The expressions of vascular endothelial growth factor and aquaporin 4 in the inner limiting membrane from eyes with diabetic macular edema_Chinese Journal of Ocular Fundus Diseases

Authors：

Chen Yiqi , Chen Huan , Wang Chenxi , Yu Jiafeng , Gao Weiqian , Zhou Congying , Tao Jiwei , Mao Jianbo ,  Shen Lijun

The Affiliated Eye Hospital of Wenzhou Medical University at Hangzhou, Hangzhou 310000, China;

Corresponding author：

Shen Lijun, Email: slj@mail.eye.ac.cn

Keywords：

Diabetic retinopathy; Macular edema; Vitrectomy; Vascular endothelial growth factors; Aquaporin 4

DOI：

10.3760/cma.j.cn511434-20201228-00643

Video：

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Objective To observe the expression of vascular endothelial growth factor (VEGF) and aquaporin 4 (AQP4) in the inner limiting membrane (ILM) of diabetic retinopathy (DR) with macular edema, and analyze the correlation between VEGF and AQP4 expression. Methods A cross-sectional study. From September 2019 to September 2020, 38 eyes of 38 patients with DR and idiopathic macular hole (iMH) who underwent vitrectomy (PPV) combined with ILM stripping at the Hangzhou campus of The Affiliated Eye Hospital of Wenzhou Medical University at Hangzhou were included in the study. Among them, there were 25 males and 13 females who aged 37-76 years old, average age was 59±10 years old; All eye included 15 right eyes and 23 left eyes. iMH and DR included 9 eyes in 9 cases and 29 eyes in 29 cases, respectively, and they were divided into iMH group and DR group. The DR group was divided into DME group and no DME group according to whether it was accompanied by diabetic macular edema (DME), with 14 eyes and 15 eyes respectively. After the stripped ILM tissue was fixed, immunofluorescence analysis was performed to obtain a picture of the fluorescence mode of AQP4 and VEGF, and the fluorescence intensity value of VEGF and AQP4 was measured by Image J software. The differences of VEGF and AQP4 immunofluorescence values in the specimens between groups were compared by one-way analysis of variance. The correlation between the fluorescence intensity of AQP4 and the fluorescence intensity of VEGF was analyzed by Pearson correlation analysis. Results The average fluorescence intensity valuesof VEGF and AQP4 in ILM specimens of DME group, no DME group and iMH group were 38.96±7.53, 28.25±3.12, 30.07±4.84 and 49.07±8.73, 37.96±6.45, 38.08±5.04, respectively. The average fluorescence intensity of VEGF and AQP4 in the ILM specimens of the DME group was significantly higher than that of the no DME group and iMH group, and the difference was statistically significant (F=13.977, 9.454; P＜0.05). The average fluorescence intensity values of VEGF and AQP4 on IML specimens in the DR group were 33.80±7.91, 43.76±9.44, respectively. The results of Pearson correlation analysis showed that the fluorescence intensity of VEGF and AQP4 in the ILM specimens of the DR group was significantly positively correlated (r=0.597, P=0.003). Conclusions The expressions of VEGF and AQP4 in ILM of eyes with DR and DME are significantly increased compared with those without DME. The expression of VEGF and AQP4 in ILM of eyes with DR is positively correlated.

Citation： Chen Yiqi, Chen Huan, Wang Chenxi, Yu Jiafeng, Gao Weiqian, Zhou Congying, Tao Jiwei, Mao Jianbo, Shen Lijun. The expressions of vascular endothelial growth factor and aquaporin 4 in the inner limiting membrane from eyes with diabetic macular edema. Chinese Journal of Ocular Fundus Diseases, 2021, 37(8): 617-622. doi: 10.3760/cma.j.cn511434-20201228-00643 Copy

1.	Ding J, Wong TY. Current epidemiology of diabetic retinopathy and diabetic macular edema[J]. Curr Diab Rep, 2012, 12(4): 346-354. DOI: 10.1007/s11892-012-0283-6.
2.	Elman MJ, Bressler NM, Qin H, et al. Expanded 2-year follow-up of ranibizumab plus prompt or deferred laser or triamcinolone plus prompt laser for diabetic macular edema[J]. Ophthalmology, 2011, 118(4): 609-614. DOI: 10.1016/j.ophtha.2010.12.033.
3.	Brown DM, Nguyen QD, Marcus DM, et al. Long-term outcomes of ranibizumab therapy for diabetic macular edema: the 36-month results from two phase Ⅲ trials: RISE and RIDE[J]. Ophthalmology, 2013, 120(10): 2013-2022. DOI: 10.1016/j.ophtha.2013.02.034.
4.	Do DV, Nguyen QD, Boyer D, et al. One-year outcomes of the da Vinci Study of VEGF Trap-Eye in eyes with diabetic macular edema[J]. Ophthalmology, 2012, 119(8): 1658-1665. DOI: 10.1016/j.ophtha.2012.02.010.
5.	Diabetic Retinopathy Clinical Research Network, Wells JA, Glassman AR, et al. Aflibercept, bevacizumab, or ranibizumab for diabetic macular edema[J]. N Engl J Med, 2015, 372(13): 1193-1203. DOI: 10.1056/NEJMoa1414264.
6.	Dehghan MH, Salehipour M, Naghib J, et al. Pars plana vitrectomy with internal limiting membrane peeling for refractory diffuse diabetic macular edema[J]. J Ophthalmic Vis Res, 2010, 5(3): 162-167.
7.	Figueroa MS, Contreras I, Noval S. Surgical and anatomical outcomes of pars plana vitrectomy for diffuse nontractional diabetic macular edema[J]. Retina, 2008, 28(3): 420-426. DOI: 10.1097/IAE.0b013e318159e7d2.
8.	Kimura T, Kiryu J, Nishiwaki H, et al. Efficacy of surgical removal of the internal limiting membrane in diabetic cystoid macular edema[J]. Retina, 2005, 25(4): 454-461. DOI: 10.1097/00006982-200506000-00010.
9.	Stefánsson E. The therapeutic effects of retinal laser treatment and vitrectomy. A theory based on oxygen and vascular physiology[J]. Acta Ophthalmol Scand, 2001, 79(5): 435-440. DOI: 10.1034/j.1600-0420.2001.790502.x.
10.	Schey KL, Wang Z, L Wenke J, et al. Aquaporins in the eye: expression, function, and roles in ocular disease[J]. Biochim Biophys Acta, 2014, 1840(5): 1513-1523. DOI: 10.1016/j.bbagen.2013.10.037.
11.	Hollborn M, Dukic-Stefanovic S, Pannicke T, et al. Expression of aquaporins in the retina of diabetic rats[J]. Curr Eye Res, 2011, 36(9): 850-856. DOI: 10.3109/02713683.2011.593108.
12.	Suzuki H, Oku H, Horie T, et al. Changes in expression of aquaporin-4 and aquaporin-9 in optic nerve after crushing in rats[J/OL]. PLoS One, 2014, 9(12): e114694[2014-12-05]. https://doi.org/10.1371/journal.pone.0114694. DOI: 10.1371/journal.pone.0114694.
13.	Dal Monte M, Nicchia GP, Cammalleri M, et al. Aquaporin 4 is required to induce retinal angiogenesis in a mouse model of oxygen‐induced retinopathy[J/OL]. Acta Ophthalmologica, 2015, 92: 253[2014-08-20]. https://doi.org/10.1111/j.1755-3768.2014.F006.x. DOI: 10.1111/j.1755-3768.2014.F006.x.
14.	Oku H, Morishita S, Horie T, et al. Nitric oxide increases the expression of aquaporin-4 protein in rat optic nerve astrocytes through the cyclic guanosine monophosphate/protein kinase G pathway[J]. Ophthalmic Res, 2015, 54(4): 212-221. DOI: 10.1159/000440846.
15.	Lassiale S, Valamanesh F, Klein C, et al. Changes in aquaporin-4 and Kir4.1 expression in rats with inherited retinal dystrophy[J]. Exp Eye Res, 2016, 148: 33-44. DOI: 10.1016/j.exer.2016.05.010.
16.	Kida T, Oku H, Horie T, et al. Implication of VEGF and aquaporin 4 mediating Müller cell swelling to diabetic retinal edema[J]. Graefe’s Arch Clin Exp Ophthalmol, 2017, 255(6): 1149-1157. DOI: 10.1007/s00417-017-3631-z.
17.	Nguyen QD, Tatlipinar S, Shah SM, et al. Vascular endothelial growth factor is a critical stimulus for diabetic macular edema[J]. Am J Ophthalmol, 2006, 142(6): 961-969. DOI: 10.1016/j.ajo.2006.06.068.
18.	Le YZ. VEGF production and signaling in Müller glia are critical to modulating vascular function and neuronal integrity in diabetic retinopathy and hypoxic retinal vascular diseases[J]. Vision Res, 2017, 139: 108-114. DOI: 10.1016/j.visres.2017.05.005.
19.	Miyamoto K, Khosrof S, Bursell SE, et al. Vascular endothelial growth factor (VEGF)-induced retinal vascular permeability is mediated by intercellular adhesion molecule-1 (ICAM-1)[J]. Am J Pathol, 2000, 156(5): 1733-1739. DOI: 10.1016/s0002-9440(10)65044-4.
20.	Miyamoto K, Khosrof S, Bursell SE, et al. Prevention of leukostasis and vascular leakage in streptozotocin-induced diabetic retinopathy via intercellular adhesion molecule-1 inhibition[J]. Proc Natl Acad Sci USA, 1999, 96(19): 10836-10841. DOI: 10.1073/pnas.96.19.10836.
21.	Sohn HJ, Han DH, Kim IT, et al. Changes in aqueous concentrations of various cytokines after intravitreal triamcinolone versus bevacizumab for diabetic macular edema[J]. Am J Ophthalmol, 2011, 152(4): 686-694. DOI: 10.1016/j.ajo.2011.03.033.
22.	Felinski EA, Antonetti DA. Glucocorticoid regulation of endothelial cell tight junction gene expression: novel treatments for diabetic retinopathy[J]. Curr Eye Res, 2005, 30(11): 949-957. DOI: 10.1080/02713680500263598.
23.	Noma H, Mimura T, Yasuda K, et al. Role of inflammation in diabetic macular edema[J]. Ophthalmologica, 2014, 232(3): 127-135. DOI: 10.1159/000364955.
24.	Funatsu H, Noma H, Mimura T, et al. Association of vitreous inflammatory factors with diabetic macular edema[J]. Ophthalmology, 2009, 116(1): 73-79. DOI: 10.1016/j.ophtha.2008.09.037.
25.	Jonas JB, Jonas RA, Neumaier M, et al. Cytokine concentration in aqueous humor of eyes with diabetic macular edema[J]. Retina, 2012, 32(10): 2150-2157. DOI: 10.1097/IAE.0b013e3182576d07.
26.	Ferrara N. VEGF and intraocular neovascularization: from discovery to therapy[J/OL]. Transl Vis Sci Technol, 2016, 5(2): 10[2016-04-11]. https://pubmed.ncbi.nlm.nih.gov/26981332/. DOI: 10.1167/tvst.5.2.10.
27.	Mu H, Zhang XM, Liu JJ, et al. Effect of high glucose concentration on VEGF and PEDF expression in cultured retinal Müller cells[J]. Mol Biol Rep, 2009, 36(8): 2147-2151. DOI: 10.1007/s11033-008-9428-8.
28.	Wang J, Xu X, Elliott MH, et al. Müller cell-derived VEGF is essential for diabetes-induced retinal inflammation and vascular leakage[J]. Diabetes, 2010, 59(9): 2297-2305. DOI: 10.2337/db09-1420.
29.	Simmons AB, Bretz CA, Wang H, et al. Correction to: gene therapy knockdown of VEGFR2 in retinal endothelial cells to treat retinopathy[J/OL]. Angiogenesis, 2018, 21(4): 765[2018-06-25]. https://pubmed.ncbi.nlm.nih.gov/29943214/. DOI: 10.1007/s10456-018-9626-5.
30.	Fu S, Dong S, Zhu M, et al. VEGF as a trophic factor for Müller glia in hypoxic retinal diseases[J]. Adv Exp Med Biol, 2018, 1074: 473-478. DOI: 10.1007/978-3-319-75402-4_58.
31.	Bressler SB, Qin H, Beck RW, et al. Factors associated with changes in visual acuity and central subfield thickness at 1 year after treatment for diabetic macular edema with ranibizumab[J]. Arch Ophthalmol, 2012, 130(9): 1153-1161. DOI: 10.1001/archophthalmol.2012.1107.
32.	Barile GR, Pachydaki SI, Tari SR, et al. The RAGE axis in early diabetic retinopathy[J]. Invest Ophthalmol Vis Sci, 2005, 46(8): 2916-2924. DOI: 10.1167/iovs.04-1409.
33.	Semeraro F, Morescalchi F, Duse S, et al. Current trends about inner limiting membrane peeling in surgery for epiretinal membranes[J/OL]. J Ophthalmol, 2015, 2015: 671905[2015-09-03]. https://pubmed.ncbi.nlm.nih.gov/26425352/. DOI: 10.1155/2015/671905.
34.	Bonnin S, Sandali O, Bonnel S, et al. Vitrectomy with internal limiting membrane peeling for tractional and nontractional diabetic macular edema: long-term results of a comparative study[J]. Retina, 2015, 35(5): 921-928. DOI: 10.1097/IAE.0000000000000433.
35.	Tenckhoff S, Hollborn M, Kohen L, et al. Diversity of aquaporin mRNA expressed by rat and human retinas[J]. Neuroreport, 2005, 16(1): 53-56. DOI: 10.1097/00001756-200501190-00013.
36.	Hamann S, Zeuthen T, La Cour M, et al. Aquaporins in complex tissues: distribution of aquaporins 1-5 in human and rat eye[J]. Am J Physiol, 1998, 274(5): C1332-C1345. DOI: 10.1152/ajpcell.1998.274.5.C1332.

1. Ding J, Wong TY. Current epidemiology of diabetic retinopathy and diabetic macular edema[J]. Curr Diab Rep, 2012, 12(4): 346-354. DOI: 10.1007/s11892-012-0283-6.
2. Elman MJ, Bressler NM, Qin H, et al. Expanded 2-year follow-up of ranibizumab plus prompt or deferred laser or triamcinolone plus prompt laser for diabetic macular edema[J]. Ophthalmology, 2011, 118(4): 609-614. DOI: 10.1016/j.ophtha.2010.12.033.
3. Brown DM, Nguyen QD, Marcus DM, et al. Long-term outcomes of ranibizumab therapy for diabetic macular edema: the 36-month results from two phase Ⅲ trials: RISE and RIDE[J]. Ophthalmology, 2013, 120(10): 2013-2022. DOI: 10.1016/j.ophtha.2013.02.034.
4. Do DV, Nguyen QD, Boyer D, et al. One-year outcomes of the da Vinci Study of VEGF Trap-Eye in eyes with diabetic macular edema[J]. Ophthalmology, 2012, 119(8): 1658-1665. DOI: 10.1016/j.ophtha.2012.02.010.
5. Diabetic Retinopathy Clinical Research Network, Wells JA, Glassman AR, et al. Aflibercept, bevacizumab, or ranibizumab for diabetic macular edema[J]. N Engl J Med, 2015, 372(13): 1193-1203. DOI: 10.1056/NEJMoa1414264.
6. Dehghan MH, Salehipour M, Naghib J, et al. Pars plana vitrectomy with internal limiting membrane peeling for refractory diffuse diabetic macular edema[J]. J Ophthalmic Vis Res, 2010, 5(3): 162-167.
7. Figueroa MS, Contreras I, Noval S. Surgical and anatomical outcomes of pars plana vitrectomy for diffuse nontractional diabetic macular edema[J]. Retina, 2008, 28(3): 420-426. DOI: 10.1097/IAE.0b013e318159e7d2.
8. Kimura T, Kiryu J, Nishiwaki H, et al. Efficacy of surgical removal of the internal limiting membrane in diabetic cystoid macular edema[J]. Retina, 2005, 25(4): 454-461. DOI: 10.1097/00006982-200506000-00010.
9. Stefánsson E. The therapeutic effects of retinal laser treatment and vitrectomy. A theory based on oxygen and vascular physiology[J]. Acta Ophthalmol Scand, 2001, 79(5): 435-440. DOI: 10.1034/j.1600-0420.2001.790502.x.
10. Schey KL, Wang Z, L Wenke J, et al. Aquaporins in the eye: expression, function, and roles in ocular disease[J]. Biochim Biophys Acta, 2014, 1840(5): 1513-1523. DOI: 10.1016/j.bbagen.2013.10.037.
11. Hollborn M, Dukic-Stefanovic S, Pannicke T, et al. Expression of aquaporins in the retina of diabetic rats[J]. Curr Eye Res, 2011, 36(9): 850-856. DOI: 10.3109/02713683.2011.593108.
12. Suzuki H, Oku H, Horie T, et al. Changes in expression of aquaporin-4 and aquaporin-9 in optic nerve after crushing in rats[J/OL]. PLoS One, 2014, 9(12): e114694[2014-12-05]. https://doi.org/10.1371/journal.pone.0114694. DOI: 10.1371/journal.pone.0114694.
13. Dal Monte M, Nicchia GP, Cammalleri M, et al. Aquaporin 4 is required to induce retinal angiogenesis in a mouse model of oxygen‐induced retinopathy[J/OL]. Acta Ophthalmologica, 2015, 92: 253[2014-08-20]. https://doi.org/10.1111/j.1755-3768.2014.F006.x. DOI: 10.1111/j.1755-3768.2014.F006.x.
14. Oku H, Morishita S, Horie T, et al. Nitric oxide increases the expression of aquaporin-4 protein in rat optic nerve astrocytes through the cyclic guanosine monophosphate/protein kinase G pathway[J]. Ophthalmic Res, 2015, 54(4): 212-221. DOI: 10.1159/000440846.
15. Lassiale S, Valamanesh F, Klein C, et al. Changes in aquaporin-4 and Kir4.1 expression in rats with inherited retinal dystrophy[J]. Exp Eye Res, 2016, 148: 33-44. DOI: 10.1016/j.exer.2016.05.010.
16. Kida T, Oku H, Horie T, et al. Implication of VEGF and aquaporin 4 mediating Müller cell swelling to diabetic retinal edema[J]. Graefe’s Arch Clin Exp Ophthalmol, 2017, 255(6): 1149-1157. DOI: 10.1007/s00417-017-3631-z.
17. Nguyen QD, Tatlipinar S, Shah SM, et al. Vascular endothelial growth factor is a critical stimulus for diabetic macular edema[J]. Am J Ophthalmol, 2006, 142(6): 961-969. DOI: 10.1016/j.ajo.2006.06.068.
18. Le YZ. VEGF production and signaling in Müller glia are critical to modulating vascular function and neuronal integrity in diabetic retinopathy and hypoxic retinal vascular diseases[J]. Vision Res, 2017, 139: 108-114. DOI: 10.1016/j.visres.2017.05.005.
19. Miyamoto K, Khosrof S, Bursell SE, et al. Vascular endothelial growth factor (VEGF)-induced retinal vascular permeability is mediated by intercellular adhesion molecule-1 (ICAM-1)[J]. Am J Pathol, 2000, 156(5): 1733-1739. DOI: 10.1016/s0002-9440(10)65044-4.
20. Miyamoto K, Khosrof S, Bursell SE, et al. Prevention of leukostasis and vascular leakage in streptozotocin-induced diabetic retinopathy via intercellular adhesion molecule-1 inhibition[J]. Proc Natl Acad Sci USA, 1999, 96(19): 10836-10841. DOI: 10.1073/pnas.96.19.10836.
21. Sohn HJ, Han DH, Kim IT, et al. Changes in aqueous concentrations of various cytokines after intravitreal triamcinolone versus bevacizumab for diabetic macular edema[J]. Am J Ophthalmol, 2011, 152(4): 686-694. DOI: 10.1016/j.ajo.2011.03.033.
22. Felinski EA, Antonetti DA. Glucocorticoid regulation of endothelial cell tight junction gene expression: novel treatments for diabetic retinopathy[J]. Curr Eye Res, 2005, 30(11): 949-957. DOI: 10.1080/02713680500263598.
23. Noma H, Mimura T, Yasuda K, et al. Role of inflammation in diabetic macular edema[J]. Ophthalmologica, 2014, 232(3): 127-135. DOI: 10.1159/000364955.
24. Funatsu H, Noma H, Mimura T, et al. Association of vitreous inflammatory factors with diabetic macular edema[J]. Ophthalmology, 2009, 116(1): 73-79. DOI: 10.1016/j.ophtha.2008.09.037.
25. Jonas JB, Jonas RA, Neumaier M, et al. Cytokine concentration in aqueous humor of eyes with diabetic macular edema[J]. Retina, 2012, 32(10): 2150-2157. DOI: 10.1097/IAE.0b013e3182576d07.
26. Ferrara N. VEGF and intraocular neovascularization: from discovery to therapy[J/OL]. Transl Vis Sci Technol, 2016, 5(2): 10[2016-04-11]. https://pubmed.ncbi.nlm.nih.gov/26981332/. DOI: 10.1167/tvst.5.2.10.
27. Mu H, Zhang XM, Liu JJ, et al. Effect of high glucose concentration on VEGF and PEDF expression in cultured retinal Müller cells[J]. Mol Biol Rep, 2009, 36(8): 2147-2151. DOI: 10.1007/s11033-008-9428-8.
28. Wang J, Xu X, Elliott MH, et al. Müller cell-derived VEGF is essential for diabetes-induced retinal inflammation and vascular leakage[J]. Diabetes, 2010, 59(9): 2297-2305. DOI: 10.2337/db09-1420.
29. Simmons AB, Bretz CA, Wang H, et al. Correction to: gene therapy knockdown of VEGFR2 in retinal endothelial cells to treat retinopathy[J/OL]. Angiogenesis, 2018, 21(4): 765[2018-06-25]. https://pubmed.ncbi.nlm.nih.gov/29943214/. DOI: 10.1007/s10456-018-9626-5.
30. Fu S, Dong S, Zhu M, et al. VEGF as a trophic factor for Müller glia in hypoxic retinal diseases[J]. Adv Exp Med Biol, 2018, 1074: 473-478. DOI: 10.1007/978-3-319-75402-4_58.
31. Bressler SB, Qin H, Beck RW, et al. Factors associated with changes in visual acuity and central subfield thickness at 1 year after treatment for diabetic macular edema with ranibizumab[J]. Arch Ophthalmol, 2012, 130(9): 1153-1161. DOI: 10.1001/archophthalmol.2012.1107.
32. Barile GR, Pachydaki SI, Tari SR, et al. The RAGE axis in early diabetic retinopathy[J]. Invest Ophthalmol Vis Sci, 2005, 46(8): 2916-2924. DOI: 10.1167/iovs.04-1409.
33. Semeraro F, Morescalchi F, Duse S, et al. Current trends about inner limiting membrane peeling in surgery for epiretinal membranes[J/OL]. J Ophthalmol, 2015, 2015: 671905[2015-09-03]. https://pubmed.ncbi.nlm.nih.gov/26425352/. DOI: 10.1155/2015/671905.
34. Bonnin S, Sandali O, Bonnel S, et al. Vitrectomy with internal limiting membrane peeling for tractional and nontractional diabetic macular edema: long-term results of a comparative study[J]. Retina, 2015, 35(5): 921-928. DOI: 10.1097/IAE.0000000000000433.
35. Tenckhoff S, Hollborn M, Kohen L, et al. Diversity of aquaporin mRNA expressed by rat and human retinas[J]. Neuroreport, 2005, 16(1): 53-56. DOI: 10.1097/00001756-200501190-00013.
36. Hamann S, Zeuthen T, La Cour M, et al. Aquaporins in complex tissues: distribution of aquaporins 1-5 in human and rat eye[J]. Am J Physiol, 1998, 274(5): C1332-C1345. DOI: 10.1152/ajpcell.1998.274.5.C1332.

Chinese Journal of Ocular Fundus Diseases

The expressions of vascular endothelial growth factor and aquaporin 4 in the inner limiting membrane from eyes with diabetic macular edema

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