The effect of Notch1 and Delta-like ligand 4 in the neovascularization of the proliferative diabetic retinopathy_Chinese Journal of Ocular Fundus Diseases

Authors：

Guo Qingmin ,  Meng Xuxia , Liu Penghui , Zhou Xianhui

Department of Ophthalmology, The Affiliated Hospital of Qingdao University, Qingdao 266500, China (Guo Qingmin, now working at Hebei Eye Hospital; Zhou Xianhui, now working at Department of Ophthalmology, Yantai YeDa Hospital);

Corresponding author：

Meng Xuxia, Email: mengxuxia@163.com

Keywords：

retinal neovascularization/etiology; Diabetic retinopathy/pathology; Receptor, Notch1; Vascular endothelial growth factor receptor-2

DOI：

10.3760/cma.j.issn.1005-1015.2017.03.013

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Objective o observe the expression of Notch1 and Delta-like ligand 4 (Dll4) on the fibrovascular membranes in proliferative diabetic retinopathy (PDR), and investigate its relationship with vascular endothelial growth factor receptor 2 (VEGFR2). Methods Fifty-seven PDR patients (60 eyes) who underwent vitrectomy were enrolled in this study. The PDR patients were divided into non-injection group (30 patients, 32 eyes) and injection group (27 patients, 28 eyes). The eyes in injection group received intravitreal injection with ranibizumab at 2 to 7 days before surgery. The preretinal fibrovascular membranes were obtained from the PDR patients during vitrectomy. Eighteen epiretinal membranes were obtained from the non-diabetic patients was served as controls. The real-time polymerase chain reaction (RT-PCR) and immunohistochemical methods were used to detecting the expression of Notch1, Dll4 and VEGFR2. In the meantime, the numbers of the nucleus of vascular endothelial cells in the membranes stained with hematoxylin were counted. Results The immunohistochemical staining revealed that there were positive expression of Notch1, Dll4 and VEGFR2 in all PDR membranes, regardless of the injection of the ranibizumab. The levels of Notch1, Dll4 and VEGFR2 protein in non-injection group were higher than those of injection group (t=3.45, 6.01, 4.08;P=0.030, 0.008, 0.023). In injection group, the number of endothelial cells in the membranes reduced (17.17±2.48) compared with that of the non-injection group (41.50±5.57). There was significant difference in the number of endothelial cells in the membranes between the two groups (t=9.58,P＜0.05). RT-PCR showed that the differences of the mRNA expression of Notch1, Dll4 and VEGFR2 were all statistically significant among the PDR group and control group (H=12.50, 12.50, 12.02;P＜0.05).The expression of Notch1, Dll4 and VEGFR2 in the PDR membranes was higher than that of epiretinal membranes from non-diabetic patients. In the PDR group, the expression of Notch1, Dll4 and VEGFR2 of non-injection group was higher than that of injection group. Spearman correlation analysis showed that the expression of mRNA between VEGFR2 and Dll4 (r=0.83), VEGFR2 and Notch1 (r=0.81), Notch1 and Dll4 (r=0.87) were all significantly correlated (P＜0.05). Conclusions The expression of Notch1 and Dll4 in the PDR membranes are higher than that of the control group, and it is positively correlated with the expression of the VEGFR2. Notch1 and Dll4 play a regulatory rule in the neovascularization in PDR, the acting way may be correlated with VEGFR2.

Citation： Guo Qingmin, Meng Xuxia, Liu Penghui, Zhou Xianhui. The effect of Notch1 and Delta-like ligand 4 in the neovascularization of the proliferative diabetic retinopathy. Chinese Journal of Ocular Fundus Diseases, 2017, 33(3): 275-280. doi: 10.3760/cma.j.issn.1005-1015.2017.03.013 Copy

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9.	Leslie JD, Ariza-McNaughton L, Bermange AL, et al. Endothelial signalling by the Notch ligand Delta-like4 restricts angiogenesis[J]. Development, 2007, 134(5): 839-844.
10.	Garcia-Pascual CM, Ferrero H, Zimmermann RC, et al. Inhibition of Delta-like 4 mediated signaling induces abortion in mice due to deregulation of decidual angiogenesis[J]. Placenta, 2014, 35(7): 501-508. DOI: 10.1016/j.placenta.2014.03.019.
11.	Wang X, He Z, Xia T, et al. Latency-associated nuclear antigen of Kaposi sarcoma-associated herpesvirus promotes angiogenesis through targeting notch signaling effector Hey1[J]. Cancer Res, 2014, 74(7): 2026-2037. DOI: 10.1158/0008-5472.CAN-13-1467.
12.	Wang H, Huang X, Zhang J, et al. The expression of VEGF and Dll4/Notch pathway molecules in ovarian cancer[J]. Clin Chim Acta, 2014, 436: 243-248. DOI: 10.1016/j.cca.2014.06.005.
13.	Jakobsson L, Franco CA, Bentley K, et al. Endothelial cells dynamically compete for the tip cell position during angiogenic sprouting[J]. Nat Cell Biol, 2010, 12(10): 943-953. DOI: 10.1038/ncb2103.
14.	Outtz HH, Tattersall IW, Kofler NM, et al. Notch1 controls macrophage recruitment and Notch signaling is activated at sites of endothelial cell anastomosis during retinal angiogenesis in mice[J]. Blood, 2011, 118(12): 3436-3439. DOI: 10.1182/blood-2010-12-327015.
15.	Zhao L, Borikova AL, Ben-Yair R, et al. Notch signaling regulates cardiomyocyte proliferation during zebra fish heart regeneration[J]. Proc Natl Acad Sci USA, 2014, 111(4): 1403-1408. DOI: 10.1073/pnas.1311705111.
16.	Battle TE, Lynch RA, Frank DA. Signal transducer and activator of transcription 1 activation in endothelial cells is a negative regulator of angiogenesis [J]. Cancer Res, 2006, 66(7): 3649-3657.
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18.	Smith LE, Shen W, Perruzzi C, et al. Regulation of vascular endothelial growth factor-dependent retinal neovascularization by insulin-like growth factor-1 receptor[J]. Nat Med, 1999, 5(12): 1390-1395.

1. Roca C, Adams RH. Regulation of vascular morphogenesis by Notch signaling[J]. Genes Dev, 2007, 21(20): 2511-2524.
2. Walshe TE, Connell P, Cryan L, et al. Microvascular retinal endothelial and pericyte cell apoptosis in vitro: role of hedgehog and Notch signaling[J]. Invest Ophthalmol Vis Sci, 2011, 52(7): 4472-4483. DOI: 10.1167/iovs.10-7061.
3. Cao R, Jensen LD, Soll I, et al. Hypoxia-induced retinal angiogenesis in zebrafish as a model to study retinopathy [J/OL]. PLoS One, 2008, 3(7): 2748[2008-07-02]. . DOI: 10.1371/journal.pone. 0002748.
4. Mailhos C, Modlich U, Lewis J, et al. Delta4, an endothelial specific notch ligand expressed at sites of physiological and tumor angiogenesis[J]. Differentiation, 2001, 69(2-3): 135-144.
5. 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.
6. American Diabetes Association. Standards of medical care in diabetes--2010[J]. Diabetes Care. 2010, 33 Suppl1: S11-61. DOI: 10.2337/dc10-S011.
7. 中华医学会眼科学会眼底病学组, 我国糖尿病视网膜病变临床诊疗指南(2014年)[J]. 中华眼科杂志, 2014, 50(11): 851-865. DOI: 10.3760/cma.j.issn.0412-4081.2014.11.014.Chinese Ocular Fundus Diseases Society, Chinese Ophthalmological Society, Chinese Medical Association. Guidelines for clinical diagnosis and treatment of diabetic retinopathy in China [J]. Chin J Ophthalmol, 2014, 50(11): 851-865. DOI: 10.3760/cma.j.issn.0412-4081.2014.11.014.
8. Chappell JC, Mouillesseaux KP, Bautch VL.Flt-1 (vascular endothelial growth factor receptor-1) is essential for the vascular endothelial growth factor-Notch feedback loop during angiogenesis [J]. Arterioscler Thromb Vasc Biol, 2013, 33(8): 1952-1959. DOI: 10.1161/ATVBAHA.113.301805.
9. Leslie JD, Ariza-McNaughton L, Bermange AL, et al. Endothelial signalling by the Notch ligand Delta-like4 restricts angiogenesis[J]. Development, 2007, 134(5): 839-844.
10. Garcia-Pascual CM, Ferrero H, Zimmermann RC, et al. Inhibition of Delta-like 4 mediated signaling induces abortion in mice due to deregulation of decidual angiogenesis[J]. Placenta, 2014, 35(7): 501-508. DOI: 10.1016/j.placenta.2014.03.019.
11. Wang X, He Z, Xia T, et al. Latency-associated nuclear antigen of Kaposi sarcoma-associated herpesvirus promotes angiogenesis through targeting notch signaling effector Hey1[J]. Cancer Res, 2014, 74(7): 2026-2037. DOI: 10.1158/0008-5472.CAN-13-1467.
12. Wang H, Huang X, Zhang J, et al. The expression of VEGF and Dll4/Notch pathway molecules in ovarian cancer[J]. Clin Chim Acta, 2014, 436: 243-248. DOI: 10.1016/j.cca.2014.06.005.
13. Jakobsson L, Franco CA, Bentley K, et al. Endothelial cells dynamically compete for the tip cell position during angiogenic sprouting[J]. Nat Cell Biol, 2010, 12(10): 943-953. DOI: 10.1038/ncb2103.
14. Outtz HH, Tattersall IW, Kofler NM, et al. Notch1 controls macrophage recruitment and Notch signaling is activated at sites of endothelial cell anastomosis during retinal angiogenesis in mice[J]. Blood, 2011, 118(12): 3436-3439. DOI: 10.1182/blood-2010-12-327015.
15. Zhao L, Borikova AL, Ben-Yair R, et al. Notch signaling regulates cardiomyocyte proliferation during zebra fish heart regeneration[J]. Proc Natl Acad Sci USA, 2014, 111(4): 1403-1408. DOI: 10.1073/pnas.1311705111.
16. Battle TE, Lynch RA, Frank DA. Signal transducer and activator of transcription 1 activation in endothelial cells is a negative regulator of angiogenesis [J]. Cancer Res, 2006, 66(7): 3649-3657.
17. Ridgway J, Zhang G, Wu Y, et al. Inhibition of Dll4 signalling inhibits tumour growth by deregulating angiogenesis[J]. Nature, 2006, 444(7122): 1083-1087.
18. Smith LE, Shen W, Perruzzi C, et al. Regulation of vascular endothelial growth factor-dependent retinal neovascularization by insulin-like growth factor-1 receptor[J]. Nat Med, 1999, 5(12): 1390-1395.

Chinese Journal of Ocular Fundus Diseases

The effect of Notch1 and Delta-like ligand 4 in the neovascularization of the proliferative diabetic retinopathy

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