The Role Of CD147 In The Pathogenesis Of Atherosclerosis In Rheumatoid Arthritis

Objective: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. Among these inflammatory cells and cytokines , CD147 , a cell surface glycoprotein , arouses great interest.Extracellular matrix metalloproteinase inducer (EMMPRIN) / CD147 is enriched on the surface of most tumor cells and shown to stimulate underlying stromal cells to produce elevated levels of MMPs. These elevated levels of MMPs become concentrated and provide a mechanism for tumor cells to invade and metastasize into the extracellular matrix. Most of the inflammatory cells in RA express CD147,indicating that CD147 may play an important role in the RA pathology.In addition, CD147 and MMP have also been shown to be expressed in human coronary atherectomy tissue, which may provide a mechanism for atherosclerotic plaque growth and potentially for lesion destabilization.Recently study showed that oxidized low-density lipoproteins stimulate CD147 release by coronary smooth muscle cells , an effect which seems to result from enhanced CD147 shedding. In turn , soluble CD147 might accelerate extracellular matrix degradation in atherosclerotic plaques and thereby promote plaque growth and plaque destabilization. Similarly, our laboratory has demonstrated that CD147 can shed from the human peripheral blood monocytes of RA patients or it can be directly secreted into the synovia, which play a role in the RA pathology.Although sCD147 expression appeared to be associated with atherosclerosis development, there is no detailed mechanism between atherosclerotic risk factors and sCD147 in RA. Studying these problems facilitated us to evaluate the atherosclerotic risk factors , pave the way of finding new therapy and decrease the risk of cardiovascular events in RA. Therefore, we aimed to :1. study the relationship between sCD147 and lipid level in serum of RA; 2. establish the Mo/Mac-derived foam cells model; 3. investigate the expression kinetics of CD147 in U937 cell-derived foam cells, and their relationship with atherosclerosis; 4. demonstrate the involvement of CD147 in the development of atherosclerosis and investigate its expression profile in monocytes, macrophages, and foam cells as well as the effects of foam-cell formation on MMP-2, 9 expression; 5. study the influence of oxidized low-density lipoproteins (ox-LDLs) on cell-associated CD147 and sCD147 expression in GM-CSF–differentiated human peripheral blood monocytes; MMP-2, 9 production by HCF after co-culturing with sCD147. Meanwhile the role of antagonistic peptide(AP-9)in the interaction of sCD147 with HCF.Methods:1. The sCD147 level in patients with RA, CAHD and healthy volunteers were detected by ELISA ; The disease activity score (DAS28) in RA patients was evaluated and the correlation between sCD147 level and DAS28 score was analyzed; The serum lipid level in patients with RA was detected by an automatic biochemical analyzer and the correlation between sCD147 and lipid level in serum was analyzed.2. GM-CSF–differentiated human peripheral blood monocytes incubated with different concentration of ox-LDL for 48 h in RA, CAHD and healthy then Mac-derived foam cells were established; After and before PMA–differentiated U937 cells incubated with different concentration of ox-LDL for 48 h then U937 Mo/Mac- derived foam cells were established.The foam cells were identified by High performance liquid phase chromatography and oil red O dyeing.3. The U937 cells were incubated with different concentrations of ox-LDL for 48 h. Then under 80mg/L ox-LDL , the U937 cells were incubated for different time duration. CD147 was analyzed at protein and mRNA levels in U937 foam cells by flow cytometry, and RT-PCR.4. U937 cells were stimulated with PMA and were allowed to differentiate into macrophages during culture for 7 d in plastic dishes. On day 7, the macrophages were incubated with various concentrations of oxidized low-density lipoprotein in a serum-free medium for 48 h to permit their development into foam cells. The protein and mRNA expression levels of CD147 in the U937 cells, macrophages, and foam cells were determined by immunofluorescence flow cytometry and RT-PCR. To assess the effects of lipid loading in macrophages on the CD147 level and MMP-2 , 9 expression in supernatants , ELISA, western blotting, and gelatin zymography was conducted.5. Sample of human peripheral blood were obtain from patients with RA(15), CAHD(17) and healthy donor volunteers(12). Monocytes were isolated from peripheral blood. GM-CSF–differentiated human peripheral blood monocytes incubated with different concentration of ox-LDL for 48 h. CD147 expression in foam cells was analyzed by Flow cytometry and western blotting; sCD147 level in supernatants was analyzed by ELISA and western blotting; CD147 mRNA in foam cells was analyzed by RT-PCR; Zymography analysed MMP-2, 9 release by HCF after co-culturing with sCD147. In additional , the influence of the inhibitor AP-9 on MMP-2, 9 was also observed.Results:1.The level of sCD147 in serum with RA patients was significantly higher than that in patients with CAHD and healthy volunteers. sCD147 level in the RA group with high DAS28 score was significantly higher than that with low or media DAS28 score. In RA patients , elevated total cholesterol(TC)and triglyceride(TG)was positively correlated with serum sCD147 level(r=0.84,P<0.05; r=0.87,P<0.05; while lightly elevated , normal TC and normal TG had no correlation with serum sCD147 level (r=0.41,P=0.21;r=0.14,P=0.57;r=0.49,P=0.87). Elevated or lightly elevated LDL-C was positively correlated with serum sCD147 level(r=0.86,P<0.05;r=0.81,P<0.05), while there was no correlation in the group with normal LDL-C level (r=0.78,P=0.22). The high density lipoprotein-cholesterol (HDL-C)level in RA patients had no correlation with serum sCD147 level(r=0.04,P=0.96;r=0.13,P=0.87).2.The CE/TC in lipid-loaded foam cells is 60% , which was higher than that in nonlipid-loaded cells(15.52%). 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.3. The expression dose-kinetics demonstrated that the CD147 showed the peak expression induced by ox-LDL 25mg/L;Time-kinetic studies revealed that the CD147 levels showed the peak expression at 6 h ;CD147 mRNA levels showed the peak expression induced by ox-LDL 25 mg/L,and the maximum mRNA level of CD147 was shown at 6h. However, ox-LDL enrichment of foam cells had no further effect on CD147 expression.4.During the PMA-induced differentiation of U937 cells into macrophages in the 7-d culture, CD147 mRNA expression increased 4- to 6-fold in the differentiated monocytes when compared with the undifferentiated ones. The mean fluorescence intensity of CD147 in the differentiated U937 cells was higher than that in the undifferentiated U937 cells (P<0.001) on days 1 and 3. On days 5 and 7, the mean fluorescence intensity of CD147 in the differentiated U937 cells was lower than that in the undifferentiated ones (P<0.05).The mean fluorescence intensity of CD147 gradually reduced in the foam cells that were treated with various concentrations of ox-LDL when compared with the untreated macrophages. In contrast, the expression of CD147, MMP-2 and MMP-9 was steadily upregulated in the corresponding culture supernatant (P<0.05), however, the mRNA expression of CD147 remained unaltered throughout the experiment.5. CD147 expression gradually reduced in the foam cells that were treated with various concentrations of ox-LDL when compared with the untreated macrophages(P<0.05). The declining trend is RA, CAHD and healthy respectively. In contrast, sCD147 was steadily upregulated in the corresponding culture supernatant. The trend of escalation is RA, CAHD and healthy respectively. In addition, sCD147 could stimulate HFC to produce elevated levels of MMP-2 and MMP-9 , which could be suppressed by AP-9.Conclusions:1. sCD147 may be involved in the pathogenesis of RA and associated with the disease activity. Elevated sCD147 level may associated with dyslipidemia in RA.2. The cell model was established in vitro and accorded with foam cells in biology and morphology.3. CD147 expression was enhanced in the U937 foam cells; CD147 expression was mainly up-regulated by low dose of ox-LDL; Ox-LDL has no further effect on CD147 mRNA expression.4. CD147 mRNA and protein expression were markedly increased 4- 6 fold on GM-CSF–induced monocyte to macrophage differentiation. Such increases in a protein maybe provide the differentiated macrophage with a mechanistic framework to perform specific functional activities at this stage of cellular maturation. In addition, lipid loading in macrophages reduced cell-associated CD147 expression, while it up-regulated the CD147 levels and secretion and activation of MMP-2, 9 in the culture supernatant. Howerver, lipid loading in macrophages had no further effect on CD147 mRNA expression. we hypothesize that the sCD147 maybe shed from the foam cells surface.5. Ox-LDLs maybe stimulate the release of sCD147 in differentiated PBMo; sCD147 stimulated MMP-2, 9 synthesis in HCF; AP-9 treatment could inhibit this process.