Flow-mediated dilatation

Flow-mediated dilatation (FMD) describes arterial dilatation in response to increased intra-luminal shear stress. In humans, this phenomenon has been described using a forearm technique, in which reactive hyperaemia following release of an arm cuff at suprasystolic pressures mediates increased brachial artery diameter⁽Reference Celermajer, Sorensen and Gooch⁹⁾. The vasodilatation can be quantified using B-mode ultrasound, and agreed protocols have been published to guide investigators using this procedure⁽Reference Corretti, Anderson and Benjamin^10, Reference Deanfield, Donald and Ferri¹¹⁾. Since the technique does not involve needles, it has proven popular with researchers and less daunting for potential volunteers.

A growing body of research has not only demonstrated impaired brachial artery FMD among patients with recognised cardiovascular risk factors⁽Reference Lekakis, Papamichael and Vemmos^12–Reference Evans, Anderson and Graham¹⁴⁾, but also suggests that the measure may serve as an independent prognostic indicator in both high-risk populations⁽Reference Brevetti, Silvestro and Schiano^15–Reference Patti, Pasceri and Melfi¹⁷⁾ and healthy volunteers⁽Reference Yeboah, Crouse and Hsu¹⁸⁾.

Brachial FMD has been employed by several groups as an endpoint during diet-related intervention trials. The effect of n-3 fatty acids on endothelial function has recently been reviewed⁽Reference Egert and Stehle¹⁹⁾, as has the effect of fruit polyphenols⁽Reference Chong, Macdonald and Lovegrove²⁰⁾, berries⁽Reference Basu, Rhone and Lyons²¹⁾ and green tea⁽Reference Moore, Jackson and Minihane^22, Reference Wolfram²³⁾ on vascular health, and these reviews include studies using FMD endpoints. In Table 1 ⁽Reference Balzer, Rassaf and Heiss^24–Reference Hermann, Spieker and Ruschitzka³⁴⁾, the intervention studies that have examined the effect of chocolate or cocoa on FMD are shown. Consumption of dark chocolate has been shown to improve brachial FMD, both acutely (six out of seven studies), and chronically (five out of six studies).

Table 1 Effect of cocoa or chocolate interventions on flow-mediated dilatation (FMD): evidence from randomised controlled trials

↑ , Significant increase; CAD, coronary artery disease; ↔ , no significant change.

While it provides a minimally invasive option for vascular function assessment, the measurement of brachial FMD does rely on considerable skill in ultrasound image acquisition and concerns about this technique's reproducibility have been expressed⁽Reference Sanderson, Sattar and Olthof³⁵⁾. De Roos et al. report substantial within-subject variability for FMD among healthy volunteers, with a CV estimated at approximately 50 %⁽Reference De Roos, Bots and Schouten³⁶⁾. Although much better CV figures (7–10 %) have been published by Deanfield's group⁽Reference Donald, Charakida and Cole^37, Reference Donald, Halcox and Charakida³⁸⁾, there remains a concern that, when employed in smaller, less experienced centres, the technique's inherent variability precludes adequate study power. In addition, the NO dependency of FMD has been questioned⁽Reference Tschakovsky and Pyke³⁹⁾, such that this response is better considered a consequence of interplay between competing dilator and constrictor influences⁽Reference Mullen, Kharbanda and Cross⁴⁰⁾.

Another potential concern is that not all prospective studies have identified brachial FMD as an independent predictor of cardiovascular morbidity. Fathi et al. report no relationship between FMD and the risk of death, acute coronary syndrome or stroke among patients with established risk factors⁽Reference Fathi, Haluska and Isbel⁴¹⁾. Austrian investigators note similar findings among patients undergoing coronary angiography⁽Reference Frick, Suessenbacher and Alber⁴²⁾ while in almost 3000 older adults, brachial artery diameter and FMD proved equally effective predictors of vascular outcome⁽Reference Yeboah, Crouse and Hsu¹⁸⁾. However, although not always shown to be an independent predictor of cardiovascular events, this does not preclude its use as an endpoint in dietary intervention studies, as long as interpretation of the result is appropriate.

Despite these reservations, brachial FMD is widely regarded as the ‘gold standard’ technique by which to assess conduit vessel function in cardiovascular research⁽Reference Deanfield, Halcox and Rabelink⁴³⁾. However, before adopting FMD as the endpoint of choice, trial investigators should consider whether conduit vessel function is the best metric with which to detect any intervention-related effect. The response quantified in standard FMD protocols depends upon forearm ischaemia-induced hyperaemic flow, which is itself a function of reduced vascular resistance. The latter is determined largely by endothelium-dependent microvascular tone and thus measures of reactive hyperaemia have been suggested as novel arterial descriptors⁽Reference Widlansky⁴⁴⁾. Analysis of brachial ultrasound recordings from over 2000 Framingham volunteers showed that Doppler-derived indices of hyperaemic shear stress correlated better with established cardiovascular risk factors than did FMD⁽Reference Mitchell, Parise and Vita⁴⁵⁾. Huang et al. have shown that lower hyperaemic flow velocities in the brachial artery following a standard period of forearm ischaemia independently predicted postoperative morbidity and mortality among patients with peripheral arterial disease undergoing elective vascular surgery⁽Reference Huang, Silver and Shvenke⁴⁶⁾.

It has been speculated that the microvascular dysfunction implied by reduced hyperaemic flow responses may be more sensitive to early atherosclerotic change than are disturbances in conduit artery vasomotion as quantified by FMD⁽Reference Philpott and Anderson⁴⁷⁾. This has particular relevance for the design of dietary intervention trials which often seek to detect subtle changes in healthy volunteers. It would therefore be considered best practice to record FMD and reactive hyperaemia concurrently, as reactive hyperaemia can be measured simultaneously with FMD, using the same equipment.

Pharmacological provocation of endothelium-dependent vasomotion

Where FMD uses mechanical shear stress to provoke arterial endothelial vasodilator production, the local infusion of appropriate agonists can produce a similar effect. While based on well-established physiological and pharmacological principles, this approach does rely on successful arterial puncture.

Furchgott & Zawadzki's description of acetylcholine-mediated endothelium-dependent vasodilatation using isolated arterial segments in vitro was soon extended to in vivo human work⁽Reference Furchgott and Zawadzki⁴⁸⁾. Within the coronary circulation, direct intra-arterial injection of acetylcholine mediated vasoconstriction among patients with significant atherosclerotic lesions but dilated angiographically normal vessels⁽Reference Ludmer, Selwyn and Shook⁴⁹⁾. Several prospective studies among patients with clinical indications for cardiac catheterisation have established an abnormal coronary response to endothelium-dependent agonists as a powerful independent predictor of cardiovascular morbidity⁽Reference Al Suwaidi, Higano and Holmes^50–Reference Targonski, Bonetti and Pumper⁵³⁾. The potential risks associated with invasive cardiac assessment limit this technique's applicability to trials conducted in healthy volunteers. However, the forearm is an accessible and relatively safe vascular bed, in which pharmacological challenges analogous to those described for epicardial vessels can be performed.

While coronary procedures have relied upon quantitative angiography, forearm studies mainly employ venous occlusion plethysmography to measure the efficacy of intra-brachial vasodilators. Concordant, proportionate responses to acetylcholine have been described in synchronously infused coronary and brachial arteries⁽Reference Hirooka, Imaizumi and Tagawa^54, Reference Tagawa, Mohri and Tagawa⁵⁵⁾. Poor forearm dilator responses to acetylcholine have been shown to predict increased rates of cardiovascular morbidity among hypertensive volunteers⁽Reference Perticone, Ceravolo and Pujia⁵⁶⁾ and patients with coronary artery disease⁽Reference Heitzer, Schlinzig and Krohn^57, Reference Fichtlscherer, Breuer and Zeiher⁵⁸⁾.

It is important to appreciate that, when infused into the brachial artery, vasoactive agents exert their influence on forearm blood flow by modulating small vessel tone and this technique is thus an assessment of microvascular function⁽Reference Wilkinson and Webb⁵⁹⁾.

Forearm blood flow response to locally infused endothelium-dependent vasodilators has been used as an endpoint in several dietary intervention trials. Healthy adults who consumed a Mediterranean-style diet for 6 weeks showed significant improvements in endothelium-dependent forearm hyperaemia⁽Reference Singh, Graves and Taylor^60, Reference Ambring, Friberg and Axelsen⁶¹⁾; however, Ambring et al. subsequently reported negative findings for a similar 4-week intervention conducted among younger volunteers using the same endpoint⁽Reference Ambring, Friberg and Axelsen⁶¹⁾. Investigators have also employed this technique to document the deleterious effects of increasing salt consumption. It was shown that 5 d of salt loading significantly reduced forearm blood flow responses to intra-brachial acetylcholine⁽Reference Tzemos, Lim and Wong⁶²⁾. Our group recently reported a significant dose-dependent relationship between increased dietary fruit and vegetable consumption and improved microvascular endothelial function, as quantified using this method⁽Reference McCall, McGartland and McKinley⁶³⁾.

However, the applicability of forearm blood flow manipulation through local vasodilator infusion to large, multi-centre clinical trials is limited by its reliance on arterial puncture, and the prospect of needle insertion is likely to prove inherently unattractive to many.

The most serious risks of brachial artery cannulation include occlusion of the artery leading to limb ischaemia, and median nerve damage due to direct trauma or compression by haematoma or infection. In reality, complications are rare, and usually involve minor bruising or local discomfort that resolve quickly, without intervention. It is, however, essential that this procedure is performed by an experienced researcher (with a background in vascular access and aseptic techniques) such as an intensivist, cardiologist or surgeon. This therefore restricts the use of this methodology to research centres with access to the aforementioned skilled operators, and also limits its use to smaller studies. The requirement for needle insertion may also deter participants, and those on oral anticoagulation or with significantly increased BMI must be excluded⁽Reference Wilkinson and Webb^59, Reference Benjamin, Calver and Collier^64, Reference Kennedy, Grocott and Schwartz⁶⁵⁾.

An alternative, less invasive, approach to pharmacological manipulation of the microvascular endothelium involves transdermal drug delivery by iontophoresis⁽Reference Morris and Shore⁶⁶⁾. A small electrical current is applied to the forearm and mediates movement of polar drugs such as acetylcholine into cutaneous vessels. Changes in skin blood flow are then quantified using laser Doppler flowmetry⁽Reference Kvandal, Stefanovska and Veber⁶⁷⁾. In a recent study, a strong correlation between this measure of skin microvasculature and FMD of the brachial artery was reported⁽Reference Debbabi, Bonnin and Ducluzeau⁶⁸⁾. Therefore, this may offer an alternative endpoint for future intervention trials, although the clinical relevance is, as yet, less established than for other methods.

A small number of trials have already used this endpoint, including a trial of fruit and vegetable purée-based drinks (a trend towards an effect shown on this endpoint in both acute and chronic settings)⁽Reference George, Niwat and Waroonphan⁶⁹⁾, a trial of a green tea polyphenol extract (no effect in the tested chronic setting)⁽Reference Frank, George and Lodge⁷⁰⁾, a study of acute fish oil consumption (effect on endpoint demonstrated)⁽Reference Armah, Jackson and Doman⁷¹⁾ and a chronic study of weight reduction and exercise (no effect demonstrated)⁽Reference Hamdy, Ledbury and Mullooly⁷²⁾. A recent study of orange juice demonstrated an acute postprandial effect of orange juice or hesperidin consumption on microvascular reactivity, but no effect on fasting reactivity after 4 weeks of consumption⁽Reference Morand, Dubray and Milenkovic⁷³⁾.

Pulse wave velocity

The velocity with which pressure pulse waves travel along an arterial segment can be mathematically related to that vessel's mechanical properties, by either the Moens–Kortweg or Bramwell–Hill equations⁽Reference Oliver and Webb⁷⁶⁾. These formulae predict that pressure pulse waves will travel faster in less distensible arteries and thus PWV is a commonly cited descriptor of ‘arterial stiffness’⁽Reference Hughes, Dixon and McVeigh⁷⁷⁾. A pressure transducer or tonometer is used to detect passage of the pulse wave between two anatomical locations. This can be done sequentially⁽Reference Schillaci, Pirro and Vaudo⁷⁸⁾ or simultaneously⁽Reference Laurent, Katsahian and Fassot⁷⁹⁾, with gating to a contemporaneously recorded electrocardiogram. The carotid and femoral arteries are commonly chosen tonometry sites, as this allows estimation of aortic PWV.

Prospective data are now available to suggest that carotid-femoral PWV (CFPWV) is an independent predictor of cardiovascular morbidity among healthy individuals⁽Reference Mattace-Raso, van der Cammen and Hofman^80, Reference Willum-Hansen, Staessen and Torp-Pedersen⁸¹⁾. While tonometry at the radial site is much more convenient for both volunteer and investigator, carotid-radial PWV did not predict coronary events or strokes among a group of patients with end-stage renal failure in whom CFPWV did have independent prognostic value⁽Reference Pannier, Guerin and Marchais⁸²⁾. This finding reflects structural distinctions between muscular forearm arteries and larger elastic vessels such as the aorta where medial cells have ectodermal rather than mesodermal origins⁽Reference Safar and Laurent⁸³⁾.

As the intra-luminal pressure within an artery increases, progressively more collagen fibres are recruited and thus its mechanical characteristics change⁽Reference Payne and Webb⁸⁴⁾. An intervention which reduces blood pressure is, therefore, also likely to reduce PWV independent of any pleiotropic effect on the arterial wall⁽Reference O'Rourke and Nichols⁸⁵⁾. This is an important caveat to the interpretation of any trial which employs PWV as an endpoint. Several recent trials have reported significant reductions in CFPWV following interventions including weight reduction⁽Reference Dengo, Dennis and Orr⁸⁶⁾, DASH⁽Reference Blumenthal, Babyak and Hinderliter⁸⁷⁾, low carbohydrate⁽Reference Keogh, Brinkworth and Noakes⁸⁸⁾ and low-glycaemic index⁽Reference Philippou, Bovill-Taylor and Rajkumar⁸⁹⁾ diets. However, in each case, significant blood pressure reductions are also recorded. Similarly, in a Na-loading study among hypertensive volunteers, changes in CFPWV were positively correlated with changes in brachial blood pressure⁽Reference Todd, Macginley and Schollum⁹⁰⁾, whilst 6 months' supplementation with conjugated linoleic acid⁽Reference Sluijs, Plantinga and de Roos⁹¹⁾ or 2 weeks of dietary salt restriction⁽Reference Dickinson, Keogh and Clifton⁹²⁾ failed to alter either blood pressure or CFPWV. However, an isoflavone intervention over 6 weeks did mediate significantly slower CFPWV in healthy volunteers without 24 h ambulatory blood pressure reduction⁽Reference Teede, McGrath and DeSilva⁹³⁾.

In summary, if an intervention mediates a significant effect on arterial blood pressure, a concordant effect on PWV will be observed. This does not imply an alteration of vascular structure or function and can be predicted from arterial physiology.

Before choosing it as endpoint, investigators should postulate a mechanism by which their proposed intervention could change CFPWV. Through an in vitro model, impairment of local endothelial NO production has been shown to increase PWV across predefined arterial segments⁽Reference Wilkinson, MacCallum and Cockcroft^94, Reference Schmitt, Avolio and Qasem⁹⁵⁾. Furthermore, brachial artery FMD has been independently associated with CFPWV in cross-sectional technique comparison studies among healthy volunteers⁽Reference McEniery, Wallace and Mackenzie⁹⁶⁾ and patients with isolated systolic hypertension⁽Reference Wallace, Yasmin and McEniery⁹⁷⁾. A significant, positive correlation between carotid-radial PWV and microvascular function in the forearm has also been described⁽Reference McCall, McGartland and Woodside⁹⁸⁾.

However, during the relatively brief trials that typify much human dietary intervention work it is questionable whether subtle alterations in levels of an endothelium-derived paracrine mediator could significantly change CFPWV which principally reflects aortic medial elastin:collagen ratios. Thus, this parameter is not ideal for use in short-term studies which aim to evaluate the possible endothelial effects of an intervention.

Pulse contour analysis

Palpation and analysis of the radial pulse as a means by which to assess systemic arterial health is a long-established practice in cardiovascular medicine⁽Reference Oparil and Izzo⁹⁹⁾. Algorithm-based reconstruction of the aortic pressure pulse from tonometer-derived radial waveforms has fuelled renewed interest in this approach⁽Reference O'Rourke and Nichols⁸⁵⁾. A commercially available device has been widely used in cardiovascular research to estimate the aortic augmentation index, a measure of arterial wave reflection⁽Reference Karamanoglu, O'Rourke and Avolio^100, Reference Chen, Nevo and Fetics¹⁰¹⁾. Since it is non-invasive, requires minimal training and generates an easily interpretable numeric output, this technique has proved popular in cardiovascular research.

Among patients undergoing cardiac catheterisation, those in the highest quartile of augmentation index had significantly more coronary disease⁽Reference Weber, Auer and O'Rourke¹⁰²⁾. Similarly, patients with end-stage renal failure who had higher augmentation indices were more likely to die during an 8-year follow-up period⁽Reference London, Blacher and Pannier¹⁰³⁾. However, a recent review of arterial stiffness by The Framingham Heart Study found that whilst a higher aortic PWV was associated with a 48 % increase in cardiovascular events, the aortic augmentation index, central pulse pressure and pulse pressure amplification showed no such correlation⁽Reference Mitchell, Hwang and Ramachandran¹⁰⁴⁾. Like PWV, the aortic augmentation index is dependent on distending arterial blood pressure⁽Reference Booth, Wallace and McEniery^105, Reference Dart, Gatzka and Cameron¹⁰⁶⁾, but additional variables, including height⁽Reference Yasmin and Brown¹⁰⁷⁾ and heart rate⁽Reference Wilkinson, MacCallum and Flint¹⁰⁸⁾, must be factored in to its interpretation.

A number of nutrition intervention studies have used pulse contour analysis as an intermediate measure of vascular function. In an acute feeding study, food and water, but not water alone, reduced the aortic augmentation index 2 h after consumption⁽Reference Ahuja, Robertson and Ball¹⁰⁹⁾. This finding is confounded by concomitant decreases in arterial blood pressure and could be argued to reflect mean blood pressure change and therefore altered vessel haemodynamics, rather than an intrinsic alteration of vascular function. After 8 weeks, both low-fat and low-carbohydrate hypoenergetic diets significantly decreased brachial blood pressure among overweight volunteers, but only the former mediated a significant reduction in aortic augmentation⁽Reference Bradley, Spence and Courtney¹¹⁰⁾.

Again, investigators should have a clear hypothesis about the mechanism through which their intervention is likely to alter pulse wave morphology before choosing aortic augmentation as the endpoint. It has been shown that pharmacological manipulation of systemic endothelial NO production significantly changes aortic pressure pulse wave morphology and thus the augmentation index⁽Reference McEniery, Wallace and Mackenzie⁹⁶⁾. In a situation analogous to the intra-brachial infusion of vasodilators and subsequent forearm blood flow measurement, systemic salbutamol (endothelium-dependent) and nitroglycerine (endothelium-independent) are administered with resulting changes in tonometry-derived aortic augmentation quantified to arrive at a ‘global endothelial function index’⁽Reference McEniery, Wallace and Mackenzie⁹⁶⁾. Such an approach may prove more sensitive to altered vascular health than resting measurements of aortic augmentation.

Biochemical measures

Since atherosclerosis is characterised by ongoing vascular inflammation, the acute-phase reactant C-reactive protein (CRP) has been proposed as a useful tool for improving disease prediction models⁽Reference Albert and Ridker¹¹¹⁾. A high-sensitivity assay is used to accurately measure the lower CRP concentrations encountered among healthy individuals. Recent data suggest that statin-mediated reductions in CRP are associated with lower rates of cardiovascular morbidity⁽Reference Ridker, Danielson and Fonseca¹¹²⁾.

The evidence regarding dietary interventions and CRP is equivocal. For example, as summarised in Table 2 ⁽Reference Berry, Mulla and Chowienczyk^113–Reference Watzl, Kulling and Moseneder¹²⁸⁾, only five out of sixteen fruit and vegetable randomised controlled trials (including Mediterranean diet and DASH trials, as fruit and vegetables are key components of these diets) have demonstrated a lowering in CRP levels. The duration of these five studies varied from 4 weeks in three studies, to 3 months in one study and 2 years in another. Of the studies, two were juice-based, one was a carotenoid-rich FV intervention and two were Mediterranean diet interventions. We refer the reader to a review by Giugliano et al. ⁽Reference Giugliano, Ceriello and Esposito¹²⁹⁾ for a broader discussion of the association between diet and inflammation.

Table 2 Effect of fruit and vegetable (FV) interventions on C-reactive protein (CRP): evidence from randomised controlled trials

↔ , No significant change; ↓ , significant decrease; DASH, Dietary Approaches to Stop Hypertension; VBA, vegetables, berries and apples; CAD, coronary artery disease.

* Orange and blackcurrant juice reduced CRP relative to sugar drink.

† CRP decreased only after Mediterranean diet with olive oil.

In mediating leucocytic infiltration of the arterial intima, glycoprotein membrane components such as intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) promote oxidative stress and ongoing arterial inflammation⁽Reference Ferrario and Strawn¹³⁰⁾. Activated endothelial cells are thought to shed these molecules into the circulation, allowing levels of their soluble form to be quantified as an estimate of ongoing arterial injury⁽Reference Hwang, Ballantyne and Sharrett¹³¹⁾. Adipocytes also express ICAM-1, and the soluble form of ICAM-1 is elevated in obese patients, being expressed in the stromal-vascular fraction of adipose tissue⁽Reference Brake, O'Brian Smith and Mersmann¹³²⁾. This is likely to contribute to the link between obesity and inflammatory complications such as atherosclerosis.

Supplementation with isoflavones for 6 weeks has been reported to significantly reduce serum levels of VCAM-1 in healthy subjects⁽Reference Teede, McGrath and DeSilva⁹³⁾, while additional dietary α-linolenic acid had a similar effect among dyslipidaemic male patients⁽Reference Rallidis, Paschos and Papaioannou¹³³⁾. Over a 24-month period, daily consumption of the phyto-oestrogen genistein significantly reduced circulating levels of both ICAM-1 and VCAM-1⁽Reference Atteritano, Marini and Minutoli¹³⁴⁾. While additional cherry consumption for 4 weeks lowered CRP levels among healthy volunteers, it had no effect on ICAM-1 concentrations⁽Reference Kelley, Rasooly and Jacob¹³⁵⁾ and similar negative results have been noted for brief fruit and vegetable interventions⁽Reference Paterson, Gordon and Niwat¹²⁵⁾.

Oxidation both contributes to and follows on from the continuous cycle of low-grade vascular inflammation which characterises atherosclerotic arterial degeneration⁽Reference Chisolm and Steinberg¹³⁶⁾. Markers of systemic oxidative stress in biological fluids have long been suggested as surrogates for vascular injury, and the isoprostanes, derived from non-enzymic arachidonic acid peroxidation, are commonly measured⁽Reference Roberts and Morrow¹³⁷⁾. A dietary intervention study has reported that urinary levels of 8-iso-PGF_2α were significantly reduced among healthy women consuming nine or ten portions of fruit and vegetables daily for 8 weeks⁽Reference Thompson, Heimendinger and Sedlacek¹³⁸⁾. However, investigators have largely reported negative findings when this endpoint has been employed to study the effects of black tea⁽Reference O'Reilly, Mallet and McAnlis¹³⁹⁾, a Mediterranean diet⁽Reference Ambring, Friberg and Axelsen⁶¹⁾, five or six portions of fruit and vegetables daily⁽Reference McCall, McGartland and Woodside^98, Reference Thompson, Heimendinger and Sedlacek¹³⁸⁾ and pure dietary flavonoids⁽Reference Loke, Hodgson and Proudfoot¹⁴⁰⁾.

Other potential biomarkers of the atherosclerotic process include the enzyme lipoprotein-associated phospholipase A₂⁽Reference Zalewski and Macphee^141, Reference Packard, O'Reilly and Caslake¹⁴²⁾, tissue plasminogen activator⁽Reference Thogerson, Jansson and Boman^143, Reference Thompson, Kienast and Pyke¹⁴⁴⁾ and plasminogen activator inhibitor-1⁽Reference Hamsten, de Faire and Walldius¹⁴⁵⁾.

Novel biochemical approaches

The array of biomarkers available and lack of an agreed ‘gold standard’ often prohibits rational study design. To quantify the biological effects of an ‘anti-inflammatory mix’ dietary supplement among obese volunteers, Bakker et al. employed a novel ‘nutrigenomics’ approach⁽Reference Bakker, van Erk and Pellis¹⁴⁶⁾. This involved measurement and integrated analysis of 120 plasma proteins, 274 plasma metabolites and peripheral blood cell transcriptomes. While such a comprehensive approach is labour intensive and statistically complex, it may represent a valuable means by which to define more subtle intervention-related changes.

More recently, circulating endothelial microparticles (EMP), endothelial progenitor cells and endothelial cells have also been used as indices of vascular health.