Objectives— Apolipoprotein C-I (apoC-I) influences lipoprotein metabolism, but little is known about its cellular effects in aortic smooth muscle cells (ASMC). Methods and Results— In cultured human ASMC, apoC-I and immunoaffinity purified apoC-I–enriched high-density lipoproteins (HDL) markedly induced apoptosis (5- to 25-fold), compared with control cells, apoC-I–poor HDL, and apolipoprotein C-III (apoC-III) as determined by 4′, 6-diamidino-2-phenylindole dihydrochloride staining and DNA ladder assay. Preincubation of cells with GW4869, an inhibitor of neutral sphingomyelinase (N-SMase), blocked apoC-I–induced apoptosis, an effect that was bypassed by C-2 ceramide. The activity of N-SMase was increased 2- to 3-fold in ASMC by apoC-I, apoC-I–enriched HDL, and tumor necrosis factor α (TNF-α) (positive control) after 10 minutes and then decreased over 60 minutes, which is a kinetic pattern not seen with controls, apoC-III, and apoC-I–poor HDL. ApoC-I and apoC-I–enriched HDL stimulated the generation of ceramide, the release of cytochrome c from mitochondria, and activation of caspase-3 greater than that found in controls, apoC-III, and apoC-I–poor HDL. GW4869 inhibited apoC-I–induced production of ceramide and cytochrome c release. Conclusions— ApoC-I and apoC-I–enriched HDL activate the N-SMase-ceramide signaling pathway, leading to apoptosis in human ASMC, which is an effect that may promote plaque rupture in vivo.
Isolated pig liver membranes contain a specific "lipoprotein binding site" that recognizes low-density lipoproteins (LDL) and apolipoprotein E (apoE) free high-density lipoprotein (HDL) [Bachorik, P. S., Kwiterovich, P. O., & Cooke, J. (1978) Biochemistry 17, 5287-5299]. We report here that a similar site exists in cultured porcine hepatocytes and that it mediates the uptake and degradation of apoE-free HDL. The binding of 125I-labeled HDL and 125I-labeled LDL (125I-HDL and 125I-LDL, respectively) at 4 degrees C and the uptake and degradation of the lipoproteins at 37 degrees C were time dependent and saturable and were not inhibited by unrelated proteins. Chloroquine (6 x 10(-5)M) inhibited the degradation of 125I-HDL by 76% and of 125I-LDL by greater than 99%; leupeptin inhibited the degradation of both lipoproteins by about 25%. 125I-HDL binding (4 degrees C), uptake, and degradation (37 degrees C) were inhibited by LDL, methyl-LDL, and methyl-HDL about as well as by unlabeled HDL but were unaltered in Pronase-treated cells or in cells that were cultured for 24 h in either lipoprotein-free medium containing HDL or LDL (200 micrograms/mL). In contrast, these conditions affected the uptake and degradation of 125I-LDL disproportionately. HDL and methyl-LDL inhibited 125I-LDL uptake by 50% or more but had little effect on degradation. 125I-LDL binding was reduced by 12% and degradation by 57% in Pronase-treated cells. Preincubation of the cells with LDL (200 micrograms/mL) reduced uptake by 35% and degradation by 68%. Similar preincubation with HDL (200 micrograms/mL) increased 125I-LDL degradation by 60% but did not affect 125I-LDL uptake. The findings indicated the presence in porcine hepatocytes of at least two distinct sites for lipoproteins. One site resembled the LDL receptor and mediated 125I-LDL degradation. A second, Pronase-insensitive site recognized both HDL and LDL. This site mediated almost all of the degradation of 125I-HDL but little if any degradation of 125I-LDL.
The mutation of serine128 to arginine in the CD 62E gene is a risk factor for coronary artery disease (CAD). We designed a new method to detect this mutation based on the observation that it is due to a transversion of nucleotide A561 to C, which abolishes a PstI recognition site. Two alleles, A and C, are easily typed when genomic DNA is amplified by PCR, digested with PstI, and separated on agarose gels. Among 153 people who underwent an elective, diagnostic arteriography in Johns Hopkins Hospital, we found that the C allele accounts for 19.5% in angiographically documented CAD patients (n = 82). It is significantly higher than the 10.6% frequency observed in normal controls (n = 71, p < 0.05). It indicates that the C allele is associated with early-onset CAD. This new method should facilitate the screening of this mutant allele in large populations and contribute to the understanding of the molecular mechanism underlying the association of this mutation with CAD.
Twelve obligate heterozygotes from two kindreds were ascertained through phytosterolemic probands homozygous for molecular defects in the ATP binding cassette (ABC) half transporter, ABCG8. The response of these heterozygotes to a Step 1 diet low in fat, saturated fat, and cholesterol, and to 2.2 g daily of plant sterols (as esters) was determined in Protocol I (16 weeks) and Protocol II (28 weeks) during three consecutive feeding periods:Step 1/placebo spread; Step 1/plant sterol spread; and Step 1/placebo spread (washout). At baseline, half the heterozygotes had moderate dyslipidemia and one-third had mildly elevated campesterol and sitosterol levels. On the Step 1/placebo spread, mean LDL cholesterol decreased significantly, 11.2% in Protocol I (n ؍ 12), and 16.0% in Protocol II (n ؍ 7). Substitution with plant sterol spread produced a significant treatment effect on LDL levels in Protocols I and II. Conversely, the mean levels of campesterol and sitosterol increased 119% and 54%, respectively, during the use of plant sterol spread for 6 weeks in Protocol I, an effect mirrored for 12 weeks in Protocol II. During the placebo spread washouts, LDL levels increased, while those of plant sterols decreased to baseline levels in both protocols. In conclusion, phytosterolemic heterozygotes respond well to a Step 1 diet, and their response to a plant sterol ester challenge appears similar to that observed in normals. Studies in humans with inherited disorders of cholesterol metabolism have provided unique and important insights into the mechanisms underlying both hypercholesterolemia and premature atherosclersosis (1). This has led to a more precise understanding of the effects of both dietary restriction of cholesterol and of pharmacological agents on reducing plasma cholesterol levels and preventing cardiovascular disease (1-4). For example, both dietary and drug treatments that lower the pool of cholesterol in the liver lead to an up-regulation of LDL receptors and to a decrease of plasma LDL cholesterol (1).The effect of dietary cholesterol on plasma cholesterol levels is modest, due in part to the incomplete absorption of cholesterol by the intestine (5, 6). For example, Bosner et al. (7) found that the mean (1 SD) cholesterol absorption in humans was 56.2 (12.1)% with a range from 29.0% to 80.1%. This protective barrier in the normal human intestine is even more efficient for dietary plant sterols, such as sitosterol and campesterol, with estimates for sitosterol absorption ranging from 0.6% to 7.5%, and for campesterol from 5.5% to 16% (6). Ostlund and coworkers (8), using serum to establish unequivocal absorption into the systemic circulation and mass spectrometry for definitive identification of labeled tracers, recently reported lower estimates of absorption, namely, 0.5% for sitosterol and 1.9% for campesterol.The plant sterols are structurally very similar to cholesterol, except that they always contain substitutions at the C24 position, and the mechanism that selectively prevents most of thei...
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