Current Medicinal Chemistry - Volume 26, Issue 9, 2019

Atherosclerotic diseases are the leading cause of mortality in industrialized countries. Correspondingly, studying the pathogenesis of atherosclerosis and developing new methods for its diagnostic and treatment remain in the focus of current medicine and health care. This review aims to discuss the mechanistic role of low-density lipoprotein (LDL) and high-density lipoprotein (HDL) in atherogenesis. In particular, the generally accepted hypothesis about the key role of oxidized LDL in atherogenesis is questioned, and an alternative concept of multiple modification of LDL is presented. The fundamental question discussed in this review is whether LDL and HDL are effectors or biomarkers, or both. This is important for understanding whether lipoproteins are a therapeutic target or just diagnostic indicators.

Background: Dyslipidaemias is a recognized risk factor for atherosclerosis, however, new evidence brought to light by trials investigating therapies to enhance HDLcholesterol have suggested an increased atherosclerotic risk when HDL-C is high. Results: Several studies highlight the central role in atherosclerotic disease of dysfunctional lipoproteins; oxidised LDL-cholesterol is an important feature, according to “oxidation hypothesis”, of atherosclerotic lesion, however, there is today a growing interest for dysfunctional HDL-cholesterol. The target of our paper is to review the functions of modified and dysfunctional lipoproteins in atherogenesis. Conclusion: Taking into account the central role recognized to dysfunctional lipoproteins, measurements of functional features of lipoproteins, instead of conventional routine serum evaluation of lipoproteins, could offer a valid contribution in experimental studies as in clinical practice to stratify atherosclerotic risk.

The “HDL hypothesis” which suggested that an elevation in HDL cholesterol (HDL-C) levels by drugs or by life style changes should be paralleled by a decrease in the risk for Cardiovascular Disease (CVD) has been challenged by recent epidemiological and clinical studies using HDL-raising drugs. HDL components such as proteins, lipids or small RNA molecules, but not cholesterol itself, possess various atheroprotective functions in different cell types and accumulating evidence supports the new hypothesis that HDL functionality is more important than HDL-C levels for CVD risk prediction. Thus, the detailed characterization of changes in HDL composition and functions in various pathogenic conditions is critically important in order to identify new biomarkers for diagnosis, prognosis and therapy monitoring of CVD. Here we provide an overview of how HDL composition, size and functionality are affected in patients with monogenic disorders of HDL metabolism due to mutations in genes that participate in the biogenesis and the remodeling of HDL. We also review the findings from various mouse models with genetic disturbances in the HDL biogenesis pathway that have been generated for the validation of the data obtained in human patients and how these models could be utilized for the evaluation of novel therapeutic strategies such as the use of adenovirus-mediated gene transfer technology that aim to correct HDL abnormalities.

Oxidatively modified low-density lipoprotein (oxLDL) is known to be involved in various diseases, including cardiovascular diseases. The presence of oxLDL in the human circulatory system and in atherosclerotic lesions has been demonstrated using monoclonal antibodies. Studies have shown the significance of circulating oxLDL in various systemic diseases, including acute myocardial infarction and diabetic mellitus. Several different enzyme-linked immunosorbent assay (ELISA) procedures to measure oxLDL were utilized. Evidence has been accumulating that reveals changes in oxLDL levels under certain pathological conditions. Since oxLDL concentration tends to correlate with low-density lipoprotein (LDL)-cholesterol, the ratio of ox-LDL and LDL rather than oxLDL concentration alone has also been focused. In addition to circulating plasma, LDL and oxLDL are found in gingival crevicular fluid (GCF), where the ratio of oxLDL to LDL in GCF is much higher than in plasma. LDL and oxLDL levels in GCF show an increase in diabetic patients and periodontal patients, suggesting that GCF might be useful in examining systemic conditions. GCF oxLDL increased when the teeth were affected by periodontitis. It is likely that oxLDL levels in plasma and GCF could reflect oxidative stress and transfer efficacy in the circulatory system.

Over the last century, many studies have demonstrated that low-density lipoprotein (LDL) is a key risk factor of cardiovascular diseases (CVD) related to atherosclerosis. Thus, for these CVD patients, LDL lowering agents are commonly used in the clinic to reduce the risk for CVD. LDL, upon modification, will develop distinct inflammatory and proatherogenic potential, leading to impaired endothelial integrity, influx of immune cells and subsequent increased foam cell formation. LDL can also directly affect peripheral monocyte composition, rendering them in a more favorable position to migrate and accumulate in the subendothelial space. It has become apparent that other lipoprotein particles, such as triglyceride- rich lipoproteins or remnants (TRL) and lipoprotein(a) [Lp(a)] may also impact on atherogenic pathways. Evidence is accumulating that Lp(a) can promote peripheral monocyte activation, eventually leading to increased transmigration through the endothelium. Similarly, remnant cholesterol has been identified to play a key role in endothelial dysfunction and monocyte behavior. In this review, we will discuss recent developments in understanding the role of different lipoproteins in the context of inflammation at both the level of the monocyte and the endothelium.

Hypercholesterolemia is one of the main risk factors for the development of atherosclerosis. Among the various lipoprotein classes, however, high density lipoproteins (HDL) are inversely associated with the incidence of atherosclerosis, since they are able to exert a series of atheroprotective functions. The central role of HDL within the reverse cholesterol transport, their antioxidant and anti-inflammatory properties and their ability to preserve endothelial homeostasis are likely responsible for HDL-mediated atheroprotection. However, drugs that effectively raise HDL-C failed to result in a decreased incidence of cardiovascular event, suggesting that plasma levels of HDL-C and HDL function are not always related. Several evidences are showing that different pathologic conditions, especially those associated with an inflammatory response, can cause dramatic alterations of HDL protein and lipid cargo resulting in HDL dysfunction. Established and investigational drugs designed to affect lipid metabolism and to increase HDL-C are only partly effective in correcting HDL dysfunction.

Protein catabolism in macrophages, which is accomplished mainly through autophagy- lysosomal degradation, ubiquitin-proteasome system, and calpains, is disturbed in atheroprone vessels. Moreover, growing evidence suggests that defects in protein catabolism interfere with cholesterol handling in macrophages. Indeed, decreases in autophagy facilitate the deposition of cholesterol in atheroprone macrophages and the subsequent development of vulnerable atherosclerotic plaques due to impaired catabolism of lipid droplets and limited efferocytic clearance of dead cells. The proteasome is responsible for the degradation of ATP-binding cassette transporters, which leads to impaired cholesterol efflux from macrophages. Overactivation of conventional calpains contributes to excessive processing of functional proteins, thereby accelerating receptor-mediated uptake of oxidized low-density lipoproteins (LDLs) and slowing cholesterol efflux. Furthermore, calpain-6, an unconventional nonproteolytic calpain in macrophages, potentiates pinocytotic uptake of native LDL and attenuates the efferocytic clearance of dead cells. Herein, we focus on recent progress in understanding how defective protein catabolism is associated with macrophage cholesterol handling and subsequent atherogenesis.

Epidemiological studies have suggested an inverse correlation between high-density lipoprotein (HDL) cholesterol levels and the risk of cardiovascular disease. HDLs promote reverse cholesterol transport (RCT) and possess several putative atheroprotective functions, associated to the anti-inflammatory, anti-thrombotic and anti-oxidant properties as well as to the ability to support endothelial physiology. The assumption that increasing HDL-C levels would be beneficial on cardiovascular disease (CVD), however, has been questioned as, in most clinical trials, HDL-C-raising therapies did not result in improved cardiovascular outcomes. These findings, together with the observations from Mendelian randomization studies showing that polymorphisms mainly or solely associated with increased HDL-C levels did not decrease the risk of myocardial infarction, shift the focus from HDL-C levels toward HDL functional properties. Indeed, HDL from atherosclerotic patients not only exhibit impaired atheroprotective functions but also acquire pro-atherogenic properties and are referred to as “dysfunctional” HDL; this occurs even in the presence of normal or elevated HDL-C levels. Pharmacological approaches aimed at restoring HDL functions may therefore impact more significantly on CVD outcome than drugs used so far to increase HDL-C levels. The aim of this review is to discuss the pathological conditions leading to the formation of dysfunctional HDL and their role in atherosclerosis and beyond.

Low-density lipoproteins (LDLs) are the major plasma carriers of cholesterol. However, LDL particles must undergo various molecular modifications to promote the development of atherosclerotic lesions. Modified LDL can be generated by different mechanisms, but as a common trait, show an increased electronegative charge of the LDL particle. A subfraction of LDL with increased electronegative charge (LDL(-)), which can be isolated from blood, exhibits several pro-atherogenic characteristics. LDL(-) is heterogeneous, due to its multiple origins but is strongly related to the development of atherosclerosis. Nevertheless, the implication of LDL(-) in a broad array of pathologic conditions is complex and in some cases anti-atherogenic LDL(-) properties have been reported. In fact, several molecular modifications generating LDL(-) have been widely studied, but it remains unknown as to whether these different mechanisms are specific or common to different pathological disorders. In this review, we attempt to address these issues examining the most recent findings on the biology of LDL(-) and discussing the relationship between this LDL subfraction and the development of different diseases with increased cardiovascular risk. Finally, the review highlights the importance of minor apolipoproteins associated with LDL(-) which would play a crucial role in the different properties displayed by these modified LDL particles.

Modified forms of LDL, both spontaneously formed in the organism or prepared in the laboratory, are immunogenic. As a consequence, antigen-antibody complexes (immune complexes, IC) formed in vivo can be measured in the peripheral blood, and their levels are strong predictors of cardiovascular disease (CVD). It has been possible to generate antibodies that recognize different LDL modifications, allowing the analysis of circulating IC constitution. Clinical studies showed that the antigenic constitution of the IC has a modulating effect on the development of CVD. Patients whose IC react strongly with antibodies to copper oxidized LDL (oxLDL) show progressive development of atherosclerosis as demonstrated by increased intima–media thickness and increased coronary calcification scores. In contrast, patients whose IC react strongly with antibodies to the heavily oxidized malondialdehyde LDL prepared in vitro (MDA-LDL) are at a high risk of acute vascular events, mainly myocardial infarction. In vitro studies have shown that while oxLDL IC induce both cell proliferation and mild to moderate macrophage apoptosis, MDA-LDL IC induce a more marked macrophage apoptosis but not cell proliferation. In addition, MDA-LDL IC induce the release of higher levels of matrix metalloproteinases and TNF than oxLDL IC. High levels of TNF are likely to be a major factor leading to apoptosis and high levels of metalloproteinases are likely to play a role in the thinning of the fibrous cap of the atheromatous plaque. The combination of apoptosis and fibrous cap thinning is a well-known characteristic of vulnerable plaques, which are more prone to rupture and responsible for the majority of acute cardiovascular events.

Oxidized LDL (ox-LDL) plays a central role in atherosclerosis by acting on multiple cells such as endothelial cells, macrophages, platelets, fibroblasts and smooth muscle cells through LOX-1. LOX-1 is a 50 kDa transmembrane glycoprotein that serves as receptor for ox-LDL, modified lipoproteins, activated platelets and advance glycation end-products. Ox- LDL through LOX-1, in endothelial cells, causes increase in leukocyte adhesion molecules, activates pathways of apoptosis, increases reactive oxygen species and cause endothelial dysfunction. In vascular smooth muscle cells and fibroblasts, they stimulate proliferation, migration and collagen synthesis. LOX-1 expressed on macrophages inhibit macrophage migration and stimulate foam cell formation. They also stimulate generation of metalloproteinases and contribute to plaque instability and thrombosis. Drugs that modulate LOX-1 are desirable targets against atherosclerosis. Many naturally occurring compounds have been shown to modulate LOX-1 expression and atherosclerosis. Currently, novel drug design techniques are used to identify molecules that can bind to LOX-1 and inhibit its activation by ox-LDL. In addition, techniques using RNA interference and monoclonal antibody against LOX-1 are currently being investigated for clinical use.

Apolipoprotein B –containing lipoproteins include triglyceride-rich lipoproteins (chylomicrons and their remnants, and very low-density lipoproteins and their remnants) and cholesterol-rich low-density lipoprotein particles. Of these, lipoproteins having sizes below 70-80 nm may enter the arterial wall, where they accumulate and induce the formation of atherosclerotic lesions. The processes that lead to accumulation of lipoprotein-derived lipids in the arterial wall have been largely studied with a focus on the low-density lipoprotein particles. However, recent observational and genetic studies have discovered that the triglyceriderich lipoproteins and their remnants are linked with cardiovascular disease risk. In this review, we describe the potential mechanisms by which the triglyceride-rich remnant lipoproteins can contribute to the development of atherosclerotic lesions, and highlight the differences in the atherogenicity between low-density lipoproteins and the remnant lipoproteins.