Sphingolipid Analysis of hyper and normotensive DBA2J mice aqueous humor and trabecular meshwork
Ontology highlight
ABSTRACT:
ORGANISM(S): Mouse Mus Musculus
DISEASE(S): Eye Disease
SUBMITTER: 
Sanjoy Bhattacharya
PROVIDER: ST000620 | MetabolomicsWorkbench | Tue Jun 06 00:00:00 BST 2017
REPOSITORIES: MetabolomicsWorkbench
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Project description:AH and TM samples were obtained from young-normotensive DBA/2J (3 and 7.5 months) and old-hypertensive DBA/2J mice (8-8.5, 10 and 12 month). Lipids were extracted using modified Bligh and Dyer method and subjected to mass spectrometric identification using appropriate class-specific lipid standards and ratiometric quantification. Corresponding aqueous phase (of extraction) protein concentrations were measured using Bradford method.
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Project description:AH and TM samples were obtained from young-normotensive DBA/2J (3 and 7.5 months) and old-hypertensive DBA/2J mice (8-8.5, 10 and 12 month). Lipids were extracted using modified Bligh and Dyer method and subjected to mass spectrometric identification using appropriate class-specific lipid standards and ratiometric quantification. Corresponding aqueous phase (of extraction) protein concentrations were measured using Bradford method.
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Project description:AH and TM samples were obtained from young-normotensive DBA/2J (3 and 7.5 months) and old-hypertensive DBA/2J mice (8-8.5, 10 and 12 month). Lipids were extracted using modified Bligh and Dyer method and subjected to mass spectrometric identification using appropriate class-specific lipid standards and ratiometric quantification. Corresponding aqueous phase (of extraction) protein concentrations were measured using Bradford method.
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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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