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We have recently reported that the human apolipoprotein A-I (apoA-I) and apolipoprotein C-III (apoC-III) genes are physically linked and that the presence of a DNA insertion in the apoA-I gene is correlated with apoA-I-apoC-III deficiency in patients with premature atherosclerosis. In addition, the presence of a polymorphic restriction endonuclease site (SacI) in the 3' noncoding region of apoC-III mRNA has been correlated with hypertriglyceridemia in humans. In this study, we report the isolation and characterization of cDNA clones containing the entire apoC-III mRNA coding sequence. The nucleotide-derived apoC-III amino acid sequence indicates that the apoC-III primary translational product contains a 20 amino acid N-terminal extension, which conforms with the general properties of known signal peptides, and is highly homologous to the recently reported rat apoC-III signal peptide. The DNA-derived apoC-III amino acid sequence differs from the previously reported apoC-III amino acid sequence at four amino acid residues. More specifically, at positions +32, +33, +37, +39, the DNA sequence predicts Glu, Ser, Gln, Ala, respectively, while the previously reported sequence specifies Ser, Gln, Ala, Gln, respectively. Finally, isolation and characterization of apoC-III cDNA clones, with or without the polymorphic SacI restriction site, indicated that the apoC-III nucleotide sequence corresponding to the Sac+ and Sac- clones differs at three nucleotide sites; however, the amino acid sequence specified by the Sac+ and Sac- alleles is identical.

ApoB is a large glycoprotein with an apparent molecular mass of 550 kDa on NaDodSO4/PAGE. It is a major constituent of most lipoproteins and plays an important role in their metabolism. Recently, apoB cDNA clones have been isolated from an expression library made with mRNA from a human hepatoma cell line. These clones, which were all 1.5-1.6 kilobases (kb) long and corresponded to the 3' end of apoB mRNA, were used to demonstrate that hepatic apoB mRNA is approximately 22 kb long. In the current report, a probe derived from one of these cDNA clones, pB8, was used for in situ hybridization experiments to map the human gene for apoB, APOB, to the distal half of the short arm of chromosome 2. This probe was also used to analyze somatic cell hybrids and, in agreement with the in situ hybridization studies, concordancy was demonstrated with chromosome 2. In addition, two hybrids with chromosome 2 translocations that contain only the short arm reacted with the pB8 probe. A third hybrid with a complex rearrangement of chromosome 2, which deleted an interstitial region and the tip of the short arm of chromosome 2, did not react. These data indicate that APOB maps to either 2p21-p23 or 2p24-pter. In further studies, DNA from normal individuals, digested with the restriction endonuclease EcoRI and subjected to Southern blot analysis with the pB8 probe, revealed a two-allele restriction fragment length polymorphism (RFLP). The major allele was 11 kb, and the minor allele was 13 kb. The minor allele was present with a frequency of 20-25%. The inheritance of the two alleles was studied in an informative family, and they segregated in a typical autosomal Mendelian fashion. The mapping studies provide the means for understanding the relationship of the APOB locus to others in the human genome, whereas the demonstration of an APOB RFLP increases our ability to assess the role of this locus in determining plasma lipoprotein levels.

Apolipoprotein C-III (apo C-III) is a 79 amino acid glycoprotein. The sugar moiety of apoC-III is attached to amino acid residue 74 and is thought to consist of 1 mole of galactose, 1 mole of N-acetyl-galactosamine, and either 0, 1, or 2 moles of sialic acid. This results in three isoproteins called C-III0, C-III1, and C-III2 designated by the number of sialic acid residues. It has been assumed, although not experimentally tested, that apoC-III0 lacks sialic acid residues but possesses the D-galactosyl-(1-3)-N-acetyl-D-galactosamine sugar backbone. To verify the structure of the three apoC-III isoproteins, we applied the method of 252Cf plasma desorption mass spectrometry to measure the exact molecular weight (Mr) of each of the isoproteins. Our data confirmed the proposed structure of apoC-III1 and apoC-III2. However, the difference in mass between apoC-III1 (9420.6, 9420.0, 9422.2 daltons) and apoC-III0 (8763.9, 8764.9, 8765.5 daltons, respectively, in three subjects) suggests that the latter is missing not just sialic acid but the entire sugar moiety. This finding may have important implications for the metabolism of apoC-III. The accuracy and reproducibility of Mr measurements described in this paper suggest that this technique holds promise for the detection of apolipoprotein amino acid substitutions or modifications undetected by conventional techniques such as isoelectric focusing.

Human diploid skin fibroblasts derived from normal individuals and those with the testicular feminization syndrome (TFM) have been shown to be killed to the same degree by dihydrotestosterone in spite of the absence of high affinity cellular androgen receptors in the TFM fibroblasts. Furthermore, several different normal fibroblast strains from various anatomical sites all showed similar amounts of androgen-induced cytotoxicity even though their respective receptor contents differed by as much as ten-fold. These results suggest that steroid-induced cytotoxicity in human fibroblasts is not correlated with receptor content, unlike murine lymphoid cells in which the receptor content has been shown to be closely related to their ability to survive hormone exposure.

This chapter outlined the methods used to study intra- and extracellular modifications of apolipoproteins. These and other related studies have shown that several of the apolipoproteins undergo a series of intra- and extracellular modifications as follows: All apolipoproteins studied contain an 18-26 long signal peptide which is cleaved cotranslationally by the signal peptidase of the rough endoplasmic reticulum. ApoE is further modified intracellularly with carbohydrate chains containing sialic acid and is secreted in the modified form designated apoEs. The modified apoE is subsequently desialated in plasma. ApoA-I is secreted in a proapoA-I form, which consists of 249 amino acids. The N-terminal hexapeptide of proapoA-I is cleaved extracellularly by a proapoA-I to plasma apoA-I converting protease. This cleavage generates the plasma apoA-I form which consists of 243 amino acids. Other known apolipoprotein modifications include the modification of apoB, apoC-III, and apoD with carbohydrate chains that contain sialic acid and the proteolytic cleavage of the proapoA-II segment. At the present time we are able to distinguish several isoprotein forms for a particular apolipoprotein. In addition, we began to understand the biochemical changes which lead to a few of these isoproteins. Future research should be directed toward a better understanding not only of the structure but most importantly of the physiological significance of the different apolipoprotein forms.

Young male Sprague-Dawley rats fed a high cholesterol, thyroid-suppressive diet were subjected to drying injury of carotid artery endothelium; animals were sacrificed at various times up to 3 months after injury, and the vessels were examined by light, scanning, and transmission electron microscopy. The diet induced marked elevation of serum cholesterol mainly present in lipoproteins of density less than 1.063. The morphology and degree of intimal thickening in the injured carotids of such animals were compared with the changes found in control groups of normolipemic rats. In the control groups, endothelium was completely regenerated between 7 and 14 days; intimal thickening was present at 14 days and at later stages and contained smooth muscle cells without lipid. In the cholesterol-fed animals, endothelial regeneration and intimal thickening occurred as in the controls with the following additional features: in the zone of intimal thickening in the injured segment, lipid was present in smooth muscle cells and, at later stages, in the extracellular matrix; undifferentiated mononuclear cells were also noted in the thickened intima and, at 3 months, were found adhering to normal and regenerated endothelium. However, no differences were found between control and hypercholesterolemic rats with respect to the degree of intimal thickening within the injured segment; enhancement of the smooth muscle proliferative response was not evident in the hypercholesterolemic rats. Our findings suggest that this form of hypercholesterolemia and its associated hyperlipoproteinemia may not be directly responsible for rat smooth muscle proliferation following endothelial denudation. They also indicate that hyperlipemia does not necessarily cause persistence of myointimal hyperplasia in arteries.

Five lines of transgenic mice, which had integrated the human apolipoprotein (apo) A-I gene and various amounts of flanking sequences, were established. Normally, apoA-I is expressed mainly in liver and intestine, but all of the transgenic lines only expressed apoA-I mRNA in liver, strongly suggesting that 256 base pairs of 5'-flanking sequence was sufficient for liver apoA-I gene expression but that 5.5 kilobase pairs was not sufficient for intestinal expression. Mean plasma levels of human apoA-I varied in different lines from approximately 0.1 to 200% of normal mouse levels. This was not dependent on the amount of flanking sequence. Lipoprotein levels were studied in detail in one of the lines with a significantly increased apoA-I pool size. In one study, the total plasma apoA-I level (mouse plus human) was 381 +/- 43 mg/dl in six animals from this line, compared to 153 +/- 17 mg/dl in matched controls. Total and high density lipoprotein cholesterol (HDL-C) levels were increased 60% in transgenic animals, compared to controls (total cholesterol: 125 +/- 12 versus 78 +/- 13 mg/dl, p = 0.0001; HDL-C 90 +/- 7 versus 55 +/- 11 mg/dl, p = 0.0001). The molar ratio of HDL-C/apoA-I was significantly lower in transgenic animals, 17 +/- 1 versus 25 +/- 2 (p = 0.0001), suggesting the increase was in smaller HDL particles. This was confirmed by native gradient gel electrophoresis. This was not due to aberrant metabolism of human apoA-I in the mouse, since human apoA-I was distributed throughout the HDL particle size range and was catabolized at the same rate as mouse apoA-I. In another study of 23 transgenic mice, HDL-C and human apoA-I levels were highly correlated (r = 0.87, p less than 0.001). The slope of the correlation line also indicated the additional HDL particles were in the smaller size range. We conclude that human apoA-I can be incorporated into mouse HDL, and excessive amounts increase HDL-C levels primarily by increasing smaller HDL particles, comparable to human HDL3 (HDL-C/apoA-I molar ratio = 18).

Endothelial cells play a critical role in thromboregulation by virtue of a surface-connected fibrinolytic system. Cultured endothelial cells synthesize and secrete tissue-type plasminogen activator (t-PA) which can bind to at least two discrete sites on the cell surface. These binding sites preserve the catalytic activity of t-PA and protect it from its physiological inhibitor (PAI-1). N-terminal glutamic acid plasminogen (Glu-PLG), the main circulating fibrinolytic zymogen, also interacts specifically with the endothelial cell surface. Binding is associated with a 12-fold increase in catalytic efficiency of plasmin generation by t-PA which may reflect conversion of Glu-PLG to its plasmin-modified form, N-terminal lysine plasminogen (Lys-PLG). Lipoprotein(a) is an atherogenic lipoprotein particle which contains the plasminogen-like apolipoprotein(a) bound to low density lipoprotein. We report here that lipoprotein(a) interferes with endothelial cell fibrinolysis by inhibiting plasminogen binding and hence plasmin generation. In addition, we demonstrate lipoprotein(a) accumulation in atherosclerotic lesions. These findings may provide a link between impaired cell surface fibrinolysis and progressive atherosclerosis.

The effect of physical exercise conditioning on fasting and postprandial lipoprotein levels was studied in six normolipidemic subjects. The study consisted of two phases: a baseline stabilization phase in which subjects maintained their regular physical activity and an exercise conditioning phase in which subjects had 29 exercise sessions during a 7-week period. Each of these sessions consisted of jogging on a treadmill for 30 minutes. The subjects averaged 15.2 miles/wk. To control for possible confounding factors, such as changes in diet composition and weight loss, we placed the subjects on a metabolic diet and increased their daily caloric intake during the exercise phase. At the end of each phase of the study, a vitamin A-fat loading test was done to specifically label and follow postprandial lipoprotein levels, and a maximum oxygen consumption test was done to evaluate the subjects' physical fitness. The exercise conditioning phase significantly increased the subjects' aerobic capacity and postheparin lipoprotein lipase activity, and the phase decreased fasting triglyceride levels. Physical exercise also significantly decreased chylomicron (Sf greater than 1,000) levels by 37%. In summary, this study suggests that physical exercise conditioning reduces fasting and postprandial lipoprotein levels by increasing the catabolism of triglyceride-rich particles. Because these particles may have a role in atherogenesis, this could be a major mechanism by which exercise prevents coronary heart disease.