Reversible and irreversible non-internalized LDL and methyl LDL accumulation by human fibroblasts

In previous in vivo animal studies, we showed that low density lipoprotein (LDL) accumulated irreversibly at the edges of healing arterial lesions rather than being internalized and degraded. To see if similar LDL accumulation occurs in vitro, fibroblasts from normal and homozygous familial hypercholesterolemic (FH) subjects were incubated at 37 degrees C with 125I-LDL and 125I-methyl LDL; the latter is not recognized by any known LDL receptor. Normal fibroblast accumulation of LDL and methyl LDL (5 microg/ml) plateaued within 1 h at 200 and 100 ng/mg, respectively. With FH cells, both LDL and methyl LDL accumulation plateaued at 100 ng/mg. Lipoprotein accumulation by both cell types rose steeply at concentrations up to 15-25 microg/ml, and less so at higher concentrations. Except for degradation of LDL by normal cells, degradation was minimal, which indicated that much of the lipoprotein accumulation was unaccompanied by internalization. The accumulation of both lipoproteins by both cell types was greater at 37 degrees C than at 4 degrees C, and was inhibited between 43 and 75% by homologous unlabeled lipoprotein. To see if any accumulation was irreversible, cells were incubated with radiolabeled lipoproteins for 3 h (pulse), then with homologous unlabeled lipoproteins for up to 20 h (chase). About 50% of intact radiolabeled lipoprotein rapidly dissociated from cells into the medium in the first 4 h of the chase period. In contrast, between 4 and 20 h, most of the remaining intact LDL and methyl LDL appeared to be irreversibly bound, because it was released at a rate of only 0-1%/h. Thus, we conclude that, under the conditions studied, both reversible and irreversible non-internalized LDL binding play a major role in LDL accumulation by cultured cells.