IL-1β-induced articular chondrocyte apoptosis. In addition, treatment with curcumin increased autophagy in articular chondrocytes and protected against IL-1β-induced apoptosis. The curcumin-mediated protection against IL-1β induced apoptosis was abolished when cells were treated with the autophagy inhibitor 3-methyladenine or transfected with Beclin-1 small interfering RNA. Furthermore, IL-1β stimulation significantly increased the phosphorylation levels of nuclear factor (NF)-κB p65 and glycogen synthase kinase-3β, and decreased the phosphorylation levels of β-catenin in articular chondrocytes, and these alterations to the phosphorylation levels were partly reversed by treatment with curcumin. Dual-luciferase and electrophoretic mobility shift assays demonstrated that IL-1β increased NF-κB p65 promoter activity in chondrocytes, and this was also reversed by curcumin. Pretreatment with the NF-κB inhibitor pyrrolidine dithiocarbamate enhanced the protective effects of curcumin on chondrocyte apoptosis, but Wnt/β-catenin inhibitor, XAV-939, did not exhibit this effect. Molecular docking and dynamic simulation studies results showed that curcumin could bound to RelA (p65) protein. These results indicate that curcumin may suppress IL-1β-induced chondrocyte apoptosis through activating autophagy and restraining NF-κB signaling pathway.     

Downregulation of protein kinase CK2 activity facilitates tumor necrosis factor-α-mediated chondrocyte death through apoptosis and autophagy

Despite the numerous studies of protein kinase CK2, little progress has been made in understanding its function in chondrocyte death. Our previous study first demonstrated that CK2 is involved in apoptosis of rat articular chondrocytes. Recent studies have suggested that CK2 downregulation is associated with aging. Thus examining the involvement of CK2 downregulation in chondrocyte death is an urgently required task. We undertook this study to examine whether CK2 downregulation modulates chondrocyte death. We first measured CK2 activity in articular chondrocytes of 6-, 21- and 30-month-old rats. Noticeably, CK2 activity was downregulated in chondrocytes with advancing age. To build an in vitro experimental system for simulating tumor necrosis factor (TNF)-α-induced cell death in aged chondrocytes with decreased CK2 activity, chondrocytes were co-treated with CK2 inhibitors and TNF-α. Viability assay demonstrated that CK2 inhibitors facilitated TNF-α-mediated chondrocyte death. Pulsed-field gel electrophoresis, nuclear staining, flow cytometry, TUNEL staining, confocal microscopy, western blot and transmission electron microscopy were conducted to assess cell death modes. The results of multiple assays showed that this cell death was mediated by apoptosis. Importantly, autophagy was also involved in this process, as supported by the appearance of a punctuate LC3 pattern and autophagic vacuoles. The inhibition of autophagy by silencing of autophage-related genes 5 and 7 as well as by 3-methyladenine treatment protected chondrocytes against cell death and caspase activation, indicating that autophagy led to the induction of apoptosis. Autophagic cells were observed in cartilage obtained from osteoarthritis (OA) model rats and human OA patients. Our findings indicate that CK2 down regulation facilitates TNF-α-mediated chondrocyte death through apoptosis and autophagy. It should be clarified in the future if autophagy observed is a consequence versus a cause of the degeneration in vivo.     

p21 WAF1 and hypoxia/reoxygenation-induced premature senescence of H9c2 cardiomyocytes

We have previously reported on hypoxia/reoxygenation-induced premature senescence in neonatal rat cardiomyocytes. In this research, we investigated the effects of p21(WAF1) (p21) in hypoxia/reoxygenation-induced senescence, using H9c2 cells. A plasmid overexpressing wild type p21(WAF1) and a plasmid expressing small hairpin RNA (shRNA) targeting p21(WAF1) were constructed, and transfected into H9c2 cells to control the p21 expression. Hypoxia/reoxygenation conditions were 1% O2 and 5% CO(2), balancing the incubator chamber with N(2) for 6 h (hypoxia 6 h), then 21% oxygen for 8 h (reoxygenation 8 h). Cell cycle was examined using flow cytometry. Senescence was assessed using β-galactosidase staining. The expression of p53, p21, p16(INK4a), and cyclin D1 was assayed using Western blotting. At hypoxia 6 h, cells overexpressing p21 had a larger G1 distribution, stronger β-galactosidase activity, and lower cyclin D1 expression compared to control cells, while the opposite results and higher p53 expression were obtained in p21-knockdown cells. At reoxygenation 8 h, p21-silenced cells had a smaller percentage of G1 cells, weaker β-galactosidase activity and lower 16(INK4a) expression, and higher cyclin D1 expression, but the overexpression group showed no difference. Taken together, this data implies that p21(WAF1) is important for the hypoxia phase, but not the reoxygenation phase, in the H9c2 senescence process.     

Wnt signaling is involved in human articular chondrocyte de-differentiation in vitro

Osteoarthritis is the most prevalent form of arthritis in the world. Certain signaling pathways, such as the wnt pathway, are involved in cartilage pathology. Osteoarthritic chondrocytes undergo morphological and biochemical changes that lead to chondrocyte de-differentiation. We investigated whether the Wnt pathway is involved in de-differentiation of human articular chondrocytes in vitro. Human articular chondrocytes were cultured for four passages in the presence or absence of IL-1 in monolayer or micromass culture. Changes in cell morphology were monitored by light microscopy. Protein and gene expression of chondrocyte markers and Wnt pathway components were determined by Western blotting and qPCR after culture. After culturing for four passages, chondrocytes exhibited a fibroblast-like morphology. Collagen type II and aggrecan protein and gene expression decreased, while collagen type I, matrix metalloproteinase 13, and nitric oxide synthase expressions increased. Wnt molecule expression profiles changed; Wnt5a protein expression, the Wnt target gene, c-jun, and in Wnt pathway regulator, sFRP4 increased. Treatment with IL-1 caused chondrocyte morphology to become more filament-like. This change in morphology was accompanied by extinction of col II expression and increased col I, MMP13 and eNOS expression. Changes in expression of the Wnt pathway components also were observed. Wnt7a decreased significantly, while Wnt5a, LRP5, β-catenin and c-jun expressions increased. Culture of human articular chondrocytes with or without IL-1 not only induced chondrocyte de-differentiation, but also changed the expression profiles of Wnt components, which suggests that the Wnt pathway is involved in chondrocyte de-differentiation in vitro.     

Pathological mechanism of chondrocytes and the surrounding environment during osteoarthritis of temporomandibular joint

Temporomandibular joint (TMJ) osteoarthritis is a common chronic degenerative disease of the TMJ. In order to explore its aetiology and pathological mechanism, many animal models and cell models have been constructed to simulate the pathological process of TMJ osteoarthritis. The main pathological features of TMJ osteoarthritis include chondrocyte death, extracellular matrix (ECM) degradation and subchondral bone remodelling. Chondrocyte apoptosis accelerates the destruction of cartilage. However, autophagy has a protective effect on condylar chondrocytes. Degradation of ECM not only changes the properties of cartilage but also affects the phenotype of chondrocytes. The loss of subchondral bone in the early stages of TMJ osteoarthritis plays an aetiological role in the onset of osteoarthritis. In recent years, increasing evidence has suggested that chondrocyte hypertrophy and endochondral angiogenesis promote TMJ osteoarthritis. Hypertrophic chondrocytes secrete many factors that promote cartilage degeneration. These chondrocytes can further differentiate into osteoblasts and osteocytes and accelerate cartilage ossification. Intrachondral angiogenesis and neoneurogenesis are considered to be important triggers of arthralgia in TMJ osteoarthritis. Many molecular signalling pathways in endochondral osteogenesis are responsible for TMJ osteoarthritis. These latest discoveries in TMJ osteoarthritis have further enhanced the understanding of this disease and contributed to the development of molecular therapies. This paper summarizes recent cognition on the pathogenesis of TMJ osteoarthritis, focusing on the role of chondrocyte hypertrophy degeneration and cartilage angiogenesis.     

PANDORA-Seq unveils the hidden small noncoding RNA landscape in atherosclerosis of LDL receptor-deficient mice

Small noncoding RNAs (sncRNAs) play diverse roles in numerous biological processes. While the widely used RNA sequencing (RNA-Seq) method has advanced sncRNA discovery, RNA modifications can interfere with the complementary DNA library construction process, preventing the discovery of highly modified sncRNAs including transfer RNA-derived small RNAs (tsRNAs) and ribosomal RNA-derived small RNAs (rsRNAs) that may have important functions in disease development. To address this technical obstacle, we recently developed a novel PANDORA-Seq (Panoramic RNA Display by Overcoming RNA Modification Aborted Sequencing) method to overcome RNA modification-elicited sequence interferences. To identify novel sncRNAs associated with atherosclerosis development, LDL receptor-deficient (LDLR^−/−) mice were fed a low-cholesterol diet or high-cholesterol diet (HCD) for 9 weeks. Total RNAs isolated from the intima were subjected to PANDORA-Seq and traditional RNA-Seq. By overcoming RNA modification-elicited limitations, PANDORA-Seq unveiled an rsRNA/tsRNA-enriched sncRNA landscape in the atherosclerotic intima of LDLR^−/− mice, which was strikingly different from that detected by traditional RNA-Seq. While microRNAs were the dominant sncRNAs detected by traditional RNA-Seq, PANDORA-Seq substantially increased the reads of rsRNAs and tsRNAs. PANDORA-Seq also detected 1,383 differentially expressed sncRNAs induced by HCD feeding, including 1,160 rsRNAs and 195 tsRNAs. One of HCD-induced intimal tsRNAs, tsRNA-Arg-CCG, may contribute to atherosclerosis development by regulating the proatherogenic gene expression in endothelial cells. Overall, PANDORA-Seq revealed a hidden rsRNA and tsRNA population associated with atherosclerosis development. These understudied tsRNAs and rsRNAs, which are much more abundant than microRNAs in the atherosclerotic intima of LDLR^−/− mice, warrant further investigations.     

Exosome-derived small non-coding RNAs reveal immune response upon grafting transplantation in Pinctada fucata (Mollusca)

Exosomes, a subset of small extracellular vesicles, carry various nucleic acids, proteins, lipids, amino acids and metabolites. They function as a mode of intercellular communication and molecular transfer. Exosome cargo molecules, including small non-coding RNAs (sncRNAs), are involved in the immune response in various organisms. However, the role of exosome-derived sncRNAs in immune responses in molluscs remains unclear. Here, we aimed to reveal the sncRNAs involved in the immune response during grafting transplantation by the pearl oyster Pinctada fucata. Exosomes were successfully extracted from the P. fucata haemolymph during graft transplantation. Abundant microRNAs (miRNAs) and PIWI-interacting RNAs (piRNAs) were simultaneously discovered in P. fucata exosomes by small RNA sequencing. The expression patterns of the miRNAs and piRNAs at the grafting and initial stages were not substantially different, but varied significantly between the initial and later stages. Target prediction and functional analysis indicate that these miRNAs and piRNAs are related to immune response upon grafting transplantation, whereas piRNAs may also be associated with transposon silencing by targeting with genome transposon elements. This work provides the basis for a functional understanding of exosome-derived sncRNAs and helps to gain further insight into the PIWI/piRNA pathway function outside of germline cells in molluscs.     

血管紧张素受体1在血管紧张素Ⅱ诱导人星形胶质细胞老化中的作用

目的通过体外细胞实验研究,探讨血管紧张素受体1在血管紧张素Ⅱ诱导人星形胶质细胞活性氧产生和细胞老化中的作用。方法人星形胶质细胞随机分为三组：血管紧张素Ⅱ＋Cand（坎地沙坦）组和血管紧张素Ⅱ＋tempol组。血管紧张素Ⅱ组是用100nM血管紧张素Ⅱ刺激人星形胶质细胞3天,血管紧张素Ⅱ＋Cand组和血管紧张素Ⅱ＋tempol组先用血管紧张素受体1阻滞剂坎地沙坦（100nM）和氧自由基清除剂tempol（3mM）预处理,再用100nM血管紧张素II刺激人星形胶质细胞3天,利用β半乳糖苷酶染色评估细胞老化。不同剂量（0、1nM、10nM、100nM、1000nM和1000nM＋坎地沙坦）的血管紧张素Ⅱ刺激人星形胶质细胞30min,DHE染色评估细胞内活性氧产生。结果血管紧张素Ⅱ引起人星形胶质细胞DHE染色表达增多和β半乳糖苷酶染色细胞增多。利用血管紧张素受体1阻滞剂坎地沙坦和氧自由基清除剂tempol预处理逆转了血管紧张素Ⅱ引起的星形胶质细胞老化。结论血紧张素Ⅱ是通过血管紧张素受体1和超氧阴离子产生引起星形胶质细胞的老化。     

EGR1 promotes the cartilage degeneration and hypertrophy by activating the Krüppel-like factor 5 and β-catenin signaling

Osteoarthritis is one of the most common orthopedic diseases in elderly people who have lost their mobility. In this study,we observed abnormally high EGR1 expression in the articular cartilage of patients with osteoarthritis. We also found significantly high EGR1 expression in the articular cartilage of mice with destabilized medial meniscus (DMM)-induced osteoarthritis and 20-month-old mice. In vitro experiments indicated that IL-1β could significantly enhance EGR1 expression in primary mouse chondrocytes. EGR1 over-expression in chondrocytes using adenovirus could inhibit COl2A1 expression and enhance MMP9 and MMP13 expression. And silencing EGR1, using RNAi, had the opposite effects. Moreover, EGR1 over-expression accelerated chondrocyte hypertrophy in vitro, and EGR1 knockdown reversed this effect. We then explored the underlying mechanism. EGR1 over-expression increased Kruppel-Like Factor 5 (KLF5) protein level without influencing its synthesis. Enhanced EGR1 expression induced its integration with KLF5, leading to suppressed ubiquitination of KLF5. Moreover, EGR1 prompted β-catenin nuclear transportation to control chondrocyte hypertrophy. Ectopic expression of EGR1 in articular cartilage aggravated the degradation of the cartilage matrix in vivo. The EGR1 inhibitor, ML264, protected chondrocytes from IL-1β-mediated cartilage matrix degradation in vitro and DMM-induced osteoarthritis in vivo. Above all, we demonstrate the effect and mechanisms of EGR1 on osteoarthritis and provide evidence that the ML264 might be a potential drug for treating osteoarthritis in the future.     

d-Galactose induces the senescence and phenotype switch of corpus cavernosum smooth muscle cells

Studies regarding age-related erectile dysfunction (ED) based on naturally aging models are limited by their high costs, especially for the acquisition of primary cells from the corpus cavernosum. Herein, d -galactose ( d -gal) was employed to accelerate cell senescence, and the underlying mechanism was explored. As predominant functional cells involved in the erectile response, corpus cavernosum smooth muscle cells (CCSMCs) were isolated from 2-month-old rats. Following this, d -gal was introduced to induce cell senescence, which was verified via β-galactosidase staining. The effects of d -gal on CCSMCs were evaluated by terminal deoxynucleoitidyl transferase dUTP nick-end labeling (TUNEL), immunofluorescence staining, flow cytometry, western blot, and quantitative real-time polymerase chain reaction (qRT-PCR). Furthermore, RNA interference (RNAi) was carried out for rescue experiments. Subsequently, the influence of senescence on the corpus cavernosum was determined via scanning electron microscopy, qRT-PCR, immunohistochemistry, TUNEL, and Masson stainings. The results revealed that the accelerated senescence of CCSMCs was promoted by d -gal. Simultaneously, smooth muscle alpha-actin (alpha-SMA) expression was inhibited, while that of osteopontin (OPN) and Krüppel-like factor 4 (KLF4), as well as fibrotic and apoptotic levels, were elevated. After knocking down KLF4 expression in d -gal-induced CCSMCs by RNAi, the expression level of cellular alpha-SMA increased. Contrastingly, the OPN expression, apoptotic and fibrotic levels declined. In addition, cellular senescence acquired partial remission. Accordingly, in the aged corpus cavernosum, the fibrotic and apoptotic rates were increased, followed by downregulation in the expression of alpha-SMA and the concurrent upregulation in the expression of OPN and KLF4. Overall, our results signaled that d -gal-induced accelerated senescence of CCSMCs could trigger fibrosis, apoptosis and phenotypic switch to the synthetic state, potentially attributed to the upregulation of KLF4 expression, which may be a multipotential therapeutic target of age-related ED.     

Hypertrophic differentiation of chondrocytes in osteoarthritis: the developmental aspect of degenerative joint disorders

Osteoarthritis is characterized by a progressive degradation of articular cartilage leading to loss of joint function. The molecular mechanisms regulating pathogenesis and progression of osteoarthritis are poorly understood. Remarkably, some characteristics of this joint disease resemble chondrocyte differentiation processes during skeletal development by endochondral ossification. In healthy articular cartilage, chondrocytes resist proliferation and terminal differentiation. By contrast, chondrocytes in diseased cartilage progressively proliferate and develop hypertrophy. Moreover, vascularization and focal calcification of joint cartilage are initiated. Signaling molecules that regulate chondrocyte activities in both growth cartilage and permanent articular cartilage during osteoarthritis are thus interesting targets for disease-modifying osteoarthritis therapies.     

AMPK induces vascular smooth muscle cell senescence via LKB1 dependent pathway

Vascular cells have a limited lifespan with limited cell proliferation and undergo cellular senescence. The functional changes associated with cellular senescence are thought to contribute to age-related vascular disorders. AMP-activated protein kinase (AMPK) has been discussed in terms of beneficial or harmful effects for aging-related diseases. However, the detailed functional mechanisms of AMPK are largely unclear. An aging model was established by stimulating vascular smooth muscle cell (VSMC) with adriamycin. Adriamycin progressively increased the mRNA and protein expressions of AMPK. The phosphorylation levels of LKB1 and acetyl-CoA carboxylase (ACC), the upstream and downstream of AMPK, were dramatically increased by adriamycin stimulation. The expressions of p53 and p21, which contribute to vascular senescence, were also increased. Inhibition of AMPK diminished senescence-associated β-galactosidase (SA-β-gal) staining, and restored VSMC proliferation. Cytosolic translocation of LKB1 by adriamycin could be a mechanism for AMPK activation in senescence. Furthermore, p53 siRNA and p21 siRNA transfection attenuated adriamycin-induced SA-β-gal staining. These results suggest that LKB1 dependent AMPK activation elicits VSMC senescence and p53–p21 pathway is a mediator of LKB1/AMPK-induced senescence.     

XIST/miR-376c-5p/OPN axis modulates the influence of proinflammatory M1 macrophages on osteoarthritis chondrocyte apoptosis

The inflammatory microenvironment in the joints is one of the critical issues during osteoarthritis (OA) and also the main factor that may aggravate symptoms. Under inflammatory microenvironment, M1 macrophages are activated and produce large numbers of proinflammatory mediators, leading to the production of degradative enzymes, the disturbance of chondrocyte apoptosis and cartilage catabolic processes, and finally the deterioration of OA. In the present study, we reveal that the overexpression of osteopontin (OPN), a cytokine, and a matrix protein involved in arthritis and chondrocyte apoptosis in OA, could exacerbate the inflammatory microenvironment in OA via promoting the production of proinflammation cytokines and the levels of degradative enzymes in M1 macrophages, therefore, enhancing the cytotoxicity of M1 macrophage on chondrocytes. XIST expression significantly increases in OA tissue specimens. XIST serves as a competing endogenous RNA for miR-376c-5p to compete with OPN for miR-376c-5p binding, thus counteracting miR-376c-5p-mediated OPN suppression. XIST knockdown could improve the inflammatory microenvironment in OA via acting on M1 macrophages, subsequently affecting the apoptosis of cocultured chondrocytes. miR-376c-5p inhibition exerts an opposing effect on M1 macrophages and cocultured chondrocytes, as well as significantly reverses the effect of XIST knockdown. As a further confirmation, XIST and OPN mRNA expression significantly increased in OA tissues and was positively correlated in tissue samples. In summary, we provide a novel mechanism of macrophages and the inflammatory microenvironment affecting chondrocyte apoptosis. XIST and OPN might be potential targets for OA treatment, which needs further in vivo experimental confirmation.     

Altered expression of the core circadian clock component PERIOD2 contributes to osteoarthritis-like changes in chondrocyte activity

In osteoarthritis, chondrocytes undergo a phenotype shift characterised by reduced expression of SOX9 (sry-box 9) and increased production of cartilage-degrading enzymes, e.g. MMP13 (matrix metalloproteinase 13) and ADAMTS5 (a disintegrin and metalloproteinase with thrombospondin motifs 5). The chondrocyte clock is also altered. Specifically, the peak level of PER2 is elevated, but peak level of BMAL1 reduced in osteoarthritic chondrocytes. The purpose of this study was to determine whether increased PER2 expression causes disease-associated changes in chondrocyte activity and to identify whether known risk factors for osteoarthritis induce changes in PER2 and BMAL1 expression. Primary human chondrocytes isolated from macroscopically normal cartilage were serum-starved overnight then re-fed with serum-replete media with/without interleukin 1β (IL-1β) (10 ng/mL), hydrogen peroxide (100 µM) or basic calcium phosphate (BCP) crystals (50 µg/mL). Peak level of BMAL1 was lower, whereas PER2 levels remained elevated for longer, in chondrocytes treated with IL-1β, hydrogen peroxide or BCP crystals compared to untreated cells. Levels of SOX9 were lower, whereas levels of ADAMTS5 and MMP13 were higher, in chondrocytes exposed to any of the three treatments compared to untreated cells. Knockdown of PER2 using siRNA partially abrogated the effects of each treatment on chondrocyte phenotype marker expression. Similarly, in chondrocytes isolated from osteoarthritic cartilage PER2 knockdown was associated with increased SOX9, reduced ADAMTS5 and reduced RNA and protein levels of MMP13 indicating partial mitigation of the osteoarthritic phenotype. Conversely, further ablation of BMAL1 expression in osteoarthritic chondrocytes resulted in a further reduction in SOX9 and increase in MMP13 expression. Overexpression of PER2 in the H5 chondrocyte cell line led to increased ADAMTS5 and MMP13 and decreased SOX9 expression. Localised inflammation, oxidative stress and BCP crystal deposition in osteoarthritic joints may contribute to disease pathology by inducing changes in the chondrocyte circadian clock.