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Graefe's Archive for Clinical and Experimental Ophthalmology

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01-05-2021 | Cornea

NF-κB, iNOS, IL-6, and collagen 1 and 5 expression in healthy and keratoconus corneal fibroblasts after 0.1% riboflavin UV-A illumination

Authors: Tim Berger, Nóra Szentmáry, Lorenz Latta, Berthold Seitz, Tanja Stachon

Published in: Graefe's Archive for Clinical and Experimental Ophthalmology | Issue 5/2021

Abstract

Purpose

To analyze the effect of riboflavin UV-A illumination on mRNA and protein expression of healthy (HCFs) and keratoconus human corneal fibroblasts (KC-HCFs), concerning the inflammatory markers NF-κB, iNOS, IL-6, and collagen 1 and 5 (Col 1/Col 5).

Methods

Keratocytes were isolated from healthy (n = 3) and keratoconus (KC) corneas (n = 3) and were cultivated in basal medium with 5% fetal calf serum, which resulted in their transformation into human corneal fibroblasts (HCFs/KC-HCFs). Cells underwent 0.1% riboflavin UV-A illumination for 250 s (CXL). NF-κB, iNOS, IL-6, Col 1, and Col 5 expression was investigated by qPCR and Western blot analysis. IL-6 concentration of the cell culture supernatant and cell lysate was determined by ELISA.

Results

In untreated KC-HCFs, NF-κB (p = 0.0002), iNOS (p = 0.0019), Col 1 (p = 0.0286), and Col 5 (p = 0.0054) mRNA expression was higher and IL-6 expression was lower (p = 0.0057), than in healthy controls. In HCFs, CXL led to an increased NF-κB (p = 0.0286) and IL-6 (p = 0.0057) mRNA expression. The IL-6 concentration in the cell culture supernatant was increased in HCFs (p = 0.0485) and KC-HCFs (p = 0.0485) after CXL. CXL increased intracellular IL-6 concentration only in KC-HCFs (p = 0.0357). In the HCF group (p = 0.0286), an increased Col 1 mRNA expression after CXL could be observed.

Conclusion

Our study confirmed altered gene expression in untreated KC-HCFs compared to untreated HCFs. Riboflavin UV-A illumination affected gene expression only in HCFs. Increased IL-6 concentration in the cell culture supernatant and cell lysate indicate a secondary inflammatory response of HCFs and KC-HCFs to riboflavin UV-A illumination.

Fan Gaskin JC, Patel DV, McGhee CNJ (2014) Acute corneal hydrops in keratoconus - new perspectives. Am J Ophthalmol 157:921–928. https://doi.org/10.1016/j.ajo.2014.01.017CrossRefPubMed

Rabinowitz YS (1998) Keratoconus. Surv Ophthalmol 42:297–319. https://doi.org/10.1016/S0039-6257(97)00119-7CrossRefPubMed

Godefrooij DA, de Wit GA, Uiterwaal CS et al (2017) Age-specific incidence and prevalence of keratoconus: a nationwide registration study. Am J Ophthalmol 175:169–172. https://doi.org/10.1016/j.ajo.2016.12.015CrossRefPubMed

Cozma I, Atherley C, James N (2005) Influence of ethnic origin on the incidence of keratoconus and associated atopic disease in Asian and white patients. Eye (Lond) 19:924–925. https://doi.org/10.1038/sj.eye.6701677CrossRef

Romero-Jiménez M, Santodomingo-Rubido J, Wolffsohn JS (2010) Keratoconus: a review. Contact Lens Anterior Eye 33:157–166. https://doi.org/10.1016/j.clae.2010.04.006CrossRefPubMed

Stachon T, Latta L, Kolev K et al (2019) Increased NF-κB and iNOS expression in keratoconus keratocytes - hints for an inflammatory component? Klin Monbl Augenheilkd. https://doi.org/10.1055/a-1002-0100

Lema I, Durán JA (2005) Inflammatory molecules in the tears of patients with keratoconus. Ophthalmology 112:654–659. https://doi.org/10.1016/j.ophtha.2004.11.050CrossRefPubMed

Karamichos D, Hutcheon A, Rich C et al (2014) In vitro model suggests oxidative stress involved in keratoconus disease. Sci Rep 4:4608. https://doi.org/10.1038/srep04608CrossRefPubMedPubMedCentral

Karamichos D, Zieske J, Sejersen H et al (2015) Tear metabolite changes in keratoconus. Exp Eye Res 132:1–8. https://doi.org/10.1016/j.exer.2015.01.007CrossRefPubMedPubMedCentral

10.

Hassell J, Birk D (2010) The molecular basis of corneal transparency. Exp Eye Res 91:326–335. https://doi.org/10.1016/j.exer.2010.06.021CrossRefPubMedPubMedCentral

11.

Hayes S, Boote C, Tuft S et al (2007) A study of corneal thickness, shape and collagen organisation in keratoconus using videokeratography and X-ray scattering techniques. Exp Eye Res 84:423–434. https://doi.org/10.1016/j.exer.2006.10.014CrossRefPubMed

12.

Alkanaan A, Barsotti R, Kirat O et al (2019) Collagen fibrils and proteoglycans of peripheral and central stroma of the keratoconus cornea - ultrastructure and 3D transmission electron tomography. Sci Rep 9:1–11. https://doi.org/10.1038/s41598-019-56529-1CrossRef

13.

Parker JS, van Dijk K, Melles GRJ (2015) Treatment options for advanced keratoconus: a review. Surv Ophthalmol 60:459–480. https://doi.org/10.1016/j.survophthal.2015.02.004CrossRefPubMed

14.

Raiskup F, Spoerl E (2013) Corneal crosslinking with riboflavin and ultraviolet A. I. principles. Ocul Surf 11:65–74. https://doi.org/10.1016/j.jtos.2013.01.002CrossRefPubMed

15.

Juarranz A, Jaén P, Sanz-Rodriguez F, Cuevas-Santos J (2008) Photodynamic therapy of cancer. Basic principles and applications. Clin Transl Oncol 10:148–154. https://doi.org/10.1007/s12094-008-0172-2CrossRef

16.

Wollensak G, Spoerl E, Seiler T (2003) Riboflavin/ultraviolet-A-induced collagen crosslinking for the treatment of keratoconus. Am J Ophthalmol 135:620–627. https://doi.org/10.1016/S0002-9394(02)02220-1CrossRefPubMed

17.

Song X, Stachon T, Wang J et al (2015) Viability, apoptosis, proliferation, activation, and cytokine secretion of human keratoconus keratocytes after cross-linking. Biomed Res Int 2015:254237. https://doi.org/10.1155/2015/254237CrossRefPubMedPubMedCentral

18.

Stachon T, Latta L, Seitz B, Szentmáry N (2020) Hypoxic stress increases NF-κB and iNOS mRNA expression in normal, but not in keratoconus corneal fibroblasts. Graefe’s Arch Clin Exp Ophthalmol. https://doi.org/10.1007/s00417-020-04900-8

19.

Gupta SC, Sundaram C, Reuter S, Aggarwal BB (2010) Inhibiting NF-κB activation by small molecules as a therapeutic strategy. Biochim Biophys Acta - Gene Regul Mech 1799:775–787. https://doi.org/10.1016/j.bbagrm.2010.05.004CrossRef

20.

Libermann TA, Baltimore D (1990) Activation of interleukin-6 gene expression through the NF-kappa B transcription factor. Mol Cell Biol 10:2327–2334. https://doi.org/10.1128/mcb.10.5.2327CrossRefPubMedPubMedCentral

21.

Morris KR, Lutz RD, Choi HS et al (2003) Role of the NF-κB signaling pathway and κB cis-regulatory elements on the IRF-1 and iNOS promoter regions in mycobacterial lipoarabinomannan induction of nitric oxide. Infect Immun 71:1442–1452. https://doi.org/10.1128/IAI.71.3.1442-1452.2003CrossRefPubMedPubMedCentral

22.

Donnelly LE, Barnes PJ (2002) Expression and regulation of inducible nitric oxide synthase from human primary airway epithelial cells. Am J Respir Cell Mol Biol 26:144–151. https://doi.org/10.1165/ajrcmb.26.1.4477CrossRefPubMed

23.

Chwa M, Atilano SR, Reddy V et al (2006) Increased stress-induced generation of reactive oxygen species and apoptosis in human keratoconus fibroblasts. Invest Ophthalmol Vis Sci 47:1902–1910. https://doi.org/10.1167/iovs.05-0828CrossRefPubMed

24.

Stachon T, Wang J, Song X et al (2015) Impact of crosslinking/riboflavin-UVA-photodynamic inactivation on viability, apoptosis and activation of human keratocytes in vitro. J Biomed Res 29:321–325. https://doi.org/10.7555/JBR.29.20130173CrossRefPubMedPubMedCentral

25.

Messmer E, Meyer P, Herwig-Carl M et al (2012) Morphological and immunohistochemical changes after corneal cross-linking. Cornea 32:111–117. https://doi.org/10.1097/ICO.0b013e31824d701bCrossRef

26.

Belin MW, Duncan JK (2016) Keratoconus: the ABCD Grading System. Klin Monbl Augenheilkd 233:701–707. https://doi.org/10.1055/s-0042-100626CrossRefPubMed

27.

Sharif R, Hjortdal J, Sejersen H et al (2017) Human in vitro model reveals the effects of collagen cross-linking on keratoconus pathogenesis. Sci Rep 7:12517. https://doi.org/10.1038/s41598-017-12598-8CrossRefPubMedPubMedCentral

Title: NF-κB, iNOS, IL-6, and collagen 1 and 5 expression in healthy and keratoconus corneal fibroblasts after 0.1% riboflavin UV-A illumination
Authors: Tim Berger
Nóra Szentmáry
Lorenz Latta
Berthold Seitz
Tanja Stachon
Publication date: 01-05-2021
Publisher: Springer Berlin Heidelberg
Published in: Graefe's Archive for Clinical and Experimental Ophthalmology / Issue 5/2021
Print ISSN: 0721-832X
Electronic ISSN: 1435-702X
DOI: https://doi.org/10.1007/s00417-020-05058-z

Keynote webinar | Spotlight on medication adherence

Springer Medicine

NF-κB, iNOS, IL-6, and collagen 1 and 5 expression in healthy and keratoconus corneal fibroblasts after 0.1% riboflavin UV-A illumination

Abstract

Purpose

Methods

Results

Conclusion

Keynote webinar | Spotlight on medication adherence

Springer Medicine

Abstract

Purpose

Methods

Results

Conclusion

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