Xinfeng Yan

Identification of Proteins with Increased Expression in Rheumatoid Arthritis Synovial Tissues

Objective. A proteomic approach was applied to discover novel rheumatoid arthritis (RA)-specific proteins by comparing the expression profiles of synovial membranes from patients with RA, osteoarthritis (OA), and ankylosing spondylitis (AS). Methods. Synovial tissues were collected from patients with RA (n = 10), OA (n = 10), or AS (n = 6), and healthy controls matched for age and sex. Proteins were separated by 2-dimensional polyacrylamide gel electrophoresis, and the proteins with significantly increased expression in the RA samples were subject to matrix-assisted laser adsorption-ionization time-of-flight spectrometry. Results were verified using Western blot and immunohistochemistry. Levels of the candidate proteins were measured within plasma and synovial fluids from the RA patients (n = 30), who had disease duration of 3–7 years, using ELISA. Levels were also measured within plasma from unmedicated RA patients (n = 41), who had disease duration of 1–6 months. Results. Compared with the OA and AS tissue samples, the proteins Ig-kappa light-chain C region, PRDX4, SOD2, TPI, and TXNDC5 were found with increased expression in synovial tissues of RA patients. PRDX4, SOD2, TPI, and TXNDC5 had 2-fold or more increase in expression in some of the early RA plasma samples (58.55%, 31.7%, 26.8%, and 36.6%, respectively) as compared with the early OA samples and control samples. TXNDC5 had 2-fold or more increase in expression in 53.3% of blood samples and 73.3% of synovial fluid samples from patients with long disease duration of RA as compared with samples from OA and AS patients. Conclusion. Functional classification indicated that these identified proteins were related with cell differentiation, glycol metabolism, immunoactivation, and endogenous antioxidant reaction.

Carbonic anhydrase I (CA1) is involved in the process of bone formation and is susceptible to ankylosing spondylitis

IntroductionAnkylosing spondylitis (AS) is characterized by abnormal bone formation in the spine and the sacroiliac joints. In vitro assays demonstrate that carbonic anhydrase I (CA1) promotes calcium precipitation. This study investigated the function of CA1 for bio-mineralization and determined if common polymorphisms in the CA1 gene might contribute to AS risk.MethodsCalcification was induced in Saos-2 cells, a human osteosarcoma cell line, with ascorbic acid and β-glycerophosphate. Calcification was determined by Alizarin Red-S (AR-S) staining. Expressions of CA1, alkaline phosphatase (ALP), bone sialoprotein (BSP), osteocalcin (OCN), osterix (OSX) and runt-related transcription factor-2 (Runx2) were determined by real-time PCR and western blotting. The cells were also treated with acetazolamide, an anti-carbonic anhydrase drug. Genotyping was performed using Illumina VeraCode microarray in a case-control study including 51 AS patients, 267 rheumatoid arthritis (RA) patients and 160 healthy controls. The result was confirmed by Taqman assay, including 258 AS patients, 288 RA patients and 288 healthy controls.ResultsFollowing the induction of calcification, Saos-2 cells produced large amounts of calcium-rich deposits. Increased transcriptions of CA1, ALP, BSP, OCN, OSX and Runx2, essential genes for ossification, were detected in the cultured cells. Following treatmen with acetazolamide, the expression of CA1 obviously declined and mineralized nodule formation was also decreased. Illumina microarray indicates that SNP at rs7841425 also showed significant differences in allelic frequency (P = 0.01396) and genotypic frequency (P = 0.005902) between AS cases and controls. In addition, SNP at rs7827474 showed significant differences in allelic frequency (P = 5.83E-04) and genotypic frequency (P = 0.000186) between RA cases and controls (P values were adjusted to multiple comparisons). The Taqman assay revealed that rs725605 demonstrated statistically significant evidence of allele frequency (P = 0.022307) and gene frequency (P = 0.007731) for association with AS. This SNP did not show significant differences in allelic frequencies and gene frequencies between RA patients and controls.ConclusionsCA1 may play an essential role in bio-mineralization and new bone formation. The gene encoding CA1 is susceptible to AS.

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.

Investigate Pathogenic Mechanism of TXNDC5 in Rheumatoid Arthritis

Hypoxia stimulates synovial hypoperfusion in rheumatoid arthritis (RA). TXNDC5 stimulates cellular proliferation in hypoxic conditions. We previously detected increased TXNDC5 expression in synovial tissues and blood from RA patients and demonstrated that the gene encoding TXNDC5 increased RA risk. The present study investigated the pathogenic roles of TXNDC5 in RA. Transgenic mice that over-expressed TXNDC5 (TXNDC5-Tg) were generated using C57BL/6J mice and treated with bovine collagen II to induce arthritis (CIA). Synovial fibroblasts from RA patients (RASFs) were cultured and incubated with TXNDC5-siRNA or CoCl2, a chemical that induces hypoxia. CIA was observed in 80% of the TXNDC5-Tg, but only 20% of the wild-type mice (WT) developed CIA. The clinical arthritis scores reached 5 in the TXNDC5-Tg, but this index only reached 2 in the control mice. CIA TXNDC5-Tg exhibited clear pannus proliferation and bone erosion in joint tissues. A significant increase in CD4 T cells was observed in the thymus and spleen of TXNDC5-Tg during CIA. Serum levels of anti-collagen II IgG, IgG1 and IgG2a antibodies were significantly elevated in the mice. Increased cell proliferation, cell migration and TXNDC5 expression were observed in RASFs following incubation with 1 µM CoCl2. However, this effect was diminished when TXNDC5 expression was inhibited with 100 nM siRNA. TNF-alpha, IL-1α, IL-1β and IL-17 levels were significantly increased in the blood of TXNDC5-Tg mice, but the levels of these cytokines declined in the supernatant of RASFs that were treated with TXNDC5 siRNA. The expression of adiponectin, a cytokine-like mediator, decreased significantly in RASFs following TXNDC5 siRNA treatment. These results suggest that TXNDC5-over-expressing mice were susceptible to CIA. This study also suggests that hypoxia induced TXCNDC5 expression, which contributed to adiponectin expression, cytokine production and the cellular proliferation and migration of fibroblasts in RA.

PADI2 Is Significantly Associated with Rheumatoid Arthritis

Citrullination, a posttranslational modification of peptidyl arginine to citrulline, plays an essential role in rheumatoid arthritis (RA). Citrullination is catalyzed by a group of peptidylarginine deiminases (PADs) including PADI 1, 2, 3, 4 and 6. Many studies have indicated that the gene encoding PADI4 is a factor in susceptibility to RA. Some studies have detected PADI2 expression in RA synovial tissues, suggesting that PADI2 also plays an important role in the disease. This study evaluated the possible association between the PADI2-encoding gene and RA. Seventeen tag SNPs across the PAD locus were genotyped using a custom-designed Illumina 96-SNP VeraCode microarray. Peripheral blood samples were collected from patients with RA (n = 267), ankylosing spondylitis (AS, n = 51) and healthy controls (n = 160). The results of genotyping were verified using Sequenom MassARRAY in an independent cohort of 307 patients with RA, 324 patients with AS and 509 healthy controls. A western blot analysis was performed using synovial tissue from patients with RA (n = 7), osteoarthritis (OA, n = 7) and AS (n = 5) to determine the levels of expression of PADI2. A microarray analysis revealed a significant association between three selected PADI2 SNPs (rs2235926, rs2057094, rs2076616) and the presence of RA. The increased susceptibility to RA associated with rs2235926 (OR = 1.706733, 95% CI = [1.576366–1.866587], p = 0.000839) and rs2057094 (OR = 1.360432, 95% CI = [1.065483–1.869482], p = 0.003291) was further confirmed by the Sequenom MassARRAY. No tag SNPs in the PADI2 locus showed a significant association with AS. Increased expression of PADI2 was detected in RA synovial tissues compared with samples from patients with OA and AS. PADI2 is significantly associated with RA and may be involved in the pathogenesis of the disease.

Screening Citrullinated Proteins in Synovial Tissues of Rheumatoid Arthritis Using 2-dimensional Western Blotting

Objective. Citrullination, a reaction converting arginine residue into citrulline residue, is essential for autoimmunity of rheumatoid arthritis (RA). We conducted 2-dimensional Western blot analyses (2-D WB) to screen for novel citrullinated proteins in synovial tissues from patients with RA. Methods. Total proteins were extracted from the synovial membranes of patients with RA (n = 10) and pooled. Four identical 2-D electrophoresis (2-DE) gels were prepared, and 2 gels were transblotted to polyvinylidene fluoride membranes that were separately probed with sera from patients with RA (n = 10) or an anticitrulline antibody. The protein profiles of the 2-DE gels were compared with the hybridization results on a global level. The immunoreactive protein spots were collected from the 2-DE gels and identified using mass spectrometry. Proteins that were detected by both RA sera and anticitrulline antibody were considered citrullinated proteins. The result was confirmed through routine WB, immunoprecipitation, and ELISA. Autoantibodies against these potential antigens were also examined in the blood of patients with RA by ELISA. Results. RA sera and the anticitrulline antibody on 2-D WB detected α-1-antitrypsin (A1AT), dynein heavy-chain 3, fibrinogen β chain, keratin type II cuticular Hb4 (KRT84), lumican, tubulin β-chain (TUBB), and vimentin. A1AT, KRT84, and TUBB had high expression in the synovial membranes (n = 5) of patients with RA and A1AT and KRT84 had high expression in RA synovial fluids (n = 40). A1AT, KRT84, and TUBB immunoprecipitated from synovial tissues showed citrullination. A high level of autoantibodies against KRT84 was detected in the blood of patients with RA (n = 92) compared to that of healthy controls (n = 92). Conclusion. Our study identified some new citrullinated proteins in RA synovial tissues using 2-D WB.

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.

Tim3+Foxp3+Treg Cells Are Potent Inhibitors of Effector T Cells and Are Suppressed in Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a chronic autoimmune disorder. Earlier studies have demonstrated that regulatory T (Treg) cells, the main cell type mediating immune tolerance, appeared to be enriched in the inflamed synovial tissues. It is still unclear why the Treg cells in RA patients are unable to limit exacerbated inflammation. Here, we found that the frequency of Tim3+Foxp3+ Treg cells, which were potent suppressors of proinflammatory responses, was downregulated in RA patients. Reduction in Tim3+Foxp3+ Treg frequency was correlated with increased RA disease activity. Furthermore, we observed that Tim3+Foxp3+ Tregs were expressed more interleukin (IL)-10 than Tim3−Foxp3+ Tregs. CD4+CD25+Tim3+ T cells had higher capability of inhibiting interferon (IFN)-γ and tumor necrosis factor (TNF)-α secretion from T cells and peripheral blood mononuclear cells (PBMCs) than CD4+CD25+Tim3− T cells. Compared to that in healthy individuals, CD4+CD25+ T cells in RA patients were less potent in suppressing IFN-γ and TNF-α production from PBMCs. Blocking Tim3 on CD4+CD25+ T cells from healthy controls resulted in an elevation of IFN-γ and TNF-α production from PBMCs, suggesting that Tim3 expression on CD4+CD25+ T cells was required for optimal Treg function. However, this phenomenon was not observed in RA patients. In conclusion, our study suggested that the CD4+CD25+Foxp3+ Treg cells from RA patients demonstrated a reduction of Tim3 and were less functional than Treg cells from healthy controls in a Tim3-related manner.

PGK1, a glucose metabolism enzyme, may play an important role in rheumatoid arthritis

BackgroundSome studies have indicated that glucose metabolism plays an important role in the pathogenesis of rheumatoid arthritis (RA). This study aimed to find the novel genes affecting glucose metabolism in RA.Materials/methodsSynovial tissues of collagen-induced arthritis (CIA) were analyzed with Rat Glucose Metabolism RT2 Profiler™ PCR Array to screen those genes with special expressions in glucose metabolism. Real-time PCR, western blotting, and ELISA were used to confirm the result in synovial tissues and blood of human RA. Culture synovial fibroblast cells (RASF) was treated with siRNA to suppress expressions of the target genes. CCK-8 cell proliferation assay and two-compartment transwell system were performed to examine cell proliferation and cell migration of the treated RASF.ResultsBoth PCR array and real-time PCR detected the up-regulation of ENO1, HK2, and PGK1 and the down-regulation of PCK1 and PDK4 in synovial tissues of CIA rats. Real-time PCR and western blotting detected the increased expression of ENO1 and PGK1 in RA synovial tissues. ELISA detected a high level of PGK1 in the blood of RA patients. Decreased cell proliferation and cell migration capabilities were significantly detected in RASF following treatment of anti-PGK1 siRNA. IL-1β and IFN-γ rather than TNF-α and IL-1α levels were significantly declined in supernatants of the treated RASF.ConclusionsPGK1, a glycolytic enzyme catalyzing the conversion of 3-phosphoglycerate into 2-phosphoglycerate, has increased expression in synovial tissues and blood of RA, which may be involved in pro-inflammation and synovial hyperplasia of the disease.

Effector T helper cell populations are elevated in the bone marrow of rheumatoid arthritis patients and correlate with disease severity

This study is to investigate the frequencies of T-helper (Th)22, Th17 and Th1 cells and the levels of related cytokines in subchondral bone marrow in patients with rheumatoid arthritis (RA). Bone marrow and peripheral blood samples were collected from RA, osteoarthritis (OA) patients and healthy controls. The frequencies of Th22, Th17, and Th1 cells were examined by flow cytometry. Levels of interleukin (IL)-22, IL-17 and IFN-γ were examined by ELISA. Disease Activity Score in 28 joints (DAS28) of RA patients were analyzed. Bone marrow Th22, Th17 and Th1 cells in RA patients were markedly increased comparing to OA or healthy controls. Each T cell subset in bone marrow was elevated comparing with that in the peripheral blood in RA patients. Consistently, plasma levels of IL-22 and IL-17 were elevated in RA patients, and the elevation was more notable in the bone marrow than in the peripheral blood. Additionally, the percentages of Th22, Th17 and Th1 cells as well as the levels of IL-22 and IL-17 in bone marrow were positively correlated with DAS28. These results suggest that local pro-inflammatory Th cells are elevated in bone marrow, which may play an important role in situ in RA and contribute to the pathogenesis of in RA.