This review represents an excerpt of a lecture given at the “Cocoa Flavanols and Cardiovascular Functions Symposium” in Lucerne, Switzerland on July 26, 2005. It is intended to provide an introduction to the concept of endothelial dysfunction, and an overview on clinical intervention studies aimed at investigating the notion of a causal relationship between flavanol consumption and improved cardiovascular function. In this context, focus was put on studies measuring endothelial function as a surrogate cardiovascular end point.
VASCULAR DISEASE AND ENDOTHELIAL FUNCTION
Measurement of endothelial function in patients has emerged as a useful tool for atherosclerosis research. In the presence of risk factors for cardiovascular disease, the endothelium loses its normal regulatory function for vessel wall homeostasis. The development and clinical manifestations of arteriosclerosis include stable and unstable angina, acute myocardial infarction, claudication, and stroke. These outcomes correlate with a loss of endothelial control of vascular tone, thrombosis, and the composition of the vascular wall. The severity of endothelial dysfunction relates to a patient's risk for experiencing an initial or recurrent cardiovascular event.1 A growing number of interventions known to decrease cardiovascular risk, including exercise, smoking cessation, weight reduction, or medication with angiotensin converting enzyme inhibitor and statins administration, will also improve endothelial function. Therefore, endothelial function serves as a “barometer” for cardiovascular health that can be used for evaluation of new therapeutic strategies.1,2
Physiologic Functions of the Vascular Endothelium
The endothelium maintains vascular homeostasis through multiple complex interactions with cells in the vessel wall and lumen. It regulates vascular tone by balancing the production of vasodilators, most importantly nitric oxide (NO), and vasoconstrictors. Endothelium-derived NO also participates in systemic physiologic functions of the endothelium, such as the control of vascular tone3 and blood clotting.4 Furthermore, the endothelium controls blood fluidity and coagulation through the production of factors that regulate platelet activity, the clotting cascade, and the fibrinolytic system. Finally, the endothelium has the capacity to produce cytokines and adhesion molecules that regulate and direct the inflammatory process.5
Pathophysiology of Endothelial Dysfunction
Under homeostatic conditions, the endothelium maintains normal vascular tone and blood flow, and there is little or no expression of proinflammatory factors. However, both traditional and novel cardiovascular disease (CVD) risk factors initiate a chronic inflammatory process that is accompanied by a loss of vasodilator and antithrombotic factors and an increase in vasoconstrictor and prothrombotic products. As outlined in Figure 1, risk factors as diverse as smoking, aging, hypercholesterolemia, hypertension, hyperglycemia, and a family history of premature atherosclerotic disease are all associated with an attenuation or loss of endothelium dependent vasodilation in both adults and children.6–12 More recently recognized risk factors such as obesity,13 elevated C-reactive protein,14 postprandial state,9 hyperhomocysteinemia,15 and chronic systemic infection16 are also associated with endothelial dysfunction. Besides abnormal vasoreactivity, there are several other imbalances present in high-risk individuals. Endothelial cells may fail to sufficiently inhibit coagulation. This may put high-risk individuals at an even more elevated risk to experience cardiovascular events.17 Interestingly, plasma NO species (RXNO), which are believed to represent a significant proportion of the circulating NO pool, are also decreased with cardiovascular risk factors.12 Furthermore, when exposed to certain pathogenic proinflammatory stimuli, the endothelium expresses leukocyte chemotactic factors, adhesion molecules, and inflammatory cytokines.18 The precise extent and order by which the normal control mechanisms are affected is not yet fully understood.
The concept of “endothelial dysfunction” refers to the above mentioned broad alteration in endothelial phenotype, which may contribute to the development and clinical expression of atherosclerosis.19 Endothelial dysfunction seems to fuel a “positive feedback loop” in which inflammatory factors promote monocyte and T-cell adhesion, foam cell formation, extracellular matrix digestion, as well as vascular smooth muscle migration and proliferation that lead to atherosclerotic plaque formation.5,20 Endothelial dysfunction is also relevant to the later stages of the disease and seems to play a role in acute coronary syndromes.21 A dysfunctional endothelium may promote plaque activation, which leads to a more vulnerable plaque and will potentially not sufficiently induce vasodilation and inhibit thrombus growth to sustain perfusion in case of plaque-rupture. Given the potential causal principle that leads from endothelial dysfunction to atherosclerosis (Fig. 1), various methods have been employed to measure endothelial dysfunction in humans.
Methods to Determine Endothelial Dysfunction in Humans
Although atherosclerosis is associated with a broad alteration in endothelial phenotype, the assessment of endothelium-dependent vasodilation has emerged as an accessible indicator of endothelial health—pars pro toto. In particular, stimuli that increase production of endothelium-derived NO have proven useful in assessing endothelium-dependent vasodilation in humans. Such stimuli include increased shear stress resulting from increased blood flow and receptor-dependent agonists, such as acetylcholine, bradykinin, or substance P. The contribution of NO to basal vascular tone and NO synthase (NOS)-dependence of vascular effects observed in intervention trials can be assessed with specific inhibitors of NOS: L-NG-monomethyl-arginine or L-NG-arginine-methylester approved for human research.
Several methods to evaluate endothelial function have been employed previously, each with its own advantages and disadvantages. The earliest studies of endothelial control of vasomotion used quantitative coronary angiography to examine the vasomotor responses of epicardial coronary arteries during infusion of acetylcholine22 or increased blood flow.23 In healthy individuals, the endothelium responds to these stimuli by releasing vasodilator factors, particularly NO. Early studies demonstrated that patients with angiographically proven coronary artery disease (CAD) display impaired flow-mediated dilation (FMD) and a vasoconstrictor response to acetylcholine rather than the normal vasodilator response, likely reflecting loss of NO and unopposed constrictor effects of acetylcholine on vascular smooth muscle.22 Invasive studies in the arm involve infusion of endothelium dependent agonists into the brachial artery and measuring the vasodilator responses of forearm resistance vessels using venous occlusion plethysmography.8,24 Similar to studies in the coronary circulation, this approach allows investigators to examine dose-response relations and use specific agonists and antagonists in a more accessible vascular bed. However, the technique requires arterial catheterization and has, therefore, only limited applicability for large-scale studies or future development as a clinical tool.
Finally, there has been considerable interest in noninvasive examination of endothelium-dependent FMD of the conduit brachial artery using vascular ultrasound.25 Briefly, FMD represents the percent diameter gain as calculated based on preischemia and postischemia (and reactive hyperemia) diameter measurements of the brachial artery. In this context, ischemia is induced through vessel occlusion by means of inflating a blood pressure cuff around the forearm or upper arm. Ischemic dilation of resistance vessels leads to increased flow in the conduit brachial artery. Sheer stress stimulates endothelial NOS and NO dilates underlying vessel wall smooth muscle cells via guanylate cyclase.26 Brachial artery FMD is in large parts mediated by NO synthesis and is used as a functional NO readout.25,27 The absolute values may vary depending on various factors including the position of the cuff, artery used measure diameter, time of ischemia, and time point of measurement after ischemia.28,29 Therefore, comparisons of FMD values should be made with caution and ideally refer to values of a control group measured with the identical set-up. This technique can safely be applied to large and diverse groups of patients. Repeated measurements can be made over time. As in the coronary circulation, endothelial function in the brachial circulation is impaired in the setting of traditional and novel risk factors and responds to interventions known to reduce CVD risk.2 Studies suggesting that endothelial function detected noninvasively in the brachial artery correlates with function in conduit coronary arteries demonstrate the systemic nature of endothelial dysfunction.30 Despite the many parallel findings, one modest-sized study suggested that, within individual subjects, brachial artery FMD does not directly correlate with resistance vessel (microvascular) function as measured by infusion studies.31 Indeed, it is likely that there is differential regulation of vascular tone in conduit and resistance vessels, and that the different measures of vascular function may have relevance to different aspects of CVD.
Although the resolution of ultrasound and image analysis systems have greatly improved, one of the major limitations of FMD is the considerable dependence on the observer requiring well-trained personnel and professional revision of ultrasound images.32 Some groups have introduced new techniques to measure endothelial function in an observer-independent way. A new method to measure endothelial function and potentially predict CAD in an observer independent way is peripheral artery tonometry (PAT).33,34 This technique only requires the investigator to apply sensors to the finger tip of study subjects and press a button. The measurement and analysis are performed automatically by a computer. The readout of PAT is the pulse wave amplitude (PWA) representing the volume changes of the finger between systole and diastole averaged over many heart cycles. The ratio of PWA values before and after ischemia of the arm is called PAT index. The PAT index correlates fairly well with FMD and it seems that it reflects both conduit artery and microvascular function.35
Studies Evaluating the Prognostic Value of Endothelial Dysfunction
Although case-control studies indicate an association between endothelial dysfunction and acute coronary syndromes,21 more convincing evidence for a pathogenic role of the former is provided by studies demonstrating that endothelial function identifies patients at increased risk for future events. Numerous published studies involving >2000 patients with atherosclerosis have proven the prognostic value of endothelial vasomotor dysfunction.1 These studies strongly and consistently demonstrate that endothelial dysfunction identifies patients who have increased risk for cardiovascular events in the short and long run. Primary-preventive and secondary-preventive therapies, for example, exercise therapy, ACE inhibitors, and statins are believed to mediate their positive prognostic effect in large parts by increasing endothelial function.
EFFECT OF FLAVANOL-RICH FOOD CONSUMPTION ON ENDOTHELIAL FUNCTION AND NO
Epidemiologic studies indicate that diets rich in fruit and vegetables are associated with a decreased incidence of adverse cardiovascular events, such as CAD and stroke.36 This effect was ascribed, at least in part, to the high content of antioxidants, in particular polyphenolic compounds, such as flavonoids, in plant-based foods. In this context, cocoa, some chocolates, red wine, and tea received much attention, because they are particularly rich in flavonoids, phytochemicals with strong antioxidant properties in vitro.37 Several lines of evidence suggest that flavanols, a major class of flavonoids, are important bioactive constituents of the above mentioned foods and that there may be a causal relationship between flavanol consumption and improvements in cardiovascular function. We and others have shown that the acute and repetitive consumption of flavanol-rich foods for up to 4 weeks can improve endothelial function (Table 1). Positive effects were observed in populations with manifest CAD, but also in healthy volunteers with or without risk factors.
Studies in Patients With CAD
In our initial study, we measured endothelium-dependent vascular reactivity as FMD after consumption of a cocoa drink with either high (176 mg, CocoaPro, Mars Inc, UK) or low (<10 mg) flavanol content in individuals with CAD or cardiovascular risk factors (hypertension, dyslipidemia, diabetes mellitus, or smoking).38 In a time course, we observed an acute reversible increase in FMD with a maximum at 2 hours after consumption, which the FMD returning to baseline at 4 to 6 hours postingestion. The time course was similar to the epicatechin plasma concentration curve previously reported by Richelle et al39 after dark chocolate consumption. To confirm this effect, we performed a larger randomized, double-blind, cross-over study (n=20).38 Besides FMD, we also measured circulating NO species (RXNO). The results demonstrate that FMD and RXNO increased at 2 hours after oral administration of the high flavanol drink, whereas both parameters remained unaltered after the ingestion of a low flavanol cocoa drink. Although baseline values before cocoa ingestion were significantly lower than healthy age matched volunteers measured in our laboratory (FMD 3.4% and RXNO 22 nmol/L), the 2 hours posthigh flavanol values (6.3% and 36 nmol/L, respectively) were comparable to the controls without CAD or cardiovascular risk factors.12 This suggests that NO-dependent endothelial function can be acutely restored in CAD patients. Both drinks were matched based on caloric intake, macronutrients, caffeine, and theobromine. Furthermore, the 2 drinks could not be distinguished by color or packaging allowing proper blinding of patients and investigators to treatment. The flavanol content of the flavanol-rich cocoa drink (176 mg) approximated the average flavanol content present in 50 g of dark chocolate, 1 to 3 apples, 1 L of red wine, or 170 mL of black tea.37 The low flavanol cocoa drink resembled a regular commercially available “drinking chocolate” with relatively low cocoa and flavanol (<10 mg) content.
Our findings corroborate results from a randomized cross-over study by Duffy et al40 showing that FMD (baseline 6.0±3.4%) increased acutely 2 hours after consumption of black tea in a group of CAD patients (450 mL, ∼477 mg of total flavonoids, FMD 9.4±3.9%, n=66). Additionally, this study showed that the repetitive, long-term consumption of black tea over 4 weeks, led to a sustained increase in FMD as measured after overnight fast (900 mL/d; ∼952 mg of total flavonoids; FMD 9.5±3.6%). A further significant increase in FMD was observed after additional acute consumption of black tea after the 4 weeks long-term consumption (450 mL, short-on-longterm, FMD 10.8±4.4%). FMD after short-on-longterm tea consumption was comparable to that of healthy volunteers in the same laboratory at baseline (FMD 11.2±5.7%). The major limitation of the study by Duffy et al40 was the lack of a sufficient placebo and water was used as the control drink. This prevented proper blinding of patients to treatment. Another study by Stein and colleagues41 demonstrated that purple grape juice (640 mL/d) consumption for 14 days improved FMD (2.2% to 6.4%) in 15 patients with CAD. One of the limitations of this study was the lack of a control group, and in contrast to the findings by Duffy et al40 and our results,38 the investigators report that nitroglycerin-induced vasodilation was also improved. Taken together, the results of the above-mentioned investigations, although based on relatively small n values, suggest that the consumption of flavanol-rich foods can cause significant increases in NO-dependent endothelial function in CAD patients. Taking into account that FMD is an established prognostic marker, which can predict future cardiovascular events in CAD patients, an increase in FMD, as observed after flavanol-rich food ingestion, would indicate a health benefit by means of potential protection from recurrent cardiovascular events (secondary prevention).
Endothelial Function in Healthy Individuals With or Without Risk Factors
To evaluate the potential of flavanols in primary prevention, more recent investigations have included assessments of healthy individuals with or without cardiovascular risk factors. We have studied young, healthy volunteers with smoking as their sole major cardiovascular risk factor.42 Young smokers seemed to be an ideal study group, because of the preserved vascular morphology with endothelial dysfunction secondary to the induction of oxidative stress.43 At baseline after a 12-hour overnight smoking cessation, our group of smokers (n=11, age 31±1 y) exhibited impaired FMD (4.5±0.8%) and decreased plasma NO species RXNO (21±3 nmol/L) as compared with an age-matched reference group of nonsmokers (FMD 7.1±0.6%, plasma RXNO 36±2 nmol/L), suggesting endothelial dysfunction due to decreased NO bioactivity.6 However, after the administration of 100 mL flavanol-rich cocoa drink (∼176 mg flavanols) to smokers, the FMD increased from 4.5±0.8% (preingestion) to 6.9±0.9% (postingestion), and RXNO were elevated by approximately 38% to plasma levels of 29±2 nmol/L, thus approaching values observed in our nonsmoking reference group. No changes with regard to FMD and circulating RXNO were seen after the ingestion of a low flavanol control drink. Interestingly, we found strong correlations between the number of cigarettes smoked on the day before the first study day (average 17±2) and the absolute increases in plasma RXNO (r=0.73, P=0.011) and FMD (r=0.69, P=0.020) 2 hours after the ingestion of the high-flavanol drink. There were no significant correlations between the cumulative life-time dose of cigarettes expressed as pack years (average: 12±2) and the increases in RXNO or FMD. This suggests that cocoa flavanols can transiently counteract endothelial dysfunction caused by chronic cigarette smoking (Fig. 2 for time course).
Although the exact mechanisms of flavanol-mediated improvements in FMD remain to be fully elucidated, NO seems to mediate a significant proportion of the observed effects. To gain further mechanistic insight, we inhibited NOS or alternatively administered ascorbic acid on top of the observed effects. NOS inhibition by intravenous infusion of the NOS inhibitor L-NG-monomethyl-arginine—mimicking acute endothelial dysfunction—abolished flavanol-related improvements of endothelial dysfunction and increases in circulating RXNO levels. Findings by other groups are also consistent with a NOS-dependent effect. Fisher et al34 were able to reverse cocoa-related increases in vasodilation, measured as PWA of the fingertip after 4 days of daily cocoa ingestion (n=27 healthy volunteers, 821 mg total flavanols/d), using L-NG-arginine-methylester, a competitive NOS inhibitor. Therefore, it can be concluded that the observed vasodilator effects are, at least in part, mediated by NOS-dependent NO production.
Ascorbic acid has previously been shown to increase endothelial function in CAD patients44 and smokers. In smokers, this effect was attributed to an increase in tetrahydrobiopterine, an essential cofactor of NOS.45 During our investigation, we administered flavanol-rich cocoa to smokers and infused 2 g of ascorbic acid after a maximal increase in FMD was obtained at 2 hours. We observed no further increase in FMD and circulating NO levels after application of ascorbic acid, although the same dose of ascorbic acid is sufficient to reverse endothelial dysfunction in smokers when applied alone.46 In summary, cocoa ingestion leads to a NOS-dependent increase in vasodilation and abolishes ascorbic acid response.
Other groups have investigated the effect of flavanol-rich dark chocolate in hypertensive patients. Taubert et al47 and Grassi et al48 demonstrated that the repetitive consumption of dark chocolate can lower blood pressure in hypertensive patients suggesting a vasodilatory effect on resistance vessels. Furthermore, Grassi et al48 also reported a significantly increased FMD in this context. In a recent study undertaken by Lauer et al,7 it was shown that an impaired FMD in essential hypertension is, in part, due to limited microvascular dilation, which in turn represents the initiating stimulus for FMD. It can be speculated, that an increased FMD in hypertensives may be secondary to improved microvascular response and enhanced the shear force on the arterial endothelium.29 Interestingly, Grassi et al49 not only reported robust effects after dark chocolate consumption on FMD and insulin resistance in their patients with essential hypertension, but they also demonstrated similar effects in healthy subjects.
What are the Bioactive Compounds?
There still exists uncertainty as to whether or not flavanols truly are the bioactive compounds mediating an enhanced vascular reactivity. This is in part due to the fact, that flavanol-rich foods and plant extracts contain a large variety of potentially bioactive compounds, and information ensuing from investigations in humans using specific, chemically pure flavanol are rare. In this context, the quite often-used strategy of providing flavanol-rich preparations/extracts as treatments and flavanol-poor extracts as controls is beginning to address the issue. However, even this approach cannot deliver a direct proof that flavanols are the bioactive modulators of cardiovascular function. The main criticism regarding studies investigating effects of flavonoid-rich foods are that the observed effects may potentially be due to compounds other than flavonoids and that flavonoids are perhaps a mere marker for some other bioactive constituents of cocoa tea, wine, or other flavanol-rich foods.
Furthermore, we have only limited data available on flavanols, and perhaps more importantly their metabolites, at the site of biologic action, especially in the context of clinical trials, nor do we have ample information on the bioactivity of flavanol metabolites in general. Most investigators limited their assessments to measurements of plasma levels of circulating flavanols after glucuronidase/sulfatase treatments of plasma extracts, thus eliminating information with respect to individual circulating flavanol metabolites.40 Therefore, we conducted a preliminary dose response study, measuring the increase in FMD in parallel with plasma levels of selected flavanols/metabolites after the consumption of increasing doses of flavanol-rich cocoa drinks (n=4 smokers, Fig. 3).42 Measurements were taken immediately before and 2 hours after ingestion of approximately 88, 176, 352, or 528 mg of flavanols (40% epicatechin/catechin, 60% procyanidins) on 4 separate days. We observed dose-dependent increases in FMD (Fig. 3). In addition, plasma levels of free, aglyconic flavanols, and those of conjugated flavanol metabolites (representing just a selected subset of all structurally-related flavanol metabolites present in plasma), increased significantly after the ingestion of >100 mL flavanol-rich cocoa containing ≥176 mg of total flavanols (Fig. 4). Among the flavanols/metabolites investigated, the increases in the plasma concentrations of epicatechin (r=0.75, P=0.005), catechin (r=0.76, P=0.004), epicatechin-7-β-D-glucuronide (r=0.69, P=0.013), 4′-O-methyl-epicatechin (r=0.65, P=0.022), and 4′-O-methyl-epicatechin-β-D-glucuronide (r=0.67, P=0.018) correlated significantly with the increase in RXNO. In addition, increases in FMD statistically correlated with the increase in epicatechin (r=0.66, P=0.020) and catechin (r=0.62, P=0.031), thus perhaps further strengthening the notion that it is indeed the flavanols that mediate the observed effects. Thus, further oral administration studies using individual, chemically pure flavanols are warranted to establish a direct causal relationship.
In the context of dietary recommendation for the general public, or the use of flavanols in therapeutic strategies to combat CVD, the human intervention studies undertaken thus far do not provide information related to the effects of long-term flavanol administrations. Thus, it is currently unknown as to whether or not flavanol-associated effects will translate into long-term health benefits such as protection from cardiovascular events. Taken together, although data on the beneficial effects of flavanols, especially in the context of cardiovascular health and disease, are accumulating and promising, additional studies aimed at understanding the molecular mechanisms, and focusing on long-term benefits and risks, are strongly indicated.
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Keywords:© 2006 Lippincott Williams & Wilkins, Inc.
endothelial function; flow-mediated dilation; flavanol; nitric oxide; cocoa