PTX3Involves In The Phagocytosis Of Apoptotic Macrophages In Advanced Lesions Of Atherosclerosis

Part Ⅰ The Cell Model of Apoptotic Macrophages in Advanced Lesions of AtherosclerosisBackground Macrophages are the most commmon cells in atherosclerosis. They involve in the development of atherosclerotic lesions. There are lots of causes to induce macrophages apoptosis in atherosclerotic plaques, the most familiar one is Ox-LDL. But Ox-LDL-induced apoptosis is associated with the type of macrophages and the dose of Ox-LDL, sometimes Ox-LDL can inhibit macrophages apoptosis. According to the characteristics of free cholesterol accumulated in macrophages in atherosclerotic plaques, cholesterol-mediated cytotoxicity maybe one important cause of macrophages apoptosis. Some studys had confirmed that free cholesterol was accumulated in primary macrophages when they were cultured with Ac-LDL and inhibitor of ACAT(compound58035). When these cells underwent apoptosis, they appeared similar characteristics with those in atherosclerotic plaques.Objective In order to discuss the mechanism of apoptosis in advanced atherosclerosis, this study will build the cell model of macrophages apoptosis in advanced atherosclerosis according to the method mentioned aboved.Methods Macrophages were obtained from mice intraperitoneally injected with0.5ml of phosphate buffered saline (PBS) containing40μg concanavalin A. Macrophages were harvested3days later by peritoneal lavage. The macrophages were cultured in DMEM supplemented with10%fetal bovine serum for24-48hours until reaching confluence. Then these cells were divided into four groups:(Ⅰ) Free cholesterol-induced apoptotic macrophages (FC-AMs) group, macrophages were incubated with acetyl-LDL and compound58035for another16-20hours (Ⅱ) Ultraviolet-induced apoptotic macrophages (UV-AMs) group, it was generated by irradiating the macrophages for15min (Ⅲ) Necrotic cells group, macrophages were induced by incubating macrophages at56℃for30min (Ⅳ) Live cells group, they were cultured normally. Apoptosis was analyzed using the Annexin V/PI assay through FACS and immunofluorescence cells chemical method.Results In LSCM, few macrophages were stained with Annexin V. The proportion of Annexin V+macrophages was approximately2.64%in live cells group. The proportion of Annexin VT macrophages in FC-AMs and UV-AMs were increased to9.67%and10.77%, respectively. FC-AMs group and UV-AMs group were compared to live cells group, the differences considered as statistically significant, P<0.005. The differences between FC-AMs and UV-AMs were not considered as statistically significant. All of the cells were labeled with Annexin V and PI in necrotic cells group. In FACS, The proportion of Annexin V+macrophages in FC-AMs were20.9%including12.7%of late apoptotic cells. The proportion of Annexin V+macrophages in live cells group were10.6%including6.6%of late apoptotic cells.Conclusion The joint role of acetyl-LDL and compound58035could induce apoptosis of peritoneal macrophages isolated from C57BL/6mice. The differences between FC-AMs and UV-AMs were not considered as statistically significant. We were successfully to construct the cell model of macrophages apoptosis in advanced atherosclerosis plaques. It prepared for clarifying the machenism of clearance of apoptotic cells in the following study. Part Ⅱ Detection And Significance of PTX3on Late Apoptotic MacrophagesBackground PTX3is a member of the pentraxin superfamily, which is a family of acute phase proteins and includes two classical short pentraxins, CRP and SAP. PTX3is produced at sites of inflammation by severa cells, primarily by dendritic cells, macrophages, fibroblasts, activated endothelial cells, and renal cells. Atherosclerosis is actually the long chronic imflammation involved by macrophages,endothelial cells and so on. The levels of PTX3were increased in advanced atherosclerotic plaques and plasm of acute myocardial infarction patients. PTX3reaches peak values after6to8hours compared with24hours for CRP. In patients with unstable angina pectoris, plasma levels are significantly elevated more than3-fold (6.20ng/mL). CRP will be replaced by PTX3for it more application value of clinic in forecasting cardiovascular events. This suggests that PTX3is associated with atherosclerosis, and may involve in the development of atherosclerosis.Objective Based on the first part of dissertation, we will discuss the relationship betweent PTX3with apoptotic macrophages in advanced atherosclerosis. Providing the evidences for the mechanism study of PTX3in atherosclerosis.Methods Building the cell model of apoptosis in advanced atherosclerosis according to the methods of first part. Detecting the levels of PTX3on FC-AMs with indirect immunofluorescence cells chemical method and FACS. Live cells group,UV-AMs group and necrotic cells group were designed to be control groups. The software of ImagePro Plus was used to analyse the results of LSCM.Results In LSCM, PTX3levels were low in live macrophages, the mean optical density of PTX3was0.0191±0.0266. Apoptotic macrophages (UV-AMs and FC-AMs) showed higher levels of PTX3, the mean optical density were0.2645±0.0682and0.2142±0.0291for UV-AMs and FC-AMs, respectively. Little or no optical density was detected in isotype control group and necrotic macrophage group. The levels of PTX3in FC-AMs group was compared to the levels of PTX3in isotype control group, the differences were considered as statistically significant, P<0.005; The levels of PTX3in FC-AMs group was compared to the levels of PTX3in live cells group, the differences were considered as statistically significant, P<0.005. The results of FACS showed that a higher percentage of apoptotic macrophages (Annexin V+) and higher PTX3levels were observed, specially for late apoptotic macrophages (Annexin V+/PI+) when they were analysed with Annexin V-FITC/PI assay and fluorescent antibody separately. Compared to Annexin V-/PI-area and Annexin V-/PI+area, Annexin V+area showed more PTX3levels.Conclusion The levels of PTX3were low in live cells group,early apoptosis group of FC-AMs and necrotic cells group. But the levels of PTX3were high in FC-AMs and UV-AMs. The mechanism may be associated with late apoptosis of macrophages. Part Ⅲ PTX3Mediates the Phagocytosis of Late Apoptotic Macrophages Background PTX3is an important component of the humoral innate immune system, which involves in the clearance of many kinds of apoptotic cells(such as Jurkat cells) by activating the classical complement pathway. PTX3plays an important role in defending pathogen infection and autoimmune diseases. Apoptosis is one continuous event in atherosclerotic plaques. In early atherosclerotic plaques, rapid phagocytic clearance of the apoptotic cells(efferocytosis) prevents subsequent cell leakage, and actually activates anti-inflammatory cell-signaling pathways. Efficient clearance of the apoptotic cells renders this process not only harmless but possibly beneficial. In advanced lesions, however, because of defective clearance of the apoptotic cells, macrophage apoptosis is associated with secondary necrosis and the increased inflammation. These enhance the formation of unstable plaques. It tells us that efferocytosis plays an important role in the development of athrosclerosis. Also, PTX3in plasm is increased obviously in acute coronary syndrome patients(ACS). As an important component of the humoral innate immune system, which involves in the clearance of many kinds of apoptotic cells, we do not know if PTX3involves in the efferocytosis in atherosclerotic lesions by now.Objective Discuss whether PTX3involves in the efferocytosis in advanced atherosclerotic lesions. In order to supply evidences to clarify the mechanism of plaque vulnerability in ACS.Methods Building the cell model of apoptosis in advanced atherosclerosis according to the methods of first part(FC-AMs). Collecting FC-AMs and staining them with red fluorescent dye DiI. Then incubating these cells with16B5(5μg/ml and50μg/ml) or isotype control(5μg/ml). The incubated FC-AMs were added to a monolayer of fresh phagocytes (they were isolated from C57BL/62hours ago). Then incubated in37℃for30min。The unswallowed FC-AMs were wished off vigorously. Phagocytosis was observed with LSCM. Calculating the ratio of phagocytosis.Results We observed that the red shrinked FC-AMs were ingested in the phagocytes, which contained some residual cellular debris. We concluded that FC-AMs were ingested by phagocytes rather than binding to the phagocytes. In16B5group (50and5μg/ml), the percentage of phagocytosis were14.63±1.56%and26.19±4.12%, respectively. In isotype control group (50μg/ml), the percentage of phagocytosis was36.4±4.25%. The differences betweent5μg/ml16B5group and50μg/ml16B5group were considered as statistically significant, P<0.05; The differences betweent50μg/ml16B5group and50μg/ml isotype control group were considered as statistically significant, P<0.005.Conclusion FC-AMs were swallowed by phagocytes isolated from C57BL/6mice2hours ago. Phagocytosis was inhibited by anti-PTX3monoclonal Ab16B5in a dose-dependent manner. PTX3mediated the phagocytosis of late apoptotic macrophages in a cell model of advanced atherosclerosis.