Molecular and Cellular PharmacologyEffect of flupirtine on cell death of human umbilical vein endothelial cells induced by reactive oxygen species
Section snippets
Materials
FBS, fungizone, l-glutamine, and HEPES were obtained from GIBCO BRL and Medium 199 was purchased from PAA Laboratories. Kanamycin monosulfate, t-BOOH, heparin, histamine, collagenase A (EC 3.4.24.3), trypsin (EC 3.4.21.4), proteinase K (EC 3.4.23.6) and RNAse A (EC 3.1.27.5) were purchased from Sigma, and fura-2-AM, fura-red-AM and H2DCF-DA from Molecular Probes. MTT (M2128), collagenase A, and ECGF were obtained from Boehringer, and the annexin-V assay apoptest was purchased from Nexins. XOD
Effect of flupirtine on viability of HUVEC
We first determined the effect of Flupirtine on the viability of confluent HUVEC. As summarised in Table 1, no significant change in cell number in a concentration range of 0.1 to 10 μg/mL of Flupirtine was observed during an incubation period of 24 hr. The amount of protein did not vary between treated and untreated cultures. The MTT assay system was applied as a further test to show that the compound did not affect cell viability. Setting the viability of untreated cultures to 100%,
Discussion
ROS are known inducers of apoptosis in neurons [42] as well as other somatic cells, e.g. endothelial cells [29], and are considered to be a main basis for neurodegenerative disorders such as β-amyloid peptide-mediated Alzheimer’s disease [43] or prion-mediated cell injury [44]. In the present study, two ROS generators, the HX/XOD and t-BOOH/Cu2+ systems, were used to induce apoptosis in HUVEC. The extent of apoptosis in HUVEC was measured both by flow cytometry and by DNA fragmentation.
Acknowledgements
This work was supported by a grant from the Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie (BMBF Verbundprojekt “Infektionsforschung”; 01 KI 94863).
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