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Connexin43 hemichannel block protects against the development of diabetic retinopathy signs in a mouse model of the disease

Journal of Molecular Medicine volume 97, pages 215–229 (2019)Cite this article

Abstract

Diabetic retinopathy (DR) is a vascular disease of the neuroretina characterised by hyperglycaemia and inflammation. Current DR therapies target late-stage vascular defects and there is evidence to suggest that they contribute to geographic atrophy and retinal ganglion cell death long term. Therefore, alternative treatments that target common upstream disease mechanisms are needed. Recent studies have shown that connexin43 hemichannel blockers can reduce inflammation and prevent vessel leak in brain and spinal cord lesions. The aim of this study was to evaluate the effectiveness of a connexin43 hemichannel blocker (Peptide5) in a mouse model of DR in which pro-inflammatory cytokines, IL-1β and TNF-α, were intravitreally injected into non-obese diabetic (NOD, hyperglycaemic) mice. Fundus and optical coherence tomography images were taken to evaluate vessel dilation and beading as well as retinal and vitreous hyper-reflective foci (HRF). Immunohistochemistry was performed to assess levels of astrogliosis, microgliosis and inflammasome activation. Results showed that Peptide5 injection lowered the incidence of vessel dilation and beading, decreased the severity of vitreous and retinal HRF, and reduced sub-retinal fluid accumulation compared to the vehicle group. Furthermore, Peptide5 led to reduced connexin43 and GFAP upregulation, inhibited microglial infiltration into the outer nuclear layer and prevented upregulation of inflammasome markers compared to vehicle. The present study provides evidence in support of Peptide5, and connexin43 hemichannel block in general, as a potential upstream approach for the treatment of DR.

Key messages

  • Connexin43 is upregulated in a novel mouse model of diabetic retinopathy (DR).

  • Connexin43 hemichannel block inhibits inflammation and inflammasome activation.

  • Connexin43 hemichannel block prevents the development of clinical DR signs.

  • Connexin43 hemichannel block is a potential upstream approach for DR treatment.

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References

  1. Abu el Asrar AM, Maimone D, Morse PH, Gregory S, Reder AT (1992) Cytokines in the vitreous of patients with proliferative diabetic retinopathy. Am J Ophthalmol 114(6):731–736

    Article  CAS  PubMed  Google Scholar 

  2. Tang J, Kern TS (2011) Inflammation in diabetic retinopathy. Prog Retin Eye Res 30(5):343–358

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Zhou J, Wang S, Xia X (2012) Role of intravitreal inflammatory cytokines and angiogenic factors in proliferative diabetic retinopathy. Curr Eye Res 37(5):416–420

    Article  CAS  PubMed  Google Scholar 

  4. Martin DF, Maguire MG, Fine SL, Ying GS, Jaffe GJ, Grunwald JE, Toth C, Redford M, Ferris FL 3rd, Comparison of Age-related Macular Degeneration Treatments Trials Research G (2012) Ranibizumab and bevacizumab for treatment of neovascular age-related macular degeneration: two-year results. Ophthalmology 119(7):1388–1398

    Article  PubMed  Google Scholar 

  5. Gemenetzi M, Lotery AJ, Patel PJ (2017) Risk of geographic atrophy in age-related macular degeneration patients treated with intravitreal anti-VEGF agents. Eye (Lond) 31(1):1–9

    Article  CAS  Google Scholar 

  6. Grunwald JE, Daniel E, Huang J, Ying GS, Maguire MG, Toth CA, Jaffe GJ, Fine SL, Blodi B, Klein ML, Martin AA, Hagstrom SA, Martin DF, Group CR (2014) Risk of geographic atrophy in the comparison of age-related macular degeneration treatments trials. Ophthalmology 121(1):150–161

    Article  PubMed  Google Scholar 

  7. Nishijima K, Ng YS, Zhong LC, Bradley J, Schubert W, Jo N, Akita J, Samuelsson SJ, Robinson GS, Adamis AP, Shima DT (2007) Vascular endothelial growth factor-a is a survival factor for retinal neurons and a critical neuroprotectant during the adaptive response to ischemic injury. Am J Pathol 171(1):53–67

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Mori R, Power KT, Wang CHM, Martin P, Becker DL (2006) Acute downregulation of connexin43 at wound sites leads to a reduced inflammatory response, enhanced keratinocyte proliferation and wound fibroblast migration. J Cell Sci 119(24):5193–5203

    Article  CAS  PubMed  Google Scholar 

  9. Oviedo-Orta E, Kwak BR, Evans WH (2013) Connexin cell communication channels: roles in the immune system and immunopathology. CRC Press, Boca Raton

    Book  Google Scholar 

  10. Qiu C, Coutinho P, Frank S, Franke S, Law L-y, Martin P, Green CR, Becker DL (2003) Targeting connexin43 expression accelerates the rate of wound repair. Curr Biol 13(19):1697–1703

    Article  CAS  PubMed  Google Scholar 

  11. Danesh-Meyer HV, Zhang J, Acosta ML, Rupenthal ID, Green CR (2016) Connexin43 in retinal injury and disease. Prog Retin Eye Res 51:41–68

    Article  CAS  PubMed  Google Scholar 

  12. Leybaert L, Lampe PD, Dhein S, Kwak BR, Ferdinandy P, Beyer EC, Laird DW, Naus CC, Green CR, Schulz R (2017) Connexins in cardiovascular and neurovascular health and disease: pharmacological implications. Pharmacol Rev 69(4):396–478

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Danesh-Meyer HV, Kerr NM, Zhang J, Eady EK, O'Carroll SJ, Nicholson LF, Johnson CS, Green CR (2012) Connexin43 mimetic peptide reduces vascular leak and retinal ganglion cell death following retinal ischaemia. Brain 135(Pt 2):506–520

    Article  PubMed  Google Scholar 

  14. Willebrords J, Crespo Yanguas S, Maes M, Decrock E, Wang N, Leybaert L, Kwak BR, Green CR, Cogliati B, Vinken M (2016) Connexins and their channels in inflammation. Crit Rev Biochem Mol Biol 51(6):413–439

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Bennett MV, Garre JM, Orellana JA, Bukauskas FF, Nedergaard M, Saez JC (2012) Connexin and pannexin hemichannels in inflammatory responses of glia and neurons. Brain Res 1487:3–15

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Decrock E, De Bock M, Wang N, Bultynck G, Giaume C, Naus CC, Green CR, Leybaert L (2015) Connexin and pannexin signaling pathways, an architectural blueprint for CNS physiology and pathology? Cell Mol Life Sci 72(15):2823–2851

    Article  CAS  PubMed  Google Scholar 

  17. Kim Y, Davidson JO, Gunn KC, Phillips AR, Green CR, Gunn AJ (2016) Role of hemichannels in CNS inflammation and the inflammasome pathway. Adv Protein Chem Struct Biol 104:1–37

    Article  CAS  PubMed  Google Scholar 

  18. De Bock M, Culot M, Wang N, Bol M, Decrock E, De Vuyst E, da Costa A, Dauwe I, Vinken M, Simon AM, Rogiers V, De Ley G, Evans WH, Bultynck G, Dupont G, Cecchelli R, Leybaert L (2011) Connexin channels provide a target to manipulate brain endothelial calcium dynamics and blood-brain barrier permeability. J Cereb Blood Flow Metab 31(9):1942–1957

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. O'Carroll SJ, Alkadhi M, Nicholson LF, Green CR (2008) Connexin 43 mimetic peptides reduce swelling, astrogliosis, and neuronal cell death after spinal cord injury. Cell Commun Adhes 15(1):27–42

    Article  CAS  PubMed  Google Scholar 

  20. Wang N, De Vuyst E, Ponsaerts R, Boengler K, Palacios-Prado N, Wauman J, Lai CP, De Bock M, Decrock E, Bol M, Vinken M, Rogiers V, Tavernier J, Evans WH, Naus CC, Bukauskas FF, Sipido KR, Heusch G, Schulz R, Bultynck G, Leybaert L (2013) Selective inhibition of Cx43 hemichannels by Gap19 and its impact on myocardial ischemia/reperfusion injury. Basic Res Cardiol 108(1):309

    Article  CAS  PubMed  Google Scholar 

  21. Davidson JO, Green CR, Nicholson LF, O'Carroll SJ, Fraser M, Bennet L, Gunn AJ (2012) Connexin hemichannel blockade improves outcomes in a model of fetal ischemia. Ann Neurol 71(1):121–132

    Article  CAS  PubMed  Google Scholar 

  22. Galinsky R, Davidson J, Bennet L, Green C, Gunn A (2015) Connexin Hemichannel blockade improves survival of striatal neurons after perinatal cerebral Ischaemia. J Paediatr Child Health 51:60

    Google Scholar 

  23. Mao Y, Tonkin RS, Nguyen T, O'Carroll SJ, Nicholson LF, Green CR, Moalem-Taylor G, Gorrie CA (2017) Systemic administration of Connexin43 mimetic peptide improves functional recovery after traumatic spinal cord injury in adult rats. J Neurotrauma 34(3):707–719

    Article  PubMed  Google Scholar 

  24. O'Carroll SJ, Gorrie CA, Velamoor S, Green CR, Nicholson LF (2013) Connexin43 mimetic peptide is neuroprotective and improves function following spinal cord injury. Neurosci Res 75(3):256–267

    Article  CAS  PubMed  Google Scholar 

  25. Orellana JA, Froger N, Ezan P, Jiang JX, Bennett MV, Naus CC, Giaume C, Saez JC (2011) ATP and glutamate released via astroglial connexin 43 hemichannels mediate neuronal death through activation of pannexin 1 hemichannels. J Neurochem 118(5):826–840

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Ormonde S, Chou CY, Goold L, Petsoglou C, Al-Taie R, Sherwin T, McGhee CN, Green CR (2012) Regulation of connexin43 gap junction protein triggers vascular recovery and healing in human ocular persistent epithelial defect wounds. J Membr Biol 245(7):381–388

    Article  CAS  PubMed  Google Scholar 

  27. Guo CX, Mat Nor MN, Danesh-Meyer HV, Vessey KA, Fletcher EL, O'Carroll SJ, Acosta ML, Green CR (2016) Connexin43 mimetic peptide improves retinal function and reduces inflammation in a light-damaged albino rat model. Invest Ophthalmol Vis Sci 57(10):3961–3973

    Article  CAS  PubMed  Google Scholar 

  28. Kim Y, Griffin JM, Harris PW, Chan SH, Nicholson LF, Brimble MA, O'Carroll SJ, Green CR (2017) Characterizing the mode of action of extracellular Connexin43 channel blocking mimetic peptides in an in vitro ischemia injury model. Biochim Biophys Acta 1861(2):68–78

    Article  CAS  Google Scholar 

  29. Mugisho OO, Rupenthal ID, Squirrell DM, Bould SJ, Danesh-Meyer HV, Zhang J, Green CR, Acosta ML (2018) Intravitreal pro-inflammatory cytokines in non-obese diabetic mice: modelling signs of diabetic retinopathy. PLoS One 13(8):e0202156. https://doi.org/10.1371/journal.pone.0202156

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Mugisho OO, Green CR, Kho DT, Zhang J, Graham ES, Acosta ML, Rupenthal ID (2018) The inflammasome pathway is amplified and perpetuated in an autocrine manner through connexin43 hemichannel mediated ATP release. Biochim Biophys Acta 1862(3):385–393

    Article  CAS  Google Scholar 

  31. Mugisho OO, Green CR, Zhang J, Binz N, Acosta ML, Rakoczy E, Rupenthal ID (2017) Immunohistochemical characterization of connexin43 expression in a mouse model of diabetic retinopathy and in human donor retinas. Int J Mol Sci 18(12):2567

    Article  CAS  PubMed Central  Google Scholar 

  32. Antonetti DA, Barber AJ, Bronson SK, Freeman WM, Gardner TW, Jefferson LS, Kester M, Kimball SR, Krady JK, LaNoue KF, Norbury CC, Quinn PG, Sandirasegarane L, Simpson IA, Group JDRC (2006) Diabetic retinopathy: seeing beyond glucose-induced microvascular disease. Diabetes 55(9):2401–2411

    Article  CAS  PubMed  Google Scholar 

  33. Coscas G, Lupidi M, Coscas F (2017) OCT-A: guided treatment of diabetic retinopathy. Acta Ophthalmol 95(S259)

  34. Mizukami T, Hotta Y, Katai N (2017) Higher numbers of hyperreflective foci seen in the vitreous on spectral-domain optical coherence tomographic images in eyes with more severe diabetic retinopathy. Ophthalmologica 238(1–2):74–80

    Article  PubMed  Google Scholar 

  35. Ferris FL 3rd, Patz A (1984) Macular edema. A complication of diabetic retinopathy. Surv Ophthalmol 28(Suppl):452–461

    Article  PubMed  Google Scholar 

  36. Chen YS, Green CR, Wang K, Danesh-Meyer HV, Rupenthal ID (2015) Sustained intravitreal delivery of connexin43 mimetic peptide by poly(D,L-lactide-co-glycolide) acid micro- and nanoparticles--closing the gap in retinal ischaemia. Eur J Pharm Biopharm 95(Pt B):378–386

    Article  CAS  PubMed  Google Scholar 

  37. Chen YS, Green CR, Teague R, Perrett J, Danesh-Meyer HV, Toth I, Rupenthal ID (2015) Intravitreal injection of lipoamino acid-modified connexin43 mimetic peptide enhances neuroprotection after retinal ischemia. Drug Deliv Transl Res 5(5):480–488

    Article  CAS  PubMed  Google Scholar 

  38. Kerr NM, Johnson CS, de Souza CF, Chee KS, Good WR, Green CR, Danesh-Meyer HV (2010) Immunolocalization of gap junction protein connexin43 (GJA1) in the human retina and optic nerve. Invest Ophthalmol Vis Sci 51(8):4028–4034

    Article  PubMed  Google Scholar 

  39. Simo-Servat O, Hernandez C, Simo R (2012) Usefulness of the vitreous fluid analysis in the translational research of diabetic retinopathy. Mediat Inflamm 2012:872978:1–11

    Article  Google Scholar 

  40. Urbancic M, Kloboves Prevodnik V, Petrovic D, Globocnik Petrovic M (2013) A flow cytometric analysis of vitreous inflammatory cells in patients with proliferative diabetic retinopathy. Biomed Res Int 2013:251528

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Feit-Leichman RA, Kinouchi R, Takeda M, Fan Z, Mohr S, Kern TS, Chen DF (2005) Vascular damage in a mouse model of diabetic retinopathy: relation to neuronal and glial changes. Invest Ophthalmol Vis Sci 46(11):4281–4287

    Article  PubMed  Google Scholar 

  42. Fletcher EL, Phipps JA, Ward MM, Puthussery T, Wilkinson-Berka JL (2007) Neuronal and glial cell abnormality as predictors of progression of diabetic retinopathy. Curr Pharm Des 13(26):2699–2712

    Article  CAS  PubMed  Google Scholar 

  43. Rungger–Brändle E, Dosso AA, Leuenberger PM (2000) Glial reactivity, an early feature of diabetic retinopathy. Invest Ophthalmol Vis Sci 41(7):1971–1980

    PubMed  Google Scholar 

  44. El-Asrar AM (2012) Role of inflammation in the pathogenesis of diabetic retinopathy. Middle East Afr J Ophthalmol 19(1):70–74

    Article  PubMed  PubMed Central  Google Scholar 

  45. Chaurasia SS, Lim RR, Parikh BH, Wey YS, Tun BB, Wong TY, Luu CD, Agrawal R, Ghosh A, Mortellaro A, Rackoczy E, Mohan RR, Barathi VA (2018) The NLRP3 Inflammasome may contribute to pathologic neovascularization in the advanced stages of diabetic retinopathy. Sci Rep 8(1):2847

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Davidson JO, Green CR, Bennet L, Nicholson LF, Danesh-Meyer H, O'Carroll SJ, Gunn AJ (2013) A key role for connexin hemichannels in spreading ischemic brain injury. Curr Drug Targets 14(1):36–46

    Article  CAS  PubMed  Google Scholar 

  47. Guo ZL, Yu SH, Chen X, Ye RD, Zhu WS, Liu XF (2016) NLRP3 is involved in ischemia/reperfusion injury. Cns Neurol Disord-Dr 15(6):699–712

    Article  CAS  Google Scholar 

  48. Ildefonso CJ, Biswal MR, Ahmed CM, Lewin AS (2016) The NLRP3 Inflammasome and its role in age-related macular degeneration. In: Retinal degenerative diseases. Springer, Berlin, pp 59–65

    Chapter  Google Scholar 

  49. Loukovaara S, Piippo N, Kinnunen K, Hytti M, Kaarniranta K, Kauppinen A (2017) NLRP3 inflammasome activation is associated with proliferative diabetic retinopathy. Acta Ophthalmol 95:803–808

    Article  CAS  PubMed  Google Scholar 

  50. Zhou R, Tardivel A, Thorens B, Choi I, Tschopp J (2010) Thioredoxin-interacting protein links oxidative stress to inflammasome activation. Nat Immunol 11(2):136–140

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

O.O.M. was supported by a doctoral scholarship from the Buchanan Ocular Therapeutics Unit, University of Auckland, New Zealand.

Funding

The study was partially funded by a Lottery Health Research Grant, Department of Internal Affairs, and an Auckland Medical Research Foundation Grant [1117015], New Zealand. C.R.G. holds the W&B Hadden Chair in Ophthalmology.

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Authors and Affiliations

  1. Buchanan Ocular Therapeutics Unit, Department of Ophthalmology and the New Zealand National Eye Centre, University of Auckland, Auckland, New Zealand

    Odunayo O. Mugisho & Ilva D. Rupenthal

  2. Department of Ophthalmology and the New Zealand National Eye Centre, University of Auckland, Auckland, New Zealand

    Colin R. Green, David M. Squirrell, Sarah Bould, Helen V. Danesh-Meyer & Jie Zhang

  3. School of Optometry and Vision Science and the New Zealand National Eye Centre, University of Auckland, Auckland, New Zealand

    Monica L. Acosta

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  1. Odunayo O. Mugisho
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Correspondence to Ilva D. Rupenthal.

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Mugisho, O.O., Green, C.R., Squirrell, D.M. et al. Connexin43 hemichannel block protects against the development of diabetic retinopathy signs in a mouse model of the disease. J Mol Med 97, 215–229 (2019). https://doi.org/10.1007/s00109-018-1727-5

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Keywords

  • Diabetic retinopathy
  • Mouse models
  • Connexin43
  • Hemichannels
  • Inflammation