Remedios Miguel

The effect of nifedipine on lipid and monocyte infiltration of the subendothelial space

PURPOSE Calcium channel blockade has been shown to inhibit experimental atherosclerosis in cholesterol-fed animals, and early clinical trials suggest its benefit in human subjects as well. METHODS To determine the effect of the calcium channel blocker nifedipine on lipid and monocyte infiltration of the subendothelial space, an endothelial cell (EC)-smooth muscle cell (SMC) bilayer model of the arterial wall was incubated for 18 hours with nifedipine (0.1 micrograms/ml). Iodine 125-labeled low-density lipoprotein (125I-LDL) (10 micrograms protein/ml) was then added to the upper-well medium. RESULTS After a 3-hour incubation period, nifedipine-treated bilayers showed an increased permeability to LDL (p < 10(-7). Nifedipine had no effect on the membrane binding or cellular uptake of LDL by the EC but did increase SMC binding and uptake (p < 0.0005). U937 monocytes were found to incorporate 125I-LDL in a concentration-dependent fashion, without saturation to 25 micrograms/ml, the highest concentration studied. Nifedipine increased monocyte uptake of LDL (10 micrograms/ml; p < 0.003 but reduced monocyte movement through the EC barrier (p < 10(-7). A study of the selective preincubation of each cell type (EC, SMC, and monocyte) with nifedipine indicated that this reduction was likely the result of a direct effect on the monocyte. CONCLUSIONS Given the potential cytotoxic effects of the monocyte within the subendothelial space, nifedipine-induced inhibition of monocyte infiltration and enhancement of lipoprotein uptake by the SMC may be protective.

Aortic endothelial and smooth muscle cell co-culture: an in vitro model of the arterial wall.

Interactions between vascular endothelial (EC) and smooth muscle cells (SMC) contribute both to the normal function of the vascular wall and to the pathogenesis of lesions such as atherosclerosis and fibrointimal hyperplasia. However, study of these interactions has been hampered by the difficulty in growing these two cell types in simultaneous culture. Methods using conditioned media, shared media, and bilayer culture have been described, but none is well suited to the study of vascular cell interactions. We report a method for EC-SMC co-culture that preserves bilayer morphology, allows independent study of the cells and their matrices after intervention, remains stable over long periods in culture, and permits study of changes in cell-cell interaction with growth of the cells to confluence. This simple bilayer co-culture system simulates the in vivo situation and may enhance our understanding of EC-SMC interactions.

Competitive inhibition of LDL binding and uptake by HDL in aortic endothelial cells

High-density lipoprotein (HDL) may inhibit the binding and cellular uptake of low-density lipoprotein (LDL) as one means of regulating the delivery of exogenous cholesterol to nonhepatic tissues. This may play an important role in atherogenesis, by altering lipid metabolism in cells of the arterial wall. To verify and better characterize this effect, endothelial cells were harvested from bovine aorta and maintained in tissue culture. Following initial preincubation in lipid-deficient culture media, these cells were incubated for 2 hr at 4 degrees C in media containing 125I-LDL (10 micrograms protein/ml) and varying concentrations of either HDL (0-400 micrograms protein/ml) or comparable amounts of Apoprotein A (Apo A), the major protein component of HDL. Intracellular and trypsin-released counts were assayed separately, as a measurement of cellular uptake and membrane bound LDL, respectively. Results of this study indicated an inhibition of LDL binding and uptake by HDL (P less than 0.005, ANOVA). A similar inhibition was found with Apo A alone (P less than 0.005). When identical studies were performed using 125I-Apoprotein B, the protein component of LDL, and Apo A, the latter was found to inhibit the binding of Apo B to the same extent (P less than 0.0006). These results indicate that HDL does inhibit LDL binding and uptake by bovine aortic endothelial cells and that, because this effect is seen equally with only the protein component of these lipoprotein particles, it is most likely due to competitive binding at the receptor level rather than to stearic hindrance or an alteration of the cell membrane.

Oxygen radicals alter LDL permeability and uptake by an endothelial-smooth muscle cell bilayer

There is evidence to suggest that the deposition of lipid within the arterial wall, which is characteristic of atherosclerosis, involves a break in the integrity of the endothelial barrier. Oxidative modification of low density lipoproteins by reactive oxygen species may enhance this process. In this study, an aortic endothelial (EC) smooth muscle cell (SMC) bilayer was briefly exposed to a free-radical generating system to determine the effect of superoxides on lipid permeability and uptake. 125I-LDL (10 micrograms/ml) was added to the EC medium at various time intervals (3, 24, and 48 hr and 9 days). The amount of lipid reaching the subendothelial space was measured. Measurements of EC and SMC binding and uptake of LDL were also obtained and compared with those of untreated cells. The results demonstrate a significant increase in the permeability of the treated EC layer to LDL (P less than 10(-6)), which was sustained over time. Superoxide exposure led to a limited initial increase in EC binding and uptake of LDL, which later returned to control values. In contrast, SMC uptake of LDL was significantly (P less than 10(-3)) and persistently increased over control values and disproportionately increased over cellular binding. Such results suggest that superoxides can increase LDL permeability of the EC barrier. Because this was not associated with a comparable increase in EC binding and uptake, it is unlikely to be due to changes in receptor-mediated, transcellular transport.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of thrombin on low-density lipoprotein permeability and uptake by an arterial endothelial smooth muscle cell bilayer

Thrombin, a mediator of thrombosis, has been shown to directly alter the function of vascular cells. We studied the effect of thrombin on low-density lipoprotein permeability and uptake by an arterial endothelial cell-smooth muscle cell bilayer to determine its potential role in atherogenesis. Confluent cell bilayers were incubated in media containing thrombin (10 or 50 units/ml) for a period of 24 hours to 9 days. Iodine 125 (125I)-LDL (10 micrograms protein/ml) was then added to the media, and after a 3-hour incubation, 125I-LDL transit through the endothelial cell layer as well as membrane binding and uptake by each cell type were measured. The lower concentration of thrombin caused a delayed increase in both the permeability (p less than 0.0001) and uptake (p less than 0.05) of LDL, but had no effect on membrane binding of the lipoprotein. The higher thrombin concentration led to an immediate increase in endothelial cell permeability to LDL (p less than 10(-7)) and a significant reduction in both cellular uptake (p less than 10(-7)) and membrane binding (p less than 0.0005). In contrast, smooth muscle cell binding and uptake were unaffected at the lower concentration of thrombin. At the higher concentration, smooth muscle cell uptake of LDL was increased (p less than 10(-7)) disproportionately to a more limited increase in membrane binding (p less than 0.05). Endothelial DNA content, reflecting cell number, was increased at 10 units/ml thrombin (p less than 0.001) but markedly reduced at 50 units/ml thrombin (p less than 0.0005), whereas smooth muscle cell DNA content remained unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)

High density lipoprotein inhibits low density lipoprotein binding and uptake by bovine aortic endothelial cells

The antiatherogenic effect of high density lipoprotein (HDL) has been attrib uted to either an inhibition of cholesterol uptake or to reversed cholesterol transport from peripheral cells. In order to determine whether HDL competi tively blocks receptor-mediated low density lipoprotein (LDL) binding and up take, bovine aortic enothelial cells (BAECs) were cultured in Dulbeccos modified Eagles Medium (DMEM) containing 10% LDL-free fetal bovine serum, and incubated with 125I-LDL in concentrations of either 10 or 25 μg protein/mL. Varying amounts of HDL (0-200 μg/mL) were added to the media. Following a twenty-four hour incubation period at 37°C, 125I-LDL binding and uptake were measured. At the lower concentration of 125I-LDL, which represents high-affinity recep tor binding, there was no significant difference in either binding or uptake within the range of HDL concentrations studied. At the higher concentration of LDL, however, there was a marked inhibition of 125I-LDL binding (p<.006) and uptake (p<,001; ANOVA), which did not saturate at the highest HDL concentrations used. These data suggest that HDL does not influence high-affinity, receptor-me diated binding and uptake of LDL but that its effect is seen at a concentration of LDL representing nonspecific binding. The lack of saturation at increasing concentrations of HDL also indicates that HDL-receptor interaction is not essen tial for the effect.