To investigate the apoptotic effects of detergent sclerosants sodium tetradecylsulphate (STS) and polidocanol (POL) on endothelial cells at sub-lytic concentrations. Human umbilical vein endothelial cells (HUVECs) were isolated and labelled with antibodies to assess for apoptosis and examined with confocal microscopy and flow cytometry. Isolated HUVECs viability was assessed using propidium iodide staining. Early apoptosis was determined by increased phosphatidylserine exposure by lactadherin binding. Caspase 3, 8, 9 and Bax activation as well as inhibitory assays with Pan Caspase (Z-VAD-FMK) and Bax (BI-6C9) were assessed to identify apoptotic pathways. Porimin activation was used to assess cell membrane permeability. Cell lysis reached almost 100 % with STS at 0.3 % and with POL at 0.6 %. Apoptosis was seen with both STS and POL at concentrations ranging from 0.075 to 0.15 %. PS exposure increased with both STS and POL and exhibited a dose-dependent trend. Active Caspase 3, 8 and 9 but not Bax were increased in HUVECs stimulated with low concentrations of both STS and POL. Inhibitory assays demonstrated Caspase 3, 8, 9 inhibition at low concentrations (0.075 to 0.6 %) with both STS and POL. Both agents increased the activation of porimin at all concentrations. Both sclerosants induced endothelial cell (EC) apoptosis at sub-lytic concentrations through a caspase-dependant pathway. Both agents induced EC oncosis.
Objectives To investigate the effects of detergent sclerosants, sodium tetradecyl sulphate and polidocanol, on endothelial cell activation and microparticle release and the effects of detergent sclerosants, sirolimus and propranolol, on apoptosis in vitro. Methods Cultured human umbilical vein endothelial cells and murine haemangioendothelioma (EOMA) cell lines were incubated with different concentrations of sodium tetradecyl sulphate and polidocanol, as well as sirolimus and propranolol. Endothelial activation was assessed using flow cytometry for CD62e (E-Selectin), CD54 (ICAM-1), CD105 (endoglin), CD144 (VE-Cadherin), CD146 (MCAM) and the release of endothelial microparticles. Cell proliferation was assessed using [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] and carboxyfluorescein succinimidyl ester assays. Apoptosis was assessed using flow cytometry for lactadherin/propidium iodide staining and for Caspase-3 expression. Results Sublytic concentrations of sodium tetradecyl sulphate and polidocanol (0.075%–0.3%) increased the expression of the activation markers CD62e and CD54. The expression of CD105 decreased in sclerosant treated cultured human umbilical vein endothelial cells. Both sodium tetradecyl sulphate and polidocanol induced the release of endothelial microparticles. All agents inhibited cell proliferation. Sodium tetradecyl sulphate and polidocanol-induced apoptosis as evidenced by increased phosphatidylserine exposure and caspase-3 expression, whereas sirolimus and propranolol increased caspase-3 expression only. Conclusion Sublytic concentrations of detergent sclerosants induce endothelial activation and the release of endothelial microparticles. All agents were anti-proliferative in EOMA cell lines, with sodium tetradecyl sulphate and polidocanol inducing cellular apoptosis.
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