Proteome Analysis of the Hyaloid Vascular System Regression During Ocular Development

Abstract

Purpose: : To investigate the differential protein expression patterns and identify the physiologically relevant angiogenic and anti–angiogenic factors involved in the hyaloid vascular system (HVS) regression during ocular development.

Methods: : Fifty newborn C57BL/6 mice were sacrificed on post–gestational days 1, 4, 8, 16, 24 and 30. The lens, pupillary membrane (PM), tunica vasculosa lentis (TVL), and primary vitreous containing vasa hyaloidea propria (VHP) were isolated. Proteins were extracted from each specimen, loaded onto nonlinear immobilized pH gradient (IPG) gel strips, and separated by isoelectric points and molecular weights. The protein expression profiles at each time point were compared using the Phoretix 2D image analysis software. Differentially–expressed proteins were isolated and identified using mass spectrometry (MS). The resulting peptide mass fingerprints were searched against the public domain NCBInr mouse protein sequence database. Functional classification was based on the classification provided in the TrEMBL and SwissProt protein knowledge database.

Results: : The generated protein expression maps showed reproducible separation of the protein spots on the 2–dimensional electrophoresis gels. Up to 700 protein spots were detected per gel. A progressive decrease in the number and intensity of the protein spots occurred from P1 to P16, particularly in the area corresponding to pI 4–7 and M_w 30–90 kDa. 161 proteins were analyzed; 127 of them were classified into functional categories and 34 were unclassified. In the protein pool differentially expressed in P16, we found two proteins which have a well–known angiogenic activity (fibroblast growth factor 22, hepatoma derived growth factor) and with anti–angiogenic activity (kininogen).

Conclusions: : Using proteomic analysis of the HVS, we have identified several proteins known to be associated with angiogenesis. This proteomic analysis of HVS may be valuable for identifying the specific proteins responsible for the early maintenance and later regression of the HVS during mouse eye development.

Keywords: proteomics • development • crystalline lens