Roles Of TIPE2Induced Apoptosis Of Macrophage In Atherosclerosis

Atherosclerosis, a disease of large arteries, has been widely recognized as an inflammatory disease and will soon become the pre-eminent heath problem worldwide, and is characterized by lipid deposition in aorta,hyperplasia of smooth muscle and connective tissue, intimal fibrous thickening and plague formation, However, its pathogenesis is still unknown today. Recent studies have indicated that local and systemic inflammation as well as immunological reactions underpin all stages of the development of atherosclerosis. Initiation and progression of vascular inflammation involves a complex cellular network including monocytes/macrophages, lymphocytes, endothelial cells, dentritic cells, mast cells and smooth muscle cells, with macrophages as major contributors. Macrophages are well known as the major immune cells in innate immune system. Activated macrophage can promote the development of AS by secreting inflammatory cytokines,matrix metalloproteinase et all.Many studies have validated that the apoptosis of macrophage plays an important role in the development of atherosclerosis, which also can cause ruptured plaque and thrombosis. Macrophges bridge innate and adaptive immunity. Emerging evidences have shown that the apoptosis of macrophage involves the deveploment of atherosclerosis in multiple levies. The efforts to elucidate immunological regulatory mechanisms will provide new insight into understanding the mechanisms of the devoplement of atherosclerosis and beneficial roles in preventing and treating this disease based on immunological strategies.TIPE2/tumor necrosis factor-alpha (TNF-a) induced protein-8-like2,was identified as a novel anti-inflammatory protein in2008. TIPE2is preferentially expressed in inflamed organs and lymphoid tissues, such as thymus, spleenm lymph node, intestine and myeloid cells. Further more, TIPE2was also detected in non-lymphoid tissues, such as endocrine tissues and reproductive organs. The study found that TIPE2-/-mice can show the symptoms such as spleen swelling,weight loss,leukocytosis et all and eventually cause death due to multiple organ inflammation. At the same time,study found that the mononuclear cell of hepatitis B patient,the expression of TIPE2was down-regulation and negative correlation with AST and ALT. TIPE2was up-regulation in glomerulus,it indicated that TIPE2may be related to diabetic nephropathy.Study also found that TIPE2was down-regulation in he mononuclear cell of SLE patients and it indicated that TIPE2may be related to SLE.TIPE2plays an essential role in the maintenance of immune homeostasis through negatively regulating innate and adaptive immunity. Our previous study found that TIPE2-/-mice can inhibit the development of atherosclerosis and the macrophage of TIPE2-/-could resist to LPS-induced cell death. The results suggest that TIPE2may play an important role in the development of atherosclerosis throught regulating the death of macrophage. Because the cell apoptosis and autophagy can both cause cell death. We studied the relation between TIPE2and the apoptosis related molecules in macrophages and the roles of TIPE2in the autophagy of macrophage in atherosclerosis by using in vitro cell test to find out the molecular mechanism that TIPE2regulates the cell death. Our work thus provides new insights into immunological mechanisms in atherogenesis and also suggests that TIPE2may be a novel potential new drug target for treating this disease.Materials and Methods1. Mouse Induction and activation of peritoneal macrophagesFor isolation of elicited peritoneal macrophages, male wild type and TIPE2deficient mice (8-10w) were injected intraperitoneally with1.0ml of6%sterile starch. Four days after the injection, cells were harvested by intraperitoneal lavage with ice-cold PBS, cultured in high-glucose version of DMEM medium (GIBCO-BRL, Carlsbad. CA,USA) supplemented with10%FBS at37℃/5%CO2 overnight. Peritoneal macrophages were then stimulated with oxLDL to investigate the regulatory role of TIPE2in the apoptosis and autophagy of macrophag, the regulatory role of TIPE2in transcription of genes which related to death receptor pathway,mitochondrial apoptotic pathway and endoplasmic reticulum and investigate related signaling pathways.2. The regulation of TIPE2on the cell death of macrophages2.1Observe the cell death of macrophagesPeritoneal macrophages were harvested as described above. Peritoneal macrophages were stimulated with50μg/ml oxLDL.Then the cells were stained by trypan blue. Observed under microscope and calculated the rate of death cells.2.2Detection the autophagy of macrophagesPeritoneal macrophages were harvested as described above. Peritoneal macrophages were stimulated with50μg/ml oxLDL. Then the cells were collected and levels of LC3and Atg5were detected by Western blot and realtime-PCR respectively.2.3Observe the apoptosis of macrophagesPeritoneal macrophages were harvested as described above. Peritoneal macrophages were stimulated with50μg/ml oxLDL.Then the cells were stained by Tunel. Observed under microscope and calculated the rate of apoptosis cells.2.3.1Detection of the macrophages apoptosisPeritoneal macrophages were harvested as described above. Peritoneal macrophages were stimulated with50μg/ml oxLDL. Cells were stained by annexinV/PI cells apoptosis assay kit. The number of macrophages stained by annexinV/PI was counted by FCM.2.3.2The regulation of TIPE2on the activation of caspase3in macrophagesPeritoneal macrophages were harvested as described above. Peritoneal macrophages were stimulated with50μg/ml oxLDL. Then the cells were collected and levels of caspase3and cleaved-caspase3were detected by Western blot.3. The regulation of TIPE2on transcription of related genes in macrophages3.1Detection extrinsic pathway of macrophage Peritoneal macrophages were harvested as described above. Peritoneal macrophages were stimulated with50μg/ml oxLDL. Then the cells were collected. RNA was extracted and reversed transcription to cDNA.Real-time quantitative PCR was used for detecting the mRNA levels of Fas,cFLIP.3.2Detection mitochondrial pathway of macrophagePeritoneal macrophages were harvested as described above. Peritoneal macrophages were stimulated with50μg/ml oxLDL. Then the cells were collected. RNA was extracted and reversed transcription to cDNA.Real-time quantitative PCR was used for detecting the mRNA levels of Bax,Bcl-2.3.3endoplasmic reticulum stress related genes of macrophagePeritoneal macrophages were harvested as described above. Peritoneal macrophages were stimulated with50μg/ml oxLDL. Then the cells were collected. RNA was extracted and reversed transcription to cDNA.Real-time quantitative PCR was used for detecting the mRNA levels of CHOP,GRP78.3.4Detection apoptosis related genes of macrophagePeritoneal macrophages were harvested as described above. Peritoneal macrophages were stimulated with50μg/ml oxLDL. Then the cells were collected. RNA was extracted and reversed transcription to cDNA.Real-time quantitative PCR was used for detecting the mRNA levels of IAP2, TNFRSF12a.4. The regulation of TIPE2on related signaling pathways in macrophagesFor isolation of elicited peritoneal macrophages, male wild type and TIPE2-deficient mice (8-10w) were injected intraperitoneally with1.0ml of6%sterile starch. Four days after the injection, cells were harvested by intraperitoneal lavage with ice-cold PBS, cultured in high-glucose version of DMEM medium (GIBCO-BRL, Carlsbad. CA,USA) supplemented with10%FBS at37℃/5%CO2overnight. Peritoneal macrophages were then stimulated with oxLDL.Then the cells were collected and levels of AKT and p-AKT were detected by Western blot.Results1. Roles of TIPE2on the cell death of macrophages 1.1Percentage of death cells were down-regulated in TIPE2-/-macrophagesPeritoneal macrophages isolated from wild type and TIPE2-/-mice were stimulated with oxLDL for0,24,48hours and stained by trypan blue. Compared with wild type cells, the percentage of death cells were significantly decreaced in TIPE2-/-macrophages.1.2Autophagy was promoted in T1PE2-/-macrophagesPeritoneal macrophages isolated from wild type and TIPE2-/-mice were stimulated with oxLDL for0,24,48hours. Expression levels of LC3was analyzed by Western blot. Compared with wild type cells, TIPE2-/-macrophages had significantly up-regulated the protein level of LC3-II,at the same time,cells were stimulated with oxLDL for0,24hours. Expression levels of Atg5was analyzed by real-time PCR. Compared with wild type cells, TIPE2-/-macrophages had significantly up-regulated the protein level of Atg5.1.3Apoptosis was inhibited TIPE2-/-macrophages1.3.1Percentage of apoptosis cells were down-regulated in TIPE2-/-macrophagesPeritoneal macrophages isolated from wild type and TIPE2-/-mice were stimulated with oxLDL for0,24hours and stained by Tunel. Compared with wild type cells, the percentage of apoptosis cells were significantly decreaced in TIPE2-/-macrophages.1.3.2Decreaced cell apoptosis of TIPE2-/-macrophagesPeritoneal macrophages isolated from wild type and TIPE2-/-mice were stimulated with oxLDL for0,24hours. The number of macrophages that can be stained by annexinV/PI was much more than that in TIPE2-/-macrophages.1.3.3Activation of caspase3was suppressed in TIPE2-/-macrophagesPeritoneal macrophages isolated from wild type and TIPE2-/-mice were stimulated with oxLDL for0,24,48hours. Expression levels of caspase3and cleave-caspase3were analyzed Western blot. Compared with wild type cells, TIPE2-/-macrophages had significantly down-regulated the protein level of cleave-caspase3,while TIPE2deficiency had no effect on level of caspase3.2. Roles of TIPE2on the expression of related genes 2.1Roles of TIPE2on extrinsic pathway in macrophagesPeritoneal macrophages isolated from wild type and TIPE2-/-mice were stimulated with oxLDL. Expression levels of Fas,cFLIP were analyzed by Real-time quantitative PCR. Compared with wild type cells, TIPE2-/-macrophages had significantly up-regulated mRNA levels of cFLIP, which was reported as anti-apoptosis genes. However,the mRNA levels of Fas,,which was reported as apoptosis genes,was down-regulated.2.2Roles of TIPE2on mitochondrial pathway in macrophagesPeritoneal macrophages isolated from wild type and TIPE2-/-mice were stimulated with oxLDL. Expression levels of Bax,Bcl2were analyzed by Real-time quantitative PCR. Compared with wild type cells, TIPE2-/-macrophages had significantly up-regulated mRNA levels of Bcl2, which was reported as anti-apoptosis genes. However,the mRNA levels of Bax,which was reported as apoptosis genes,was down-regulated.2.3TIPE2-/-macrophages are resistant to ERSPeritoneal macrophages isolated from wild type and TIPE2-/-mice were stimulated with oxLDL. Expression levels of CHOP and GRP78were analyzed by Real-time quantitative PCR. Compared with wild type cells, TIPE2-/-macrophages had significantly down-regulated mRNA levels of CHOP and GRP78.2.4Roles of TIPE2on the expression of apoptosis-related genes in macrophagesPeritoneal macrophages isolated from wild type and TIPE2-/-mice were stimulated with oxLDL. Expression levels of IAP2,TNFRSF12a were analyzed by real-time quantitative PCR. Compared with wild type cells, T1PE2-/-macrophages had significantly down-regulated mRNA levels of TNFRSF12a, which was reported as apoptosis genes.However, the mRNA levels of IAP2,which was reported as anti-apoptosis genes,was up-regulated.3. Positive regulation of AKT pathway by TIPE2in oxLDL-stimulated macrophagesPeritoneal macrophages isolated from wild type and TIPE2-/-mice were stimulated with oxLDL for0,5,15,30minutes. Expression levels of AKT and p-AKT were analyzed Western blot. Compared with wild type cells, TIPE2-/-macrophages had significantly up-regulated the protein level of p-AKT,while TIPE2deficiency had no effect on level of AKT.Indicated that the activation of AKT signal pathway was enhanced in TIPE2-/-macrophage.Conclusions1. This topic further confirmed that TIPE2can anti-atherosclerosis by inducing the death of macrophage.2. TIPE2can promote the apoptosis of macrophage by regulating the death receptor pathway,mitochondrial apoptotic pathway and endoplasmic reticulum stress,etc.3. The molecular mechanism that TIPE2promotes the death of macrophage may be connected with that TIPE2can negatively regulate the activation of AKT.4. We also discovered that the level of autophagy was enhanced in TIPE2-/-macrophage.This indicate that the autophagy of macrophage was inhibited in atherosclerosis.And the effect on atherosclerosis awaits father investigation.Innovations and significances1. It is the first time to investigate the relationship between TIPE2and the apoptosis of macrophage and indicate that TIPE2can promote macrophage apoptosis.2. The molecular mechanisms by which TIPE2promote macrophage apoptosis may be associated with its regulatory effects on the extrinsic pathway,mitochondrial pathway,endoplasmic reticulum stress and the activation of AKT.3. Our results may provide theoretical and experimental guidance for further research. It may provide a new target for the treatment of atherosclerosis.