Paolo Puddu

Hypercholesterolaemic individuals display an extracellular deposition of amorphous and membranous lipids, which precedes the subsequent phase, that is accumulation within the subendothelial space of T cells and of macrophages full of cholesterol; these are foamy cells that make up the so-called “fatty streaks”. These lesions, which are usually inflammatory, may be found in the aorta starting from the first decade of life, in the coronaries as from the second, and in cerebral arteries in the third and fourth decade. Normally, they have no clinical relevance, but they represent the earliest stage, the forerunners of the most advanced lesions, that is the plaques.
Fibrolipidic plaques are produced during the silent phase of atherosclerosis, which may last for decades. They contain variable amounts of a connective matrix produced by the smooth muscle cells, of lipids, either free or within the cytoplasm of the foamy cells, and of T cells. The plaques may be solid, almost entirely made up of connective tissue, or they may have a central part, called core and made up of extracellular lipids, which may occupy over 60% in volume of the plaque itself.
Most individuals display plaques having a heterogeneous makeup, varying between these extremes, also at a coronary artery level. Around the lipidic core there are numerous macrophages full of lipids, and there also are great amounts of inflammatory cytokines acting as an alpha tumorous necrosis factor (TNF-alfa), produced by the macrophages themselves, which also express other factors. These include the pro-coagulative tissue factor and certain metalloproteinases activated by plasmin, which may bring about the degradation of all the collagenous matrix components.

Left anterior descending coronary artery sectioned along its length to reveal narrowing of the lumen, most pronounced in the proximal portion at the left, from advanced atherosclerosis, gross.

This shows why the plaques ought to be regarded as dynamic structures, involving the existence of inflammatory phenomena and of a high rate of connective tissue exchange. The plaque is subject to calcification, to ulceration of the endoluminal surface and to haemorrhages from the small vessels surrounding it. It may grow up to the point of reducing the vasal lumen or it may incur complications such as breakage or fissures, as well as the forming of a thrombus, which may result in acute vase obstruction. This is the typical case of myocardial infarction or ictus. According to this morphological outline, things may appear quite simple. However the events associated with atherosclerosis are extremely complex. It is therefore important to recall the biochemical and molecular mechanisms regulating this atherogenetic process. First of all, it has been observed that inflammatory blood cells, and in particular monocytes/macrophages, as well as endothelial cells, play a leading role throughout the atherosclerosis progress.

(ATS) Role of Endothelial Activation

The acknowledgement of the importance of endothelial cells in the pathogenesis of the atherosclerotic disease dates back to the seventies, when experts observed that their mechanical removal dramatically increased the possibility of inducing lesions in animals subject to a hyperlipidic diet. Therefore Russel Ross formulated the “response to damage” hypothesis. Its most recent version focuses on a dysfunction rather than on a physical endothelium loss, and this is confirmed by many observations both on animal models and on man.
The endothelium is regarded today as an actual organ or system, provided with autocrine, paracrine and endocrine functions. It weighs 1.8 kilos and its surface covers an area of 700 m2. In addition to its property as a selective permeability barrier, it displays other functional properties capable of modulating the muscular tone, the proliferation of smooth vascular muscle cells, haemostasis, thrombolysis, platelet aggregation, monocyte adhesiveness, inflammation, immune response and the production of free radicals. Relaxing vascular factors are: nitric oxide, prostacycline, bradychinin and the hyperpolarizing factors.
On the other hand endotheline-l, thromboxane and the activation of angiotensine II are vasoconstrictors. The main vasodilator is the nitric oxide (NO) radical, which also has an anti-platelet action and an inhibiting property as regards the growth of smooth muscular cells.
All risk factors may, through various mechanisms, produce an alteration of the endothelial function and an increase in the production of reactive oxygen species by the endothelium and by the vascular smooth muscle cells.
In particular, systemic factors, such as hypercholesterolaemia, hyperglycaemia and hyperhomocysteinemia, as well as local factors, such as the activation of the macrophages and of the T cells and the shear stress, may contribute to oxidative stress, expressed through the hyperformation of oxygen-reactive metabolites, especially of the superoxide anion. The increase in superoxide anion produces a decrease in the environmental levels of nitric oxide through a radical/radical reaction. The endothelial dysfunction has various causes and consequences. Among these, the inactivation of NO is an early phenomenon and experimental data indicate that this contributes to the pathogenesis of the disease. It is known that hypercholesterolaemia brings about an increase in the endothelial production of superoxide, and this increase can also be observed in hypertension, in diabetes mellitus, in hyperhomocysteinemia, etc. Based on the above, we can infer that oxidative stress represents a common element for many risk factors. The first consequences resulting from the reaction between superoxide anion and nitric oxide is the formation of peroxinitric, with a reduction in NO bioactivity.
Peroxinitric is a strongly oxidizing molecule, which decomposes into two powerful free radicals, OH and NO2. Its action in not limited to a very weak activation of the guanylcyclase and to hence playing an absolutely secondary role compared to NO. Peroxinitric may also initiate lipidic peroxidation and oxidize the thiolic groups or the tyrosine residues.
The formation of lipoperoxides, and in particular of oxidized low-density lipoproteins (LDL), in turn produces, at an experimental level, various negative effects:
1) cytotoxic effect on endothelial cells;
2) promotion of the recruitment of inflammatory cells on the vascular wall and increase in their local production of oxygen free radicals;
3) decrease in the levels of nitric oxide synthetase (eNOS) in the endothelial cells;
4) interference with products of lipidic peroxidation, such as lysophosphatidylcholine, with the transduction of the signal and the receptor-dependent stimulation of the eNOS activity and with the activation of the guanylcyclase.
These experimental data are indirectly tested also in man, but a direct link between lipid peroxidation and endothelial dysfunction has not yet been demonstrated. In this regard, it is interesting to mention the discovery that the use of antioxidants, such as alpha-tocopherol and ascorbic acid, may improve the bioactivity of NO. An antioxidant effect has also been demonstrated with certain hypolipidemizing drugs belonging to the statin class, which produce a favourable effect on the endothelial dysfunction.

Role of Blood Inflammatory Cells

The minimally oxidized LDLs, the CD/40 ligands, the platelet growth factor (PDGF) and interleukin-1 beta (IL-l beta) stimulate and promote atherogenesis and induce, in endothelial cells, in smooth muscular cells (SMC) and in the monocytes, the expression of chemotactic cytokines [in particular MCP- 1 (monocyte chemoattracting protein 1), which causes the recruitment and the transmigration of the monocytes (but not of the neutrophils) through the endothelial barrier.
The monocytes and the T cells adhere to the endothelium, thanks to the action of selectines produced by the monocytes and of ICAM adhesion molecules, of the endothelial VCA-4 and VCAM-l integrines. In order to be internalised by the macrophages, the LDLs need to be highly oxidized, and this process is carried out by the ROSs (a species which reacts to oxygen) produced by the endothelium and by the macrophages. Several enzymes, which are found in human ATS lesions, are also involved, and among these are myeloperoxidase, sphingomyelinase and a secretory phospholipase. The rapid uptake of the highly oxidized LDLs (or of modified LDLs, such as the glycated ones) by the macrophages, which become foamy cells, is mediated by a group of “scavenger” receptors, which recognise many ligands. The expression of the scavengers is regulated by the gamma PPAR, whose ligands inhibit oxidized fat acids, and by cytokines, such as the alpha tumorous necrosis factor and gamma interferon.
The foamy cell formation process is inhibited by the apo E, secreted by the macrophages, which promotes the outflow of cholesterol towards the HDLs and is therefore capable of inhibiting the transformation of the macrophages into foamy cells. The foamy cells stretched by the lipids die, and the lipids are released into the extracellular spaces and form the lipidic core. The SMCs proliferate and form a collagen capsule around the core.
This process involves the action of the inflammatory proteins and of the modified LDLs. Once the plaque has formed, a further endothelial inflammatory damage has occurred: its cells flow into the focal areas, thus exposing the sub-endothelial matrix, on which the platelets adhere, to form a thrombus. A thrombus may also form as a result of the breakage of a plaque that is poor of SMC and rich of lipids and activated macrophages, with a strong expression of the tissue factor and with a thin capsule and unorganised collagen structure. The plaques bearing such features, which are called vulnerable plaques, are highly exposed to breakage; this can be caused by the release of metalloproteinases by the activated macrophages, which destroy the connectival framework. The thrombosis precipitates acute ischemic events. These phenomena are inhibited by hypolipidemizing drugs belonging to the statin class.

Role of the activation of peroxisomial receptors (PPAR)

An action similar to that of statins also appears to be carried out by PPAR agonists, which can modulate the functions of the vascular walls, thus directly influencing the cellular mechanisms underlying the atherosclerotic disease. In particular, the alpha PPAR synthetic activators-ligands, such as the fibrates (and the F ANSs) stimulate the oxidization of the lipids, alter the metabolism of the lipoproteins and inhibit the inflammation of the vascular walls, as well as the expression of the IL 1-6 and of the cycloxygenase 2 in the monocytes/macrophages.
Lastly, they prevent the expression of endotheline-l (ET-1), which takes part in the atherogenesis by activating the chemotactic properties of the monocytes and inducing the adhesion molecules into the endothelial cells. On the smooth muscle cells they carry out an anti-inflammatory action by negatively interfering on the kB nuclear factor (FN-kB). On the contrary, the gamma PPAR, whose activators are the thiazolidenadione drugs, appear to favour experimental atherogenesis, but also appear to have beneficial effects by acting on insulin-resistance and by regulating certain pro-inflammatory genes. Further controlled clinical studies will prove useful in solving the query as to whether the activation of the PPARs actually carries out an anti-atherosclerotic activity on man.

Inflammation Biohumoral Markers, Potentially Predictive of a Coronary Syndrome Risk

The ascertainment that inflammation is intimately involved in the development of atherosclerosis and in acute coronary syndromes emerges from a number of experimental and clinical studies, based on the search for inflammation biochemical markers, both in the plasma and in the atherosclerotic tissue. In particular, these researches have confirmed the presence of inflammation also in coronary atherosclerotic diseases. It is therefore some time that the makers are thought to have a potential role in early coronary atherosclerotic risk prediction and also in predicting the risk of explosion of acute cardiovascular events in patients who are known to suffer from coronary diseases. Experts have advanced the hypothesis that the estimate of chronic inflammation non-specific markers may predict the risk of acute myocardial infarction even 10 to 20 years earlier. These researchers are coupled by those that consider the predictive value of the so-called “traditional” risk factors, among which smoke, diabetes, hypertension and dyslipidosis, especially if combined. However, experts have observed that the estimate of traditional factors is not entirely sufficient for an overall evaluation of risk.
Therefore new, non-traditional, risk factors, which may be regarded as additional, have been taken into account. Some of these are correlated to coagulative processes: the VIIc factor, 1 inhibitor of the plasminogen activator (P AI -1) and the von Willebrand factor. Other factors are of a metabolic nature: lipoprotein (a), homocysteine and insulin. Lastly, inflammatory factors have attracted considerable attention. Among these, certain acute phase proteins, such as interleukin, the A amyloid serum protein, the vascular adhesion molecules, the alpha TNF and the fibrinogen. In particular the C reactive protein may play a leading role in the pathogenesis of acute complications of coronary atherosclerosis, such as variant angina and myocardial infarction.
Ever since 1985-1989, experts have proved that in atherosclerotic lesions you can find activated components of the complement and of the C reactive protein. Experts have expressed the belief that the C reactive protein and the complement represent the most effective inflammation mediators in atherosclerotic plaques.
Bhadki’s équipe of Mainz University, in Germany, has demonstrated that lipoproteinaceous particles isolated from human plaques, called “complement activators existing in the lesions”, can activate an alternative process for the complement. These activators do not appear to be directly correlated to the oxidized LDLs, which probably play a minor role. On the other hand, another complement activation process appears to be represented by the C reactive protein, which places itself with the C5b-9 complement in early atherosclerotic lesions. The correlations between the complement system and atherogenesis are emphasised by certain observations that have demonstrated that the complement activates endothelial cells, by inducing a pro-inflammatory response. In addition, shear stress, which in vivo carries out a powerful anti-atherogenic function, antagonizes, through the activation of clusterin, the complement effects on the endothelium. It has also been demonstrated that the activated complement has chemotactic properties and may damage the cells, thus fostering the development of intimal lesions and the recruitment of the monocytes in the seat where the atheroma has formed.
Ever since 1988, our Bologna University group has detected in patients affected by severe atherosclerotic lesions an increase in the components of the complement system, associated with an increase in IgAs; these, in the form of enzymatic complexes containing lipoproteins, have been detected in the patients affected by dyslipidosis with atherosclerosis and xanthomatosis. In a study carried out in 1995 and published on “The American Journal of Medicine”, the authors demonstrated that high levels of the third complement component (C3) of the serum of males who had not previously suffered from ischemic events are independently associated with the risk of myocardial infarction.
This proves that circulating levels of C3 are positive indicators of the risk of infarction. In addition, in 1998, researchers found that C3 in the serum, produced in response to interleukin-1, which is an acute-phase cytokine and protein, by the macrophages, by the liver and by the adipose tissue, is associated with a number of traditional infarction risk factors.
Among these, in particular, are plasmatic insulin levels before meals and apoliprotein B. A research published in the year 2000, has confirmed that C3, when associated with insulin, represents a pro-atherogenic metabolic unbalance marker, which coincides at least in part with insulin-resistence. Continuation of these studies in the year 2001 has made it possible to establish that treatment with ACE inhibitors, statins and beta-blockers appears to exert an excellent control over traditional risk factors in patients who have previously suffered from infarction, but is not effective in producing a reduction in average levels of C3 and of homocysteine.
Only a combined treatment associating atorvastatin and vitamin E for three months has succeeded in lowering C3 in individuals displaying persistently high values of this molecule, as well as cholesterol and triglycerides. These and other researches confirm that C3 and the C reactive protein can play a major role in atherogenesis. They also suggest that C3 plasmatic levels represent a powerful risk indicator for acute coronary events, such as myocardial infarction.
The development of pharmacological agents capable of modulating the complement system may assist us in better understanding its multiple physiologic and pathologic functions and represent an important area for future research.
(trad.Interpres-Giussano)

Paolo Puddu
Professore Ordinario di Medicina Interna
Dipartimento di Medicina Interna e Cardioangiologia
Università degli Studi di Bologna