Volume 17, Issue 5, Supplement, Pages S24-S31 (May 2007)

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ABSTRACT

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Mechanism by which Alcohol and Wine Polyphenols Affect Coronary Heart Disease Risk

The reduction in coronary heart disease (CHD) from moderate alcohol intake may be mediated, in part, by increased fibrinolysis; endothelial cell (EC)–mediated fibrinolysis should decrease acute atherothrombotic consequences (eg, plaque rupture) of myocardial infarction (MI). We have shown that alcohol and individual polyphenols modulate EC fibrinolytic protein (t-PA, u-PA, PAI-1, u-PAR and Annexin-II) expression at the cellular, molecular, and gene levels to sustain increased fibrinolytic activity. Herein we describe the sequence of molecular events by which EC t-PA expression is increased through common activation of p38 MAPK signaling. Up-regulation of t-PA gene transcription, through specific alcohol and polyphenol transcription factor binding sites in the t-PA promoter, results in increased in vitro fibrinolysis and in vivo clot lytic activity (using real-time fluorescence [Fl] imaging of Cy5.5-labeled fibrin clot lysis in a mouse model). Fl-labeled fibrin clots injected into untreated C56Bl/6 wild-type control mice are lysed in approximately 2 hours and clot lytic rates significantly increased in mice treated with either alcohol, catechins, or quercetin (4–6 weeks). Fl-labeled clot lysis in ApoE knock-out mice (atherosclerosis model) showed impaired in vivo clot lysis that was “normalized” to wild-type control levels by treatment with alcohol, catechin, or quercetin for 6 to 8 weeks.

Key words: Coronary Heart Disease Prevention, Fibrinolysis, Endothelial Cell

Selected Abbreviations and Acronyms: CHD, coronary heart disease, EC, endothelial cell, EMSA, electrophoretic mobility shift assay, FDP, fibrin degradation products, Fl, fluorescence imaging, HDL, high-density lipoprotein, LDL, low-density lipoprotein, MI, myocardial infarction, PMG, plasminogen, SMC, smooth muscle cell, TF, tissue factor

Article Outline

• Abstract

• Introduction

• Normal hemostasis

• Regulation of fibrinolysis

• Expression of t-PA mRNA, in vivo, in rat thoracic aorta (in situ hybridization)

• MAPK signaling pathways involved in alcohol- and polyphenol-induced t-PA expression

• Regulation of t-PA gene transcription

• Real-time fluorescence imaging analysis of Cy5.5-labeled fibrin clot lyses, in vivo, using a mouse model

• Molecular mechanisms by which alcohol and polyphenols induce increased t-PA mediated fibrinolysis

• Acknowledgment

• References

• Copyright

Introduction

Cardiovascular disease, in particular coronary heart disease (CHD), and the ensuing acute atherothrombotic complications resulting in myocardial infarction (MI), is the leading cause of death among adults in the Western world 1, 2, 3, 4, 5. Epidemiological studies have shown that light-to-moderate drinkers of alcoholic beverages or wine, particularly red wine (1 to 4 drinks per day), have significantly lower mortality rates (20%–40%) than nondrinkers or heavier drinkers 6, 7, 8, 9, 10. These reduced rates appear to be due largely to a reduced risk for CHD-related mortality attributed, at least in part, to an attenuation in acute CHD-related vascular thrombotic and atherothrombotic complications 11, 12, 13, 14. CHD is a complex multifactorial disease, yet significant insights have been gained in our understanding of the etiology underlying the initiation, progression, and acute consequences of CHD. The pathogenesis of CHD and acute atherothrombotic complications resulting in MI involves the complex interplay between multiple altered cellular and molecular functions. Functions are typically affected at the level of the heart muscle (cardiomyocytes), blood vessels (endothelial cells [ECs] and smooth muscle cells [SMC]), blood cells (platelets and monocytes), and plasma components (lipoproteins, clotting factors, fibrinogen, etc.). Consequently, any systemic factors (such as alcohol or wine components) that will reduce, minimize, or restore these altered CHD-related functions will reduce the overall risk for CHD-related mortality.

Alcohol affects a diverse array of vascular and biochemical functions that have potential cardioprotective benefits. Induced changes in lipid profile, including increases in high-density lipoprotein (HDL) cholesterol and its subtypes (HDL2, HDL3), are thought to represent a major mechanism to reduce the risk for CHD-related mortality 15, 16, 17. However, other changes in vascular, myocardial, hemostatic, and EC functions may be equally important in collectively contributing to reducing this overall risk. Identified functions include decreased platelet aggregation/function 18, 19; decreased myocardial ischemia-reperfusion injury 20, 21; increased EC-dependent vasorelaxation (22); simultaneous activation of EC antiapoptotic and proapoptotic pathways (23); decreased plasma levels of factor VII (24) and fibrinogen 17, 25; increased fibrinolysis 26, 27, 28, 29, 30, 31; increased levels of atrial natriuretic peptide (32); and inhibition of initiation/progression of atherosclerotic lesion formation in hyperlipidemic mice (33).

Similarly, unfractionated red wine, dealcoholized red wine, and individual red wine components (ie, polyphenols) have been associated with reduced CHD mortality 7, 8, 10, 34. Functions affected include increased vasorelaxation of human and rat aortic rings 35, 36, 37; antiatherogenic effects in apoE-deficient mice (reduced LDL oxidation/aggregation, foam cell formation, lesion progression) 38, 39; inhibition of endothelin-1 synthesis in bovine aortic ECs 40, 41; down-regulation of tissue factor (TF) gene transcription in cultured human ECs and monocytes 42, 43, 44; reduced thrombosis (45); reduced inflammation 46, 47; inhibition of platelet aggregation/function 48, 49; inhibition of SMC proliferation 50, 51; and increased fibrinolysis and up-regulation of fibrinolytic protein gene transcription in cultured human ECs 52, 53.

Fig. 1 schematically summarizes described mechanisms by which moderate alcohol and/or wine polyphenols can affect different vascular, cellular, and hemostatic functions that likely act in combination or perhaps synergistically to provide potential protective benefits that may reduce overall risk for CHD.

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Figure 1 Mechanisms by which moderate alcohol and/or wine polyphenols may provide cardiovascular disease protection. The central theme of this presentation is focused on endothelial cell (EC) fibrinolysis (blood clot lysis) (highlighted by bolded arrows).

Normal hemostasis

ECs play a major role in maintaining normal hemostasis and vascular patency by regulating the balance between the synthesis and interaction of coagulation proteins to generate thrombin and promote blood clotting (fibrin/thrombosis) and fibrinolytic proteins to generate plasmin and facilitate blood clot lysis. Under normal physiological conditions, the amount of blood clot formed is readily degraded or lysed by the fibrinolytic system, thereby limiting any thrombotic complications. However, if the fibrinolytic system is overwhelmed by an acute event, such as the rupture of an unstable plaque, causing sudden exposure and release of factors (ie, TF) leading to increased activation of the coagulation system, this would result in the rapid formation of an atherothrombus and the ensuing potential of an MI.

Described studies will focus specifically on the sequence of molecular mechanisms by which alcohol and principal red wine polyphenols (catechin and quercetin) can individually modulate the expression of the EC fibrinolytic proteins at the cellular, molecular and gene levels to sustain increased fibrinolysis (blood clot lytic or “busting” activity). Components of the EC fibrinolytic system include tissue-type plasminogen activator (PA), t-PA; urokinase-type PA, u-PA; type-1 PA inhibitor, PAI-1; receptor/binding proteins for t-PA/plasminogen (Pmg), Annexin-II (Ann-II); and u-PA receptor, u-PAR. Increased fibrinolytic activity will be expected to decrease the risk for both early thrombotic as well as later acute CHD-related (plaque rupture-induced) atherothrombotic consequences of MI and hence the overall risk for CHD-related mortality.

Regulation of fibrinolysis

Fibrinolysis requires the conversion of the inactive circulating proenzyme (Pmg) to its active two-chain plasmin form by t-PA and u-PA (54). Plasmin will degrade fibrin/blood clots to maintain normal hemostasis by forming fibrin degradation products (FDPs), which are subsequently cleared by the liver. ECs are a major site of synthesis of the fibrinolytic proteins, t-PA, u-PA and the major physiological regulator of fibrinolysis, PAI-1 54, 55, 56. EC-mediated fibrinolysis is further regulated by and localized to the EC surface via specific receptors or binding proteins for t-PA and Pmg (Ann II) (57) and u-PA (u-PAR) (58). To maintain the required normal level of EC-mediated fibrinolytic activity necessitates the regulated synthesis and complex multicomponent interactions of all of these different fibrinolytic components. Consequently, systemic factors that alter the expression/interaction of one or more of these EC fibrinolytic components to decrease EC fibrinolytic activity will increase the risk for thrombotic/atherothrombotic complications. Conversely, circulating systemic factors, such as alcohol or wine components (polyphenols) that alter the expression/interaction of one or more of these EC fibrinolytic components to increase fibrinolytic activity, will be expected to significantly reduce the overall risk for acute thrombotic/atherothrombotic events and complications associated with CHD, including MI. The individual fibrinolytic components and their interactions, resulting in the regulated expression of surface-localized EC fibrinolysis are depicted schematically in Fig. 2.

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Figure 2 Regulation of EC fibrinolysis and maintenance of hemostasis.

The demonstrated ability of alcohol and polyphenols to increase the expression and interaction of EC fibrinolytic proteins, to increase fibrinolysis and provide protection in vitro and in vivo (as detailed below), is remarkably consistent with epidemiological studies that have led to the suggestion that moderate alcohol consumption may mediate additional cardioprotection by promoting fibrinolysis through changes in the activity, level, or interaction of one or more of the components of the fibrinolytic system 26, 30.

Alcohol increases t-PA in bovine aortic ECs (59) and simultaneously increases t-PA and decreases PAI-1 in cultured human ECs 60, 61. We have previously shown that low concentrations of ethanol (0.01%–0.1%, vol/vol, ≤25 mM) induces sustained (∼24 hours) increased (∼2- to 3-fold) surface-localized fibrinolytic activity in cultured human ECs 28, 29, 62. Induced, increased EC fibrinolytic activity is concomitant with the up-regulation of t-PA, u-PA, u-PAR, and Ann-II antigen/activity, mRNA and gene transcription 63, 64, 65 and simultaneous down-regulation of PAI-1 antigen/mRNA and gene transcription 66, 67. We have also shown that individual polyphenols, catechin and quercetin, similarly induce increased surface-localized fibrinolytic activity in cultured human ECs and is associated with both increased t-PA and u-PA mRNA and gene transcription (53).

Expression of t-PA mRNA, in vivo, in rat thoracic aorta (in situ hybridization)

These studies were carried out to verify our in vitro results on the effects of alcohol and polyphenols on fibrinolysis and t-PA/u-PA mRNA expression in cultured ECs. Initial animal studies were carried out, using a rat model, to determine whether direct delivery (gavage, by stomach tube) of a moderate amount of ethanol (present in 2 drinks) or individual polyphenols (equivalent to catechin or quercetin concentrations present in 2 glasses of red wine) would affect the expression of t-PA and/or u-PA mRNA, in vivo, in thoracic aortic endothelium. In situ hybridization analysis demonstrated that ethanol and each of the individual polyphenols rapidly (∼ 6 hours) increased both t-PA and u-PA mRNA expression in the thoracic aortic endothelium, as shown in Fig. 3. In situ t-PA/u-PA mRNA results were further confirmed by reverse-transcriptase polymerase chain reaction (RT-PCR) measurement of t-PA/u-PA mRNA levels in adjacent tissue sections to those simultaneously analyzed by in situ hybridization. These results clearly demonstrated that ingestion of moderate levels of alcohol or catechin or quercetin rapidly achieved sufficiently high blood concentrations for each individual component to induce demonstrable gene activation to confirm our previous in vitro results with cultured human ECs. Similar results were obtained with alcohol-/polyphenol-induced increased expression of u-PA mRNA, in vivo, in rat thoracic aortic endothelium (data not shown).

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Figure 3 Alcohol- and polyphenol-induced in vivo expression of t-PA mRNA in rat aortic endothelium. Top, In situ hybridization; note increased mRNA expression at endothelium surface (dark line), compared with control; bottom, real-time PCR analysis of t-PA, u-PA, and PAI-1 mRNA levels in adjacent sections.

MAPK signaling pathways involved in alcohol- and polyphenol-induced t-PA expression

To address the question whether alcohol and polyphenols may act through activation of similar cellular kinases we determined whether the MAPK cascades (ie, extracellular signal regulated kinases, ERK1 and ERK2; c-Jun N-terminal kinase [JNK]; and, p38 MAPK) were involved in mediating alcohol- and polyphenol-induced effects on fibrinolytic protein gene transcription in cultured human ECs. Low alcohol levels, quercetin or catechin each transiently activated (phosphorylated) all 3 of the MAPKs, p38, JNK and ERK1/2, in a time-dependent manner (Western blot). Preincubation of cultured human ECs with the physiologic inhibitors of MAPK pathways prior to induction with ethanol, catechins, or quercetin indicated that inhibition of only p38 MAPK attenuated the alcohol-, catechin- or quercetin-induced expression of both t-PA mRNA (RT-PCR) and antigen levels (Western blot). Inhibition of the JNK pathway had minimal effect on quercetin-induced t-PA expression, and no effect on the alcohol-and catechin-induced t-PA gene expression. ERK inhibition had no effect on alcohol- or polyphenol-induced t-PA expression. Therefore, these findings suggest that the p38 MAPK is the signaling pathway upstream of alcohol and polyphenol transcription factors (68). These combined results provide a novel mechanism by which alcohol or individual wine components (i.e., alcohol, catechin, and quercetin) can act through a common shared signaling pathway in the t-PA promoter to increase t-PA gene transcription, concomitant with t-PA–mediated increased EC fibrinolysis.

Regulation of t-PA gene transcription

We have previously demonstrated that the PCR amplified 3.63-kb promoter and 5′ flanking region(s) of the t-PA gene (ligated to a pGL-3 luciferase [luc] reporter gene) contained regulatory sequences important in the transcriptional regulation by alcohol (63) and polyphenols, including catechin and quercetin (53). Further studies focusing on the identification of regulatory elements and associated transcription factors in this 3.63-kb t-PA promoter fragment, responsive to alcohol and individual polyphenols, are currently being finalized. These studies involve generating overlapping 5′-3′ promoter deletion fragments, each ligated into the reporter gene to generate 5′ promoter deletion constructs from the 3.63-kb t-PA/luc construct. A combination of transient transfection studies and electrophoretic mobility assays are being used to identify the possible involved cis regulatory elements. This region contains putative binding sites for a number of transcription factors including, Sp1 and NFκB. Both these transcription factors have been shown to regulate basal and induced t-PA transcription. Therefore, we hypothesize that alcohol and polyphenols regulate t-PA gene transcription via the activation of one or both these factors. A combination of oligonucleotide mutational analysis, electrophoretic mobility shift assays (EMSAs), and super EMSAs were used to identify a distinct combination of NFκB and Sp1 binding sites for alcohol, catechin and quercetin, activation of t-PA gene transcription 69, 70.

Real-time fluorescence imaging analysis of Cy5.5-labeled fibrin clot lyses, in vivo, using a mouse model

We have modified an established pulmonary microembolism mouse model, using 125I-labeled fibrin clots to evaluate in vivo clot lysis (71), and developed a novel real-time in vivo clot lyses assay, using Cy5.5 fluorescence (Fl)–labeled fibrin clots to examine the effects of alcohol and individual polyphenols on in vivo fibrinolysis (72). Briefly, Fl-labeled microclots (3.2 μg of protein) are injected into the tail vein of a mouse and immediately become lodged in the lung. This allows for the quantitative measurement of in vivo clot lysis at 10-minute scan intervals (usually up to 120 minutes) in each individual mouse, using the GE Explore Optix™ acquisition imaging system. The sequence of actual real-time Fl-imaging analyses (10-minute scans, 10–90 minutes) of Cy5.5-labeled fibrin clot lysis in the WT mouse lung, captured with the GE Explore Optix™ acquisition imaging system, is shown in Fig. 4 (left). The quantitative measurement of a representative time-dependent change in spontaneous Fl-labeled clot lysis, in vivo, in the lung of a WT mouse, under these specific experimental conditions described above, is shown graphically in Fig. 4 (right). At the end of the scan period, the mice are sacrificed and the thoracic aorta and plasma harvested for measurement of changes in fibrinolytic protein mRNA and protein levels.

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Figure 4 Real-time fluorescence imaging analysis of spontaneous Cy5.5-labeled fibrin clot lysis, in vivo, in a C57Bl/6 mouse model.

Analyses of the time-dependent change in quantitative measurements of spontaneous clot lysis in vivo demonstrated that untreated C56Bl/6 WT control mice rapidly lysed their clots in approximately 2 hours. The rate of in vivo clot lysis was significantly increased in mice treated with either alcohol, catechin or quercetin for 4 to 6 weeks (72). This alcohol- and polyphenol-induced increase in clot lyses was concomitant with an increase in expression of thoracic aortic t-PA mRNA levels, in vivo (real-time PCR).

Real-time analysis of Fl-labeled clot lysis in an apoE-deficient (k/o) mouse model of atherosclerosis demonstrated that in vivo clot lytic rates were significantly impaired in these mice, compared with WT control mice. However, we have recently shown that this impaired clot lytic activity in apoE k/o mice can be “restored” or “normalized” to WT control levels, in vivo, by treatment with alcohol, catechins, or quercetin for 6 weeks.

These results demonstrate that alcohol and different individual polyphenols can independently stimulate and promote increased blood clot lysis, in vivo, in WT control mice. Furthermore, alcohol or polyphenols can also induce the “restoration” or “normalization” of impaired blood clot lysis, in vivo, in an atherosclerosis setting. These combined results strongly suggest that moderate consumption of alcoholic beverages, containing only an alcohol component or alcohol plus polyphenol components (i.e., red wine) will be expected to similarly promote and sustain increased blood clot lysis, in vivo, to substantially reduce the risk for acute CHD-related atherothrombotic consequences of MI and hence the overall risk for CHD-related mortality.

Molecular mechanisms by which alcohol and polyphenols induce increased t-PA mediated fibrinolysis

Described studies have identified and defined a molecular mechanism by which moderate levels of alcohol or individual wine components (i.e., principal red wine polyphenols, catechins, and quercetin) induce increased EC t-PA expression, resulting in increased EC fibrinolysis. Increased EC fibrinolysis will promote increased blood clot lysis and will be expected to contribute, in part, to reducing the risk for early thrombotic events and later acute atherothrombotic (following plaque rupture) consequences of MI. The possible molecular events that interact sequentially to initiate and promote increased t-PA–mediated blood clot lysis are shown schematically in Fig. 5. The proposed molecular activation sequences include: alcohol- and/or catechin- and/or quercetin-induced activation of p38 MAPK signaling which can lead to; activation and nuclear translocation of specific transcription factors, NFκB and Sp1; binding of transcription factors to their inducer-specific, separate individual binding sites in the t-PA promoter to activate gene transcription; translation of increased t-PA mRNA to increased t-PA protein expression and transport to the EC surface; localization of increased t-PA at the EC surface by binding to the increased, simultaneously expressed t-PA binding protein, Ann-II; increased blood clot lysis and formation of fibrin degradation products (FDPs). In addition to the increased expression of t-PA and Ann-II, the concurrent decreased expression of inhibitor PAI-1 and its regulatory action is also shown.

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Figure 5 Proposed molecular mechanisms by which moderate alcohol and wine polyphenols induce increased EC t-PA-mediated fibrinolysis to promote increased blood clot lysis.

This work was supported, in part, by Grant HL070610 (National Heart Lung Blood Institute) and Grant AA11674 (National Institute on Alcohol Abuse and Alcoholism).

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From the University of Alabama at Birmingham

Address correspondence to: Edlue M. Tabengwa, PhD, University of Alabama at Birmingham, 845 19th St South, BBRB 809, Birmingham, AL 35294-2170. Tel.: (205) 934-4296; fax: (205) 975-7121.

This work has been supported, in part, by Grant HL070610 (National Heart, Lung and Blood Institute) and Grant AA 11674 (National Institute on Alcohol Abuse and Alcoholism).

PII: S1047-2797(07)00008-7

doi:10.1016/j.annepidem.2007.01.006

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