Weijuan Ma

Comparative Analysis of Gene Expression Profiles Between Primary Knee Osteoarthritis and an Osteoarthritis Endemic to Northwestern China, Kashin-Beck Disease

OBJECTIVE To investigate the differences in gene expression profiles of adult articular cartilage from patients with Kashin-Beck disease (KBD) versus those with primary knee osteoarthritis (OA). METHODS The messenger RNA expression profiles of articular cartilage from patients with KBD, diagnosed according to the clinical criteria for KBD in China, were compared with those of cartilage from patients with OA, diagnosed according to the Western Ontario and McMaster Universities OA Index. Total RNA was isolated separately from 4 pairs of the KBD and OA cartilage samples, and the expression profiles were evaluated by Agilent 4x44k Whole Human Genome density oligonucleotide microarray analysis. The microarray data for selected transcripts were confirmed by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) amplification. RESULTS For 1.2 x 10(4) transcripts, corresponding to 58.4% of the expressed transcripts, 2-fold changes in differential expression were revealed. Expression levels higher in KBD than in OA samples were observed in a mean + or - SD 6,439 + or - 1,041 (14.6 + or - 2.4%) of the transcripts, and expression levels were lower in KBD than in OA samples in 6,147 + or - 1,222 (14.2 + or - 2.8%) of the transcripts. After application of the selection criteria, 1.85% of the differentially expressed genes (P < 0.001 between groups) were detected. These included 233 genes, of which 195 (0.4%) were expressed at higher levels and 38 (0.08%) were expressed at lower levels in KBD than in OA cartilage. Comparisons of the quantitative RT-PCR data supported the validity of our microarray data. CONCLUSION Differences between KBD and OA cartilage exhibited a similar pattern among all 4 of the pairs examined, indicating the presence of disease mechanisms, mainly chondrocyte matrix metabolism, cartilage degeneration, and apoptosis induction pathways, which contribute to cartilage destruction in KBD.

Mitochondrial function is altered in articular chondrocytes of an endemic osteoarthritis, Kashin-Beck disease.

OBJECTIVE Kashin-Beck disease (KBD) is an endemic degenerative osteoarthritis (OA) associated with extracellular matrix degradation and chondrocyte necrosis in the articular and growth plate cartilage. The role of mitochondria in degenerative diseases is widely recognized but its function in KBD is unknown. The aim of this investigation was to evaluate mitochondrial function to understand the mitochondria-mediated caspase activation and apoptosis in adult KBD chondrocytes. METHODS Mitochondrial function was evaluated by analyzing the activities of respiratory chain enzyme complexes and citrate synthase (CS), intracellular adenosine triphosphate (ATP) contents, as well as changes in mitochondrial membrane potential (DeltaPsim). Apoptotic cell death was evaluated by analyzing the cytochrome c release from mitochondria to the cytosol, caspase-9 and 3 activities, and the apoptosis rate of KBD articular chondrocytes. RESULTS Activities of complexes II, III, IV and V were reduced in KBD articular chondrocytes compared with cells from normal controls. However, the mitochondrial mass was increased in KBD samples. Cultured KBD chondrocytes had a reduction of cellular ATP levels and contained a higher proportion of cells with de-energized mitochondria. Mitochondrial cytochrome c release and activation of caspase-9 and 3 were also observed. The percentages of positive apoptotic chondrocytes from the KBD patient group stained by Hoechst nuclear stain and Annexin V/PI for flow cytometry exhibited higher levels than that of the healthy controls. CONCLUSION These findings suggest the involvement of mitochondrial function and apoptotic cell death in the pathophysiology of KBD. The dysfunction of the mitochondria may play an important role in KBD articular chondrocytes apoptosis.

Recent advances in the research of an endemic osteochondropathy in China: Kashin-Beck disease

Kashin-Beck disease (KBD) is an endemic chronic osteochondral disease, which has a high prevalence and morbidity in the Eastern Siberia of Russia, and in the broad diagonal, northern-east to southern-west belt in China and North Korea. In 1990s, it was estimated that in China 1-3 million people had some degree of symptoms of the disease, although even higher estimates have been presented. In China, the extensive prevalence peaked in the late 1950s, but since then, in contrast to the global trend of the osteoarthritis (OA), the number of cases has been dramatically falling. Up to 2013, there are 0.64 millions patients with the KBD and 1.16 millions at risk in 377 counties of 13 provinces or autonomous regions. This is obviously thanks to the preventive efforts carried out, which include providing millions of people with dietary supplements and clean water, as well as relocation of whole villages in China. However, relatively little is known about the molecular mechanisms behind the cartilage damage, the genetic and the environmental risk factors, and the rationale of the preventive effects. During the last decade, new data on a cellular and molecular level has begun to accumulate, which hopefully will uncover the grounds of the disease.

Proteomic changes in articular cartilage of human endemic osteoarthritis in China

Kashin–Beck disease (KBD) is a chronic endemic osteochondropathy with unclear pathogenesis. It is a degenerative disease similar to osteoarthritis, but with different manifestations of cartilage damage. The aim of this investigation was to show the protein changes in KBD cartilage and to identify the candidate proteins in order to understand the pathogenesis of the disease. Proteins were extracted from the media of primary cell cultures of KBD and normal chondrocytes, and separated by two‐dimensional fluorescence difference gel electrophoresis (2‐D DIGE). MALDI‐TOF/TOF analysis revealed statistically significant differences in 27 proteins from KBD chondrocyte cultures, which consisted of 17 up‐regulated and ten down‐regulated proteins. The results were further validated by Western blot analysis. The proteins identified are mainly involved in cellular redox homeostasis and stress response (MnSOD, Hsp27, Peroxiredoxin‐1, and Cofilin‐1), glycolysis (PGK‐1, PGM‐1, α‐enolase), and cell motility and cytoskeletal organization (Actin, Calponin‐2, and Keratin). These KBD‐associated proteins indicate that cytoskeletal remodeling, glycometabolism, and oxidative stress are abnormal in KBD articular cartilage.

Altered Aggrecan Synthesis and Collagen Expression Profiles in Chondrocytes from Patients with Kashin—Beck Disease and Osteoarthritis

OBJECTIVES: To investigate cell morphology, aggrecan expression, and type I, II, III and X collagen expression in chondrocytes from adults with Kashin—Beck disease or osteoarthritis (OA). METHODS: Samples of knee articular cartilage were taken during surgery; cartilage samples obtained from fresh cadavers without arthritic disease were used as controls. Samples were digested with collagenase; isolated chondrocytes were cultured in monolayers. Aggrecan was detected by toluidine blue staining; collagen and aggrecan protein levels were evaluated by immuno cytochemistry and immunofluorescence staining. RESULTS: Samples were obtained from six participants per group. Aggrecan and type II collagen levels in chondrocytes from patients were significantly lower than those from controls, but levels of type I, III and X collagen were enhanced in patients compared with controls. Production of type III and X collagen was higher in chondrocytes from patients with Kashin—Beck disease than in those from OA patients. CONCLUSIONS: Biochemical and morphological mechanisms underlying Kashin—Beck disease and OA include enhanced dedifferentiation and hypertrophy of chondrocytes, increased type I, III and X collagen levels, and suppressed type II collagen and aggrecan production compared with control samples.

Exome Sequencing and Functional Analysis Identifies a Novel Mutation in EXT1 Gene That Causes Multiple Osteochondromas

Multiple osteochondromas (MO) is an inherited skeletal disorder, and the molecular mechanism of MO remains elusive. Exome sequencing has high chromosomal coverage and accuracy, and has recently been successfully used to identify pathogenic gene mutations. In this study, exome sequencing followed by Sanger sequencing validation was first used to screen gene mutations in two representative MO patients from a Chinese family. After filtering the data from the 1000 Genome Project and the dbSNP database (build 132), the detected candidate gene mutations were further validated via Sanger sequencing of four other members of the same MO family and 200 unrelated healthy subjects. Immunohistochemisty and multiple sequence alignment were performed to evaluate the importance of the identified causal mutation. A novel frameshift mutation, c.1457insG at codon 486 of exon 6 of EXT1 gene, was identified, which truncated the glycosyltransferase domain of EXT1 gene. Multiple sequence alignment showed that codon 486 of EXT1 gene was highly conserved across various vertebrates. Immunohistochemisty demonstrated that the chondrocytes with functional EXT1 in MO were less than those in extragenetic solitary chondromas. The novel c.1457insG deleterious mutation of EXT1 gene reported in this study expands the causal mutation spectrum of MO, and may be helpful for prenatal genetic screening and early diagnosis of MO.

Regulatory gene networks and signaling pathways from primary osteoarthritis and Kashin-Beck disease, an endemic osteoarthritis, identified by three analysis software.

Three new software systems, Ingenuity pathway analysis(IPA, TranscriptomeBrowser and MetaCore, were compared by analyzing chondrocyte microarray data of Kashin-Beck disease (KBD) and primary knee osteoarthritis(OA) to understand the pathway or network analysis software which has a superior function to identify target genes with easy operation and effective for differential diagnosis and treatment of KBD and OA. RNA was isolated from cartilage samples taken from KBD patients and OA ones. Agilent 44K human whole genome oligonucleotide microarrays were used to detect differentially expressed genes. From IPA, we identified one significant canonical pathway and two significant networks. From GeneHub analysis, we got three networks. One significant canonical pathway and one significant network were obtained from TranscriptomeBrowser analysis. POSTN and LEF1 which were got from IPA, RAC2 which was identified by both of the IPA and TranscriptomeBrowser may be most closely related to the etiopathogenesis of KBD. According to our data analysis, IPA and TranscriptomeBrowser are suitable for pathway analysis, while, TranscriptomeBrowser is suitable for network analysis. The significant genes obtained from IPA and TranscriptomeBrowser analysis may thus provide a better understanding of the molecular details in the pathogenesis of KBD and also provide useful pathways and network maps for future research in osteochondrosis.

Expression profile analysis of mycotoxin-related genes in cartilage with endemic osteochondropathy kashin-beck disease

BackgroundKashin-Beck Disease (KBD) is an endemic osteochondropathy. Mycotoxins are believed to play an important role in the pathogenesis of KBD. Because the molecular mechanism of mycotoxin-induced cartilage lesions remains unclear, there is not effective treatment for KBD now. To identify key genes involved in the mycotoxin-induced cartilage lesions, we compared the expression profiles of mycotoxin-related genes (MRG) between KBD cartilage and healthy cartilage.MethodsTotal RNA was isolated from cartilage samples, following by being amplified, labeled and hybridized to Agilent human whole genome microarray chip. qRT-PCR was conducted to validate the microarray data. 1,167 MRG were derived from the environmentally related genomic database Toxicogenomics. The microarray data of MRG was subjected to single gene and gene ontology (GO) expression analysis for identifying differently expressed genes and GO.ResultsWe identified 7 up-regulated MRG and 2 down-regulated MRG in KBD cartilage, involved in collagen, apoptosis, metabolism and growth & development. GO expression analysis found that 4 apoptosis-related GO and 5 growth & development-related GO were significantly up-regulated in KBD cartilage.ConclusionsBased on the results of previous and our studies, we suggest that mycotoxins might contribute to the development of KBD through dysfunction of MRG involved in collagen, apoptosis and growth & development in cartilage.

MALDI-TOF-MS serum protein profiling for developing diagnostic models and identifying serum markers for discogenic low back pain

BackgroundThe identification of the cause of chronic low back pain (CLBP) represents a great challenge to orthopedists due to the controversy over the diagnosis of discogenic low back pain (DLBP) and the existence of a number of cases of CLBP of unknown origin. This study aimed to develop diagnostic models to distinguish DLBP from other forms of CLBP and to identify serum biomarkers for DLBP.MethodsSerum samples were collected from patients with DLBP, chronic lumbar disc herniation (LDH), or CLBP of unknown origin, and healthy controls (N), and randomly divided into a training set (n = 30) and a blind test set (n = 30). Matrix-assisted laser desorption ionization time-of-flight mass spectrometry was performed for protein profiling of these samples. After the discriminative ability of two most significantly differential peaks from each two groups was assessed using scatter plots, classification models were developed using differential peptide peaks to evaluate their diagnostic accuracy. The identity of peptides corresponding to three representative differential peaks was analyzed.ResultsThe fewest statistically significant differential peaks were identified between DLBP and CLBP (3), followed by CLBP vs. N (5), DLBP vs. N (9), LDH vs. CLBP (20), DLBP vs. LDH (23), and LDH vs. N (43). The discriminative ability of two most significantly differential peaks was poor in classifying DLBP vs. CLBP but good in classifying DLBP vs. LDH. The accuracy of models for classification of DLBP vs. CLBP was not very high in the blind test (forecasting ability, 67.24%; sensitivity, 70%), although a higher accuracy was observed for classification of DLBP vs. LDH and LDH vs. N (forecasting abilities, ~90%; sensitivities, >90%). A further investigation of three representative differential peaks led to the identification of two peaks as peptides of complement C3, and one peak as a human fibrinogen peptide.ConclusionsOur findings benefit not only the diagnosis of CLBP but also the understanding of the differences between different forms of DLBP. The ability to distinguish between different causes of CLBP and the identification of serum biomarkers may be of great value to diagnose different causes of DLBP and predict treatment efficacy.

In vitro effects of sodium hyaluronate on the proliferation and the apoptosis in chondrocytes from patients with Kashin-Beck disease and osteoarthritis

Abstract Objective To identify the in vitro effects of sodium hyaluronate(HA) on the proliferation and the apoptosis of chondrocytes from patients with Kashin-Beck disease(KBD) and osteoarthritis(OA). Methods Samples of articular cartilages from KBD and OA patients, as well as healthy volunteers(6 subjects in each of the 3 groups) were dissected, digested with collagenase and the cells cultured in monolayers. Chondrocytes from each sample were assigned to an untreated group and two HA-treated groups: H0(no HA), H100(HA, 0.1 g/L) and H500(HA, 0.5 g/L). The first passage chondrocytes were used to observe proliferation using the MTT assay, and apoptosis by flow cytometry through Annexin V/PI staining. Results HA promoted proliferation of chondrocytes in all the three groups, and in KBD and OA groups, for cells cultured for 4 and 6 days, H500 significantly promoted the cell proliferation. The apoptotic rates of both KBD and OA group chondrocytes were in the order H500 Conclusion Sodium hyaluronate administration has a dose-dependent in vitro effect to promote proliferation and inhibit apoptosis of chondrocytes from patients with KBD and OA.