Effect of ATR gene knockdown on senescence and function of rat bone marrow endothelial progenitor cells

doi:10.12307/2022.375

Abstract

Abstract: BACKGROUND: Endothelial progenitor cells play an important role in vascular endothelial renewal and repair. The transplantation of autologous endothelial progenitor cells expanded and cultured in vitro to promote angiogenesis in ischemic sites has become a new strategy for the prevention and treatment of ischemic cardiovascular diseases. However, the endothelial progenitor cells derived from the elderly body will show signs of cellular senescence when expanded in vitro. The ability of aging endothelial progenitor cells to repair vascular endothelium is difficult to exert. Studies have shown that DNA damage is closely related to cell aging, and DNA damage detection points are an important checkpoint for regulating DNA damage.
OBJECTIVE: Lentiviral RNA interference technology was used to knockdown ATM and Rad3-related kinase (ATR) gene in rat bone marrow endothelial progenitor cells, and to explore its role in the aging process of endothelial progenitor cells.
METHODS: Endothelial progenitor cells from bone marrow in rats were isolated, cultured and identified. After the isolated endothelial progenitor cells were cultured to the third generation, they were counted and randomly divided into three groups: non-infection control group (blank group), negative control group, and gene knockdown group (ATR-RNAi group). The non-infection control group was cultured normally, and the negative control group and gene knockdown group were stably transfected with lentiviral RNAi and lentiviral ATR-RNAi to day 3 and day 6, respectively. The level of ATR mRNA in each group of cells was detected by qRT-PCR. The protein expression of ATR in each group of cells was detected by western blotting. The senescence of each group of cells was detected by β-galactosidase staining. MTT colorimetric method was used to detect cell proliferation activity in each group. Transwell chamber and in vitro angiogenesis kit were used to detect cell migration ability and tubule formation of each group. Flow cytometry was applied to detect the cell cycle change in each group.
RESULTS AND CONCLUSION: (1) Compared with the non-infection control group, the expression levels of ATR mRNA and ATR protein in the endothelial progenitor cells in ATR-RNAi group were decreased significantly (P < 0.01), indicating that ATR gene expression was effectively reduced. (2) Compared with the non-infection control group, the number of blue-stained cells in the ATR-RNAi group decreased, indicating that the degree of aging was significantly reduced (P < 0.01). (3) Compared with the non-infection control group, cell proliferation, migration and tubule formation in the ATR-RNAi group were significantly enhanced (P < 0.05). (4) Compared with the non-infection control group, the percentages of cells in the G1 and G2 phases in the ATR-RNAi group were decreased significantly, and the percentage of cells in the S phase was increased significantly (P < 0.05). (5) These results indicate that knockdown of the expression of ATR genes could delay the aging process of endothelial progenitor cells, promotes the function of endothelial progenitor cells, and reduces the stagnation of cell cycles.

Key words: endothelial progenitor cells, bone marrow, ATM and Rad3-related kinase, lentiviral transfection, cell senescence, functional activity, cell cycle

CLC Number:

Tang Mi, Qin Zhen, Wei Zhengxin, Ni Ming, Chai Xiaokang. Effect of ATR gene knockdown on senescence and function of rat bone marrow endothelial progenitor cells[J]. Chinese Journal of Tissue Engineering Research, 2022, 26(19): 2985-2990.

Figures/Tables 6

References

[1] XIA WH, LI J, SU C, et al. Physical exercise attenuates age-associated reduction in endothelium-reparative capacity of endothelial progenitor cells by increasing CXCR4/JAK-2 signaling in healthy men. Aging Cell. 2012;11(1):111-119.
[2] ASAHARA T, MUROHARA T, SULLIVAN A, et al. Isolation of putative progenitor endothelial cells for angiogenesis. Science. 1997;275(5302): 964-967.
[3] CHILLÀ A, MARGHERI F, BIAGIONI A, et al. Mature and progenitor endothelial cells perform angiogenesis also under protease inhibition: the amoeboid angiogenesis. J Exp Clin Cancer Res. 2018;37(1):74.
[4] XUE C, HUANG Q, ZHANG T, et al. Matrix stiffness regulates arteriovenous differentiation of endothelial progenitor cells during vasculogenesis in nude mice. Cell Prolif. 2019;52(2):e12557.
[5] HU Z, WANG H, FAN G, et al. Danhong injection mobilizes endothelial progenitor cells to repair vascular endothelium injury via upregulating the expression of Akt, eNOS and MMP-9. Phytomedicine. 2019;61: 152850.
[6] FANG J, GUO Y, TAN S, et al. Autologous Endothelial Progenitor Cells Transplantation for Acute Ischemic Stroke: A 4-Year Follow-Up Study. Stem Cells Transl Med. 2019;8(1):14-21.
[7] 秦臻,韦正新,宰青青,等.黄精降低活性氧水平促进衰老内皮祖细胞功能的研究[J].中国药理学通报,2019,35(1):123-127.
[8] 朱光旭,黄岚,武晓静,等.不同年龄段大鼠血清影响内皮祖细胞向成熟内皮诱导分化[J].中国动脉硬化杂志,2007,15(9): 647-652.
[9] OU HL, SCHUMACHER B. DNA damage responses and p53 in the aging process. Blood. 2018;131(5):488-495.
[10] 秦臻,韦正新,许键炜.黄精对衰老大鼠内皮祖细胞 DNA 损伤检测点ATM/ATR 通路的影响[J].中药新药与临床药理,2019,30(5):529-534.
[11] NGANGUEM NZALLE YRANNEY BRICE,林慕之,毛善永,等.大鼠骨髓源内皮祖细胞的分离与鉴定[J].贵州医科大学学报,2020,45(3): 265-269.
[12] 李丽玲,计晓娟,余更生,等.1种新来源的内皮祖细胞提取分离方法[J].免疫学杂志,2019,35(3):269-276.
[13] 罗伟,李翔翮,杨先腾,等.小鼠骨髓源性内皮祖细胞的分离培养与鉴定[J].中华创伤骨科杂志,2018,20(6):523-528.
[14] 秦臻,黄水清.当归补血汤含药血清对内皮祖细胞功能及其 PI3K/Akt 通路影响的研究[J].中国药理学通报,2013,29(9):1320-1323.
[15] 胡洁,陈庆伟,贺无恙.增龄与动脉粥样硬化性心血管疾病临床类型的相关性分析[J].临床心血管病杂志,2018,34(11):1081-1085.
[16] 饶璇,李元建.内皮细胞损伤与修复的研究进展[J].中国动脉硬化杂志,2017,25(5):531-535.
[17] FAZEL S, CIMINI M, CHEN L, et al. Cardioprotective c-kit+ cells are from the bone marrow and regulate the myocardial balance of angiogenic cytokines. J Clin Invest. 2006;116(7):1865-1877.
[18] QIAO J, QI K, CHU P, et al. Infusion of endothelial progenitor cells ameliorates liver injury in mice after haematopoietic stem cell transplantation. Liver Int. 2015;35(12):2611-2620.
[19] 宰青青,秦臻,叶兰.黄精对自然衰老大鼠内皮祖细胞功能及端粒酶活性的影响[J].中国中西医结合杂志,2016,36(12):1480-1485.
[20] NAKAMURA AJ, CHIANG YJ, HATHCOCK KS, et al. Both telomeric and non-telomeric DNA damage are determinants of mammalian cellular senescence. Epigenetics Chromatin. 2008;1(1):6.
[21] 李文瑞,刘新光,周中军. DNA 损伤与细胞衰老关系的研究进展[J].国际检验医学杂志,2010,31(6):576-578.
[22] MAYNARD S, FANG EF, SCHEIBYE-KNUDSEN M, et al. DNA Damage, DNA Repair, Aging, and Neurodegeneration. Cold Spring Harb Perspect Med. 2015;5(10):a025130.
[23] MARÉCHAL A, ZOU L. DNA damage sensing by the ATM and ATR kinases. Cold Spring Harb Perspect Biol. 2013;5(9):a012716.
[24] 乔田奎,陈伟,袁素娟.ATM 蛋白激酶依赖性信号转导通路研究进展[J].复旦学报(医学版),2012,39(4):433-437.
[25] FALCK J, COATES J, JACKSON SP. Conserved modes of recruitment of ATM, ATR and DNA-PKcs to sites of DNA damage. Nature. 2005; 434(7033):605-611.
[26] SANCAR A, LINDSEY-BOLTZ LA, UNSAL-KAÇMAZ K, et al. Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints. Annu Rev Biochem. 2004;73:39-85.
[27] REAPER PM, GRIFFITHS MR, LONG JM, et al. Selective killing of ATM- or p53-deficient cancer cells through inhibition of ATR. Nat Chem Biol. 2011;7(7):428-430.