Therapeutic regulation of VE-cadherin with a novel oligonucleotide drug for diabetic eye complications using retinopathy mouse models
A major feature of diabetic retinopathy is breakdown of the blood–retinal barrier, resulting in macular oedema. We have developed a novel oligonucleotide-based drug, CD5-2, that specifically increases expression of the key junctional protein involved in barrier integrity in endothelial cells, vascular-endothelial-specific cadherin (VE-cadherin). CD5-2 prevents the mRNA silencing by the pro-angiogenic microRNA, miR-27a. CD5-2 was evaluated in animal models of ocular neovascularisation and vascular leak to determine its potential efficacy for diabetic retinopathy.
CD5-2 was tested in three mouse models of retinal dysfunction: conditional Müller cell depletion, streptozotocin-induced diabetes and oxygen-induced retinopathy. Vascular permeability in the Müller cell-knockout model was assessed by fluorescein angiography. The Evans Blue leakage method was used to determine vascular permeability in streptozotocin- and oxygen-induced retinopathy models. The effects of CD5-2 on retinal neovascularisation, inter-endothelial junctions and pericyte coverage in streptozotocin- and oxygen-induced retinopathy models were determined by staining for isolectin-B4, VE-cadherin and neural/glial antigen 2 (NG2). Blockmir CD5-2 localisation in diseased retina was determined using fluorescent in situ hybridisation. The effects of CD5-2 on VE-cadherin expression and in diabetic retinopathy-associated pathways, such as the transforming growth factor beta (TGF-β) and wingless/integrated (WNT) pathway, were confirmed using western blot of lysates from HUVECs, a mouse brain endothelial cell line and a VE-cadherin null mouse endothelial cell line.
CD5-2 penetrated the vasculature of the eye in the oxygen-induced retinopathy model. Treatment of diseased mice with CD5-2 resulted in reduced vascular leak in all three animal models, enhanced expression of VE-cadherin in the microvessels of the eye and improved pericyte coverage of the retinal vasculature in streptozotocin-induced diabetic models and oxygen-induced retinopathy models. Further, CD5-2 reduced the activation of retinal microglial cells in the streptozotocin-induced diabetic model. The positive effects of CD5-2 seen in vivo were further confirmed in vitro by increased protein expression of VE-cadherin, SMAD2/3 activity, and platelet-derived growth factor B (PDGF-B).
CD5-2 has therapeutic potential for individuals with vascular-leak-associated retinal diseases based on its ease of delivery and its ability to reverse vascular dysfunction and inflammatory aspects in three animal models of retinopathy.
KeywordsBlood–retinal barrier Diabetic retinopathy Microglia MicroRNA Müller cells Neovascularisation Oligonucleotide Pericytes VE-cadherin
Diabetic macular oedema
Endothelial nitric oxide synthase
Ganglion cell layer
Inner nuclear layer
LDL receptor-related protein 6
Neural/glial antigen 2
Outer nuclear layer
7/12/14/17 days postnatal
Platelet-derived growth factor B
Platelet-derived growth factor receptor β
Proliferative diabetic retinopathy
Smad family member
Vascular endothelial growth factor
We give special thanks to J. Hunter (Centenary Institute, Sydney, Australia) for preparing the human endothelial cells and to the imaging and animal facility staff at the Centenary Institute for their technical assistance. We thank E. Dejana (Italian Foundation for Cancer Research [FIRC], Institute of Molecular Oncology [IFOM], Milan, Italy) for supplying the VEC-null endothelial cells and G. Grau (Department of Pathology, Sydney Medical School, University of Sydney, Sydney, NSW, Australia) for the mouse brain endothelial cells.
KKT designed the study, acquired and analysed the data, drafted and approved the final version of the manuscript. JRG and MAV contributed to the conception of the study, and drafted and approved the final version of the manuscript. MG and TM contributed to the conception and design of the study. TC-L was involved in interpretation of the data. WYS, MY, YZ, JL and PC acquired data, revised the article’s intellectual content and approved the final version. MG, TM and TC-L revised the article’s intellectual content and approved final version. KKT had full access to all the data, excluding those relating to the Müller cell transgenic model in this study. KKT and JRG are responsible for the integrity of this study.
This research was supported by grants from the National Health and Medical Research Council (NHMRC) of Australia (no. 571408). JRG holds the Wenkart Chair of the Endothelium at the Centenary Institute and the Sydney Medical School, University of Sydney.
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
- 3.Speiser P, Gittelsohn AM, Patz A (1968) Studies on diabetic retinopathy. 3. Influence of diabetes on intramural pericytes. Arch Ophthalmol 80(3):332–337. https://doi.org/10.1001/archopht.1968.00980050334007 CrossRefPubMedGoogle Scholar
- 12.Aiello LP, Northrup JM, Keyt BA, Takagi H, Iwamoto MA (1995) Hypoxic regulation of vascular endothelial growth factor in retinal cells. Arch Ophthalmol 113(12):1538–1544. https://doi.org/10.1001/archopht.1995.01100120068012 CrossRefPubMedGoogle Scholar
- 22.Nielsen LB, Wang C, Sorensen K et al (2012) Circulating levels of microRNA from children with newly diagnosed type 1 diabetes and healthy controls: evidence that miR-25 associates to residual beta-cell function and glycaemic control during disease progression. Exp Diabetes Res 2012:896362PubMedPubMedCentralGoogle Scholar
- 26.Lange C, Ehlken C, Stahl A, Martin G, Hansen L, Agostini HT (2009) Kinetics of retinal vaso-obliteration and neovascularisation in the oxygen-induced retinopathy (OIR) mouse model. Graefes Arch Clin Exp Ophthalmol 247(9):1205–1211. https://doi.org/10.1007/s00417-009-1116-4 CrossRefPubMedGoogle Scholar
- 43.Omri S, Behar-Cohen F, de Kozak Y et al (2011) Microglia/macrophages migrate through retinal epithelium barrier by a transcellular route in diabetic retinopathy: role of PKCζ in the Goto Kakizaki rat model. Am J Pathol 179(2):942–953. https://doi.org/10.1016/j.ajpath.2011.04.018 CrossRefPubMedPubMedCentralGoogle Scholar
- 47.Monickaraj F, McGuire PG, Nitta CF, Ghosh K, Das A (2016) Cathepsin D: an Mϕ-derived factor mediating increased endothelial cell permeability with implications for alteration of the blood-retinal barrier in diabetic retinopathy. FASEB J 30(4):1670–1682. https://doi.org/10.1096/fj.15-279802 CrossRefPubMedGoogle Scholar