| It was first reported in 1959 that atherosclerosis (AS) could be caused by ionizing radiation, which was one of the independent risk factors for AS as high blood pressure, high blood fats, diabetes, smoking, lack of exercise, overweight, and aging. Ionizing radiation, together with other risk factors, can accelerate AS. Large and medium-sized arteries are involved in AS, resulting in vascular lumen blockage of different degrees, distal ischemia in blocked artery, and local tissue necrosis in the end. Atherosclerosis can also bring about coronary heart disease. If the coronary artery stenosis is 50%, serious diseases such as angina pectoris, acute myocardial infarction, or arrhythmia may occur clinically, resulted from myocardial ischemia or necrosis. Ionizing radiation can directly damage the irradiated cells and the radiated cells transfer the response reaction to the normal irradiated cells through communication connected by cell medium or cell gap, so that normal cells also exhibit biological damage similar to irradiated effects, a radiobiological phenomenon known as the bystander effect.With the wide use of radiation therapy of tumor, late survival of tumor patients after radiotherapy became a focus. Therefore, not only the direct damage from radiation but also the far later damage induced by bystander effect is gaining social concerns. Research has shown that death from cardiovascular and cerebrovascular diseases radiation-induced AS is the most common complications that lead to death of tumor patients beside cancer. But the mechanism of ionizing radiation induced cardiovascular disease still remains unclear. Its mechanism varies in relation to age. As the population receiving radiation increases graually in the world, it is necessary to uncover the mechanism of radiation-induced AS in order to provide an experimental basis for preventing ionizing radiation induced cardiovascular diseases. Most patients receive radiation therapy with part of body exposed to radiation. After receiving radiation, the monocytes/macrophages in blood vessel of the irradiated part will release a large number of bioactive factors since macrophages are important immune cells, leading to bystander effect. Thereafter, it is necessary to further the study on ionizing radiation induced AS and the impact of bystander effect upon irradiation induced AS, so as to provide a new idea about prevention.In this study, we took U937 cells into radiation, built a co-culturing model for irradiated U937 cells and unirradiated HUVEC, and observed that the irradiated U937 cells can lead to DNA damage to irradiated HUVEC and micronucleus formation. The NO concentration of irradiated U937 cells increased, and the expression of iNOS protein in cells also increased. Exogenous SNP treatment of different concentrations to HUVECs cells would also lead to HUVECs micronucleus increase relevant to the concentration of SNP, whereas adding NO scavenger c-PTIO would reduce the increasing of HUVECs micronucleus. Pretreatment of U937 cells with iNOS inhibitor SMT and adding NO scavenger c-PTIO to the co-culturing system can inhibit HUVECs micronucleus. Meanwhile, the irradiated U937 cells can cause obvious increase of apoptosis rate of unirradiated HUVECs and remarkable decrease of clone rate. In comparison, using SMT to pretreat U937 cells and c-PTIO to treat the two co-culturing cells can totally inhibit the apoptosis of HUVECs induced by irradiated U937 cells and recover the survival of HUVECs partially. It suggests that irradiated U937 cells can induce bystander effects such as DNA damage and cell apoptosis by releasing NO radiation activated co-cultured HUVECs through iNOS. Due to the significant increase of phosphorylation of p38 protein in co-cultured HUVECs, after treating the irradiated HUVECs with p38 inhibitor SB203580, the DNA damage and cells apoptosis rate of HUVECs as adjacent cells decreases significantly, while cells’ survival rate reinforces. The expression of the corcultured HUVECs cell surface VCAM-1 and the U937 cell adhesion ability enhance. However, SMT and c-PTIO can remarkably inhibit the expression of VCAM-1 of HUVECs. The expression of VCAM-1 is the important factor to adjust the adhesion of U937 to HUVECs, which lowers the adhesion of HUVECs to the irradiated U937 cells. If SB203580 is used to pretreat HUVECs cells, and then is co-cultured with irradiated U937 cells, the VCAM-1 expression of HUVECs is totally inhibited, and its adhesion to irradiated U937 decreases to its bottom line, In addition, the irradiated U937 cells obviously promoted the release of active MMP-9 and MMP-2 of HUVECs. But when the irradiated target cells U937 are treated by U937 SMT, c-PTIO, or HUVECs adjacent cells are pretreated by SB203580, the ability of HUVECs cells to release active MMP-9 and MMP-2 decreases significantly. It suggests that the irradiated U937 cells release large amounts of NO expression by activating iNOS prompt, and then activate the co-cultured unirradiated HUVECs, producing a series of inflammation of the bystander effect, in which the p38/MAPK pathway plays an important role.To further the study, we took the smooth muscle cells, which play an important role in the atherosclerotic process, as the adjacent cells, build two co-culturing cell models to simulate the actual phenomenon by using Transwell chamber. One model is to co-culture irradiated U937 with unirradiated HUSMC:U937 were planted in the chamber while HUSMC on six-hole board. The other is to co-culture irradiated U937 and unirradiated HUSMC and HUVECs:U937 were planted in the chamber while HUVECs were planted on the chamber, HUSMC on six-hole board.. The irradiated U937 cells induce the proliferation HUSMC by releasing cytokine, TNF-a involved. The proliferation ability of HUSMC in the system of two cells co-cultured is stronger than the three cells co-cultured. When using Exogenous TNF-a to treat HUSMC, we found that IL-6 mRNA gene of HUSMC increases. In the co-culturing systems of two cells and three cells, we compared irradiated U937 cells with corresponding groups, and found that it can lead to the increase of MCP-1, IL-6 in the supernatant in the co-culture system. Comparing two-cell system with three-cell system, the gene expression of IL-6 and TNF-a of HUSMC increases significantly while IL-6 in the supernatant in the three-cell system is higher than two-cell system, MCP-1 in the supernatant in the two-cell system is higher than three-cell system. This proves that the release of IL-6 partially comes from HUVECs in the co-culturing system. MCP-1 mainly comes from HUSMC, and the inflammatory factor TNF-a, MCP-1, IL-6 in the co-culturing system increases. Briefly, this study shows that the irradiated macrophages can lead to the damage of co-cultured endothelial cells, and enhance the adhesion ability of endothelial cells to macrophages. This process is relevant to NO secreted by endothelial cells. The irradiated U937 cells can lead to the enhancement of the proliferative ability of unirradiated HUSMC. During these processes, a series of inflammatory factors were involved. These experiments prove that bystander effects can cause the damage of endothelial cells, change of smooth muscle cell proliferation and migration ability. Therefore, we suggest that bystander effects are involved in radiation-induced AS. |
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