Published online 2018 May
Alexandros Tsoupras | Ronan Lordan | Ioannis Zabetakis
Since the Seven Countries Study, dietary cholesterol and the levels of serum cholesterol in relation to the development of chronic diseases have been somewhat demonised. However, the principles of the Mediterranean diet and relevant data linked to the examples of people living in the five blue zones demonstrate that the key to longevity and the prevention of chronic disease development is not the reduction of dietary or serum cholesterol but the control of systemic inflammation. In this review, we present all the relevant data that supports the view that it is inflammation induced by several factors, such as platelet-activating factor (PAF), that leads to the onset of cardiovascular diseases (CVD) rather than serum cholesterol. The key to reducing the incidence of CVD is to control the activities of PAF and other inflammatory mediators via diet, exercise, and healthy lifestyle choices. The relevant studies and data supporting these views are discussed in this review.
1.1. Biological Significance of Cholesterol—Circulating Blood Cholesterol
Cholesterol, an unsaturated alcohol of the steroid family, is essential for the normal function of all animal cells. It is also a fundamental element for the normal structural makeup and the fluidity of all cell membranes. Cholesterol interacts with phospholipid bilayers in the cell membrane and increases membrane packing. Cholesterol also takes part in signal transduction, intracellular transport, nerve conduction, and signalling pathways through lipid rafts and caveolae. Cholesterol has various other biological functions, i.e., it is a precursor molecule for several biochemical pathways such as the synthesis of vitamin D, steroid hormones (e.g., cortisol, aldosterone, and adrenal androgens), and sex hormones (e.g., testosterone, oestrogens, and progesterone). Cholesterol is also a constituent of bile salts, which are crucial constituents of digestion, as they facilitate the absorption of lipids, fats, and fat-soluble vitamins A, D, E, and K .
Since cholesterol is mostly a lipophilic molecule, it does not dissolve well in blood. For this reason, it is packed into lipoproteins that are composed of a lipid core (which can contain cholesterol esters and triglycerides) and a hydrophilic outer membrane comprising phospholipids, apolipoprotein, and free cholesterol. This allows for the transport of the nonpolar lipid molecules such as cholesterol and triglycerides around the body through the blood to cells that require them. Plasma lipoproteins are separated into five major classes: chylomicrons, very-low-density lipoproteins (VLDL), intermediate-density lipoproteins (IDL), low-density lipoproteins (LDL), and high-density lipoproteins (HDL) [1,2].
Cholesterol can enter the blood through the digestion of dietary fat via chylomicrons. However, since cholesterol has an important role in cellular function, it can also be directly synthesised by each cell in the body. Notably, LDL particles are thought to act as a major transporter of cholesterol to the peripheral tissues, as at least two-thirds of circulating cholesterol resides in LDL. Conversely, HDL molecules are thought to do the opposite. They take excess cholesterol and return it to the liver for excretion [1,2].
Recent evidence suggests that dietary intake of cholesterol can influence plasma and serum levels, but not significantly. However, this is still subject to debate and further study . Plasma cholesterol levels along with the levels of LDL cholesterol, HDL cholesterol, and serum triglycerides are currently used as biomarkers of the so-called standard ‘lipid profile’ for each individual. The standard lipid profile has been widely used as a traditional biomarker, not only for cardiovascular health but also for other lipid-related abnormalities and disorders .
1.2. Cholesterol Levels: Demonising a Risk Factor but Not the Causative Mechanisms of Chronic Diseases
Several modifiable and non-modifiable risk factors (genetic, environmental, nutrition, and lifestyle, etc.) are thought to influence the balance between health and disease by inducing mechanisms related to disease onset, development, and the manifestations of symptoms. The presence or coexistence of these risk factors seem to trigger underlying molecular and cellular mechanistic pathways that can lead to continuous chronic manifestations and the long-term loss of tissue homoeostasis and tissue dysfunction. These continuous chronic manifestations can develop over time before cellular disturbances manifest and cause tissue disorders, while, if not counterbalanced by our immune system and by specific preventive measures such as a healthy diet and lifestyle, the subsequent symptomatic disease finally appears, and medical treatment may be required to reduce the risk of mortality. Elucidating these molecular and cellular mechanistic pathways and acquiring the mechanistic evidence of the underlying multifactorial causes of a chronic disease can lead to suitable preventive targets against these diseases with fewer side effects, which is an ongoing difficult and demanding task. Such difficulties have misled the scientific and medical community to often and lightly extrapolate the easily acquired observed statistical and epidemiological correlations of traditional risk factors to several chronic diseases, towards matching these risk factors as the causative agents of these diseases.
According to the ‘cholesterol hypothesis’, high blood cholesterol is a major risk factor, while lowering cholesterol levels can reduce risk . Dyslipidaemias (i.e., hypercholesterolaemia or hyperlipidaemia) are abnormalities of lipid metabolism characterised by increased circulating levels of serum total cholesterol, LDL cholesterol, triglycerides, and decreased levels of serum HDL cholesterol. High levels of LDL cholesterol and non-HDL cholesterol have been associated with cardiovascular risk, while other cholesterol-related serum markers, such as the small dense LDL cholesterol, lipoprotein(a), and HDL particle measurements, have been proposed as additional significant biomarkers for CVD risk factors to add to the standard lipid profile . HDL cholesterol has been considered as the atheroprotective ‘good’ cholesterol because of its strong inverse correlation with the progression of CVD ; however, it is the functionality of HDL cholesterol, rather than its concentration that is more important for the preventative qualities of HDL cholesterol in CVD. In general, dyslipidaemias have been ranked as significant modifiable risk factors contributing to prevalence and severity of several chronic diseases including aging, hypertension, diabetes, and CVD. High serum levels of these lipids have been associated with an increased risk of developing atherosclerosis .
Furthermore, dyslipidaemias have been characterised by several studies not only as a risk factor but as a “well-established and prominent cause” of cardiovascular morbidity and mortality worldwide . Even though such an extrapolation is not adequate, it was, however, not surprising that this was made, because since the term arteriosclerosis was first introduced by pioneering pathologists of the 19th century, it has long been believed that atherosclerosis merely involved the passive accumulation of cholesterol into the arterial walls for the formation of foam cells. This process was considered the hallmark of atherosclerotic lesions and subsequent CVD. Moreover, one-sided interpretations of several epidemiological studies, such as the Seven Countries Study (SCS), have highlighted outcomes that mostly concerned correlations between saturated fat intake, fasting blood cholesterol concentrations, and coronary heart disease mortality [10,11,12,13]. Such epidemiological correlations between dyslipidaemias and atherosclerosis led to the characterisation of atherosclerosis as primarily a lipid disorder, and the “lipid hypothesis” was formed, which would dominate thinking for much of the 20th century.
In the clinical setting, in order to address the lipid hypothesis, the levels of cholesterol related plasma lipoproteins and triglycerides (lipid profile) have been used as traditional biomarkers for cardiovascular risk, but also for dietary and treatment guideline designs . Dietary and medical guidelines have focused on the reduction of cholesterol and lipid levels as the best way to prevent chronic diseases such as CVD [5,9]. Such guidelines suggest the application of statin therapies in order to reduce the levels of cholesterol (through inhibition of cholesterol synthesis by HMG-CoA reductase inhibitors); however, numerous side effects have been reported, including the development of other chronic diseases such as diabetes mellitus . Moreover, specific dietary strategies for reducing cholesterol intake are the mainstay of management in most cases of dyslipidaemia, prior to, or simultaneously with, the initiation of a lipid lowering agent . Dietary fats, cholesterol, and the levels of serum cholesterol in relation to the development of CVD have been somewhat demonised.
On the other hand, since cholesterol is an essential biomolecule for the normal function of all our cells, an emerging question has recently surfaced: “how much do we need to lower the levels of cholesterol”? Furthermore, given the fact that cholesterol plays a crucial role in several of our cellular and tissue mechanisms, it is not surprising that there are several consequences due to the aggressive reduction of cholesterol levels in the body, which has been common practice over the last few decades. In addition, targeting cholesterol and fat intake by introducing diets with low-fat products and by reducing the intake of high-fat foods can lead to less absorption and lower bioavailability of other lipids containing high value nutrients, such as several lipid soluble vitamins (especially vitamin D) and other lipid molecules. Such lipids have exhibited a plethora of beneficial bioactivities, not only related to reducing the risk of chronic diseases but also through a wide range of important bio-functionalities and anti-inflammatory properties . Therefore, lower cholesterol levels do not equate to better health, or to lower risk of chronic diseases such as CVD. Homeostasis must be maintained, even with regard to cholesterol, both HDL and LDL .
Moreover, recent systematic reviews and meta-analyses have started to question the validity of the lipid hypothesis, as there is lack of an association or an inverse association between LDL cholesterol and both all-cause and CVD mortality in the elderly  and several cancers such as lung, prostate, and breast cancer [16,17,18]. Such studies provide the rationale for more research about the causes (and not only the risk factors) of chronic diseases such as atherosclerosis, CVD, and cancer, but also for a re-evaluation of the guidelines for cardiovascular prevention, in particular because the benefits of statin treatments have been exaggerated .
Statistical and epidemiological extrapolations often lack fully clarified biochemical mechanistic evidence, while associations and correlations do not necessarily mean causation. In addition, a follow-up by systematic reviews and meta-analyses often present contradictory outcomes against the initial results that were introduced by early stage epidemiological studies lacking consistency, biological gradient, and coherence. Thus, such extrapolations can lead to one-sided, premature targeting of risk factors accompanied with consequences, often without the desirable outcomes. Targeting a risk factor such as high serum cholesterol may decrease the probabilities for a disease, but usually cannot prevent the causation of chronic diseases.
1.3. Revisiting the Lipid Hypothesis: Outcomes of the Mediterranean Diet against Inflammation
Previous epidemiological and observational studies, such as the SCS in which the lipid hypothesis was mostly based, have been re-evaluated. For example, even though within the SCS the strength of the association between serum cholesterol and cardiovascular mortality were similar in different cultures, the absolute risks differed substantially. Kromhout reported that at a serum cholesterol level of 200 mg/dL, the 25-year cardiovascular mortality rate was five times higher in the Northern European populations of the SCS compared to the Southern Mediterranean populations , and thus the relations between diet, serum cholesterol, and cardiovascular mortality are more complex than originally thought. This is because it is not only dietary cholesterol involved, but other lipids and antioxidants may play a role in the onset and prevention of atherosclerosis . Such a low prevalence of cardiovascular mortality in the Mediterranean cohorts of SCS is now attributed to their lifestyle and especially to their dietary habits, namely the traditional Mediterranean diet (Med-diet) [10,20]. A common feature of the diet amongst populations in the Mediterranean is a relatively high dietary intake of vegetables, fruits, legumes, whole grains, monounsaturated fats, and nuts, followed by moderate consumption of fish, dairy products (mainly cheese and yogurt), alcohol, and low consumption of red and processed meats .
The major outcomes of the SCS and other similar epidemiological studies (i.e., studies trying to decipher the ‘French Paradox’ ) concerning the protective effects of dietary patterns, such as the Med-diet against chronic diseases, were initially either neglected or misinterpreted. CVD and cardiovascular mortality occurred in much relatively lower rates in the Southern European populations (i.e., Italy and Greece) despite a rather high dietary intake of saturated fats and cholesterol [10,20,23]. A recent systematic review and meta-analysis revealed that Med-diet can actually reduce the incidence of cardiovascular events, breast cancer, and type II diabetes mellitus, without any restriction on fat intake .
Over the last 2 years there has been a significant number of studies referring to adoption of the Med-diet pattern and its associated beneficial outcomes in a plethora of several chronic diseases that are either directly or indirectly related to inflammation. These studies refer to heart failure, CVD , cancer [26,27], obesity , metabolic syndrome [29,30,31], diabetes [31,32,33,34], and other subsequent manifestations such as diabetic retinopathy , asthma , autoimmune diseases such as rheumatoid arthritis , incident frailty risk , non-alcoholic fatty liver disease [39,40], inflammatory bowel disease , cognitive health, the risk of Alzheimer’s disease and dementia [42,43,44], and age-related macular degeneration .
In addition, the Med-diet has also been associated with beneficial outcomes, even in secondary CVD prevention . When patients suffering from CVD or diabetes follow the Mediterranean dietary pattern, the incidence of recurrent myocardial infarction and cerebrovascular events is reduced. The protective effect of this dietary pattern can be maintained for up to four years after the first infarction (Lyon Diet Heart Study) . Moreover, in contrast to the contradictions of lipid hypothesis and mortality in elderly people , the HALE project has also shown that individuals aged 70 to 90 years following a Med-diet and healthy lifestyle have a 50% lower rate of all-cause and cause-specific mortality . Followers of the Med-diet are also less likely to suffer sudden cardiac death and age-related cognitive decline .
The inverse association between Med-diet and all causes of diseases and cardiovascular-mortality has been attributed to several of its pleiotropic protective effects. For instance, the Med-diet can beneficially influence several risk factors such as lowering BMI, blood pressure, reducing insulin resistance, reducing lipid levels (i.e., the ratio of cholesterol/HDL cholesterol), and improving HDL-cholesterol functionality [50,51,52,53,54]. However, the main beneficial impact of Med-diet is on the improvement of endothelial function and the decrease of the inflammatory milieu, inflammation-related mediators, biomarkers such as platelet-activating factor (PAF), and several cytokines. It is also suggested that there is an improvement of oxidative stress, with lower concentrations of oxidised LDL and improved apolipoprotein profiles, and, finally, there is evidence of beneficial effects against platelet aggregation and blood coagulation [3,55,56,57,58].
The overall outcomes and beneficial effects of Med-diet have radically shifted the attention from the lipid-centric model that is characterised by the desired reduction of cholesterol levels to more effective targeting against the factual causative factors of chronic diseases, which are inflammation and its related manifestations. Prevention is key to reducing global mortality due to chronic diseases such as CVD; therefore, it is imperative to separate the underlying causes and processes of the disease from the risk factors and symptoms of disease. The clarification of the key roles and interplay of various cells, inflammatory mediators, and pathways during chronic inflammatory manifestations related to the onset of several chronic diseases is of great importance and may lead to a plethora of novel potential targets for fine-tuning of the inflammatory response during the chronic smouldering of inflammation that characterises these disorders
2. Re-Discovering Chronic Inflammation as the Cause for Chronic Diseases
Inflammation is a physiological reaction of the innate immune system that maintains a constant internal milieu while being exposed to continuously changing environmental pressures, irrespective of whether the initial causes originate from mechanical, physical, chemical, infectious, immunological, or reactive natural traumatic injury or metabolic dysfunction. The inflammatory response aims to reduce the agent that causes tissue injury and/or minimise these effects, to induce appropriate wound healing and to restore tissue homeostasis. Inflammatory responses are initiated by innate sensing mechanisms that detect the presence of microbial infection, stressed or dying cells, loss of cellular integrity, barrier breach, etc. A cascade of inflammatory pathways and mechanistic effects is supposedly well-orchestrated by the immune system in order to eradicate the causative agent.
Several immune cells can change their number, morphology, and nature depending on the stage and type of inflammation. Biochemically, inflammation is denoted by a local increase of numerous tissue hormones, transmitters, complement components, cytokines, and lipid mediators such as PAF and eicosanoids. Most of these products are autacoids that are synthesised at the site of inflammation in order to resolve the inflammatory process by removing or inhibiting the actions of the triggering agent . Provided that the immune response succeeds in eliminating the infectious agent or to repair the initial tissue injury, the inflammatory process will be terminated in a timely fashion and thus only affects tissue function transiently.
However, in cases where the inflammation fails to resolve due to the persistence of the triggering agent or due to unsuccessful repair of the initial tissue injury or dysfunction, a sustained underlying inflammatory process develops, leading to further tissue dysfunction and detrimental consequences. Several traditional and emerging risk factors are thought to influence our health and, especially, inflammation-related chronic diseases, by their interrelation with underlying molecular and cellular manifestations that result in chronic inflammatory responses leading to the loss of tissue homoeostasis and dysfunction. Apart from dyslipidaemias, other well-established risk factors include hypertension, diabetes, smoking, excessive food intake, previous infections (influenza, oral pathogens) or underlying autoimmune diseases such as lupus or rheumatoid arthritis, pollution, and genetic abnormalities . It is now well established that a common junction of such risk factors is chronic and unresolved inflammatory manifestations. Inflammation that causes endothelial dysfunction seems to be the key causative underlying mechanistic player, at the molecular and cellular level, for the onset and development of subsequent inflammation-related chronic disorders such as atherosclerosis and subsequent CVD, ischemic and renal disorders, cancer metastasis, diabetes, infections, and comorbidities [8,57,58,59,60,61,62].
For example, in cases of dyslipidaemia, increased cholesterol levels are not the causative agent or the underlying biochemical mechanism responsible for endothelial dysfunction and atherosclerosis development. The accumulation of excess plasma LDL cholesterol is addressed by the innate immune system as an undesired event. Therefore, an inflammatory response at the endothelial wall is promoted to reduce the threat by the removal of excess LDL and oxidised-LDL (Ox-LDL) cholesterol from the blood stream to the subendothelium, where they are engulfed by comigrated monocytes for final removal [63,64]. During chronic inflammatory diseases, inflammation and infections can also induce a variety of alterations in lipid metabolism, including decreases in serum HDL cholesterol, increases in triglycerides, lipoprotein(a), and LDL levels. These changes of the lipid levels may initially dampen inflammation or fight infection; however, the sustained inflammation can contribute to the increased risk of atherosclerosis . In addition to affecting serum lipid levels, inflammation also adversely effects lipoprotein function; LDL is more easily oxidised, as the ability of HDL to prevent the oxidation of LDL is diminished, while several steps in the reverse cholesterol transport pathway are also adversely affected during inflammation. The greater the severity of the underlying inflammatory disease, the more consistently these abnormalities in lipids and lipoproteins are observed . Thus, it is not serum cholesterol and lipoproteins that influence the endothelium but the inflammatory response that affects the well integrity and functionality of the endothelium.
Apart from the effects of inflammation on plasma lipids, it is now well established that more important soluble and cellular immune factors associated with chronic inflammation can promote inflammation-related endothelial dysfunction and atherogenesis, either during dyslipidaemia or independently of dyslipidaemia . Even though atherosclerosis and CVD were previously viewed as lipid storage disorders, we now recognise that inflammation drives much of endothelial dysfunction and mechanisms of clinical complications with these diseases and related comorbidities, such as sepsis [67,68], human immunodeficiency virus (HIV) infection [69,70,71,72,73,74], periodontal diseases [75,76,77], kidney disorders [78,79,80,81], healthy ageing, and inflammatory autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis, independently of traditional cardiovascular risk factors such as serum lipid levels [66,82,83].
Inflammation plays a key role in all stages of the formation of vascular lesions maintained and exacerbated by several risk factors such as unhealthy diet and lifestyle, smoking, hyperlipidaemia/hypercholesterolaemia, hypertension, autoimmune diseases, etc. The consequence of chronic inflammation is endothelial dysfunction that sets in, and we can define it as an integrated marker of the damage to arterial walls by classic risk factors. Endothelial dysfunction is usually characterised by an inflammation-related milieu acting on leukocytes and endothelial cells, through an interplay with other immune cells such as T lymphocytes, mast cells, dendritic cells (DC), and platelets [57,58,66,84,85]. The orchestrated overexpression and increased production of pro-inflammatory cytokines occurs, including interleukin-6 (IL-6), tumour necrosis factor (TNF) and its receptor, high-sensitivity C-reactive protein (hsCRP), type I interferons (IFN-α, IFN-β), adhesion molecules, chemokines, and lipid inflammatory mediators such as PAF and eicosanoids. Other linked events include the increased generation of reactive oxygen species (ROS), the increased oxidation of LDL cholesterol, and the reduction of protective nitric oxide levels.
Therefore, the mechanistic pathways and key players implicated in the inflammatory crosstalk taking place throughout the onset, development, and progression of chronic diseases is of great importance, in order to unravel putative preventive and therapeutic targets with less side effects. The inverse effects of the Med-diet with chronic diseases is mostly related to the pleiotropic effects and interplay of its food constituents on all these inflammation-related pathways; following a Mediterranean dietary pattern leads to the reduction of several inflammatory mediators and biomarkers related to the endothelial functionality, such as decreases in hsCRP, IL-6, and intracellular adhesion molecule-1 (ICAM-1) .
In this review, we clarify the roles of risk factors, such as plasma cholesterol and the importance of causative agents for chronic diseases, namely chronic and unresolved inflammation and its manifestations. Instead of cholesterol, targeting and treatment of inflammation will lead to lower side effects in chronic disorders. The overall outcomes and the extensive paradigms of the beneficial effects of the Mediterranean diet against the inflammatory milieu, without any reported side effects so far, have radically shifted attention away from the lipid-centric hypotheses and the subsequent trends for targeting cholesterol towards more effective approaches against inflammation, which is the causative factors of chronic diseases.
Therefore, the causative role of inflammation in the onset and progression of several chronic disorders is summarised with respect to the role of PAF and its related inflammatory cascades, which seem to serve as common junctions of the inflammatory milieu. The coexistence of several risk factors seems to upstream pro-inflammatory stimuli (i.e., cytokines, oxidative stress, PAF itself, etc.), leading to increased levels of PAF that, through the PAF/PAF-R pathways, attenuate the initial signal, while downstream inflammatory cascades, in combination with inactivation of homeostatic mechanisms such as PAF catabolism, can result in chronic and unresolved inflammatory manifestations and related chronic disorders.
Common junctions, such as PAF and its related inflammatory pathways, seem to be promising therapeutic targets for the prevention and treatment of the onset and progression of inflammation-related chronic diseases, particularly CVD. Implementation of healthy lifestyle choices based on appropriate dietary interventions and exercise have exhibited beneficial outcomes and health benefits, without noticeable side effects. Adoption of dietary patterns such as the Med-diet provides bioactive food microconstituents with pleiotropic beneficial effects that are not limited to decreasing co-absorption of cholesterol and increasing plasma HDL levels and functionality, but mainly by providing better stability against oxidation and inflammation. Therefore, microconstituents such as polar lipids and vitamins present in foods of the Med-diet beneficially affect the levels, activities, and metabolism of key inflammatory mediators implicated in chronic diseases, including the PAF pathway, towards reducing inflammation and acquiring homeostasis, which can lead to reduced risk of inflammation-related chronic disorders.
Nature has provided us with a wide range of dietary weapons, which, if appropriately combined in dietary patterns such as the Med-diet, can beneficially contribute to improving our quality of life, health, and life expectancy by equilibrating the inflammatory milieu to normal levels and thus preventively reducing the risk of inflammation-related chronic disorders. Let us not forget the words of Hippocrates of Kos (460-377 BC), who is universally recognised as the father of modern medicine: “Let food be thy medicine and medicine be thy food”.