Single-molecule imaging reveals stoichiometry of vascular endothelial growth factor receptor 2 in retinal vascular endothelial cells_Chinese Journal of Ocular Fundus Diseases

Authors：

Yang Songyang , Zhang Mingliang , Hu Lanlan , Wang Liming , Zhang Xiaomin ,  Li Xiaorong

Tianjin Key Laboratory of Retinal Functions and Diseases, Tianjin International Joint Research and Development Centre of Ophthalmology and Vision Science, Eye Institute and School of Optometry, Tianjin Medical University Eye Hospital, Tianjin 300384, China;

Corresponding author：

Li Xiaorong, Email: xiaorli@163.com

Keywords：

Vascular endothelial growth factor receptor-2; Retinal vessels; Endothelial cells; Single-molecule imaging

DOI：

10.3760/cma.j.cn511434-20200608-00265

Video：

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Objective To observe the stoichiometry of vascular endothelial growth factor receptor 2 (VEGFR2) on the retinal vascular endothelial cell membrane by single-molecule fluorescence imaging.Methods Rhesus monkey retinal vascular endothelial cells (RF/6A) were divided into blank control group (normal culture) and plasmid transfection group [transfected with VEGFR2-green fluorescent protein (GFP) recombinant plasmid]. The expression of GFP in the plasmid transfected group was observed by confocal microscope, and the expression of VEGFR2 in the cells was detected by real-time fluorescent quantitative polymerase chain reaction (qPCR) and Western blot. The fluorescence intensity distribution and bleaching steps of single VEGFR2-GFP molecule on the cell membrane were recorded by single-molecule imaging. The distribution of fluorescence intensity and the number of fluorescence bleaching steps of GFP were recorded.Results GFP green fluorescence was observed in the transfected cells 12 hours after transfection. qPCR results showed that the expression of VEGFR2 and GFP mRNA in the plasmid transfected group was significantly higher than that in the blank control group (t=11.240, 12.330; P＜0.001, 0.001). Western blot results showed that the expression of VEGFR2 protein in the plasmid transfected group was significantly higher than that in the blank control group (t=8.346, P＜0.01). The results of single-molecule imaging showed that the fluorescence intensity distribution of VEGFR2-GFP on the surface of RF/6A cell membrane without ligand stimulation was bimodal, in which monomer and dimer were 86.0% and 14.0% respectively. By counting the steps of GFP fluorescence bleaching, the proportions of receptor monomer, dimer, trimer, and tetramer were 81.4%, 12.9%, 5.5%, and 0.3% respectively.Conclusion In the absence of ligands, VEGFR2 coexists in the form of monomers and dimers on the surface of RF/6A cell membrane, and monomers are dominant.

Citation： Yang Songyang, Zhang Mingliang, Hu Lanlan, Wang Liming, Zhang Xiaomin, Li Xiaorong. Single-molecule imaging reveals stoichiometry of vascular endothelial growth factor receptor 2 in retinal vascular endothelial cells. Chinese Journal of Ocular Fundus Diseases, 2021, 37(2): 138-144. doi: 10.3760/cma.j.cn511434-20200608-00265 Copy

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11.	Chen J, Smith LEH. Retinopathy of prematurity[J]. Angiogenesis, 2007, 10(2): 133-140. DOI: 10.1007/s10456-007-9066-0.
12.	Kempen JH, O'Colmain BJ, Leske MC, et al. The prevalence of diabetic retinopathy among adults in the United States[J]. Arch Ophthalmol, 2004, 122(4): 552-563. DOI: 10.1001/archopht.122.4.552.
13.	Friedman DS, O'Colmain BJ, Muñoz B, et al. Prevalence of age-related macular degeneration in the United States[J]. Arch Ophthalmol, 2004, 122(4): 564-572. DOI: 10.1001/archopht.122.4.564.
14.	Ferrara N. VEGF and the quest for tumour angiogenesis factors[J]. Nat Rev Cancer, 2002, 2(10): 795-803. DOI: 10.1038/nrc909.
15.	Gille H, Kowalski J, Li B, et al. Analysis of biological effects and signaling properties of Flt-1 (VEGFR-1) and KDR (VEGFR-2). A reassessment using novel receptor-specific vascular endothelial growth factor mutants[J]. J Biol Chem, 2001, 276(5): 3222-3230. DOI: 10.1074/jbc.M002016200.
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24.	Da Rocha-Azevedo B, Lee S, Dasgupta A, et al. Heterogeneity in VEGF receptor-2 mobility and organization on the endothelial cell surface leads to diverse models of activation by VEGF[J/OL]. Cell Rep, 2020, 32(13): 108187[2020-09-29]. https://linkinghub.elsevier.com/retrieve/pii/S2211-1247(20)31176-1. DOI:10.1016/j.celrep.2020.108187.
25.	Busbee BG, Ho AC, Brown DM, et al. Twelve-month efficacy and safety of 0.5 mg or 2.0 mg ranibizumab in patients with subfoveal neovascular age-related macular degeneration[J]. Ophthalmology, 2013, 120(5): 1046-1056. DOI: 10.1016/j.ophtha.2012.10.014.
26.	Heier JS, Brown DM, Chong V, et al. Intravitreal aflibercept (VEGF Trap-Eye) in wet age-related macular degeneration[J]. Ophthalmology, 2012, 119(12): 2537-2548. DOI: 10.1016/j.ophtha.2012.09.006.
27.	Hsu M, Hung Y, Hwang D, et al. Detection of aqueous VEGF concentrations before and after intravitreal injection of anti-VEGF antibody using low-volume sampling paper-based ELISA[J/OL]. Sci Rep, 2016, 6: 34631[2016-10-11]. http://europepmc.org/article/MED/27725716. DOI:10.1038/srep34631.
28.	Lu D, Shen J, Vil MD, et al. Tailoring in vitro selection for a picomolar affinity human antibody directed against vascular endothelial growth factor receptor 2 for enhanced neutralizing activity[J]. J Biol Chem, 2003, 278(44): 43496-43507. DOI: 10.1074/jbc.m307742200.
29.	de Moraes Neto JE, Pereira F, Neves RL, et al. Preclinical assessment of intravitreal ramucirumab: in vitro and in vivo safety profile[J/OL]. International Journal of Retina and Vitreous, 2020, 6(1): 40[2020-09-04]. https: //journalretinavitreous.biomedcentral.com/articles/10.1186/s40942-020-00243-y. DOI: 10.1186/s40942-020-00243-y.

1. Zhang SX, Ma J. Ocular neovascularization: implication of endogenous angiogenic inhibitors and potential therapy[J]. Prog Retin Eye Res, 2007, 26(1): 1-37. DOI: 10.1016/j.preteyeres.2006.09.002.
2. Campochiaro PA. Molecular pathogenesis of retinal and choroidal vascular diseases[J]. Prog Retin Eye Res, 2015, 49: 67-81. DOI: 10.1016/j.preteyeres.2015.06.002.
3. Hubbard SR. Juxtamembrane autoinhibition in receptor tyrosine kinases[J]. Nat Rev Mol Cell Biol, 2004, 5(6): 464-471. DOI: 10.1038/nrm1399.
4. Dosch DD, Ballmer-Hofer K. Transmembrane domain-mediated orientation of receptor monomers in active VEGFR-2 dimers[J]. FASEB J, 2010, 24(1): 32-38. DOI: 10.1096/fj.09-132670.
5. Ruch C, Skiniotis G, Steinmetz MO, et al. Structure of a VEGF-VEGF receptor complex determined by electron microscopy[J]. Nat Struct Mol Biol, 2007, 14(3): 249-250. DOI: 10.1038/nsmb1202.
6. 张哲, 刘竹青, 刘巨平, 等. 二甲双胍联合抗血管内皮生长因子药物治疗糖尿病视网膜病变的可能协同作用[J]. 中华眼底病杂志, 2018, 34(5): 453-457. DOI: 10.3760/cma.j.issn.1005-1015.2018.05.008.Zhang Z, Liu ZQ, Liu JP, et al. The synergistic effect of metformin and anti-vascular endothelial growth factor in the treatment of diabetic retinopathy[J]. Chin J Ocul Fundus Dis, 2018, 34(5): 453-457. DOI: 10.3760/cma.j.issn.1005-1015.2018.05.008.
7. Zhang W, Jiang Y, Wang Q, et al. Single-molecule imaging reveals transforming growth factor-beta-induced type Ⅱ receptor dimerization[J]. Proc Natl Acad Sci U S A, 2009, 106(37): 15679-15683. DOI: 10.1073/pnas.0908279106.
8. Zhang M, He K, Wu J, et al. Single-molecule imaging reveals the stoichiometry change of epidermal growth factor receptor during transactivation by β2-adrenergic receptor[J]. Science China Chemistry, 2017, 60(10): 1310-1317. DOI: 10.1007/s11426-017-9072-5.
9. 程茗, 李楠, 方晓红. 应用全内反射单分子荧光成像研究蛋白复合物亚基组成[J]. 中国科学:化学, 2012, 42(12): 1663-1671. DOI: 10.1360/032012-322.Cheng M, Li N, Fang XH. Study of protein stoichiometry by single-molecule imaging with total internal reflection fluorescence microscopy[J]. Scientia Sinica Chimica, 2012, 42(12): 1663-1671. DOI: 10.1360/032012-322.
10. Yang W, Lu J, Weng J, et al. Prevalence of diabetes among men and women in China[J]. N Engl J Med, 2010, 362(12): 1090-1101. DOI: 10.1056/NEJMoa0908292.
11. Chen J, Smith LEH. Retinopathy of prematurity[J]. Angiogenesis, 2007, 10(2): 133-140. DOI: 10.1007/s10456-007-9066-0.
12. Kempen JH, O'Colmain BJ, Leske MC, et al. The prevalence of diabetic retinopathy among adults in the United States[J]. Arch Ophthalmol, 2004, 122(4): 552-563. DOI: 10.1001/archopht.122.4.552.
13. Friedman DS, O'Colmain BJ, Muñoz B, et al. Prevalence of age-related macular degeneration in the United States[J]. Arch Ophthalmol, 2004, 122(4): 564-572. DOI: 10.1001/archopht.122.4.564.
14. Ferrara N. VEGF and the quest for tumour angiogenesis factors[J]. Nat Rev Cancer, 2002, 2(10): 795-803. DOI: 10.1038/nrc909.
15. Gille H, Kowalski J, Li B, et al. Analysis of biological effects and signaling properties of Flt-1 (VEGFR-1) and KDR (VEGFR-2). A reassessment using novel receptor-specific vascular endothelial growth factor mutants[J]. J Biol Chem, 2001, 276(5): 3222-3230. DOI: 10.1074/jbc.M002016200.
16. Shibuya M, Claesson-Welsh L. Signal transduction by VEGF receptors in regulation of angiogenesis and lymphangiogenesis[J]. Exp Cell Res, 2006, 312(5): 549-560. DOI: 10.1016/j.yexcr.2005.11.012.
17. Kisko K, Brozzo MS, Missimer J, et al. Structural analysis of vascular endothelial growth factor receptor-2/ligandcomplexes by small-angle X-ray solution scattering[J]. FASEB J, 2011, 25(9): 2980-2986. DOI: 10.1096/fj.11-185397.
18. Fantl WJ, Johnson DE, Williams LT. Signalling by receptor tyrosine kinases[J]. Annu Rev Biochem, 1993, 62: 453-481. DOI: 10.1146/annurev.bi.62.070193.002321.
19. Sarabipour S, Ballmer-Hofer K, Hristova K. VEGFR-2 conformational switch in response to ligand binding[J/OL]. Elife, 2016, 5: e13876[2016-04-07]. http://europepmc.org/article/MED/27052508. DOI:10.7554/eLife.13876.
20. Sarabipour S, Hristova K. Mechanism of FGF receptor dimerization and activation[J/OL]. Nat Commun, 2016, 7: 10262[2016-01-04]. http://europepmc.org/article/MED/26725515. DOI:10.1038/ncomms10262.
21. Johnson DS, Jaiswal JK, Simon S. Total internal reflection fluorescence (TIRF) microscopy illuminator for improved imaging of cell surface events[J/OL]. Curr Protoc Cytom, 2012, 12: 12-29[2012-07-01]. https://currentprotocols.onlinelibrary.wiley.com/doi/10.1002/0471142956.cy1229s61. DOI:10.1002/0471142956.cy1229s61.
22. Ulbrich MH, Isacoff EY. Subunit counting in membrane-bound proteins[J]. Nat Methods, 2007, 4(4): 319-321. DOI: 10.1038/nmeth1024.
23. Fuh G, Li B, Crowley C, et al. Requirements for binding and signaling of the kinase domain receptor for vascular endothelial growth factor[J]. J Biol Chem, 1998, 273(18): 11197-11204. DOI: 10.1074/jbc.273.18.11197.
24. Da Rocha-Azevedo B, Lee S, Dasgupta A, et al. Heterogeneity in VEGF receptor-2 mobility and organization on the endothelial cell surface leads to diverse models of activation by VEGF[J/OL]. Cell Rep, 2020, 32(13): 108187[2020-09-29]. https://linkinghub.elsevier.com/retrieve/pii/S2211-1247(20)31176-1. DOI:10.1016/j.celrep.2020.108187.
25. Busbee BG, Ho AC, Brown DM, et al. Twelve-month efficacy and safety of 0.5 mg or 2.0 mg ranibizumab in patients with subfoveal neovascular age-related macular degeneration[J]. Ophthalmology, 2013, 120(5): 1046-1056. DOI: 10.1016/j.ophtha.2012.10.014.
26. Heier JS, Brown DM, Chong V, et al. Intravitreal aflibercept (VEGF Trap-Eye) in wet age-related macular degeneration[J]. Ophthalmology, 2012, 119(12): 2537-2548. DOI: 10.1016/j.ophtha.2012.09.006.
27. Hsu M, Hung Y, Hwang D, et al. Detection of aqueous VEGF concentrations before and after intravitreal injection of anti-VEGF antibody using low-volume sampling paper-based ELISA[J/OL]. Sci Rep, 2016, 6: 34631[2016-10-11]. http://europepmc.org/article/MED/27725716. DOI:10.1038/srep34631.
28. Lu D, Shen J, Vil MD, et al. Tailoring in vitro selection for a picomolar affinity human antibody directed against vascular endothelial growth factor receptor 2 for enhanced neutralizing activity[J]. J Biol Chem, 2003, 278(44): 43496-43507. DOI: 10.1074/jbc.m307742200.
29. de Moraes Neto JE, Pereira F, Neves RL, et al. Preclinical assessment of intravitreal ramucirumab: in vitro and in vivo safety profile[J/OL]. International Journal of Retina and Vitreous, 2020, 6(1): 40[2020-09-04]. https: //journalretinavitreous.biomedcentral.com/articles/10.1186/s40942-020-00243-y. DOI: 10.1186/s40942-020-00243-y.

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Single-molecule imaging reveals stoichiometry of vascular endothelial growth factor receptor 2 in retinal vascular endothelial cells

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