Clinical aspects of assessment of endothelial function

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1 Pharmacological Reports 2006, 58, suppl., ISSN Copyright 2006 by Institute ofpharmacology Polish Academy ofsciences Review Clinical aspects of assessment of endothelial function Jaros³aw D. Kasprzak, Magdalena K³osiñska, Jaros³aw Dro d Department of Rapid Cardiac Diagnostics, II Chair of Cardiology, Medical University of ódÿ, Bieganski Hospital, Kniaziewicza 1/5, PL ódÿ, Poland Correspondence: Jaros³aw D. Kasprzak, kasprzak@ptkardio.pl Abstract: Prevention and treatment of atherosclerosis is an important priority. Measurement of endothelial function has been reported as a useful tool for atherosclerosis research. Endothelium appear to integrate the injury of commonly known risk factors and others, genetic and environmental as yet undiscovered. The assessment of endothelium-dependent vasodilation has emerged as indicator of endothelial health. Invasive methods examine coronary or brachial artery vasomotion in response to infusion of acetylcholine. Noninvasive ultrasound assessment of flow-mediated vasodilation (FMD) of peripheral artery is more often used. However, despite its wide-spread use, there are technical and interpretive limitations of this technique. Recent studies have shown that the severity of endothelial dysfunction relates to cardiovascular risk. Endothelial function might be also used to monitor the effectiveness of risk reduction therapy. This article will review the growing literature in an effort to evaluate methods of assessment of endothelial function and its clinical utility. Key words: endothelial function, acetylcholine, flow-mediated dilation, atherosclerosis, prognosis Abbreviations: BP blood pressure, CD40L CD40 ligand, CRP c-reactive protein, FMD flow-mediated dilation, ICAM-1 intercellular adhesion molecule 1, IFN interferon, Il-1 interleukin 1, LDL low density lipoprotein, MCP-1 monocyte chemoattractant protein 1, NO nitric oxide, PAD peripheral arterial disease, PAI-1 plasminogen activator inhibitor 1, RH-PAT reactive hyperemia peripheral arterial tonometry, TNF tumor necrosis factor, t-pa tissue plasminogen activator, VCAM-1 vascular cell adhesion molecule 1 Introduction Cardiovascular events are a leading cause of death in most industrialized societies. The underlying disorder in the majority of these cases is atherosclerosis. Prevention of the development of atheroscerotic lesions and their early detection for intensive pharmacology are one of the most important challenges in medicine. There is growing evidence that the earliest manifestation of atherosclerosis is endothelial dysfunction, occuring even in the absence of angiographic evidence of disease [11]. Patient with established endothelial dysfunction in any vascular bed are at risk of experiencing a cardiovascular event [15, 38]. The endothelium is a monolayer of cells located between the blood lumen and the vascular smooth muscle cells. This dynamic endocrine organ plays a critical role in vascular homeostasis by secreting substances that not only regulate vascular tone, plate- Pharmacological Reports, 2006, 57, suppl.,

2 Hypertension Causes and consequences of endothelial dysfunction Diabetes Smoking LDL Homocysteine Estrogen deficiency Oxydative stress / toxicity affecting endothelial cells and vascular smooth muscle Apoptosis ENDOTHELIAL DYSFUNCTION Leukocyte adhesion Lipid deposition markers have been used as indicators of abnormal function of the endothelium, such as E-selectin, ICAM-1, VCAM-1, CD40L, CRP, Il-1, TNF, IFN, MCP-1, von Willebrand factor, t-pa, PAI-1, microalbuminuria and tests of apoptosis [1, 27, 36, 43]. Clinical utility of these measurements is limited, because of their non-specific character. However, they allow insight into multiple facets of endothelial physiology and represent good complement to imaging assessment of endothelial function. The structural integrity of endothelium is a basis of its normal function. Augmented apoptotic rate is possibly involved in endothelial denudation and subsequent dysfunction. There are more than 30 identified inducers of apoptosis, including a net of neurohormones, cytokines and second messengers [19, 25]. Proapoptotic activity of serum can be studied in endothelial cell lines in vitro. The findings cannot be fully extrapolated to in vivo clinical conditions, but they show interaction between the bloodstream and human endothelium. An important characteristics of endothelial dysfunction is decreased synthesis, release and bioavailability of endothelium-derived relaxing factor nitric oxide (NO). It has been widely used as a clinical marker of endothelial function determined by measuring the vasomotor response to physiological and pharmacological stimuli. Acetylcholine via muscarinic endothelial membrane receptors, mediates the release of the NO that counteracts the direct vasoconstrictor effect by mus- Vasoconstriction VSMC growth Thrombosis atherogenesis and complications (stroke, infarction, PAD) Fig. 1. Transition from risk factors to atherosclerosis mediated by endothelial dysfunction let activity and coagulation factors, but also influence vascular inflammation, cell migration and proliferation [31]. Healthy endothelium promotes vasodilation, antithrombosis and antiinflammation. Risk factors for atherosclerosis, such as aging, hypercholesterolemia, hypertension, diabetes, cigarette smoking impair endothelial function [7] (Fig. 1). Dysfunction of endothelial cells is associated with vasoconstriction, inflammation and thrombogenicity, which increase development of atherosclerotic plaques and disposition to rupture. Elevated peripheral resistance intensifies afterload and contributes to tissue hypoperfusion. Because endothelium is also a front line organ for novel, as yet undiscovered damaging agents, endothelial function reflects the overall impact of multiple environmental and genetic factors on the vascular wall (Fig. 1). On the other hand, improvement of endothelial function is one of the earliest clinical markers after effective modification of atherogenic risk factors [23, 44]. For these reasons, assessment of endothelial function could serve as a useful diagnostic and prognostic test. Assessment of endothelial function The pathophysiology of endothelial dysfunction includes abnormal vasomotor function, proinflammatory and prothrombotic state. A number of laboratory 34 Pharmacological Reports, 2006, 57, suppl., 33 40

3 clinical aspects of endothelial function Jaros³aw D. Kasprzak et al. carinic receptors on the smooth muscle layer [42]. Quantitative coronary angiography after intraarterial infusion of acetylcholine is a clinical gold standard for the assessment of endothelial function in coronary arteries. It is a direct test, in situ, relatively operator-independent. Vasoconstriction or even no arterial diameter change after acetylcholine stimulation disclose endothelial dysfunction and indicate hot places, at risk of atherosclerosis [33]. Alternatively, cold pressor test or mental stress test can be applied to elicit sympathetic activation [24, 47], which is a more physiological stimulus for NO release. Increased blood flow due to augmented tissue demand, provokes shear stress, which activates endothelial nitric oxide synthase [10, 22]. Direct invasive assessment of coronary vasomotor response caries a definite risk of myocardial infarction, stroke or death. It should not be performed unless coronary angiography becomes clinically necessary and even at that time is rarely applied in clinical practice. Endothelial dysfunction as a systemic disorder has been demonstrated not only in the epicardial coronary arteries but also in the peripheral conduit vessels [2]. Concordant responses in coronary and peripheral vasculature might allow for less invasive methods of endothelial function testing. We can assess vasodilator response to infused acetylcholine in more accessible vessels such as brachial artery. In this case blood flow measurement by strain-gauge plethysmography is more common than quantitative angiography. However, there is still a need for the catheterization of vessels, with risk of infection, arteries or nerves injury. Non-invasive measurements of vascular disease are more promising methods for wide application in clinical practice. A high resolution ultrasound imaging of brachial artery flow-mediated dilation (FMD) is the most often used test for assessing endothelial function [8]. The primary hemodynamic determinant of FMD is induced wall shear stress, caused by the increased blood flow reactive, following transient ischemia (inflation and deflation of a sphygmomanometer cuff). It is a sensitive and reproducible technique, and a close relationship with coronary vasomotor response to acetylcholine [2]. As a safe and quick method, FMD can be conducted among children and young adults, especially if repeated measurements are required [9]. Despite its attractive character, there are a few technical and interpretive limitations. First of all, the normal values range is not well defined, because of inverse relation between the degree of vasodilation and baseline brachial diameter [9]. Subjects with small size of brachial arteries may have normal arterial dilation, even in the presence of endothelial dysfunction. On the other hand patients with very large brachial arteries appear to show impairment of FMD despite normal endothelial function [2]. This phenomenon precludes comparison of vasodilator responses between individuals with different baseline diameters. Reporting absolute change in diameter will minimize this problem [9]. Mean FMD values vary considerably across studies in similar populations: in coronary heart disease ranging from 1.3 to 14%, in diabetes mellitus from 0.75 to 12%. Even in healthy volunteers the range of reported FMD values from 0.2 to 19.2% [5]. In part, such differences are due to methodological variations, including arterial occlusion times, positioning of the occlusion cuff and the time point of vessel measurement following cuff release [3]. An occlusion duration 4.5 min is minimal to elicit maximal dilator response. Shorter arterial occlusion decreases FMD (mean difference 1.3%) [5]. In addition to occlusion time, cuff position may also determines of the wide variation in FMD values reported. Compared with forearm occlusion, upper arm cuff position elicits greater change in arterial diameter (mean differences in FMD 2.47%) [5]. This may depend on the quantity of downstream resistance vessels involved with each cuff placement. Ulnar collateral arteries, which supply muscles of the forearm are not occluded with cuff placement on the forearm [3]. In this situation subsequent increase in blood flow is not so intense. Direct vasodilator effect of ischemia on the brachial artery during upper arm occlusion is also possibile [9]. Because the peak of FMD occurs later if upper arm occlusion is used (71 s after release of the occlusive cuff) [3], conventional time window for FMD measurement (45 60 s after deflation) is inappropriate and falsifies the obtained measurement. The time of day chosen for FMD measurement is also important. Otto et al. [29] demonstrated that endothelial function is reduced in the early morning, around the time of waking (6 AM), compared with measurements obtained before sleep (9 PM) and later in the day (11 AM) in healthy humans. FMD at 6 AM (4.4%) was markedly decreased, compared with the measurements at 9 PM (7.5%) and 11 AM (7.7%). Specific mechanisms responsible for the morning-related endothelial impairment are unknown, but this phenomenon needs to be recognized in clinical studies. Pharmacological Reports, 2006, 57, suppl.,

4 Even though the technical aspects of ultrasound assessment of brachial FMD in the study are well established [9], placement of calipers using visual inspection as a crucial point of diameter measurement is operator-dependent and susceptible to error. It may be not enough precision for measurement such a fine post-stimulus diameter change, which is generally on the order of 0.3 mm and is even less in the presence of impaired endothelial function [46]. Thus, in order to promote endothelial function testing in clinical practice we need new, easy, operator-independent methods. One of them is reactive hyperemia peripheral arterial tonometry (RH-PAT). It is a non-invasive technique to assess peripheral microvascular endothelial function by measuring changes in digital pulse volume during reactive hyperemia. Normal response is characterized by a distinct increase in the signal amplitude after cuff released, compared with baseline. The RH-PAT data are analyzed by a computer in an operator-independent manner. As previously demonstrated [4], digital hyperemic response is attenuated in patients with established coronary endothelial dysfunction. This suggests a role for RH-PAT as a noninvasive, bedside test to identify patients with this disorder. Prognostic value of endothelial dysfunction Endothelial dysfunction, an early marker of vascular pathology, might represent a useful prognostic indicator. A study in 157 patients with nonobstructive coronary artery disease (lesions < 40% lumen diameter) found that severe coronary endothelial dysfunction, defined as the lowest tertile of coronary responses to acetylcholine, significantly increased the risk of cardiac events, during 28 months of follow up [35]. In contrast, patients with mild dysfunction or normal endothelial function experienced no cardiac events. This severe endothelial dysfunction in even mildly diseased coronary arteries predicts adverse long-term outcomes. Intravascular ultrasound parameters assessing coronary atherosclerosis in this study were not helpful in predicting cardiac events. Evidence from subsequent studies reinforced the concept that coronary endothelial function may be a useful prognostic indicator. Schächinger et al. [33] assessed coronary vasoreactivity in 147 patients (57% with angiographic evidence of atherosclerosis) using 3 different stimuli: acetylcholine, cold pressor testing and increased blood flow. During a follow-up period of about 8 years, the authors showed that responses to each of these tests independently predicted of cardiovascular events. Predictive power of endothelial dysfunction was not limited to a specific method of assessment. Importantly, coronary endothelial dysfunction remained significant, even after adjustment for traditional cardiovascular risk factors or angiographic evidence of coronary atherosclerosis. Even if atherosclerotic lesions are present, coronary endothelial function is still important. In an larger study, Halcox et al. [15] monitored 308 patients after intracoronary acetylcholine test, with mean duration of follow-up 46 months. 132 patients had medically managed coronary artery disease of mild to moderate severity and 176 had angiographically smooth coronary arteries. Epicardial and microvascular coronary endothelial dysfunction independently predicted cardiovascular events in patients with and without coronary artery disease. When only unpredictable acute cardiovascular events, including death, myocardial infarction, stroke and unstable angina were analysed, event-free survival was also significantly related to endothelial function. This study provides next important clinical lesson: angiographically smooth epicardial coronary arteries can be associated with endothelial dysfunction, which predicts a worse long-term outcome in this low-risk population. This finding was confirmed in later investigation [34]. Epicardial coronary vasoreactivity in response to cold pressor test was assessed in 130 patients with normal coronary angiograms. Over a mean follow-up period of 45 months cardiovascular events occurred only in patients with impaired vasoreactivity (absent vasodilation or vasoconstriction) in response to sympathetic stimulation. After adjustment for known risk factors for coronary artery disease, abnormal vasoreactivity remained significant as predictor of worse outcome. The incidence of cardiovascular events increased significantly with higher vasoconstrictor response. Not only the presence of endothelial dysfunction was important, but also the degree of this impairment. This observation is in keeping with the result of previous study [35]. Assessment of peripheral vascular function may also provide prognostic variables in cardiovascular diseases [37]. Previous clinical studies have demonstrated that peripheral endothelium-dependent vasodi- 36 Pharmacological Reports, 2006, 57, suppl., 33 40

5 clinical aspects of endothelial function Jaros³aw D. Kasprzak et al. Tab. 1. Significance of endothelial function assessment: a clinician s viewpoint 1. Endothelial function integrates the influences of numerous risk factors (hereditary, environmental, lifestyle-related) 2. Endothelial dysfunction precedes morphologic changes in vascular system and clinical manifestation of cardiovascular disease indicating need for early and effective prevention 3. Endothelial dysfunction is reversible by healthy lifestyle change or medical therapy 4. Noninvasive assessment of endothelial dysfunction provides independent prognostic information in various cardiovascular conditions lation, measured in response to acetylcholine or as flow-mediated dilation, has independent prognostic implications. Heitzer et al. [16] evaluated forearm blood flow in response to acetylcholine in 276 patients with angiographically documented coronary artery disease. Patients who experienced cardiovascular events over a mean follow-up of 4.5 years where characterized by impaired endothelial vasodilator response. However, the majority of events in this study were revascularization procedures, which, unlike spontaneous cardiovascular events are more likely to be influenced by non-biological factors. In another study [26] 73 patients who underwent cardiac catheterization due to chest pain and the assessment of brachial artery FMD by ultrasound, were observed over a mean of 5 years. Cardiovascular events occurred more often in patients with impaired (< 10%) versus preserved (> 10%) FMD. The issue is limited by relatively small sample size, heterogeneous mix of stable and unstable patients and dominance of revascularization procedures as adverse outcome. Reduced brachial artery vasoreactivity was demonstrated as an independent predictor for increased cardiovascular risk in patients with peripheral arterial disease (PAD) of the lower limbs [6]. In this study 131 patients with intermittent claudication after measurement of the FMD and the ankle-brachial pressure index were observed for a mean 23 months. Median FMD was significantly lower in patients with an event (5.8%) than in those without (7.6%). More importantly, measurement of brachial artery FMD improved the prognostic value of ankle-brachial pressure index, which is the most powerful prognostic indicator in PAD. Therefore, FMD may be particularly useful to identify a subgroup of PAD patients at very high risk. In addition to the studies that examined the predictive value of peripheral endothelial function in established atherosclerosis, Perticone et al. [30] assessed the forearm blood flow in response to acetylcholine in subjects with risk factors, but no known atherosclerosis. There were 225 untreated hypertensive patients, without history of angina or myocardial infarction. During a mean follow-up of 31.5 months patients with blunted forearm microvascular response were more likely to experience cardiovascular events. Endothelial dysfunction has been demonstrated in coronary and peripheral arteries in patients with chronic heart failure [20, 41]. Tentolouris et al. [39] showed that forearm vasodilatatory response to reactive hyperemia was significantly lower in ischemic and dilated cardiomyopathy, compared to patients with coronary artery disease and normal left ventricular function. Endothelial dysfunction found in both types of heart failure, was more pronounced in patients with ischemic type than in dilated cardiomyopathy. Underlying atherosclerosis can be responsible for this difference. Peripheral vasoconstriction, increasing afterload and myocardial microcirculation abnormalities, may contribute to augmented cardiac damage and accelerated progression of heart failure. 67 patients with heart failure (different etiology) were observed during a median follow-up of 45.7 months [13]. As expected, a significantly better clinical outcome was in patients with FMD of the radial artery above the median (6.2%), compared with those with FMD below this value. A number of interventions have been shown to restore endothelial function measured a flow-dependent vasodilation. Many of these same interventions are known to limit cardiovascular risk. First of all, there is a strong and consistent evidence for beneficial effects of lipid-lowering therapy by statins [12, 40]. Treatement with statins significantly improves endothelial function, even before appreciable reduction in plasma LDL cholesterol. Lipid-independent improvement of endothelial function was observed after one day of treatment [21] and increased during this therapy [28]. This phenomenon can be responsible for exceeding benefits, that would be expected from the observed reduction in cholesterol in large trials with statins [32]. Pharmacological Reports, 2006, 57, suppl.,

6 In hypertension, reduction of blood pressure (BP) per se does not seem to restore endothelial function. Modena et al. [23] examined 400 postmenopausal women with newly diagnosed hypertension and impaired brachial artery FMD ( 10%). Patients had increased risk of cardiovascular events over the next five years, when endothelial dysfunction was not reversed (FMD 10%) by six months of antihypertensive therapy, while optimal control of blood pressure was achieved (BP < 140/90 mmhg). The type of drugs and degree of BP lowering did not explain the difference in prognosis. However, the effect of antihypertensive agents on endothelial function is different. It has been shown [17], that in patients with essential hypertension, forearm blood flow during reactive hyperemia was significantly greater in the angiotensinconverting enzyme inhibitor-treated group than in the group treated with calcium antagonists, -blockers or diuretic agents. BP did not differ with respect to the antihypertensive agent used. Reactive hyperemia was similar in the calcium antagonist, -blockers, diuretic and untreated groups, although BP was significantly less in treated groups. A Medline search shows that, every year several hundred articles are published on the role of different therapeutic interventions aimed at influencing endothelial function. Those data reveal the enormous interest in the endothelium as the new target for cardiovascular therapeutics. Nevertheless, the clinician is principally interested in the prognostic role of reversible endothelial dysfunction. Studies in postmenopausal women have repeatedly shown that hormone replacement therapy improves endothelial function [14]. Despite the apparent beneficial effects on endothelial function, clinical trials have failed to show a benefit from hormone replacement therapy [18, 45]. 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