, Volume 62, Issue 2, pp 322–334 | Cite as

Therapeutic regulation of VE-cadherin with a novel oligonucleotide drug for diabetic eye complications using retinopathy mouse models

  • Ka Ka Ting
  • Yang Zhao
  • Weiyong Shen
  • Paul Coleman
  • Michelle Yam
  • Tailoi Chan-Ling
  • Jia Li
  • Thorleif Moller
  • Mark Gillies
  • Mathew A. VadasEmail author
  • Jennifer R. GambleEmail author



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.


Blood–retinal barrier Diabetic retinopathy Microglia MicroRNA Müller cells Neovascularisation Oligonucleotide Pericytes VE-cadherin 



Blood–brain barrier


Blood–retinal barrier


Diabetic macular oedema


Endothelial nitric oxide synthase


Glyceraldehyde-3-phosphate dehydrogenase


Ganglion cell layer


Inner nuclear layer




LDL receptor-related protein 6




Neural/glial antigen 2


Oxygen-induced retinopathy


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-specific cadherin


VE-cadherin null


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.

Contribution statement

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.

Supplementary material

125_2018_4770_MOESM1_ESM.pdf (15.9 mb)
ESM (PDF 16291 kb)


  1. 1.
    Antonetti DA, Klein R, Gardner TW (2012) Diabetic retinopathy. N Engl J Med 366(13):1227–1239. CrossRefPubMedGoogle Scholar
  2. 2.
    Cunha-Vaz J, Bernardes R, Lobo C (2011) Blood-retinal barrier. Eur J Ophthalmol 21(Suppl 6):S3–S9CrossRefGoogle Scholar
  3. 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. CrossRefPubMedGoogle Scholar
  4. 4.
    Leal EC, Martins J, Voabil P et al (2010) Calcium dobesilate inhibits the alterations in tight junction proteins and leukocyte adhesion to retinal endothelial cells induced by diabetes. Diabetes 59(10):2637–2645. CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Navaratna D, McGuire PG, Menicucci G, Das A (2007) Proteolytic degradation of VE-cadherin alters the blood-retinal barrier in diabetes. Diabetes 56(9):2380–2387. CrossRefPubMedGoogle Scholar
  6. 6.
    Roy S, Ha J, Trudeau K, Beglova E (2010) Vascular basement membrane thickening in diabetic retinopathy. Curr Eye Res 35(12):1045–1056. CrossRefPubMedGoogle Scholar
  7. 7.
    Semeraro F, Cancarini A, dell’Omo R, Rezzola S, Romano MR, Costagliola C (2015) Diabetic retinopathy: vascular and inflammatory disease. J Diabetes Res 2015:582060CrossRefGoogle Scholar
  8. 8.
    Zeng HY, Green WR, Tso MO (2008) Microglial activation in human diabetic retinopathy. Arch Ophthalmol 126(2):227–232. CrossRefPubMedGoogle Scholar
  9. 9.
    Aiello LP, Avery RL, Arrigg PG et al (1994) Vascular endothelial growth factor in ocular fluid of patients with diabetic retinopathy and other retinal disorders. N Engl J Med 331(22):1480–1487. CrossRefPubMedGoogle Scholar
  10. 10.
    Gavard J, Gutkind JS (2006) VEGF controls endothelial-cell permeability by promoting the beta-arrestin-dependent endocytosis of VE-cadherin. Nat Cell Biol 8(11):1223–1234. CrossRefPubMedGoogle Scholar
  11. 11.
    Sidibe A, Imhof BA (2014) VE-cadherin phosphorylation decides: vascular permeability or diapedesis. Nat Immunol 15(3):215–217. CrossRefPubMedGoogle Scholar
  12. 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. CrossRefPubMedGoogle Scholar
  13. 13.
    Das A, Stroud S, Mehta A, Rangasamy S (2015) New treatments for diabetic retinopathy. Diabetes Obes Metab 17(3):219–230. CrossRefPubMedGoogle Scholar
  14. 14.
    Lampugnani MG, Orsenigo F, Gagliani MC, Tacchetti C, Dejana E (2006) Vascular endothelial cadherin controls VEGFR-2 internalization and signaling from intracellular compartments. J Cell Biol 174(4):593–604. CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Davidson MK, Russ PK, Glick GG, Hoffman LH, Chang MS, Haselton FR (2000) Reduced expression of the adherens junction protein cadherin-5 in a diabetic retina. Am J Ophthalmol 129(2):267–269. CrossRefPubMedGoogle Scholar
  16. 16.
    Yan Y, Chang Q, Li Q et al (2015) Identification of plasma vascular endothelia-cadherin as a biomarker for coronary artery disease in type 2 diabetes mellitus patients. Int J Clin Exp Med 8(10):19466–19470PubMedPubMedCentralGoogle Scholar
  17. 17.
    Lee CS, Kim YG, Cho HJ et al (2016) Dipeptidyl peptidase-4 inhibitor increases vascular leakage in retina through VE-cadherin phosphorylation. Sci Rep 6(1):29393. CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    He J, Wang H, Liu Y, Li W, Kim D, Huang H (2015) Blockade of vascular endothelial growth factor receptor 1 prevents inflammation and vascular leakage in diabetic retinopathy. J Ophthalmol 2015:605946CrossRefGoogle Scholar
  19. 19.
    Young JA, Ting KK, Li J et al (2013) Regulation of vascular leak and recovery from ischemic injury by general and VE-cadherin-restricted miRNA antagonists of miR-27. Blood 122(16):2911–2919. CrossRefPubMedGoogle Scholar
  20. 20.
    Zhao Y, Ting KK, Li J et al (2017) Targeting vascular endothelial-cadherin in tumor-associated blood vessels promotes T cell-mediated immunotherapy. Cancer Res 77(16):4434–4447. CrossRefPubMedGoogle Scholar
  21. 21.
    Zhou Q, Gallagher R, Ufret-Vincenty R, Li X, Olson EN, Wang S (2011) Regulation of angiogenesis and choroidal neovascularization by members of microRNA-23~27~24 clusters. Proc Natl Acad Sci U S A 108(20):8287–8292. CrossRefPubMedPubMedCentralGoogle Scholar
  22. 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
  23. 23.
    Karolina DS, Tavintharan S, Armugam A et al (2012) Circulating miRNA profiles in patients with metabolic syndrome. J Clin Endocrinol Metab 97(12):E2271–E2276. CrossRefPubMedGoogle Scholar
  24. 24.
    Shen W, Fruttiger M, Zhu L et al (2012) Conditional Muller cell ablation causes independent neuronal and vascular pathologies in a novel transgenic model. J Neurosci 32(45):15715–15727. CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Smith LE, Wesolowski E, McLellan A et al (1994) Oxygen-induced retinopathy in the mouse. Invest Ophthalmol Vis Sci 35(1):101–111PubMedGoogle Scholar
  26. 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. CrossRefPubMedGoogle Scholar
  27. 27.
    Sasongko MB, Wong TY, Nguyen TT, Cheung CY, Shaw JE, Wang JJ (2011) Retinal vascular tortuosity in persons with diabetes and diabetic retinopathy. Diabetologia 54(9):2409–2416. CrossRefPubMedGoogle Scholar
  28. 28.
    Hammes HP, Lin J, Renner O et al (2002) Pericytes and the pathogenesis of diabetic retinopathy. Diabetes 51(10):3107–3112. CrossRefPubMedGoogle Scholar
  29. 29.
    Robinson R, Barathi VA, Chaurasia SS, Wong TY, Kern TS (2012) Update on animal models of diabetic retinopathy: from molecular approaches to mice and higher mammals. Dis Model Mech 5(4):444–456. CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Gerhardinger C, Dagher Z, Sebastiani P, Park YS, Lorenzi M (2009) The transforming growth factor-beta pathway is a common target of drugs that prevent experimental diabetic retinopathy. Diabetes 58(7):1659–1667. CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Chen Y, Hu Y, Zhou T et al (2009) Activation of the Wnt pathway plays a pathogenic role in diabetic retinopathy in humans and animal models. Am J Pathol 175(6):2676–2685. CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Gerhardt H, Betsholtz C (2003) Endothelial-pericyte interactions in angiogenesis. Cell Tissue Res 314(1):15–23. CrossRefPubMedGoogle Scholar
  33. 33.
    Bentley K, Franco CA, Philippides A et al (2014) The role of differential VE-cadherin dynamics in cell rearrangement during angiogenesis. Nat Cell Biol 16(4):309–321. CrossRefPubMedGoogle Scholar
  34. 34.
    Taddei A, Giampietro C, Conti A et al (2008) Endothelial adherens junctions control tight junctions by VE-cadherin-mediated upregulation of claudin-5. Nat Cell Biol 10(8):923–934. CrossRefPubMedGoogle Scholar
  35. 35.
    Luo Y, Xiao W, Zhu X et al (2011) Differential expression of claudins in retinas during normal development and the angiogenesis of oxygen-induced retinopathy. Invest Ophthalmol Vis Sci 52(10):7556–7564. CrossRefPubMedGoogle Scholar
  36. 36.
    Dalkara T, Gursoy-Ozdemir Y, Yemisci M (2011) Brain microvascular pericytes in health and disease. Acta Neuropathol 122(1):1–9. CrossRefPubMedGoogle Scholar
  37. 37.
    McGuire PG, Rangasamy S, Maestas J, Das A (2011) Pericyte-derived sphingosine 1-phosphate induces the expression of adhesion proteins and modulates the retinal endothelial cell barrier. Arterioscler Thromb Vasc Biol 31:e107–e115CrossRefGoogle Scholar
  38. 38.
    Cunha SI, Pietras K (2011) ALK1 as an emerging target for antiangiogenic therapy of cancer. Blood 117(26):6999–7006. CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Rudini N, Felici A, Giampietro C et al (2008) VE-cadherin is a critical endothelial regulator of TGF-beta signalling. EMBO J 27(7):993–1004. CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Li Q, Verma A, Han PY et al (2010) Diabetic eNOS-knockout mice develop accelerated retinopathy. Invest Ophthalmol Vis Sci 51(10):5240–5246. CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Fu S, Dong S, Zhu M et al (2015) Muller glia are a major cellular source of survival signals for retinal neurons in diabetes. Diabetes 64(10):3554–3563. CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Hnik P, Boyer DS, Grillone LR, Clement JG, Henry SP, Green EA (2009) Antisense oligonucleotide therapy in diabetic retinopathy. J Diabetes Sci Technol 3(4):924–930. CrossRefPubMedPubMedCentralGoogle Scholar
  43. 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. CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Krady JK, Basu A, Allen CM et al (2005) Minocycline reduces proinflammatory cytokine expression, microglial activation, and caspase-3 activation in a rodent model of diabetic retinopathy. Diabetes 54(5):1559–1565. CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    da Fonseca AC, Matias D, Garcia C et al (2014) The impact of microglial activation on blood-brain barrier in brain diseases. Front Cell Neurosci 8:362CrossRefGoogle Scholar
  46. 46.
    Ryu JK, McLarnon JG (2009) A leaky blood-brain barrier, fibrinogen infiltration and microglial reactivity in inflamed Alzheimer’s disease brain. J Cell Mol Med 13(9a):2911–2925. CrossRefPubMedGoogle Scholar
  47. 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. CrossRefPubMedGoogle Scholar
  48. 48.
    Deliyanti D, Talia DM, Zhu T et al (2017) Foxp3(+) Tregs are recruited to the retina to repair pathological angiogenesis. Nat Commun 8(1):748. CrossRefPubMedPubMedCentralGoogle Scholar
  49. 49.
    Amadio M, Govoni S, Pascale A (2016) Targeting VEGF in eye neovascularization: What’s new? A comprehensive review on current therapies and oligonucleotide-based interventions under development. Pharmacol Res 103:253–269. CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Ka Ka Ting
    • 1
  • Yang Zhao
    • 1
  • Weiyong Shen
    • 2
  • Paul Coleman
    • 1
  • Michelle Yam
    • 2
  • Tailoi Chan-Ling
    • 3
  • Jia Li
    • 1
  • Thorleif Moller
    • 4
  • Mark Gillies
    • 2
  • Mathew A. Vadas
    • 1
    Email author
  • Jennifer R. Gamble
    • 1
    Email author
  1. 1.Centre for the Endothelium Vascular Biology Program Centenary InstituteThe University of SydneyNewtownAustralia
  2. 2.Save Sight InstituteThe University of SydneySydneyAustralia
  3. 3.Discipline of Anatomy and Histology, School of Medical Sciences Bosch InstituteThe University of SydneySydneyAustralia
  4. 4.Ranger Biotechnologies A/SÅrslevDenmark

Personalised recommendations