Complement-regulatory protein expression and activation of complement cascade on erythrocytes from patients with rheumatoid arthritis (RA)

It has been previously reported that the expression of the complement receptor, CR1, on erythrocytes is reduced in patients with RA and that the reduced expression of CR1 is related to disease activity. In this study we investigate the role of other regulatory proteins, i.e. decay-accelerating factor (DAF) and CD59 (membrane inhibitor of reactive lysis), in the pathogenesis of RA by checking the expression of DAF and CD59 on erythrocytes of RA patients to establish whether reduced expression of DAF and CD59 on erythrocytes could be related to increased ability of erythrocytes to activate complement in RA. Flow cytometry was used to measure the expression of DAF and CD59 on erythrocytes from RA patients as well as the deposition of C3 fragments occurring in vivo or after in vitro complement activation. Significantly reduced expression of DAF and CD59 was observed on erythrocytes of RA patients. A significant inverse relationship was observed between DAF expression and in vitro complement activation, whereas no significant relationship between CD59 and complement activation was observed. Finally, we demonstrated an inverse relationship between CH50 activity and DAF expression. Thus, determination of DAF on erythrocytes can emerge as an additional tool in the assessment of extent of complement activation in RA

MICRORNA-33A REGULATES CHOLESTEROL SYNTHESIS AND CHOLESTEROL EFFLUX RELATED GENES IN OSTEOARTHRITIC CHONDROCYTES

Introduction: Several studies have shown that osteoarthritis (OA) is strongly associated with metabolism-related disorders, highlighting OA as the fifth component of the metabolic syndrome (MetS). On the basis of our previous findings on dysregulation of cholesterol homeostasis in OA, we were prompted to investigate whether microRNA-33a (miR-33a), one of the master regulators of cholesterol and fatty acid metabolism, plays a key role in OA pathogenesis. Methods: Articular cartilage samples were obtained from 14 patients with primary OA undergoing total knee replacement surgery. Normal cartilage was obtained from nine individuals undergoing fracture repair surgery. Bioinformatics analysis was used to identify miR-33a target genes. miR-33a and sterol regulatory element-binding protein 2 (SREBP-2) expression levels were investigated using real-time PCR, and their expression was also assessed after treatment with transforming growth factor-beta 1 (TGF-beta 1) in cultured chondrocytes. Akt phosphorylation after treatment with both TGF-beta 1 and miR-33a inhibitor or TGF-beta 1 and miR-33a mimic was assessed by Western blot analysis. Furthermore, we evaluated the effect of miR-33a mimic and miR-33a inhibitor on Smad7, a negative regulator of TGF-beta signaling, on cholesterol efflux-related genes, ATP-binding cassette transporter A1 (ABCA1), apolipoprotein A1 (ApoA1) and liver X receptors (LXR alpha and LXR beta), as well as on matrix metalloproteinase-13 (MMP-13), using real-time PCR. Results: We found that the expression of miR-33a and its host gene SREBP-2 was significantly elevated in OA chondrocytes compared with normal chondrocytes. Treatment of cultured chondrocytes with TGF-beta 1 resulted in increased expression of both miR-33a and SREBP-2, as well as in rapid induction of Akt phosphorylation, whereas TGF-beta-induced Akt phosphorylation was enhanced by miR-33a and suppressed by inhibition of miR-33a, as a possible consequence of Smad7 regulation by miR-33a. Moreover, treatment of normal chondrocytes with miR-33a resulted in significantly reduced ABCA1 and ApoA1 mRNA expression levels and significantly elevated MMP-13 expression levels, promoting the OA phenotype, whereas miR-33a's suppressive effect was reversed using its inhibitor. Conclusions: Our findings suggest, for the first time to our knowledge, that miR-33a regulates cholesterol synthesis through the TGF-beta 1/Akt/SREBP-2 pathway, as well as cholesterol efflux-related genes ABCA1 and ApoA1, in OA chondrocytes, pointing to its identification as a novel target for ameliorating the OA phenotype