The Retinal Proteome in Experimental Diabetic Retinopathy

Abstract

Diabetic retinopathy is the leading cause of blindness in working age persons. Targeted studies have uncovered several components of the pathophysiology of the disease without unveiling the basic mechanisms. This study describes the use of complementary proteomic and genomic discovery methods that revealed that the proteins of the crystallin superfamily are increased dramatically in early diabetic retinopathy. Orthogonal methods confirmed that the amplitude of the up-regulation is greater than other changes described so far in diabetic retinopathy. A detailed time course study during diabetes showed differential up-regulation of the different isoforms of the crystallins superfamily. α- and β-crystallins were regulated primarily at the translation level, whereas γ-crystallins were also regulated transcriptionally. We also demonstrated cell-specific patterns of expression of the different crystallins in normal and diabetic rat retinas. In addition, systemic and periocular insulin treatments restored retinal crystallin protein expression during diabetes, indicating effects of phosphoinositide 3-kinase/Akt activity. Altogether this work shows the importance of proteomics discovery methods coupled with targeted approaches to unveil new disease mechanistic details and therapeutic targets. Diabetic retinopathy is the leading cause of blindness in working age persons. Targeted studies have uncovered several components of the pathophysiology of the disease without unveiling the basic mechanisms. This study describes the use of complementary proteomic and genomic discovery methods that revealed that the proteins of the crystallin superfamily are increased dramatically in early diabetic retinopathy. Orthogonal methods confirmed that the amplitude of the up-regulation is greater than other changes described so far in diabetic retinopathy. A detailed time course study during diabetes showed differential up-regulation of the different isoforms of the crystallins superfamily. α- and β-crystallins were regulated primarily at the translation level, whereas γ-crystallins were also regulated transcriptionally. We also demonstrated cell-specific patterns of expression of the different crystallins in normal and diabetic rat retinas. In addition, systemic and periocular insulin treatments restored retinal crystallin protein expression during diabetes, indicating effects of phosphoinositide 3-kinase/Akt activity. Altogether this work shows the importance of proteomics discovery methods coupled with targeted approaches to unveil new disease mechanistic details and therapeutic targets. Diabetic retinopathy is the leading cause of blindness in working age persons, and despite numerous studies, the pathophysiological mechanisms, especially during the early stages of diabetes, remain to be elucidated. Diabetic retinopathy develops, to some degree, in nearly all patients with diabetes and is the most common cause of new cases of blindness among adults. The predominant causes of vision loss are clinically significant macular edema and proliferative diabetic retinopathy, but vision impairment can be prevented or minimized if the retinopathy is identified in its early stages. Diabetic retinopathy includes microvascular and neuronal, glial, and microglial cell defects early in the course of the disease before clinically visible vascular lesions. Photoreceptor and ganglion cell death occurs as early as 2–4 weeks after the onset of diabetes (1Barber A.J. Lieth E. Khin S.A. Antonetti D.A. Buchanan A.G. Gardner T.W. Neural apoptosis in the retina during experimental and human diabetes. Early onset and effect of insulin.J. Clin. Investig. 1998; 102: 783-791Crossref PubMed Scopus (1038) Google Scholar, 2Martin P.M. Roon P. Van Ells T.K. Ganapathy V. Smith S.B. Death of retinal neurons in streptozotocin-induced diabetic mice.Investig. Ophthalmol. Vis. Sci. 2004; 45: 3330-3336Crossref PubMed Scopus (374) Google Scholar, 3Park S.H. Park J.W. Park S.J. Kim K.Y. Chung J.W. Chun M.H. Oh S.J. Apoptotic death of photoreceptors in the streptozotocin-induced diabetic rat retina.Diabetologia. 2003; 46: 1260-1268Crossref PubMed Scopus (250) Google Scholar). Because of the difficulty of studying the mechanisms of the early stages of diabetes in humans, rodent models of type 1 and type 2 diabetes have been developed. Those models have been used to study various specific aspects of early stages of diabetic retinopathy including blood retinal barrier leakage (4Antonetti D.A. Barber A.J. Khin S. Lieth E. Tarbell J.M. Gardner T.W. 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. 1998; 47: 1953-1959Crossref PubMed Scopus (503) Google Scholar), growth factor signaling such as the insulin/insulin-like growth factor receptor and vascular endothelial cell receptor pathways (5Reiter C.E. Gardner T.W. Functions of insulin and insulin receptor signaling in retina: possible implications for diabetic retinopathy.Prog. Retin. Eye Res. 2003; 22: 545-562Crossref PubMed Scopus (88) Google Scholar, 6Reiter C.E. Wu X. Sandirasegarane L. Nakamura M. Gilbert K.A. Singh R.S. Fort P.E. Antonetti D.A. Gardner T.W. Diabetes reduces basal retinal insulin receptor signaling: reversal with systemic and local insulin.Diabetes. 2006; 55: 1148-1156Crossref PubMed Scopus (143) Google Scholar), and oxidative stress mechanisms such as reactive oxygen species and advanced glycation end product production (7Adamis A.P. Is diabetic retinopathy an inflammatory disease?.Br. J. Ophthalmol. 2002; 86: 363-365Crossref PubMed Scopus (315) Google Scholar, 8Bonnardel-Phu E. Wautier J.L. Schmidt A.M. Avila C. Vicaut E. Acute modulation of albumin microvascular leakage by advanced glycation end products in microcirculation of diabetic rats in vivo.Diabetes. 1999; 48: 2052-2058Crossref PubMed Scopus (53) Google Scholar). These targeted studies provide valuable information on different aspects of the pathology and are useful in new therapeutic development, but more detailed discovery research is also needed to understand the full range of metabolic dysregulation that leads to diabetes complications. Proteome profiling using two-dimensional (2D) 1The abbreviations used are: 2D, two-dimensional; iTRAQ, isobaric tag for relative and absolute quantitation; STZ, streptozotocin; GFAP, glial fibrillary acidic protein; mRNA, messenger ribonucleic acid; INL, inner nuclear layer; GCL, ganglion cell layer; NFL, nerve fiber layer; SCX, strong cation exchange; NCBI, National Center for Biotechnology Information. 1The abbreviations used are: 2D, two-dimensional; iTRAQ, isobaric tag for relative and absolute quantitation; STZ, streptozotocin; GFAP, glial fibrillary acidic protein; mRNA, messenger ribonucleic acid; INL, inner nuclear layer; GCL, ganglion cell layer; NFL, nerve fiber layer; SCX, strong cation exchange; NCBI, National Center for Biotechnology Information. DIGE and/or isobaric tag for relative and absolute quantitation (iTRAQ) are methods that can characterize new pathophysiological components and potential therapeutic targets. Several studies have used 2D DIGE to gain a better understanding of diabetic complications in the heart and retina (9Quin G.G. Len A.C. Billson F.A. Gillies M.C. Proteome map of normal rat retina and comparison with the proteome of diabetic rat retina: new insight in the pathogenesis of diabetic retinopathy.Proteomics. 2007; 7: 2636-2650Crossref PubMed Scopus (43) Google Scholar, 10Wang Y.D. Wu J.D. Jiang Z.L. Wang Y.B. Wang X.H. Liu C. Tong M.Q. Comparative proteome analysis of neural retinas from type 2 diabetic rats by two-dimensional electrophoresis.Curr. Eye Res. 2007; 32: 891-901Crossref PubMed Scopus (28) Google Scholar, 11Warda M. Kim H.K. Kim N. Youm J.B. Kang S.H. Park W.S. Khoa T.M. Kim Y.H. Han J. Simulated hyperglycemia in rat cardiomyocytes: a proteomics approach for improved analysis of cellular alterations.Proteomics. 2007; 7: 2570-2590Crossref PubMed Scopus (10) Google Scholar, 12Zabel C. Sagi D. Kaindl A.M. Steireif N. Klare L. J. Comparative proteomics in and proteins in Proteome Res. 2006; PubMed Scopus Google Scholar). These approaches revealed changes in the retina including changes in the expression of and were described as proteins and are in are crystallin the and the to the protein and protein and/or and apoptosis and the human PubMed Scopus Google Scholar, J. V. The protein 2003; PubMed Scopus Google Scholar, J.D. J.W. A.J. of by the 2006; PubMed Scopus Google Scholar, Liu and to and to during Death 2004; PubMed Scopus Google Scholar). The of the and β-crystallins are the are in apoptosis and vascular in the retina C. L. Liu P. J. X. associated protein and fiber cell Ophthalmol. Vis. Sci. 2007; 48: PubMed Scopus Google Scholar, C. P. C. M. S. D. A potential for and γ-crystallins in the vascular of the PubMed Scopus Google Scholar). the use of complementary discovery 2D DIGE and iTRAQ, by and using targeted We with proteomics methods a and increased expression of and isoforms in the retina of diabetic We also the cellular of the crystallins and the ganglion cell-specific expression of the γ-crystallins in retina during diabetes. that the increased crystallin expression is regulated the isoforms at and rats were used in all were a with to a rat and were in with the for Research in and on the and of Diabetes by of in and rats of as described (1Barber A.J. Lieth E. Khin S.A. Antonetti D.A. Buchanan A.G. Gardner T.W. Neural apoptosis in the retina during experimental and human diabetes. Early onset and effect of insulin.J. Clin. Investig. 1998; 102: 783-791Crossref PubMed Scopus (1038) Google rats were diabetic blood after diabetes insulin during the of the of diabetes by a insulin to of for the of the (1Barber A.J. Lieth E. Khin S.A. Antonetti D.A. Buchanan A.G. Gardner T.W. Neural apoptosis in the retina during experimental and human diabetes. Early onset and effect of insulin.J. Clin. Investig. 1998; 102: 783-791Crossref PubMed Scopus (1038) Google Scholar). A after weeks for insulin of a of of of for before the were insulin of a of insulin in and with in the other This on the of studies of the retinal insulin receptor signaling without blood The and diabetes studies were to increased cell microvascular microglial cell and insulin receptor signaling (1Barber A.J. Lieth E. Khin S.A. Antonetti D.A. Buchanan A.G. Gardner T.W. Neural apoptosis in the retina during experimental and human diabetes. Early onset and effect of insulin.J. Clin. Investig. 1998; 102: 783-791Crossref PubMed Scopus (1038) Google Scholar, D.A. Barber A.J. Khin S. Lieth E. Tarbell J.M. Gardner T.W. 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. 1998; 47: 1953-1959Crossref PubMed Scopus (503) Google Scholar, A.J. Antonetti D.A. Gardner T.W. expression of retinal occludin and glial fibrillary acidic protein in experimental diabetes. The Penn State Retina Research Ophthalmol. Vis. Sci. Google Scholar, C.E. Sandirasegarane L. M. Barber A.J. Antonetti D.A. M. Gardner T.W. of insulin signaling in rat retina in and J. 2003; PubMed Scopus Google Scholar, and microglial in the retina of streptozotocin-induced diabetic PubMed Scopus Google Scholar). rats were with of and by loss for of retinas. were in and at analysis of the blood and of to for the different of and diabetic used for this systemic local systemic and local in a new 2D DIGE as detailed J. A.G. proteomic of J. PubMed Scopus Google Scholar). 2D DIGE methods and are in information a proteomics were in 1 1 1 and by as described C.E. Wu X. Sandirasegarane L. Nakamura M. Gilbert K.A. Singh R.S. Fort P.E. Antonetti D.A. Gardner T.W. Diabetes reduces basal retinal insulin receptor signaling: reversal with systemic and local insulin.Diabetes. 2006; 55: 1148-1156Crossref PubMed Scopus (143) Google Scholar). were with the and all were for protein were from and by and by using the protein were to a and and of were using or of protein used for a for of protein were on and by on of the with and all were on a of using to and relative expression as described J. A.G. proteomic of J. PubMed Scopus Google Scholar). to be regulated were from the using a and were to and of from a by of were in a approach and using an The with were in the and with a to of of and of The most with a to of that were on an and of human were to without in a range from to the with a of for using and using were the of the on all studies were with rat retinal the In the and a of greater than isoforms of the protein were identified the with the that of the an identified to that or the the and on of in of were and reduced by the to of and were at for 1 to a of and for at from were with of at were with the for 2 and by of of of the different were by of and The in of for were on a using a at a of 1 A in in The A at and at to A to for the The of all were and were of were to the and to the in of and to by a using a on a and The and The and an of 2 The a for a of were in a on an and were at the of analysis of using the from the 1 for the and the of the in that for the were from in the and with with a to of at and a of A to for analysis such that on the of with of in the range from the with a of for after at a to of or or were for and quantitation were using the as in were the of the on with a protein a of at and a local discovery of than The local discovery from the of the of using the from for local discovery from Proteome Res. 7: PubMed Scopus Google Scholar). were as described for 2D analysis to to using analysis with and γ-crystallins by were as described but using X. C.E. Antonetti D.A. Gardner T.W. rat retinal cell in a 2004; PubMed Scopus Google and the were by the using an as described (5Reiter C.E. Gardner T.W. Functions of insulin and insulin receptor signaling in retina: possible implications for diabetic retinopathy.Prog. Retin. Eye Res. 2003; 22: 545-562Crossref PubMed Scopus (88) Google Scholar). were in and in with after from experimental were on the The were with before at crystallins were the used for using and or were used with Research methods S.H. L. J.B. changes in PubMed Scopus Google Scholar), and and were using the with an analysis as described A expression of 2007; PubMed Scopus Google Scholar). using the and and and the analysis were used to relative of product using as an were to be in an absolute all the were to the as before of models with for the of the were to the to diabetic rats with or without insulin and The and are were using analysis of by for We used the 2D DIGE and analysis on protein from diabetic and to proteome of diabetic and rat retina with or without insulin The use of and a range of revealed expression of more than of the proteins showed a increased most of the changes revealed protein expression in the diabetic proteins were in a of the to a range and and a and analysis of the from this revealed that the proteins to different proteins or isoforms of the crystallin analysis of the proteome differential expression during diabetes revealed that all the crystallin protein identified during this study were in diabetic of of and and and and were to during the course of diabetes These are that retinal protein at this time by E. M. S. L. S. and in expression of the of the demonstrated in diabetic rats P. expression of and in streptozotocin-induced diabetic PubMed Scopus Google Scholar), but this is the time that of the were also to be 2 shows that analysis confirmed the increased crystallin expression in the retina during diabetes. the of changes in crystallin expression are the and most retinal protein changes in experimental proteins identified in rat retina by using the 2D DIGE and relative changes in A A A A in a new proteins identified in rat retina by and relative changes in diabetes. expression is as relative to that of the the and of the crystallin during diabetes, were using and and of the by to were at various in all a significant in α- and γ-crystallins expression in retinas from diabetic rats A and the increased of expression of β-crystallins after weeks of diabetes This the by and proteome analysis is to the that the isoforms from the whereas 2D analysis the methods different isoforms with on a This is a common with of proteomics and methods have to be used to course of the relative of the expression of β-crystallins and γ-crystallins during early stages of diabetes diabetic and diabetic with systemic insulin different from different from diabetic better understand the possible implications of increased crystallin the expression of the different crystallins during diabetes. This detailed time course study during the early of diabetes onset showed that the α- and β-crystallins have a up-regulation of expression during diabetes A and up-regulation of expression at 2 and weeks of diabetes by a of expression to at β-crystallins were as early as 1 after diabetes before to after 2 of expression at weeks of diabetes. γ-crystallins were and weeks after the onset of diabetes, in a This time course study also the in the of up-regulation of the different crystallins during diabetes. the α- and expression were the of expression were increased after weeks of diabetes. These the crystallins and cellular the cellular of the different crystallins in the retina of diabetic and an study on retinal from and diabetic rats using the used for for in the and the and the ganglion fiber of normal with a vascular cell with an increased in diabetes in the increased in diabetic retina the blood of the as as in the glial but in the in the ganglion cell a of the of the inner nuclear and the in the with a glial cell that β-crystallins are by a of or with a nerve fiber and an indicating that the in the to the of the ganglion or the A of the on from diabetic rat retinas with of the in some of the and the in the the INL, and the photoreceptors The in the with a nerve fiber indicating that γ-crystallins are in the ganglion cell in the retinal of diabetic rats that the in the is specific to the ganglion cell and the the increased crystallin expression patterns in retinal neurons to a in the of neurons to the of diabetes. are regulated at the in the and mechanisms of during Retin. Eye Res. 2007; PubMed Scopus Google Scholar), so the expression of crystallin using and specific time methods to the up-regulation of the crystallins in diabetes We have expression of retinal at and weeks of diabetes Barber A.J. Antonetti D.A. Gardner T.W. of the retinal to diabetes a inflammatory PubMed Google Scholar). revealed that crystallin were among the most after weeks of diabetes in the rat using specific for and We that and were in retinas of diabetic at or weeks after the onset of diabetes and showed a of but significant changes at or weeks of diabetes messenger showed an increased of and and weeks after the onset of diabetes This up-regulation is more than the protein at and weeks for γ-crystallins that are of crystallin in the identified in rat retina by and relative changes in diabetes changes in a new insulin using hyperglycemia and prevented loss by diabetes We showed that basal retinal insulin receptor signaling is in diabetes C.E. Wu X. Sandirasegarane L. Nakamura M. Gilbert K.A. Singh R.S. Fort P.E. Antonetti D.A. Gardner T.W. Diabetes reduces basal retinal insulin receptor signaling: reversal with systemic and local insulin.Diabetes. 2006; 55: 1148-1156Crossref PubMed Scopus (143) Google so the effects of insulin on retinal crystallin expression in the diabetic rat insulin prevented or the up-regulation of α- and γ-crystallins and A and but or the up-regulation Because the insulin were for the of the of diabetes, the for γ-crystallins a reversal of the up-regulation or of that the at the time systemic insulin be the of crystallins by the and/or a effect of the insulin insulin insulin receptor without blood C.E. Wu X. Sandirasegarane L. Nakamura M. Gilbert K.A. Singh R.S. Fort P.E. Antonetti D.A. Gardner T.W. Diabetes reduces basal retinal insulin receptor signaling: reversal with systemic and local insulin.Diabetes. 2006; 55: 1148-1156Crossref PubMed Scopus (143) Google so the that a local insulin also the crystallin insulin for the of the study the blood but the up-regulation of α- and γ-crystallins after weeks of diabetes rats were insulin and an effect of the treatments for with These that up-regulation of α- and γ-crystallins is to retinal specific insulin receptor as as the systemic metabolic with that systemic and local insulin insulin receptor signaling the phosphoinositide 3-kinase/Akt C.E. Wu X. Sandirasegarane L. Nakamura M. Gilbert K.A. Singh R.S. Fort P.E. Antonetti D.A. Gardner T.W. Diabetes reduces basal retinal insulin receptor signaling: reversal with systemic and local insulin.Diabetes. 2006; 55: 1148-1156Crossref PubMed Scopus (143) Google Scholar). We the retinal proteome and of and diabetic the comparison revealed the of isoforms of the crystallin during diabetes. The are as and isoforms are with diabetes. This by proteomics discovery methods DIGE and and confirmed by the of increased expression of the the time and of expression among the different have an up-regulation of and in diabetic rats after weeks of diabetes P. expression of and in streptozotocin-induced diabetic PubMed Scopus Google Scholar), but this is the of an up-regulation of isoforms of the protein during diabetes. This is also the and cell-specific study of the expression of the crystallins in the retina in a disease such as diabetes. This work shows that systemic and local insulin can retinal crystallin been that the expression of crystallins in the retina from during the retinal of to retinal changes in after Vis. 2007; Google Scholar). the of retinas were with a of the during the is to that of the to crystallins by several of that retinal the in this work the of crystallin in the retinal We also the expression of crystallin in retinal and different retinal cell that retina crystallin the differential retinal demonstrated by also crystallin expression by retinal cells. expression are increased in other retinal models such as retinal and experimental M. J.W. changes the crystallin in Eye Res. 2003; PubMed Scopus Google Scholar, changes in expression after in the rat Ophthalmol. Vis. Sci. 2004; 45: PubMed Scopus Google Scholar, analysis of retinal expression in the of Ophthalmol. Vis. Sci. 2006; 47: PubMed Scopus Google Scholar). expression in a specific associated with macular and in photoreceptors by Ophthalmol. 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Because diabetes vascular endothelial growth factor expression and retinal vascular is that the increased crystallin expression be to vascular in diabetes C. P. C. M. S. D. A potential for and γ-crystallins in the vascular of the PubMed Scopus Google Scholar). We and expression in whereas were in in the blood retinal A study S. of during retinal crystallin 2007; PubMed Scopus Google showed that β-crystallins be and a on ganglion cell is possible that of crystallins to crystallin to Barber A.J. and other in the retinal ganglion of diabetic mice.Investig. Ophthalmol. Vis. Sci. PubMed Scopus Google have that diabetes of retinal in the time in Several studies of showed the possible of in the inflammatory in the of during diabetes occurs early in the course of the disease at the time as and GFAP, of glial the indicating an of in the disease S. M. M. glial the inflammatory in Proteome Res. 2007; PubMed Scopus Google Scholar). This with the of in from patients and with experimental J.M. D.A. 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The of crystallins in cell during and during diabetes be of a of or the of for the of the The of crystallin expression in diabetes cell death by apoptosis or cell We showed that systemic and local insulin α- and We also showed that insulin the of the retinal insulin 3-kinase/Akt (5Reiter C.E. Gardner T.W. Functions of insulin and insulin receptor signaling in retina: possible implications for diabetic retinopathy.Prog. Retin. Eye Res. 2003; 22: 545-562Crossref PubMed Scopus (88) Google Scholar, 6Reiter C.E. Wu X. Sandirasegarane L. Nakamura M. Gilbert K.A. Singh R.S. Fort P.E. Antonetti D.A. Gardner T.W. Diabetes reduces basal retinal insulin receptor signaling: reversal with systemic and local insulin.Diabetes. 2006; 55: 1148-1156Crossref PubMed Scopus (143) Google Scholar), that this is or in the of α- and expression in the We demonstrated that this can also the increased apoptosis in diabetic retinopathy, the possible of crystallins in the of this in diabetic retinopathy. studies to the of the different crystallins in the retina are the 2D DIGE and analysis that α- and β-crystallins are to several that or such as in the M. M. Smith Smith J.B. changes in human crystallins identified by two-dimensional and Eye Res. 1998; PubMed Scopus Google Scholar, M. of β-crystallins during of Vis. 1998; Google Scholar). A study also showed that γ-crystallins also be targeted for such as in the retina from diabetic rats X. X. J.W. of in diabetic rat 7: PubMed Scopus Google Scholar). of be to in the The in this study the importance of methods such as 2D DIGE and methods by detailed specific studies for the of disease mechanisms. The discovery methods used in this work also showed a of expression of a that is We the Penn State Retina Research and the Penn State Eye Center for and the for and from the Penn State and from the Penn State and of the Penn State Diabetes Research