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.

An increased expression of E‐selectin has been observed in the arterial endothelium interacting with lymphocytes and macrophages in human atherosclerotic lesions. We examined whether a polymorphism in the E‐selectin gene, due to a G to T mutation (G98T) in the untranslated region of exon 2, was associated with premature coronary artery disease (CAD). Other lipid and nonlipid risk factors including a Ser to Arg (S128R) substitution in the E‐selectin gene were also assessed. In patients with premature CAD (men ≤45 years old and women ≤55 years old, N=51) who underwent an elective diagnostic coronary arteriography, the frequency of the mutation was significantly higher than in controls (N=50, 0.22 vs. 0.10, p=0.024). After controlling for other CAD risk factors (plasma total cholesterol, triglyceride, LDL‐apolipoprotein B, cigarette smoking and the S128R mutation) by multiple logistic analysis, the G98T mutation in the E‐selectin gene was still a significant predictor of premature CAD [p=0.022, odds ratio (95%, CI)=3.58 (1.20–10.67)].

The use of serial analysis of gene expression (SAGE) to determine gene expression profiles is increasing because the technique can provide absolute transcript numbers in a digital format and identify new genes. We developed a miniSAGE technique, which uses only 1 μg total RNA and reduces the amount of the starting material by 250- to 500-fold. Unlike the other modified SAGE methods, the miniSAGE technique does not require the additional PCR amplifications. The additional PCR amplifications potentially introduce bias and compromise the quantitative aspects of the SAGE method. Three key modifications in the miniSAGE technique are: (i) using the phase lock gel (PLG, Eppendorf) to increase the recovery and the purity of DNA material after each phenol extraction step; (ii) reducing the amount of linkers in the ligation, thereby minimizing their interference with SAGE ditag amplification and increasing the SAGE ditag yield; and (iii) employing the mRNA capture kit (Boehringer Mannheim) to allow the first five steps: mRNA isolation, cDNA synthesis, enzyme cleavage of cDNA, binding of the cleaved biotin–cDNA to the streptavidin-magnetic beads, ligating linkers to the bound cDNA, and the release of cDNA tags to occur within one tube to significantly reduce the loss of material between successive steps. Two fibroblast SAGE libraries have been successfully prepared. The preliminary analysis of 3838 tags from one library demonstrated a typical fibroblast gene expression pattern. This miniSAGE technique will permit a broader application of SAGE.

The stimulatory effects of three normal human serum basic proteins (BP), BP I (M r 14,000, pI 9.10), BP II (M r 27, 500, pI 8.48), and BP III (M r 55,000, pI 8.73) on cellular triglyceride and cholesterol formation require intact protein-tyrosine kinase phosphorylation (TKP). Here we examined whether there is an abnormality in TKP in cultured fibroblasts from 11 patients with hyperapobetalipoproteinemia (hyperapoB) that manifest two acylation-stimulatory defects, decreased stimulation of triglyceride synthesis by BP I but enhanced formation of cholesterol by BP II. Soluble and insoluble proteins in Triton X-100 extracts were isolated by immunoprecipitation with a monoclonal anti-phosphotyrosine antibody (MAPA) bound to agarose beads and by ultracentrifugation, respectively, from confluent fibroblasts after incubation for 24 h in supplemented serum-free and lipid-free medium (DMEM/F12). Western blots of insoluble proteins showed that group (Gp) II (M r 36,000–55,000) and Gp III (M r 14,000–35,000) from hyperapoB cells, grown in DMEM/F12 medium without BP, had significantly decreased reactivity to MAPA. No significant differences in reactivity to MAPA were detected between normal and hyperapoB cells for Gp I (M r97–120,000). BP II, but not BP I or BP III, reversed the decreased reactivity of Gp II and Gp III to MAPA in hyperapoB cells. Sodium vanadate, an inhibitor of phosphotyrosine phosphatases, did not reverse the deficiency in TKP or the 50% deficiency in the stimulation of mass triglyceride by BP I in hyperapoB cells. Tyrosine-phosphorylated Erk-2, a mitogen-activated protein kinase, identified as one of the proteins in Gp II, was significantly decreased in hyperapoB cells. These results provide further evidence for abnormal protein TKP in hyperapoB cells and suggest a possible link between atherosclerotic changes in hyperapoB patients and growth factors upstream from mitogen-activated protein kinase.

Hyperapobetalipoproteinemia (hyperapoB), a familial lipoprotein disorder characterized by an increase in small, dense, low-density lipoprotein (LDL) particles, is strongly associated with coronary artery disease. There are two metabolic defects in hyperapoB: an increased synthesis of a very-low-density lipoprotein in liver, resulting in an overproduction of LDL, and a delayed clearance of post-prandial triglyceride and free fatty acids. To date, defects in the apolipoprotein B gene do not appear to explain the hyperapoB phenotype. Defect(s) in the uptake or intracellular metabolism of free fatty acids have been found in cells from hyperapoB patients. Three basic proteins (BPs)--BP I (Mr 14,000, pI 9.10), BP II (Mr 27,500, pI 8.48), and BP III (Mr 55,000, pI 8.73)--were isolated from normal human serum. Compared with normal fibroblasts, cultured hyperapoB fibroblasts incubated with BP I, which appears to be the same protein as acylation-stimulating protein (ASP), showed 50% less stimulation of triglyceride acylation and cholesterol esterification, whereas BP II markedly stimulated cholesteryl ester formation, and BP III caused no difference in response vs normal fibroblasts. However, in cultured normal human monocyte macrophages, BP III, but not BP I or BP II, stimulated cholesteryl esterification two- to threefold. BP I, BP II, and BP III may provide new insights into normal metabolism of lipids, lipoproteins, and free fatty acids and the pathophysiology of hyperapoB.