Construction, phenotype and functional identification of vascular endothelial cell-specific PDHA1 knockout mice

doi:10.12307/2022.622

Abstract

Abstract: BACKGROUND: PDHA1 gene is an indispensable gene in the aerobic respiratory chain. The energy metabolism of vascular endothelial cells is related to many diseases. Construction of vascular endothelial cell-specific PDHA1 knockout mice helps to study the role of energy metabolism in diseases related to vascular endothelial cells.
OBJECTIVE: To breed vascular endothelial cell-specific PDHA1 knockout mice and identify their phenotypes.
METHODS: PDHA1(iΔEC/ iΔEC) mice were co-bred with PDHA1(iΔEC/-) mice, and more PDHA1(iΔEC/ iΔEC) mice were obtained for subsequent experiments. PCR and agarose gel electrophoresis were used for gene identification. RT-PCR and western blot were used to detect the mRNA and protein expression of genes related to energy metabolism after PDHA1 knockout, respectively. The protein expression of PDHA1 after PDHA1 conditional knockout was determined by double immunofluorescence staining of VE-cadherin and PDHA1.
RESULTS AND CONCLUSION: Genotypes of offspring mice were successfully detected by agarose gel electrophoresis, including 15 PDHA1(iΔEC/ iΔEC) and 2 PDHA1(iΔEC/-) mice. Results of reverse transcription PCR and western blot assay showed that the transcription and translation levels of PDHA1 were decreased, the protein and mRNA expressions of HK2 in the glycolysis pathway were increased, and the protein and mRNA expression of IDH in the aerobic phosphorylation pathway were decreased. Double immunofluorescence staining of VE-cadherin and PDHA1 showed a decrease in the expression of PDHA1.

Key words: PDHA1, double immunofluorescence, CreERT2 gene, phenotypic validation, gene identification, energy metabolism, vascular endothelial cell, pulmonary fibrosis

CLC Number:

Hao Wei, Sun Yue, Yang Anning, Liu Taiyang, Bao Rui, Liu Yaoyang, Wang Qiushi, Wang Meng, Chang Sirong, Li Yuanyuan, Liu Zhihong. Construction, phenotype and functional identification of vascular endothelial cell-specific PDHA1 knockout mice[J]. Chinese Journal of Tissue Engineering Research, 2022, 26(20): 3207-3211.

Figures/Tables 5

References

[1] LAUFS U, LIAO JK. Post-transcriptional regulation of endothelial nitric oxide synthase mRNA stability by Rho GTPase. J Biol Chem. 1998;273(37):24266-24271.
[2] SENONER T, DICHTL W. Oxidative Stress in Cardiovascular Diseases: Still a Therapeutic Target? Nutrients. 2019;11(9):2090.
[3] XIE T, WANG C, JIN Y, et al. CoenzymeQ10-Induced Activation of AMPK-YAP-OPA1 Pathway Alleviates Atherosclerosis by Improving Mitochondrial Function, Inhibiting Oxidative Stress and Promoting Energy Metabolism. Front Pharmacol. 2020;11:1034.
[4] ZHANG J, MURI J, FITZGERALD G, et al. Endothelial Lactate Controls Muscle Regeneration from Ischemia by Inducing M2-like Macrophage Polarization. Cell Metab. 2020;31(6):1136-1153.
[5] 安南,陈子琦,黄敏.血管内皮细胞代谢与肿瘤血管新生研究进展[J].药学学报,2020,55(7):1373-1381.
[6] 陈伟才,欧文斌.丙酮酸脱氢酶活性缺失与疾病发生的研究进展[J].生命的化学,2017,37(5):751-760.
[7] GOODE DJ, MOLLIVER DC. Regulation of Mitochondrial Function by Epac2 Contributes to Acute Inflammatory Hyperalgesia. J Neurosci. 2021;41(13): 2883-2898.
[8] NUMATA-UEMATSU Y, UEMATSU M, YAMAMOTO T, et al. Leigh syndrome-like MRI changes in a patient with biallelic HPDL variants treated with ketogenic diet. Mol Genet Metab Rep. 2021;29:100800.
[9] XU L, LI Y, ZHOU L, et al. SIRT3 elicited an anti-Warburg effect through HIF1α/PDK1/PDHA1 to inhibit cholangiocarcinoma tumorigenesis. Cancer Med. 2019;8(5):2380-2391.
[10] LIU L, CAO J, ZHAO J, et al. PDHA1 Gene Knockout In Human Esophageal Squamous Cancer Cells Resulted In Greater Warburg Effect And Aggressive Features In Vitro And In Vivo. Onco Targets Ther. 2019;12:9899-9913.
[11] HORGA A, WOODWARD CE, MILLS A, et al. Differential phenotypic expression of a novel PDHA1 mutation in a female monozygotic twin pair. Hum Genet. 2019;138(11-12):1313-1322.
[12] CHEN J, GUCCINI I, DI MITRI D, et al. Compartmentalized activities of the pyruvate dehydrogenase complex sustain lipogenesis in prostate cancer. Nat Genet. 2018;50(2):219-228.
[13] DING BS, CAO Z, LIS R, et al. Divergent angiocrine signals from vascular niche balance liver regeneration and fibrosis. Nature. 2014;505(7481):97-102.
[14] 王婷婷,卜歆,李金奎,等.DDR2血管内皮细胞条件性基因敲除小鼠的复制、鉴定及表型分析[J].中国现代医学杂志,2019,29(21):1-6.
[15] 丁燕,赖丽芬,奈文青,等.血管内皮细胞敲除Rheb基因杂合子小鼠模型建立及意义[J].山东医药,2017,57(36):34-36.
[16] 郑雅婷.利用基因敲入方法制备在Vegfr3位点表达Cre~(ERT2)重组酶的转基因小鼠及其应用[D].苏州:苏州大学,2018.
[17] 蔺文轩,孙岳,杨安宁,等.辅酶Q10对小鼠矽肺纤维化的作用[J].环境与职业医学,2019,36(10):949-954.
[18] CAO Z, YE T, SUN Y, et al. Targeting the vascular and perivascular niches as a regenerative therapy for lung and liver fibrosis. Sci Transl Med. 2017; 9(405):eaai8710.
[19] 王肖,赖舒畅,叶艳诗,等.利用Cre/lox P系统构建胰岛β细胞特异性敲除PDHA1基因的小鼠模[J].中国组织工程研究,2019,23(31):5023-5029.
[20] 丁燕,孟碧莹,向光大.建立血管内皮细胞敲除DEPTOR基因小鼠模型及鉴定[J].中国组织工程研究,2019,23(23):3698-3704.
[21] EBINA M, SHIMIZUKAWA M, SHIBATA N, et al. Heterogeneous increase in CD34-positive alveolar capillaries in idiopathic pulmonary fibrosis. Am J Respir Crit Care Med. 2004;169(11):1203-1208.
[22] CAO Y, ZHANG X, WANG L, et al. PFKFB3-mediated endothelial glycolysis promotes pulmonary hypertension. Proc Natl Acad Sci U S A. 2019;116(27): 13394-13403.
[23] TROTSYUK AA, CHEN K, KWON SH, et al. Inhibiting Fibroblast Mechanotransduction Modulates Severity of Idiopathic Pulmonary Fibrosis. Adv Wound Care (New Rochelle). 2021. doi: 10.1089/wound.2021.0077.
[24] 马越.丹贝益肺汤对肺纤维化大鼠血管内皮细胞HSPA12B表达的影响[D].哈尔滨:黑龙江省中医药科学院,2019.
[25] 尹晓敏,高义军,彭解英.血管内皮细胞与组织纤维化[J].国外医学(口腔医学分册),2003,30(2):129-131.
[26] LI X, CARMELIET P. Targeting angiogenic metabolism in disease. Science. 2018;359(6382):1335-1336.