Limits...
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.

Show MeSH

Related in: MedlinePlus

Examples of differentially expressed proteins identified by DIGE.Retinal protein isolated from three-month diabetic and age-matched control rats was separated and quantitated by DIGE analysis with identification by MS/MS. Two examples of confidently identified, differentially regulated protein spots are shown. The upper panels depict spot location and signal intensity, while the lower panels depict three-dimensional representations of the densitometric spot volumes used for quantitation. Both Cryaa and Crybb2 crystallin isoforms were significantly increased in expression (by 491% and 305%, respectively) in diabetic rats compared to controls. * p<0.05, two-tailed t-test, n = 8/group.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3020973&req=5

pone-0016271-g002: Examples of differentially expressed proteins identified by DIGE.Retinal protein isolated from three-month diabetic and age-matched control rats was separated and quantitated by DIGE analysis with identification by MS/MS. Two examples of confidently identified, differentially regulated protein spots are shown. The upper panels depict spot location and signal intensity, while the lower panels depict three-dimensional representations of the densitometric spot volumes used for quantitation. Both Cryaa and Crybb2 crystallin isoforms were significantly increased in expression (by 491% and 305%, respectively) in diabetic rats compared to controls. * p<0.05, two-tailed t-test, n = 8/group.

Mentions: Labeled retinal proteins from control and diabetic rats were separated by isoelectric point and molecular weight, producing consistent spot patterns on analytical and preparative gels (Figure 2). 1231 protein spots were matched across >6 analytical gels, yielding n = 6–8/group for differential expression analyses. After subtracting local background and normalizing to Cy-2 signals for inter-gel comparisons, 48 protein spots were determined to be significantly different between control and diabetic rats (≥1.1-fold change, two-tailed t-test, p<0.05). Differential protein expression represented both inductions and reductions with diabetes, ranging from approximately −200% to 500% of control levels.


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 differentially expressed proteins identified by DIGE.Retinal protein isolated from three-month diabetic and age-matched control rats was separated and quantitated by DIGE analysis with identification by MS/MS. Two examples of confidently identified, differentially regulated protein spots are shown. The upper panels depict spot location and signal intensity, while the lower panels depict three-dimensional representations of the densitometric spot volumes used for quantitation. Both Cryaa and Crybb2 crystallin isoforms were significantly increased in expression (by 491% and 305%, respectively) in diabetic rats compared to controls. * p<0.05, two-tailed t-test, n = 8/group.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3020973&req=5

pone-0016271-g002: Examples of differentially expressed proteins identified by DIGE.Retinal protein isolated from three-month diabetic and age-matched control rats was separated and quantitated by DIGE analysis with identification by MS/MS. Two examples of confidently identified, differentially regulated protein spots are shown. The upper panels depict spot location and signal intensity, while the lower panels depict three-dimensional representations of the densitometric spot volumes used for quantitation. Both Cryaa and Crybb2 crystallin isoforms were significantly increased in expression (by 491% and 305%, respectively) in diabetic rats compared to controls. * p<0.05, two-tailed t-test, n = 8/group.
Mentions: Labeled retinal proteins from control and diabetic rats were separated by isoelectric point and molecular weight, producing consistent spot patterns on analytical and preparative gels (Figure 2). 1231 protein spots were matched across >6 analytical gels, yielding n = 6–8/group for differential expression analyses. After subtracting local background and normalizing to Cy-2 signals for inter-gel comparisons, 48 protein spots were determined to be significantly different between control and diabetic rats (≥1.1-fold change, two-tailed t-test, p<0.05). Differential protein expression represented both inductions and reductions with diabetes, ranging from approximately −200% to 500% of control levels.

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