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E. Albe, E. Escalona, R. Rajagopal, T.Y. Chung, J.A. Javier, J.H. Chang, D.T. Azar; Proteomic Identification of ALK1 as a Differentially Expressed Protein During Hyaloid Vascular System Regression . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3145.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: The activin receptor–like kinase 1 (ALK1), a TGF–beta1 type I receptor, plays an inhibitory role in angiogenesis and vascular development. Mutations of ALK1 gene are linked to human type II hereditary hemorrhagic telangiectasia. Our purpose was to develop a mouse model to study the differential expression of inhibitory proteins during the phase of Hyaloid Vascular System (HVS) regression and determine the role of ALK1 in this model. Methods: Thirty–two newborn C57BL/6 mice were sacrificed on post–gestational days 1 (n= 20 eyes), 4 (n= 20 eyes), 8 (n= 12 eyes), and 16 (n= 12 eyes). The lens, the Pupillary Membrane (PM), the Tunica Vasculosa Lentis (TVL) and the primary vitreous containing the 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. Protein expression profile at each time point was compared using the Phoretix 2D image analysis software. Proteins from differentially expressed protein spots were isolated and identified using Mass Spectrometry (MS). Immunohistochemistry was performed to determine the expression of ALK1 during the HVS regression phase. Results: The generated protein expression maps showed reproducible separation of the protein spots on the 2 Dimension Electrophoresis gels. Up to 1400 proteins spots were detected per gel. 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 Mr 30 –90 kDa. Twenty protein spots in the P16 gels were not present in the P1 gels. MS revealed 39 differentially expressed proteins ( PP 16 – 1 to 39) in the P16 specimens. ALK1 was identified as PP16–31 (spot n°17). The warping of P16 with P1, P4 and P8 showed the presence of this spot only in P4 and P8. Immunohistochemistry of the cornea, PM, and TVL using anti ALK1 antibody confirmed the presence of ALK1 in the TVL at P4 and P16. Conclusions: The synthesis or degradation of one or more proteins, present at P16 (PP16 –1 to 39), may be related to the regression of HVS in the mouse. Identification of ALK1 by proteomic analysis and immunohistochemistry in this model suggests that the TGF–beta1 pathway may be involved in this process.
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