Atherosclerosis and the Benefits of Red Wine

Raquel Zielinski 

            Coronary artery disease (CAD) is the leading cause of death in developed countries (1).  According to the World health Organization, an estimated 16.6 million global deaths are due to cardio-vascular disease (2).  Many factors such as diets high in animal fats, smoking, diabetes, sedentary lifestyle, and hypercholesterolemia have been positive indicators for coronary heart disease.  The French have diets high in animal fats such as butter and lard, and they have higher serum cholesterol levels and blood pressure than Americans. However, this population has 50% less CAD related deaths than other European nations and the United States (1,3). This has been termed the “French Paradox.”   A possible explanation is that the French consume larger quantities of red wine, however molecular explanations and possible mechanisms remain unclear.  It is believed that the polyphenols in red wine are responsible for this health benefit.  Much has been said about the anti-oxidative affects of polyphenols (4,5).  Anti-oxidative affects alone cannot explain the benefits of drinking wine (8).  CAD, specifically atherosclerosis, is characterized by endothelial cell (EC) dysfunction, due to chemical or mechanical injury.  This is accompanied by oxidation of LDL, foam cell formation from macrophages, migration and proliferation of vascular smooth muscle cells (VSMC) at the site of a lesion, and increased extracellular matrix  (6,7).  The focus of this paper will concentrate on mechanisms and inhibition of vascular smooth muscle migration (VSMC) and proliferation by red wine as a possible explanation of the “French Paradox.”

Atherosclerosis is a progressive degenerative arterial disease that has a complex pathogenesis.  Atherosclerosis has also been characterized as an inflammatory disease due to the similarities with other inflammatory responses (8).  It is a response to injury, which goes too far, eventually becoming a disease state (6,8).  A blood vessel consists of a monolayer of endothelial cells in contact with blood, an intimal layer that is the innermost layer, the media where smooth-

 muscle cells are located and the adventitia (figure 1.)  At onset, perhaps in response to injury, monocyte adhesion glycoprotein molecules appear on the surface of endothelial cells promoting adhesion of monocytes and T-lymphocytes.  The monocytes and T-cells migrate to the intima, where monocytes differentiate into phagocytic macrophages that ingest lipids, especially oxidized lipids.  The lipid-filled macrophages, (foam cells) are rich in cholesterol.  They accumulate beneath the vascular endothelium and together with lymphocytes, form a fatty streak (figure 2.)  This is the first sign of a lesion (6-8).  Dead foam cells contribute to a growing mass of lipids and debris in the lesion (9). This is followed by migration of vascular smooth muscle cells (VSMC) from the media into the intima where they proliferate and become part of the lipid streak.  Smooth muscle cells also produce the extracellular matrix comprised of connective tissue, which is a major part of the lesion (6,7,9).


As the response to injury continues, the inflammatory process becomes the disease.   Proliferation of VSMC coupled with the lipid layer and increased VSMC matrix deposition, result in the formation of a fibrous plaque (6).  The growing plaque bulges into the lumen and impedes blood flow.  The endothelial cells are also damaged and unable to release the messenger, nitric oxide that causes smooth muscle cells to relax (vessel dilation).  Additionally, the arterial cells are deprived of nutrients due to the thick plaque, resulting in degeneration of the arterial tissue.  Fibroblasts begin to accumulate in the damaged area (scar tissue forming cells) forming a collagen-rich cap over the plaque.  In advanced stages, calcium precipitates in the plaque and causes hardening of the arteries.  The plaque may eventually rupture through the endothelium allowing platelets, which normally do not adhere to smooth muscle, to attach to the now exposed collagen.  The growing aggregation of platelets will cause further blockage, possibly completely occluding the vessel or breaking away and completely blocking another blood vessel (thromboembolism.)

The migration and proliferation of VSMC in atherosclerosis is activated by platelet-derived growth factor (PDGF).  PDGF exists as a dimer of polypeptide chains termed A and B.  PDGF dimers are known as PDGF-AA, PDGF-BB and PDGF-AB.  These ligands have different binding affinities to two types of PDGF receptors (PDGFR), aPDGFR and bPDGFR.  The aPDGFR can bind B-chain and A-chain polypeptides of PDGF; bPDGFR can only bind a B-chain (18).   Association of PDGF-BB to bPDGFR has been shown to elicit strong chemotactic and mitogenic responses in VSMC (1,19).  Growth factors are released by platelets, VSMC, T-cells macrophages, and endothelial cells (6-9).  Although the mechanisms of atherosclerosis and the migration of VSMC into the intima are not well understood, experiments have shown that PDGF and bPDGFR on the surface of VSMC play major roles in VSMC activity (10,11).  It has been shown in a baboon that PDGF is involved in intimal lesion development at sites of vascular injury (10).  When an anti-bPDGFR antibody was used, the migration of VSMC into the intima was inhibited.  These results show that inhibition of VSMC migration via inhibition of bPDGFR is an important factor in decreasing intimal lesion development (10).  Further evidence of the role of bPDGFR in VSMC proliferation and recruitment was provided in experiments involving Apolipoprotein E deficient mice that were placed on a high fat diet.  It was shown that bPDGFR played a major role in atherosclerotic lesion formation.  Inhibition of bPDGFR with an antibody resulted in inhibition of lesion formation via decreased VSMC proliferation and migration. This was a result of the inhibition of autophosphorylation of bPDGFR and bPDGFR mediated pathways (11).  These pathways are not yet well understood.  Blockade of bPDGFR in mice with advanced lesions prevented the progression of the lesion by as much as 33% compared with the control (11). 

When the ligand binds to bPDGFR, a series of PDGF-dependent activities occur.  VSMC migration and proliferation are a result of theses PDGF-dependent activities.  bPDGFR is a transmembrane receptor tyrosine kinase.  Upon the binding of PDGF, bPDGFR dimerizes and autophosphorylation of tyrosine residues occurs.  Next, intracellular signaling proteins bind to their specific phosphotyrosines at their SH2 domains (figure 3).   These include Src kinases, the GTPase activating protein of Ras (RasGAP), phospholipase Cg (PLCg), phosphatidylinositol 3-kinase (PI3K), and phosphotyrosine phosphatase SHP-2.  Various signal cascades follow.  The cascades result in a series of PDGF mediated responses including mitogen- activated protein (MAP) kinase activation.  Figure 3 generally illustrates one of the cascades that results in DNA replication and subsequent VSMC proliferation.  The SH2 domain of the protein, Grb-2, binds to the receptor.  Grb2 is not catalytic but acts as a link for the protein, Sos, and the receptor.  Sos will then bind Ras inducing Ras to exchange GDP for GTP.  Ras is a small monomeric G-protein that is activated when bound to GTP and inactive when bound to GDP.  Activated Ras will mediate a cascade of MAP kinases, which ultimately activates the transcription factor (TF) and the transcription promoter, resulting in the production of mRNA or DNA replication.  Increased replication, results in VSMC proliferation.  RasGAP is also activated by the receptor.  RasGAP eventually turns off the signal response by accelerating the hydrolysis of GTP to GDP. 

The cascade that results in migration is not well known. The binding of PDGF to the bPDGFR is believed to trigger several signaling pathways that involve Src kinases, Ras, Cg (PLCg), and (PI3K) that lead to cell migration (1, 21, 22,23).   Figure 3 illustrates some possible details of the cascade.

 

Red wine and its components have been shown to reduced the risk of heart disease by several mechanisms including decreased platelet aggregation, decreased low-density lipid oxidation, and decreased VSMC proliferation and migration  (1,8,13-16).  Several studies have focused specifically on the inhibitory effects of red wine on VSMC proliferation and migration via inhibition of PDGF signaling and associated cascades (1,13,16).  One such study found that decreased VSMC proliferation might be a possible mechanism of decreased atherogenic effect of red wine.  Rat aorta VSMC, bovine endothelial cells (EC) and human (EC) and VSMC were treated with red wine polyphenols (RWP). RWP specifically inhibited VSMC proliferation and DNA synthesis in a dose-dependent manner, but had no effect on EC proliferation or DNA synthesis except at much higher concentrations (13).  Decreased proliferation was due to the downregulation of the cyclin A gene. Cyclin A plays an important role in cell cycle regulation.  It is involved in G1-S phase transition and in DNA replication during S phase (13, 20).  Cyclin A also helps drive the cell from G2 to mitosis.  RWP inhibited the activating transcription factor (ATF), cyclin A promoter activity, and cyclin A mRNA expression in VSMC (13).  The suppressed expression of cyclin A mRNA suppresses cyclin A dependent cellular activities, including mitosis.  Mitosis is required for cell proliferation.  Although, this model did not show evidence for inhibition of VSMC chemotaxis other experiments have.

In vitro VSMC were pre-treated with red wine, white wine, ethanol, and the wine components, tannic acid and quercetin. VSMC were then treated with PDGF-BB.  Only red wine and its components inhibited tyrosine phosphorylation of bPDGFR.  This was due to inhibition of PDGF-BB binding to the receptor in a dose dependent manner (1).  When VSMC were treated with PDGF-BB, there was a great increase in cell migration and DNA synthesis.  However, when VSMC were treated with PDGF-BB and red wine, migration and DNA synthesis were inhibited in a dose-dependent manner.  White wine did not exhibit an inhibitory effect (1).  Therefore, red wine inhibited the binding of PDGF-BB to the receptor and consequently inhibited PDGF dependent cellular responses.  This included the inhibition of bPDGFR binding to signaling molecules, RasGAP, PLCg, PI3K, SHP-2 as well as the inhibition of downstream signaling events such as phosphorylation of MAPK, and activation of transcription factors and genes. 

What components of wine inhibit CAD?  There are over 500 components of wine (12,17).  Red wine has a significantly higher content of polyphenols than white wine (12). Polyphenolic compounds in wine are classified as either flavonoids, or non-flavonoids.  Flavonoids contain a characteristic double phenyl ring structure and are divided into three groups: flavanols, anthocyanins, and flavan-3-ols (catechins and tannins).  These are non-alcoholic compounds found in wine, fruits, vegetables and chocolate.  The fact that red wine but not white had an inhibitory effect on PDGF dependent chemotaxis and proliferation suggests that non-alcoholic components present in red wine but not in white had the inhibitory effect.  HPLC analysis revealed that red wine has considerably higher concentrations of polyphenols than white especially, catechin flavonoids and gallic acid (1).  Gallic acid is another flavonoid found in red wine.  The catechin family flavoniods (+)catechin, (-)epicatechin, and (-)epigallocatechin-3-O gallate, individually had a smaller inhibitory effect on bPDGFR tyrosine phosphorylation and associated down stream cascades than red wine.  Therefore, the inhibitory effects of the components of red wine appear to be additive. Gallic acid had no effect on proliferation or migration of VSMC (1).  This is supported by a study, which found that a combination of grape seed and grape skin extracts had a greater inhibitory effect on platelet aggregation than the individual extracts alone (15).  Therefore, the effects of polyphenols are additive.

The fermentation process accounts for differences in the PDGF inhibitory effects between red and white wines.  The red wine fermentation mash contains grape skins, seeds and stems, which contain the greatest concentration of flavonoids (12).  However, white wine is not fermented with its skin or seeds.  When white wine is subjected to similar mash fermentation as red wine, it exhibits dose dependent inhibitory effects on VSMC migration and proliferation (1). 

The pathogenesis of atherosclerosis is extremely complex involving endothelial cell dysfunction, accumulation and adhesion of monocytes, oxidation of LDL, platelet aggregation, macrophage activation and the deposition of extracellular matrix.  The pathogenesis also involves the proliferation and migration of VSMC from the media into the intima of blood vessels.  The initiation and advancement of the disease involves many signal transduction pathways and cascades.  Epidemiological studies of the French population have suggested that red wine may have a cardio-protective benefit due to lower incidence of CAD mortality and higher red wine consumption by the French.  Studies have found that there is some correlation between red wine polyphenols and inhibition of some of the pathogenic aspects of atherosclerosis.  These have included decreased platelet aggregation, decreased oxidation of LDL, and decreased proliferation and migration of VSMC.  This paper focuses on the mitogenic and chemotactic effects of PDGF and the inhibitory effects of red wine polyphenols.  The inhibition of PDGF by inhibition of PDGFR has been shown to be one possible molecular explanation for the “French Paradox.”

 

 

Acknowledgements:

            Thank you to thank April Miles for her drawings of the blood vessels and Amber Zielinski for her help with the reference page.

 

References:

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Shanmuganayagam, D., Beahm, M.R., Osman, H.E., Krueger, C.G., Reed, J.D., Folts, J.D.  Grape Seed and Grape Skin Extracts Elicit a Greater Antiplatelet Effect When Used in Combination than When Used Individually in Dogs and Humans.  J. Nutr. (2002) 132: 3592-3598.

 

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Copyright 2003 Raquel Zielinski and Koni Stone