Modulation of intracellular Ca2+ concentration in brain microvascular endothelial cells actively induced by brain targeted liposomes

AIMS The aim of our study is to evaluate the interaction at the neurovascular unit of liposomes (mApoE-PA-LIP) functionalized with ApoE-derived peptide (mApoE) and phosphatidic acid (PA). In light of our previous results (Re et al.,2010), we assessed mApoE-PA-LIP activities on human cerebral microvascular cells (hCMEC/D3) as an in vitro model of human blood-brain barrier (BBB). METHODS The intracellular Ca2+ concentration was measured by digital imaging microscopy in hCMEC/D3 maintained in a low-profile chamber in presence of physiological salt solution or PSS (NaCl 150 mM; KCl 6 mM; MgCl2 1mM; CaCl2 1.5mM; HEPES 10mM; Glucose 10mM). We pre-incubated hCMEC/D3 cells with 4µm acetoxy-methyl-ester Fura-2 AM for 30 minutes at 37°C. Afterwards, we perfused the cells with mApoE-LIP or mApoE-PA-LIP (in PSS) to evaluate ATP (50µM)-evoked intracellular calcium waves. RESULTS The interaction of mApoE-PA-LIP with the hCMEC/D3 modulated the duration of ATP-induced intracellular calcium waves. We found an increase (mean ± se, 136 ± 3.75 sec, n=50, p-value <0.05) of the duration of the ATP-evoked calcium waves in presence of mApoE-PA-LIP in comparison to mApoE-LIP perfusion (mean ± se, 125 ± 2.13 sec). mApoE-LIP and “PSS alone” did not prolong ATP-evoked calcium waves. CONCLUSIONS Our data confirm that the specific liposome functionalization with phosphatidic acid may be linked to the enhanced intracellular calcium waves evoked in hCMEC/D3 by ATP. This finding suggests an intriguing issue involving PA intracellular pathways and its possible implications in the modulation of calcium waves duration in hCMEC/D3 cells.