Using a macrophage cell line that constitutively expresses a human apolipoprotein E (apoE) cDNA, we have investigated the post-translational metabolism of endogenously produced apoE. Inhibition of lysosomal or cysteine proteases led to significant inhibition of apoE degradation but did not increase apoE secretion, indicating that cellular degradation is not limiting for apoE secretion in macrophages. Treatment of macrophages with inhibitors of proteoglycan synthesis (4-methylumbelliferyl-beta-D-xyloside) or sulfation (sodium chlorate) enhanced the release of apoE from cells and significantly attenuated the increase in secretion produced by incubation with phosphatidylcholine vesicles (PV). These observations suggested that a significant fraction of the apoE retained by cells (and released by incubation with PV) was associated with proteoglycans. Treatment of cells with exogenous heparinase led to a greater than 4-fold increase in apoE secretion and similarly attenuated the response to PV, suggesting that apoE was trapped in an extracellular proteoglycan matrix. This conclusion was confirmed in studies showing that PV could enhance the release of apoE from cells during an incubation at 4 degrees C, but this enhanced release was abolished in proteoglycan-depleted cells. Incubation with lactoferrin at 4 or 37 degrees C produced a similar decrement in cellular apoE, again indicating the existence of a cell surface pool of apoE. Pulse-chase studies showed that the apoE trapped in the proteoglycan matrix was susceptible to rapid cellular degradation such that net synthesis of apoE (secreted plus cell-associated) was increased significantly in proteoglycan-depleted cells compared with control cells as early as 45 min during a chase period.

Metaphase karyotype analysis of fetal cells obtained by amniocentesis or chorionic villus sampling is the current standard for prenatal cytogenetic diagnosis, particularly for the detection of trisomy 21. We previously demonstrated that large quantities of cell-free fetal DNA (cffDNA) are easily extracted from amniotic fluid (AF). In this study, we explored potential clinical applications of AF cffDNA by testing its ability to hybridize to DNA microarrays for comparative genomic hybridization (CGH) analysis. cffDNA isolated from 11 male fetuses showed significantly increased hybridization signals on SRY and decreased signals on X-chromosome markers, compared with female reference DNA. cffDNA isolated from six female fetuses showed the reverse when compared with male reference DNA. cffDNA from three fetuses with trisomy 21 had increased hybridization signals on the majority of the chromosome 21 markers, and cffDNA from a fetus with monosomy X (Turner syndrome) had decreased hybridization signals on most X-chromosome markers, compared with euploid female reference DNA. These results indicate that cffDNA extracted from AF can be analyzed using CGH microarrays to correctly identify fetal sex and aneuploidy. This technology facilitates rapid screening of samples for whole-chromosome changes and may augment standard karyotyping techniques by providing additional molecular information.

Macrophages are a significant source of lipoprotein lipase (LPL) and apolipoprotein E (apo E) in the developing arterial wall lesion, and each of these proteins can importantly modulate lipid and lipoprotein metabolism by arterial wall cells. LPL and apo E share a number of cell surface binding sites, including proteoglycans, and we have previously shown that proteoglycans are important for modulating net secretion of apoprotein E from macrophages. We therefore evaluated a potential role for LPL in modulating net secretion of macrophagederived apo E. In pulse-chase experiments, addition of LPL during the chase period produced a decrease in secretion of apoprotein E from human monocyte-derived macrophages, from the human monocytic THP1 cell line, and from J774 cells transfected to constitutively express a human apo E cDNA. LPL similarly reduced apo E secretion when it was prebound to the macrophage cell surface at 4°C. A native LPL particle was required to modulate apo E secretion; addition of monomers and aggregates did not produce the same effect. Depletion of cell surface proteoglycans by a 72-h incubation in 4-methylumbelliferyl-␤-D-xyloside did not attenuate the ability of LPL to reduce apo E secretion. However, addition of receptor-associated protein attenuated the effect of LPL on apo E secretion. Although LPL could mediate removal of exogenously added apo E from the culture medium, detailed pulse-chase analysis suggested that it primarily prevented release of newly synthesized apo E from the cell layer. Cholesterol loading of cells or antibodies to the low density lipoprotein receptor attenuated LPL effects on apo E secretion. We postulate that LPL sequesters endogenously synthesized apo E at the cell surface by a low density lipoprotein receptor-dependent mechanism. Such post-translational regulation of macrophage apo E secretion by LPL could significantly influence apo E accumulation in arterial vessel wall lesions.Macrophages synthesize and secrete both apo E 1 and LPL (1, 2). These cells, therefore, can be a significant source of each of these proteins in the arterial wall. Both proteins have important roles in systemic lipoprotein metabolism (3, 4) as well as in lipoprotein metabolism at the level of the individual cell (5-14). Regional accumulation of each of these proteins has been detected at sites of atherosclerotic lesion development (15); apo E has been found predominantly on the surface of macrophages and in the matrix surrounding macrophages in atherosclerotic lesions, whereas LPL is also associated with arterial smooth muscle cells. apo E expression in the vessel wall has been shown to be important for modulating vessel wall cholesterol homeostasis (16,17). In fact, macrophage-specific expression of apo E in apo E-null mice has been shown to protect against atherosclerotic lesion development even in the presence of high levels of circulating atherogenic lipoproteins (17). As noted above, LPL also accumulates in diseased vessel wall. A number of cell surface binding sites for LPL have been id...

We have previously shown that expression of a human apoE cDNA in J774 macrophages enhances cholesterol efflux to HDL 3 . We have also shown that endogenous apoE expression produces a cell surface pool of apoE associated with proteoglycans. In this series of experiments, we first demonstrate the presence of a cell surface proteoglycan-associated apoE pool in human monocyte-derived macrophages. We then examine the hypothesis that endogenous expression of apoE modulates HDL 3 binding to macrophages, thereby, accounting for enhanced cholesterol efflux to HDL 3 , specifically examining a role for the cell surface pool. Enhanced binding of apoE-free human HDL 3 to apoE-expressing macrophages, compared to non-expressing macrophages, was observed at 37 Њ C and 4 Њ C. The enhanced binding was not due to apoE secreted into the medium, as determined by experiments utilizing conditioned medium from apoE-secreting cells. Removal of the cell surface pool of apoE, however, substantially reduced the incremental HDL 3 binding produced by apoE expression. Cellular cholesterol mass measurements demonstrated that experimental conditions that reduced HDL 3 binding to apoE-expressing macrophages, did not substantially reduce cholesterol efflux to HDL 3 .In summary, our results document a clear role for cell surface pool of apoE in modulating HDL 3 interaction with macrophages. The enhanced binding, however, does not appear to be a major mechanism contributing to the increased cholesterol efflux to HDL 3 , which results from endogenous macrophage expression of apoE.-Lin, C-Y., M. Lucas, and T. Mazzone. Endogenous apoE expression modulates HDL 3 binding to macrophages.

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