Xiaotian Chang

Increased expression of carbonic anhydrase I in the synovium of patients with ankylosing spondylitis

BackgroundOne of the most distinctive features of ankylosing spondylitis (AS) is new bone formation and bone resorption at sites of chronic inflammation. Previous studies have indicated that the hyperplasia and inflammation of synovial tissues are significantly related to the pathogenic process of AS. The present study used a proteomic approach to identify novel AS-specific proteins by simultaneously comparing the expression profiles of synovial membranes from patients with AS, rheumatoid arthritis (RA) and osteoarthritis (OA).MethodsSynovial tissues were collected from the hip joints of patients with AS and knee joints of patients with RA or OA (n = 10 for each disease) during joint replacement surgery. Proteins extracted from the synovial tissues were separated by 2-D electrophoresis (2-DE), and the proteins with significantly increased expression in the AS samples were subjected to MALDI-TOF/TOF-MS analysis. The results were verified using western blotting and immunohistochemistry. Levels of the candidate proteins in synovial fluids from knee joints (n = 40 for each disease) were measured using ELISA.ResultsThe proteomic approach revealed significantly increased expression of carbonic anhydrase I (CA1) in the synovial membrane of patients with AS as compared with the RA and OA tissue samples. Immunohistochemistry and western blotting analysis confirmed the findings described above. The ELISA detected a higher level of CA1 in synovial fluids from patients with AS than those with OA. The mean value of the CA1 level was also higher in AS patients as compared with RA patients. This study also detected increased expression of alpha-1-antitrypsin in the synovial tissues from AS patients, which is in agreement with other reports.ConclusionIn vitro experiments by other groups indicated that CA1 catalyzes the generation of HCO3- through the hydration of CO2, which then combines with Ca2+ to form a CaCO3 precipitate. Calcification is an essential step of bone formation. Substantial evidence indicates that carbonic anhydrase also stimulates bone resorption. Hence, overexpression of CA1 in the synovial tissues of AS patients may promote improper calcification and bone resorption in AS.

Investigating a pathogenic role for TXNDC5 in rheumatoid arthritis.

IntroductionExpression of TXNDC5, which is induced by hypoxia, stimulates cell proliferation and angiogenesis. Our previous study detected increased TXNDC5 expression in the synovial tissues of rheumatoid arthritis (RA) patients using proteomic methods. The current study investigated a pathogenic role for TXNDC5 in RA.MethodExpression of TXNDC5 in synovial membranes was quantitatively analyzed by immunohistochemistry, Western blotting and real-time polymerase chain reaction (PCR). Serum TXNDC5 levels and serum anti-TXNDC5 antibody levels were determined using sandwich enzyme-linked immunosorbent assay (ELISA). A total of 96 single nucleotide polymorphisms (SNPs) in or near the TXNDC5 gene were genotyped using custom-designed Illumina 96-SNP VeraCode microassay. Allele frequencies and genotype frequencies of SNPs were assessed using a case-control design in a cohort of 267 Chinese patients with RA, 51 patients with ankylosing spondylitis (AS) and 160 healthy controls. Additional genotyping of 951 patients with RA and 898 healthy controls was performed for four SNPs (rs2277105, rs369086, rs443861 and rs11962800) using the TaqMan method.ResultsReal-time PCR, Western blotting and immunohistochemistry detected significantly higher TXNDC5 expression in the synovial tissues of RA patients compared to samples from patients with osteoarthritis (OA) or AS. ELISA detected significantly higher levels of TXNDC5 in the blood of RA patients compared to OA, AS and systemic lupus erythematosus patients, and healthy controls. ELISA did not detect significantly different levels of anti-TXNDC5 antibody in the blood of RA, OA and AS patients and healthy controls. A total of 9 SNPs (rs9505298, rs41302895, rs1225936, rs1225938, rs372578, rs443861, rs408014, rs9392189 and rs2743992) showed significant association with RA, while 16 SNPs (rs1044104, rs1225937, rs1225938, rs372578, rs89715, rs378963, rs1225944, rs1225947, rs1238994, rs369086, rs408014, rs368074, rs1225954, rs1225955, rs13209404 and rs3812162) showed significant association with AS. Taqman SNP assay demonstrated that rs443861 has an association with RA, which correlates with the microassay results.ConclusionsTXNDC5 is up-regulated in synovial tissues of RA patients. TXNDC5 has a genetic effect on the risk of RA and AS.

Expression and citrullination of keratin in synovial tissue of rheumatoid arthritis

Keratin is the main component of cellular intermediate filaments, and its post-translational modification plays an important role in cell differentiation and apoptosis, as well as disease states. The conversion of peptidylarginine to citrulline, termed citrullination, is particularly involved in the pathogenesis of rheumatoid arthritis (RA). Immunohistochemistry using an antibody mixture that broadly recognized various keratin forms detected cytokeratin in many cells in the area lining the synovial membrane of RA. Furthermore, double immuno-florescent labeling showed that the cells expressing cytokeratin were also positive for citrulline when they were in the vicinity of extracellular deposits or approached the exterior of the synovial membrane. Western blot analysis demonstrated citrullination of keratin purified from RA synovial tissue by immuno-precipitation. The above results indicate the presence of citrullinated cytokeratin in synovial membranes in RA.