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Multi-modal proteomic analysis of retinal protein expression alterations in a rat model of diabetic retinopathy.

VanGuilder HD, Bixler GV, Kutzler L, Brucklacher RM, Bronson SK, Kimball SR, Freeman WM - PLoS ONE (2011)

Bottom Line: The aim of this study was to identify retinal proteomic alterations associated with functional dysregulation of the diabetic retina to better understand diabetic retinopathy pathogenesis and that could be used as surrogate endpoints in preclinical drug testing studies.Alterations in pro-inflammatory, signaling and crystallin family proteins were confirmed by orthogonal methods in multiple independent animal cohorts.These proteins, especially those not normalized by insulin therapy, may also be useful in preclinical drug development studies.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Penn State College of Medicine, Hershey, Pennsylvania, United States of America.

ABSTRACT

Background: As a leading cause of adult blindness, diabetic retinopathy is a prevalent and profound complication of diabetes. We have previously reported duration-dependent changes in retinal vascular permeability, apoptosis, and mRNA expression with diabetes in a rat model system. The aim of this study was to identify retinal proteomic alterations associated with functional dysregulation of the diabetic retina to better understand diabetic retinopathy pathogenesis and that could be used as surrogate endpoints in preclinical drug testing studies.

Methodology/principal findings: A multi-modal proteomic approach of antibody (Luminex)-, electrophoresis (DIGE)-, and LC-MS (iTRAQ)-based quantitation methods was used to maximize coverage of the retinal proteome. Transcriptomic profiling through microarray analysis was included to identify additional targets and assess potential regulation of protein expression changes at the mRNA level. The proteomic approaches proved complementary, with limited overlap in proteomic coverage. Alterations in pro-inflammatory, signaling and crystallin family proteins were confirmed by orthogonal methods in multiple independent animal cohorts. In an independent experiment, insulin replacement therapy normalized the expression of some proteins (Dbi, Anxa5) while other proteins (Cp, Cryba3, Lgals3, Stat3) were only partially normalized and Fgf2 and Crybb2 expression remained elevated.

Conclusions/significance: These results expand the understanding of the changes in retinal protein expression occurring with diabetes and their responsiveness to normalization of blood glucose through insulin therapy. These proteins, especially those not normalized by insulin therapy, may also be useful in preclinical drug development studies.

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Examples of a differentially-expressed protein identified by iTRAQ.Retinal protein isolated from four diabetic and four age-matched control rats was labeled with isobaric tags prior to separation, identification, and quantitation by 8-plex iTRAQ using LC/MS/MS. An example MS/MS spectrum and the corresponding quantitation data (magnification of iTRAQ signal region) for a confidently identified (>95%) peptide component of Anxa5 are depicted. The signal intensities of all confidently identified peptides unique to Anxa5 were compiled to quantify Anxa5 expression. Anxa5 was significantly increased by approximately two-fold in three-month diabetic rats compared to controls. * p<0.05, two-tailed t-test, n = 4/group.
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pone-0016271-g003: Examples of a differentially-expressed protein identified by iTRAQ.Retinal protein isolated from four diabetic and four age-matched control rats was labeled with isobaric tags prior to separation, identification, and quantitation by 8-plex iTRAQ using LC/MS/MS. An example MS/MS spectrum and the corresponding quantitation data (magnification of iTRAQ signal region) for a confidently identified (>95%) peptide component of Anxa5 are depicted. The signal intensities of all confidently identified peptides unique to Anxa5 were compiled to quantify Anxa5 expression. Anxa5 was significantly increased by approximately two-fold in three-month diabetic rats compared to controls. * p<0.05, two-tailed t-test, n = 4/group.

Mentions: In the quantitative LC-MS/MS analysis with isobaric tags, 455 proteins were confidently detected with at least two peptides identified with greater than the 95% confidence required for accurate quantitation. A total of 27 proteins were significantly regulated (t-test, two tailed) with a fold change filter of ≥1.1 or ≤0.9. Differential expression was distributed equally between down- (14 proteins) and up- (13 proteins) regulated species (Table 4). Proteins exhibiting expression differences included signaling, cytoskeletal, and neuronal proteins. An example of iTRAQ data is provided in Figure 3 with a representative MS/MS spectrum and quantitation data for a confidently identified peptide component of Anxa5. Anxa5, Hspa1b, Marcks, Dbi, Gnat1, and Rcn were chosen for confirmational analysis. The finding of reduced VAMP2 protein expression after three months of hyperglycemia agrees with our previous observations in this model [13]. The full iTRAQ data set is available in Table S3.


Multi-modal proteomic analysis of retinal protein expression alterations in a rat model of diabetic retinopathy.

VanGuilder HD, Bixler GV, Kutzler L, Brucklacher RM, Bronson SK, Kimball SR, Freeman WM - PLoS ONE (2011)

Examples of a differentially-expressed protein identified by iTRAQ.Retinal protein isolated from four diabetic and four age-matched control rats was labeled with isobaric tags prior to separation, identification, and quantitation by 8-plex iTRAQ using LC/MS/MS. An example MS/MS spectrum and the corresponding quantitation data (magnification of iTRAQ signal region) for a confidently identified (>95%) peptide component of Anxa5 are depicted. The signal intensities of all confidently identified peptides unique to Anxa5 were compiled to quantify Anxa5 expression. Anxa5 was significantly increased by approximately two-fold in three-month diabetic rats compared to controls. * p<0.05, two-tailed t-test, n = 4/group.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3020973&req=5

pone-0016271-g003: Examples of a differentially-expressed protein identified by iTRAQ.Retinal protein isolated from four diabetic and four age-matched control rats was labeled with isobaric tags prior to separation, identification, and quantitation by 8-plex iTRAQ using LC/MS/MS. An example MS/MS spectrum and the corresponding quantitation data (magnification of iTRAQ signal region) for a confidently identified (>95%) peptide component of Anxa5 are depicted. The signal intensities of all confidently identified peptides unique to Anxa5 were compiled to quantify Anxa5 expression. Anxa5 was significantly increased by approximately two-fold in three-month diabetic rats compared to controls. * p<0.05, two-tailed t-test, n = 4/group.
Mentions: In the quantitative LC-MS/MS analysis with isobaric tags, 455 proteins were confidently detected with at least two peptides identified with greater than the 95% confidence required for accurate quantitation. A total of 27 proteins were significantly regulated (t-test, two tailed) with a fold change filter of ≥1.1 or ≤0.9. Differential expression was distributed equally between down- (14 proteins) and up- (13 proteins) regulated species (Table 4). Proteins exhibiting expression differences included signaling, cytoskeletal, and neuronal proteins. An example of iTRAQ data is provided in Figure 3 with a representative MS/MS spectrum and quantitation data for a confidently identified peptide component of Anxa5. Anxa5, Hspa1b, Marcks, Dbi, Gnat1, and Rcn were chosen for confirmational analysis. The finding of reduced VAMP2 protein expression after three months of hyperglycemia agrees with our previous observations in this model [13]. The full iTRAQ data set is available in Table S3.

Bottom Line: The aim of this study was to identify retinal proteomic alterations associated with functional dysregulation of the diabetic retina to better understand diabetic retinopathy pathogenesis and that could be used as surrogate endpoints in preclinical drug testing studies.Alterations in pro-inflammatory, signaling and crystallin family proteins were confirmed by orthogonal methods in multiple independent animal cohorts.These proteins, especially those not normalized by insulin therapy, may also be useful in preclinical drug development studies.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Penn State College of Medicine, Hershey, Pennsylvania, United States of America.

ABSTRACT

Background: As a leading cause of adult blindness, diabetic retinopathy is a prevalent and profound complication of diabetes. We have previously reported duration-dependent changes in retinal vascular permeability, apoptosis, and mRNA expression with diabetes in a rat model system. The aim of this study was to identify retinal proteomic alterations associated with functional dysregulation of the diabetic retina to better understand diabetic retinopathy pathogenesis and that could be used as surrogate endpoints in preclinical drug testing studies.

Methodology/principal findings: A multi-modal proteomic approach of antibody (Luminex)-, electrophoresis (DIGE)-, and LC-MS (iTRAQ)-based quantitation methods was used to maximize coverage of the retinal proteome. Transcriptomic profiling through microarray analysis was included to identify additional targets and assess potential regulation of protein expression changes at the mRNA level. The proteomic approaches proved complementary, with limited overlap in proteomic coverage. Alterations in pro-inflammatory, signaling and crystallin family proteins were confirmed by orthogonal methods in multiple independent animal cohorts. In an independent experiment, insulin replacement therapy normalized the expression of some proteins (Dbi, Anxa5) while other proteins (Cp, Cryba3, Lgals3, Stat3) were only partially normalized and Fgf2 and Crybb2 expression remained elevated.

Conclusions/significance: These results expand the understanding of the changes in retinal protein expression occurring with diabetes and their responsiveness to normalization of blood glucose through insulin therapy. These proteins, especially those not normalized by insulin therapy, may also be useful in preclinical drug development studies.

Show MeSH
Related in: MedlinePlus