Fig. 4. Fractional distribution of the most abundant proteins in human vitreous. (A) Chart showing a summary of the relative amounts of highly abundant proteins in PDR vitreous. (B) Table showing the mean percent of number of total peptides for the 15 most abundant proteins identified in NDM, noDR, and PDR samples relative to the number of total peptides detected from respective samples. Reprinted with permission from Gao et al. [37]. Copyright 2008 American Chemical Society.

SUMMARY AND CONCLUSIONS

Mass spectrometry–based proteomics has identified at least several hundred proteins from human vitreous. Diabetic retinopathy is associated with about a fourfold increase in total vitreous protein content and increased protein diversity compared

Fig. 5. Comparison of proteins abundance in noDR and PDR vitreous relative to NDM vitreous. Ratio of the mean total peptides detected in noDR or PDR groups relative to the NDM group. The absence of protein detection in a group is indicated by >20-fold. Reprinted with permission from Gao et al. [37]. Copyright 2008 American Chemical Society.

with NDM control vitreous. Most of these increases in protein in diabetic retinopathy appear to be due to the infiltration of plasma proteins and contributions from intraocular hemorrhage and cell lysis. Once in the vitreous, a limited number of these plasma and intracellular proteins have been shown to exert potent effects on retinal functions. These findings suggest that the loss of blood retinal barrier function in diabetes may promote further increases in RVP as diabetic retinopathy progresses. While the number of proteins identified by vitreous proteomics is increasing rapidly, the relative significance and biological functions of most of these proteins within the vitreous milieu are unknown. Direct functional analyses of protein action in the vitreous are needed to elucidate their potential effects in diabetic retinopathy. In addition, further characterization of the vitreous proteome may reveal biomarkers that correlate with clinical characteristics and could provide new insights into disease progression and responses to therapies.

ACKNOWLEDGMENTS

This work was supported in part by the US National Institutes of Health (grants EY019029, DK 36836) and the Juvenile Diabetes Research Foundation.

186 Feener

REFERENCES

1. Cheung N, Mitchell P, Wong TY. Diabetic retinopathy. Lancet. 2010;376(9735):124–36.

2. Le Goff MM, Bishop PN. Adult vitreous structure and postnatal changes. Eye (Lond). 2008;22(10):1214–22.

3. Ponsioen TL, van Luyn MJ, van der Worp RJ, van Meurs JC, Hooymans JM, Los LI. Collagen distribution in the human vitreoretinal interface. Invest Ophthalmol Vis Sci. 2008;49(9): 4089–95.

4. Shui YB, Holekamp NM, Kramer BC, Crowley JR, Wilkins MA, Chu F, et al. The gel state of the vitreous and ascorbate-dependent oxygen consumption: relationship to the etiology of nuclear cataracts. Arch Ophthalmol. 2009;127(4):475–82.

5. Mitry D, Fleck BW, Wright AF, Campbell H, Charteris DG. Pathogenesis of rhegmatogenous retinal detachment: predisposing anatomy and cell biology. Retina. 2010;30(10): 1561–72.

6. Dernouchamps JP, Vaerman JP, Michiels J, Heremans JF. Transferrins in rabbit ocular fluids. Ophthalmologica. 1975;170(1):72–83.

7. Van Bockxmeer FM, Martin CE, Constable IJ. Iron-binding proteins in vitreous humour. Biochim Biophys Acta. 1983;758(1):17–23.

8. Burke JM, Smith JM. Retinal proliferation in response to vitreous hemoglobin or iron. Invest Ophthalmol Vis Sci. 1981;20(5):582–92.

9. Forrester JV, Prentice CR, Williamson J, Forbes CD. Fibrinolytic activity of the vitreous body. Invest Ophthalmol. 1974;13(11):875–9.

10. Shimada K. The complement components and their inactivators in the intraocular fluids of the guinea pig. Invest Ophthalmol. 1970;9(4):307–15.

11. Raymond L, Jacobson B. Isolation and identification of stimulatory and inhibitory cell growth factors in bovine vitreous. Exp Eye Res. 1982;34(2):267–86.

12. Jacobson B, Dorfman T, Basu PK, Hasany SM. Inhibition of vascular endothelial cell growth and trypsin activity by vitreous. Exp Eye Res. 1985;41(5):581–95.

13. Taylor CM, Weiss JB. Partial purification of a 5.7K glycoprotein from bovine vitreous which inhibits both angiogenesis and collagenase activity. Biochem Biophys Res Commun. 1985;133(3):911–6.

14. Preis I, Langer R, Brem H, Folkman J. Inhibition of neovascularization by an extract derived from vitreous. Am J Ophthalmol. 1977;84(3):323–8.

15. Glaser BM, D’Amore PA, Michels RG. The effect of human intraocular fluid on vascular endothelial cell migration. Ophthalmology. 1981;88(9):986–91.

16. Glaser BM, D’Amore PA, Michels RG, Brunson SK, Fenselau AH, Rice T, et al. The demonstration of angiogenic activity from ocular tissues. Preliminary report. Ophthalmology. 1980;87(5):440–6.

17. Glaser BM, D’Amore PA, Lutty GA, Fenselau AH, Michels RG, Patz A. Chemical mediators of intraocular neovascularization. Trans Ophthalmol Soc U K. 1980;100(3):369–73.

18. Miller JW, Adamis AP, Shima DT, D’Amore PA, Moulton RS, O’Reilly MS, et al. Vascular endothelial growth factor/vascular permeability factor is temporally and spatially correlated with ocular angiogenesis in a primate model. Am J Pathol. 1994;145(3):574–84.

19. Adamis AP, Miller JW, Bernal MT, D’Amico DJ, Folkman J, Yeo TK, et al. Increased vascular endothelial growth factor levels in the vitreous of eyes with proliferative diabetic retinopathy. Am J Ophthalmol. 1994;118(4):445–50.

20. Aiello LP, Avery RL, Arrigg PG, Keyt BA, Jampel HD, Shah ST, et al. Vascular endothelial growth factor in ocular fluid of patients with diabetic retinopathy and other retinal disorders. N Engl J Med. 1994;331(22):1480–7.

21. Aiello LP, Bursell SE, Clermont A, Duh E, Ishii H, Takagi C, et al. Vascular endothelial growth factor-induced retinal permeability is mediated by protein kinase C in vivo and suppressed by an orally effective beta-isoform-selective inhibitor. Diabetes. 1997;46(9):1473–80.

22. Funatsu H, Yamashita H, Sakata K, Noma H, Mimura T, Suzuki M, et al. Vitreous levels of vascular endothelial growth factor and intercellular adhesion molecule 1 are related to diabetic macular edema. Ophthalmology. 2005;112(5):806–16.

23. Diabetic Retinopathy Clinical Research Network, Elman MJ, Aiello LP, Beck RW, Bressler NM, Bressler SB, et al. Randomized trial evaluating ranibizumab plus prompt or deferred laser or triamcinolone plus prompt laser for diabetic macular edema. Ophthalmology. 2010;117(6):1064–77.

24. Nguyen QD, Shah SM, Khwaja AA, Channa R, Hatef E, Do DV, et al. Two-year outcomes of the ranibizumab for edema of the mAcula in diabetes (READ-2) study. Ophthalmology. 2010;117(11):2146–51.

25. Funatsu H, Noma H, Mimura T, Eguchi S, Hori S. Association of vitreous inflammatory factors with diabetic macular edema. Ophthalmology. 2009;116(1):73–9.

26. Yoshimura T, Sonoda KH, Sugahara M, Mochizuki Y, Enaida H, Oshima Y, et al. Comprehensive analysis of inflammatory immune mediators in vitreoretinal diseases. PLoS One. 2009;4(12):e8158.

27. Praidou A, Klangas I, Papakonstantinou E, Androudi S, Georgiadis N, Karakiulakis G, et al. Vitreous and serum levels of platelet-derived growth factor and their correlation in patients with proliferative diabetic retinopathy. Curr Eye Res. 2009;34(2):152–61.

28. Simo R, Hernandez C, Segura RM, Garcia-Arumi J, Sararols L, Burgos R, et al. Free insulinlike growth factor 1 in the vitreous fluid of diabetic patients with proliferative diabetic retinopathy: a case-control study. Clin Sci (Lond). 2003;104(3):223–30.

29. Yamane K, Minamoto A, Yamashita H, Takamura H, Miyamoto-Myoken Y, Yoshizato K, et al. Proteome analysis of human vitreous proteins. Mol Cell Proteomics. 2003;2(11):1177– 87.

30. Garcia-Ramirez M, Canals F, Hernandez C, Colome N, Ferrer C, Carrasco E, et al. Proteomic analysis of human vitreous fluid by fluorescence-based difference gel electrophoresis (DIGE): a new strategy for identifying potential candidates in the pathogenesis of proliferative diabetic retinopathy. Diabetologia. 2007;50(6):1294–303.

31. Shitama T, Hayashi H, Noge S, Uchio E, Oshima K, Haniu H, et al. Proteome profiling of vitreoretinal diseases by cluster analysis. Proteomics Clin Appl. 2008;2(9):1265–80.

32. Simo R, Vidal MT, Garcia-Arumi J, Carrasco E, Garcia-Ramirez M, Segura RM, et al. Intravitreous hepatocyte growth factor in patients with proliferative diabetic retinopathy: a casecontrol study. Diabetes Res Clin Pract. 2006;71(1):36–44.

33. Krogsaa B, Lund-Andersen H, Mehlsen J, Sestoft L, Larsen J. The blood-retinal barrier permeability in diabetic patients. Acta Ophthalmol (Copenh). 1981;59(5):689–94.

34. Plehwe WE, Sleightholm MA, Kohner EM. Does vitreous fluorophotometry reflect severity of early diabetic retinopathy? Br J Ophthalmol. 1989;73(4):255–60.

35. Kim T, Kim SJ, Kim K, Kang UB, Lee C, Park KS, et al. Profiling of vitreous proteomes from proliferative diabetic retinopathy and nondiabetic patients. Proteomics. 2007;7(22): 4203–15.

36. Gao BB, Clermont A, Rook S, Fonda SJ, Srinivasan VJ, Wojtkowski M, et al. Extracellular carbonic anhydrase mediates hemorrhagic retinal and cerebral vascular permeability through prekallikrein activation. Nat Med. 2007;13(2):181–8.

37. Gao BB, Chen X, Timothy N, Aiello LP, Feener EP. Characterization of the vitreous proteome in diabetes without diabetic retinopathy and diabetes with proliferative diabetic retinopathy. J Proteome Res. 2008;7(6):2516–25.

38. Nakanishi T, Koyama R, Ikeda T, Shimizu A. Catalogue of soluble proteins in the human vitreous humor: comparison between diabetic retinopathy and macular hole. J Chromatogr B Analyt Technol Biomed Life Sci. 2002;776(1):89–100.

39. Koyama R, Nakanishi T, Ikeda T, Shimizu A. Catalogue of soluble proteins in human vitreous humor by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrospray ionization mass spectrometry including seven angiogenesis-regulating factors. J Chromatogr B Analyt Technol Biomed Life Sci. 2003;792(1):5–21.

40. Ouchi M, West K, Crabb JW, Kinoshita S, Kamei M. Proteomic analysis of vitreous from diabetic macular edema. Exp Eye Res. 2005;81(2):176–82.

41. Kim SJ, Kim S, Park J, Lee HK, Park KS, Yu HG, et al. Differential expression of vitreous proteins in proliferative diabetic retinopathy. Curr Eye Res. 2006;31(3):231–40.

42. Aebersold R, Mann M. Mass spectrometry-based proteomics. Nature. 2003;422(6928): 198–207.

43. Gao BB, Phipps JA, Bursell D, Clermont AC, Feener EP. Angiotensin AT1 receptor antagonism ameliorates murine retinal proteome changes induced by diabetes. J Proteome Res. 2009;8(12):5541–9.

44. Kim K, Kim SJ, Yu HG, Yu J, Park KS, Jang IJ, et al. Verification of biomarkers for diabetic retinopathy by multiple reaction monitoring. J Proteome Res. 2010;9(2):689–99.

45.Gao BB, Stuart L, Feener EP. Label-free quantitative analysis of one-dimensional PAGE LC/ MS/MS proteome: application on angiotensin II-stimulated smooth muscle cells secretome. Mol Cell Proteomics. 2008;7(12):2399–409.

46. Mann M, Kelleher NL. Precision proteomics: the case for high resolution and high mass accuracy. Proc Natl Acad Sci USA. 2008;105(47):18132–8.

47. Mueller LN, Brusniak MY, Mani DR, Aebersold R. An assessment of software solutions for the analysis of mass spectrometry based quantitative proteomics data. J Proteome Res. 2008;7(1):51–61.

48. Bantscheff M, Schirle M, Sweetman G, Rick J, Kuster B. Quantitative mass spectrometry in proteomics: a critical review. Anal Bioanal Chem. 2007;389(4):1017–31.

49. Kumar C, Mann M. Bioinformatics analysis of mass spectrometry-based proteomics data sets. FEBS Lett. 2009;583(11):1703–12.

50. Garcia-Ramirez M, Hernandez C, Villarroel M, Canals F, Alonso MA, Fortuny R, et al. Interphotoreceptor retinoid-binding protein (IRBP) is downregulated at early stages of diabetic retinopathy. Diabetologia. 2009;52(12):2633–41.

51. Simo R, Higuera M, Garcia-Ramirez M, Canals F, Garcia-Arumi J, Hernandez C. Elevation of apolipoprotein A-I and apolipoprotein H levels in the vitreous fluid and overexpression in the retina of diabetic patients. Arch Ophthalmol. 2008;126(8):1076–81.

52. Bhutto IA, Kim SY, McLeod DS, Merges C, Fukai N, Olsen BR, et al. Localization of collagen XVIII and the endostatin portion of collagen XVIII in aged human control eyes and eyes with age-related macular degeneration. Invest Ophthalmol Vis Sci. 2004;45(5):1544–52.