Alexandra A. Melnichenko

Modified Low Density Lipoprotein and Lipoprotein-Containing Circulating Immune Complexes as Diagnostic and Prognostic Biomarkers of Atherosclerosis and Type 1 Diabetes Macrovascular Disease

In atherosclerosis; blood low-density lipoproteins (LDL) are subjected to multiple enzymatic and non-enzymatic modifications that increase their atherogenicity and induce immunogenicity. Modified LDL are capable of inducing vascular inflammation through activation of innate immunity; thus, contributing to the progression of atherogenesis. The immunogenicity of modified LDL results in induction of self-antibodies specific to a certain type of modified LDL. The antibodies react with modified LDL forming circulating immune complexes. Circulating immune complexes exhibit prominent immunomodulatory properties that influence atherosclerotic inflammation. Compared to freely circulating modified LDL; modified LDL associated with the immune complexes have a more robust atherogenic and proinflammatory potential. Various lipid components of the immune complexes may serve not only as diagnostic but also as essential predictive markers of cardiovascular events in atherosclerosis. Accumulating evidence indicates that LDL-containing immune complexes can also serve as biomarker for macrovascular disease in type 1 diabetes.

Anti-Atherosclerotic Therapy Based on Botanicals

Natural products including botanicals for both therapy of clinical manifestations of atherosclerosis and reduction of atherosclerosis risk factors are topics of recent patents. Only a few recent patents are relevant to the direct antiatherosclerotic therapy leading to regression of atherosclerotic lesions. Earlier, using a cellular model we have developed and patented several anti-atherosclerotic drugs. The AMAR (Atherosclerosis Monitoring and Atherogenicity Reduction) study was designed to estimate the effect of two-year treatment with time-released garlic-based drug Allicor on the progression of carotid atherosclerosis in 196 asymptomatic men aged 40-74 in double-blinded placebo-controlled randomized clinical study. The primary outcome was the rate of atherosclerosis progression, measured by high-resolution B-mode ultrasonography as the increase in carotid intima-media thickness (IMT) of the far wall of common carotid arteries. The mean rate of IMT changes in Allicor-treated group (-0.022±0.007 mm per year) was significantly different (P = 0.002) from the placebo group in which there was a moderate progression of 0.015±0.008 mm at the overall mean baseline IMT of 0.931±0.009 mm. A significant correlation was found between the changes in blood serum atherogenicity (the ability of serum to induce cholesterol accumulation in cultured cells) during the study and the changes in intima-media thickness of common carotid arteries (r = 0.144, P = 0.045). Thus, the results of AMAR study demonstrate that long-term treatment with Allicor has a direct anti-atherosclerotic effect on carotid atherosclerosis and this effect is likely to be due to serum atherogenicity inhibition. The beneficial effects of other botanicals including Inflaminat (calendula, elder and violet), phytoestrogen- rich Karinat (garlic powder, extract of grape seeds, green tea leafs, hop cones, β-carotene, α-tocopherol and ascorbic acid) on atherosclerosis have also been revealed in clinical studies which enforces a view that botanicals might represent promising drugs for anti-atherosclerotic therapy.

Low density lipoprotein-containing circulating immune complexes : role in atherosclerosis and diagnostic value

It has been suggested that low density lipoprotein-containing circulating immune complexes (LDL-CIC) play a role in atherogenesis and are involved in the formation of early atherosclerotic lesion. These complexes, as well as anti-LDL autoantibodies, have been found in the blood and in the atherosclerotic lesions of patients with different cardiovascular diseases, as well as in the blood of animals with experimental atherosclerosis. It can be suggested that the presence of anti-LDL antibodies in the blood is a result of immune response induced by lipoprotein modification. LDL-CIC differs from native LDL in many aspects. It has much lower sialic acid content, smaller diameter, and higher density and is more electronegative than native LDL. Fraction of LDL-CICs is fundamental to the serum atherogenicity manifested at the cellular level. LDL-CIC, unlike native LDL, is able to induce intracellular accumulation of neutral lipids, especially esterified cholesterol, in cells cultured from uninvolved human aortic intima and in macrophage cultures. After removal of LDL-CIC, the CHD patients sera lose their atherogenic properties. Titer of LDL-CIC in blood serum significantly correlates with progression of atherosclerosis in human in vivo and has the highest diagnostic value among other measured serum lipid parameters. Elevated CIC-cholesterol might well be a possible risk factor of coronary atherosclerosis.

Mechanisms of foam cell formation in atherosclerosis

Low-density lipoprotein (LDL) and cholesterol homeostasis in the peripheral blood is maintained by specialized cells, such as macrophages. Macrophages express a variety of scavenger receptors (SR) that interact with lipoproteins, including SR-A1, CD36, and lectin-like oxLDL receptor-1 (LOX-1). These cells also have several cholesterol transporters, including ATP-binding cassette transporter ABCA1, ABCG1, and SR-BI, that are involved in reverse cholesterol transport. Lipids internalized by phagocytosis are transported to late endosomes/lysosomes, where lysosomal acid lipase (LAL) digests cholesteryl esters releasing free cholesterol. Free cholesterol in turn is processed by acetyl-CoA acetyltransferase (ACAT1), an enzyme that transforms cholesterol to cholesteryl esters. The endoplasmic reticulum serves as a depot for maintaining newly synthesized cholesteryl esters that can be processed by neutral cholesterol ester hydrolase (NCEH), which generates free cholesterol that can exit via cholesterol transporters. In atherosclerosis, pro-inflammatory stimuli upregulate expression of scavenger receptors, especially LOX-1, and downregulate expression of cholesterol transporters. ACAT1 is also increased, while NCEH expression is reduced. This results in deposition of free and esterified cholesterol in macrophages and generation of foam cells. Moreover, other cell types, such as endothelial (ECs) and vascular smooth muscle cells (VSMCs), can also become foam cells. In this review, we discuss known pathways of foam cell formation in atherosclerosis.

Small Dense Low-Density Lipoprotein as Biomarker for Atherosclerotic Diseases

Low-density lipoprotein (LDL) plays a key role in the development and progression of atherosclerosis and cardiovascular disease. LDL consists of several subclasses of particles with different sizes and densities, including large buoyant (lb) and intermediate and small dense (sd) LDLs. It has been well documented that sdLDL has a greater atherogenic potential than that of other LDL subfractions and that sdLDL cholesterol (sdLDL-C) proportion is a better marker for prediction of cardiovascular disease than that of total LDL-C. Circulating sdLDL readily undergoes multiple atherogenic modifications in blood plasma, such as desialylation, glycation, and oxidation, that further increase its atherogenicity. Modified sdLDL is a potent inductor of inflammatory processes associated with cardiovascular disease. Several laboratory methods have been developed for separation of LDL subclasses, and the results obtained by different methods can not be directly compared in most cases. Recently, the development of homogeneous assays facilitated the LDL subfraction analysis making possible large clinical studies evaluating the significance of sdLDL in the development of cardiovascular disease. Further studies are needed to establish guidelines for sdLDL evaluation and correction in clinical practice.

How do macrophages sense modified low-density lipoproteins?

In atherosclerosis, serum lipoproteins undergo various chemical modifications that impair their normal function. Modification of low density lipoprotein (LDL) such as oxidation, glycation, carbamylation, glucooxidation, etc. makes LDL particles more proatherogenic. Macrophages are responsible for clearance of modified LDL to prevent cytotoxicity, tissue injury, inflammation, and metabolic disturbances. They develop an advanced sensing arsenal composed of various pattern recognition receptors (PRRs) capable of recognizing and binding foreign or altered-self targets for further inactivation and degradation. Modified LDL can be sensed and taken up by macrophages with a battery of scavenger receptors (SRs), of which SR-A1, CD36, and LOX1 play a major role. However, in atherosclerosis, lipid balance is deregulated that induces inability of macrophages to completely recycle modified LDL and leads to lipid deposition and transformation of macrophages to foam cells. SRs also mediate various pathogenic effects of modified LDL on macrophages through activation of the intracellular signaling network. Other PRRs such Toll-like receptors can also interact with modified LDL and mediate their effects independently or in cooperation with SRs.

Pluronic block copolymers inhibit low density lipoprotein self-association

Little is known about exogenous inhibitors of low-density lipoprotein (LDL) aggregation. The search for nontoxic and bioavailable inhibitors of LDL aggregation is of interest, especially considering that the suppression of the aggregation of LDL might represent a therapeutic approach. We hypothesized that amphiphilic copolymers of propylene oxide and ethylene oxide, the so-called Pluronic block copolymers, can be used to influence the aggregation of LDL. In this work we used Pluronic® P85, L61 and F68. A comparative study of the effects of Pluronic block copolymers with various hydrophilic–lipophilic properties on the aggregation process of LDL showed that Pluronic copolymers with strong hydrophobic properties (P85 and L61) at concentrations close to or greater than the respective critical concentration of micelle formation inhibited the aggregation process of LDL; however, the “hydrophilic” Pluronic F68 had no effect on the aggregation of LDL at any concentration. Thus, the study demonstrated for the first time that Pluronic® block copolymers inhibit LDL self-association. The possibility of modulating the aggregation of LDL by various Pluronic copolymers can be regarded as a prerequisite in the creation of new types of anti-atherosclerotic drugs.

The role of mitochondrial dysfunction in cardiovascular disease: a brief review

Abstract Cardiovascular disease (CVD) is a leading cause of mortality worldwide. Proper mitochondrial function is necessary in tissues and organs that are of high energy demand, including the heart. Mitochondria are very sensitive to nutrient and oxygen supply and undergo metabolic adaptation to the changing environment. In CVD, such an adaptation is impaired, which, in turn, leads to a progressive decline of the mitochondrial function associated with abnormalities in the respiratory chain and ATP synthesis, increased oxidative stress, and loss of the structural integrity of mitochondria. Uncoupling of the electron transport chain in dysfunctional mitochondria results in enhanced production of reactive oxygen species, depletion of cell ATP pool, extensive cell damage, and apoptosis of cardiomyocytes. Mitophagy is a process, during which cells clear themselves from dysfunctional and damaged mitochondria using autophagic mechanism. Deregulation of this process in the failing heart, accumulation of dysfunctional mitochondria makes the situation even more adverse. In cardiac pathology, aberrations of the activity of the respiratory chain and ATP production may be considered as a core of mitochondrial dysfunction. Indeed, therapeutic restoration of these key functional properties can be considered as a primary goal for improvement of mitochondrial dysfunction in CVD. Key messages Mitochondrial dysfunction plays a crucial role in cardiovascular disease pathogenesis. Cardiovascular disease is associated with altered mithochondrial biogenesis and clearance. In cardiovascular disease, impaired mitochondrial function results in decreased ATP production and enhanced ROS formation.

The Interaction of Plasma Sialylated and Desialylated Lipoproteins with Collagen from the Intima and Media of Uninvolved and Atherosclerotic Human Aorta

We have evaluated the binding of sialylated and desialylated lipoproteins to collagen isolated from the proteoglycan and musculoelastic layers of intima and media of uninvolved human aorta and atherosclerotic lesions. Comparing various collagen preparations from the uninvolved intima-media, the binding of sialylated apoB-containing lipoproteins was best to collagen from the intimal PG-rich layer. Binding of sialylated apoB-containing lipoproteins to collagen from this layer of fatty streak and fibroatheroma was 1.4- and 3.1-fold lower, respectively, in comparison with normal intima. Desialylated VLDL versus sialylated one exhibited a greater binding (1.4- to 3.0-fold) to all the collagen preparations examined. Desialylated IDL and LDL showed a higher binding than sialylated ones when collagen from the intimal layers of fibroatheroma was used. Binding of desialylated HDL to collagen from the intimal PG-rich layer of normal tissue, initial lesion, and fatty streak was 1.2- to 2.0-fold higher compared with sialylated HDL.

Desialylation Decreases the Resistance of Apo B-Containing Lipoproteins to Aggregation and Increases Their Atherogenic Potential

Subfractions of apo B-containing lipoproteins (VLDL and intermediate-density lipoproteins) with reduced content of sialic acid were found in human blood. These lipoproteins are characterized by high capacity to spontaneous association (aggregation) and stimulated accumulation of cholesterol in smooth muscle cells of human aortic intima. In vitro treatment of apo B-containing lipoproteins with α-2,6-sialidase and α-2,3-sialidase stimulated aggregation and increased the ability of these particles to potentiate cholesterol accumulation in smooth muscle cells of the intact human aortic intima. Probably, desialylation of various apo B-containing lipoproteins can occur in the blood; this process decreases their resistance to aggregation, and increases the ability of these particles to stimulate accumulation of cholesterol in human aortic intima cells, i.e. increases their atherogenic potential.