Up-regulation of p21 activated kinase 4 expression in the retina of diabetes mice and its effects on the behavior and mitochondrial function in retinal vascular endothelial cells_Chinese Journal of Ocular Fundus Diseases

Authors：

Jiao Mingfei , Li Hui , Cao Jingjing , Kou Zhenyu , Wu Guijia , Liu Aihua ,  Dong Lijie

Tianjin Key Laboratory of Retinal Functions and Diseases, Tianjin Branch of National Clinical Research Center for Ocular Disease, Eye Institute and School of Optometry, Tianjin Medical University Eye Hospital, Tianjin 300384, China;

Corresponding author：

Dong Lijie, Email: aitaomubang@126.com

Keywords：

Diabetic retinopathy; P21 activated kinase 4; Retinal vascular endothelial cells; Mitochondrial function; Cell migration; Leukocyte adhesion; Animal experiment; Cell experiment

DOI：

10.3760/cma.j.cn511434-20220418-00224

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Objective To observe the effects of p21 activated kinase 4 (PAK4) on the mitochondrial function and biological behavior in retinal vascular endothelial cells. Methods The experimental study was divided into two parts: in vivo animal experiment and in vitro cell experiment. In vivo animal experiments: 12 healthy C57BL/6J male mice were randomly divided into normal control group and diabetes group, with 6 mice in each group. Diabetes mice were induced by streptozotocin to establish diabetes model. Eight weeks after modeling, quantitative real-time polymerase chain reaction and Western blots were performed to detect the expression of PAK4 in diabetic retinas. In vitro cell experiments: the human retinal microvascular endothelial cells (hRMEC) were divided into three groups: conventional cultured cells group (N group), empty vector transfected (Vector group); pcDNA-PAK4 eukaryotic expression plasmid transfected group (PAK4 group). WB and qPCR were used to detect transfection efficiency, while scratching assay, cell scratch test was used to detect cell migration in hRMEC of each group. In vitro white blood cell adhesion experiment combined with 4 ', 6-diamino-2-phenylindole staining was used to detect the number of white blood cells adhering to hRMEC in each group. The Seahorse XFe96 cell energy metabolism analyzer measures intracellular mitochondrial basal respiration, adenosine triphosphate (ATP) production, maximum respiration, and reserve respiration capacity. The t-test was used for comparison between the two groups. Single factor analysis of variance was used for comparison among the three groups. Results In vivo animal experiments: compared with normal control group, the relative expression levels of PAK4 mRNA and protein in retina of diabetic mice were significantly increased, with statistical significance (t=25.372, 22.419, 25.372; P＜0.05). In vitro cell experiment: compared with the N group and Vector group, the PAK4 protein, mRNA relative expression and cell mobility in the hRMEC of PAK4 group were significantly increased, with statistical significance (F=36.821, 38.692, 29.421; P＜0.05). Flow cytometry showed that the adhesion number of leukocytes on hRMEC in PAK4 group was significantly increased, and the difference was statistically significant (F=39.649, P＜0.01). Mitochondrial pressure measurement results showed that the capacity of mitochondrial basic respiration, ATP production, maximum respiration and reserve respiration in hRMEC in PAK4 group was significantly decreased, with statistical significance (F=27.472, 22.315, 31.147, 27.472; P＜0.05). Conclusion Over-expression of PAK4 impairs mitochondrial function and significantly promotes leukocyte adhesion and migration in retinal vascular endothelial cells.

Citation： Jiao Mingfei, Li Hui, Cao Jingjing, Kou Zhenyu, Wu Guijia, Liu Aihua, Dong Lijie. Up-regulation of p21 activated kinase 4 expression in the retina of diabetes mice and its effects on the behavior and mitochondrial function in retinal vascular endothelial cells. Chinese Journal of Ocular Fundus Diseases, 2023, 39(5): 401-407. doi: 10.3760/cma.j.cn511434-20220418-00224 Copy

1.	王勇, 曹靖靖, 李辉, 等. 干扰素基因刺激蛋白抑制剂改善视网膜血管内皮细胞白细胞粘附及糖酵解水平[J]. 中华眼底病杂志, 2022, 38(12): 1013-1019. DOI: 10.3760/cma.j.cn511434-20220520-00312.Wang Y, Cao JJ, Li H, et al. Interferon gene stimulating protein inhibitor improves leukocyte adhesion and glycolysis of retinal vascular endothelial cells[J]. Chin J Ocul Fundus Dis, 2022, 38(12): 1013-1019. DOI: 10.3760/cma.j.cn511434-20220520-00312.
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1. 王勇, 曹靖靖, 李辉, 等. 干扰素基因刺激蛋白抑制剂改善视网膜血管内皮细胞白细胞粘附及糖酵解水平[J]. 中华眼底病杂志, 2022, 38(12): 1013-1019. DOI: 10.3760/cma.j.cn511434-20220520-00312.Wang Y, Cao JJ, Li H, et al. Interferon gene stimulating protein inhibitor improves leukocyte adhesion and glycolysis of retinal vascular endothelial cells[J]. Chin J Ocul Fundus Dis, 2022, 38(12): 1013-1019. DOI: 10.3760/cma.j.cn511434-20220520-00312.
2. 刘巨平, 洪亚茹, 姚旭阳, 等. 骨形成蛋白4促进视网膜血管内皮细胞增生和迁移[J]. 中华眼底病杂志, 2022, 38(4): 304-309. DOI: 10.3760/cma.j.cn511434-20201109-00539.Liu JP, Hong YR, Yao XY, et al. Bone morphogenetic protein 4 promotes proliferation and migration of retinal vascular endothelial cells[J]. Chin J Ocul Fundus Dis, 2022, 38(4): 304-309. DOI: 10.3760/cma.j.cn511434-20201109-00539.
3. Mookerjee SA, Gerencser AA, Nicholls DG, et al. Quantifying intracellular rates of glycolytic and oxidative ATP production and consumption using extracellular flux measurements[J]. J Biol Chem, 2017, 292(17): 7189-7207. DOI: 10.1074/jbc.M116.774471.
4. Barth T, Helbig H. [Diabetic retinopathy][J]. Klin Monbl Augenheilkd, 2021, 238(10): 1143-1159. DOI: 10.1016/S0140-6736(09)62124-3.
5. Won SY, Park JJ, Shin EY, et al. PAK4 signaling in health and disease: defining the PAK4-CREB axis[J]. Exp Mol Med, 2019, 51(2): 1-9. DOI: 10.1038/s12276-018-0204-0.
6. Fu Y, Fang L, Yin Q, et al. Interfering with PAK4 protein expression affects osteosarcoma cell proliferation and migration[J/OL]. Biomed Res Int, 2021, 2021: 9977001[2021-12-30]. https://europepmc.org/article/MED/35005025. DOI: 10.1155/2021/9977001.
7. Ashraf U, Ding Z, Deng S, et al. Pathogenicity and virulence of Japanese encephalitis virus: neuroinflammation and neuronal cell damage[J]. Virulence, 2021, 12(1): 968-980. DOI: 10.1155/2021/9977001.
8. Yu B, Song C, Feng CL, et al. Effects of gradient high-field static magnetic fields on diabetic mice[J]. Zool Res, 2023, 44(2): 249-258. DOI: 10.24272/j.issn.2095-8137.2022.460.
9. Shao Y, Dong LJ, Takahashi Y, et al. miRNA-451a regulates RPE function through promoting mitochondrial function in proliferative diabetic retinopathy[J]. Am J Physiol Endocrinol Metab, 2019, 316(3): 443-452. DOI: 10.1152/ajpendo.00360.2018.
10. Dong L, Li W, Lin T, et al. PSF functions as a repressor of hypoxia-induced angiogenesis by promoting mitochondrial function[J]. Cell Commun Signal, 2021, 19(1): 14. DOI: 10.1186/s12964-020-00684-w.
11. Zhou P, Xie W, Meng X, et al. Notoginsenoside R1 ameliorates diabetic retinopathy through PINK1-dependent activation of mitophagy[J]. Cells, 2019, 8(3): 213. DOI: 10.3390/cells8030213.
12. Zhao CC, Zhan MN, Liu WT, et al. Combined LIM kinase 1 and p21-activated kinase 4 inhibitor treatment exhibits potent preclinical antitumor efficacy in breast cancer[J]. Cancer Lett, 2020, 493: 120-127. DOI: 10.1016/j.canlet.2020.08.006.
13. Wright GM, Gimbrone NT, Sarcar B, et al. CDK4/6 inhibition synergizes with inhibition of P21-Activated Kinases (PAKs) in lung cancer cell lines[J/OL]. PLoS One, 2021, 16(6): e0252927[2021-06-17]. https://europepmc.org/article/MED/34138895. DOI: 10.1371/journal.pone.0252927.
14. Zhang X, Zhang X, Li Y, et al. PAK4 regulates G6PD activity by p53 degradation involving colon cancer cell growth[J]. Cell Death Dis, 2017, 8(5): 2820. DOI: 10.1038/cddis.2017.85.
15. Naїja A, Merhi M, Inchakalody V, et al. The role of PAK4 in the immune system and its potential implication in cancer immunotherapy[J/OL]. Cell Immunol, 2021, 367: 104408[2021-07-01]. https://linkinghub.elsevier.com/retrieve/pii/S0008-8749(21)00127-1. DOI: 10.1016/j.cellimm.2021.104408.
16. Ma W, Wang Y, Zhang R, et al. Targeting PAK4 to reprogram the vascular microenvironment and improve CAR-T immunotherapy for glioblastoma[J]. Nat Cancer, 2021, 2(1): 83-97. DOI: 10.1038/s43018-020-00147-8.
17. Mao K, Lei D, Zhang H, et al. MicroRNA-485 inhibits malignant biological behaviour of glioblastoma cells by directly targeting PAK4[J]. Int J Oncol, 2017, 51(5): 1521-1532. DOI: 10.3892/ijo.2017.4122.
18. Singh AD, Kulkarni YA. Vascular adhesion protein-1 and microvascular diabetic complications[J]. Pharmacol Rep, 2022, 74(1): 40-46. DOI: 10.1007/s43440-021-00343-y.
19. Han J, Li Y, Liu X, et al. Metformin suppresses retinal angiogenesis and inflammation in vitro and in vivo[J/OL]. PLoS One, 2018, 13(3): e0193031[2018-03-07]. https://europepmc.org/article/MED/29513760. DOI: 10.1371/journal.pone.0193031.
20. Shao Y, Chen J, Freeman W, et al. Canonical Wnt signaling promotes neovascularization through determination of endothelial progenitor cell fate via metabolic profile regulation[J]. Stem Cells, 2019, 37(10): 1331-1343. DOI: 10.1002/stem.3049.
21. Gu X, Ma Y, Liu Y, et al. Measurement of mitochondrial respiration in adherent cells by Seahorse XF96 Cell Mito Stress Test[J/OL]. STAR Protoc, 2021, 2(1): 100245[2020-12-30]. https://linkinghub.elsevier.com/retrieve/pii/S2666-1667(20)30232-X. DOI: 10.1016/j.xpro.2020.100245.

Chinese Journal of Ocular Fundus Diseases

Up-regulation of p21 activated kinase 4 expression in the retina of diabetes mice and its effects on the behavior and mitochondrial function in retinal vascular endothelial cells

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