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Quantitative proteomic analysis of aqueous humor in a juvenile rabbit lensectomy model reveals surgery stage specific differences in coagulation, complement, and ocular immunosuppressive proteins

ABSTRACT: Purpose: To identify and quantify the proteins associated with the robust postoperative immune response in a juvenile rabbit model of lensectomy with intraocular lens (IOL) insertion. Methods: Twenty-six 6-7 week-old New Zealand White rabbits underwent unilateral portions of lensectomy with IOL insertion including: anterior chamber paracentesis, corneal incision with wound suture, lensectomy only, and lensectomy with IOL insertion. Aqueous humor (AH) was collected immediately prior and three days after each procedure. Semi-quantitative discovery of proteins was achieved using liquid chromatography tandem mass spectrometry (LC-MS/MS). Based on discovery results, targeted quantitation utilizing stable isotopically labeled (SIL) peptides for 3 proteins of interest, fibrinogen-beta chain, transforming growth factor beta-2, and retinol binding protein 3, were used to confirm the observed quantitative trends.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Oryctolagus Cuniculus (ncbitaxon:9986)

SUBMITTER: Iris Kassem

PROVIDER: MSV000084150 | MassIVE | Mon Jul 29 08:38:00 BST 2019

REPOSITORIES: MassIVE

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Project description:Impaired drainage of aqueous humor through the trabecular meshwork (TM) culminating in increased intraocular pressure is a major risk factor for glaucoma, a leading cause of blindness worldwide. Regulation of aqueous humor drainage through the TM, however, is poorly understood. The role of RhoA GTPase-mediated contractile activity, cell adhesive interactions, and gene expression in regulation of aqueous humor outflow was investigated using adenoviral vector-driven expression of constitutively active RhoA (RhoAV14). Organ cultured anterior segments from porcine eyes expressing RhoAV14 exhibited significant reduction of aqueous humor outflow. Cultured TM cells expressing RhoAV14 revealed strong contractile cell morphology, increased actin stress fibers and focal adhesions, along with increased levels of phosphorylated myosin II, and collagen IV, fibronectin and laminin. cDNA microarray analysis of RNA extracted from RhoAV14 expressing human TM cells revealed a significant increase in the expression of genes encoding extracellular matrix (ECM) proteins, cytokines, integrins, cytoskeletal proteins and signaling proteins. Conversely, various ECM proteins stimulated robust increases in phosphorylation of myosin II, paxillin and focal adhesion kinase, and activated Rho GTPase and actin stress fiber formation in TM cells, indicating a potential regulatory feedback interaction between ECM-induced mechanical strain and Rho GTPase-induced isometric tension in TM cells. Collectively, these data demonstrate that sustained activation of Rho GTPase signaling in the aqueous humor outflow pathway increases resistance to aqueous humor outflow through the trabecular pathway by influencing the contractile force, cell adhesive interactions, and the expression of ECM proteins and cytokines in TM cells. Keywords: Gene Expression Two condition experiment: Human trabecular mesh work cells infected with Adenivirus expressing GFP Vs Adenovirus expressing GFP and constitutively active RhoAV14

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Quantitative proteomic analysis of aqueous humor in a juvenile rabbit lensectomy model reveals surgery stage specific differences in coagulation, complement, and ocular immunosuppressive proteins

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