Examining endothelial function in humans in vivo: improving guidelines and exploring novel measures

Chapter 1 introduced the old and new measurement of endothelial function, and vascular health. The first part of this thesis focussed on the “old” flow-mediated dilation measurement, introduced in 1992, which relies on brachial artery vasodilation after a hyperaemic stimulus (e.g. after occlusion of a pneumatic cuff), and the susceptibility of the FMD measurement to variability. In Chapter 2 we sought for factors that might help to improve the reproducibility of the FMD measurement. We performed an analysis including 672 participants with repeated FMD. Overall we found an acceptable reproducibility, with 33% of the FMD measurements showing an excellent-to-moderate reproducibility. We identified several factors that independently increased the variation of the FMD, including the presence of hypertension, a lower resting FMD%, a larger baseline artery diameter, a longer time between subsequent measurements, and less laboratory experience with the measurement. Future studies should take these factors into consideration, as certain measures may lower variability of the FMD or more subjects should be included in individual studies when variation relates to non-modifiable factors. Unfortunately, we also found that a large proportion of our study population demonstrated a moderate-to-poor reproducibility, despite all included studies adhered to expert-consensus guidelines. In Chapter 3, we described the relation between adherence to the expertconsensus guidelines and reproducibility of the FMD. In this meta-analysis, we combined data from twenty-seven studies, comprising 48 study groups, with a total of 1537 subjects. Adherence to expert guidelines was inversely related to the measurement error and adopting the guidelines (with specific notification of the use of a stereotactic probe-holder, continuous diameter recording and the use of automated wall-detection and analysis software) was crucial for improving the reproducibility of the FMD. The second part of this thesis introduced the “new” carotid artery reactivity (CAR) measurement, which depends on carotid artery vasomotor responses following sympathetic stimulation (e.g. after a cold pressor test, CPT). The carotid artery appears to mirror coronary responses to CPT, as it shows dilation in healthy participants, whilst those at risk demonstrate a constriction. In this thesis, we sought to understand the relation of CAR with risk factors, and the similarity with the coronary responses. An attempt was made to unravel the underlying physiological mechanism of CAR. Finally, the prognostic value of CAR was examined in peripheral arterial disease patients, to further investigate to clinical potential of the CAR test. In Chapter 4 we explored the relation of CAR with cardiovascular risk, followed by assessing the similarity in response to sympathetic stimulation between the carotid artery and coronary arteries. We first compared CAR between 50 young and 44 older participants to assess relationships between CAR and traditional cardiovascular risk factors. We found that CAR was lower in participants with ≥2 risk factors, compared to those with lesser risk factors. Secondly, we compared left anterior descending (LAD) artery velocity with carotid artery diameter in a subgroup of 33 participants, to assess similarity between coronary and carotid artery responses. We found that CAR correlated well with coronary artery velocity. This implies that CAR is related to increased CV risk and may represent a surrogate measure for coronary vascular health. In Chapter 5, the physiological mechanism underlying the CAR was further explored, by examining carotid artery responses to different sympathetic stimuli (e.g. the cold pressor test [CPT] and the lower body negative pressure test [LBNP]), exploring the role of α1-receptors, (nor) epinephrine receptors contributing to vasoconstriction, and assessing similarity between carotid and coronary arteries. First, 10 participants underwent both sympathetic tests in randomized order, whilst concurrently measuring CAR and coronary artery velocity. We found distinct carotid artery responses to different tests of sympathetic stimulation (e.g. dilation in response to CPT, and constriction following LBNP). Second, when measurements were repeated following α1-receptor blockade by Prazosin, we found α1-receptors partly contributed to CPT-induced responses. Finally, we found agreement between carotid and coronary artery responses, during both types of sympathetic nerve stimulation as well as during α1-receptor blockade. These data indicate strong similarity between carotid and coronary responses to sympathetic tests and the role of α1-receptors. Since the CAR test was newly introduced, the prognostic value of the test remained unknown and this question is highly relevant to understand its clinical utility. Therefore, in Chapter 6, we examined whether CAR predicts (cardiovascular) events in patients with peripheral arterial disease. A total of 172 PAD patients were included, and we recorded cardiac and cerebrovascular events, mortality and clinical progression to percutaneous transluminal angioplasty or loss of patency during a 12-months follow-up. We found that patients with carotid constriction showed a four-fold higher risk for cardiovascular events and two-fold increased risk for clinical deterioration, even after adjustment for other risk factors. This indicates that CAR provides a simple, novel strategy to predict CV events and progression in PAD patients, which has stronger prognostic value than current techniques. Chapter 7 summarizes, discusses, and explains the findings of these studies, and aims to provide recommendations and implications. We discuss future prospects, and provide perspective for future vascular health assessment

Assessing the perceived quality of brachial artery Flow Mediated Dilation studies for inclusion in meta-analyses and systematic reviews: Description of data employed in the development of a scoring tool based on currently accepted guidelines

Brachial artery Flow Mediated Dilation (FMD) is widely used as a non-invasive measure of endothelial function. Adherence to expert consensus guidelines on FMD measurement has been found to be of vital importance to obtain reproducible data. This article lists the literature data which was considered in the development of a tool to aid in the objective judgement of the extent to which published studies adhered to expert guidelines for FMD measurement. Application of this tool in a systematic review of FMD studies © 2016 . (http://dx.doi.org/10.1016/j.atherosclerosis.2016.03.011) (Greyling et al., 2016 [1]) indicated that adherence to expert consensus guidelines is strongly correlated to the reproducibility of FMD data

Carotid Artery Reactivity Predicts Events in Peripheral Arterial Disease Patients.

OBJECTIVE: Patients with peripheral arterial disease (PAD) have increased risk on future cerebro- and cardiovascular events. Our aim was to examine whether carotid artery reactivity (CAR; a novel, simple procedure to examine endothelial function) predicts cardiovascular events in PAD patients. BACKGROUND: Increased risk for future cardiovascular events in PAD patients is likely related to endothelial dysfunction, highlighting the necessity for simple assessment of endothelial function. METHODS: A total of 172 PAD patients (68 ± 10 years, 67% male) underwent the CAR, which involves ultrasound measurement of carotid artery diameter during sympathetic stimulation produced by 90-second hand immersion in 4°C ice-water (ie, cold pressor test). CAR-responses were dichotomized into carotid constriction or dilation. We recorded cardiac and cerebrovascular events, mortality, and clinical progression to percutaneous transluminal angioplasty or loss of patency during 12-month follow-up. RESULTS: Eighty-two PAD patients demonstrated carotid constriction and 90 patients demonstrated dilation. PAD patients with carotid constriction showed more cardiovascular events compared to patients with dilation (Kaplan-Meier Log rank; P < 0.05). Cox proportional hazard models showed that patients with carotid constriction continued to show higher risk for cardiovascular events [hazard ratio: 4.1; 95% confidence interval (CI), 1.3-12.5] and clinical progression (hazard ratio: 2.0; 95% CI, 1.2-3.3), even after adjustment for other risk factors. Ankle brachial pressure index and carotid intima-medial thickness alone did not predict (cardiovascular) event or improve risk assessment beyond that provided by CAR. CONCLUSION: Carotid vasoconstriction identifies PAD patients with a 4-fold increased risk for future cardiovascular events and 2-fold increased risk for clinical deterioration. CAR provides a simple, novel strategy to predict cardiovascular events and progression in PAD patients. CLINICAL TRIAL REGISTRATION: www.trialregister.nl/trialreg/index.asp, NTR-4117

Correlation of carotid artery reactivity with cardiovascular risk factors and coronary artery vasodilator responses in asymptomatic, healthy volunteers.

OBJECTIVES: Carotid artery reactivity (CAR%), involving carotid artery diameter responses to a cold pressor test (CPT), is a noninvasive measure of conduit artery function in humans. This study examined the impact of age and cardiovascular risk factors on the CAR% and the relationship between CAR% and coronary artery vasodilator responses to the CPT. METHODS: Ultrasound was used to measure resting and peak carotid artery diameters during the CPT, with CAR% being calculated as the relative change from baseline (%). We compared CAR% between young (n = 50, 24 ± 3 years) and older participants (n = 44, 61 ± 8 years), and subsequently assessed relationships between CAR% and traditional cardiovascular risk factors in 50 participants (44 ± 21 years). Subsequently, we compared left anterior descending (LAD) artery velocity (using transthoracic Doppler) with carotid artery diameter (i.e. CAR%) during the CPT (n = 33, 37 ± 17 years). RESULTS: A significantly larger CAR% was found in young versus older healthy participants (4.1 ± 3.7 versus 1.8 ± 2.6, P < 0.001). Participants without cardiovascular risk factors demonstrated a higher CAR% than those with at least two risk factors (2.9 ± 2.9 versus 0.5 ± 2.9, P = 0.019). Carotid artery diameter and LAD velocity increased during CPT (P < 0.001). Carotid diameter and change in velocity correlated with LAD velocity (r = 0.486 and 0.402, P < 0.004 and 0.02, respectively). CONCLUSION: Older age and cardiovascular risk factors are related to lower CAR%, while CAR% shows good correlation with coronary artery responses to the CPT. Therefore, CAR% may represent a valuable technique to assess cardiovascular risk, while CAR% seems to reflect coronary artery vasodilator function

Plasma levels of the cardiovascular protective endogenous nucleoside adenosine are reduced in patients with primary aldosteronism without affecting ischaemia-reperfusion injury: A prospective case-control study.

BACKGROUND: Patients with primary aldosteronism (PA) experience more cardiovascular events compared to patients with essential hypertension (EHT), independent from blood pressure levels. In animals, mineralocorticoid receptor antagonists limit ischaemia-reperfusion (IR) injury by increasing extracellular adenosine formation and adenosine receptor stimulation. Adenosine is an endogenous compound with profound cardiovascular protective effects. Firstly, we hypothesized that patients with PA have lower circulating adenosine levels which might contribute to the observed increased cardiovascular risk. Secondly, we hypothesized that by this mechanism, patients with PA are more susceptible to IR compared to patients with EHT. DESIGN: In our prospective study in 20 patients with PA and 20 patients with EHT, circulating adenosine was measured using a pharmacological blocker solution that halts adenosine metabolism after blood drawing. Brachial artery flow-mediated dilation (FMD) before and after forearm IR was used as a well-established method to study IR injury. RESULTS: Patients with PA had a 33% lower adenosine level compared to patients with EHT (15.3 [13.3-20.4] vs 22.7 [19.4-36.8] nmol/L, respectively, P < .01). The reduction in FMD after IR, however, did not differ between patients with PA and patients with EHT (-1.0 ± 2.9% vs -1.6 ± 1.6%, respectively, P = .52). CONCLUSIONS: As adenosine receptor stimulation induces various powerful protective cardiovascular effects, its lower concentration in patients with PA might be an important novel mechanism that contributes to their increased cardiovascular risk. We suggest that modulation of the adenosine metabolism is an exciting novel pharmacological opportunity to limit cardiovascular risk in patients with PA that needs further exploration

Expert consensus and evidence-based recommendations for the assessment of flow-mediated dilation in humans.

Endothelial dysfunction is involved in the development of atherosclerosis, which precedes asymptomatic structural vascular alterations as well as clinical manifestations of cardiovascular disease (CVD). Endothelial function can be assessed non-invasively using the flow-mediated dilation (FMD) technique. Flow-mediated dilation represents an endothelium-dependent, largely nitric oxide (NO)-mediated dilatation of conduit arteries in response to an imposed increase in blood flow and shear stress. Flow-mediated dilation is affected by cardiovascular (CV) risk factors, relates to coronary artery endothelial function, and independently predicts CVD outcome. Accordingly, FMD is a tool for examining the pathophysiology of CVD and possibly identifying subjects at increased risk for future CV events. Moreover, it has merit in examining the acute and long-term impact of physiological and pharmacological interventions in humans. Despite concerns about its reproducibility, the available evidence shows that highly reliable FMD measurements can be achieved when specialized laboratories follow standardized protocols. For this purpose, updated expert consensus guidelines for the performance of FMD are presented, which are based on critical appraisal of novel technical approaches, development of analysis software, and studies exploring the physiological principles underlying the technique. Uniformity in FMD performance will (i) improve comparability between studies, (ii) contribute to construction of reference values, and (iii) offer an easy accessible and early marker of atherosclerosis that could complement clinical symptoms of structural arterial disease and facilitate early diagnosis and prediction of CVD outcomes

Traditional and Nontraditional Cardiovascular Risk Factors in Active Octogenarians Who Develop Cardiovascular Events.

Cardiovascular disease (CVD) is highly prevalent and contributes to disease burden and mortality in older adults. 1 The American Heart Association concluded that CVD is the leading cause of death in octogenarians, followed by cancer and Alzheimer’s disease. 1 Studies questioned the validity of traditional CVD risk factors to predict CVD-related events in older adults. 2 ,3 Therefore, we explored differences in nontraditional risk factors between octogenarians with vs without cardiovascular events across a 4.5-year follow-up

Expert consensus and evidence-based recommendations for the assessment of flow-mediated dilation in humans

Endothelial dysfunction is involved in the development of atherosclerosis, which precedes asymptomatic structural vascular alterations as well as clinical manifestations of cardiovascular disease (CVD). Endothelial function can be assessed non-invasively using the flow-mediated dilation (FMD) technique. Flow-mediated dilation represents an endothelium-dependent, largely nitric oxide (NO)-mediated dilatation of conduit arteries in response to an imposed increase in blood flow and shear stress. Flow-mediated dilation is affected by cardiovascular (CV) risk factors, relates to coronary artery endothelial function, and independently predicts CVD outcome. Accordingly, FMD is a tool for examining the pathophysiology of CVD and possibly identifying subjects at increased risk for future CV events. Moreover, it has merit in examining the acute and long-term impact of physiological and pharmacological interventions in humans. Despite concerns about its reproducibility, the available evidence shows that highly reliable FMD measurements can be achieved when specialized laboratories follow standardized protocols. For this purpose, updated expert consensus guidelines for the performance of FMD are presented, which are based on critical appraisal of novel technical approaches, development of analysis software, and studies exploring the physiological principles underlying the technique. Uniformity in FMD performance will (i) improve comparability between studies, (ii) contribute to construction of reference values, and (iii) offer an easy accessible and early marker of atherosclerosis that could complement clinical symptoms of structural arterial disease and facilitate early diagnosis and prediction of CVD outcomes