Therapeutic efficiency of lipoprotein(a) reduction by low-density lipoprotein immunoapheresis

This study was performed to investigate the effect of low-density lipoprotein (LDL) immunoapheresis on lipoprotein(a) [Lp(a)] reduction in patients with heterozygous and homozygous familial hyperlipidemia (N=16) and insufficient response to lipid-lowering agents. By desorption of approximately 5,700+/-500 mL of plasma, a mean reduction in total cholesterol of 62% (P < .001) and in LDL-cholesterol of 70% (P < .001) was achieved. Lp(a), which was elevated at study entry in seven of these patients (82.1+/-34.3 mg/dL; range, 48 to 148 mg/dL), was reduced during the initial LDL-apheresis procedure by 74.8%+/-14.1% (P < .001). Long-term apheresis treatment performed at weekly intervals resulted in an mean reduction in Lp(a) pretreatment values to 39.1+/-28.5 mg/dL (-54%; P < .001). Desorbed Lp(a) was measured at the waste of the columns for 31 apheresis treatments. Lp(a) concentration of the column waste was higher in patients with elevated serum Lp(a) pretreatment values as compared with those with Lp(a) serum values within the normal range (elevated Lp(a), 1,420+/-380 mg; without elevated Lp(a), 235+/-190 mg; P < .001). The rate of return of Lp(a) following apheresis treatment scheduled at weekly intervals was comparable to that of LDL-cholesterol.     

Apolipoprotein(a) binds via its C-terminal domain to the protein core of the proteoglycan decorin. Implications for the retention of lipoprotein(a) in atherosclerotic lesions

Although it is known that lipoprotein(a) (Lp(a)) binds to proteoglycans, the mechanism for this binding has not been fully elucidated. In order to shed light on this subject, we examined the interactions of decorin, a proteoglycan with a well defined protein core and a single glycosaminoglycan (GAG) chain, with Lp(a) and derivatives, namely Lp(a) deprived of apo(a), or Lp(a-), free apo(a), and the two main proteolytic fragments, F1 and F2. By circular dichroism criteria, the decorin preparations used had the same secondary structure as that previously reported for native decorin. Authentic low density lipoprotein from the same human donor was used as a control. In a solid phase system, Lp(a-)and low density lipoprotein bound to decorin in a comparable manner. This binding required Ca2+/Mg2+ ions, was lysine-mediated, and was markedly decreased in the presence of GAG-depleted decorin, suggesting the ionic nature of the interaction likely involving apoB100 and the GAG component of decorin. Free apo(a) also bound to decorin; however, the binding was neither cation-dependent nor lysine-mediated, unaffected by sialic acid depletion of apo(a), and markedly decreased when either reduced and alkylated apo(a) or reduced and alkylated decorin was used in the assay. Of note, the binding of apo(a) was unaffected when it was incubated with a spectrally native decorin that had been renatured from either 4 M guanidine hydrochloride by extensive dialysis or cooled from 65 to 25 degrees C. On the other hand, the binding significantly increased when decorin was depleted of GAGs, which by themselves had no affinity for apo(a). The binding of apo(a) to the decorin protein core was also elicited by the C-terminal domain of apo(a), and it was favored by high NaCl concentrations, 1 to 2 M. No binding was exhibited by the N-terminal domain accounting for the lack of effect of apo(a) size polymorphism on the binding. In the case of whole Lp(a), the binding to immobilized decorin was mostly GAG-dependent and ionic in nature. A minor contribution by apo(a) was detected when GAG-depleted decorin was used in the assay. Our results indicate that the binding of Lp(a) to decorin involves interactions both electrostatic (apoB100-GAG) and hydrophobic (apo(a)-decorin protein core), and that the binding of apo(a) requires decorin protein core to be in its native state.     

Apolipoprotein(a) genetic polymorphism and serum lipoprotein(a) concentration in patients with peripheral vascular disease

Serum lipoprotein(a) (Lp(a)) levels were measured in 89 men with peripheral vascular disease (PVD) and 129 (100 male and 29 woman) healthy controls. Apolipoprotein(a) genetic polymorphism was determined by immunoblotting in all subjects. Patients with PVD had significantly higher serum Lp(a) levels than controls. Apolipoprotein(a) phenotype frequencies in patients with PVD did not differ from those of the control group. Both patients and controls with phenotype S2 had higher serum Lp(a) levels than those with phenotype S4. It should be emphasized that serum Lp(a) levels were significantly higher in PVD patients than controls for those with phenotype S2, S3/S4 and S4. Raised serum Lp(a) levels together with other lipoprotein abnormalities in patients with PVD imply a high cardiovascular risk. Genetic polymorphism clearly influences serum Lp(a) levels both in patients and controls. In patients with PVD, environmental and/or other genetic factors must play a role in raising Lp(a) levels.     

Gender differences in intima-media permeability to low-density lipoprotein at atherosclerosis-prone aortic sites in rabbits. Lack of effect of 17 beta-estradiol

Premenopausal women are protected from coronary heart disease, and premenopausal nonhuman primates are protected from atherosclerosis, the underlying cause of coronary heart disease. Estrogen is thought to account for this protection in females, and part of this protection is independent of the effects on risk factors, including lipoprotein levels. This study considered the hypothesis that reduced intima-media permeability to low-density lipoproteins (LDL) may account for the protection from atherosclerosis and coronary heart disease in premenopausal females and that this effect might be mediated by estrogen. Intima-media permeability to LDL was determined in male and female rabbits made hypercholesterolemic by feeding them 0.5% cholesterol for 8 days. The diet of half of the female rabbits was supplemented with 17 beta-estradiol (4 mg/d) during cholesterol feeding and the preceding 4 weeks. Estrogen treatment in the female rabbits did not influence the intima-media permeability to LDL. However, intima-media permeability to LDL for branch sites of the abdominal aorta and aortic arch (regions highly susceptible to atherosclerosis) was 43% and 38% lower, respectively, in male rabbits than in female rabbits: (2.93 +/- 0.39 microL/h/g, (n = 8), vs 6.28 +/- 0.86 microL/h/g, (n = 16), P < .001, and 4.69 +/- 0.28 microL/h/g, (n = 8) vs 7.57 +/- 0.75 microL/h/g, (n = 16), P < .02). In contrast, intima-media permeability to LDL in 7 of 8 aortic sites relatively resistant to atherosclerosis did not differ between male and female rabbits. These data suggest that the protection from atherosclerosis associated with female sex and estrogen is mediated by mechanism(s) other than reduction in intima-media permeability to LDL.     

Apolipoprotein (a)

Apo (a) consists of multiple tandem repeat of kringle 4, which resembles a counterpart of plasminogen. Plasma Lp (a) levels are genetically determined primarily by alleles at the apo (a) gene. Apo (a) shows size heterogeneity on the analysis of the protein and the mRNA. Pulse field gel electrophoresis revealed that size heterogeneity is largely due to different numbers of kringle 4-encoding sequences in the apo (a) gene. The cloning of apo (a) gene and identification of the 5'-flanking promoter region provide tools to study the regulation of apo (a) gene. The development of transgenic mice expressing human apo (a) offered a good model for understanding of atherosclerosis associated with elevated plasma levels of Lp (a).     

Linkage of low-density lipoprotein size to the lipoprotein lipase gene in heterozygous lipoprotein lipase deficiency

Small low-density lipoprotein (LDL) particles are a genetically influenced coronary disease risk factor. Lipoprotein lipase (LpL) is a rate-limiting enzyme in the formation of LDL particles. The current study examined genetic linkage of LDL particle size to the LpL gene in five families with structural mutations in the LpL gene. LDL particle size was smaller among the heterozygous subjects, compared with controls. Among heterozygous subjects, 44% were classified as affected by LDL subclass phenotype B, compared with 8% of normal family members. Plasma triglyceride levels were significantly higher, and high-density lipoprotein cholesterol (HDL-C) levels were lower, in heterozygous subjects, compared with normal subjects, after age and sex adjustment. A highly significant LOD score of 6.24 at straight theta=0 was obtained for linkage of LDL particle size to the LpL gene, after adjustment of LDL particle size for within-genotype variance resulting from triglyceride and HDL-C. Failure to adjust for this variance led to only a modest positive LOD score of 1.54 at straight theta=0. Classifying small LDL particles as a qualitative trait (LDL subclass phenotype B) provided only suggestive evidence for linkage to the LpL gene (LOD=1. 65 at straight theta=0). Thus, use of the quantitative trait adjusted for within-genotype variance, resulting from physiologic covariates, was crucial for detection of significant evidence of linkage in this study. These results indicate that heterozygous LpL deficiency may be one cause of small LDL particles and may provide a potential mechanism for the increase in coronary disease seen in heterozygous LpL deficiency. This study also demonstrates a successful strategy of genotypic specific adjustment of complex traits in mapping a quantitative trait locus.     

DALI-the first human whole-blood low-density lipoprotein and lipoprotein (a) apheresis system in clinical use: procedure and clinical results

BACKGROUND: The DALI low-density lipoprotein (LDL) apheresis system is the first whole-blood apheresis system in regular clinical use. DALI stands for direct adsorption of lipoproteins, which describes the basic principle of operation of this newly developed LDL apheresis procedure. METHODS: The selective removal of LDLs and lipoprotein (a) [Lp(a)] is performed in human whole blood by adsorption onto polyacrylate-coated polyacrylamide beads in an adsorber. This article describes the results of the first open multicentre clinical trial in 14 patients in whom the safety and the efficacy of the system were tested. All patients were treated on average 17 times on a weekly basis. In total, 238 sessions were carried out during the study without severe side-effects. On average, 7675 mL of the patients' whole blood was processed in about 2 h. Anticoagulation in the extracorporeal system was carried out by first giving a heparin bolus followed by continuous addition of an acid citrate dextrose (ACD-A) infusion during the treatment. RESULTS: The processing of nearly 1.6 times the patient blood volumes resulted in a reduction in the median LDL-cholesterol level by 66-77% (dependent on the system configuration). The Lp(a) concentrations were reduced by 59-73% (dependent on the system configuration). HDL-cholesterol, blood cell count and the other clinical parameters were not significantly affected. CONCLUSION: Based on this short-term evaluation, the DALI apheresis system is a well-tolerated, effective and simple way of reducing LDL and Lp(a) in human whole blood. The system has been introduced to clinical practice. However, to use the DALI apheresis system in clinical routine, further evaluation of long-term effects is required.     

Apolipoprotein E Sendai (arginine 145-->proline): a new variant associated with lipoprotein glomerulopathy

Lipoprotein glomerulopathy (LPG) is a novel disease characterized by proteinuria, lipoprotein thrombi in the glomeruli, and increased concentration of plasma apolipoprotein (apo) E. It is believed that a genetic disorder of apo E may be present and associated with the disease. Three patients with LPG were examined in this study. The patients' DNA sequences were analyzed, and a nucleotide G to C point mutation in exon 4 of the apo E gene was confirmed in each patient. This missense mutation denotes amino acid substitution of the proline residue for arginine residue at position 145 of apo E. This variant (apo E Sendai) may cause a marked molecular conformational change of the apo E. These findings suggest that a novel variant is etiologically related to LPG.     

Apolipoprotein E genotype is a determinant of low-density lipoprotein cholesterol and of its response to a low-cholesterol diet in Type 1 diabetic patients with elevated urinary albumin excretion

BACKGROUND: Immunoablative high-dose cyclophosphamide without stem-cell rescue induces durable, complete remission in most patients with aplastic anemia. OBJECTIVE: To determine the efficacy of high-dose cyclophosphamide in various refractory, severe autoimmune diseases. DESIGN: Prospective phase II study. SETTING: Johns Hopkins University (Baltimore, Maryland) and Hahnemann University (Philadelphia, Pennsylvania). PATIENTS: Eight patients with refractory, severe autoimmune disease. Intervention: Immunoablative high-dose cyclophosphamide (50 mg/kg of body weight per day) for 4 consecutive days. MEASUREMENTS: Clinical and laboratory variables of autoimmune disease. RESULTS: Seven patients improved markedly: Five achieved complete remission and two achieved partial remission. Four patients have remained in continuous complete remission for 3 to 21 months, and two patients in partial remission continue to improve after 14 and 19 months of follow-up. High-dose cyclophosphamide was well tolerated; median times to a neutrophil count of 0.5 x 10(9) cells/L and platelet transfusion independence were 17 and 16 days, respectively. CONCLUSIONS: Immunoablative high-dose cyclophosphamide without stem-cell rescue can induce complete remission in patients with refractory, severe autoimmune disease. Reemergence of marrow function is similar to that seen after autologous transplantation and does not carry the risk for reinfusion of autoaggressive lymphocytes with the autograft.     

Hyperbetalipoproteinemia with small low-density lipoprotein, a characteristic disorder of lipoprotein in essential hypertension

The purpose of the present study was to elucidate the characteristic lipoprotein disorder in essential hypertension. Twenty-six patients with essential hypertension (HT) but without diabetes mellitus or obesity and 24 healthy subjects (control) were recruited into this study. Lipoproteins of HT and controls were separated by ultracentrifugation to very-low-density lipoprotein (VLDL), intermediate density lipoprotein (IDL), low-density liproprotein (LDL), and (HDL) fractions. Cholesterol and triglycerides were determined with enzyme assay, and apoB were determined by highly sensitive latex agglutination (Kyowa-hakko Co. LD). There was no difference in age (mean +/- SE; HT, 63 +/- 2 versus control, 60 +/- 2 years) or body-mass index (22.7 +/- 0.4 versus 21.7 +/- 0.5 kg/m2) between HT and controls. Blood pressure in HT and controls was 158 +/- 2/87 +/- 12 mm Hg and 123 +/- 3/72 +/- 2 mm Hg, respectively. Cholesterol did not change significantly in plasma (192.1 +/- 7.0 versus 176.4 +/- 4.2 mg/dL), VLDL (15.2 +/- 2.4 versus 11.8 +/- 1.7 mg/dL), IDL (14.8 +/- 2.4 versus 10.7 +/- 1.6 mg/dL), LDL (93.7 +/- 4.6 versus 83.1 +/- 3.9 mg/dL), nor in HDL (51.9 +/- 2.7 versus 58.1 +/- 3.2 mg/dL). Triglycerides (TG) increased in plasma (120.0 +/- 10.0 versus 87.5 +/- 9.3 mg/dL, p < 0.05), although TG did not change in all subfractions. ApoB increased in plasma (105.5 +/- 5.1 versus 85.6 +/- 3.6 mg/dL, p < 0.01), IDL (9.0 +/- 1.3 versus 5.4 +/- 0.6 mg/dL, p < 0.05), and LDL (76.3 +/- 4.3 versus 59.4 +/- 3.7 mg/dL, p < 0.01) in HT compared with controls. The ratio of cholesterol to apoB in LDL decreased (1.27 +/- 0.06 versus 1.48 +/- 0.08, p < 0.05). In essential HT, number of apoB containing lipoproteins (IDL, LDL) increased. Low ratio of cholesterol to apoB was noted in LDL, indicating the presence of small, dense LDL. As cholesterol in LDL was normal, hyperbetalipoproteinemia is also a characteristic disorder of essential HT.     

The low-density lipoprotein receptor-related protein (LRP) mediates clearance of coagulation factor Xa in vivo

In response to fibroblast growth factor (FGF), FGF receptor-1 (FGFR-1) (flg) becomes tyrosine phosphorylated and associates with phospholipase C gamma (PLC gamma) and a 90 kDa protein. We report here that in cells transformed by v-Src, FGFR-1 becomes phosphorylated on tyrosine; however, neither PLC gamma nor p90 was found to be associated with tyrosine-phosphorylated FGFR-1. Instead, there was a strong constitutive association of FGFR-1 with the adaptor proteins Shc and Grb2 and the Ras guanine nucleotide exchange factor Sos. Association with Shc and Grb2 and Sos was not observed in response to FGF. Suramin did not prevent either tyrosine phosphorylation or Shc/Grb2/Sos association, indicating a non-autocrine mechanism. Thus, in cells transformed by v-Src, tyrosine phosphorylation of FGFR-1 results not in the expected association with PLC gamma and p90, but rather in the recruitment of the Ras activating Shc/Grb2/Sos complex. These data suggest a mechanism for Ras activation by v-Src involving phosphorylation of novel tyrosine(s) on FGFR-1.     

Smoking cessation increases the resistance of low-density lipoprotein to oxidation

The effects of smoking cessation on the susceptibility to oxidation of low-density lipoprotein (LDL) was investigated in 14 men who quit smoking for 3 months. LDL was isolated and susceptibility of LDL to V-70 (4-methoxy-2,4-dimethylvalerinitrile)-mediated oxidation was assessed by measuring conjugated diene production at 234 nm, the lag phase being a measure of the resistance of LDL to oxidation. The mean duration of the lag phase became 1.9-fold longer after 3 months (P < 0.001). The result suggests that the increase in resistance of LDL to oxidation contributes to the reduction of the risk of coronary heart disease by smoking cessation.     

Different mechanisms are progressively recruited to promote Cu(II) reduction by isolated human low-density lipoprotein undergoing oxidation

The kinetics of Cu(II) reduction and its relationship to the process of low density lipoprotein (LDL) oxidation were investigated in isolated human LDL incubated with CuSO4 by using the Cu(I) chelator and indicator dye bathocuproine disulfonate (BC). The inclusion of BC in the incubation medium containing isolated LDL and different concentrations of CuSO4 revealed a biphasic kinetics of Cu(II) reduction consisting of an early phase followed by a plateau phase and a subsequent extensive reduction phase. The amount of Cu(I) formed during the early phase, as well as the rate of its generation, were strictly dependent on both the level of Cu(II) available (saturation was observed at 20 and 50 microM CuSO4) and the concentration of alpha-tocopherol within native LDL particles. Artificial enrichment of LDL with different concentrations of alpha-tocopherol led to a parallel increase of both the amount of Cu(II) reduced and the rate of reduction. The late phase of Cu(II) reduction was strictly related to the availability of copper but was largely independent from alpha-tocopherol. Neither the amount of Cu(I) generated nor the rate of generation were saturated at concentrations of copper up to 100 microM. Comparable results were obtained by adding BC at different time-points to the LDL-copper mixture, in order to measure at the same time-points both the true rate of Cu(II) reduction and the generation of TBARS during the dynamic process of LDL oxidation. The rate of Cu(II) reduction was already high during the lag-phase of the LDL oxidation profile and progressively decreased as alpha-tocopherol concentration decreased. The subsequent increase in the rate of Cu(II) reduction paralleled the formation of TBARS during the extensive LDL oxidation phase. These results suggest that different mechanisms of Cu(II) reduction, namely alpha-tocopherol-dependent and independent (likely lipid peroxide-dependent), are progressively recruited during copper-promoted LDL oxidation.     

Passive smoking induces atherogenic changes in low-density lipoprotein

BACKGROUND: According to the American Heart Association, passive smoking is an important risk factor for coronary heart disease (CHD), but the mechanisms underlying this association are not fully understood. We studied the acute effect of passive smoking on the factors that influence the development of CHD: the antioxidant defense of human serum, the extent of lipid peroxidation, and the accumulation of LDL cholesterol in cultured human macrophages, the precursors of foam cells in atherosclerotic lesions. METHODS AND RESULTS: Blood samples were collected during 2 ordinary working days from healthy, nonsmoking subjects (n=10) before and after (up to 5.5 hours) spending half an hour in a smoke-free area (day 1) or in a room for smokers (day 2). Passive smoking caused an acute decrease (1.5 hours after exposure) in serum ascorbic acid (P<.001) and in serum antioxidant defense (P<.001), a decreased capacity of LDL to resist oxidation (P<.01), and the appearance of increased amounts of lipid peroxidation end products in serum (P<.01). Finally, LDL isolated from subjects after passive smoking was taken up by cultured macrophages at an increased rate (P<.05). CONCLUSIONS: Exposure of nonsmoking subjects to secondhand smoke breaks down the serum antioxidant defense, leading to accelerated lipid peroxidation, LDL modification, and accumulation of LDL cholesterol in human macrophages. These data provide the pathophysiological background for the recent epidemiological evidence about the increased CHD risk among passive smokers.     

Sequences within apolipoprotein(a) kringle IV types 6-8 bind directly to low-density lipoprotein and mediate noncovalent association of apolipoprotein(a) with apolipoprotein B-100

Lipoprotein(a) [Lp(a)] particle formation is a two-step process in which initial noncovalent interactions between apolipoprotein(a) [apo(a)] and the apolipoprotein B-100 (apoB-100) component of low-density lipoprotein (LDL) precede disulfide bond formation. To identify kringle (K) domains in apo(a) that bind noncovalently to apoB-100, the binding of a battery of purified recombinant apo(a) [r-apo(a)] species to immobilized human LDL has been assessed. The 17K form of r-apo(a) (containing all 10 types of kringle IV sequences) as well as other truncated r-apo(a) derivatives exhibited specific binding to a single class of sites on immobilized LDL, with Kd values ranging from approximately 340 nM (12K) to approximately 7900 nM (KIV5-8). The contribution of kringle IV types 6-8 to the noncovalent interaction of r-apo(a) with LDL was demonstrated by the decrease in binding affinity observed upon sequential removal of these kringle domains (Kd approximately 700 nM for KIV6-P, Kd approximately 2000 nM for KIV7-P, Kd approximately 5100 nM for KIV8-P, and no detectable specific binding of KIV9-P). Interestingly, KIV9 also appears to participate in the noncovalent binding of apo(a) to LDL since the binding of KIV5-8 (Kd approximately 7900 nM) was considerably weaker than that of KIV5-9 (Kd approximately 2000 nM). Finally, it is demonstrated that inhibition of Lp(a) assembly by proline, lysine, and lysine analogues, as well as by arginine and phenylalanine, is due to their ability to inhibit noncovalent association of apo(a) and apoB-100 and that these compounds directly exert their effects primarily through interactions with sequences contained within apo(a) kringle IV types 6-8. On the basis of the obtained data, a model is proposed for the interaction of apo(a) and LDL in which apo(a) contacts the single high-affinity binding site on apoB-100 through multiple, discrete interactions mediated primarily by kringle IV types 6-8.     

Testosterone decreases lipoprotein(a) in men

We administered testosterone, with or without the aromatase inhibitor testolactone, to determine the effects of testosterone and its aromatization to estradiol on Lp(a) levels in normal men. Average Lp (a) values decreased by 37% during testosterone alone and by 28% when testosterone and testolactone were combined, suggesting that testosterone reduces Lp(a) in men primarily by an androgenic effect and not by its conversion to estradiol.