Effect of Delta-like ligand 4 on pathological structure of retina in early diabetic rats and its relationship with vascular endothelial growth factor receptor-2_Chinese Journal of Ocular Fundus Diseases

Authors：

Yu Chuan , Hu Die ,  Meng Xuxia , Zhou Xianhui , Cao Bowen , Li Xiaoqing , Chen Ya’nan

Department of Ophthalmology, The Affiliated of Qingdao University, Qingdao 266500, China (Hu die, now working at the Qingdao Eye Hospital；Zhou Xianhui, now working at the Department of Ophthalmology, Yantai YeDa Hospital；Cao Bowen,now studying at the University of Tuebingen, Germany);

Corresponding author：

Meng Xuxia, Email: mengxuxia@163.com

Keywords：

Diabetic retinopathy; Detal like 4; Vascular endothelial growth factor receptor 2

DOI：

10.3760/cma.j.issn.1005-1015.2018.06.012

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ObjectiveTo observe the effect of Delta-like ligand 4 (Dll-4) on the pathological structure of retina in early diabetic rats (DM) and its relationship with vascular endothelial growth receptor-2 (VEGFR-2).MethodsA total of 70 male Sprague-Dawley rats were randomly divided into normal group and DM group, with 10 and 60 rats in each group, respectively. The rats of DM group was induced by intraperitoneal injection of streptozotocin to established DM model. The rats with blood glucose recovery and death were excluded, and the final 60 rats were included in the statistics. Rats in the normal group were injected with an equal volume of citric acid-sodium citrate buffer. Rats in the DM group were divided into DM 1 month (DM 1m) group, DM 2 months (DM 2m) group, DM 3 months (DM 3m) group and DM 3m + Anti group, DM 3m + phosphate buffer solution (PBS) group by random number table method, and 10 rats in each group. In the DM 3m+Anti group, 4 μl of anti-Dll-4 polyclonal antibody was injected into the vitreous cavity, and the antibody concentration was 0.25 mg/ml. The DM 3m+PBS group was intravitreally injected with an equal volume of PBS. Five days after the injection, the rats were sacrificed. Rats in the DM 3m group and the normal group were not treated, and were sacrificed 3 months after the model was established. The structure and microvascular changes of the retina were observed by hematoxylin-eosin staining, and the total thickness of the retina was measured. The expression of Dll-4 and VEGFR-2 in the retina was detected by immunohistochemistry and fluorescence quantitative polymerase chain reaction (PCR). One-way analysis of variance was used to compare the expression of Dll-4 and VEGFR-2 in the retina of each group. The least significant difference t test was used to compare the two groups.ResultsLight microscopy showed that the retinal ganglion cells layer in the DM 3m group were obviously edematous, the inner and outer nuclear layers were thinner, the number of cells was reduced, the arrangement was disordered, the edema of outer plexiform layer was obvious, and the microvessels were abnormally dilated. In the DM 3m+Anti group, the edema of outer plexiform layer was lessened than that of the DM 3m group, and the other layers were not significantly different from the DM 3m group. Compared with the normal group, the total retinal thickness of the DM 3m group, the DM 3m+Anti group and the DM 3m+PBS group increased (t=5.596, 3.290, 4.286; P=0.000, 0.008, 0.002). Immunohistochemical staining showed that a small amount of Dll4 was positively expressed in the retinal ganglion cell layer of the normal group; a small amount of VEGFR-2 was positively expressed in the ganglion cell layer and the inner and outer nuclear layers. The positive expression of Dll-4 and VEGFR-2 in retinal vascular endothelial cells of DM 3m group increased significantly. The expression of Dll-4 was significantly decreased in the retinal layers and vascular endothelial cells of DM 3m+Anti group, while the expression of VEGFR-2 was significantly increased. There was no significant difference between the positive expression of Dll4 and VEGFR-2 in the DM 3m+PBS group and the DM 3m group. The results of real-time PCR showed that the relative expression of Dll-4 and VEGFR-2 mRNA in the DM 3m group was significantly higher than that in the normal group (t=6.705, 20.871; P＜0.05). Compared with DM 3m group, the relative expression of Dll-4 mRNA in DM 3m+Anti group decreased, and the relative expression of VEGFR-2 mRNA increased (t=2.681, 3.639;P＜0.05). The relative expressions of Dll-4 and VEGFR-2 mRNA in the DM 3m+PBS group and DM 3m group were not statistically significant (t=0.513, 0.657; P＜0.05).ConclusionsThe expression of Dll-4 in retinal vascular endothelial cells is gradually increased during the early retinopathy of DM rats. The expression of Dll-4 is inhibited, the expression of VEGFR-2 is up-regulated, and the plexus edema is alleviated.

Citation： Yu Chuan, Hu Die, Meng Xuxia, Zhou Xianhui, Cao Bowen, Li Xiaoqing, Chen Ya’nan. Effect of Delta-like ligand 4 on pathological structure of retina in early diabetic rats and its relationship with vascular endothelial growth factor receptor-2. Chinese Journal of Ocular Fundus Diseases, 2018, 34(6): 586-591. doi: 10.3760/cma.j.issn.1005-1015.2018.06.012 Copy

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9.	Lobov IB, Renard RA, Papadopoulos N, et al. Delta-like ligand 4 (Dll4) is induced by VEGF as a negative regulator of angiogenic sprouting[J]. Proc Natl Acad Sci USA, 2007, 104(9): 3219-3224. DOI: 10.1073/pnas.0611206104.
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14.	Liu ZJ, Shirakawa T, Li Y, et al. Regulation of Notch1 and Dll4 by vascular endothelial growth factor in arterial endothelial cells: implications for modulating arteriogenesis and angiogenesis[J]. Mol Cell Biol, 2003, 23(1): 14-25.
15.	Suchting S, Freitas C, le Noble F , et al. The Notch ligand Delta-like 4 negatively regulates endothelial tip cell formation and vessel branching[J]. Proc Natl Acad Sci USA, 2007, 104(9): 3225-3230. DOI: 10.1073/pnas.0611177104.
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18.	Harrington LS, Sainson RC, Williams CK, et al. Regulation of multiple angiogenic pathways by Dll4 and Notch in human umbilical vein endothelial cells[J]. Microvasc Res, 2008, 75(2): 144-154. DOI: 10.1016/j.mvr.2007.06.006.
19.	Aboualizadeh E, Sorenson CM, Schofield AJ, et al. Temporal diabetes-induced biochemical changes in distinctive layers of mouse retina[J]. Sci Rep, 2018, 8(1): 1096. DOI: 10.1038/s41598-018-19425-8.

1. Shutter JR, Scully S, Fan W, et al. Dll4, a novel Notch ligand expressed in arterial endothelium [J]. Genes Dev, 2000, 14(11): 1313-1318.
2. 胡迭, 孟旭霞, 付浴东, 等. Notch-1及Delta样配体4在糖尿病大鼠早期视网膜中的表达[J]. 中国糖尿病杂志, 2016, 24(9): 840-844. DOI: 10.3969/j.issn.1006-6187.2016.09.016.Hu D, Meng XX, Fu YD, et al. Expression of Notch-1 and Dll-4 in retina of diabetic rats at early stage[J]. Chin J Diabetes, 2016, 24(9): 840-844. DOI: 10.3969/j.issn.1006-6187.2016.09.016.
3. 郭庆敏, 孟旭霞, 刘鹏辉, 等. Notch受体蛋白1、Delta样配体4在增生型糖尿病视网膜病变新生血管形成中的作用[J]. 中华眼底病杂志, 2017, 33(3): 275-280. DOI: 10.3760/cma.j.issn.1005-1015.2017.03.013.Guo QM, Meng XX, Liu PH, et al. The effect of Notch1 and Delta-like ligand 4 in the neovascularization of the proliferative diabetic retinopathy[J]. Chin J Ocul Fundus Dis, 2017, 33(3): 275-280. DOI: 10.3760/cma.j.issn.1005-1015.2017.03.013.
4. Ruszkowska-Ciastek B, Sokup A, Socha MW, et al. A preliminary evaluation of VEGF-A, VEGFR1 and VEGFR2 in patients with well-controlled type 2 diabetes mellitus[J]. J Zhejiang Univ Sci B, 2014, 15(6): 575-581. DOI: 10.1631/jzus.B1400024.
5. Tremolada G, Del Turco C, Lattanzio R, et al. The role of angiogenesis in the development of proliferative diabetic retinopathy: impact of intravitreal anti-VEGF treatment[J/OL]. Exp Diabetes Res, 2012, 2012: 728325[2012-04-11]. http://dx.doi.org/10.1155/2012/728325. DOI: 10.1155/2012/728325.
6. Wang X, Wang G. Intravitreous vascular endothelial growth factor and hypoxia-inducible factor 1a in patients with proliferative diabetic retinopathy[J]. Am J Ophthalmol, 2009, 148(6): 883-889. DOI: 10.1016/j.ajo.2009.07.007.
7. Dureau P, Bonnel S, Menasche M, et al. Quantitative analysis of intravitreal injections in the rat[J]. Curr Eye Res, 2001, 22(1): 74-77.
8. Shi S, Li X, Li Y, et al. Expression and function of Delta-like ligand 4 in a rat model of retinopathy of prematurity[J]. Neural Regen Res, 2013, 8(8): 723-730. DOI: 10.3969/j.issn.1673-5374.2013.08.007.
9. Lobov IB, Renard RA, Papadopoulos N, et al. Delta-like ligand 4 (Dll4) is induced by VEGF as a negative regulator of angiogenic sprouting[J]. Proc Natl Acad Sci USA, 2007, 104(9): 3219-3224. DOI: 10.1073/pnas.0611206104.
10. Ferrara N.Vascular endothelial growth factor: molecular and biological aspects[J]. Curr Top Microbiol Immunol, 1999, 237: 1-30.
11. Olsson AK, Dimberg A, Kreuger J. VEGF receptor signalling - in control of vascular function [J]. Nat Rev Mol Cell Biol, 2006, 7(5): 359-371. DOI: 10.1038/nrm1911.
12. Hiratsuka S, Minowa O, Kuno J, et al. Flt-1 lacking the tyrosine kinase domain is sufficient for normal development and angiogenesis in mice[J]. Proc Natl Acad Sci USA, 1998, 95(16): 9349-9354.
13. Park JE, Chen HH, Winer J, et al. Placenta growth factor Potentiation of vascular endothelial growth factor bioactivity, in vitro and in vivo, and high affinity binding to Flt-1 but not to Flk-1/KDR[J]. J Biol Chem, 1994, 269(41): 25646-25654.
14. Liu ZJ, Shirakawa T, Li Y, et al. Regulation of Notch1 and Dll4 by vascular endothelial growth factor in arterial endothelial cells: implications for modulating arteriogenesis and angiogenesis[J]. Mol Cell Biol, 2003, 23(1): 14-25.
15. Suchting S, Freitas C, le Noble F , et al. The Notch ligand Delta-like 4 negatively regulates endothelial tip cell formation and vessel branching[J]. Proc Natl Acad Sci USA, 2007, 104(9): 3225-3230. DOI: 10.1073/pnas.0611177104.
16. Trindade A, Djokovic D, Gigante J, et al. Endothelial Dll4 overexpression reduces vascular response and inhibits tumor growth and metastasization in vivo[J]. BMC Cancer, 2017, 17(1): 189. DOI: 10.1186/s12885-017-3171-2.
17. Williams CK, Li JL, Murga M, et al. Up-regulation of the Notch ligand Delta-like 4 inhibits VEGF-induced endothelial cell function[J]. Blood, 2006, 107(3): 931-939. DOI: 10.1182/blood-2005-03-1000.
18. Harrington LS, Sainson RC, Williams CK, et al. Regulation of multiple angiogenic pathways by Dll4 and Notch in human umbilical vein endothelial cells[J]. Microvasc Res, 2008, 75(2): 144-154. DOI: 10.1016/j.mvr.2007.06.006.
19. Aboualizadeh E, Sorenson CM, Schofield AJ, et al. Temporal diabetes-induced biochemical changes in distinctive layers of mouse retina[J]. Sci Rep, 2018, 8(1): 1096. DOI: 10.1038/s41598-018-19425-8.

Chinese Journal of Ocular Fundus Diseases

Effect of Delta-like ligand 4 on pathological structure of retina in early diabetic rats and its relationship with vascular endothelial growth factor receptor-2

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