Browsing by keyword "Given, Curtis A. 2nd"
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Cell confluence-dependent remodeling of endothelial membranes mediated by cholesterolThe plasma membranes of endothelial cells reaching confluence undergo profound structural and functional modifications, including the formation of adherens junctions, crucial for the regulation of vascular permeability and angiogenesis. Adherens junction formation is accompanied by the tyrosine dephosphorylation of adherens junctions proteins, which has been correlated with the strength and stability of adherens junctions. Here we show that cholesterol is a critical determinant of plasma membrane remodeling in cultures of growing cow pulmonary aortic endothelial cells. Membrane cholesterol increased dramatically at an early stage in the formation of confluent cow pulmonary aortic endothelial cell monolayers, prior to formation of intercellular junctions. This increase was accompanied by the redistribution of caveolin from a high density to a low density membrane compartment, previously shown to require cholesterol, and increased binding of the annexin II-p11 complex to membranes, consistent with other studies indicating cholesterol-dependent binding of annexin II to membranes. Furthermore, partial depletion of cholesterol from confluent cells with methyl-beta-cyclodextrin both induced tyrosine phosphorylation of multiple membrane proteins, including adherens junctions proteins, and disrupted adherens junctions. Both effects were dramatically reduced by prior complexing of methyl-beta-cyclodextrin with cholesterol. Our results reveal a novel physiological role for cholesterol regulating the formation of adherens junctions and other plasma membrane remodeling events as endothelial cells reach confluence.
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Visualization of clot lysis in a rat embolic stroke model: application to comparative lytic efficacyBACKGROUND AND PURPOSE: The purpose of this study was to develop a novel MRI method for imaging clot lysis in a rat embolic stroke model and to compare tissue plasminogen activator (tPA)-based clot lysis with and without recombinant Annexin-2 (rA2). METHODS: In experiment 1 we used in vitro optimization of clot visualization using multiple MRI contrast agents in concentrations ranging from 5 to 50 muL in 250 muL blood. In experiment 2, we used in vivo characterization of the time course of clot lysis using the clot developed in the previous experiment. Diffusion, perfusion, angiography, and T1-weighted MRI for clot imaging were conducted before and during treatment with vehicle (n=6), tPA (n=8), or rA2 plus tPA (n=8) at multiple time points. Brains were removed for ex vivo clot localization. RESULTS: Clots created with 25 muL Magnevist were the most stable and provided the highest contrast-to-noise ratio. In the vehicle group, clot length as assessed by T1-weighted imaging correlated with histology (r=0.93). Clot length and cerebral blood flow-derived ischemic lesion volume were significantly smaller than vehicle at 15 minutes after treatment initiation in the rA2 plus tPA group, whereas in the tPA group no significant reduction from vehicle was observed until 30 minutes after treatment initiation. The rA2 plus tPA group had a significantly shorter clot length than the tPA group at 60 and 90 minutes after treatment initiation and significantly smaller cerebral blood flow deficit than the tPA group at 90 minutes after treatment initiation. CONCLUSIONS: We introduce a novel MRI-based clot imaging method for in vivo monitoring of clot lysis. Lytic efficacy of tPA was enhanced by rA2.