Abstract
Purpose :
The intraocular pressure (IOP) of rabbits varies during pubertal development. The regulators of these fluctuations are unknown. The aim of this study is to compare the proteome profile in rabbit aqueous humor (AH) before, during and after puberty to identify and characterize proteins that may contribute to IOP homeostasis.
Methods :
Male Dutch-belted rabbits were divided into two groups and studied between the ages of 10 and 46 weeks (w). Body weight, testicular volume, and serum testosterone were monitored to identify puberty. Before (18w), during (26w) and after puberty (34w) IOP was measured by pneumatonometry in one group (n=20) and AH (10-30 ul) was collected in the other (n=6). The AH was pooled and stored at -80oC until assayed. Total protein (500ng) from the pooled AH was analyzed by liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) and protein identification was done using Scaffold.
Results :
IOPs before, during and post puberty were 29.2±1.75, 26.7±1.49 and 29.5±1.90 mmHg respectively. MS analysis identified 119 to 174 proteins in each AH sample. Found were proteins belonging to signaling ligands (TGFβ2); extracellular matrix and matrix associated proteins (lumican, vitronectin, SPARC, cochlin); and other proteins (transferrin, autotaxin, osteopontin, complement factors, apolipoproteins). Three proteins in the AH decreased during puberty and increased post-puberty following the course of IOP fluctuations. These proteins are 1) gelsolin (5-fold) - actin binding protein involved in clearing of actin filaments, 2) clusterin (3fold) – chaperone protein involved in folding of secretory proteins, and 3) histidine-rich glycoprotein (HRG) (1.3-fold) – adaptor protein involved in cell-cell adhesion. Interestingly, α2-macroglobulin - a matrix-metalloprotease-2 and -9 inhibitor-decreased in concentration (5-fold) when other proteins were increasing.
Conclusions :
Though the ocular tissues that secrete these proteins are not known, based on our analysis and earlier studies finding gelsolin, clusterin and HRG in different forms of glaucoma, one may speculate their involvement in the regulation of IOP homeostasis during development and aging. We are currently testing the relevance of these proteins in the regulation of IOP homeostasis. Understanding the physiological lowering of IOP during puberty and the proteins involved in its regulation may provide insights into new pathways to lower IOP during pathology.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.