THE PAF IMPLICATED ATHEROSCLEROSIS THEORY

Platelet activating factor - a molecular link between atherosclerosis theories
Constantinos A. Demopoulos^a, Haralabos C. Karantonis^a, Smaragdi Antonopoulou^b
a Faculty of Chemistry, National and Kapodistrian University of Athens, Athens, Greece
^b Department of Science of Nutrition-Dietetics, Harokopio University, Athens, Greece

Our team has developed a new theory for the initiation of atheromatosis (atherogenesis) and most importantly offers a biochemical explanation of the epidemiological data showing that the Mediterranean diet can act protectively against atherogenesis, atherosclerosis and cardiovascular diseases.
    This theory is a new approach to this scientific issue and unifies the so far proposed theories (Inflammation, Oxidation and Response-to-Retention) combining our new experimental data with the existing experimental data of the scientific community. Considering atherogenesis as an inflammatory process, our new theory proposes an integrated biochemical mechanism and it reveals the pivotal role of the inflammatory mediator Platelet-Activating Factor (PAF) for the initiation of atheromatosis.
    The structure of PAF has been elucidated in 1979 by C.A.Demopoulos¹. Since then an International Conference for PAF is organized every four years worldwide. The proposed biochemical mechanism² for the initiation of atheromatosis combines our new experimental data with the existing ones and it interprets the epidemiological observations demonstrating the protective role of the Mediterranean diet against atherogenesis, atherosclerosis and cardiovascular diseases.

    The proposed mechanism from our team, is the following:
Oxidation of LDL (Low Density Lipoprotein, rich in cholesterol) takes place in human blood and it is dependent on several factors such as diet (total intake of antioxidants) and life style factor (such as smoking, sedentary lifestyle etc). Even though PAF production is under strict control, unregulated production of PAF is observed during LDL oxidation. Our team isolated and identified, both chemically and biologically, the PAF produced during LDL oxidation³.
    When PAF levels increased irregularly either by LDL oxidation or by pathological reasons (e.g. diseases), then PAF – among others- can initiate a rapid, local inflammatory response in the vessel, which leads to damage of the endothelium and to a dysfunctional endothelium with increased permeability, permitting blood cells and oxidized-LDL to move into sub endothelial space, foam cells formation and proliferation of smooth muscle cells. All the above steps are well known biological activities of PAF. Foam cells together with platelets, smooth muscle cells and lipids, such as cholesterol, adhere and this complex mixture of cells and lipids composes the atheromatic plaque.
    Taking into consideration the pivotal role of PAF for the initiation of atheromatosis and the protective effects of the Mediterranean diet on atheromatosis, our team investigated the presence of PAF inhibitors in foods of the Mediterranean diet.
    Initially, we investigated the presence of PAF inhibitors in olive oil⁴ and we demonstrated the presence of lipid molecules exerting -despite their low amounts in olive oil- strong anti-PAF in vitro activity (as PAF-inhibitors or PAF-antagonists) in washed rabbit platelets. The same PAF inhibitors were detected in olive pomace too. Similar lipid compounds were also found in seed oils. However, their concentration was much lower than those found in olive oil. The structure of the main biologically active compounds from the above sources had been studied and we elucidated their structure^5,6.
    PAF inhibitors were also found in several other foods tested, such as honey⁷, yoghurt⁸, wine^9,10,11,12, fish^{13,14,15,16,17,18}, seafood¹⁹ and plants²⁰. In addition, Vitamin E, an antioxidant and antiatherogenic compound existing in plant foods, also inhibits PAF actions²¹. Moreover, European scientists demonstrated the existence of PAF inhibitors in garlic and onion, which are both widely consumed throughout the Mediterranean region.

    We also isolated PAF inhibitors from human blood²², which is the first pool of food nutrients and play a significant role in various diseases such as coronary artery diseases.

    According to our theory, the epidemiological evidence for the protective role of the Mediterranean diet against atheromatosis and cardiovascular diseases can be explained in a biochemical way from our research since most of the Mediterranean foods contain lipid molecules which exert strong inhibitory activities against PAF (the inflammatory mediator of atherogenesis and cardiovascular diseases), thus preventing the initiation of atheromatosis. We also have to mention the significant role of the antioxidants which are found in high quantities in Mediterranean diet foods since they prevent - partly - LDL oxidation and therefore PAF production.
Obviously, suitable in vivo data are the only unambiguous way to prove a theory like the one we propose. And to our satisfaction, the results of our in vivo studies seem to be unambiguous.
    We conducted dietary intervention studies with animals in the Laboratory of Experimental Surgery and Surgical Research “N.S.Christeas”, School of Medicine, National & Kapodistrian University of Athens collaborating with Despina Perrea, Associate Professor and Director of the Laboratory. The results of those experiments have shown that the protective role of olive oil against cardiovascular diseases are also exerted by the olive oil fraction with the potent PAF inhibitors (the polar lipid fraction) and not by the olive oil fraction containing mainly the antioxidants and the unsaturated fatty acids (the neutral lipid fraction)²³. We also found that atherogenesis in hypercholesterolaemic rabbits is inhibited by PAF antagonists from olive pomace²⁴.
    Using other suitable studies on experimental animals, we proved that PAF-antagonists derived from olive oil and olive pomace, cause atherosclerosis regression, in equal proportion to that caused by statins²⁵, which are also PAF-inhibitors ²⁶.

    Investigation of PAF-antagonists presence in different stages of both olive oil production and olive pomace elaboration (concerning olive pomace oil production), revealed that these biologically active (anti-atherosclerotic) ingredients are, mainly, present in olive mill wastewater (OMW)²⁷. OMW constitutes a watery by-product of the olive oil industry and due to its high phenolic compound content is toxic for the environment; therefore its disposal is an important issue of the olive oil industry, since the necessary financial aid is not granted for the safe environmental disposal of olive oil waste water ²⁸.

    The existence of biologically active lipids with antiatherogenic properties in olive oil is superior, since these lipids exist in the diet and are of natural origin, so that their use implies no risks for human health as the side effects of the drugs do.
    Among all biochemical parameters only the decreased PAF-induced platelet aggregability (which means decreased PAF-biological action in blood, due to the high amounts of food PAF inhibitors in blood) is correlated with the inhibition of atherogenesis²³.
    According to our results PAF is the main etiological factor of atherosclerosis in contrast to the wide belief that saturated fats and cholesterol play this role. High levels of cholesterol or saturated fatty acids can augment the oxidation of LDL and therefore the production of PAF. PAF inhibitors found in food of the Mediterranean diet can protect us from high levels of saturated fatty acids and cholesterol effects. This suggestion is supported by the fact that Cretans have low mortality rates due to cardiovascular diseases despite their relatively high levels of cholesterol.
    Moreover, the proposed mechanism which suggests PAF as the main etiological factor of atherosclerosis and not cholesterol is enforced by experimental data from other investigators who have shown that administration of PAF inhibitors in combination with a hypercholesterolaemic diet prevents the progression of atheromatosis in rabbits and hamsters. Among other inhibitors used, a PAF specific inhibitor was included, from the Chinese herbal plant Ginkgo biloba which is well known to the Chinese for its protective role against cardiovascular diseases.

    In addition, dietary intervention studies have shown that the administration of traditional Mediterranean diet meals of Olympic Catering²⁹ or Goody’s³¹ to either normal volunteers or type 2 diabetic patients (who have a predisposition to cardiovascular diseases) resulted in the characteristic lower PAF activity in blood (measured as PAF-induced platelet aggregability), which correlates with the inhibition of atherogenesis according to our animal experiments²³. Our theory is therefore also indirectly confirmed in humans, since one could not study the phenomenon directly (inducing atherogenesis in humans).

    We should mention that our theory also explains the so-called “French paradox” or the lower mortality rates of French due to cardiovascular diseases despite their high consumption of saturated fat and cholesterol ³⁰. Since this observation was attributed to the daily moderate consumption of red wine by the French we searched, for the presence of PAF inhibitors in different varieties of wines of Domaine Hatzimichalis^9,10,11,12. The amounts of PAF inhibitors were not always found higher in red wines compared to white ones. Cabernet Sauvignon red wine and Ambelon white wine (Robola) contained the highest amounts of PAF inhibitors among all wines tested. Similar molecules exerting PAF inhibitory properties were found in musts and wine marcs³².

    Finally our theory can explain the results of the “Seven Countries Study” (USA, Finland, Netherlands, Italy, Yugoslavia, Japan and Greece) which shows that the increased consumption of olive oil by the population of Crete and Corfu is inversely correlated with the prevalence of coronary arteries atherogenesis in contrast to the population of the USA where the higher rates of cardiovascular diseases were attributed to the increased consumption of saturated fat. On the other hand the low rates of cardiovascular diseases in the Japanese population were attributed to the high consumption of seafood. Therefore, our team investigated the presence of PAF inhibitors in various seafood (fish, cephalopods) and we isolated potent PAF inhibitors^{13,14,15,16,17,18,19}. We also found that atherogenesis in hypercholesterolaemic rabbits is inhibited by PAF antagonists from fish ³³. Our current research is focused on quantitative and qualitative differences in PAF inhibitors between wild and farmed fish³⁴ and their effect on atherogenesis.

    The aforementioned research is made solely by Greek investigators in Greece and it has been funded by Greek Universities and the General Secretariat of Research and Development.
    Since 1994, C.A.Demopoulos, Professor of Biochemistry and Food Chemistry, Director of the Laboratory of Biochemistry, Faculty of Chemistry, National & Kapodistrian University of Athens, and Smaragdi Antonopoulou, Associate Professor of Biochemistry, Director of the Laboratory of Biochemistry, Chairman of the Department of Nutrition-Dietetics and Vice Rector of Harokopio University of Athens, are working on the subject with significant help from undergraduate and postgraduate students and in collaboration with several other Greek researchers.
    Preliminary results of our research were announced at the 16th Greek Chemistry Conference in 1995 by S. Antonopoulou³⁵ while additional results were presented in 1997 in INFORM²⁰ which is the official magazine of the American Oil Chemical Society (AOCS). Subsequently, our result presented after invitation at the 93rd World Conference of AOCS (2002) in Canada by our postgraduate student Charalabos Karantonis³⁶.
Our research was also presented³⁷ at the 71st European Atherosclerosis Society Congress (1999) followed by an online publication (commentary) of our results on the official website of the International Atherosclerosis Society³⁸.
    In 2003, a review article (after invitation) describing our theory was published in the European Journal of Lipid Science and Technology², which is published for the European Federation of Lipids.
    C.A. Demopoulos presented this theory as a guest speaker of the 45th International Conference of the Bioscience of Lipids³⁹, which was supported by the Federation of the European Biochemical Societies (FEBS) and the International Union of Biochemistry and Molecular Biology (IUBMB). During this conference C.A. Demopoulos was awarded for his long and significant contribution to the basic research of lipids.
    In 2005, the Hellenic Atherosclerosis Society nominated C.A.Demopoulos as “honorary member” in recognition of his research in atherosclerosis
   In 2006, the International Atherosclerosis Society invited us to comment, on the official website, on our recent results concerning the in vivo inhibition of atherogenesis from olive oil⁴⁰.

    In conclusion, the Seven Countries Study along with newer studies has shown that the Mediterranean diet is the most appropriate diet for a healthy life. This diet may express its protective role to cardiovascular diseases through some minor food constituents with anti-PAF biological activity ⁴¹, offering new insight into the mechanism of the Mediterranean diet's beneficial effects

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