Donna G. Virgil

Apolipoprotein C-I induces apoptosis in human aortic smooth muscle cells via recruiting neutral sphingomyelinase

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 &agr; (TNF-&agr;) (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.

Reversible high affinity uptake of apo E-free high density lipoproteins in cultured pig hepatocytes

We examined the high affinity binding, uptake, and degradation of apo E-free 125I-high density Iipoprotein (HDL) in cultured pig hepatocytes. At steady state, the cells degraded 9.4% of cell-associated 125I-HDL/hour, compared with 41.7%/hour for 125I-LDL. Pulse-chase experiments at 4° C revealed that high affinity 125I-HDL binding was reversible. Similar experiments at 37° C revealed that about 70% of the cell-associated 125I-HDL was released as a macromolecule; the remainder was degraded to acidsoluble products. In contrast, over 75% of the 125I-LDL that was released had been degraded to acid soluble products. The amount of macromolecular 125I-HDL released at 37° C was similar to the amount that was bound to the cell surface, as estimated from measurements of trypsin-releasable radioactivity. Density gradient ultracentrifugation and SDS-polyacrylamide gel electrophoretic analysis of macromolecular 125I-HDL released to the medium revealed an increase in density, and the apparent partial proteoiysis of apo A-l (Mr 25,000) to products of apparent Mr 12,000–14,000. The findings suggest that high affinity 125I-HDL uptake had a reversible component in which HDL was concentrated temporarily at the cell surface, modified, and then released as a somewhat denser Iipoprotein particle. Measurement of 125I-HDL and 125I-LDL degradation in cell homogenates revealed no difference in the inherent susceptibility of the two lipoproteins to proteoiysis by lysosomai enzymes. The overall slower rate of degradation of 125I-HDL compared to 125I-LDL was therefore due in part to the smaller fraction of HDL that was committed to irreversible catabolism. The rate of catabolism of this fraction, however, was considerable. Cells pulsed at 4° C and subsequently warmed to 37° C released one-half the acid-soluble products from 125I-HDL within about 4 hours, compared with 2 hours for cells pulsed with 125I-LDL. These findings indicate that HDL was internalized, transported to lysosomes, and degraded at about one-half the rate of LDL.

Enzymatic analysis of total- and HDL-cholesterol: Comparison with the standardized Liebermann-Burchard method used by the lipid research clinics program

We compared two enzymatic cholesterol methods with the standardized chemical method used in the Lipid Research Clinics (LRC) program. The methods were used to measure total cholesterol and high density lipoprotein (HDL) cholesterol in heparin-MnCl2 supernatants of 1,812 sera collected over a 16-mth period from subjects who were sampled as part of the Hispanic Health and Nutrition Examination Survey. Thirty percent of the subjects had fasted for 12 h or more before venepuncture. The enzymatic total cholesterol values were 1.4-1.8% lower than the LRC method and both enzymatic methods correlated highly with the LRC method (r greater than 0.97). The enzymatic HDL cholesterol values were 2.4 and 6.4% higher than the LRC method, and the correlation between the enzymatic and LRC methods was greater than 0.93. The differences between the enzymatic and LRC methods were the same in samples from fasting and non-fasting subjects.

Interrelationships between the concentration and size of the largest high-density lipoprotein subfraction and apolipoprotein C-I in infants at birth and follow-up at 2–3 months of age and their parents

BACKGROUND Lipoprotein subfractions in infants may predict the risk of cardiovascular disease factors in children. OBJECTIVE To examine the relationships between lipid and nonlipid factors and lipoprotein subfractions in infants at birth and follow-up (FU) and in their parents. METHODS Prospective study in a community-based hospital of 103 families ascertained through a pregnant mother at 36 weeks gestation or older. Of 103 infants studied at birth, 85 were sampled at FU at 2-3 months of age, along with 76 fathers. Lipids, lipoproteins, and their subclasses were determined by nuclear magnetic resonance spectroscopy. Correlations of lipid-related parameters were calculated using Spearman rank correlations. RESULTS Female gender in infants and use of formula only were the only nonlipid variables associated with lipoprotein subfractions. LDL parameters were significantly correlated between infants at birth and FU. The largest high-density lipoprotein subfraction, H5C, was the only lipid variable significantly associated between mothers and infants at birth. Paternal low-density lipoprotein size was significantly correlated with that of infants at FU but not at birth. In each of the four groups, markedly inverse interrelationships were found between H5C and small LDL particles. At birth and at FU, apoC-I was strongly related with H5C but not TG. Conversely, apoC-I in the parents was strongly related with TG but not H5C. CONCLUSION Significant relationships were found between lipoprotein subfractions within infants at birth and FU and their parents. ApoC-I and H5C levels very early in life may affect the development of dyslipidemia and obesity in childhood.

Abnormal Protein Tyrosine Phosphorylation in Fibroblasts from Hyperapobetalipoproteinemia Subjects

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.