[Frontiers In Bioscience, Landmark, 23, 1220-1240, January 1, 2018]

Modified low-density lipoproteins as biomarkers in diabetes and metabolic syndrome

Andrea Rivas-Urbina 1,2, Sonia Benitez1, Antonio Perez3,4, Jose Luis Sanchez-Quesada1,4

1Cardiovascular Biochemistry Group, Research Institute of the Hospital de Sant Pau (IIB Sant Pau), Barcelona, Spain, 2Biochemistry and Molecular Biology Department, Universitat Autònoma de Barcelona, Cerdanyola, Spain, 3Endocrinology and Nutrition Department, Hospital de la Santa Creu I Sant Pau, Barcelona, Spain, 4CIBERDEM. Institute of Health Carlos III, Spain

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Non-enzymatic glycosylation
4. Oxidative stress
5. Effects of oxidation and non-enzymatic glycosylation on lipoprotein function
6. Other modifications affecting LDL
6.1. Enzymatic modifications
6.2. Carbamylated LDL
6.3. Nitrated LDL
6.4. Desialylated LDL
6.5. NEFA-loaded LDL
7. Electronegative LDL. A pool of modified forms of LDL in blood
8. Why does diabetic dyslipidemia stimulate lipoprotein modification?
9. sdLDL as a biomarker of CVR
10. Association of modified LDLs with CVR
11. LDL(-) as a biomarker of CVR
12. Modified LDL as a biomarker of CV risk in DM
13. Summary and perspectives
14. Acknowledgements
15. References

1. ABSTRACT

Cardiovascular disease of atherosclerotic origin is the main cause of death in diabetes mellitus and metabolic syndrome. One of the mechanisms involved in such increased risk is the high incidence of lipoprotein modification in these pathologies. Increased glycosylation, oxidative stress or high non-esterified fatty acid levels in blood, among other factors, promote the modification and subsequent alteration of the properties of lipoproteins. Since the modification of low-density lipoprotein (LDL) is the triggering factor in the development of atherosclerosis, considerable research has been focused on the quantification of modified LDLs in blood to be used as biomarkers of cardiovascular risk. The present review deals with the main molecular mechanisms involved in the modification of LDL in diabetes and metabolic syndrome and briefly describe the atherogenic effects that these modified LDLs exert on the arterial wall. The possibility of using the high levels of modified LDLs or their immunocomplexes as a predictive tool for cardiovascular risk in diabetes-related pathologies is also discussed.

2. INTRODUCTION

Metabolic syndrome and type 2 diabetes mellitus confer an increased risk of cardiovascular disease (CVD). Compared with non-diabetic individuals, diabetic patients have 2 to 4 times increased risk for stroke and death from heart disease (1). Glucose intolerance and type 2 diabetes are core components of metabolic syndrome. A major underlying cause of CVD in patients with MS or diabetes is the presence of a characteristic form of atherogenic dyslipidemia (2), but other characteristics of this disease contribute synergistically to the increase of the cardiovascular risk (CVR). Among these characteristics, two phenomena, non-enzymatic glycosylation and oxidative stress, are exacerbated in diabetes and affect the function of a number of macromolecules including lipoproteins. Both phenomena are closely interconnected and play a relevant role in the development of atherosclerosis in patients with diabetes (3). Lipoproteins modified by non-enzymatic glycosylation and/or oxidation change their native properties. Thus, high-density lipoproteins (HDL) loss their antiatherogenic potential whereas low-density lipoproteins (LDL) acquire proinflammatory, proapoptotic and proatherogenic characteristics. Besides these modifications, lipoproteins can also be affected by other chemical processes, described in detail below, which lead to the formation of modified LDL particles. The involvement of modified LDL in the development of the atheromatous plaque suggests that its quantification in plasma could reflect the evolution of atherosclerotic lesions, representing a valuable tool for the prediction and stratification of CVR (4).

3. NON-ENZYMATIC GLYCOSYLATION

As a result of hyperglycemia, proteins, lipids and nucleic acids are glycosylated by non-enzymatic processes. Regarding proteins, glucose reacts with the amino groups of lysine and arginine residues, forming an unstable by-product (Schiff base) and, later, the stable Amadori product. Structural proteins with a long half-life, such as collagen, are the most affected by non-enzymatic glycosylation processes but other proteins in blood, such as albumin or immunoglobulins, are also glycosylated and their quantification (fructosamine, i.e. glycosylated proteins in blood) is used as an indicator of glycemic control. Of course, the protein moiety of all lipoproteins can be also glycosylated during their lifetime in circulation and, as a consequence, the normal function of lipoproteins is compromised (5).

Non-enzymatic glycosylation also induces the formation of oxygen free radicals, a phenomenon known as glycoxidation (6). This process generates a rearrangement of molecular bonds and leads to the formation of advanced glycation end-products (AGE), which irreversibly change the function of proteins. The formation of AGE requires longer period of time than the formation of Amadori products and it is generally assumed that it affects mainly to structural proteins. However, AGEs associated to proteins with relatively short mean life, such as apolipoprotein B (apoB) in LDL, have been detected in blood circulation (7).

The modification by methylglyoxal (MG) or other highly reactive aldehydes is another type of modification related to hyperglycemia, that does not directly involve glucose (8). MG is a glucose metabolite of the dicarbonyl type with a high reducing power. These metabolites rapidly react with arginine residues of proteins forming a heterocyclic compound (hydroimidazolone) which is part of the heterogeneous family of AGE compounds. Thornalley and coworkers have demonstrated the existence of MG-modified LDL (MG-LDL) in blood and have observed that their concentration is increased in patients with diabetes and decreases after treatment with metformin (9, 10).

4. OXIDATIVE STRESS

Increasing evidence from experimental and clinical studies suggests that systemic oxidative stress plays a major role in the pathogenesis of diabetes mellitus and atherosclerosis (7). Besides the glycosylation-associated oxidation (glycoxidation) of proteins, described above, another major cause of increased oxidative stress in diabetes is that, as a result of hyperglycemia, there is an increase in mitochondrial activity that favors the production of reactive oxygen species (ROS), such as the superoxide anion (O2-)or the hydroxyl radical (·OH) (11). Therefore, alterations of the oxidative stress-related parameters are frequent in the plasma of these individuals. Oxidative stress is particularly relevant in the intima layer of the arterial wall, a microenvironment surrounded by metabolically active cells (smooth muscle cells, macrophages, endothelial cells) that generate ROS and that does not have the abundant antioxidant defenses present in blood. The primary cellular damage resulting from this free radical reactivity, which mainly affects cellular membranes, is a process known as lipid peroxidation. Oxidative modification can damage all macromolecules in the subendothelial space, but lipoproteins are especially affected by oxidation due to their high content of lipids (12). ROS mainly oxidize the unsaturated fatty acids of the phospholipids located on the surface of the lipoproteins, being LDL highly sensitive to this modification. As a result, a number of oxidized lipids (lipoperoxides, oxidized phospholipids, oxidized fatty acids, oxidized cholesterol) and derived products (lysophosphatidylcholine, aldehydes, ketones) are formed in lipoproteins, having most of them proinflammatory, proliferative and apoptotic properties (13).

5. EFFECTS OF OXIDATION AND NON-ENZYMATIC GLYCOSYLATION ON LIPOPROTEIN FUNCTION

The knowledge gathered from three decades of research has shown that the modification of LDL is a key event in the development of atherosclerosis (13). By far, oxidative modification is the most studied mechanism, but alternative modifications are gaining strength as putative mechanisms involved in the development of atherosclerosis. Figure 1 shows different mechanisms of modification that could affect LDL in diabetes and metabolic syndrome. The oxidatively-modified form of low density lipoprotein (oxLDL) is a proinflammatory and proatherogenic particle containing protein adducts and inflammatory lipids that promotes atherosclerosis by different mechanisms (14, 15). First, oxidation generates lipid-derived molecules, such as malondialdehyde (MDA), which promotes the derivatization of lysine and arginine residues in apolipoprotein B. This provokes the loss of affinity for the LDL receptor, and the increased binding to scavenger receptors (SR). As a consequence, oxLDL is able to induce massive intracellular accumulation of cholesterol esters by macrophages (16, 17). In addition, the oxidation-derived lipid products generated in oxLDL induce the different cell types in the arterial wall to express cytokines, chemokines and growth factors. In this way, oxLDL promotes the chronic inflammatory and cell proliferation processes which are characteristic of atherosclerosis (18-22). Ox-LDL is also cytotoxic and apoptotic, favoring the formation of the necrotic nucleus of advanced atheromatous lesions. In fact, ox-LDL is a mixture of particles with different degrees of oxidation whose atherogenic properties change depending on the oxidative stage of LDL (23). Thus, minimally oxidized LDL is much more inflammatory than extensively oxidized particles but it has less capacity to induce foam cell formation.

Regarding glycosylation, it must be distinguished between glycosylated LDL (gl-LDL), LDL-modified with AGE (AGE-LDL) and LDL modified with MG (MG-LDL) (3, 24). The main atherogenic property of gl-LDL is a loss of its affinity for the LDL receptor (25) but it is not very inflammatory, in contrast with AGE-LDL that, since is originated from oxidative processes, is inflammatory, apoptotic and induces foam cell formation (5, 6). Concerning MG-LDL, it has been reported that has smaller particle size, greater susceptibility to aggregation and greater affinity for binding to proteoglycans of the arterial wall (26). Of note, the short half-life of circulating LDL (2.5.-3.5. days) has been an argument against the formation of gl-LDL or AGE-LDL in blood since, in absence of reducing agents, 6-7 days are necessary for glucose to modify proteins. Therefore, it has been implicitly assumed that the formation of these modified particles would occur mainly in LDL retained in injured areas of the arterial wall for a period longer than its plasma lifetime and their presence in the blood would be a reflection of the development of arteriosclerotic lesions (24). In contrast, the modification by MG does not directly involve glucose but this metabolite with a high reducing power rapidly reacts with arginine residues. Thus, the relevance of MG-LDL, within the AGE-LDL family, is that it could be formed during LDL plasma lifetime.

6. OTHER MODIFICATIONS AFFECTING LDL

Besides oxidation and glycosylation, other modifications of LDL have been described to occur in vivo (27). Some of them would occur mainly in the intima layer of the arterial wall while others are probably more relevant during blood circulation. Table 1 summarizes the different modifications that could affect lipoproteins and the most probable environment where this modification occurs.

6.1. Enzymatic modifications

Regarding modifications occurring in the arterial wall, during the development of atherosclerotic lesions there is a hyperexpression of proteolytic and lipolytic enzymes such as phospholipase A2 (PLA2), sphingomyelinase (SMase), cholesterol esterase (CEase), metalloproteinases (MMPs) or cathepsins (28-32). Therefore, it is presumed that LDL retained in the arterial wall is affected not only by oxidative or glycosylative processes but also by proteases and lipases. As a consequence, lipoproteins isolated from the arterial wall show protein fragmentation and a high content of enzyme-mediated lipid degradation products such as lysophosphatidylcholine, ceramide or free cholesterol (33). Nowadays, although it is still accepted that lipid peroxidation has a role in atherogenesis, it is considered that other enzyme-related mechanisms of LDL modification, such as degradation by lipases or proteases, could have an even more predominant role in the generation of modified LDL in the arterial wall (34, 35). The main atherogenic effect of enzymatic modifications is that these processes trigger LDL aggregation and fusion, favoring its subendothelial retention (36). Hence, retained LDL is exposed to undergo further modifications by other mechanisms such as oxidation or glycosylation. Other effect of enzyme-mediated lipolysis is the formation of lipids, such as lysophosphatidylcholine or ceramide, which would not be formed by oxidation or glycosylation and display apoptotic and inflammatory properties.

6.2. Carbamylated LDL

Enzymatic processes preferentially occur in the artery wall, but LDL can also be modified by other mechanisms in the blood circulation. Recently, the presence of carbamylated LDL in plasma has been reported (37). The carbamylation of LDL occurs due to spontaneous, non-enzymatic chemical modification of the amine-containing residues in apoB by urea-derived cyanate (38). This modification is especially relevant to smokers, since tobacco smoke favors the formation of thiocyanate, and also in patients with chronic uremia due to severe renal insufficiency (39). Then, carbamylation of LDL could have a role in the development of atherosclerosis in patients with diabetes that also present kidney disease. Among the atherogenic properties of carbamylated LDL, it has been reported that is immunogenic, prothrombotic, proliferative and that induces endothelial dysfunction (40-42).

6.3. Nitrated LDL

Although less studied than ROS-mediated modification, LDL can also be modified by reactive nitrogen species (RNS), a process known as nitration. This phenomenon is closely related to oxidative modification because the main reactive molecule is peroxynitrite (ONOO-), which derives from nitric oxide (·NO) and superoxide anion O2-. Besides promoting lipoperoxidation, nitration of apoB in LDL results in the derivatization of tyrosine and oxidation of cysteine, which alters apoB structure (43). As occurs with carbamylation, nitration of LDL is a process that occurs in plasma (44) and is potentiated in smokers and in patients with severe kidney disease (45, 46).

6.4. Desialylated LDL

Other form of modified LDL detected in plasma is desialylated LDL, which has a reduced content in sialic acid, the final carbohydrate in the apoB-enzymatic glycosylation chains. Desialylated LDL is increased in patients with diabetes and it has the capacity to induce the formation of foam cells being, therefore, potentially atherogenic (47). The desialylation process has been attributed to oxidative processes since these favor the non-enzymatic hydrolysis of sialic acid bound to apoB (48). Desialylated LDL shares some properties with oxidized LDL; thus, it could be a reflection of oxidative stress (49).

6.5. NEFA-loaded LDL

Although it cannot be strictly considered a chemical modification, overloading of LDL with non-esterified fatty acids (NEFA) confer some atherogenic properties to LDL (50, 51). NEFA are usually transported in circulation by albumin, however, when NEFA concentration rises and the capacity of albumin to bind NEFA is exceeded, these lipids bind to other macromolecules, mainly lipoproteins. This phenomenon could have especial relevance in situations of insulin resistance such as metabolic syndrome or diabetes and it has been reported that LDL from diabetic patients has a high NEFA content (52). LDL with an increased NEFA content is inflammatory and its structure is altered favoring its aggregation (53-56). This could explain the observations that diabetic LDL is more inflammatory than LDL from subjects without diabetes, despite not having increased rates of lipoperoxidation (57).

7. ELECTRONEGATIVE LDL: A POOL OF MODIFIED FORMS OF LDL IN BLOOD

A common property of the different forms of modified LDL is an increase of the electric charge of these particles (58, 59). According to this property, total LDL can be subfractionated by anion exchange chromatography into two populations, a major subfraction of non-modified native LDL and a minor subfraction of electronegative LDL (LDL(-)). This minor subfraction accounts for 2-10% of total LDL in normolipidemic subjects. LDL(-) is heterogeneous in terms of size, density, lipid and protein content (60-62). The most widely accepted idea is that LDL(-) is a pool of LDL particles modified by several mechanisms. However, only a small part of LDL(-) would consist of oxidized, glycosylated, nitrated, desialylated or carbamylated LDL because LDL(-) proportion is much higher than that described for these modified LDLs (0.1.-1%) (63).

Besides the chemical modifications previously described, other alterations in the composition of LDL also contribute to the presence of LDL(-). It has been reported that NEFA content is a major contributor to the electronegativity of LDL particles (56). In addition, both small dense and very large LDL particles also present an increased electronegative charge (64). The same occurs with LDL particles that contains other apolipoproteins different than apoB (65). Indeed, LDL(-) is characterized by abnormal size (small or large) and increased content of NEFA and minor apolipoproteins that include, among others, apoA-I, apoE, apoC-III, apoD, apoJ or apoF (56, 60, 65, 66). Therefore, an increased proportion of LDL(-) in blood would reflect a range of metabolic abnormalities, which are associated with high CVR and systemic inflammation. Accordingly, the proportion of LDL(-) is increased in a number of metabolic diseases with increased CVR, such as familial hypercholesterolemia, hypertriglyceridemia, diabetes, metabolic syndrome, severe renal disease, non-alcoholic fatty liver disease and also in patients with angiographically-established coronary disease (58, 67, 68). Moreover, LDL(-) proportion dramatically increases during the early phase of acute myocardial infarction and after cerebral ischemia (69, 70).

Since LDL(-) is a mixture of modified LDL particles and has an abnormal composition, it shows inflammatory, apoptotic and proliferative properties (58, 68, 71, 72). These atherogenic characteristics displayed by LDL(-) isolated from normolipemic and normoglycemic subjects are exacerbated in LDL(-) isolated from diabetic subjects, especially when these subjects are in poor glycemic control(73). Then, LDL(-) from diabetics would be more atherogenic than LDL(-) from normoglycemic subjects. This could be due to the confluence in poorly-controlled diabetic patients LDL of multiple factors including, increased oxidation/nitration, increased glycosylation, increased NEFA content, smaller size and, if kidney disease is also present, increased carbamylation.

8. WHY DOES DIABETIC DYSLIPIDEMIA STIMULATE LIPOPROTEIN MODIFICATION?

A common metabolic abnormality associated with diabetes is a specific dyslipidemia that includes a spectrum of quantitative and qualitative changes in lipids and lipoproteins (74). This anomalous lipid profile, known as diabetic or atherogenic dyslipidemia, is characterized by high levels of triglycerides and apoB, low concentration of high density lipoprotein (HDL) cholesterol, and increased postprandial lipidemia. This abnormal lipid profile is typical of diabetes but it is also present in pre-diabetic situations such as insulin resistance and metabolic syndrome (1, 75, 76).

The origin of diabetic dyslipemia comes from an increased hepatic production of very low density lipoprotein (VLDL) due to the high plasma concentration of NEFA. In this situation, VLDL particles are very large due to a very high content of triglycerides (77, 78). Hypertriglyceridemia alters some enzymatic activities related to VLDL catabolism, specifically the enzymes cholesteryl ester transfer protein (CETP) and hepatic lipase (HL). Hypertriglyceridemia stimulates the enzymatic activity of CETP, which facilitates the transfer of triglycerides from triglyceride-rich lipoproteins (i.e. VLDL) to HDL and LDL in exchange for cholesteryl esters (79). This leads to an increase in the triglyceride content of HDL and LDL (80). Triglyceride-enriched HDL particles are subjected to increased catabolism; consequently, they have a short plasma half-life. In addition, triglyceride-enriched LDL particles undergo subsequent hydrolysis via HL, thereby reducing the LDL particle size (81).

In contrast to HDL, which has atheroprotective properties, LDL and VLDL are considered atherogenic, being apoB their main protein component. Despite that 80-90% of apoB is associated with LDL and that apoB concentration is high in diabetes, the LDL cholesterol levels are usually normal in these patients (82). This peculiarity is explained by the prevalence of LDL particles of small size (small, dense LDL, sdLDL). sdLDL have lower relative cholesterol content and higher relative apoB and triglyceride content than normal LDL particles (81). Thus, an increase in triglyceride-rich lipoproteins is commonly associated with a reduction in HDL concentration and an increase in sdLDL levels. This means that at a given LDL cholesterol concentration, diabetic patients have a greater number of LDL particles (83, 84).

sdLDL particles are more atherogenic than large buoyant LDL due to several characteristics that facilitate their modification (81, 84, 85). First, sdLDL has a lower affinity for the LDL receptor, which implies a lower rate of plasma clearance and longer time in the circulation; this would expedite LDL modification by different mechanisms such as oxidation, glycosylation, desialylation or carbamylation. Second, sdLDL crosses the endothelial barrier easier than native LDL since this is a process mainly dependent on the size of the lipoprotein particle. In addition, sdLDL also binds with greater affinity to the proteoglycans that constitute the intima layer of the arterial wall, favoring subendothelial retention of lipoproteins. Third, sdLDL has a greater susceptibility to be modified by oxidative mechanisms and also by non-enzymatic glycosylation(86).

To these intrinsic pro-atherogenic properties of sdLDL, it must be added the qualitative alterations in HDL function in a diabetic dyslipidemia situation. The antiatherogenic role of HDL goes beyond that its classic role in the reverse transport of cholesterol. HDL has a determinant action in the protection of LDL against modifications, some enzymes and apolipoproteins associated to HDL, such as apoA-I, apoJ, paraoxonase, platelet-activating factor acetylhydrolase (PAF-AH) or lecithin-cholesterol acyl transferase (LCAT) act synergistically preventing the oxidation of LDL (87, 88). However, the glycosylation and oxidation of these proteins also affect their functionality, compromising the antioxidant and anti-inflammatory capacity of HDL. Therefore, the concentration of HDL is not only diminished in patients with diabetes, but it is also dysfunctional. In this way, the impairment of the HDL anti-atherogenic properties in diabetes favors the formation of modified LDL.

9. sdLDL AS A BIOMARKER OF CVR

It is well documented that small dense LDL (sdLDL) levels are elevated in conditions linked to atherosclerosis, such as metabolic syndrome, disease in which sdLDL has been reported to be an independent predictive factor for cardiovascular events (89, 90). Other studies concur with the concept that sdLDL cholesterol (sdLDL-C) is a better marker for predicting CVR than total LDL cholesterol (91, 92). However, not all the studies agree; these discrepancies could depend on the methods used to measure sdLDL. The recent use of homogeneous assays has allowed to evaluate sdLDL in large clinical trials (90). In a large prospective study using these assays it was found that sdLDL-C is associated with coronary heart disease even in patients with low CVR based on their LDLc levels (93). The value of sdLDL-C as an independent CVR factor has also been suggested by comparing with intima media thickness measurement (94). sdLDL has been associated with poor outcome after angioplasty in peripheral artery disease (95). Several atherogenic properties have been ascribed to sdLDL, which can be further modified in plasma by several mechanisms, such as desialylation, glycation, and oxidation (90), as described above. These modifications would confer more atherogenic properties to this LDL and, consequently, a closer relation with CV events.

10. ASSOCIATION OF MODIFIED LDLS WITH CVR

Owing to its known role in atherosclerosis, different forms of modified LDLs have been proposed as biomarkers for CVR and for detecting the vulnerability of atherosclerotic plaques. Most studies using modified LDLs as biomarkers have been conducted with oxLDL (96-99). Holvoet et al. developed the first immunoassay to detect the presence of oxLDL in plasma, which was reported to be increased in atherosclerotic patients (100). Since then, a multitude of studies have associated oxLDL concentration with different expressions of vascular disease, and its concentration is increased in pathologies with increased CVR. Thus, oxLDL is increased in patients with atherosclerosis and correlates with the severity of coronary disease (4, 101-105). Moreover, oxLDL increases after acute myocardial infarction and is associated with plaque instability (90). oxLDL also acutely increases after percutaneous coronary intervention (93, 106, 107). It has been recently reported that oxLDL also increases in ischemic stroke (108). In stroke, particularly in large artery atherosclerosis subtype, an increased oxLDL concentration in acute phase was associated with higher mortality or worse outcome. These observations suggest that oxLDL in blood comes from the arterial wall and could be a biomarker of atherosclerotic plaque vulnerability.

Regarding its predictive value as a CVR marker, numerous studies have described increased concentrations of oxLDL in diseases with high vascular risk, such as hypercholesterolemia, hypertension, chronic heart failure, peripheral arterial disease, diabetes, metabolic syndrome, obesity and renal disease (4, 109-117). In spite of this, some studies cast doubts on the usefulness of oxLDL as an independent predictive biomarker of future cardiovascular events (118, 119). The main concern comes from the fact that oxLDL values strongly correlate with other known lipid risk factors, including total and LDL cholesterol (120). Thus, even though the involvement of oxLDL in atherosclerosis has been clearly established, its value as an independent biomarker of CVR is moderate. This is due to different factors. On the one hand, some conflicting results have been reported; for instance, not all studies have found the association of oxLDL levels with the burden of atherosclerotic lesions (121, 122). Choi et al also reported that statin therapy increased the titers of oxLDL measured by two independent ELISAs but found no quantitative changes in coronary angiography (123). Several factors could underlie these conflicting results. First, there is no international standardization in oxLDL determination, due to the use of different antibodies that recognize epitopes of different stages of LDL oxidation (120). Second, at least three commercial ELISAs have been widely used, but each antibody recognizes different epitopes that could reflect different processes, thereby preventing an adequate comparison of the results obtained by different groups. On the other hand, oxLDL represents only a part of the modified LDL particles that can be found in circulation. In specific pathologies other forms of modified LDL could be more relevant than oxLDL. For instance, carbamylated LDL possibly plays an important role in patients with severe renal disease. Unfortunately, there are no established guidelines for modified LDL evaluation that allow its application to the clinical practice and its usefulness as a predictive biomarker. Regarding diabetes, the usefulness of AGE-LDL as biomarker is discussed in detail below.

11. LDL(-) AS A BIOMARKER OF CVR

An alternative to oxLDL as a biomarker could be LDL(-). Modified lipoproteins such as oxLDL and AGE-LDL are considered biomarkers for active atherosclerotic lesions, as they are supposed to be generated in the arterial wall. But, as discussed above, LDL(-) represents not only a pool of modified LDLs but also reflects the existence of metabolic abnormalities leading to alterations in the composition of the lipoprotein. Therefore, the quantification of LDL(-) would be especially useful in asymptomatic patients (58, 63). LDL(-) proportion is high in several groups of subjects with enhanced CVR, such as familial hypercholesterolemia, hypertriglyceridemia, diabetes, renal disease, and non-alcoholic fatty liver disease (68, 90). Furthermore, LDL(-) proportion highly increases after stroke (70) and myocardial infarction (69), being its concentration higher in acute than in chronic coronary disease (124). Moreover, LDL(-) levels are associated with the severity of coronary disease angiographically-determined (125) and with the carotid intima-media thickness (126).

Specifically in diabetes, several studies have confirmed by different methods an increased proportion of LDL(-) compared to healthy subjects (57, 72, 127-131). Interestingly, the elevated proportion of LDL(-) decreases after insulin therapy in type 1 but not in type 2 diabetes, which suggests that non-enzymatic glycosylation has a more relevant role in LDL(-) generation in type 1 than in type 2 diabetics. In the same context, it was observed that the oral antihyperglycemic agent pioglitazone decreases the negative charge of LDL (130). Also in prediabetic insulin-resistant subjects the proportion of LDL(-) is increased (132). The clinical importance of LDL(-) in the metabolic syndrome has been studied by Chen and co-workers. These authors found a correlation between L5, the most electronegative form of LDL, plasma levels and the number of metabolic syndrome criteria (according to the Framingham score) in asymptomatic patients (133).

Unfortunately, there are no standardized methods for LDL(-) quantification in large-population studies. The usual methods are size exclusion chromatography combined with ultracentrifugation or capillary electrophoresis. But these methodologies are not applicable to a large number of subjects and are not accessible to most of clinical chemistry laboratories. Abdalla and coworkers have developed monoclonal antibodies to perform immunoassays for LDL(-) measurement in population studies (134, 135). However, these methods are not commercially available and it is unknown if all modifications affecting LDL(-) could be detected.

Measuring the presence of auto-antibodies to LDL(-) could be an alternative approach for LDL(-) quantification. Similarly to oxLDL or AGE-LDL, LDL(-) shows immunogenic properties, and it has been proposed that LDL(-) is internalized by macrophages in the subendothelial space, thereby promoting anti-LDL(-) production by B cells. Auto-antibodies for LDL(-) and immunocomplexes (IC), consisting of LDL(-) and anti-LDL(-) antibodies, could be released to the lumen space. The presence of these auto-antibodies and IC has been detected in plasma. Moreover, the proportion of auto-antibodies for LDL(-) is increased in coronary disease, particularly in the acute phase of unstable angina (124, 134, 135). However, the role of these auto-antibodies is controversial since it has been described that administration of anti-LDL(-) protects from atherosclerosis development in mice (136). Further studies are necessary to elucidate the possible role of auto-antibodies anti LDL(-) in the evolution of atherosclerotic plaques and during acute vascular events.

12. MODIFIED LDL AS A BIOMARKER OF CV RISK IN DM AND METABOLIC SYNDROME

As discussed throughout this review, lipoproteins from diabetic patients are subjected to a number of modifications. Accordingly, diabetics have increased concentrations in blood of several modified LDLs, including oxLDL, glycLDL, AGE-LDL, MG-LDL and LDL(-). Table 2 summarizes the main studies on the relation of modified LDL and CVR in subjects with diabetes, metabolic syndrome or related diseases. Diabetic patients with phenotype B of LDL subfraction (predominance of sdLDL) have even higher plasma levels of oxLDL and glycLDL than those with phenotype A (predominance of large LDL) (137, 138). It has also been described that patients with poor glycemic control have increased concentration of different types of modified LDL (130, 139), and that glycemic optimization decreases these levels (131). Hypolipemic treatment also decreases the concentration of modified LDL (70); this suggests that the determination of these modified forms of LDL as independent CVR biomarkers is rather controversial, particularly in type 2 diabetic patients, who usually have an altered lipid profile. Thus, some studies suggested that in the context of diabetes, oxLDL is a factor that predicts CV events, whereas others did not find this association when corrected by lipid profile (101, 140). However, an independent association of oxLDL with atherosclerosis progression is found in some studies in which intima-media thickness (IMT) or nephropaty are evaluated (141, 142).

In diabetic patients, besides the presence of modified LDL, autoantibodies anti-modified LDL (anti-oxLDL, anti-AGE-LDL, anti-MDA-LDL) forming immuno-complexes (IC) with modified LDLs are detected in blood. Recently, anti-ribosylated-glycated-LDL has also been found in type 1 and type 2 diabetic patients (143). Although data from the studies of ICs are sometimes difficult to interpret, it is generally accepted that IgM antibodies would have a protective effect, whereas IgG antibodies would be directly related with atherosclerosis (144). Thus, IgM anti-AGE-LDL concentration has been reported to protect from CVR both in diabetic and non-diabetic subjects (145, 146). In contrast, some studies show a positive relationship of IgG autoantibodies titers with the development of atherosclerosis (124, 147, 148). Other authors, however, disagree with this direct relation; thus, Asciutto and coworkers reported that low levels of IgG anti-MDA-LDL correlates with high risk of postoperative death after carotid endarterectomy (149), whereas high levels of these antibodies are associated with decreased plaque inflammation (150). Moreover, studies performed in diabetic mice shows that treatments with anti-oxLDL IgG (151) or AGE-LDL immunization (152) protect against atherosclerosis, thereby suggesting a protective role for IgG autoantibodies. Regarding LDL(-) autoantibodies and diabetes, there is a higher concentration of auto-antibodies anti-LDL(-) in type 1 and type 2 diabetic patients, as well as in those with impaired glucose tolerance, than in control subjects (153).

The above described discrepancies could come from the difficulty of measuring modified LDL and autoantibodies, which could be due to the fact that both molecules are strongly associated in IC (154). The evaluation of IC is technically difficult, since it requires a previous precipitation of IC before the quantification of modified LDL or autoantibodies by immunoassay. IC-oxLDL and IC-AGE-LDL have been detected mainly in type 1 diabetes (155). In this regard, are noteworthy the studies of Virella and Lopes-Virella et al. describing that in these subjects the major part of oxLDL and AGE-LDL is associated to antibodies as IC (156). IC-LDLs have a higher atherogenic effect than modified LDL alone and seem to be a solid predictor of CVR (157-160). Sobenin et al. reported that, even in the absence of clinical manifestations, elevated levels of IC-LDL are increased in early carotid atherosclerosis, measured as IMT (161). Orekhov et al. suggested that IC-LDL can be considered biomarkers for macrovascular disease in type 1 diabetes (162). Several studies performed in large populations of type 1 diabetic subjects (DCCT/EDIC cohort) have shown that IC-oxLDL and IC-AGE-LDL concentrations are associated, independently of other risk factors, with IMT and atherosclerosis progression (163, 164), coronary calcification (165), risk of nephropathy (166) and progression of retinopathy (167). Similar studies conducted in type 2 diabetic patients (VADT cohort) have shown that high levels of IC-MDA-LDL are associated with myocardial infarction and acute CV events (168), retinopathy (169), and macroalbuminuria (170).

13. SUMMARY AND PERSPECTIVES

Diabetic patients have increased plasma concentrations of LDLs modified by different mechanisms. In general, this concentration correlates with other lipid risk factors attributed to diabetic dyslipemia, which somehow prevents its use as an independent biomarker of cardiovascular risk. However, some studies show that modified LDL and, more specifically immunocomplexes of modified LDLs, could be independent risk factors, being its plasma concentration associated with atherosclerosis progression in type 1 and type 2 diabetic patients. Therefore, the detection of modified LDL and immunocomplexes could help to better predict cardiovascular risk in diabetes and probably in other pathologies related to cardiovascular disease. It would be very useful to develop a specific profile of modified LDL that, combined to genetics of patients with diabetes or metabolic disease, could predict the risk of cardiovascular disease and to personalize therapy. However, more mechanistic studies are warranted to gain insight into the molecular processes leading to LDL modification and their consequences for atherosclerosis development in patients with diabetes.

14. ACKNOWLEDGMENTS

The authors of this study have been funded with FEDER funds and by the Instituto de Salud Carlos III from the Ministry of Health (CIBERDEM, FIS PI13-00364, FIS PI16-0471) and by AGAUR from the Generalitat de Catalunya (2014-SGR-246).

15. REFERENCES

1. J. A. Farmer: Diabetic dyslipidemia and atherosclerosis: evidence from clinical trials. Curr Diab Rep, 8(1), 71-7 (2008)
DOI: 10.1007/s11892-008-0013-2

2. K. G. Alberti, P. Zimmet and J. Shaw: Metabolic syndrome--a new world-wide definition. A Consensus Statement from the International Diabetes Federation. Diabet Med, 23(5), 469-80 (2006)
DOI: 10.1111/j.1464-5491.2006.01858.x

3. M. Brownlee: The pathobiology of diabetic complications: a unifying mechanism. Diabetes, 54(6), 1615-25 (2005)
DOI: 10.2337/diabetes.54.6.1615

4. A. E. Fraley and S. Tsimikas: Clinical applications of circulating oxidized low-density lipoprotein biomarkers in cardiovascular disease. Curr Opin Lipidol, 17(5), 502-9 (2006)
DOI: 10.1097/01.mol.0000245255.40634.b5

5. A. Cerami, H. Vlassara and M. Brownlee: Role of advanced glycosylation products in complications of diabetes. Diabetes Care, 11 Suppl 1, 73-9 (1988)

6. M. Brownlee, A. Cerami and H. Vlassara: Advanced products of nonenzymatic glycosylation and the pathogenesis of diabetic vascular disease. Diabetes Metab Rev, 4(5), 437-51 (1988)
DOI: 10.1002/dmr.5610040503

7. F. Giacco and M. Brownlee: Oxidative stress and diabetic complications. Circ Res, 107(9), 1058-70 (2010)
DOI: 10.1161/CIRCRESAHA.110.223545

8. N. Rabbani and P. J. Thornalley: Methylglyoxal, glyoxalase 1 and the dicarbonyl proteome. Amino Acids, 42(4), 1133-42 (2012)
DOI: 10.1007/s00726-010-0783-0

9. N. Rabbani, M. V. Chittari, C. W. Bodmer, D. Zehnder, A. Ceriello and P. J. Thornalley: Increased glycation and oxidative damage to apolipoprotein B100 of LDL cholesterol in patients with type 2 diabetes and effect of metformin. Diabetes, 59(4), 1038-45 (2010)
DOI: 10.2337/db09-1455

10. N. Rabbani, L. Godfrey, M. Xue, F. Shaheen, M. Geoffrion, R. Milne and P. J. Thornalley: Glycation of LDL by Methylglyoxal Increases Arterial Atherogenicity: A Possible Contributor to Increased Risk of Cardiovascular Disease in Diabetes. Diabetes, 60, 1-8 (2011)
DOI: 10.2337/db11-0085

11. U. Hink, H. Li, H. Mollnau, M. Oelze, E. Matheis, M. Hartmann, M. Skatchkov, F. Thaiss, R. A. Stahl, A. Warnholtz, T. Meinertz, K. Griendling, D. G. Harrison, U. Forstermann and T. Munzel: Mechanisms underlying endothelial dysfunction in diabetes mellitus. Circ Res, 88(2), E14-22 (2001)
DOI: 10.1161/01.RES.88.2.e14

12. P. Klatt and H. Esterbauer: Oxidative hypothesis of atherogenesis. J Cardiovasc Risk, 3(4), 346-51 (1996)
DOI: 10.1097/00043798-199608000-00002
DOI: 10.1177/174182679600300402

13. D. Steinberg: The LDL modification hypothesis of atherogenesis: an update. J Lipid Res, 50 Suppl, S376-81 (2009)

14. M. Navab, J. A. Berliner, A. D. Watson, S. Y. Hama, M. C. Territo, A. J. Lusis, D. M. Shih, B. J. Van Lenten, J. S. Frank, L. L. Demer, P. A. Edwards and A. M. Fogelman: The Yin and Yang of oxidation in the development of the fatty streak. A review based on the 1994 George Lyman Duff Memorial Lecture. Arterioscler Thromb Vasc Biol, 16(7), 831-42 (1996)
DOI: 10.1161/01.ATV.16.7.831

15. D. Li and J. L. Mehta: Oxidized LDL, a critical factor in atherogenesis. Cardiovasc Res, 68(3), 353-4 (2005)
DOI: 10.1016/j.cardiores.2005.09.009

16. T. J. Van Berkel, M. Van Eck, N. Herijgers, K. Fluiter and S. Nion: Scavenger receptor classes A and B. Their roles in atherogenesis and the metabolism of modified LDL and HDL. Ann N Y Acad Sci, 902, 113-26; discussion 126-7 (2000)
DOI: 10.1111/j.1749-6632.2000.tb06306.x

17. Y. Yamada, T. Doi, T. Hamakubo and T. Kodama: Scavenger receptor family proteins: roles for atherosclerosis, host defence and disorders of the central nervous system. Cell Mol Life Sci, 54(7), 628-40 (1998)
DOI: 10.1007/s000180050191

18. J. A. Berliner, M. Navab, A. M. Fogelman, J. S. Frank, L. L. Demer, P. A. Edwards, A. D. Watson and A. J. Lusis: Atherosclerosis: basic mechanisms. Oxidation, inflammation, and genetics. Circulation, 91(9), 2488-96 (1995)
DOI: 10.1161/01.CIR.91.9.2488

19. M. Navab, G. M. Ananthramaiah, S. T. Reddy, B. J. Van Lenten, B. J. Ansell, G. C. Fonarow, K. Vahabzadeh, S. Hama, G. Hough, N. Kamranpour, J. A. Berliner, A. J. Lusis and A. M. Fogelman: The oxidation hypothesis of atherogenesis: the role of oxidized phospholipids and HDL. J Lipid Res, 45(6), 993-1007 (2004)
DOI: 10.1194/jlr.R400001-JLR200

20. M. Navab, S. Y. Hama, S. T. Reddy, C. J. Ng, B. J. Van Lenten, H. Laks and A. M. Fogelman: Oxidized lipids as mediators of coronary heart disease. Curr Opin Lipidol, 13(4), 363-72 (2002)
DOI: 10.1097/00041433-200208000-00003

21. R. Ross: Atherosclerosis--an inflammatory disease. N Engl J Med, 340(2), 115-26 (1999)
DOI: 10.1056/NEJM199901143400207

22. J. L. Young, P. Libby and U. Schonbeck: Cytokines in the pathogenesis of atherosclerosis. Thromb Haemost, 88(4), 554-67 (2002)

23. I. Levitan, S. Volkov and P. V. Subbaiah: Oxidized LDL: diversity, patterns of recognition, and pathophysiology. Antioxid Redox Signal, 13(1), 39-75 (2010)
DOI: 10.1089/ars.2009.2733

24. N. Younis, R. Sharma, H. Soran, V. Charlton-Menys, M. Elseweidy and P. N. Durrington: Glycation as an atherogenic modification of LDL. Curr Opin Lipidol, 19(4), 378-84 (2008)
DOI: 10.1097/MOL.0b013e328306a057

25. X. Wang, R. Bucala and R. Milne: Epitopes close to the apolipoprotein B low density lipoprotein receptor-binding site are modified by advanced glycation end products. Proc Natl Acad Sci U S A, 95(13), 7643-7 (1998)
DOI: 10.1073/pnas.95.13.7643

26. N. Rabbani, L. Godfrey, M. Xue, F. Shaheen, M. Geoffrion, R. Milne and P. J. Thornalley: Glycation of LDL by Methylglyoxal Increases Arterial Atherogenicity: A Possible Contributor to Increased Risk of Cardiovascular Disease in Diabetes. Diabetes (2011) 60(7):1973-80
DOI: 10.2337/db11-0085

27. J. L. Sanchez-Quesada and S. Villegas: Modified forms of LDL in plasma. In: Atherogenesis. Ed S. Parthasarathy. InTech, (2011)

28. K. Oorni, M. O. Pentikainen, M. Ala-Korpela and P. T. Kovanen: Aggregation, fusion, and vesicle formation of modified low density lipoprotein particles: molecular mechanisms and effects on matrix interactions. J Lipid Res, 41(11), 1703-14 (2000)

29. K. J. Williams and I. Tabas: Lipoprotein retention--and clues for atheroma regression. Arterioscler Thromb Vasc Biol, 25(8), 1536-40 (2005)
DOI: 10.1161/01.ATV.0000174795.62387.d3

30. I. Tabas, Y. Li, R. W. Brocia, S. W. Xu, T. L. Swenson and K. J. Williams: Lipoprotein lipase and sphingomyelinase synergistically enhance the association of atherogenic lipoproteins with smooth muscle cells and extracellular matrix. A possible mechanism for low density lipoprotein and lipoprotein(a) retention and macrophage foam cell formation. J Biol Chem, 268(27), 20419-32 (1993)

31. M. Klouche, S. Gottschling, V. Gerl, W. Hell, M. Husmann, B. Dorweiler, M. Messner and S. Bhakdi: Atherogenic properties of enzymatically degraded LDL: selective induction of MCP-1 and cytotoxic effects on human macrophages. Arterioscler Thromb Vasc Biol, 18(9), 1376-85 (1998)
DOI: 10.1161/01.ATV.18.9.1376

32. M. Torzewski, P. Suriyaphol, K. Paprotka, L. Spath, V. Ochsenhirt, A. Schmitt, S. R. Han, M. Husmann, V. B. Gerl, S. Bhakdi and K. J. Lackner: Enzymatic modification of low-density lipoprotein in the arterial wall: a new role for plasmin and matrix metalloproteinases in atherogenesis. Arterioscler Thromb Vasc Biol, 24(11), 2130-6 (2004)
DOI: 10.1161/01.ATV.0000144016.85221.66

33. H. C. Stary: Natural history and histological classification of atherosclerotic lesions: an update. Arterioscler Thromb Vasc Biol, 20(5), 1177-8 (2000)
DOI: 10.1161/01.ATV.20.5.1177

34. I. Tabas: Nonoxidative modifications of lipoproteins in atherogenesis. Annu Rev Nutr, 19, 123-39 (1999)
DOI: 10.1146/annurev.nutr.19.1.123

35. S. Bhakdi, K. J. Lackner, S. R. Han, M. Torzewski and M. Husmann: Beyond cholesterol: the enigma of atherosclerosis revisited. Thromb Haemost, 91(4), 639-45 (2004)
DOI: 10.1160/TH03-12-0733

36. I. Tabas, K. J. Williams and J. Boren: Subendothelial lipoprotein retention as the initiating process in atherosclerosis: update and therapeutic implications. Circulation, 116(16), 1832-44 (2007)
DOI: 10.1161/CIRCULATIONAHA.106.676890

37. E. O. Apostolov, D. Ray, A. V. Savenka, S. V. Shah and A. G. Basnakian: Chronic uremia stimulates LDL carbamylation and atherosclerosis. J Am Soc Nephrol, 21(11), 1852-7 (2010)
DOI: 10.1681/ASN.2010040365

38. M. Alique, C. Luna, J. Carracedo and R. Ramírez: LDL biochemical modifications: a link between atherosclerosis and aging. Food & Nutrition Research, 59, 10.3.402/fnr.v59.2.9240 (2015)

39. F. H. Verbrugge, W. H. Tang and S. L. Hazen: Protein carbamylation and cardiovascular disease. Kidney Int, 88(3), 474-8 (2015)
DOI: 10.1038/ki.2015.166

40. T. Speer, F. O. Owala, E. W. Holy, S. Zewinger, F. L. Frenzel, B. E. Stahli, M. Razavi, S. Triem, H. Cvija, L. Rohrer, S. Seiler, G. H. Heine, V. Jankowski, J. Jankowski, G. G. Camici, A. Akhmedov, D. Fliser, T. F. Luscher and F. C. Tanner: Carbamylated low-density lipoprotein induces endothelial dysfunction. Eur Heart J, 35(43), 3021-32 (2014)
DOI: 10.1093/eurheartj/ehu111

41. E. O. Apostolov, E. Ok, S. Burns, S. Nawaz, A. Savenka, S. Shah and A. G. Basnakian: Carbamylated-oxidized LDL: proatherosclerotic effects on endothelial cells and macrophages. J Atheroscler Thromb, 20(12), 878-92 (2013)
DOI: 10.5551/jat.14035

42. E. O. Apostolov, A. G. Basnakian, E. Ok and S. V. Shah: Carbamylated low-density lipoprotein: nontraditional risk factor for cardiovascular events in patients with chronic kidney disease. J Ren Nutr, 22(1), 134-8 (2012)
DOI: 10.1053/j.jrn.2011.10.023

43. R. T. Hamilton, L. Asatryan, J. T. Nilsen, J. M. Isas, T. K. Gallaher, T. Sawamura and T. K. Hsiai: LDL protein nitration: implication for LDL protein unfolding. Arch Biochem Biophys, 479(1), 1-14 (2008)
DOI: 10.1016/j.abb.2008.07.026

44. E. Torres-Rasgado, G. Fouret, M. A. Carbonneau and C. L. Leger: Peroxynitrite mild nitration of albumin and LDL-albumin complex naturally present in plasma and tyrosine nitration rate-albumin impairs LDL nitration. Free Radic Res, 41(3), 367-75 (2007)
DOI: 10.1080/10715760601064706

45. A. Bakillah, F. Tedla, I. Ayoub, D. John, A. J. Norin, M. M. Hussain and C. Brown: Plasma Nitration of High-Density and Low-Density Lipoproteins in Chronic Kidney Disease Patients Receiving Kidney Transplants. Mediators Inflamm, 2015, 352356 (2015)
DOI: 10.1155/2015/352356

46. Y. Yamaguchi, J. Haginaka, S. Morimoto, Y. Fujioka and M. Kunitomo: Facilitated nitration and oxidation of LDL in cigarette smokers. Eur J Clin Invest, 35(3), 186-93 (2005)
DOI: 10.1111/j.1365-2362.2005.01472.x

47. I. A. Sobenin, V. V. Tertov, T. Koschinsky, C. E. Bunting, E. S. Slavina, Dedov, II and A. N. Orekhov: Modified low density lipoprotein from diabetic patients causes cholesterol accumulation in human intimal aortic cells. Atherosclerosis, 100(1), 41-54 (1993)
DOI: 10.1016/0021-9150(93)90066-4

48. E. R. Zakiev, V. N. Sukhorukov, A. A. Melnichenko, I. A. Sobenin, E. A. Ivanova and A. N. Orekhov: Lipid composition of circulating multiple-modified low density lipoprotein. Lipids Health Dis, 15(1), 134 (2016)

49. Z. Ozturk, H. Sonmez, F. M. Gorgun, H. Ekmekci, D. Bilgen, N. Ozen, V. Sozer, T. Altug and E. Kokoglu: The Relationship Between Lipid Peroxidation and LDL Desialylation in Experimental Atherosclerosis. Toxicol Mech Methods, 17(5), 265-73 (2007)
DOI: 10.1080/15376510600992608

50. S. Benitez, J. L. Sanchez-Quesada, L. Lucero, R. Arcelus, V. Ribas, O. Jorba, A. Castellvi, E. Alonso, F. Blanco-Vaca and J. Ordonez-Llanos: Changes in low-density lipoprotein electronegativity and oxidizability after aerobic exercise are related to the increase in associated non-esterified fatty acids. Atherosclerosis, 160(1), 223-32 (2002)
DOI: 10.1016/S0021-9150(01)00565-2

51. S. Jayaraman, D. L. Gantz and O. Gursky: Effects of phospholipase A(2) and its products on structural stability of human LDL: relevance to formation of LDL-derived lipid droplets. J Lipid Res, 52(3), 549-57 (2011)
DOI: 10.1194/jlr.M012567

52. C. Phillips, D. Owens, P. Collins and G. H. Tomkin: Low density lipoprotein non-esterified fatty acids and lipoprotein lipase in diabetes. Atherosclerosis, 181(1), 109-14 (2005)
DOI: 10.1016/j.atherosclerosis.2004.12.033

53. M. Lu, D. L. Gantz, H. Herscovitz and O. Gursky: Kinetic analysis of thermal stability of human low density lipoproteins: a model for LDL fusion in atherogenesis. J Lipid Res, 53(10), 2175-85 (2012)
DOI: 10.1194/jlr.M029629

54. S. Benitez, M. Camacho, R. Arcelus, L. Vila, C. Bancells, J. Ordonez-Llanos and J. L. Sanchez-Quesada: Increased lysophosphatidylcholine and non-esterified fatty acid content in LDL induces chemokine release in endothelial cells. Relationship with electronegative LDL. Atherosclerosis, 177(2), 299-305 (2004)

55. S. Benitez, V. Villegas, C. Bancells, O. Jorba, F. Gonzalez-Sastre, J. Ordonez-Llanos and J. L. Sanchez-Quesada: Impaired binding affinity of electronegative low-density lipoprotein (LDL) to the LDL receptor is related to nonesterified fatty acids and lysophosphatidylcholine content. Biochemistry, 43(50), 15863-72 (2004)
DOI: 10.1021/bi048825z

56. J. W. Gaubatz, B. K. Gillard, J. B. Massey, R. C. Hoogeveen, M. Huang, E. E. Lloyd, J. L. Raya, C. Y. Yang and H. J. Pownall: Dynamics of dense electronegative low density lipoproteins and their preferential association with lipoprotein phospholipase A(2). J Lipid Res, 48(2), 348-57 (2007)
DOI: 10.1194/jlr.M600249-JLR200

57. S. Benitez, A. Perez, J. L. Sanchez-Quesada, A. M. Wagner, M. Rigla, R. Arcelus, O. Jorba and J. Ordonez-Llanos: Electronegative low-density lipoprotein subfraction from type 2 diabetic subjects is proatherogenic and unrelated to glycemic control. Diabetes Metab Res Rev, 23(1), 26-34 (2007)
DOI: 10.1002/dmrr.643

58. A. P. Mello, I. T. da Silva, D. S. Abdalla and N. R. Damasceno: Electronegative low-density lipoprotein: origin and impact on health and disease. Atherosclerosis, 215(2), 257-65 (2011)
DOI: 10.1016/j.atherosclerosis.2010.12.028

59. J. L. Sanchez-Quesada, S. Benitez and J. Ordonez-Llanos: Electronegative low-density lipoprotein. Curr Opin Lipidol, 15(3), 329-35 (2004)
DOI: 10.1097/00041433-200406000-00014

60. J. L. Sanchez-Quesada, S. Benitez, C. Otal, M. Franco, F. Blanco-Vaca and J. Ordonez-Llanos: Density distribution of electronegative LDL in normolipemic and hyperlipemic subjects. J Lipid Res, 43(5), 699-705 (2002)

61. J. L. Sanchez-Quesada, S. Villegas and J. Ordonez-Llanos: Electronegative low-density lipoprotein. A link between apolipoprotein B misfolding, lipoprotein aggregation and proteoglycan binding. Curr Opin Lipidol, 23(5), 479-86 (2012)
DOI: 10.1097/MOL.0b013e328357c933

62. L. Y. Ke, N. Stancel, H. Bair and C. H. Chen: The underlying chemistry of electronegative LDL’s atherogenicity. Curr Atheroscler Rep, 16(8), 428 (2014)
DOI: 10.1007/s11883-014-0428-y

63. J. L. Sanchez-Quesada, M. Estruch, S. Benítez and J. Ordonez-Llanos: Electronegative LDL: a useful biomarker of cardiovascular risk? Clin Lipidol, 7(3), 345-359 (2012)
DOI: 10.2217/clp.12.26

64. S. Lund-Katz, P. M. Laplaud, M. C. Phillips and M. J. Chapman: Apolipoprotein B-100 conformation and particle surface charge in human LDL subspecies: implication for LDL receptor interaction. Biochemistry, 37(37), 12867-74 (1998)
DOI: 10.1021/bi980828m

65. C. Bancells, F. Canals, S. Benitez, N. Colome, J. Julve, J. Ordonez-Llanos and J. L. Sanchez-Quesada: Proteomic analysis of electronegative low-density lipoprotein. J Lipid Res, 51(12):3508-15 (2010)
DOI: 10.1194/jlr.M009258

66. H. H. Chen, B. D. Hosken, M. Huang, J. W. Gaubatz, C. L. Myers, R. D. Macfarlane, H. J. Pownall and C. Y. Yang: Electronegative LDLs from familial hypercholesterolemic patients are physicochemically heterogeneous but uniformly proapoptotic. J Lipid Res, 48(1), 177-84 (2007)
DOI: 10.1194/jlr.M500481-JLR200

67. J. L. Sanchez-Quesada and A. Perez: Modified lipoproteins as biomarkers of cardiovascular risk in diabetes mellitus. Endocrinol Nutr, 60(9), 518-28 (2013)
DOI: 10.1016/j.endonu.2012.12.007

68. S. Akyol, J. Lu, O. Akyol, F. Akcay, F. Armutcu, L. Y. Ke and C. H. Chen: The role of electronegative low-density lipoprotein in cardiovascular diseases and its therapeutic implications. Trends Cardiovasc Med, 27(4), 239-246 (2017)
DOI: 10.1016/j.tcm.2016.11.002

69. T. C. Yang, P. Y. Chang and S. C. Lu: L5-LDL from ST-elevation myocardial infarction patients induces IL-1beta production via LOX-1 and NLRP3 inflammasome activation in macrophages. Am J Physiol Heart Circ Physiol, 312(2), H265-H274 (2017)
DOI: 10.1152/ajpheart.00509.2016

70. M. Y. Shen, F. Y. Chen, J. F. Hsu, R. H. Fu, C. M. Chang, C. T. Chang, C. H. Liu, J. R. Wu, A. S. Lee, H. C. Chan, J. R. Sheu, S. Z. Lin, W. C. Shyu, T. Sawamura, K. C. Chang, C. Y. Hsu and C. H. Chen: Plasma L5 levels are elevated in ischemic stroke patients and enhance platelet aggregation. Blood, 127(10), 1336-45 (2016)
DOI: 10.1182/blood-2015-05-646117

71. M. Estruch, J. L. Sanchez-Quesada, J. Ordonez Llanos and S. Benitez: Electronegative LDL: a circulating modified LDL with a role in inflammation. Mediators Inflamm, 2013, 181324 (2013)
DOI: 10.1155/2013/181324

72. C. Y. Yang, H. H. Chen, M. T. Huang, J. L. Raya, J. H. Yang, C. H. Chen, J. W. Gaubatz, H. J. Pownall, A. A. Taylor, C. M. Ballantyne, F. A. Jenniskens and C. V. Smith: Pro-apoptotic low-density lipoprotein subfractions in type II diabetes. Atherosclerosis, 193(2), 283-91 (2007)
DOI: 10.1016/j.atherosclerosis.2006.08.059

73. M. Estruch, I. Minambres, J. L. Sanchez-Quesada, M. Soler, A. Perez, J. Ordonez-Llanos and S. Benitez: Increased inflammatory effect of electronegative LDL and decreased protection by HDL in type 2 diabetic patients. Atherosclerosis (2017)
DOI: 10.1016/j.atherosclerosis.2017.07.015

74. L. Wu and K. G. Parhofer: Diabetic dyslipidemia. Metabolism, 63(12), 1469-79 (2014)
DOI: 10.1016/j.metabol.2014.08.010

75. S.-Q. J. L. Wagner Fahlin A.M., Pérez A.: Diabetes mellitus y lipemia posprandial. Endocrinol Nutr, 47, 311-21 (2000)

76. S. M. Grundy, J. I. Cleeman, S. R. Daniels, K. A. Donato, R. H. Eckel, B. A. Franklin, D. J. Gordon, R. M. Krauss, P. J. Savage, S. C. Smith, Jr., J. A. Spertus and F. Costa: Diagnosis and management of the metabolic syndrome: an American Heart Association/National Heart, Lung, and Blood Institute Scientific Statement. Circulation, 112(17), 2735-52 (2005)
DOI: 10.1161/CIRCULATIONAHA.105.169404

77. A. D. Mooradian: Dyslipidemia in type 2 diabetes mellitus. Nat Clin Pract Endocrinol Metab, 5(3), 150-9 (2009)
DOI: 10.1038/ncpendmet1066

78. B. Verges: Abnormal hepatic apolipoprotein B metabolism in type 2 diabetes. Atherosclerosis, 211(2), 353-60 (2010)
DOI: 10.1016/j.atherosclerosis.2010.01.028

79. M. Guerin, W. Le Goff, T. S. Lassel, A. Van Tol, G. Steiner and M. J. Chapman: Atherogenic role of elevated CE transfer from HDL to VLDL(1) and dense LDL in type 2 diabetes : impact of the degree of triglyceridemia. Arterioscler Thromb Vasc Biol, 21(2), 282-8 (2001)
DOI: 10.1161/01.ATV.21.2.282

80. R. M. Krauss: Lipids and lipoproteins in patients with type 2 diabetes. Diabetes Care, 27(6), 1496-504 (2004)
DOI: 10.2337/diacare.27.6.1496

81. M. R. Diffenderfer and E. J. Schaefer: The composition and metabolism of large and small LDL. Curr Opin Lipidol, 25(3), 221-6 (2014)
DOI: 10.1097/MOL.0000000000000067

82. C. C. Cowie, B. V. Howard and M. I. Harris: Serum lipoproteins in African Americans and whites with non-insulin-dependent diabetes in the US population. Circulation, 90(3), 1185-93 (1994)
DOI: 10.1161/01.CIR.90.3.1185

83. M. A. Austin and K. L. Edwards: Small, dense low density lipoproteins, the insulin resistance syndrome and noninsulin-dependent diabetes. Curr Opin Lipidol, 7(3), 167-71 (1996)
DOI: 10.1097/00041433-199606000-00010

84. R. M. Krauss: Lipoprotein subfractions and cardiovascular disease risk. Curr Opin Lipidol, 21(4), 305-11 (2010)
DOI: 10.1097/MOL.0b013e32833b7756

85. R. Carmena, P. Duriez and J. C. Fruchart: Atherogenic lipoprotein particles in atherosclerosis. Circulation, 109(23 Suppl 1), III2-7 (2004)
DOI: 10.1161/01.CIR.0000131511.50734.44

86. N. N. Younis, H. Soran, P. Pemberton, V. Charlton-Menys, M. M. Elseweidy and P. N. Durrington: Small dense LDL is more susceptible to glycation than more buoyant LDL in Type 2 diabetes. Clin Sci (Lond), 124(5), 343-9 (2013)
DOI: 10.1042/CS20120304

87. A. Kontush and M. J. Chapman: Functionally defective high-density lipoprotein: a new therapeutic target at the crossroads of dyslipidemia, inflammation, and atherosclerosis. Pharmacol Rev, 58(3), 342-74 (2006)

DOI: 10.1124/pr.58.3.1

88. A. Kontush and M. J. Chapman: Why is HDL functionally deficient in type 2 diabetes? Curr Diab Rep, 8(1), 51-9 (2008)
DOI: 10.1007/s11892-008-0010-5

89. M. Rizzo, V. Pernice, A. Frasheri, G. Di Lorenzo, G. B. Rini, G. A. Spinas and K. Berneis: Small, dense low-density lipoproteins (LDL) are predictors of cardio- and cerebro-vascular events in subjects with the metabolic syndrome. Clin Endocrinol (Oxf), 70(6), 870-5 (2009) x
DOI: 10.1111/j.1365-2265.2008.03407.x

90. E. A. Ivanova, V. A. Myasoedova, A. A. Melnichenko, A. V. Grechko and A. N. Orekhov: Small Dense Low-Density Lipoprotein as Biomarker for Atherosclerotic Diseases. Oxid Med Cell Longev, 2017, 1273042 (2017) 042

91. M. Ai, S. Otokozawa, B. F. Asztalos, Y. Ito, K. Nakajima, C. C. White, L. A. Cupples, P. W. Wilson and E. J. Schaefer: Small dense LDL cholesterol and coronary heart disease: results from the Framingham Offspring Study. Clin Chem, 56(6), 967-76 (2010)
DOI: 10.1373/clinchem.2009.137489

92. H. Arai, Y. Kokubo, M. Watanabe, T. Sawamura, Y. Ito, A. Minagawa, T. Okamura and Y. Miyamato: Small dense low-density lipoproteins cholesterol can predict incident cardiovascular disease in an urban Japanese cohort: the Suita study. J Atheroscler Thromb, 20(2), 195-203 (2013)
DOI: 10.5551/jat.14936

93. R. C. Hoogeveen, J. W. Gaubatz, W. Sun, R. C. Dodge, J. R. Crosby, J. Jiang, D. Couper, S. S. Virani, S. Kathiresan, E. Boerwinkle and C. M. Ballantyne: Small dense low-density lipoprotein-cholesterol concentrations predict risk for coronary heart disease: the Atherosclerosis Risk In Communities (ARIC) study. Arterioscler Thromb Vasc Biol, 34(5), 1069-77 (2014)
DOI: 10.1161/ATVBAHA.114.303284

94. H. Shen, L. Xu, J. Lu, T. Hao, C. Ma, H. Yang, Z. Lu, Y. Gu, T. Zhu and G. Shen: Correlation between small dense low-density lipoprotein cholesterol and carotid artery intima-media thickness in a healthy Chinese population. Lipids Health Dis, 14, 137 (2015)
DOI: 10.1186/s12944-015-0143-x

95. V. Jacomella, P. A. Gerber, K. Mosimann, M. Husmann, C. Thalhammer, I. Wilkinson, K. Berneis and B. R. Amann-Vesti: Small dense low density lipoprotein particles are associated with poor outcome after angioplasty in peripheral artery disease. PLoS One, 9(9), e108813 (2014)
DOI: 10.1371/journal.pone.0108813

96. M. G. Valle Gottlieb, I. B. da Cruz, M. M. Duarte, R. N. Moresco, M. Wiehe, C. H. Schwanke and L. C. Bodanese: Associations among metabolic syndrome, ischemia, inflammatory, oxidatives, and lipids biomarkers. J Clin Endocrinol Metab, 95(2), 586-91 (2010)
DOI: 10.1210/jc.2009-1592

97. V. Sigurdardottir, B. Fagerberg and J. Hulthe: Circulating oxidized low-density lipoprotein (LDL) is associated with risk factors of the metabolic syndrome and LDL size in clinically healthy 58-year-old men (AIR study). J Intern Med, 252(5), 440-7 (2002)
DOI: 10.1046/j.1365-2796.2002.01054.x

98. P. Holvoet, S. B. Kritchevsky, R. P. Tracy, A. Mertens, S. M. Rubin, J. Butler, B. Goodpaster and T. B. Harris: The metabolic syndrome, circulating oxidized LDL, and risk of myocardial infarction in well-functioning elderly people in the health, aging, and body composition cohort. Diabetes, 53(4), 1068-73 (2004)
DOI: 10.2337/diabetes.53.4.1068

99. T. Ueba, S. Nomura, T. Nishikawa, M. Kajiwara and K. Yamashita: Circulating oxidized LDL, measured with FOH1a/DLH3 antibody, is associated with metabolic syndrome and the coronary heart disease risk score in healthy Japanese. Atherosclerosis, 203(1), 243-8 (2009)
DOI: 10.1016/j.atherosclerosis.2008.05.048

100. P. Holvoet, G. Perez, Z. Zhao, E. Brouwers, H. Bernar and D. Collen: Malondialdehyde-modified low density lipoproteins in patients with atherosclerotic disease. J Clin Invest, 95(6), 2611-9 (1995)
DOI: 10.1172/JCI117963

101. P. Holvoet, T. B. Harris, R. P. Tracy, P. Verhamme, A. B. Newman, S. M. Rubin, E. M. Simonsick, L. H. Colbert and S. B. Kritchevsky: Association of high coronary heart disease risk status with circulating oxidized LDL in the well-functioning elderly: findings from the Health, Aging, and Body Composition study. Arterioscler Thromb Vasc Biol, 23(8), 1444-8 (2003)
DOI: 10.1161/01.ATV.0000080379.05071.22

102. P. Holvoet, A. Mertens, P. Verhamme, K. Bogaerts, G. Beyens, R. Verhaeghe, D. Collen, E. Muls and F. Van de Werf: Circulating oxidized LDL is a useful marker for identifying patients with coronary artery disease. Arterioscler Thromb Vasc Biol, 21(5), 844-8 (2001)
DOI: 10.1161/01.ATV.21.5.844

103. P. Holvoet, J. Van Cleemput, D. Collen and J. Vanhaecke: Oxidized low density lipoprotein is a prognostic marker of transplant-associated coronary artery disease. Arterioscler Thromb Vasc Biol, 20(3), 698-702 (2000)
DOI: 10.1161/01.ATV.20.3.698

104. P. Holvoet, D. Collen and F. Van de Werf: Malondialdehyde-modified LDL as a marker of acute coronary syndromes. JAMA, 281(18), 1718-21 (1999)
DOI: 10.1001/jama.281.18.1718

105. P. Holvoet, J. M. Stassen, J. Van Cleemput, D. Collen and J. Vanhaecke: Oxidized low density lipoproteins in patients with transplant-associated coronary artery disease. Arterioscler Thromb Vasc Biol, 18(1), 100-7 (1998)
DOI: 10.1161/01.ATV.18.1.100

106. S. Ehara, M. Ueda, T. Naruko, K. Haze, A. Itoh, M. Otsuka, R. Komatsu, T. Matsuo, H. Itabe, T. Takano, Y. Tsukamoto, M. Yoshiyama, K. Takeuchi, J. Yoshikawa and A. E. Becker: Elevated levels of oxidized low density lipoprotein show a positive relationship with the severity of acute coronary syndromes. Circulation, 103(15), 1955-60 (2001)
DOI: 10.1161/01.CIR.103.15.1955

107. S. Tsimikas, C. Bergmark, R. W. Beyer, R. Patel, J. Pattison, E. Miller, J. Juliano and J. L. Witztum: Temporal increases in plasma markers of oxidized low-density lipoprotein strongly reflect the presence of acute coronary syndromes. J Am Coll Cardiol, 41(3), 360-70 (2003)
DOI: 10.1016/S0735-1097(02)02769-9

108. A. Wang, Y. Yang, Z. Su, W. Yue, H. Hao, L. Ren, Y. Wang, Y. Cao and Y. Wang: Association of Oxidized Low-Density Lipoprotein With Prognosis of Stroke and Stroke Subtypes. Stroke, 48(1), 91-97 (2017)
DOI: 10.1161/STROKEAHA.116.014816

109. P. Holvoet, G. Theilmeier, B. Shivalkar, W. Flameng and D. Collen: LDL hypercholesterolemia is associated with accumulation of oxidized LDL, atherosclerotic plaque growth, and compensatory vessel enlargement in coronary arteries of miniature pigs. Arterioscler Thromb Vasc Biol, 18(3), 415-22 (1998)
DOI: 10.1161/01.ATV.18.3.415

110. S. Tsimikas, P. Clopton, E. S. Brilakis, S. M. Marcovina, A. Khera, E. R. Miller, J. A. de Lemos and J. L. Witztum: Relationship of oxidized phospholipids on apolipoprotein B-100 particles to race/ethnicity, apolipoprotein(a) isoform size, and cardiovascular risk factors: results from the Dallas Heart Study. Circulation, 119(13), 1711-9 (2009)
DOI: 10.1161/CIRCULATIONAHA.108.836940

111. J. Frostegard, R. Wu, C. Lemne, T. Thulin, J. L. Witztum and U. de Faire: Circulating oxidized low-density lipoprotein is increased in hypertension. Clin Sci (Lond), 105(5), 615-20 (2003)
DOI: 10.1042/CS20030152

112. U. P. Jorde, P. C. Colombo, K. Ahuja, A. Hudaihed, D. Onat, T. Diaz, D. S. Hirsh, E. A. Fisher, C. H. Tseng and T. J. Vittorio: Exercise-induced increases in oxidized low-density lipoprotein are associated with adverse outcomes in chronic heart failure. J Card Fail, 13(9), 759-64 (2007)
DOI: 10.1016/j.cardfail.2007.06.724

113. Y. Hurtado-Roca, H. Bueno, A. Fernandez-Ortiz, J. M. Ordovas, B. Ibanez, V. Fuster, F. Rodriguez-Artalejo and M. Laclaustra: Oxidized LDL Is Associated With Metabolic Syndrome Traits Independently of Central Obesity and Insulin Resistance. Diabetes, 66(2), 474-482 (2017)
DOI: 10.2337/db16-0933

114. L. Morell-Azanza, S. Garcia-Calzon, T. Rendo-Urteaga, N. Martin-Calvo, M. Chueca, J. A. Martinez, M. C. Azcona-Sanjulian and A. Marti: Serum oxidized low-density lipoprotein levels are related to cardiometabolic risk and decreased after a weight loss treatment in obese children and adolescents. Pediatr Diabetes (2016)

115. A. L. Norris, J. Steinberger, L. M. Steffen, A. M. Metzig, S. J. Schwarzenberg and A. S. Kelly: Circulating oxidized LDL and inflammation in extreme pediatric obesity. Obesity (Silver Spring), 19(7), 1415-9 (2011)
DOI: 10.1038/oby.2011.21

116. E. Kassi, M. Dalamaga, E. Faviou, G. Hroussalas, K. Kazanis, C. Nounopoulos and A. Dionyssiou-Asteriou: Circulating oxidized LDL levels, current smoking and obesity in postmenopausal women. Atherosclerosis, 205(1), 279-83 (2009)
DOI: 10.1016/j.atherosclerosis.2008.11.006

117. T. A. Takamura, T. Tsuchiya, M. Oda, M. Watanabe, R. Saito, R. Sato-Ishida, H. Akao, Y. Kawai, M. Kitayama and K. Kajinami: Circulating malondialdehyde-modified low-density lipoprotein (MDA-LDL) as a novel predictor of clinical outcome after endovascular therapy in patients with peripheral artery disease (PAD). Atherosclerosis, 263, 192-197 (2017)
DOI: 10.1016/j.atherosclerosis.2017.06.029

118. W. Koenig, M. Karakas, A. Zierer, C. Herder, J. Baumert, C. Meisinger and B. Thorand: Oxidized LDL and the risk of coronary heart disease: results from the MONICA/KORA Augsburg Study. Clin Chem, 57(8), 1196-200 (2011)
DOI: 10.1373/clinchem.2011.165134

119. T. Wu, W. C. Willett, N. Rifai, I. Shai, J. E. Manson and E. B. Rimm: Is plasma oxidized low-density lipoprotein, measured with the widely used antibody 4E6, an independent predictor of coronary heart disease among U.S. men and women? J Am Coll Cardiol, 48(5), 973-9 (2006)
DOI: 10.1016/j.jacc.2006.03.057

120. A. Trpkovic, I. Resanovic, J. Stanimirovic, D. Radak, S. A. Mousa, D. Cenic-Milosevic, D. Jevremovic and E. R. Isenovic: Oxidized low-density lipoprotein as a biomarker of cardiovascular diseases. Crit Rev Clin Lab Sci, 52(2), 70-85 (2015)
DOI: 10.3109/10408363.2014.992063

121. S. Toshima, A. Hasegawa, M. Kurabayashi, H. Itabe, T. Takano, J. Sugano, K. Shimamura, J. Kimura, I. Michishita, T. Suzuki and R. Nagai: Circulating oxidized low density lipoprotein levels. A biochemical risk marker for coronary heart disease. Arterioscler Thromb Vasc Biol, 20(10), 2243-7 (2000)
DOI: 10.1161/01.ATV.20.10.2243

122. A. Segev, B. H. Strauss, J. L. Witztum, H. K. Lau and S. Tsimikas: Relationship of a comprehensive panel of plasma oxidized low-density lipoprotein markers to angiographic restenosis in patients undergoing percutaneous coronary intervention for stable angina. Am Heart J, 150(5), 1007-14 (2005)
DOI: 10.1016/j.ahj.2004.12.008

123. S. H. Choi, A. Chae, E. Miller, M. Messig, F. Ntanios, A. N. DeMaria, S. E. Nissen, J. L. Witztum and S. Tsimikas: Relationship between biomarkers of oxidized low-density lipoprotein, statin therapy, quantitative coronary angiography, and atheroma: volume observations from the REVERSAL (Reversal of Atherosclerosis with Aggressive Lipid Lowering) study. J Am Coll Cardiol, 52(1), 24-32 (2008)
DOI: 10.1016/j.jacc.2008.02.066

124. J. A. Oliveira, A. Sevanian, R. J. Rodrigues, E. Apolinario and D. S. Abdalla: Minimally modified electronegative LDL and its autoantibodies in acute and chronic coronary syndromes. Clin Biochem, 39(7), 708-14 (2006)
DOI: 10.1016/j.clinbiochem.2006.05.002

125. G. Niccoli, M. Baca, M. De Spirito, T. Parasassi, N. Cosentino, G. Greco, M. Conte, R. A. Montone, G. Arcovito and F. Crea: Impact of electronegative low-density lipoprotein on angiographic coronary atherosclerotic burden. Atherosclerosis, 223(1), 166-70 (2012)
DOI: 10.1016/j.atherosclerosis.2012.04.005

126. B. Zhang, N. Maeda, K. Okada, M. Tatsukawa, Y. Sawayama, A. Matsunaga, K. Kumagai, S. Miura, T. Nagao, J. Hayashi and K. Saku: Association between fast-migrating low-density lipoprotein subfraction as characterized by capillary isotachophoresis and intima-media thickness of carotid artery. Atherosclerosis, 187(1), 205-12 (2006)
DOI: 10.1016/j.atherosclerosis.2005.09.005

127. R. Gambino, E. Pisu, G. Pagano and M. Cassader: Low-density lipoproteins are more electronegatively charged in type 1 than in type 2 diabetes mellitus. Lipids, 41(6), 529-33 (2006)
DOI: 10.1007/s11745-006-5001-1

128. E. Moro, P. Alessandrini, C. Zambon, S. Pianetti, M. Pais, G. Cazzolato and G. B. Bon: Is glycation of low density lipoproteins in patients with Type 2 diabetes mellitus a LDL pre-oxidative condition? Diabet Med, 16(8), 663-9 (1999)
DOI: 10.1046/j.1464-5491.1999.00136.x

129. J. L. Sanchez-Quesada, A. Perez, A. Caixas, J. Ordonmez-Llanos, G. Carreras, A. Payes, F. Gonzalez-Sastre and A. de Leiva: Electronegative low density lipoprotein subform is increased in patients with short-duration IDDM and is closely related to glycaemic control. Diabetologia, 39(12), 1469-76 (1996)
DOI: 10.1007/s001250050600

130. J. L. Sanchez-Quesada, A. Perez, A. Caixas, M. Rigla, A. Payes, S. Benitez and J. Ordonez-Llanos: Effect of glycemic optimization on electronegative low-density lipoprotein in diabetes: relation to nonenzymatic glycosylation and oxidative modification. J Clin Endocrinol Metab, 86(7), 3243-9 (2001)
DOI: 10.1210/jc.86.7.3243

131. J. L. Sanchez-Quesada, I. Vinagre, E. D. Juan-Franco, J. Sanchez-Hernandez, F. Blanco-Vaca, J. Ordonez-Llanos and A. Perez: Effect of Improving Glycemic Control in Patients With Type 2 Diabetes Mellitus on Low-Density Lipoprotein Size, Electronegative Low-Density Lipoprotein and Lipoprotein-Associated Phospholipase A2 Distribution. Am J Cardiol, 110, 67-71 (2012)
DOI: 10.1016/j.amjcard.2012.02.051

132. B. Zhang, T. Kaneshi, T. Ohta and K. Saku: Relation between insulin resistance and fast-migrating LDL subfraction as characterized by capillary isotachophoresis. J Lipid Res, 46(10), 2265-77 (2005)
DOI: 10.1194/jlr.M500192-JLR200

133. J. F. Hsu, T. C. Chou, J. Lu, S. H. Chen, F. Y. Chen, C. C. Chen, J. L. Chen, M. Elayda, C. M. Ballantyne, S. Shayani and C. H. Chen: Low-density lipoprotein electronegativity is a novel cardiometabolic risk factor. PLoS One, 9(9), e107340 (2014)
DOI: 10.1371/journal.pone.0107340

134. N. R. Damasceno, A. Sevanian, E. Apolinario, J. M. Oliveira, I. Fernandes and D. S. Abdalla: Detection of electronegative low density lipoprotein (LDL-) in plasma and atherosclerotic lesions by monoclonal antibody-based immunoassays. Clin Biochem, 39(1), 28-38 (2006)
DOI: 10.1016/j.clinbiochem.2005.09.014

135. E. Faulin Tdo, K. C. de Sena-Evangelista, D. B. Pacheco, E. M. Augusto and D. S. Abdalla: Development of immunoassays for anti-electronegative LDL autoantibodies and immune complexes. Clin Chim Acta, 413(1-2), 291-7 (2011)
DOI: 10.1016/j.cca.2011.10.004

136. D. M. Grosso, S. Ferderbar, A. C. Wanschel, M. H. Krieger, M. L. Higushi and D. S. Abdalla: Antibodies against electronegative LDL inhibit atherosclerosis in LDLr-/- mice. Braz J Med Biol Res, 41(12), 1086-92 (2008)
DOI: 10.1590/S0100-879X2008001200007

137. J. L. Sanchez-Quesada, I. Vinagre, E. de Juan-Franco, J. Sanchez-Hernandez, F. Blanco-Vaca, J. Ordonez-Llanos and A. Perez: Effect of improving glycemic control in patients with type 2 diabetes mellitus on low-density lipoprotein size, electronegative low-density lipoprotein and lipoprotein-associated phospholipase A2 distribution. Am J Cardiol, 110(1), 67-71 (2012)
DOI: 10.1016/j.amjcard.2012.02.051

138. J. L. Sanchez-Quesada, I. Vinagre, E. De Juan-Franco, J. Sanchez-Hernandez, R. Bonet-Marques, F. Blanco-Vaca, J. Ordonez-Llanos and A. Perez: Impact of the LDL subfraction phenotype on Lp-PLA2 distribution, LDL modification and HDL composition in type 2 diabetes. Cardiovasc Diabetol, 12, 112 (2013)
DOI: 10.1186/1475-2840-12-112

139. M. F. Lopes-Virella, N. L. Baker, K. J. Hunt, J. Lachin, D. Nathan and G. Virella: Oxidized LDL immune complexes and coronary artery calcification in type 1 diabetes. Atherosclerosis, 214(2), 462-7 (2011)
DOI: 10.1016/j.atherosclerosis.2010.11.012

140. K. Shimada, H. Mokuno, E. Matsunaga, T. Miyazaki, K. Sumiyoshi, A. Kume, K. Miyauchi and H. Daida: Predictive value of circulating oxidized LDL for cardiac events in type 2 diabetic patients with coronary artery disease. Diabetes Care, 27(3), 843-4 (2004)
DOI: 10.2337/diacare.27.3.843

141. N. Ujihara, Y. Sakka, M. Takeda, M. Hirayama, A. Ishii, O. Tomonaga, T. Babazono, C. Takahashi, K. Yamashita and Y. Iwamoto: Association between plasma oxidized low-density lipoprotein and diabetic nephropathy. Diabetes Res Clin Pract, 58(2), 109-14 (2002)
DOI: 10.1016/S0168-8227(02)00134-1

142. K. Gokulakrishnan, R. Deepa, K. Velmurugan, R. Ravikumar, K. Karkuzhali and V. Mohan: Oxidized low-density lipoprotein and intimal medial thickness in subjects with glucose intolerance: the Chennai Urban Rural Epidemiology Study-25. Metabolism, 56(2), 245-50 (2007)
DOI: 10.1016/j.metabol.2006.10.002

143. F. Akhter, M. Salman Khan, M. Faisal, A. A. Alatar and S. Ahmad: Detection of Circulating Auto-Antibodies Against Ribosylated-LDL in Diabetes Patients. J Clin Lab Anal, 31(2) (2017)
DOI: 10.1002/jcla.22039

144. M. F. Lopes-Virella and G. Virella: Clinical significance of the humoral immune response to modified LDL. Clin Immunol, 134(1), 55-65 (2010)
DOI: 10.1016/j.clim.2009.04.001

145. D. Engelbertsen, J. Vallejo, T. D. Quach, G. N. Fredrikson, R. Alm, B. Hedblad, H. Bjorkbacka, T. L. Rothstein, J. Nilsson and E. Bengtsson: Low Levels of IgM Antibodies against an Advanced Glycation Endproduct-Modified Apolipoprotein B100 Peptide Predict Cardiovascular Events in Nondiabetic Subjects. J Immunol, 195(7), 3020-5 (2015)
DOI: 10.4049/jimmunol.1402869

146. D. Engelbertsen, D. V. Anand, G. N. Fredrikson, D. Hopkins, R. Corder, P. K. Shah, A. Lahiri, J. Nilsson and E. Bengtsson: High levels of IgM against methylglyoxal-modified apolipoprotein B100 are associated with less coronary artery calcification in patients with type 2 diabetes. J Intern Med, 271(1), 82-9 (2012)
DOI: 10.1111/j.1365-2796.2011.02411.x

147. S. Tsimikas, W. Palinski and J. L. Witztum: Circulating autoantibodies to oxidized LDL correlate with arterial accumulation and depletion of oxidized LDL in LDL receptor-deficient mice. Arterioscler Thromb Vasc Biol, 21(1), 95-100 (2001)
DOI: 10.1161/01.ATV.21.1.95

148. R. Laczik, P. Szodoray, K. Veres, E. Szomjak, I. Csipo, S. Sipka, Jr., Y. Shoenfeld, Z. Szekanecz and P. Soltesz: Assessment of IgG antibodies to oxidized LDL in patients with acute coronary syndrome. Lupus, 20(7), 730-5 (2011)
DOI: 10.1177/0961203311398884

149. G. Asciutto, N. V. Dias, A. Edsfeldt, R. Alm, G. N. Fredrikson, I. Goncalves and J. Nilsson: Low levels of IgG autoantibodies against the apolipoprotein B antigen p210 increases the risk of cardiovascular death after carotid endarterectomy. Atherosclerosis, 239(2), 289-94 (2015)
DOI: 10.1016/j.atherosclerosis.2015.01.023

150. G. Asciutto, M. Wigren, G. N. Fredrikson, I. Y. Mattisson, C. Gronberg, R. Alm, H. Bjorkbacka, N. V. Dias, A. Edsfeldt, I. Goncalves and J. Nilsson: Apolipoprotein B-100 Antibody Interaction With Atherosclerotic Plaque Inflammation and Repair Processes. Stroke, 47(4), 1140-3 (2016)
DOI: 10.1161/STROKEAHA.116.012677

151. Y. Li, Z. Lu, Y. Huang, M. F. Lopes-Virella and G. Virella: F(ab’)2 fragments of anti-oxidized LDL IgG attenuate vascular inflammation and atherogenesis in diabetic LDL receptor-deficient mice. Clin Immunol, 173, 50-56 (2016)
DOI: 10.1016/j.clim.2016.07.020
PMid:27455858

152. L. Zhu, Z. He, F. Wu, R. Ding, Q. Jiang, J. Zhang, M. Fan, X. Wang, B. Eva, N. Jan, C. Liang and Z. Wu: Immunization with advanced glycation end products modified low density lipoprotein inhibits atherosclerosis progression in diabetic apoE and LDLR null mice. Cardiovasc Diabetol, 13, 151 (2014)
DOI: 10.1186/s12933-014-0151-6
PMid:25391642 PMCid:PMC4234834

153. E. Apolinario, S. Ferderbar, E. C. Pereira, M. C. Bertolami, A. Faludi, O. Monte, A. R. Gagliardi, H. T. Xavier and D. S. Abdalla: Minimally modified (electronegative) LDL- and anti-LDL- autoantibodies in diabetes mellitus and impaired glucose tolerance. Int J Atheroscler, 1(1), 42-47 (2006)

154. G. Virella, J. Colglazier, C. Chassereau, K. J. Hunt, N. L. Baker and M. F. Lopes-Virella: Immunoassay of modified forms of human low density lipoprotein in isolated circulating immune complexes. J Immunoassay Immunochem, 34(1), 61-74 (2013)
DOI: 10.1080/15321819.2012.683500
PMid:23323982

155. E. Korpinen, P. H. Groop, H. K. Akerblom and O. Vaarala: Immune response to glycated and oxidized LDL in IDDM patients with and without renal disease. Diabetes Care, 20(7), 1168-71 (1997)
DOI: 10.2337/diacare.20.7.1168
PMid:9203457

156. G. Virella, R. E. Carter, A. Saad, E. G. Crosswell, B. A. Game and M. F. Lopes-Virella: Distribution of IgM and IgG antibodies to oxidized LDL in immune complexes isolated from patients with type 1 diabetes and its relationship with nephropathy. Clin Immunol, 127(3), 394-400 (2008)
DOI: 10.1016/j.clim.2008.02.005
PMid:18533284 PMCid:PMC2601558

157. M. F. Lopes-Virella, N. Binzafar, S. Rackley, A. Takei, M. La Via and G. Virella: The uptake of LDL-IC by human macrophages: predominant involvement of the Fc gamma RI receptor. Atherosclerosis, 135(2), 161-70 (1997)
DOI: 10.1016/S0021-9150(97)00157-3

158. G. Virella and M. F. Lopes-Virella: The Pathogenic Role of the Adaptive Immune Response to Modified LDL in Diabetes. Front Endocrinol (Lausanne), 3, 76 (2012)
DOI: 10.3389/fendo.2012.00076
PMid:22715334 PMCid:PMC3375400

159. A. F. Saad, G. Virella, C. Chassereau, R. J. Boackle and M. F. Lopes-Virella: OxLDL immune complexes activate complement and induce cytokine production by MonoMac 6 cells and human macrophages. J Lipid Res, 47(9), 1975-83 (2006)
DOI: 10.1194/jlr.M600064-JLR200
PMid:16804192

160. I. A. Sobenin, J. T. Salonen, A. V. Zhelankin, A. A. Melnichenko, J. Kaikkonen, Y. V. Bobryshev and A. N. Orekhov: Low density lipoprotein-containing circulating immune complexes: role in atherosclerosis and diagnostic value. Biomed Res Int, 2014, 205697 (2014)
DOI: 10.1155/2014/205697
PMid:25054132 PMCid:PMC4087281

161. I. A. Sobenin, V. P. Karagodin, A. C. Melnichenko, Y. V. Bobryshev and A. N. Orekhov: Diagnostic and prognostic value of low density lipoprotein-containing circulating immune complexes in atherosclerosis. J Clin Immunol, 33(2), 489-95 (2013)
DOI: 10.1007/s10875-012-9819-4
PMid:23073618

162. A. N. Orekhov, Y. V. Bobryshev, I. A. Sobenin, A. A. Melnichenko and D. A. Chistiakov: Modified low density lipoprotein and lipoprotein-containing circulating immune complexes as diagnostic and prognostic biomarkers of atherosclerosis and type 1 diabetes macrovascular disease. Int J Mol Sci, 15(7), 12807-41 (2014)
DOI: 10.3390/ijms150712807
PMid:25050779 PMCid:PMC4139876

163. M. F. Lopes-Virella, K. J. Hunt, N. L. Baker, J. Lachin, D. M. Nathan and G. Virella: Levels of oxidized LDL and advanced glycation end products-modified LDL in circulating immune complexes are strongly associated with increased levels of carotid intima-media thickness and its progression in type 1 diabetes. Diabetes, 60(2), 582-9 (2011)
DOI: 10.2337/db10-0915
PMid:20980456 PMCid:PMC3028359

164. K. J. Hunt, N. Baker, P. Cleary, J. Y. Backlund, T. Lyons, A. Jenkins, G. Virella, M. F. Lopes-Virella and D. E. R. Group: Oxidized LDL and AGE-LDL in circulating immune complexes strongly predict progression of carotid artery IMT in type 1 diabetes. Atherosclerosis, 231(2), 315-22 (2013)
DOI: 10.1016/j.atherosclerosis.2013.09.027
PMid:24267245 PMCid:PMC3924569

165. M. F. Lopes-Virella, K. J. Hunt, N. L. Baker, G. Virella and T. Moritz: The levels of MDA-LDL in circulating immune complexes predict myocardial infarction in the VADT study. Atherosclerosis, 224(2):526-31 (2011)
DOI: 10.1016/j.atherosclerosis.2012.08.006
PMid:22963984 PMCid:PMC4240617

166. M. F. Lopes-Virella, R. E. Carter, N. L. Baker, J. Lachin and G. Virella: High levels of oxidized LDL in circulating immune complexes are associated with increased odds of developing abnormal albuminuria in Type 1 diabetes. Nephrol Dial Transplant, 27(4), 1416-23 (2012)
DOI: 10.1093/ndt/gfr454
PMid:21856760 PMCid:PMC3471550

167. M. F. Lopes-Virella, N. L. Baker, K. J. Hunt, T. J. Lyons, A. J. Jenkins, G. Virella and D. E. S. Group: High concentrations of AGE-LDL and oxidized LDL in circulating immune complexes are associated with progression of retinopathy in type 1 diabetes. Diabetes Care, 35(6), 1333-40 (2012)
DOI: 10.2337/dc11-2040
PMid:22511260 PMCid:PMC3357232

168. M. F. Lopes-Virella, K. J. Hunt, N. L. Baker, G. Virella, T. Moritz and V. Investigators: The levels of MDA-LDL in circulating immune complexes predict myocardial infarction in the VADT study. Atherosclerosis, 224(2), 526-31 (2012)
DOI: 10.1016/j.atherosclerosis.2012.08.006

169. G. N. Fredrikson, D. V. Anand, D. Hopkins, R. Corder, R. Alm, E. Bengtsson, P. K. Shah, A. Lahiri and J. Nilsson: Associations between autoantibodies against apolipoprotein B-100 peptides and vascular complications in patients with type 2 diabetes. Diabetologia, 52(7), 1426-33 (2009)
DOI: 10.1007/s00125-009-1377-9

170. M. F. Lopes-Virella, K. J. Hunt, N. L. Baker, G. Virella and V. G. o. Investigators: High levels of AGE-LDL, and of IgG antibodies reacting with MDA-lysine epitopes expressed by oxLDL and MDA-LDL in circulating immune complexes predict macroalbuminuria in patients with type 2 diabetes. J Diabetes Complications, 30(4), 693-9 (2016)
DOI: 10.1016/j.jdiacomp.2016.01.012

Key Words: Diabetes, metabolic syndrome, atherosclerosis, cardiovascular risk, biomarkers, modified LDL, oxidized LDL, AGE-LDL, glycosylated LDL, electronegative LDL

Send correspondence to: Jose Luis Sanchez-Quesada, Cardiovascular Biochemistry Group, Research Institute of the Hospital de Sant Pau (IIB Sant Pau), Barcelona, Spain, Tel: 3435537588, Fax: 3435537287, E-mail: jsanchezq@santpau.cat