, Volume 62, Issue 12, pp 2365–2374 | Cite as

Neutrophil elastase contributes to the pathological vascular permeability characteristic of diabetic retinopathy

  • Haitao Liu
  • Emma M. Lessieur
  • Aicha Saadane
  • Sarah I. Lindstrom
  • Patricia R. Taylor
  • Timothy S. KernEmail author



Levels of neutrophil elastase, a serine protease secreted by neutrophils, are elevated in diabetes. The purpose of this study was to determine whether neutrophil elastase (NE) contributes to the diabetes-induced increase in retinal vascular permeability in mice with streptozotocin-induced diabetes, and, if so, to investigate the potential role of IL-17 in this process.


In vivo, diabetes was induced in neutrophil elastase-deficient (Elane−/−), Il-17a−/− and wild-type mice. After 8 months of diabetes, Elane−/− mice and wild-type age-matched control mice were injected with FITC-BSA. Fluorescence microscopy was used to assess leakage of FITC-BSA from the retinal vasculature into the neural retina. The level of NE in Il-17a−/− diabetic retina and sera were determined by ELISA. In vitro, the effect of NE on the permeability and viability of human retinal endothelial cells and the expression of junction proteins and adhesion molecules were studied.


Eight months of diabetes resulted in increased retinal vascular permeability and levels of NE in retina and plasma of wild-type animals. All of these abnormalities were significantly inhibited in mice lacking the elastase. The diabetes-induced increase in NE was inhibited in mice lacking IL-17. In vitro, NE increased retinal endothelial cell permeability, which was partially inhibited by a myeloid differentiation primary response 88 (MyD88) inhibitor, NF-κB inhibitor, and protease-activated receptor (PAR)2 inhibitor. NE degraded vascular endothelial-cadherin (VE-cadherin) in a concentration-dependent manner.


IL-17 regulates NE expression in diabetes. NE contributes to vascular leakage in diabetic retinopathy, partially through activation of MyD88, NF-κB and PAR2 and degradation of VE-cadherin.


Diabetic retinopathy Elane IL-17 Neutrophil elastase Vascular permeability 



Human retinal endothelial cell


Intercellular adhesion molecule 1


Inner nuclear layer


Inner plexiform layer


Myeloid differentiation primary response 88


Neutrophil elastase


Outer nuclear layer


Outer plexiform layer


Protease-activated receptor


Spectral-domain optical coherence tomography


Toll-like receptor 4


Vascular endothelial cadherin




Zona occludens 1



The authors thank D. A. Antonetti and X. Liu (University of Michigan, Ann Arbor, MI, USA) for the retinal whole mount immunostaining. Chieh Allen Lee, Katie Franke and Heather Butler (Case Western Reserve University, Cleveland, OH, USA) who maintained the mouse colonies. Dawn Smith (Case Western Reserve University) who maintained the hRECs.

Contribution statement

HL performed molecular analyses and wrote the manuscript. EML analysed data and wrote the manuscript. AS and PRT acquired data, were involved in the analysis and interpretation of data and reviewed the manuscript. SIL performed ELISA, was involved in the analysis and interpretation of data and edited the manuscript. TSK designed experiments, acquired data and reviewed/edited the manuscript. All the authors approved the final version of the manuscript to be published. TSK is the guarantor of this work.


This work was supported by NIH grants RO1 EY022938 and R24 EY024864 (to TSK), and grants BX003604 (to TSK) and BX003403 (to PRT) from the Department of Veterans Affairs, and core grant P30 EY011373 to Case Western Reserve University. 81900884 (to HL) from The First Affiliated Hospital of Dalian Medical University. This research received no specific grant from any funding agency in commercial or not-for-profit sectors.

Duality of interest

The authors declare that there is no duality of interest associated with this manuscript.

Supplementary material

125_2019_4998_MOESM1_ESM.pdf (121 kb)
ESM Figure (PDF 121 kb)


  1. 1.
    Lee R, Wong TY, Sabanayagam C (2015) Epidemiology of diabetic retinopathy, diabetic macular edema and related vision loss. Eye Vis 2(1):17. CrossRefGoogle Scholar
  2. 2.
    Ogurtsova K, da Rocha Fernandes JD, Huang Y et al (2017) IDF Diabetes Atlas: global estimates for the prevalence of diabetes for 2015 and 2040. Diabetes Res Clin Pract 128:40–50. CrossRefPubMedGoogle Scholar
  3. 3.
    Solomon SD, Chew E, Duh EJ et al (2017) Diabetic retinopathy: a position statement by the American Diabetes Association. Diabetes Care 40(3):412–418. CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Engelgau MM, Geiss LS, Saaddine JB et al (2004) The evolving diabetes burden in the United States. Ann Intern Med 140(11):945–950. CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Klaassen I, Van Noorden CJ, Schlingemann RO (2013) Molecular basis of the inner blood-retinal barrier and its breakdown in diabetic macular edema and other pathological conditions. Prog Retin Eye Res 34:19–48. CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Joussen AM, Smyth N, Niessen C (2007) Pathophysiology of diabetic macular edema. Dev Ophthalmol 39:1–12PubMedGoogle Scholar
  7. 7.
    Tien T, Barrette KF, Chronopoulos A, Roy S (2013) Effects of high glucose-induced Cx43 downregulation on occludin and ZO-1 expression and tight junction barrier function in retinal endothelial cells. Invest Ophthalmol Vis Sci 54(10):6518–6525. CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    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
  9. 9.
    Li G, Veenstra AA, Talahalli RR et al (2012) Marrow-derived cells regulate the development of early diabetic retinopathy and tactile allodynia in mice. Diabetes 61(12):3294–3303. CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Joussen AM, Poulaki V, Le ML et al (2004) A central role for inflammation in the pathogenesis of diabetic retinopathy. FASEB J 18(12):1450–1452. CrossRefPubMedGoogle Scholar
  11. 11.
    Veenstra AA, Tang J, Kern TS (2013) Antagonism of CD11b with neutrophil inhibitory factor (NIF) inhibits vascular lesions in diabetic retinopathy. PLoS One 8(10):e78405. CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Wang Y, Xiao Y, Zhong L et al (2014) Increased neutrophil elastase and proteinase 3 and augmented NETosis are closely associated with beta-cell autoimmunity in patients with type 1 diabetes. Diabetes 63(12):4239–4248. CrossRefPubMedGoogle Scholar
  13. 13.
    Korkmaz B, Horwitz MS, Jenne DE, Gauthier F (2010) Neutrophil elastase, proteinase 3, and cathepsin G as therapeutic targets in human diseases. Pharmacol Rev 62(4):726–759. CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Lee WL, Downey GP (2001) Leukocyte elastase: physiological functions and role in acute lung injury. Am J Respir Crit Care Med 164(5):896–904. CrossRefPubMedGoogle Scholar
  15. 15.
    Moraes TJ, Chow CW, Downey GP (2003) Proteases and lung injury. Crit Care Med 31(Suppl):S189–S194. CrossRefGoogle Scholar
  16. 16.
    Owen CA (2008) Roles for proteinases in the pathogenesis of chronic obstructive pulmonary disease. Int J Chron Obstruct Pulmon Dis 3:253–268. CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Shapiro SD (2002) Proteinases in chronic obstructive pulmonary disease. Biochem Soc Trans 30(2):98–102. CrossRefPubMedGoogle Scholar
  18. 18.
    Walsh DE, Greene CM, Carroll TP et al (2001) Interleukin-8 up-regulation by neutrophil elastase is mediated by MyD88/IRAK/TRAF-6 in human bronchial epithelium. J Biol Chem 276(38):35494–35499. CrossRefPubMedGoogle Scholar
  19. 19.
    Devaney JM, Greene CM, Taggart CC, Carroll TP, O’Neill SJ, McElvaney NG (2003) Neutrophil elastase up-regulates interleukin-8 via toll-like receptor 4. FEBS Lett 544(1-3):129–132. CrossRefPubMedGoogle Scholar
  20. 20.
    Soh UJ, Dores MR, Chen B, Trejo J (2010) Signal transduction by protease-activated receptors. Br J Pharmacol 160(2):191–203. CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Ritchie E, Saka M, Mackenzie C et al (2007) Cytokine upregulation of proteinase-activated-receptors 2 and 4 expression mediated by p38 MAP kinase and inhibitory kappa B kinase beta in human endothelial cells. Br J Pharmacol 150(8):1044–1054. CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Hung DT, Wong YH, Vu TK, Coughlin SR (1992) The cloned platelet thrombin receptor couples to at least two distinct effectors to stimulate phosphoinositide hydrolysis and inhibit adenylyl cyclase. J Biol Chem 267(29):20831–20834PubMedGoogle Scholar
  23. 23.
    Rahman A, True AL, Anwar KN, Ye RD, Voyno-Yasenetskaya TA, Malik AB (2002) Gαq and Gβγ regulate PAR-1 signaling of thrombin-induced NF-κB activation and ICAM-1 transcription in endothelial cells. Circ Res 91(5):398–405. CrossRefPubMedGoogle Scholar
  24. 24.
    Veenstra A, Liu H, Lee CA, Du Y, Tang J, Kern TS (2015) Diabetic retinopathy: retina-specific methods for maintenance of diabetic rodents and evaluation of vascular histopathology and molecular abnormalities. Curr Protoc Mouse Biol 5(3):247–270. CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Liu H, Tang J, Du Y et al (2015) Retinylamine benefits early diabetic retinopathy in mice. J Biol Chem 290(35):21568–21579. CrossRefPubMedGoogle Scholar
  26. 26.
    Liu H, Tang J, Du Y et al (2016) Photoreceptor cells influence retinal vascular degeneration in mouse models of retinal degeneration and diabetes. Invest Ophthalmol Vis Sci 57(10):4272–4281. CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Antonetti DA, Barber AJ, Khin S, Lieth E, Tarbell JM, Gardner TW (1998) Vascular permeability in experimental diabetes is associated with reduced endothelial occludin content: vascular endothelial growth factor decreases occludin in retinal endothelial cells. Penn State Retina Research Group. Diabetes 47(12):1953–1959. CrossRefPubMedGoogle Scholar
  28. 28.
    Du Y, Cramer M, Lee CA et al (2015) Adrenergic and serotonin receptors affect retinal superoxide generation in diabetic mice: relationship to capillary degeneration and permeability. FASEB J 29(5):2194–2204. CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Muthusamy A, Lin CM, Shanmugam S, Lindner HM, Abcouwer SF, Antonetti DA (2014) Ischemia-reperfusion injury induces occludin phosphorylation/ubiquitination and retinal vascular permeability in a VEGFR-2-dependent manner. J Cereb Blood Flow Metab 34(3):522–531. CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Nooteboom A, Hendriks T, Otteholler I, van der Linden CJ (2000) Permeability characteristics of human endothelial monolayers seeded on different extracellular matrix proteins. Mediat Inflamm 9(5):235–241. CrossRefGoogle Scholar
  31. 31.
    Tonade D, Liu H, Palczewski K, Kern TS (2017) Photoreceptor cells produce inflammatory products that contribute to retinal vascular permeability in a mouse model of diabetes. Diabetologia 60(10):2111–2120. CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Sigurdardottir S, Zapadka TE, Lindstrom SI, Liu H, Lee CA, Kern TS, Taylor PR (2019) Diabetes-mediated IL-17A enhances retinal inflammation, oxidative stress, and vascular permeability. Cell Immunol 341:103921. CrossRefPubMedGoogle Scholar
  33. 33.
    Frey T, Antonetti DA (2011) Alterations to the blood-retinal barrier in diabetes: cytokines and reactive oxygen species. Antioxid Redox Signal 15(5):1271–1284. CrossRefPubMedGoogle Scholar
  34. 34.
    Vinores SA, Derevjanik NL, Mahlow J, Berkowitz BA, Wilson CA (1998) Electron microscopic evidence for the mechanism of blood-retinal barrier breakdown in diabetic rabbits: comparison with magnetic resonance imaging. Pathol Res Pract 194(7):497–505. CrossRefPubMedGoogle Scholar
  35. 35.
    Beltramo E, Porta M (2013) Pericyte loss in diabetic retinopathy: mechanisms and consequences. Curr Med Chem 20(26):3218–3225. CrossRefPubMedGoogle Scholar
  36. 36.
    Berkowitz BA, Bissig D, Ye Y, Valsadia P, Kern TS, Roberts R (2012) Evidence for diffuse central retinal edema in vivo in diabetic male Sprague Dawley rats. PLoS One 7(1):e29619. CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Owen CA, Campbell MA, Sannes PL, Boukedes SS, Campbell EJ (1995) Cell surface-bound elastase and cathepsin G on human neutrophils: a novel, non-oxidative mechanism by which neutrophils focus and preserve catalytic activity of serine proteinases. J Cell Biol 131(3):775–789. CrossRefPubMedGoogle Scholar
  38. 38.
    Yu X, Akbarzadeh R, Pieper M et al (2018) Neutrophil adhesion is a prerequisite for antibody-mediated proteolytic tissue damage in experimental models of epidermolysis bullosa acquisita. J Investig Dermatol 138(9):1990–1998. CrossRefPubMedGoogle Scholar
  39. 39.
    Hashemi M, Naderi M, Rashidi H, Ghavami S (2007) Impaired activity of serum alpha-1-antitrypsin in diabetes mellitus. Diabetes Res Clin Pract 75(2):246–248. CrossRefPubMedGoogle Scholar
  40. 40.
    Joussen AM, Doehmen S, Le ML et al (2009) TNF-α mediated apoptosis plays an important role in the development of early diabetic retinopathy and long-term histopathological alterations. Mol Vis 15:1418–1428PubMedPubMedCentralGoogle Scholar
  41. 41.
    Mecham RP, Broekelmann TJ, Fliszar CJ, Shapiro SD, Welgus HG, Senior RM (1997) Elastin degradation by matrix metalloproteinases. Cleavage site specificity and mechanisms of elastolysis. J Biol Chem 272(29):18071–18076. CrossRefPubMedGoogle Scholar
  42. 42.
    Boxio R, Wartelle J, Nawrocki-Raby B et al (2016) Neutrophil elastase cleaves epithelial cadherin in acutely injured lung epithelium. Respir Res 17(1):129. CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Peterson MW, Walter ME, Nygaard SD (1995) Effect of neutrophil mediators on epithelial permeability. Am J Respir Cell Mol Biol 13(6):719–727. CrossRefPubMedGoogle Scholar
  44. 44.
    Champagne B, Tremblay P, Cantin A, Pierre Y (1998) Proteolytic cleavage of ICAM-1 by human neutrophil elastase. J Immunol 161:6398–6405PubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Haitao Liu
    • 1
    • 2
  • Emma M. Lessieur
    • 3
  • Aicha Saadane
    • 3
  • Sarah I. Lindstrom
    • 4
  • Patricia R. Taylor
    • 4
    • 5
  • Timothy S. Kern
    • 3
    • 5
    • 6
    Email author
  1. 1.Department of Biology, School of MedicineCase Western Reserve UniversityClevelandUSA
  2. 2.Department of OphthalmologyThe First Affiliated Hospital of Dalian Medical UniversityDalianPeople’s Republic of China
  3. 3.Center for Translational Vision Research, Department of Ophthalmology, Gavin Herbert Eye Institute, School of MedicineUniversity of California-IrvineIrvineUSA
  4. 4.Department of Ophthalmology and Visual SciencesCase Western Reserve UniversityClevelandUSA
  5. 5.Veterans Administration Medical Center Research Service 151ClevelandUSA
  6. 6.Veterans Administration Medical Center Research ServiceLong BeachUSA

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