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.
OBJECTIVE
The aim of this study was to compare the protein profile of vitreous fluid from diabetic patients with proliferative diabetic retinopathy (PDR) with that from non-diabetic patients with idiopathic macular holes (MH). The mRNA of proteins differentially produced was also assessed in the retinas from diabetic and non-diabetic donors.


METHODS
Vitreous humour from type 1 diabetic patients with PDR (n = 8) and from non-diabetic patients with MH (n = 10) closely matched in terms of age were studied. The comparative proteomic analysis was performed using fluorescence-based difference gel electrophoresis (DIGE). Differentially produced proteins (abundance ratio >1.4, p < 0.05) were identified by mass spectrometry. Expressions of mRNA were measured by real-time RT-PCR in retinas from ten human eyes obtained at post-mortem (five eyes from diabetic subjects and five eyes from non-diabetic subjects).


RESULTS
Eight proteins were highly produced in PDR patients in comparison with non-diabetic subjects: zinc-alpha(2)-glycoprotein (ZAG), apolipoprotein (apo) A1, apoH, fibrinogen A, and the complement factors C3, C4b, C9 and factor B). We found three proteins that were underproduced in PDR subjects: pigment epithelial derived factor (PEDF), interstitial retinol-binding protein (IRBP) and inter-alpha-trypsin inhibitor heavy chain (ITIH2). There was no overlap in the vitreous levels of the above-mentioned proteins between PDR patients and non-diabetic control subjects. The differential production of ZAG, C3, factor B, PEDF and IRBP was further confirmed by western blot, and was in agreement with mRNA levels detected in the retina.


CONCLUSIONS
Proteomic analysis by DIGE, which permits an accurate quantitative comparison, was useful in identifying new potential candidates involved in the pathogenesis of PDR.
García-Ramírez
M
M
Diabetes Research Unit, Endocrinology Division, Vall d'Hebron University Hospital Research Institute, Passeig Vall d'Hebron 119-129, 08035 Barcelona, Spain.
Canals
F
F
Hernández
C
C
Colomé
N
N
Ferrer
C
C
Carrasco
E
E
García-Arumí
J
J
Simó
R
R
D002102:Cadaver; D003922:Diabetes Mellitus, Type 1 / Q000235:genetics*; D003930:Diabetic Retinopathy / Q000235:genetics* / Q000473:pathology; D004586:Electrophoresis; D005136:Eye Proteins / Q000235:genetics* / Q000302:isolation & purification; D006454:Hemoglobins / Q000235:genetics; D006801:Humans; D040901:Proteomics; D012016:Reference Values; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014822:Vitreous Body / Q000473:pathology / Q000502:physiology*
D005136:Eye Proteins; D006454:Hemoglobins