Effect Of Cyclophilin A On Monocyte-derived Foam Cell

Aim:Rheumatoid arthritis (RA) is a chronic destructive autoimmune disease characterized by the inflammation and progressive destruction of distal joints. Until now, the pathogenesis of RA has not been understood well and the therapy of RA just controls the symptom and development of RA, but doesn’t cure it thoroughly. Studies suggest that the mortality rate is approximately twice as high in patients with RA compared with the general population. Cardiovascular disease accounts for 70% of excess mortality in RA patients. Especially, accelerated atherosclerosis could be responsible for it. Systemic inflammatory response in RA is central to the accelerated atherogenesis.Cyclophilin A (CypA) has been classified as an immunophilin and has a variety of intracellular functions. It is a recently reported cytokine that acts as a chemoattractant to human monocytes, neutrophils, eosinophils, and T cells. It is also the major target of immunosuppressive drug cyclosporin A (CsA). It has multiple functions, such as peptidyle-prolyl-isomer-ase activity (PPIase) for intracellular signaling, protein trafficking, and the regulation of other proteins activity and contributes to inflammation. CypA is a secreted molecule that has a physiological and pathological role in cardiovascular diseases, making it a potential biomarker and mediator in cardiovascular diseases, such as vascular stenosis, atherosclerosis, and abdominal aortic aneurysms. Previous studies showed CD147 which is the receptor of CyPA overexpression on synoviocytes in RA synovium enhances the production of MMP-2,-9 to promote the cartilage destruction in RA.Recently study showed that upon coincubation with platelets, CD34+ progenitor cells have the potential to differentiate into foam cells. EMMPRIN is upregulated during the differentiation process. If the CypA/EMMPRIN activation pathway enhances the production of MMP may play a relevant role in promoting the vulnerability of atherosclerotic plaques? So in this study, we want to identify whether CypA can regulate the differentiate process of foam cells to promote the progression of atherosclerosis.Methods:1. Established THP-1 derived foam cells model.Sphorbol12-myristate13-acetate (PMA)-differentiated THP-1 cells and incubated with ox-LDL for 48h then THP-1 derived foam cells were established. The foam cells were identified by oil red O dyeing.2. CypA mediated adhesion of the foam cells differentiates process.Flow cytometric analysis of membrane-associated CD54 and CD147 expression on the surface of monocytes, macrophages and foam cells with different concentration of CypA. Cell invasion assay detected the adhesion ability during the differentiate process of foam cells both in the absence or presence of CypA. CsA , c7b8f10 or Hab18mab was used to interrupt the interaction between CypA and CD147,in order to identify whether CD147 facilitates CypA’s function. 3. CypA stimulation to MMP-2,-9 expressions and mediated invasion ability during foam cells differentiates process.Foam cells were stimulated by different concentration of CypA, the cells were run RT-PCR for MMP-2,-9 mRNA levels. The supernatants were collected for detection of MMP-2,-9 protein activity by gelatin zymography. At the same time, CsA , c7b8f10 or Hab18mab was used to interrupt the interaction between CypA and CD147 to identify whether CD147 facilitates CypA’s function.Invasion assay experiment was used to observer invasive ability of foam cells differentiates process under CypA stimulation. CsA, c7b8f10 or Hab18mab was used to interrupt the interaction between CypA and CD147 to identify whether CD147 facilitates CypA’s function.Results:1.Successfully Established THP-1 derived foam cells model. After lipid-loaded foam cells treated with Oil red O, many red pellets were found in the plasma of the cells. Thus the cell model accorded with foam cell in biology and morphology.2. CypA increased adhesion ability and antagonists inhibitory effect of foam cells.The Flow cytometry analysis revealed that the MFI of membrane associated CD54 and CD147 were significantly increased in the foam cells when compared with the monocytes and macrophages (P<0.05); In addition to this, CypA significantly up-regulated CD54 expression in foam cells (P<0.05). Accordingly, we examined adhesion of foam cells with CypA stimulation to confirm that the ability of adhesion of THP-1 cells differentiation into the foam cells were increased and CypA increased the ability of adhesion of foam cells(P<0.05); We found that CsA、c7b8f10 and HAb18mAb decreased the ability of foam cells adhesion(P<0.05). 3. CypA stimulation to MMP-2,-9 expressions and invasion ability and antagonists inhibitory effect during foam cells differentiates process.After differentiation, MMP-2, 9 production were significantly increased in macrophages-derived foam cells(P<0.05); CypA significantly increased MMP-2, 9 expression in the foam cells(P<0.05); We found that different antibody respectively decreased the foam cells MMP-2, 9 expression, both in the absence or presence of CypA(P<0.05).A higher number of foam cells (cells/filter) which differentiated from monocytes were found to have invaded through transwell chambers than that of monocytes and macrophages(P<0.05); The number of invading foam cells in CypA’s stimulation group was much bigger than that in negative control group(P<0.05); With or without CypA stimulation, the number of invading cells were both decreased when the monocytes or macrophages were pretreated with different antibody respectively(P<0.05).Conclusions:In conclusion, our data suggest that the ability of adhesion and invasion and MMPs production were increased during the process of THP-1 cells differentiation into the foam cells. In addition, CypA increased the above biological activity of foam cells in atherosclerosis. These findings indicating that during RA process, CypA might participate in the regulation of foam cells differentiation, inducing MMP production, activation and secretion, and enhance cell adhesion and invasion, thus participate in atherosclerosis occurrence and development. Different antibody like CsA, c7b8f10 and HAb18mAb inhibitory effect contributes to further clarify the mechanism of atherosclerosis occurrence and development and explore new ways of treatment..