Roles Of TNFAIP8L1 On Atherosclerosis

BackgroundWith the large development of economy and the living standard of people, cardiovascular diseases have become one of the main diseases which threaten the health of people in the worldwide. As the most common cardiovascular disease, atherosclerosis has been largely acknowledged as a progressive disease in the arterial wall. Cardiovascular diseases caused by atherosclerosis have been the disease which have the highest ratio of causing death, as well as in Chinese. The originating factor of the atherosclerosis is the dysfunction of endothelial cells. The integrity of function of endothelial cells (ECs) plays an important role in the development of atherosclerosis. So keeping the integrity of function is vital for resisting the development of atherosclerosis. Dysfunction of endothelial cells caused by atherosclerosis can lead to apoptosis of endothelial cells and meanwhile, the event of endothelial cell apoptosis accelerated has been found in atherosclerosis plaque in previous experiments. Factors resulting in atherosclerosis include environmental factors and genic factors, and oxidative stress has been acknowledged as a vitally important factor to induce atherosclerosis.Oxidative stress is a series of adaptive reactions caused by disequilibrium between reactive oxygen species (ROS) and antioxidant system of bodies. ROS consists of hydrogen peroxide (H2O2), hydroxyl radical (OH-), superoxide (O2-) and Hypochlorite (OCl-). The increase of ROS leads to dysfunction of endothelial cells and vascular inflammation and develop into atherosclerosis eventually. Oxidative stress reacts so sensitively for ECs which is a key factor to contribute to AS. H2O2 could induce oxidative stress directly and superabundant ROS will give rise to damage of lipid、DNA and protein directly and apoptosis of cells finally. The apoptosis of vascular cells can increase the ratio of atherosclerotic generating.MAPK family including Erks、JNKs and P38 MAPK, as well as PI3K/Akt/ survivor signaling pathway、Fox03a pathway and NF-κB pathway plays a critical role of mediator in ROS induced-oxidative stress.TIPE1 (TNFAIP8L1, TNF-α-induced protein 8-like 1) as a member of tumor necrosis factor-alpha-induced protein 8 (TNFAIP8) family is not known very well,however, as we know now. Meanwhile, TIPE2 (TNFAIP8L1, TNF-α-induced protein 8-like 2) as one of other members of TNFAIP8 family has been acknowledged as a negative regulator of innate immunity and cell immunity and could resist atherosclerosis. TIPE1 expresses in various murine tissues and human cell lines, and it may regulate cell death,but its mechanism is not clear at present, so it should been explored urgently. Hence, we will apply TIPE1 to our study to definite the role in atherosclerosis.Methods1. Effects of TIPE1 on vascular endothelial cells induced by oxidative stress in vitro1.1 To obtain the human umbilical vein endothelial cells (HUVECs)HuVECs were obtained by health Newborn infants in Qilu Hospital. The generation of cells we used are third to tenth and cells are cultured with M199 culture medium with 10% FBS.1.2 The expression of TIPE1 is enhanced exposing to H2O2 in endothelial cells.The expression of TIPE1 was detected in endothelial cell ls with H2O2 stimulation using RT-PCR and real time PCR and western blot in ECs stimulating with H2O2.1.3 Effects of TIPE1 on vascular endothelial cells induced by oxidative stress1.3.1 The effects of TIPE1 overexpression on the production of ROS and expression of antioxidant molecules stimulated by H2O2 inducingExcessive ROS was detected in endothelial cells transfected with pRK5-TIPE1 or pRK5 controls by Flow Cytometry.The expression of antioxidant mlecules such as CAT、SOD2、GPX、and FoxO3a were determined in endothelial cells using RT-PCR and real time PCR and western blot by H2O2 stimulating.1.3.2 Effects of TIPE1 overexpression on endothelial cells growthThe growth of endothelial cells were detected by CCK-8. Endothelial cells were divided into four groups including pRK5、pRK5-TIPE1、pRK5-100 μmol/L H2O2、 pRK5-TIPE1 100 μmol/L H2O2. After stimulated 0h、6h、12h、24h by H2O2 stimulating, we measured the OD value by 450nm and drew growth curve.1.3.3 Effects of TIPE1 overexpression on endothelial cells deathThe endothelial cells death were detected by trypan blue staining、Annexin V/PI and analyzed cell cycle by PI staining. The expression of Bax、Bcl2 and caspase3 were also detected by western blot.1.3.4 TIPE1 overexpression increases the uptake of ox-LDL in endothelial cells.We treated endothelial cells with ox-LDL and ox-LDL could be stained by oil-O-red, so we measured the OD value (500nm) of oil-O-red.1.4 The effects of TIPE1 knockdown on endothelial cells functionEndothelial cells were transfected by small interference of TIPE1 and control small interference. We detected ROS production by Flow Cytometry and detected cell growth death by CCK-8 and trypan blue staining respectively.1.5 The effects of TIPE1 on endothelial cells signaling pathways in oxidative stressAkt、MAPK and IκB signaling pathways were detected by western blot.2. In VivoWe used ApoE-/- mice to build the model of atherosclerosis and were divided into control group and experimental group. They were fed with high-fat diet for 9 weeks, and meanwhile were injected with control lentivirus and TIPE1 lentivirus two times respectively in the second and fifth week. The main methods were frozen section、HE staining、oil-O-red staining、general oil-O-red staining and lipid metabolism detecting.Results1. The expression of TIPE1 is enhanced exposing to H2O2 in endothelial cells.The expression of TIPE1 were detected in endothelial cell using RT-PCR and real time PCR and western blot by H2O2. The expression of TIPE1 increased significantly in both a dose-dependent and a time-dependent when stimulated by H2O2 manner. 2. TIPE1 enhanced the production of ROS with H2O2 stimulating.The production of ROS were detected when TIPE1 overexpression by Flow Cytometry. We got that TIPE1 promotes the production of ROS with H2O2 stimulating.3. TIPE1 decreases the expression of antioxidant moleculesThe expression of antioxidant enzymes, such as CAT、SOD2、GPx were detected by PCR and FoxO3a was detected by western blot. And we found the overexpression of TIPE1 decreases the expression of antioxidant enzyme.4. TIPE1 decreases endothelial cells growthThe growth of endothelial cells were detected by CCK-8 and the OD value was measured by 450nm and we drew growth curve to detect growth of endothelial cells. We found that the endothelial cells whose TIPE1 overexpressed grew more slowly than control ECs. Furthermore, we got that TIPE1 overexpressing could decreases endothelial cells growth stimulated by 100 μmol/L H2O2 sharply than transfected with pRK5 by the same stimulation. So we got TIPE1 decreases endothelial cells growth.5. TIPE1 enhances endothelial cells death induced by oxidative stress5.1 TIPE1 overexpression enhances H2O2-induced cell death.The death of endothelial cells were detected by PI and we got the subdiploid peak in cell cycle. We can find that TIPE1 overexpression enhances cell death with H2O2 stimulation.5.2 TIPE1 overexpression promotes endothelial cells death in oxidative stress by trypan blue staining.Endothelial cells were stained by trypan blue, we counted the quantity of blue cells representing died cells and the sum of cells and specific value between the two, so we can get cell death rate. We found that TIPE1 overexpression promotes endothelial cells death.5.3 TIPE1 overexpression enhances endothelial cells death in oxidative stress by Annexin V/PI.The death of endothelial cells were detected by Annexin V/PI and got the ratio of FITC+PI+ to total cells. We found that TIPE1 overexpression enhanced endothelial cells death in oxidative stress.5.4 TIPE1 overexpression upregulates expression of caspase3 and Bax and downregulates the activation of Bcl2 in oxidative stress6. TIPE1 overexpression increases uptake of ox-LDL in endothelial cells.Endothelial cells were treated with ox-LDL and ox-LDL could be stained by oil-O-red, so we got that TIPE1 overexpression increases the uptake of ox-LDL in endothelial cells.7. TIPE1 enhances endothelial cells death by regulating the activation of Akt、NF-κB、 MAPK signaling pathway in oxidative stress.By western blot, we found that TIPE1 overexpression can increase the activation of p38 and JNK and decrease the activation of Akt、Erk and IκB.8.TIPE1 enhances the plaque forming in vivo.No matter in aortic root or abdominal aorta, the area of plaque forming is obviously larger in treated with TIPE1 lentiviruses than it in treated with control lentiviruses. It is clear that TIPE1 accelerates the plaque deposits in vitro.9. TIPE1 accelerates secretion of triglyceride and free fatty acid.TIPE1 accelerating secretion of triglyceride and free fatty acid can show us TIPE1 increases atherosclerosis by accelerating secretion of triglyceride and free fatty.Conclusion1 TIPE1 accelerates atherosclerosis in mice.2 Mechanism of TIPE1 action in atherosclerosis is based on its promoting-role on oxidative stress-induced cell death in vascular endothelial cells. TIPE1 is upregulated by H2O2 in both dose-and time-dependent manners, and subsequently accelerates cell death and ox-LDL uptake.3 TIPE1 enhances oxidative stress-induced activation of p38 and JNK but decreases the phosphorylation of Akt, Erk and IκB.Innovations and significances1 It is the first time to investigate the regulating roles of TIPE1 on atherosclerosis, providing a novel target for AS therapy.2 It is the first time to uncover the role of TIPE1 in the regulation of endothelial cells function, which is great helpful to further investigate biological functions of TIPEl protein under pathophysiological conditions.