Gunther M. Fless

Lipoproteins inhibit the secretion of tissue plasminogen activator from human endothelial cells.

We studied the effect of lipoprotein(a) [Lp(a)], low-density lipoprotein (LDL), and high-density lipoprotein (HDL) on tissue plasminogen activator (TPA) secretion from human endothelial cells. At 1 mumol/L, Lp(a) inhibited constitutive TPA secretion by 50% and phorbol myristate acetate- and histamine-enhanced TPA secretion by 40%. LDL and HDL also depressed TPA secretion by 45% and 35% (constitutive) and 40% to 60% (stimulated). TPA mRNA levels were also examined and found to change in parallel with antigen secretion. In contrast to TPA, plasminogen activator inhibitor type-1 secretion and mRNA levels were not affected by any of the three lipoproteins. These results suggest that the interaction of lipoproteins with certain cell-surface binding sites may interfere with the proper production and/or secretion of TPA.

Rhesus monkey lipoprotein(a) binds to lysine Sepharose and U937 monocytoid cells less efficiently than human lipoprotein(a). Evidence for the dominant role of kringle 4(37).

Rhesus lipoprotein(a) (Lp[a]) binds less efficiently than human Lp(a) to lysine-Sepharose and to cultured U937 cells. Studies using elastase-derived plasminogen fragments indicated that neither kringle 5 nor the protease domain of Lp(a) are required in these interactions pointing at an involvement of the K4 region. Comparative structural analyses of both the human and simian apo(a) K4 domain, together with molecular modeling studies, supported the conclusion that K4(37) plays a dominant role in the lysine binding function of apo(a) and that the presence of arginine 72 rather than tryptophan in this kringle can account for the functional deficiency observed with rhesus Lp(a). These in vitro results suggest that rhesus Lp(a) may be less thrombogenic than human Lp(a).

Plasma lipoprotein (a) protein concentration and coronary artery disease in black patients compared with white patients

PURPOSE This study examines the relation between lipoprotein (a) protein levels and other lipid parameters and coronary artery disease in white and black patients. PATIENTS AND METHODS Plasma lipoprotein (a) protein levels were measured prior to coronary angiography in a population of 127 white and 111 black patients. Each angiogram was given a total coronary artery disease score based on the number and severity of atherosclerotic coronary lesions. RESULTS White and black patients exhibited no differences in total plasma cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, and triglycerides. Black patients had higher lipoprotein (a) protein levels than white patients (8.6 versus 4.0 mg/dL; p < 0.0001). The extent and severity of coronary artery disease was the same in white and black patients. White and black patients with coronary artery disease had higher lipoprotein (a) levels than patients without coronary lesions (4.37 versus 1.99 mg/dL, p = 0.027 for white; 9.23 versus 6.87 mg/dL, p = 0.072 for black). In both groups of patients, there was a weak but significant positive correlation between lipoprotein (a) protein levels and coronary artery disease score. CONCLUSION Lipoprotein (a) is higher in patients with coronary artery disease. Black patients have higher plasma lipoprotein (a) protein levels than white patients and a comparable degree of coronary artery disease. It follows that the cardiovascular pathogenicity of lipoprotein (a) is not significantly greater in black patients despite higher lipoprotein (a) levels.

Serum low density lipoproteins with mitogenic effect on cultured aortic smooth muscle cells

Low density lipoprotein (LDL) subspecies of different size and lipid mass were isolated by density gradient ultracentrifugation from the serum of male rhesus monkeys (Macaca mulatta) fed both a low fat, low cholesterol commercial primate ration, and cholesterol-supplemented high-fat diets, as well as from the serum of human donors. The mitogenic effect of these lipoproteins was examined using primary cultures of rhesus aortic smooth muscle cells. It was observed that the smaller LDL (molecular weight 2.7 X 10(6) from normolipidemic monkeys and a small LDL (molecular weight 2.6 X 10(6) occurring in some normal human subjects exhibited no mitogenic action. In turn, the larger LDL subspecies (molecular weight greater than 3.0 X 10(6), and buoyant density less than 1.030 g/ml), whether from normolipidemic or hyperlipidemic monkeys, or from some normal human subjects, had a marked proliferative action. The results indicate that both hyperlipidemic and normal sera (both human and rhesus) contain mitogenic LDL species although in different amounts. LDL-III, the rhesus equivalent of human Lp(a) was not mitogenic despite its similarity on size and lipid composition to the stimulating particles. However, on the removal of most of its large sialic acid moiety, a clear mitogenic action was observed. The mechanisms responsible for the proliferative effect are unclear and may involve LDL mass, lipid composition, and surface charge although other speculations cannot at present be ruled out. Furthermore, since the small LDL subspecies of either rhesus or human origin were nonmitogenic and similar in mass to the LDL found in calf serum, the mitogenic response of the smooth muscle cells to large LDLs may depend on their early conditioning with the LDL of calf serum.

Structural and functional changes of rhesus serum low density lipoproteins during cycles of diet-induced hypercholesterolemia.

Over the course of a 2-year study, two male rhesus monkeys underwent episodes of diet-induced hypercholesterolemia (from a diet supplemented with 25% coconut oil and 2% cholesterol) followed by regression phases in which the animals received a low fat Purina chow diet. During the induction of hypercholesterolemia, serum cholesterol, apo B, saturation of low density lipoprotein (LDL) cholesteryl ester fatty acyl chains, and the ability of the serum to stimulate cholesterol esterification by smooth muscle cells rose immediately and in parallel, whereas there was a lag period before the serum became mitogenic to smooth muscle cells. Concurrently, there were important changes in the density, size, chemistry, and concentration of the LDL species in the rhesus serum; induced LDL shifted from the LDL-II to the LDL-I density region with increasing cholesterol concentration. Both structural and functional changes were reversed upon return to a normal Purina chow diet, although at different rates. Serum cholesterol, apo B, and the rate of cholesterol esterification in smooth muscle cells promoted by the serum declined in parallel while the mitogenicity of the serum to smooth muscle cells and the degree of saturation of LDL cholesteryl ester fatty acids took longer to return to normal values. In fact, there was an immediate and dramatic rise in saturation upon reversal before the LDL cholesteryl ester fatty acyl chains returned to their normal composition. The Lp(a) particles did not increase in either concentration or size in response to the test diet, although the change in their lipid composition was similar to those of the other LDL species. The studies indicate that dietary manipulations affect the physicochemicai properties of the LDL particles, and that the resultant structural alterations are accompanied by changed in vitro cellular response, suggestive of a greater atherogenicity.

Binding and degradation of lipoprotein(a) and LDL by primary cultures of human hepatocytes. Comparison with cultured human monocyte-macrophages and fibroblasts.

Although lipoprotein(a) (Lp[a]) has structural similarities to low-density lipoprotein (LDL) that include the presence of apolipoprotein B100, there is some disagreement over the strength of its interaction with the LDL receptor and its cellular catabolism by the LDL receptor-mediated pathway. To clarify this subject we evaluated LDL receptor-mediated binding and degradation of Lp(a) and LDL in three human cell lines. The binding of 50 nmol/L Lp(a) at 37 degrees C to the LDL receptor of primary hepatocytes, macrophages, and fibroblasts was only 10%, 29%, and 29% of the respective value obtained with 50 nmol/L LDL. Analysis of 4 degrees C binding curves indicated that Lp(a) and LDL had equal affinities for the LDL receptor of fibroblasts, whereas maximal binding of Lp(a) was remarkably lower than that of LDL. LDL receptor-mediated degradation of 50 nmol/L Lp(a) in hepatocytes, macrophages, and fibroblasts was only 17%, 22%, and 26%, respectively, of the value obtained with 50 nmol/L LDL and varied greatly among the cells in that it was lowest in hepatocytes, an order of magnitude greater in macrophages, and two orders of magnitude greater in fibroblasts. In contrast, the nonspecific degradation rate of Lp(a) was similar to that of LDL in each of the three tested cell lines. However, the proportion of the degradation of Lp(a) that was nonspecific varied greatly, being 76%, 58%, and 33% in hepatocytes, macrophages, and fibroblasts, respectively. These studies indicate that not only is Lp(a) recognized by the LDL receptor but also that, in fibroblasts, Lp(a) and LDL have equal affinities for the LDL receptor, although Lp(a) has a much lower receptor occupancy than LDL. Additionally, they show that there are great cellular differences in the LDL receptor-mediated degradation of Lp(a). If these results can be extrapolated in vivo, where normal LDL levels are 40- to 50-fold higher than those of Lp(a), it would be unlikely that the hepatic LDL receptor is significantly involved in the degradation of Lp(a).

Polymorphic forms of Lp(a) with different structural and functional properties: cold-induced self-association and binding to fibrin and lysine-Sepharose☆

Two different Lp(a) polymorphs were isolated from the same individual and shown to have important differences both in their solution properties and in interaction with lysine Sepharose and fibrin. One Lp(a) particle (d-Lp(a)) with a large apo(a) isoform had a density of 1.087 g/ml and a molecular weight of 3.17 million, while the other Lp(a) particle with a small apo(a) isoform having a mobility faster than that of apoB was larger and had a molecular weight of 3.75 million and a density of 1.054 g/ml. D-Lp(a) underwent cold-induced self-association and also had a higher affinity for lysine Sepharose, whereas the other Lp(a) polymorph did not. Both Lp(a) particles bound fibrin via two different binding sites, one of which involved fibrin lysine residues which are also recognized by plasminogen. Lysine-mediated binding of d-Lp(a) by fibrin was ten times stronger than that of the other Lp(a) particle, whereas non-lysine-mediated binding of either Lp(a) species by fibrin was of equal strength. At saturation, 80% of d-Lp(a) bound fibrin at sites that did not involve lysine residues, whereas only 33% of the other Lp(a) polymorph bound to these sites. These findings indicate that the binding of Lp(a) to fibrin is more complex than previously thought and imposes another layer of difficulty on our understanding of how Lp(a) regulates and/or impairs fibrinolysis.

Effect of Glycation on the Properties of Lipoprotein(a)

Lipoprotein(a) [Lp(a)] was glycated by incubation in vitro with glucose (0 to 200 mmol/L), and its properties were compared with native Lp(a) and native and glycated LDL. Glucose was incorporated into Lp(a) in proportions that mirrored the distribution of lysines between apolipoprotein (apo) B-100 and apo(a). Because the kringle IV domains of apo(a) are lysine poor, only 10% of glucose bound to apo(a), whereas 90% was attached to the apoB-100 of Lp(a). Approximately 3% of the lysines of both Lp(a) and LDL were modified, which is a level comparable with that observed in LDL isolated from diabetic individuals. Glucose uptake by Lp(a) and LDL was almost identical and was linear as a function of concentration and time. Glycation increased the negative charge of Lp(a) and LDL as monitored by electrophoresis and ion-exchange chromatography and also reduced the affinity of Lp(a) and LDL for heparin-Sepharose. Glycation did not affect the lysine-binding property of Lp(a) or generate measurable malondialdehyde oxidation adducts. The catabolism of glycated Lp(a) by human monocyte-derived macrophages (HMDMs), like that of native Lp(a), was largely LDL receptor independent. Both glycated Lp(a) and LDL were degraded at a comparatively faster rate and stimulated greater cholesteryl ester formation than their unmodified counterparts. However, the degradation rate of glycated Lp(a) was approximately four- to fivefold slower and its stimulation of cholesteryl ester formation was ninefold lower than that of either form of LDL. These results show that Lp(a) can be glycated nonenzymatically in vitro, that the incorporation of glucose is dependent on the distribution of lysines between apo(a) and apoB-100, and that glycation does not affect the lysine-binding properties of Lp(a). Furthermore, glycation produced modest increases in the degradation rate of Lp(a) and associated cholesteryl ester synthesis by HMDMs. Based on these data, glycation does not appear to significantly enhanced the atherogenic potential of unmodified Lp(a).

Attenuation of immunologic reactivity of lipoprotein(a) by thiols and cysteine-containing compounds. Structural implications.

Samples of human plasma having lipoprotein(a) (Lp[a]) protein levels between 5 and 15 mg/dl and a single apolipoprotein(a) (apo[a]) isoform were incubated in vitro at pH 7.7 with various concentrations (1-20 mM) of N-acetylcysteine, homocysteine, 2-mercaptoethanol (2ME), and dithiothreitol (DTT) for 1 hour at 37 degrees C under a nitrogen atmosphere. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblot analyses using a polyclonal antibody specific for apo(a) showed a progressive decrease in apo(a) immunoreactivity as a function of reductant concentration. This decrease of apo(a) immunoreactivity was corroborated by enzyme-linked immunosorbent assay (ELISA) using anti-apo(a) as the capture antibody and either anti-apo B or anti-apo(a) as the developing antibody. In turn, there was no significant decrease in the immunoreactivity of apo B-100, as assessed by ELISA using anti-apo B as both the capture and the detecting antibody. In the case of high concentrations of DTT the plasma samples had to be diluted to prevent gel formation on addition of the reductant. A progressive drop in immunoreactivity as a function of reagent concentration was also observed in pure preparations of Lp(a) incubated with the reducing agents at pH 7.7. At equivalent stoichiometries the changes were more marked than those observed with whole plasma, suggesting a quenching effect by the plasma proteins on the activity of the reductants. The changes in immunoreactivity were attended by dissociation of apo(a) from Lp(a) as assessed by Western blotting. This dissociation, which we interpret as the result of cleavage of the interchain disulfide bond(s), was complete at 5 mM DTT and 100 mM 2ME.(ABSTRACT TRUNCATED AT 250 WORDS)

Postprandial lipoprotein (a) response to a single meal containing either saturated or ω-3 polyunsaturated fatty acids in subjects with hypoalphalipoproteinemia

We have recently reported that the apolipoprotein (apo) B-100-apo(a) complex, the protein moiety of lipoprotein(a) [Lp(a)], has a high affinity for triglyceride(TG)-rich particles (TRP) and that this complex can affiliate with endogenous TG-rich lipoproteins. To shed more light on the apo B-100-apo(a) complex associated with plasma TRP during postprandial lipidemia, we fed five male subjects presenting with primary hypoalphalipoproteinemia (HP) and four male controls a single fat meal (60 g/m2) containing saturated fatty acids (SFA) and, 6 weeks later, an isocaloric meal containing omega-3 polyunsaturated fatty acids. The subjects were phenotyped for plasma Lp(a) and apo C-III levels, apo(a) and apo E isoforms, and lipoprotein lipase and hepatic lipase activities. Vitamin A was included in the meal as a marker of intestinally derived TRP. Following the SFA meal, three of the HP subjects showed a decrease in plasma levels of Lp(a) that lasted 10 to 12 hours in the presence of an increased hypertriglyceridemic response. Two HP subjects who had low preprandial lipoprotein lipase activity and elevated plasma apo C-III levels showed an increase in plasma Lp(a) levels along with the hypertriglyceridemic excursion. However, in all cases, inclusive of the controls, there was an elevation in plasma levels of TRP of Sf greater than 1,000 that contained apo B-100-apo(a) 6 to 8 hours after the meal. This TRP excursion appeared not to be related to the basal levels of plasma Lp(a), high-density lipoprotein (HDL) cholesterol, TGs, or apo(a) and apo E isoforms, and it did not coincide with the retinyl ester peak.(ABSTRACT TRUNCATED AT 250 WORDS)