Atherosclerosis is the leading cause of mortality in developed countries. This complex disease can be described as an excessive inflammatory, fibro fatty, proliferate response to damage of the artery wall. Many believe that it can be induced from simple dysfunction of endothelial lining as occurs with hyperlipidemia, hypertension or cigarette smoke 1 2 3 . Many risk factors are known for this disease and lots of biochemical markers are used to predict and so prevent it in susceptible people. Recent studies have shown that inflammation also plays a key role in atherosclerosis. C-reactive protein (CRP) has been recommended as the marker of choice to monitor cardiovascular risk, being a stronger predictor of atherosclerosis than even plasma low density lipoprotein (LDL) concentration 4 5 . Inflammation is a prominent feature of atherosclerosis 6 7 , and it is postulated that as an acute-phase protein, elevation of plasma CRP may signal the underlying atherosclerotic process.

There is a relationship between some factors of coagulation system such as (fibrinogen, factor VII) and fibrinolytic factors [tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1)] and the clinical manifestations of atherosclerosis. The relationship between fibrinogen and risk for cardiovascular events is highly proved rather than the factor VII plasma concentration. It has been proved that high levels of factor VII activity effects on the patients with high risk for coronary artery disease and myocardial infarction survivors. It seems that reduced fibrinolytic activity improve the risk of cardiovascular disease 8 .

Endothelial dysfunction has been also proved to be a key variable in the pathogenesis of atherosclerosis and its complications. Nitric oxide (NO) which is produced from transformation of arginin to cytroline by iNOS and eNOS enzymes (isoformes of nitric oxide synthetase) is the most important and well known endothelium derived mediator that is responsible for endothelial dysfunction 9 . NO release by the endothelium regulates blood flow, inflammation and platelet aggregation and consequently its disruption during endothelial dysfunction can decrease plaque stability and encourage the formation of atherosclerotic lesions and thrombi 10 . In states of inflammation, NO production by the vasculature increases considerably and contributes to oxidative stress 11 12 13 .

Flavonoids and phenolic compounds widely distributed in plants which have been reported to exert multiple biological effect, including antioxidant, free radical scavenging abilities, anti-inflammatory, anticarcinogenic, etc. Recently there has been an upsurge of interest in the therapeutic potentials of medicinal plants as antioxidants in reducing such free radical induced tissue injury 14 . Apple is one of the main sources of dietary flavonoids that show the strongest associations with decreased mortality. Also, apple is a good source of antioxidants. It contains appreciable amounts of vitamin C and of various phenolic compounds (catechins, phenolics acids, quercetin and phloretin), which also have protective effects. Epidemiological studies have linked the consumption of apples with reduced risk of some cancers, cardiovascular disease, asthma, and diabetes, lipid oxidation, cholesterol and atherosclerosis perogression. In addition quercetin has demonstrated significant anti-inflammatory activity 15 . This study was performed to determine the effects of apple juice on markers of inflammation, coagulation, endothelial and progression of atherosclerosis.

The significant decrease in CRP, fibrinogen, factor VII, TC, TG, LDL-C and the significant increase in nitrite, nitrate and HDL-C in rabbits receiving apple juice as compared to the high-cholesterol group represent that this extract is effective in moderating the dyslipidemic condition arising from a high cholesterol diet.

Histological results indicate that apple juice significantly reduced atherosclerotic lesions of coronary arteries, when compared to the high-cholesterol groups.

In our study, nitrite and nitrate increased in high cholesterol control compared to normal diet. It has been suggested that enhanced NO synthesis might be a defense mechanism to compensate for continuous inactivation of NO and protection against damaging factors 20 . The proposed mechanism responsible for the increase of nitrite and nitrate may be a significant increase in overall NOS synthesis by other cell types (than endothelium) in advanced lesions composed of the endothelial, neuronal and inducible isoforms of nitric oxide synthase (iNOS) enzymes 21 .

Both low and high-doses apple juice with cholesterolemic diet caused significant increase in nitrite and nitrate compared with high cholesterolemic diet.

The release of nitric oxide (NO) by a healthy vascular endothelium, which consists of a single layer of endothelial cells, prevents the adherence of platelets and leukocytes to the arterial wall. Therefore, reducing platelet activity could reduce athero-thrombotic events 22 . In addition to its antiatherogenic properties, NO also stimulates the vascular smooth muscle to relax and produce vasodilation. However, the endothelium can be damaged by many of the known vascular disease risk factors such as hypercholesterolemia, hypertension, and diabetes, thus reducing the bioavailability of NO 23 . The release of ROS is increased in states of oxidative stress and neutralizes the beneficial effects of NO by converting it to peroxynitrite 24 . The increase in NO and decrease in ROS attenuate multiple mechanisms that are central to the development and progression of atherosclerosis. The biological activity of NO can be effectively increased by the scavengers of oxygen-free radicals 25 . However, polyphenolic compounds can increase the NO releasing by two mechanisms: 1-Stimulation of NO synthase activity and preservation or stabilization of NO release under basal conditions. 2- Protection of NO from destruction by superoxides and other free radicals 26 . Schuldt EZ et al reported that polyphenols were able to scavenge free oxygen radicals to a limited extent resulting in an increased NO level 26 .

Diets rich in dealcoholyzed red wine (DRW), quercetin or catechin induced endothelium-dependent vasorelaxation in rat aorta in a resting state through the enhancement of NO production, without modifying O2- generation, thus the bioavailability of NO was increased. The increase in the NO-cyclic GMP pathway explains the beneficial effect of flavonoids at vascular level 27 .

Following concurrent use of 5 and 10 ml apple juice with cholesterolemic diet, fibrinogen and factor VII levels were significantly decreased in comparison with high-cholesterol group.

Studies show that red wine consumption caused significant decreases in fibrinogen and factor VII 28 29 .

In a study by Grenett HE et al in rats, the impact of polyphenols on expression of fibrinolytic protein [urokinase-pA(u-PA), (t-PA), (PAI-1)] mRNAs in endothelial cells of aorta was evaluated. The expression of t-PA and u-PA mRNA was up-regulated, but conversely PAI-1 mRNA expression was down-regulated by both ethanol and polyphenols including catechin and quercetin. As a result, the firbrinolysis was increased in rat endothelial cells, an effect which might exert a beneficial cardio protective role 30 .

The individual polyphenols of apple juice and ciders were examined for their ability to inhibit tissue activator, urokinase and plasmin. Neither phloridzin nor chlorogenic acid had any inhibitory activity at concentrations of 500 micrograms/ml while epicatechin had only a slight inhibitory effect at this concentration 31 .

Using both doses of apple juice with cholesterolemic diet induced a significant decrease in CRP compared to hypercholesterolemic diet. In high cholesterol group, CRP was increased significantly compared to the normal diet group; however it has been shown in another studies with hypercholesterolemic rabbits 32 33 .

C-RP may actively promote atherogenesis 34 , causing lesion formation via mechanisms such as endothelial dysfunction and leukocyte activation 35 and changes in plaque structure resulting in stability reduction and enhancing rupture. Many reviews aimed to study the effects of flavonoids on inflammatory processes 36 which showed that they may inhibit some active enzymes during the inflammatory process 37 . Isoforms of iNOS and of cyclooxygenase (COX-2) are responsible for the production of prostaglandins and nitric oxide biosynthesis which act as a mediators. In vitro studies have demonstrated that nitric oxide production and the expression of iNOS inhibit by flavonoid quercetin 38 . Flavonoids can modulate the molecular events cascade in several critical steps resulted in iNOS or COX-2 over expression. It seems that activation of a transcription essential for the expression of proinflammatory genes, the nuclear factor kappa B (NF-kappa B)converge the iNOS and COX-2 induction pathways 39 .

Some of inducible transcription factors can constitute potential key targets for the treatment of the inflammation and NFkappaB is one of the most important of them which can trigger a cascade of molecular events by its modulation. It is showed that CRP protein level in hepatic cells may reduce by diverse flavonoides and that the effect is dose-dependent 40 . Studies indicated that CRP can be induced by IL-6 mechanism that involves NF-kappaB activation 41 42 . An inhibition of the nuclear NF-kappaB translocation and of CRP synthesis in hepatocytes can cause by alterations in the intracellular state redox 43 . So it is possible that effects of flavonoids on CRP expression could be mediated, at least partly, by the modulation of the NF-kappaB-dependent pathway.

Results of Golzarand M et al showed that ingestion of apple cider vinegar in patients with type 2 diabetes had no effect on CRP but it reduced IL-6 concentrations 44 .

Findings of Chung OK, et al demonstrated that intake of dietary flavonoids is inversely associated with serum CRP concentrations in U.S. adults. Intake of flavonoid-rich foods may reduce inflammation-mediated chronic diseases 45 .

Studies of Terra X, et al indicated that the procyanidins of grape seed decrease plasma CRP levels in rat. The decrease in plasma CRP in hypercholesterolemic rats is related to a down-regulation of CRP mRNA expression in the liver 46 .

We showed that dietary consumption of 5 and 10 ml apple juice with cholesterol diet significantly decreased serum TC and TG. Aprikian O, et al [2001] found that when cholesterol fed rats were supplemented with lyophilized apples, there was a significant drop in plasma cholesterol and liver cholesterols and an increase in high-density lipoproteins (HDL). Furthermore, they found that cholesterol excretion increased in the feces of rats fed apples, suggesting reduced cholesterol absorption 47 . Also, Aprikian O, et al [2003] in more recent studies, found that combined apple pectin and apple phenolic fractions lowered plasma and liver cholesterol, triglycerides, and apparent cholesterol absorption to a much greater extent than either apple pectin alone or apple phenolics alone 48 .

Histological results indicate that apple juice intake reduced atherosclerotic lesion in coronary vessels significantly with compared to hypercholesterolemic group. Because inflammation has an important role in atherosclerosis development, significant reduction in inflammatory lesion may be due to anti-inflammatory effect of apple juice.

The protective effects of flavonoids against chronic diseases have been attributed to their free radical-scavenging property. In the case of CVD, flavonoids have been shown to reduce low density lipoprotein (LDL) oxidation, an important step in atherogenesis 49 50 . Hung MT, et al 51 has suggested that polyphenolic compounds inhibit proinflammatory cytokines expression including IL-6, IL-1 and CRP expression. Apple was identified as a major dietary source of flavonoids especially flavan-3-ols (catechins) in the epidemiologic studies that inhibits cyclooxygenase enzymes 52 53 that produce inflammation and stimulate inflammatory responses in body 54 .

Studies by Decorde K, et al indicated that phenols of grape, apple and their juices reduce atherosclerosis lesion in hamster. This value was reduced by %93 in grape juice,%78 in purple grape,%60 in apple juice and %48 in apple 16 .

In study performed by Da Luz PL, et al on the effect of red wine on experimental atherosclerosis, the rabbits that given red wine had significantly less atherosclerotic plaque development in their arteries than those not given wine at all 55 .

In conclusion, results of this study suggest that hypercholesterolemic atherosclerosis is associated with an increase in oxidative stress in coronary arteries and that apple juice is effective in reducing hypercholesterolemic atherosclerosis by lowering levels of fibrinogen, VII, CRP, TC, TG, LDL-C and raising levels of nitrite, nitrate and HDL-C. Therefore, apple juice may be useful in preventing hypercholesterolemic atherosclerosis and lowering the related risk of coronary artery disease.

The authors declare that they have no competing interests.

AHR participated in the sequence alignment and drafted the manuscript, NE carried out the laboratory tests. AE participated in the sequence alignment. MS participated in the design of the study and performed the statistical analysis. SA conceived the study, and participated in its design and coordination. All authors read and approved the final manuscript.