Abstract
There are several ways of demonstrating the major role not only of total cholesterol but mainly its low density lipoprotein fraction on the origin of the fatty streaks that can evolve into atherosclerotic plaques. Different approaches lead to the same assertion as can be verified by levels of research that approach the topic. The first studies were experimental. Conversion of fatty streaks into fibrous plaques: during long periods of feeding animals with cholesterol, there is finally deposition of connective tissue and development of a fibrous layer. The Framingham study was the one with the longest longitudinal follow-up time, establishing the concepts of cardiovascular epidemiology. Conceived in the city of Framingham, it followed a cohort of 6,000 individuals in order to investigate the risk factors correlated with the development of atherosclerotic disease. Over the course of 20 years, the study confirmed the importance of high levels of low-density lipoprotein cholesterol (LDL-C) and reduction of high-density lipoprotein cholesterol (HDL-C). Probably one of the first indications that coronary artery disease was related to cholesterol came from anecdotal case reports of children with xanthomas, who had sudden death or myocardial infarction before the age of 10. The first cases of homozygous familial hypercholesterolemia were described. Several researchers have described familial hypercholesterolemia as a genetic, monogenic alteration with a risk of early coronary artery disease, secondary to serum cholesterol elevations. The importance of LDL-C in atherogenesis can be explained in various contexts as represented by experimental researches, epidemiological data and genetic studies.
Abbreviations
HDL-C: High Density Lipoprotein Cholesterol
LDL-C: Low Density Lipoprotein Cholesterol
Introduction
Although the scientific literature is abundant on cholesterol’s role in atherosclerosis, speculations on its importance is still demeaned by many media communications. In this article we reaffirm the concept by describing the various levels of evidence of this approach.
Atherosclerotic disease and its clinical manifestations, which include acute myocardial infarction and ischemic stroke, are the leading cause of morbidity and mortality in the world. Among the atherogenic risk factors, the most well-documented and that determines a causal relationship with atherosclerotic disease are the values of low-density lipoprotein cholesterol (LDL-C) [1]. There is multiple evidence, from experimental studies to large intervention studies, that have established not only the role of LDL-C, but also of apolipoprotein B (Apo B), including triglyceride-rich lipoproteins and their remnants and lipoprotein (a) [(Lp(a)] as active participants in the atherogenic process [1]. Despite the accumulation of evidence over decades, there is some skepticism as to the causal nature of LDL-C and atherosclerotic disease. It is essential to identify LDL-C as a therapeutic target to reduce cardiovascular risk, especially after the emergence of new drugs that further reduce LDL-C levels, with additional risk reduction [1].
This article aims to demonstrate that LDL-C is an important atherogenic risk factor and that any mechanism of reduction in plasma LDL-C concentrations reduces the risk of events proportional to the absolute reduction of LDL-C and the cumulative time of exposure to it.
Experimental evidence
The fundamental role of cholesterol in the pathogenesis of atherosclerosis was proposed more than 100 years ago by Nikolai N. Anitschkow, a young pathologist from St. Petersburg, who when feeding rabbits a diet rich in cholesterol, observed the appearance of arterial lesions that resembled the atherosclerotic lesions of humans, publishing this experiment in 1913 [2]. The blood of these animals showed an increase in cholesterol and probably this "lipoid" content was deposited in the arterial wall [2].
Anitschkow went beyond the initial observations, not least because it would take time for this animal model to be accepted as an experimental model of human atherosclerosis. In the 20 years following his first article, Anitschkow described the fundamentals related to the pathophysiology and progression of atherosclerosis, published in 1933 [3]:
Foamy cells: in the initial lesions – the fatty streaks – most of the lipids are found inside the cells in the form of multiple and small lipid droplets. Because lipids are extracted during routine preparation of tissue samples, multiple lipid droplets are seen as empty vacuoles; hence the designation "foam cells".
Cholesterol accumulation: in tissue sections, lipid droplets are birefringent. Anitschkow recognized birefringence as a characteristic property of liquid crystals of cholesterol esters.
White blood cell recruitment: cholesterol-laden foamy cells are white blood cells that have infiltrated the artery wall. Thus, Anitschkow anticipated that inflammation could play a role in the development of the lesion.
Structurally intact endothelium: the monolayer of endothelial cells over the lesions appears to be intact, indicating that the invading blood cells must have penetrated through the endothelial cells. Endothelial denudation, although it occurred later, was not a necessary antecedent for the formation of the lesion.
Anatomical and non-random distribution of lesions: there is a characteristic and reproducible pattern of distribution of lesions. They occur most commonly and at arterial branching points. Anitschkow correctly assumed that this location was determined by hemodynamic factors.
Conversion of fatty streaks into fibrous plaques: during long periods of feeding the animals with cholesterol (months), there is finally deposition of connective tissue (conversion of the fatty band into fibrous plaque) and development of a fibrous layer. In human disease, it is the rupture of this fibrous layer that precipitates thrombosis and acute myocardial infarction; neither the rabbit model nor other animal models reproduce this terminal thrombotic event with any regularity.
Reversibility: initial lesions are partially reversible, but reversal is slow; late lesions resolve even more slowly. Most, but not all, lipids can be mobilized from advanced lesions, abandoning the fibrous layer and some cholesterol crystals.
Severity of the lesions is proportional to the increase in the level of cholesterol in the blood. The extent and severity of the lesions are proportional to the degree of elevation of cholesterol in the blood and the duration of exposure to it. Anitschkow was well aware that it was the level of cholesterol in the blood that determined the size and extent of the lesions, and not necessarily the amount of cholesterol ingested.
Notion of multicausality: high cholesterol in the blood is necessary, but not always sufficient, to develop atherosclerosis.
In the 1933 review, Anitschkow points out the etiological nature of atherosclerosis [3]. For the author, although the degree of atherosclerosis was more evident depending on the degree of elevation of cholesterol in the blood, this process could be affected by other factors such as blood pressure, toxic substances and local arterial alterations. In his animal model, however, such additional insults or insults were not necessary; hypercholesterolemia was sufficient. The accuracy of this conclusion was confirmed by Watanabe's discovery in 1980, where rabbits of a certain strain had blood cholesterol levels of 600 mg/dl and uniformly developed atherosclerosis after a regular diet [4]. These rabbits had mutations in the LDL-C receptor gene similar to those found in individuals with familial hypercholesterolemia, resulting in high plasma LDL-C values, which is a sufficient cause of atherosclerosis [4]. However, as Anitschkow acknowledged, the rate of progression of lesions, at any LDL-C level, was significantly slowed or accelerated by other factors, such as hypertension or immune system disorders.
Defending the importance of hypercholesterolemia in human atherosclerosis was an uphill battle [5]. Anitschkow was a scientist far ahead of his time. He was born in 1885 and wrote his classic article in 1913. The general acceptance of the lipid theory would have to wait more than 60 years to be recognized, when in 1984, the National Heart Institute (NHI) concluded the first large-scale, randomized, double-blind clinical trial, showing that lowering serum cholesterol would significantly reduce the risk of myocardial infarction [6,7]. This primary prevention trial with cholestyramine, a bile salt scavenger that lasted 7 years, was a milestone in cardiovascular prevention. This and other evidence that implicated cholesterol as a causal agent of atherosclerosis formed the basis for the first National Heart Institute Consensus [8] and the formulation of national guidelines for the control of high cholesterol levels.
Epidemiological evidence
Previous studies have shown that blood cholesterol levels were determined largely by the amount of fat in the diet.
The study of the seven countries was the first epidemiological evidence that associated the increase in cholesterol with cardiovascular events. This study selected seven countries ranging from Japan, where the average cholesterol values were the lowest (160 mg/dl) to countries such as Finland, with very high average cholesterol values (260 mg/dl). The saturated fat content was 2.5% (of the total caloric value) in Japan and 20% in Finland. During the 10-year segment, for every 1,000 individuals followed, 70 deaths from myocardial infarction were recorded in Finland and only five in Japan. This study demonstrated that the risk of fatal and non-fatal coronary events was proportional to serum cholesterol values, which in turn were proportional to saturated fat intake [9,10].
The Framingham study was the one with the longest longitudinal segment time, establishing the concepts of cardiovascular epidemiology. Conceived in the city of Framingham, it followed a cohort of 6,000 individuals in order to investigate the risk factors correlated with the development of atherosclerotic disease. Over the course of 20 years, the study confirmed the importance of high LDL-C levels and reduction of high-density lipoprotein cholesterol (HDL-C), in addition to the additive effect of other risk factors such as smoking, diabetes mellitus, hypertension, obesity, sedentary lifestyle, metabolic syndrome, and excessive alcohol intake, as factors strongly correlated with atherosclerosis and its clinical manifestations, especially coronary artery disease and cerebrovascular disease [11,12]. The Framingham data had a major impact on epidemiological research on ischemic heart disease, more than any other epidemiological study alone, laying the foundations of cardiovascular prevention. After 60 years, the study continues to follow generations of descendants of those who participated from the beginning [13].
Several meta-analyses of prospective epidemiological studies have confirmed the consistent relationship between the magnitude of exposure to elevated LDL-C values and cardiovascular risk.
The Emerging Risk Factors Collaboration (ERFC) evaluated individual data from 302,430 participants with no previous cardiovascular disease from 68 prospective studies, including 58 cohort studies, four case-control studies, and six randomized intervention studies. It was observed that the linear elevation of plasma LDL-C values was associated with an increased risk of non-fatal myocardial infarction or death from coronary heart disease. Although the authors reported an association between non-HDL cholesterol concentration and coronary heart disease risk in the primary analysis, any regression model that includes terms for non-HDL, HDL-C, and triglycerides is a simple mathematical rearrangement of a model that includes terms for LDL-C, HDL-C, and calculated triglycerides. Therefore, in the analysis of the Emerging Risk Factors Collaboration, the effect of both LDL-C and non-HDL-C were equal in predicting the risk of coronary artery disease. To confirm this fact, in a sub-sample of eight studies involving 44,234 individuals, the impact of LDL-C values (measured directly) on the risk of ischemic heart disease was similar to the impact of non-HDL cholesterol [14].
Genetic evidence
Probably one of the first indications that coronary artery disease was related to cholesterol came from anecdotal case reports of children with xanthomas (large deposits of lipids just below the skin or attached to tendon sheaths, on the back of the hands or ankles), who had sudden death or myocardial infarction before the age of 10. The first cases of homozygous familial hypercholesterolemia were described [15].
Several researchers have described familial hypercholesterolemia as a genetic alteration, monogenic and with a risk of early coronary artery disease, secondary to serum cholesterol elevations [15,16]. The nature of the gene involved was discovered by Michael S. Brown and Joseph, Goldstein, who identified the LDL-C receptor as the causative gene for familial hypercholesterolemia, demonstrating its critical role in determining blood LDL-C levels, as reported: "Our approach to unraveling the genetic defect in familial hypercholesterolemia was to apply cell culture techniques. Our studies led to the discovery of a cell surface receptor for LDL and the elucidation of the mechanism by which this receptor transports LDL particles into cells, through depressions and coated vesicles. Within the cell, LDL-C-derived cholesterol triggers several regulatory functions, including feedback inhibition of cholesterol synthesis. Therefore, we discovered that familial hypercholesterolemia is caused by genetic defects in the LDL receptor. These defects disrupt the normal regulation of cholesterol metabolism. In addition, LDL-C receptor studies have provided clear evidence for the selective uptake of macromolecules into cells, giving rise to the concept of receptor-mediated endocytosis" [17].
Familial hypercholesterolemia is an autosomal codominant disease mediated by mutation with loss of function in the LDL-C receptor gene (LDLR), causing increased levels of circulating LDL [18]. Other less frequent mutations have been described, such as mutation in the gene encoding apolipoprotein B resulting in loss of function and non-recognition of the LDL receptor by apolipoprotein B- and mutation in gain-of-function genes encoding the PCSK9 protein (proprotein convertase subtilisin/kexin type 9), which degrades the LDL receptor, raising plasma LDL-C levels. Regardless of the underlying genetic defect, familial hypercholesterolemia is an autosomal dominant disease, characterized by markedly elevated LDL-C levels and premature atherosclerosis, particularly coronary artery disease [18,19].
The most common form is heterozygous familial hypercholesterolemia, which affects between 1:200 and 1:300 individuals and which, if left untreated, markedly increases the risk of early atherosclerotic disease in adults [20,21]. Homozygous familial hypercholesterolemia is a rare condition with an extreme phenotype, characterized by plasma LDL-C levels above 500 mg/dl from birth. If left untreated, it leads to the development, in almost all patients, of premature atherosclerosis in childhood or adolescence [19,22,23].
In conclusion, although the phenotypic expression of familial hypercholesterolemia is variable, the risk of cardiovascular events is proportional to the absolute magnitude and duration of exposure to elevated LDL-C levels.
Acknowledgments
None.
Conflict of interest
None.
