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The influence of adiponectin, a protein secreted by adipocytes, on the activation of transendothelial LDL transport, the initial event of atherogenesis, was studied. The addition of adiponectin to the cultured endothelial hybridoma EA.hy926 cells did not affect both basal and TNF-stimulated transendothelial transport of LDL. In addition, adiponectin affects neither expression levels of CAV1, SCARB1, and ACVRL1 genes encoding proteins involved in transendothelial LDL transport, nor the MMP secretion by the EA.hy926cells. At the same time, adiponectin suppressed the TNF-stimulated IL-8 production and expression of the adhesion molecule gene ICAM1 in these cells. Thus, adiponectin reduces proinflammatory activation of EA.hy926 cells, which is not accompanied by changes in the transendothelial LDL transport. We speculate that anti-inflammatory action of adiponectin is the base for the influence of this adipokine on atherogenesis., (© 2024. Springer Science+Business Media, LLC, part of Springer Nature.)

Adiponectin is an adipose tissue hormone, participating in energy metabolism and involved in atherogenesis. Previously, it was found that adiponectin increases expression of the APOA1 (apolipoprotein A-1) gene in hepatocytes, but the mechanisms of this effect remained unexplored. Our aim was to investigate the role of adiponectin receptors AdipoR1/R2, AMP-activated protein kinase (AMPK), nuclear peroxisome proliferator-activated receptor alpha (PPARα) and liver X receptors (LXRs) in mediating the action of adiponectin on hepatic APOA1 expression in human hepatoma HepG2 cells. The level of APOA1 expression was determined by RT-qPCR and ELISA. We showed that the siRNA-mediated knockdown of genes coding for AdipoR1, AdipoR2, AMPK, PPARα, and LXRα and β prevented adiponectin-induced APOA1 expression in HepG2 cells and demonstrated that interaction of PPARα and LXRs with the APOA1 gene hepatic enhancer is important for the adiponectin-dependent APOA1 transcription. The results of this study point out to the involvement of both types of adiponectin receptors, AMPK, PPARα, and LXRs in the adiponectin-dependent upregulation of the APOA1 expression.

Background: Adiponectin (AN) is a protein synthesized by adipocytes that has regulatory effects on lipid and lipoprotein metabolism, increases tissue sensitivity to insulin, and modulates endothelial functions and inflammatory response. However, its involvement in the processes of atherogenesis remains poorly understood., Objective: To determine the localization and sources of AN in atherosclerotic and normal human aortic intima., Material and Methods: Immunohistochemical study was performed on sections of atherosclerotic and normal human aorta obtained during autopsy. Reverse transcription real-time PCR was performed using biopsies of para-aortic and abdominal adipose tissue, intima-media of the thoracic aorta, atherosclerotic plaques of the human carotid and femoral arteries, as well as on endothelial cells isolated from the human thoracic aorta. Transendothelial transport of AN was evaluated in a two-chamber model using a monolayer of human endothelial cell hybridoma EA.Hy926., Results: It has been established that AN is present in atherosclerotic but not in normal human aortic intima. At the same time, AN ADIPOQ mRNA was not detected either in the intima media of the human aorta, nor in isolated endothelial cells of the aorta, nor in cells of atherosclerotic plaques of the carotid and femoral arteries. AN slowly penetrated the endothelial monolayer in vitro, but this transport was significantly enhanced by the action of tumor necrosis factor-alpha (TNFa)., Conclusion: Obtained data indicate that AN is present in atherosclerotic but not in normal aortic intima. We assume that AN is not synthesized by the cells of normal and atherosclerotic arterial walls, but permeates from the plasma. Transendothelial transport of AN, like many other plasma proteins, is activated during the development of atherosclerotic lesions, apparently under the action of pro-inflammatory cytokines, in particular, TNFα.

Background: Metabolic syndrome, a cluster of interrelated disorders including abdominal obesity, insulin resistance, dyslipidemia, and hypertension (HTN) plays an important role in development of atherosclerotic lesions in arterial wall. Dysregulation of adipose tissue hormones (adipokines) production is a possible link between abdominal obesity and other manifestations of metabolic syndrome. Adiponectin is a well-known adipokine which affects metabolism and inflammatory response. However, data on its role in atherogenesis are still controversial. The aim of this study is to investigate whether adiponectin is present in atherosclerotic lesions of human aorta., Methods: Thirty-five autopsy segments from abdominal, thoracic aortas, and aortic arch of four men (mean age: 57 years) were fixed and stained for lipids [Oil Red O (ORO)], cells [hematoxylin-eosin (H&E)], and adiponectin [indirect immunoperoxidase assay (IPA) method]. Samples of both stable and unstable plaques were selected for analysis. Human adipose tissue, THP-1 monocytes/macrophages, and human endothelial hybrid cell line (EA.hy926) were chosen for detection of adiponectin messenger ribonucleic acid (mRNA) using reverse transcription polymerase chain reaction (RT-PCR)., Results: Adiponectin accumulations were found inside endothelial cells covering both stable and unstable atherosclerotic plaques. Focal depositions of adiponectin were also found in fibrous caps of stable lesions and atheromatous core of both stable and unstable plaques and also in adventitia. RT-PCR revealed mRNA expression of adiponectin gene in adipose tissue, but not in mononuclears and endothelial cells., Conclusion: Adiponectin is present in aortic plaques of humans, but is not synthesized in endothelial cells and mononuclears, at least in culture conditions. Detection of adiponectin in atherosclerotic lesions can serve as indirect evidence of possible participation of this adipokine in atherogenesis., (© 2019 Isfahan Cardiovascular Research Center & Isfahan University of Medical Sciences.)

Copper is an essential micronutrient, because it is a catalytic and structural cofactor of enzymes that control the basic processes in all cells, and moreover it is a participant in signaling pathways. The toxic properties of copper ions, due to their chemical nature, are manifested when the cellular and/or organism systems for copper homeostasis are disturbed. Aim of the work was to study the relationships between the diet caloric and the copper status in the blood serum, the copper metabolism in the liver and white adipose tissue (WAT) of rats., Material and Methods: The work was performed on three groups (each n=5) of white outbred rats (average body weight 220±15 g), kept for 75 days on a standard, low-calorie (LCR) or high-calorie (high-fat) (HCR) rations. mRNA concentration was measured by qRT-PCR technology. The сeruloplasmin (CP) content was determined by the method of immune electrophoresis, immune blotting and by oxidase activity. The copper concentration was measured by atomic absorption spectrometry., Results and Discussion: It has been shown that serum level of triglycerides increased in rats fed LCR. The main indicators of copper status (concentration of atomic copper, the level of holo-CP, and the content of immunoreactive CP) decreased in rats fed HCR. In the liver, none of the diets affected Cp gene expression level. In the cells of the subcutaneous fatty tissue, the concentration of both splice-forms of CP-mRNA significantly increased in rats fed LCR. In visceral adipose tissue the concentration of Cp-mRNA encoding the secretory CP did not change in LCR-rats, but the level of mRNA, encoding CP anchored to plasma membrane, dropped to almost zero as compared to the control group. There was no significant change in the level of both splice-forms of CP-mRNA in HCR-rats. The features of copper metabolism in the cells of the liver and WAT, due to the caloric content of ration, have been discussed., Conclusions: In rats' liver, the link between copper metabolism and calorie intake is manifested in changes in the expression of the CP gene at the translation level, and in white adipose tissue - at the level of transcription and post-transcriptional maturation of the pre-mRNA of this gene., Competing Interests: The authors declare no overt and potential conflict of interest related to the publication of this article., (Copyright© GEOTAR-Media Publishing Group.)

The study included 79 patients with coronary artery disease, 25 individuals with preclinical atherosclerosis and 59 healthy controls. Key lipid parameters were examined in all the participants. Levels of antibodies (Abs) against (IgG and IgM) LDL modified by malondialdehyde (MDA), acetic anhydride and hypochlorite, were determined by the enzyme-linked immunosorbent assay (ELISA). Abs specificity was tested by competitive ELISA. Circulating immune complexes (CIC) were isolated by precipitation in polyethylene glycol. Abs to hypochlorite-modified low density lipoprotein (hypochlorite-LDL) were detected in the serum samples. These Abs did not demonstrate cross-reactivity with MDA-modified LDL (MDA-LDL) and acetylated LDL (acetyl-LDL). Patients with coronary artery disease had increased levels of CIC (p<0.0001) and decreased levels of Abs (IgM) to hypochlorite-LDL, compared with healthy controls and patients with preclinical atherosclerosis (p=0.006). A correlation between the levels of Abs (IgG) to the hypochlorite-LDL and Abs to MDA- and acetyl-LDL was found. There was a correlation between the content of the Abs (IgM) to MDA- and acetyl-LDL and the concentration of CIC-cholesterol. Lipid parameters did not correlate with Abs levels.

Aim: The role of adipose tissue hormones, adipokines, in formation of metabolic disorders in schizophrenia is not fully understood. The aim was to investigate the association of leptin and adiponectin plasma levels with metabolic parameters in antipsychotic treated patients with schizophrenia and in the group of age, gender and body mass index matched mental healthy persons., Methods: One hundred patients with diagnosis of schizophrenia, who took antipsychotic medication, and equal number of control subjects, were enrolled for cross-sectional evaluation. Fasting blood plasma levels of glucose, lipids, insulin, adiponectin, leptin concentrations and insulin resistance HOMA index were determined., Results: In both groups plasma leptin concentration positively correlated with body mass index, insulin plasma level and HOMA index, while adiponectin level had negative correlations with adiposity measures and positive associations with high density lipoprotein cholesterol content. At the same time, in schizophrenia group, but not in control subjects, leptin level positively associated with cholesterol and triglycerides concentrations and adiponectin negatively correlated with plasma insulin content, HOMA index and triglycerides levels. After controlling for confounders significant correlations remained for leptin concentration with HOMA index and plasma triglycerides level in schizophrenic patients and for adiponectin concentration with plasma high density lipoprotein cholesterol concentrations in both studied groups., Conclusions: Both adipokines associate with metabolic parameters in antipsychotic treated patients with schizophrenia. Leptin can play more specific role in pathogenesis of metabolic syndrome in schizophrenic persons than in mental healthy subjects., (Copyright © 2015 Diabetes India. Published by Elsevier Ltd. All rights reserved.)

Bradykinin B2 receptor is involved in many processes, including the regulation of blood pressure and smooth muscle contraction, vasodilation, inflammation, edema, cell proliferation, pain. It is suggested that this receptor may be one of the factors that have cardioprotective and infarct-limiting effects. It is assumed that certain genetic variants in both coding and non-coding regions ofBDKRB2 gene may influence its expression. In the 3'-untranslated region of BDKRB2 exon 3 the minisatellite repeat B2-VNTR is located. B2-VNTR has previously been shown to affect the BDKRB2 mRNA stability. Therefore, it is important to perform the molecular genetic analysis of this minisatellite in patients with different forms of coronary heart disease in order to reveal possible associations between specific B2-VNTR alleles and certain clinical forms of coronary heart disease. In the present study, a comparative analysis of the allele and genotype frequencies of B2-VNTR was carried out in groups of healthy individuals and patients with two clinical forms of coronary heart disease (angina pectoris and myocardial infarction), ethnically Russian. The results of the B2-VNTR length polymorphism analysis indicate that this tandem repeat may be attributed to a class of low polymorphic and non-hypervariable minisatellite. In all analyzed groups we revealed three B2-VNTR alleles, consisting of 43, 38 and 33 repeat units. Alleles of 43 and 33 repeats were major in all investigated groups. No statistically significant differences were found in the B2-VNTR allele and genotype frequencies between men and women in control group, and also between healthy men and men with angina pectoris and myocardial infarction. Thus, B2-VNTR length polymorphism was not associated with these clinical forms of coronary heart disease in Russian men. However, we do not exclude the possibility of association between the B2-VNTR short alleles (38 and 33 repeats) and cardioprotective effects of bradykinin B2 receptor in women with coronary heart disease. This hypothesis requires further investigation.

Accumulation of cholesterol in arterial wall macrophages is a main hallmark of atherosclerosis. The ABCG1 transporter mediates cholesterol efflux to high density lipoproteins (HDL) and plays an important role in macrophage foam cell formation. The goal of our study was to investigate the potential role of ABCG1 in atherosclerosis development in humans. ABCG1 gene expression has been examined in leukocytes, monocytes and monocyte-derived macrophages of patients with atherosclerosis and in the control group. Real time PCR and Western blotting were used to determine ABCG1 mRNA and ABCG1 protein levels. Monocyte ABCG1 mRNA level was inversely correlated with the rate of artery occlusion (r = -0.45, P = 0.016). Patients with 100% artery occlusions had decreased monocyte ABCG1 mRNA levels compared to patients who had smaller plaques and controls (P < 0.05). ABCG1 mRNA (P < 0.001) and ABCG1 protein (P < 0.05) levels in macrophages of patients with coronary artery stenosis were significantly reduced compared to the control group. No significant correlation between the ABCG1 gene expression in mononuclear cells and HDL cholesterol concentration has been found. Our study suggests that decrease in the ABCG1 gene expression in macrophages is associated with atherosclerosis.

Interrelationship between total, multimer (MM) and oligomer (OM) adiponectin blood concentrations with some metabolic parameters has been investigated in 49 men and 49 women (mean age of 57.3 +/- 10.1 years). We have found negative correlations between total blood adiponectin and its MM form content with body mass index, waist circumference, the insulin resistance index HOMA, with blood concentrations of insulin, glucose, free fatty acids, triglycerides and also positive correlations with high density lipoprotein cholesterol content. There was a poor correlation between OM adiponectin concentration with any parameter studied. According to the regression analysis, concentration of total adiponectin but not its MM form was an independent determinant of the HOMA index in women and free fatty acid concentration in men. In the group of men with the low level of adiponectin its MM form but not total adiponectin reversely correlated with the HOMA index and was its independent determinant. Thus, correlation between blood adiponectin concentration and metabolic parameters is associated with its MM rather than OM form. Study of the role of adiponectin in development of metabolic disorders may be limited to determination of total blood adiponectin concentration except a group of male patients characterized by a low level of this adipokine. In these patients concentrations of the MM form should be determined.

ABCA1 transporter is known to play important role in the cholesterol transport from peripheral tissues. However its contribution in atherosclerosis development remains not completely understood. Using Real Time PCR, a significant reduction of ABCA1 mRNA level in leukocytes of patients with atherosclerosis was determined when compared with controls. Mean ABCA1 expression levels in leukocytes for the group of patients and for the control group are 0.57 +/- 0.28 and 0.93 +/- 0.14 (p = 0.02). At the same time we detected a significant increase of ABCA1 mRNA level in macrophages of patients when compared with controls. Mean ABCA1 expression levels in macrophages for the group of patients and for the control group are 1.32 +/- 0.10 and 0.90 +/- 0.14 (p = 0.014). In summary, we suggest that expression level of ABCA1 gene may contribute to the development of atherosclerosis.

Asymmetric dimethylarginine (ADMA) is an endogenous competitive inhibitor of NO synthetase. Elevated ADMA levels are accompanied by impaired endothelium-dependent vasodilation in the brachial artery and increased intima-media thickness in the elastic arteries. The purpose of the study was to develop a procedure for qualitative and quantitative determination of the plasma content of free ADMA and symmetric dimethylarginine by reverse-phase high performance liquid chromatography in an isocratic elution mode, by applying an electrochemical detector. Ortho-phthalic aldehyde with the sulfur-containing component sodium sulfite was used as a derivation reagent. The mobile phase is a system that consists of acetonitrile, sodium hydrophosphate, and sodium dihydrophosphate. The retention time for the detectable substance on the column was 21.7 min. The total time of the analysis was 31 min. In patients with the clinical manifestations of atherosclerosis and in healthy individuals, the plasma ADMA concentration measured by the devised method was 0.69-0.30 and 0.13-0.04 microM, respectively.

Aim of the study was to elucidate possible role of adiponectin in development of clinico-metabolic derangements in metabolic syndrome (MS). We examined 35 men and 45 women (mean age 58.0+/-8.8 years). Adiponectin level was significantly lower (p<0.01) in patients with MS (5.8+/-4.6) compared with subjects without MS (9.2+/-5.3 mkg/ml). Body mass index (BMI), waist circumference (WC), index of insulin resistance , levels of glucose, insulin, free fatty acids (FFA), triglycerides (TG) were lower in 3rd tertile of distribution of adiponectin blood content compared with 1st tertile. Significant negative correlations were found between adiponectin content and BMI (r=-0.36), WC (r=-0.34), index (r=-0.31), level of diastolic arterial pressure (r=-0.34), concentrations of glucose (r=-0.24), insulin (r=-0.26), FFA (r=-0.24), TG (r=-0.45), uric acid (r=-0.29). Correlation between adiponectin and level of high density lipoprotein cholesterol was positive (I=0.26). However after adjustment for MI, sex, and age adiponectin content correlated significantly only with concentration of TG (r=-0.27) and diastolic arterial pressure (r=-0.26). Regression analysis revealed independent from factors presented above role of adiponectin in determination of blood level of TG (beta=-0.30, p=0.02).

Examination of low-density lipoprotein (LDL) receptor, its promoter, and major exon-intron boundaries from a sample of patients with familial hypercholesterolemia (FH) from 74 probands of St. Petersburg revealed 34 mutations and 8 widely spread polymorphisms at this locus. Only four mutations were considered silent, while the other 30 are likely associated with familial hypercholesterolemia (FH). Mutations in the LDL receptor gene, inducing the disease, were identified in 41 (55%) out of 74 families with FH. Mutation R3500Q in apolipoprotein B (APOB) gene was not detected in all probands. Therefore in the families lacking mutations hypercholesterolemia was induced by mutations in the introns of the LDL receptor gene or by other genetic factors. Nineteen mutations causing disease progression were described in St. Petersburg for the first time, while 18 of them are specific for Russia. Among Ashkenazi Jews, major mutation G197del was detected in 30% (7 out of 22) of patients with FH. In the Slavic population of St. Petersburg, no major mutations were detected. Only five mutations were identified in two families, while 24 were found in isolated families. These data are indicative of the lack of a strong founder effect for FH in the St. Petersburg population.

LDLP-antibody immune complexes and free LDLP were isolated from the serum and aortal wall of people who had died of myocardial infarction; their atherogenicity was compared on the basis of their capture by mouse peritoneal macrophages. The experiment showed that the capture of serum and aortal autoimmune LDPT-antibody complexes was approximately 2.5 times higher than that of free serum lipoproteins (mostly native ones). However, the capture of free (mostly modified) aortal LDLP was approximately 3.6 times higher than that of aortal immune complexes. According to the data received, the comparative atherogenicity of immune complexes and free lipoproteins can be ranked in the following order: modified LDLP --> modified LDLP-IgG complex --> native LDLP. The study shows that LDLP-antibody immune complexes formed in a rabbit's organism get extremely quickly eliminated from bloodstream, probably due to their active capture by reticuloendothelial system cells. The authors suggest that the formation of antilipoprotein autoantibodies and lipoprotein-antibody complexes is a protective response of the organism to appearance of highly atherogenic modified lipoproteins directed towards the weakening of the atherogenicity of such lipoproteins and the intensification of their elimination from bloodstream.

The concentration of high-density lipoprotein cholesterol and triglycerides in rat serum sharply decreased after psychic trauma caused by life hazard. The content of these substances remained unchanged for not less than 1 week after trauma. The concentration of high-density lipoprotein cholesterol was low, while serum content of triglycerides increased 6 weeks after trauma. The concentration of high-density lipoprotein cholesterol significantly decreased after repeated psychic trauma. These changes were accompanied by a sharp increase in the concentration of triglycerides in the serum. Total cholesterol concentration in the liver decreased under these conditions.

Background: Familial hypercholesterolemia is a human monogenic disease caused by population-specific mutations in the low density lipoprotein (LDL) receptor gene. Despite thirteen different mutations of the LDL receptor gene were reported from Russia prior to 2003, the whole spectrum of disease-causing gene alterations in this country is poorly known and requires further investigation provided by the current study., Methods: Forty-five patients with clinical diagnosis of FH were tested for the apolipoprotein B (apoB) mutation R3500Q by restriction fragment length analysis. After exclusion of R3500Q mutation high-sensitive fluorescent single-strand conformation polymorphism (SSCP) analysis and automatic DNA sequencing were used to search for mutations in the LDL receptor gene., Results: We found twenty one rare sequence variations of the LDL receptor gene. Nineteen were probably pathogenic mutations, and two (P518P, T705I) were considered as neutral ones. Among the mutations likely to be pathogenic, eight were novel (c.670-671insG, C249X, c.936-940del5, c.1291-1331del41, W422X, c.1855-1856insA, D601N, C646S), and eleven (Q12X, IVS3+1G>A, c.651-653del3, E207X, c.925-931del7, C308Y, L380H, c.1302delG, IVS9+1G>A, V776M, V806I) have already been described in other populations. None of the patients had the R3500Q mutation in the apoB gene., Conclusions: Nineteen pathogenic mutations in the LDL receptor gene in 23 probands were identified. Two mutations c.925-931del7 and L380H are shared by St.-Petersburg population with neighbouring Finland and several other mutations with Norway, Sweden or Denmark, i.e. countries from the Baltic Sea region. Only four mutations (c.313+1G>A, c.651-653del3, C308Y and W422X) were recurrent as all those were found in two unrelated families. By this study the number of known mutations in the LDL receptor gene in St.-Petersburg area was increased nearly threefold. Analysis of all 34 low density lipoprotein receptor gene mutations found in St.-Petersburg argues against strong founder effect in Russian familial hypercholesterolemia.

The cytopathogenic effect of various modified lipoproteins on cultured peritoneal macrophages was studied in vitro. After minor peroxide modification lipoproteins induced apoptosis of macrophages. Immune complexes containing oxidized lipoproteins caused cell necrosis.

A method for the simultaneous measurement of two biologically important thiol compounds cysteine and homocysteine and five amino acids including neurotransmitters aspartate and glutamate is reported. This method utilized derivatization of compounds with o-phthalaldehyde in the presence of 2-mercaptoethanol following alkylation of the free sulfydryl group with iodoacetic acid followed by separation using reversed-phase high-performance liquid chromatography. These o-phthalaldehyde-2-mercaptoethanol-labeled compounds were separated within 30 min on a Spherisorb ODS-2 column with isocratic elution using 17% methanol, 0.04 M sodium phosphate buffer (pH 7.0), 0.002 M Na2EDTA and detected fluorimetrically (excitation 340 nm, emission 450 nm). Using this method, the concentrations of homocysteine, cysteine, glutamic acid. aspartic acid, asparagine, serine and glutamine in human plasma were determined.

With the goal of developing non-viral techniques for exogenous gene delivery into mammalian cells, we have studied receptor-mediated gene transfer using complexes of plasmid DNA and galactosylated poly-L-lysine, poly(L-Lys)Gal. To evaluate the optimal parameters for efficient gene transfer into human hepatoma HepG2 cells by the DNA-poly(L-Lys)Gal complexes, the bacterial reporter genes lacZ and cat were used. Examination of the reporter gene expression level showed that the efficiency of DNA delivery into the cells depends on the structure of DNA--poly(L-Lys)Gal complexes formed at various ionic strength values. The efficiency of DNA transfer into the cells also depends on DNA/poly(L-Lys)Gal molar ratio in the complexes. Plasmid vector carrying human apolipoprotein A-I (apoA-I) gene was injected as its complex with poly(L-Lys)Gal into rat tail vein. Some level of ApoA-I was detected in the serum of the injected rats. Also, the human apoA-I-containing plasmid was found to be captured specifically by the rat liver cells and transported into the cell nuclei, where it can persist as an episome-like structure for at least a week. After repeated injections of DNA--poly(L-Lys)Gal complexes, the level of human ApoA-I in rat serum increases, probably, due to accumulation of functional human apoA-I gene in the liver cell nuclei. The data seem to be useful for the development of non-viral approaches to gene therapy of cardiovascular diseases.

Hyperhomocysteinemia is an independent risk factor for atherosclerosis and vascular occlusive diseases. Measurements of total homocysteine (Hcy) require an accurate reproducible method. A high-performance liquid chromatographic (HPCL) method for evaluating plasma Hcy after reduction with 2-mercaptoethanol and precolumn derivatization with iodoacetic acid and o-phthaldialdehyde is described. The resultant derivatives were separated on reverse-phase column C18 in the isocratic HPCL mode with fluorimetric detection. The detection limit for Hcy is 0.8 mumol/liter. The concentration of Hcy after overnight fasting was increased significantly (p < 0.02) in the patients in comparison with the control (11.7 +/- 1.1 vs. 8.3 +/- 0.5 mumol/liter). The method can be used for measuring the concentrations of asparaginic acid, glutamic acid, cysteine, asparagine, serine, and glutamine.

The influence of human and bovine serum albumin on the proton-mediated lysis of human, rabbit and sheep erythrocytes was studied. An essential acceleration of hemolysis (2-7-fold) by the action of albumin was observed only at pH values below 4.0, i.e. at values at which the transition of albumin to the E conformation occurs. It was shown in the experiments with fatty-acid-free albumin and the protein loaded by palmitate that the accelerating effect of albumin was due to the hemolytic action of fatty acids released from the protein. As a result of the proton potentiation of detergent-dependent hemolysis, fatty acids released from E-albumin become more potent hemolytic agents.

Familial hypercholesterolemia (FH), a monogenic disease known to be caused by low-density lipoprotein receptor (LDLR) gene mutations, results in the development of premature atherosclerosis and coronary artery disease in affected individuals. The spectrum of LDLR gene mutations in Russia is poorly known. Using polymerase chain reaction (PCR)-single-strand conformational polymorphism (SSCP) analysis, followed by DNA sequencing, we have screened selected exons of the LDLR gene in 80 unrelated St. Petersburg FH patients for the presence of mutations. Two new LDLR gene mutations, 347delGCC and E397X, were characterized among individuals with familial hypercholesterolemia in St. Petersburg. The carriers of both mutations possessed highly elevated blood serum cholesterol. Cosegregation of E397X mutation and LDLR gene RFLP haplotypes with hyperlipidemia was demonstrated by family study. Both mutations seem to be specific to Slavic patients., (Copyright 1998 Academic Press.)

Using PCR-single-strand conformation polymorphism analysis, followed by sequencing of the abnormal samples, two novel point mutations in the 5' end of the fourth exon of the low-density lipoprotein receptor gene were found in two Russian families with familial hypercholesterolemia. These missense mutations consist of C127W and C139G transitions and result in a loss of one of three disulfide bonds in the fourth cysteine-rich repeat of the ligand-binding domain of the low-density lipoprotein receptor. Hypercholesterolemia segregated with the identified mutations.

To study structure-function relationships in low density lipoprotein receptor (LDLR), a key protein in human cholesterol metabolism, it is reasonable to operate with separate protein domains. To obtain highly purified functionally active LDLR ligand-binding domain, we have cloned the corresponding LDLR cDNA fragment in two expression plasmid vectors of Escherichia coli. We have developed methods to purify fusion and practically individual recombinant proteins and characterized the obtained products biochemically. Antibodies raised against fused with beta-galactosidase and individual recombinant protein have been shown to be efficient in identification of LDLR protein in crude lysates of human fibroblasts (cell line HT-1080).

Offspring of rabbits fed with cholesterol diet during pregnancy and/or lactation was more sensitive to fat load versus the offspring of rabbits fed with standard diet. Such enhancement of hyperlipidemic reaction is predominantly due to the enhanced rate of blood cholesterol increase (i.e., secretion of cholesterol-containing lipoproteins) but not to the decreased rate of cholesterol elimination from the blood stream. This was confirmed by monitoring the fate of human low-density lipoproteins intravenously injected in rabbits. The data indicate certain alterations of cholesterol metabolism regulation in the offspring of rabbits with alimentary hypercholesterolemia. Hypercholesterolemia of pregnant and lactating rabbits is important for the formation of the lipid metabolism in their offspring because cholesterol levels in the serum of female rabbits positively correlate with the expression of hypercholesterolemic and hypertriglyceridemia reactions of their offspring in response to fat load and with kinetic parameters which characterize the increase in cholesterol and its elimination from the blood stream during fat load.

Immunomodulating drug tactivin was used for treatment of 29 IHD patients with low T-suppressor activity and high cell sensitization to apoprotein B-containing lipoproteins and with lipoprotein-antibody immune complex detected in the blood. Tactivin was injected subcutaneously in the dose 10 micrograms (5 injections for 12 days). Tactivin treatment normalized T-suppressor activity, lowered concentration of plasma lipoprotein-antibody immune complex and improved clinical course of IHD. The beneficial effect of tactivin was observed in 70% of patients during 2 months. 30% of patients needed a second course of treatment of recover normal immunological and clinical indices. The treatment with tactivin of IHD patients must be combined with application of routine antianginal drugs.

The efficacy of extracorporeal cryohemosorption was estimated in the treatment of atherosclerotic patients in terms of the autoimmune theory of the pathogenesis of this disease. There was a slight decrease in the plasma levels of total cholesterol and triglycerides, there was over 2-fold reduction in the plasma levels of fibrinogen and fibronectin. All major components of apo B-containing lipoproteins and immunoglobulin G were found as part of the cryoprecipitate. An extremely high concentration of lipid hydroperoxides in the precipitate suggest that cryoprecipitation removes not only autoimmune complexes, but highly-atherogenic peroxide-modified lipoproteins. The three-fold decrease in the levels of lipoprotein-antibody complexes resulted in lower atherogenicity of apo B-containing lipoproteins. It is suggested that the mechanism responsible for beneficial clinical action of cryohemosorption sessions is largely associated with the removal of autoimmune lipoprotein-antibody complexes and peroxide-modified apo B-containing lipoproteins than correction of lipid metabolism.

Jejunoileostomy effects on blood lipids and cardiovascular system were investigated in obese patients. They were found to lose 30% of their body mass within 6 postoperative months with parallel reduction in concentrations of total cholesterol, triglycerides, LDL and VLDL cholesterol and apoprotein B. These changes persisted for 4.5 years of the follow-up. The surgery in males has a more pronounced effect on blood lipids than in females. Significant changes were not registered in post- and preoperative levels of HDL cholesterol and apolipoprotein A-1. Jejunoileostomy promoted a fall in blood pressure, a rise in exercise tolerance, alleviation of atherosclerosis symptoms.

Intravenous ethyl oleate-induced blockade of the reticulo-endothelial apparatus (REA) in rabbits was found to reduce the elimination rate of both native and 125I-modified (acetylated) low lipoproteins. Lower lipoprotein catabolism is likely to result in hyperlipidemia observed in REA blockade. At the same time the latter was shown to induce the changes in low lipoproteins, which diminished their capacity to interact with fibroblasts, on the one hand, and to stimulate their macrophage uptake.

The level of lipids and apolipoproteins was studied in the serum of patients with various degrees of obesity without hormonal impairments in order to find interrelationship between obesity and atherosclerosis development. 14 men and 42 women aged 19-59 years were examined. Concentration of triglycerides and very low density lipoprotein (VLDL) cholesterol was distinctly higher in obese patients of both sexes than in normal weight persons. However, content of apo A-I and apo B was similar in blood of both obese and normal persons, while concentration of apo E was higher in obesity. In obesity, the ratio HDL cholesterol/A-I was decreased, thus indicating that the HDL3/HDL2 ratio was altered, showing a decreased content of the latter subfraction. The findings suggest that impairment in metabolism of mainly HDL and VLDL subfractions occurred in patients with obesity. These impairments were atherogenic and may be responsible for a high risk for atherosclerosis and heart ischemic disease in obese patients.

A comprehensive clinicoimmunological examination was made of 105 male patients aged 35-65 years who suffered from coronary heart disease. The authors identified a group of 33 patients whose unstable course of angina pectoris was associated with pronounced apolipoprotein B sensitization, as well as with the presence of the autoimmune lipoprotein-antibody complex and abnormalities in the cellular link of immunity. All 33 patients received antianginal agents. Out of them, 15 patients took additionally a course of T-activin therapy to modulate immunological shifts. The results of the examination demonstrated that the immunomodulator exerted a beneficial effect both on the course of CHD (reduction in the number of anginal episodes, improvement of left ventricular contractility) and the immune system (recovery of T-suppressor function, disappearance of lipoprotein sensitization, decrease in the patient's blood detection rates of the autoimmune lipoprotein-antibody complexes from 83 to 28%). The highest effect was reached by T-activin 1.5-2 months following termination of the course therapy. Immunomodulating therapy is regarded as an additional approach to the treatment of CHD patients with marked lipoprotein sensitization.

It is shown that lipoproteins possessing autoantigenic properties may appear in blood serum of patients with atherosclerosis complicated by coronary disease. Developing sensitization to these lipoproteins, accompanied by the formation of an autoimmune complex may be the principal factor in the formation of arterial atherosclerotic lesions. Sensitization to vascular wall antigens is viewed as a factor contributing to rapid progress of atherosclerosis.

The methods for isolation of pure apolipoproteins A-I, A-II and E from the blood plasma of donors for preparation of monospecific rabbit antisera against these apolipoproteins and their estimation in human blood plasma using immunoelectrophoresis are described. It was found that the average content of apolipoprotein A-I (apo A-I) in the blood plasma of healthy males is 126.6 mg%, that of apolipoprotein A-II (apo A-II) is 56.8 mg%, that of apolipoprotein E (apo E) is 10.2 mg%. The apo A-I content in blood plasma is increased in hyper-alpha-lipoproteinemic patients and is decreased in hypo-alpha-lipoproteinemic ones, i. e. there is a direct relationship between the changes in concentration of high density lipoproteins (HDL) and apo A-I. The concentration of apo A-II in dis-alpha-lipoproteinemias varies within a narrow range. A considerable increase of the alpha-cholesterol/apo A-I ratio suggesting an increased capacity of HDL to transport cholesterol in hyper-alpha-lipoproteinemic patients is observed. There exists an indirect correlation between the changes in the contents of apo A-I and apo E in dis-alpha-lipoproteinemic patients.

An immune complex of lipoprotein-antibody was found in human blood plasma and in tissue liquid of aorta, impaired by atherosclerosis. In the majority of cases the antigen component of the immune complex was lipoprotein of very low density. After dissociation of the complex at acid pH, immunoglobulin G was isolated, which was identified by double immunodiffusion in agar gel; the immunoglobulin reacted in RBS both with autological and, in some cases, with isological lipoproteins.

Electrophoresis in the polyacrylamide gel and ultracentrifugation with a multistage density gradient demonstrated virtually the whole of the blood plasma lipoproteids spectrum to be contained in the human aortal wall. An immunological identity of the whole beta- and alpha-lipoproteids and also of their individual apoproteins in the aortal wall and blood plasma was ascertained. Moreover, the ratio of etherified cholesterol/total cholesterol in the vascular and plasmatic lipoproteids proved to be quiet close. In determining the content of the summary pre-beta-and beta-lipoproteids in the aortal wall the quantity of the said lipoproteids in atherosclerotic lesions was found several times as high as in a normal aorta, whereas the lipoproteids concentration in an intact aortal wall correlated with the intensity of atherosclerosis in the same aorta. The data obtained bear evidence to a plasmatic origin of lipoproteids in the vascular wall and to their important role in atherogenesis.

The immunocompetent tissues were studied on the clinical and necropsy material, taking into consideration the development of sensitization to the atheorogenic lipids (VLDL, LDL). The signs of the sensitization to VLDL or LDL were observed in 83% patients. It is shown that the T-type immune response resulting from the sensitization to the lipoproteins is formed when the exacerbation of the atherosclerosis occurs. The vascular wall antigens in the lymph nodes regional to the arteries produce a local B-type immune response. The leucocyte migration test in combination with the staining of the blood smears for cation proteins can be used in the clinical practice for the detection of the atherosclerosis patients with a specific sensitization.

Immune complexes consisting of human [125I]LDL or [125I]VLDL and anti-apo B IgG were prepared in vitro. After intravenous administration of these complexes or free LDL to normal rabbits the elimination rate for complexes was 2-3-fold higher than for free lipoproteins. The liver/spleen radioactivity ratio after administration of immune complexes was 34% less than after administration of free lipoproteins. Investigations carried out on the cell cultures have shown that during 4-h incubation the uptake of [125I]LDL-anti-apo B IgG complex by human lung fibroblasts was lower than uptake of free [125I]LDL, whereas mouse peritoneal macrophages took up immune complex more actively than free LDL. During 3 days of incubation of macrophages with LDL-anti-apo B IgG the transformation of macrophages into foam cells was observed. This process was accompanied by almost a 60-fold increase of cholesteryl ester content in the cell. It is suggested that excessive uptake of lipoprotein-antibody complexes by macrophages leading to formation of foam cells may play an important role in atherogenesis.

A study was made of a fatty acid composition of individual lipid fractions in the lipoproteids of the blood serum of a very low, low and high density, and the wall of the aorta affected with atherosclerosis. There was a close similarity between a fatty acid composition of phospholipids, triglycerides, and cholesterol esters of all the lipoproteid classes of the vascular wall and blood plasma. The fatty acid compostion of individual fractions proved to be similar in all the lipoproteid fractions. The lipids of the vascular wall not included into the lipoproteid composition differed considerably by their fatty acid composition from the lipids isolated from lipoproteids; lipids of the wall of the aorta contained much less unsaturated fatty acids than lipoproteids.

The content of A1, A2, and E apolipoproteins in blood plasma of males (40-59 years of age) with normo-, hyper-, and hypoalphalipoproteinemia was determined by rocket immunoelectrophoresis. Considerable changes were revealed in the alphalipoprotein cholesterol/A1-apo ratio which were evidence of increased capacity of HDLP for transporting cholesterol in hyperalphalipoproteinemia and a decrease in this HDLP function in hypoalphalipoproteinemia irrespective of their concentration in blood plasma. A reverse correlative connection was revealed between the A1-apo content and the E-apo content in dysalphalipoproteinemia. It is suggested that the changes in the content of A1-apo and E-apo in blood plasma are significant in lipoprotein metabolism in dysalphalipoproteinemia.

It is shown that the LDL peroxide lipid products level is 1.6-4 times higher in patients with the ischemic heart diseases (IHD) than in normal subjects. At the same time the LDL uptake by macrophages was identical in case of normal subjects and of IHD patients. It is suggested that the LDL premodification in the blood flow in the IHD patients promotes the further modification of LDL particles after their penetration into the vessel wall.