Korean J Ophthalmol.  2016 Jun;30(3):163-171. 10.3341/kjo.2016.30.3.163.

Angiogenin for the Diagnosis and Grading of Dry Eye Syndrome

• Kim WS
• Wee SW
• Lee SH
• Kim JC

• KMID: 2373971
• DOI: http://doi.org/10.3341/kjo.2016.30.3.163

Abstract

PURPOSE
To investigate the properties of angiogenin (ANG) as a potential tool for the diagnosis and grading of dry eye syndrome (DES) by analyzing tear protein profiles.
METHODS
Tear samples were collected with capillary tubes from 52 DES patients and 29 normal individuals as controls. Tear protein profiles were analyzed with an immunodot blot assay as a screening test. To confirm that the tear ANG levels were in inverse proportion to the disease severity grade, the ANG and lactoferrin (LF) tear contents of normal controls and DES patients were compared in an enzyme-linked immunosorbent assay.
RESULTS
In the immunodot blot assay, the ANG area was lower in patients with grades 3 and 4 DES than in normal controls. The areas of basic fibroblast growth factor, transforming growth factor β2, and interleukin 10 were significantly greater than those of normal controls only in grade 4 DES patients, but these proteins were not linearly correlated with dry eye severity. Upon enzyme-linked immunosorbent assay analysis, the mean concentrations of ANG and LF decreased significantly as dry eye severity increased, except between grades 1 and 2. In addition, the ratios of ANG and LF to total tear proteins were correlated significantly with DES severity.
CONCLUSIONS
ANG level was significantly lower in DES patients than in normal controls, and was significantly correlated with the worsening severity of DES, except between grades 1 and 2, as was LF. Therefore, ANG may be a useful measure of DES severity through proteomic analysis.

Keyword

Angiogenin; Dry eye syndrome; Lactoferrin; Tear

MeSH Terms

Adult
Aged
Angiogenesis Inducing Agents/pharmacology
Dry Eye Syndromes/*diagnosis/metabolism
Enzyme-Linked Immunosorbent Assay
Female
Follow-Up Studies
Humans
Immunoblotting
Male
Middle Aged
Proteomics/methods
Ribonuclease, Pancreatic/*pharmacology
Severity of Illness Index
Tears/chemistry
Young Adult
Angiogenesis Inducing Agents
Ribonuclease, Pancreatic

Figure

• 

  Fig. 1 Immunodot blot assay of tear proteins in dry eye syndrome (DES) patients and normal participants as controls. (A) The relative amount of angiogenin tended to decrease as the DES grade increased. The densities of basic fibroblast growth factor (bFGF), transforming growth factor (TGF)-β2, and interleukin (IL)-10 increased remarkably only in grade 4 DES patients compared to normal controls. (B) Custom Human Cytokines Antibody Array Map. pos = positive; neg = negative; SDF-1α = stromal cell derived factor-1alpha; VEGF = vascular endothelial growth factor.

• 

  Fig. 2 The relative density, presented as the average amount of each cytokine expressed in each dry eye syndrome (DES) severity grade group relative to that in the normal control group. (A-C) The areas of basic fibroblast growth factor (bFGF) (A), interleukin (IL)-10 (B), and transforming growth factor (TGF)-β2 (C) were significantly greater than those of normal controls only in grade 4 DES patients. (D) There was no significant difference of tumor necrosis factor (TNF)-α expression according to the grades of DES. (E) The areas of angiogenin in DES grade 3 and 4 patients were significantly lower than those of normal controls and DES patients of grades 1 and 2 (*p < 0.05).

• 

  Fig. 3 Enzyme-linked immunosorbent assay analysis of (A) angiogenin (ANG) and (B) lactoferrin (LF) in dry eye syndrome (DES) patients (classified according to grade) and normal controls. The percentage concentrations of ANG and LF relative to total tear proteins were measured. Statistically significant correlations between DES severity and the percentage concentrations of both ANG (A) and LF (B) were noted. The group-to-group differences of ANF and LF also were statistically significant, except between DES grades 1 and 2 (*p < 0.05).

• 

  Fig. 4 Scatter plot showing the correlation between angiogenin (ANG) and lactoferrin (LF) in enzyme-linked immunosorbent assay analysis. In the correlation analysis, the percentage concentrations of ANG and LF relative to total proteins were significantly correlated with the dry eye syndrome grade (r = 0.661, p < 0.001).

Reference

1. Javadi MA, Feizi S. Dry eye syndrome. J Ophthalmic Vis Res. 2011; 6:192–198.

2. Bhavsar AS, Bhavsar SG, Jain SM. A review on recent advances in dry eye: pathogenesis and management. Oman J Ophthalmol. 2011; 4:50–56.

3. Stern ME, Gao J, Siemasko KF, et al. The role of the lacrimal functional unit in the pathophysiology of dry eye. Exp Eye Res. 2004; 78:409–416.

4. Pflugfelder SC. Anti-inf lammatory therapy of dry eye. Ocul Surf. 2003; 1:31–36.

5. Solomon A, Dursun D, Liu Z, et al. Pro- and anti-inflammatory forms of interleukin-1 in the tear fluid and conjunctiva of patients with dry-eye disease. Invest Ophthalmol Vis Sci. 2001; 42:2283–2292.

6. Rolando M, Barabino S, Mingari C, et al. Distribution of conjunctival HLA-DR expression and the pathogenesis of damage in early dry eyes. Cornea. 2005; 24:951–954.

7. Farris RL. Tear osmolarity: a new gold standard? Adv Exp Med Biol. 1994; 350:495–503.

8. Gao J, Schwalb TA, Addeo JV, et al. The role of apoptosis in the pathogenesis of canine keratoconjunctivitis sicca: the effect of topical Cyclosporin A therapy. Cornea. 1998; 17:654–663.

9. de Souza GA, Godoy LM, Mann M. Identification of 491 proteins in the tear fluid proteome reveals a large number of proteases and protease inhibitors. Genome Biol. 2006; 7:R72.

10. Green-Church KB, Nichols KK, Kleinholz NM, et al. Investigation of the human tear film proteome using multiple proteomic approaches. Mol Vis. 2008; 14:456–470.

11. Versura P, Nanni P, Bavelloni A, et al. Tear proteomics in evaporative dry eye disease. Eye (Lond). 2010; 24:1396–1402.

12. Weremowicz S, Fox EA, Morton CC, Vallee BL. Localization of the human angiogenin gene to chromosome band 14q11, proximal to the T cell receptor alpha/delta locus. Am J Hum Genet. 1990; 47:973–981.

13. Gao X, Xu Z. Mechanisms of action of angiogenin. Acta Biochim Biophys Sin (Shanghai). 2008; 40:619–624.

14. Lee SH, Kim KW, Min KM, et al. Angiogenin reduces immune inflammation via inhibition of TANK-binding kinase 1 expression in human corneal fibroblast cells. Mediators Inflamm. 2014; 2014:861435.

15. Sack RA, Conradi L, Krumholz D, et al. Membrane array characterization of 80 chemokines, cytokines, and growth factors in open- and closed-eye tears: angiogenin and other defense system constituents. Invest Ophthalmol Vis Sci. 2005; 46:1228–1238.

16. The definition and classification of dry eye disease: report of the Definition and Classification Subcommittee of the International Dry Eye WorkShop (2007). Ocul Surf. 2007; 5:75–92.

17. McMonnies CW. Key questions in a dry eye history. J Am Optom Assoc. 1986; 57:512–517.

18. Nichols KK, Nichols JJ, Mitchell GL. The lack of association between signs and symptoms in patients with dry eye disease. Cornea. 2004; 23:762–770.

19. Aebersold R, Mann M. Mass spectrometry-based proteomics. Nature. 2003; 422:198–207.

20. Oh JY, Kim MK, Choi HJ, et al. Investigating the relationship between serum interleukin-17 levels and systemic immune-mediated disease in patients with dry eye syndrome. Korean J Ophthalmol. 2011; 25:73–76.

21. Zittermann SI, Issekutz AC. Basic fibroblast growth factor (bFGF, FGF-2) potentiates leukocyte recruitment to inflammation by enhancing endothelial adhesion molecule expression. Am J Pathol. 2006; 168:835–846.

22. Pflugfelder SC, Stern ME. Symposium Participants. Immunoregulation on the ocular surface: 2nd Cullen Symposium. Ocul Surf. 2009; 7:67–77.

23. Danjo Y, Lee M, Horimoto K, Hamano T. Ocular surface damage and tear lactoferrin in dry eye syndrome. Acta Ophthalmol (Copenh). 1994; 72:433–437.

24. Tsai PS, Evans JE, Green KM, et al. Proteomic analysis of human meibomian gland secretions. Br J Ophthalmol. 2006; 90:372–377.

25. Conneely OM. Antiinflammatory activities of lactoferrin. J Am Coll Nutr. 2001; 20:5 Suppl. 389S–395S.

26. Brock JH. Lactoferrin: 50 years on. Biochem Cell Biol. 2012; 90:245–251.

27. Dogru M, Matsumoto Y, Yamamoto Y, et al. Lactoferrin in Sjogren's syndrome. Ophthalmology. 2007; 114:2366–2367.

28. Murata M, Wakabayashi H, Yamauchi K, Abe F. Identification of milk proteins enhancing the antimicrobial activity of lactoferrin and lactoferricin. J Dairy Sci. 2013; 96:4891–4898.

29. Schmaldienst S, Oberpichler A, Tschesche H, Horl WH. Angiogenin: a novel inhibitor of neutrophil lactoferrin release during extracorporeal circulation. Kidney Blood Press Res. 2003; 26:107–112.

30. Skeie JM, Zeng S, Faidley EA, Mullins RF. Angiogenin in age-related macular degeneration. Mol Vis. 2011; 17:576–582.

31. Marek N, Raczynska K, Siebert J, et al. Decreased angiogenin concentration in vitreous and serum in proliferative diabetic retinopathy. Microvasc Res. 2011; 82:1–5.

32. Cho P, Brown B, Chan I, et al. Reliability of the tear breakup time technique of assessing tear stability and the locations of the tear break-up in Hong Kong Chinese. Optom Vis Sci. 1992; 69:879–885.

33. Abusharha AA, Pearce EI. The effect of low humidity on the human tear film. Cornea. 2013; 32:429–434.