Abstract 546: L-NAME Attenuates the Cardiotonic Steroids-induced Monolayer Permeability in Lymphatic Endothelial Cells
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Objective: Preeclampsia (preE) is a hypertensive disorder unique to pregnancy. Cardiotonic steroids (CTS) such as marinobufagenin (MBG), cinobufotalin (CINO), and ouabain (OUB) are Na + /K + ATPase inhibitors. MBG is elevated in a rat model and patients with preE. MBG causes a vascular leak syndrome in vivo and increases endothelial cell monolayer permeability. Edema is a common syndrome of preE. To assess whether CTS are involved in the leakage of lymphatic endothelial cells (LECs) lining during preE, we evaluated the effect of these CTS on monolayer permeability of LECs in culture. Methods: LECs were isolated from a rat mesenteric collecting lymphatic vessel. The cells were treated with DMSO (vehicle), MBG, CINO, or OUB (1, 10 or 100 nM). Some LECs were pretreated with L-NAME (N-Nitro-L-Arginine Methyl Ester) at a concentration of 1M before treatment with 100 nM MBG or CINO. Monolayer permeability of CTS-induced LECs was measured by using a fluorescent dye that was quantified on a fluorescence plate reader. The expression of -catenin and VE-cadherin in the CTS-treated LECs was measured by immunofluorescence. Western blot was performed to measure -catenin, VE-cadherin, and LYVE-1 protein levels. Statistical comparisons were performed using analysis of variance with Dunnett's post hoc tests. Results: MBG ( 1 nM, p<0.05) and CINO ( 10 nM, p<0.05) significantly increased the monolayer permeability of LECs compared to DMSO while OUB had no effect. Pretreatment of LECs with 1M L-NAME attenuated the monolayer permeability of LECs treated with either 100 nM of MBG (p<0.05) or 100 nM of CINO (p<0.05). The -catenin protein expression in LECs was downregulated by both MBG (p<0.05) and CINO (p<0.05) treatment. However, CTS did not cause any disruption of the LECs tight junctions. CINO (p<0.05) downregulated the VE-cadherin and LYVE-1 protein expression, but MBG did not. Conclusions: We have demonstrated that bufadienolides, MBG and CINO, caused an increase in the monolayer permeability of LECs which was attenuated by L-NAME pretreatment. Moreover, the -catenin protein expression was downregulated by MBG and CINO treatment with no significant effect on tight junctions. These data suggest that CTS may be involved in the vascular leak syndrome in the LEC lining in preE.
