The role of apoptosis and mitosis in LDL transport across endothelial cell monolayers

We have previously shown that leaky junctions associated with dying or dividing cells are the dominant pathway for LDL transport under convective conditions in vitro, accounting for more than 90% of the transport. To explore the role of apoptosis in the leaky junction pathway, TNFalpha and cycloheximide (TNFalpha/CHX) were used to induce an elevated rate of apoptosis in cultured bovine aortic endothelial cell (BAEC) monolayers and the convective fluxes of LDL and water were measured. Control monolayers had an average apoptosis rate of 0.30%. Treatment with TNFalpha/CHX induced a 18.3-fold increase in apoptosis and a 4.4-fold increase in LDL permeability (Pe). Increases in apoptosis and permeability were attenuated by treatment with the caspase inhibitor Z-VAD-FMK. Water flux (Jv) increased by 2.7-fold after treatment with TNFalpha/CHX, and this increase was not attenuated by treatment with Z-VAD-FMK. Immunostaining of the tight junction protein ZO-1 showed that TNFalpha/CHX treatment disrupts the tight junction in addition to inducing apoptosis. This disruption is present even when Z-VAD-FMK is used to inhibit apoptosis, and likely accounts for the increase in water flux. We found a strong correlation between the rate of apoptosis and the permeability of BAEC monolayers to LDL.;To explore the role of mitosis in the leaky junction pathway, the microtubule stabilizing agent paclitaxel was used to alter the rate of mitosis and the fluxes of LDL and water were measured. Control monolayers had an average mitosis rate of 0.029%. Treatment with paclitaxel (2.5mM) for 1.5, 3, 4.5 or 6 hours yielded increasing rates of mitosis ranging from 0.099% to 1.03%. Pe increased up to 5-fold, while Jv increased up to 3-fold over this range of mitosis rates. We found a strong correlation between the mitosis rate and both the convective LDL permeability and the water flux. These results demonstrate the potential of manipulating endothelial monolayer permeability by altering the rates of apoptosis and mitosis pharmacollogicaly. This has implications for the treatment of atherosclerosis.