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

Project description:Pseudoexfoliation syndrome (PEX) is a systemic disorder that manifests as a fluffy, proteinaceous fibrillar material throughout the body. In the eye, such deposits result in glaucoma (PEXG), due to impeding aqueous humor outflow. When a patient presents acute glaucoma, it is necessary to remove some of the aqueous fluid within the eye to relief pain and pressure. This label free proteomics dataset was collected from human donors during cataract surgery. The aqueous humor was collected during essential ophthalmic procedures that allowed paracentesis after obtaining informed consents from human subjects without collecting identifiers, but all disease and medication history were collected. The sample collection included non-glaucomatous controls (CTL-GC), those with pseudoexfoliation syndrome (PEX-GC), and synthesized GC-Globulin pure protein (GC-Pure). Approximately 50-120 ul volume of AH was collected by paracentesis and stored in -80C immediately upon acquisition until analysis. Protein extraction was carried out by homogenization of the tissue in extraction buffer (TEAB, NaCl and SDS). Protein amounts were estimated and normalized to 10 ug across experimental samples. Samples were reduced using TCEP, alkylated with iodoacetamide and digested overnight with trypsin. Untargeted liquid chromatography-mass spectrometry was performed on an Easy nLC 1000 liquid chromatograph coupled to a QExactive mass spectrometer (LC-MS/MS). Data analysis was performed using Proteome Discoverer 3.0 and Graph Pad Prism 10. Each sample was run three separate times. Raw mass spectrometry data files were analyzed using Proteome Discoverer 3.0. The human proteome was downloaded from UniProt and used as the target database for protein identification. Max missed cleavage site was set to 2 and minimum peptide length to 6. Precursor Mass Tolerance was set to 10ppm and Fragment Mass Tolerance to 0.02 Da. Post-translational modifications for experimental proteins included oxidation, acetylation, and carbamidomethylation. The normalization was set to total peptide amount and confidence to low.

Project description:Glaucoma is a neurodegenerative disease in which vision is lost as a result of the apoptosis of retinal ganglion cells. When the trabecular meshwork cells, that regulate aqueous humor outflow, are stressed as they are in some forms of glaucoma, they considerably up-regulated secretion of a protein called myocilin into the aqueous. The function of this protein is unclear; but since some aqueous humor flows to the posterior of the eye, the effects of myocilin will also be felt by the retinal cells. We have a transgenic mouse that secretes large amounts of myocilin into aqueous humor. In these mice, there is a deposition of myocilin on membranes of certain cells suggesting myocilin might have some signaling functions. This signaling function could explain why stresses that increase intraocular pressures can increase the likelihood for glaucoma, cause the myocilin to be secreted at very high levels by the trabecular meshwork. Myocilin may be important in treating glaucoma. This experiment will determine if myocilin is altering gene expression in the retina. The results should show variations in expression levels and will give us some indication of the pathways that are important to preserve retinal ganglion cells after a diagnosis of glaucoma. Our hypothesis is that myocilin is acting like a signaling protein in the eye. It acts not only in the anterior segment but also in retina as a result of the flow of some aqueous to the back of the eye. This signaling function is protective to certain ocular cells. This function of myocilin may influence cells in the retina and may counter the apoptotic signals in the retinal ganglion cells that occur during glaucoma. Retinas from transgenic mice and from mice of the same strain that do not have the transgene will be dissected. The mice will be three weeks of age. Because of the small size of the retina, samples will be pooled to obtain the RNA necessary to run the microarray. Three control and three transgenic samples will be run and compared with each other. We have data indicating that myocilin is at high levels in the aqueous humor of the transgenic animals.

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Homo sapiens

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