Skip to main content
Erschienen in: Graefe's Archive for Clinical and Experimental Ophthalmology 1/2017

25.10.2016 | Basic Science

Arginase activity, urea, and hydroxyproline concentration are reduced in keratoconus keratocytes

verfasst von: Tanja Stachon, Krasimir Kolev, Zsuzsa Flaskó, Berthold Seitz, Achim Langenbucher, Nóra Szentmáry

Erschienen in: Graefe's Archive for Clinical and Experimental Ophthalmology | Ausgabe 1/2017

Einloggen, um Zugang zu erhalten

Abstract

Purpose

Keratoconus (KC) is a disease characterized by thinning and deformation of the cornea, but its etiology remains unknown. Seventy percent of the corneal stroma consists of collagen, which is composed of three intertwined polypeptide chains with glycine-hydroxyproline-proline repeats along their sequence. Arginase is a cytoplasmatic enzyme and catalyzes the conversion of arginine to urea and ornithine, which serves as a precursor for the endogenous synthesis of proline and hydroxyproline. The purpose of this study was to analyze arginase activity, as well as collagen and urea formation in normal and KC-keratocytes and to determine the impact of urea on keratocyte viability and proliferation in vitro.

Methods

Primary human keratocytes were isolated by digestion in collagenase (1.0 mg/mL) from surgically removed corneas of eight keratoconus patients and eight normal human corneal buttons and cultured in DMEM/Ham’s F12 medium supplemented with 5 % fetal calf serum. Arginase activity and urea concentration were measured in cell-lysates, hydroxyproline concentration in supernatant of cultured keratocytes using colorimetric assay. Cell viability and cell proliferation of cultured keratocytes were assessed after treatment with urea at concentrations up to10 mM for 24 h using assays for metabolic activity and DNA replication.

Results

Arginase activity and urea concentration in KC-keratocytes decreased by about 50 % compared to normal keratocytes (p = 0.003 and p = 0.008). Hydroxyproline synthesized by cultured KC-keratocytes was also approximately 50 % less compared to normal keratocytes (p = 0.02) and this difference decreased following treatment with 5.0 or 10.0 mM urea (p = 0.02; 0.03), without any change in cell viability (p > 0.09). However, the urea treatment increased modestly (by 20 %) the proliferation rate of KC-keratocytes (p = 0.04; 0.04; 0.04), without any effect on normal cultured keratocytes (p > 0.09).

Conclusions

We identified suppressed arginase activity in the metabolic program of cultured keratoconus keratocytes. The level of urea, as one product of the enzyme arginase was also decreased. This results in impaired collagen synthesis, evidenced in the culture by reduced hydroxyproline concentration. In addition, our data showed that the other product of the arginase reaction, urea supports the proliferation of KC-keratocytes, without changes in their viability. The metabolic reprogramming of keratoconus keratocytes and its impact on development of a clinically detectable keratoconus disease has to be further analyzed.
Literatur
1.
Zurück zum Zitat Krachmer JH, Feder RS, Belin MW (1984) Keratoconus and related noninflammatory corneal thinning disorders. Surv Ophthalmol 28:293–322CrossRefPubMed Krachmer JH, Feder RS, Belin MW (1984) Keratoconus and related noninflammatory corneal thinning disorders. Surv Ophthalmol 28:293–322CrossRefPubMed
6.
Zurück zum Zitat Collier SA (2001) Is the corneal degradation in keratoconus caused by matrix-metalloproteinases? Clin Experiment Ophthalmol 29:340–344CrossRefPubMed Collier SA (2001) Is the corneal degradation in keratoconus caused by matrix-metalloproteinases? Clin Experiment Ophthalmol 29:340–344CrossRefPubMed
8.
Zurück zum Zitat Gloor M, Fluhr J, Lehmann L et al (2002) Do urea/ammonium lactate combinations achieve better skin protection and hydration than either component alone? Skin Pharmacol Appl Skin Physiol 15:35–43CrossRefPubMed Gloor M, Fluhr J, Lehmann L et al (2002) Do urea/ammonium lactate combinations achieve better skin protection and hydration than either component alone? Skin Pharmacol Appl Skin Physiol 15:35–43CrossRefPubMed
11.
12.
Zurück zum Zitat Koshiyama Y, Gotoh T, Miyanaka K et al (2000) Expression and localization of enzymes of arginine metabolism in the rat eye. Curr Eye Res 20:313–321CrossRefPubMed Koshiyama Y, Gotoh T, Miyanaka K et al (2000) Expression and localization of enzymes of arginine metabolism in the rat eye. Curr Eye Res 20:313–321CrossRefPubMed
15.
Zurück zum Zitat Jager K, Kielstein H, Dunse M et al (2013) Enzymes of urea synthesis are expressed at the ocular surface, and decreased urea in the tear fluid is associated with dry-eye syndrome. Graefes Arch Clin Exp Ophthalmol 251:1995–2002. doi:10.1007/s00417-013-2391-7 CrossRefPubMed Jager K, Kielstein H, Dunse M et al (2013) Enzymes of urea synthesis are expressed at the ocular surface, and decreased urea in the tear fluid is associated with dry-eye syndrome. Graefes Arch Clin Exp Ophthalmol 251:1995–2002. doi:10.​1007/​s00417-013-2391-7 CrossRefPubMed
22.
Zurück zum Zitat Mace M, Galiacy SD, Erraud A et al (2011) Comparative transcriptome and network biology analyses demonstrate antiproliferative and hyperapoptotic phenotypes in human keratoconus corneas. Invest Ophthalmol Vis Sci 52:6181–6191. doi:10.1167/iovs.10-70981 CrossRefPubMed Mace M, Galiacy SD, Erraud A et al (2011) Comparative transcriptome and network biology analyses demonstrate antiproliferative and hyperapoptotic phenotypes in human keratoconus corneas. Invest Ophthalmol Vis Sci 52:6181–6191. doi:10.​1167/​iovs.​10-70981 CrossRefPubMed
Metadaten
Titel
Arginase activity, urea, and hydroxyproline concentration are reduced in keratoconus keratocytes
verfasst von
Tanja Stachon
Krasimir Kolev
Zsuzsa Flaskó
Berthold Seitz
Achim Langenbucher
Nóra Szentmáry
Publikationsdatum
25.10.2016
Verlag
Springer Berlin Heidelberg
Erschienen in
Graefe's Archive for Clinical and Experimental Ophthalmology / Ausgabe 1/2017
Print ISSN: 0721-832X
Elektronische ISSN: 1435-702X
DOI
https://doi.org/10.1007/s00417-016-3520-x

Weitere Artikel der Ausgabe 1/2017

Graefe's Archive for Clinical and Experimental Ophthalmology 1/2017 Zur Ausgabe

Neu im Fachgebiet Augenheilkunde

Update Augenheilkunde

Bestellen Sie unseren Fach-Newsletter und bleiben Sie gut informiert.