Effect of astrocyte-derived microparticles on intracellular free calcium concentration in umbilical vein endothelial cells

Abstract

Objective To investigate the effect of astrocyte-derived microparticles on intracellular free calcium concentration in umbilical vein endothelial cells (UVECs). Methods Brain astrocytes were first isolated from 24 h new-born Sprague-Dawley rats and cultured in vitro. Then, they were stimulated with calcium ionophore, A23187, and microparticles were extracted by fractional centrifugation and verified by electron microscopy. UVECs cultured in vitro were divided into control group, nimotop group, and low, median and high concentrations of microparticles groups; cells from nimotop group were pretreated with 10 μL nimotop for 10 min; cells from the later 4 groups were given 1×108, 0.5×108, 1×108, and 2×108/mL microparticles respectively; cells from the control group were given the same volume of medium. Ten min after cultivation, they were loaded with fluorescent probe Fluo3-AM, respectively. Later, the concentration of Ca2+ in UVECs was measured by confocal laser scanning microscope and flow cytometry. Results The isolation and in vitro culture methods for rat astrocytes provided stable and reliable results. With A23187 stimulation and fractional centrifugation, astrocyte derived microparticles were available for extraction. Laser scanning confocal microscope indicated that the intracellular fluorescence intensity in the control group was the lowest; after incubating UVECs with low, median and high concentrations of astrocyte-derived microparticles, the intracellular fluorescence intensity increased, and it increased following the concentrations of astrocyte-derived microparticles; pretreatment with nimotop could moderately decrease the intracellular fluorescence intensity, but that in the nimotop group was still higher than that in the control group. The mean fluorescence values for intracellular free Ca2+ were 51 866, 57 996 and 73 630, respectively, in the low, median and high concentrations of microparticles groups, which showed statistically significant increase as compared with that from the control group (45 759, P<0.05). When nimotop was applied to the cells, it blocked the influx of calcium ions and the measured value was significantly changed to 49843, enjoying significant difference as compared with that from the low, median and high concentrations of microparticles groups (P<0.05). Conclusion Astrocyte-derived microparticles are capable of increasing the intracellular free calcium concentration in UVECs, and the effects can be blocked by nimotop. Key words: Traumatic brain injury; Blood brain barrier; Microparticle; Free calcium