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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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Confirmation of discovery findings in independent animal sets.Targets identified in the proteomic and transcriptomic analyses were confirmed at the level of protein expression by immunoblotting in two independent animal sets (Experiments 3 & 4). mRNA expression was also examined in samples from Experiment 3 by qPCR. Elevated levels of retinal crystallin, alpha B (Cryab); crystallin, beta A3 (Cryba3), crystallin, beta B2 (Crybb2), diazepam-binding inhibitor (Dbi), and annexin 5 (Anxa5) protein were confirmed in two independent animal sets. Crystallin, alpha A (Cryaa) was significantly elevated in one set of animals but did not reach significance in the other. For these six proteins there was no concomitant change in mRNA expression. C = Control, D = Diabetic, * p<0.05, two-tailed t-test.
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pone-0016271-g006: Confirmation of discovery findings in independent animal sets.Targets identified in the proteomic and transcriptomic analyses were confirmed at the level of protein expression by immunoblotting in two independent animal sets (Experiments 3 & 4). mRNA expression was also examined in samples from Experiment 3 by qPCR. Elevated levels of retinal crystallin, alpha B (Cryab); crystallin, beta A3 (Cryba3), crystallin, beta B2 (Crybb2), diazepam-binding inhibitor (Dbi), and annexin 5 (Anxa5) protein were confirmed in two independent animal sets. Crystallin, alpha A (Cryaa) was significantly elevated in one set of animals but did not reach significance in the other. For these six proteins there was no concomitant change in mRNA expression. C = Control, D = Diabetic, * p<0.05, two-tailed t-test.

Mentions: To confirm discovery findings, immunoblot confirmation analysis was performed using two sets of animals, independent from the discovery experiments (Experiments 3 & 4, Table 1) on selected targets generated from each discovery method. To complement the protein confirmations, mRNA target expression was also examined in the Experiment 3 samples. Targeted protein and mRNA quantitation exhibited high confirmation rates of statistically significant differential expression in both validation experiments. Two patterns of expression were evident with one set of proteins regulated at both the transcript and protein levels (Cp, Lgals3, Fgf2, and Stat3) (Figure 5) and another set with altered protein expression but no significant changes in transcript expression (Cryaa, Cryab, Cryba3, Crybb2, Dbi, and Anxa5) (Figure 6). In all cases the significant differences in protein or mRNA agreed with the discovery findings. Targets from each of the discovery findings (Luminex - Fgf2; DIGE - Cryaa, Crybb2; iTRAQ - Anxa5, Dbi; microarray - Cp, Lgals3, Fgf2, and Stat3) were confirmed by orthogonal methods suggesting the quantitative validity of the discovery methods and reproducibility of the animal model.


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)

Confirmation of discovery findings in independent animal sets.Targets identified in the proteomic and transcriptomic analyses were confirmed at the level of protein expression by immunoblotting in two independent animal sets (Experiments 3 & 4). mRNA expression was also examined in samples from Experiment 3 by qPCR. Elevated levels of retinal crystallin, alpha B (Cryab); crystallin, beta A3 (Cryba3), crystallin, beta B2 (Crybb2), diazepam-binding inhibitor (Dbi), and annexin 5 (Anxa5) protein were confirmed in two independent animal sets. Crystallin, alpha A (Cryaa) was significantly elevated in one set of animals but did not reach significance in the other. For these six proteins there was no concomitant change in mRNA expression. C = Control, D = Diabetic, * p<0.05, two-tailed t-test.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3020973&req=5

pone-0016271-g006: Confirmation of discovery findings in independent animal sets.Targets identified in the proteomic and transcriptomic analyses were confirmed at the level of protein expression by immunoblotting in two independent animal sets (Experiments 3 & 4). mRNA expression was also examined in samples from Experiment 3 by qPCR. Elevated levels of retinal crystallin, alpha B (Cryab); crystallin, beta A3 (Cryba3), crystallin, beta B2 (Crybb2), diazepam-binding inhibitor (Dbi), and annexin 5 (Anxa5) protein were confirmed in two independent animal sets. Crystallin, alpha A (Cryaa) was significantly elevated in one set of animals but did not reach significance in the other. For these six proteins there was no concomitant change in mRNA expression. C = Control, D = Diabetic, * p<0.05, two-tailed t-test.
Mentions: To confirm discovery findings, immunoblot confirmation analysis was performed using two sets of animals, independent from the discovery experiments (Experiments 3 & 4, Table 1) on selected targets generated from each discovery method. To complement the protein confirmations, mRNA target expression was also examined in the Experiment 3 samples. Targeted protein and mRNA quantitation exhibited high confirmation rates of statistically significant differential expression in both validation experiments. Two patterns of expression were evident with one set of proteins regulated at both the transcript and protein levels (Cp, Lgals3, Fgf2, and Stat3) (Figure 5) and another set with altered protein expression but no significant changes in transcript expression (Cryaa, Cryab, Cryba3, Crybb2, Dbi, and Anxa5) (Figure 6). In all cases the significant differences in protein or mRNA agreed with the discovery findings. Targets from each of the discovery findings (Luminex - Fgf2; DIGE - Cryaa, Crybb2; iTRAQ - Anxa5, Dbi; microarray - Cp, Lgals3, Fgf2, and Stat3) were confirmed by orthogonal methods suggesting the quantitative validity of the discovery methods and reproducibility of the animal model.

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