3 research outputs found
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Rapid changes in the coagulant proteins on saphenous vein endothelium in response to arterial flow
Healthy endothelium provides a nonthrombogenic surface. In this study the authors investigated the effect of arterial flow on the saphenous vein endothelial expression of proteins controlling thrombosis. Human saphenous vein segments, freshly excised from patients, were placed in a validated in vitro circuit with flow conditions shown to simulate arterial or venous circulations. In separate experiments, placement of an external polytetrafluoroethylene (PTFE) stent was used to differentiate the effects of pulsatile wall deformation and shear stress, while addition of drugs to the vein perfusate allowed study of the role of ion channels in transducing the response of the vein to arterial flow. Endothelial concentrations of thrombomodulin, nitric oxide synthase, tissue factor, and tissue plasminogen activator were assessed by quantitative immunohistochemistry and Western blotting of endothelial cell lysates, in paired vein samples, in comparison to control proteins. Arterial flow conditions caused a rapid and significant reduction in the endothelial concentration of thrombomodulin: The immunostaining area decreased from 80.1 ± 7.0 to 48.3 ± 5.0 and 32.9 ± 3.0% at 45 and 90 minutes respectively, p = 0.01. These findings were confirmed by Western blotting. The reduction in thrombomodulin concentration was unaffected by eliminating vein wall deformation by placement of an external PTFE stent or by including the K+ channel blocker tetraethylammonium (TEA) in the vein perfusate. In contrast, thrombomodulin concentrations remained high when blockers of stretch-activated cation and calcium channels were included in the vein perfusate. The endothelial concentration of nitric oxide synthase increased after 90 minutes of arterial flow and this change was abolished when TEA was included in the vein perfusate. Arterial flow induced rapid changes in saphenous vein antithrombotic proteins. Different cation channels mediated the flow-induced changes in thrombomodulin and nitric oxide synthase
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Arterial flow conditions downregulate thrombomodulin on saphenous vein endothelium
Background - The antithrombogenic properties of venous endothelium may be attenuated when vein is implanted in the arterial circulation. Such changes may facilitate thrombosis, which is the final common pathway for saphenous vein arterial bypass graft occlusion. Methods and Results - Using human saphenous vein in a validated ex vivo flow circuit, we investigated (1) the possibility that arterial flow conditions (mean pressure, 100 mm Hg, 90 cpm, ?200 mL/min) alter the concentration of proteins involved in regulating thrombosis at the vessel wall and (2) the influence of ion channel blockade on such effects. Concentrations of thrombomodulin and tissue factor were quantified by Western blotting (ratio of yon Willebrand factor staining) and immunohistochemistry (as a percentage of CD31-staining area). Thrombomodulin concentrations after 90 minutes of venous and arterial flow conditions were quantified by immunostaining (68.9±4.8% and 41.0±3.0% CD31, respectively; P<0.01) and by Western blotting (1.35±0.20 and 0.15±0.03 ratio of yon Willebrand factor, respectively; P<0.01). The ability of endothelial cells to generate activated protein C also decreased from 62±14 to 19±10 ng · min- 1 · 1000 cells-1 (P=0.01). The significant reduction in thrombomodulin was attenuated if calcium was removed from the perfusate but not by external vein stenting. Inclusion in the vein perfusate of drugs that reduce calcium entry (including Gd3+, to block stretch-activated ion channels, and nifedipine) abolished the reduction in thrombomodulin concentration observed after arterial flow conditions. In freshly excised vein, negligible concentrations of tissue factor were detected on the endothelium and concentrations did not increase after 90 minutes of arterial flow conditions, although the inclusion of nifedipine caused the immunostaining to increase from 3.0±0.4% to 8.5±0.7% CD31 (P<0.02). Conclusions - In saphenous vein endothelium exposed to arterial flow conditions, there is rapid downregulation of thrombomodulin, sufficient to limit protein C activation, by a calcium-dependent mechanism
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Human saphenous vein endothelial cells express a tetrodotoxin- resistant, voltage-gated sodium current
Whole-cell patch-clamp electrophysiological investigation of endothelial cells cultured from human saphenous vein (HSVECs) has identified a voltage- gated Na+ current with a mean peak magnitude of -595 ± 49 pA (n = 75). This current was inhibited by tetrodotoxin (TTX) in a concentration-dependent manner, with an IC50 value of 4.7 µM, suggesting that it was of the TTX- resistant subtype. An antibody directed against the highly conserved intracellular linker region between domains III and IV of known Na+ channel a-subunits was able to retard current inactivation when applied intracellularly. This antibody identified a 245-kDa protein from membrane lysates on Western blotting and positively immunolabeled both cultured HSVECs and intact venous endothelium. HSVECs were also shown by reverse transcription-polymerase chain reaction to contain transcripts of the hH1 sodium channel gene. The expression of Na+ channels by HSVECs was shown using electrophysiology and cell-based enzyme-linked immunosorbent assay to be dependent on the concentration and source of human serum. Together, these results suggest that TTX-resistant Na+ channels of the hill isoform are expressed in human saphenous vein endothelium and that the presence of these channels is controlled by a serum factor