Pulse pressure is a predictor of vascular endothelial function in middle-aged subjects with no apparent heart disease

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1 Pulse pressure is a predictor of vascular endothelial function in middle-aged subjects with no apparent heart disease Vascular Medicine 15(4) The Author(s) 2010 Reprints and permission: sagepub. co.uk/journalspermissions.nav DOI: / X Roy Beigel 1,2, Danny Dvir 2,3, Yaron Arbel 2,4, Alon Shechter 1,2, Micha S Feinberg 1,2 and Michael Shechter 1,2 Abstract Elevated pulse pressure (PP) is increasingly being recognized as a cardiovascular risk factor. To investigate whether PP is associated with endothelial function in subjects with no apparent heart disease we prospectively assessed brachial flowmediated dilation (FMD) in 525 consecutive subjects with no apparent heart disease [323 (61%) men, mean age 52 ± 11 years, mean body mass index (BMI) 26 ± 4 kg/m 2 ]. Following an overnight fast and discontinuation of all medications for 12 hours, the FMD and endothelium-independent, nitroglycerin-mediated vasodilation (NTG) were assessed using highresolution linear array ultrasound. Univariate linear analysis revealed a significant inverse association between FMD and PP (r = 0.65, p < 0.01), systolic blood pressure (r = 0.52, p < 0.01) and age (r = 0.21, p < 0.05). Multivariate analysis showed that PP was the strongest independent predictor of FMD. We therefore divided the study population into two groups: group A (n = 290) the median PP, and group B (n = 235) > the median PP of 50 mmhg. Male sex, hypertension, diabetes, BMI, heart rate, and the use of aspirin, long-acting nitrates, calcium channel blockers, angiotensin-converting enzyme inhibitors and beta blockers were significantly more common in Group B compared with Group A. FMD but not NTG was significantly greater in patients with PP the median PP, compared with > the median PP (14.9 ± 7.9% vs 10.8 ± 8.8%, p < and 16.1 ± 9.6% vs 14.8 ± 8.4%, p = 0.38; respectively). Thus, PP is inversely associated with brachial FMD in middle-aged subjects with no apparent heart disease, suggesting a potential mechanism whereby elevated PP contributes to cardiovascular disease. Long-term follow-up is warranted to elucidate the incidence of coronary artery disease in both study groups. Keywords atherosclerosis; coronary artery disease; endothelial function; hypertension; prognosis; pulse pressure Introduction Pulse pressure (PP), defined as the difference between systolic and diastolic blood pressure, represents a well-established independent predictor for cardiovascular morbidity and mortality. 1,2 A possible explanation for the association between PP and adverse cardiovascular outcomes may be provided by the concept of bi-directionality: increased PP is both a cause and a consequence of atherosclerosis. 3 The vascular endothelium is a large paracrine organ which plays a critical role in vascular homeostasis by secreting several mediators regulating vessel tone and diameter, coagulation factors, vascular inflammation, cell proliferation and migration, platelet and leukocyte interaction, and activity and thrombus formation. 4,5 Endothelial dysfunction is therefore recognized as a major factor in the development of atherosclerosis, 6,7 hypertension 8 and heart failure. 9 Flow-mediated dilation (FMD) of the brachial artery by ultrasound is widely used as a measure of endothelial function. 5 Vascular endothelial dysfunction is an independent risk factor for cardiovascular events, and provides important prognostic data in addition to the classic cardiovascular risk factors. 10,11 Recent studies have shown an inverse association between PP elevation and endothelial dysfunction 1 Leviev Heart Center, Chaim Sheba Medical Center, Tel Hashomer, Israel 2 Sackler Faculty of Medicine, Tel Aviv University, Ramat Aviv, Israel 3 Department of Cardiology, Rabin Medical Center, Petach Tiqva, Israel 4 Department of Internal Medicine D, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel Corresponding author: Michael Shechter Leviev Heart Center, Chaim Sheba Medical Center Tel Hashomer Israel Fax: shechtes@netvision.net.il Part of this study was presented as an abstract at the 57th Annual Scientific Session of the American College of Cardiology, Chicago, IL, USA, April 2008.

2 300 Vascular Medicine 15(4) assessed by acetylcholine reactivity in intact carotid arteries of rabbits, 12 and naïve hypertensive patients. 13 Thus, the aim of the current study was to detect the association of peripheral vascular endothelial function assessed by brachial artery FMD and PP in middle-aged subjects with no apparent heart disease. Methods Study design and population We prospectively recruited 525 consecutive subjects from the Endothelial Function Assessment Laboratory of the Leviev Heart Center at the Chaim Sheba Medical Center. All patients were referred either by primary care physicians or through direct patient request for risk factor evaluation via a primary prevention clinic. No patient was referred due to chest pain. Primary care physicians send patients to our laboratory for coronary artery disease (CAD) assessment (low versus high) to facilitate treatment management decisions of patients with risk factors for CAD. All patients undergo a full consultation by a cardiologist who performs lipid-lowering therapy testing according to the updated National Cholesterol Education Program (NCEP) ATP III, and all-risk factor management according to the AHA/ ACC/ESC guidelines. 14 A detailed letter is given to both the patient and the treating physician. Inclusion criteria included subjects without known cardiovascular disease (i.e. no history of angina pectoris, myocardial infarction, coronary artery bypass grafting, coronary angiography with angioplasty and/or stenting, or any lesion > 50% of the coronary artery luminal diameter, cerebrovascular accident, or peripheral vascular disease) with normal electrocardiograms and echocardiography. Exclusion criteria included atrial fibrillation, sinus bradycardia (heart rate < 50 bpm) without pacemaker, sick sinus syndrome, second or third degree AV block, intolerance to nitrates, renal failure with serum creatinine 2 mg/dl, history of drug or alcohol abuse, chronic liver disease, or refusal to sign the informed consent form. Cardiac risk factors assessed included age, sex, tobacco smoking, diabetes, hypercholesterolemia, hypertension, and obesity [body mass index (BMI) > 30 kg/ m 2 ]. Smokers included subjects with a current or prior history of tobacco use. Diabetes was defined as a fasting blood glucose level of > 126 mg/dl or treatment with dietary modification or oral hypoglycemic agents at the time of the study. Hypercholesterolemia was defined as fasting serum total cholesterol > 200 mg/dl or if the subject was being treated with lipid-lowering medication or dietary modification. Hypertension was defined as a seated systolic blood pressure > 140 mmhg or diastolic > 90 mmhg on at least three occasions, or if such a diagnosis had been made in the past and the patient was being treated with medications or lifestyle modification. BMI was calculated as weight (kg) divided by height (m 2 ). Following an overnight fast and the discontinuation of all medications for 12 hours, a physical examination, brachial artery reactivity testing, and blood tests for measurements of lipids, blood cell count, electrolytes, fasting glucose, homocysteine, and high-sensitivity C-reactive protein (hs- CRP), were performed. Blood samples were centrifuged immediately for 15 minutes at 3000/min. The serums were stored at 20 C. All blood samples were tested at the end of the study in the same laboratory and by the same operator who was blinded to the patients clinical status and endothelial function results. The hospital review board approved the study, and all participants gave written informed consent. Vascular function protocol Endothelial function in the form of endothelium-dependent FMD in the brachial artery was measured as previously described. 5,15 21 Briefly, FMD was assessed in the subject s right arm in the recumbent position in a temperaturecontrolled room (22 C) after a 10-minute equilibration period by a single ultrasonographer blinded to the subject s clinical status. Using a 15-6 MHz linear array (15-6L HP) ultrasound (HP SON0S 7500 CV system; Agilent Technologies Inc., Andover, MA, USA), the brachial artery was longitudinally imaged approximately 5 cm proximal to the antecubital crease. An electrocardiogram (ECG) was monitored continuously and blood pressure was taken in the left arm at every minute throughout the study. Study phases Endothelium-dependent FMD Following a 2-minute baseline period, a longitudinal image of 3 cm of vessel without color flow was obtained and frozen for 5 seconds. The image was then unfrozen and switched to a pulse wave Doppler for 5 seconds at a sweep speed of 50 mm/second. A pneumatic tourniquet (Hokanson AG101; Bellevue, WA, USA), placed around the left upper arm proximal to the target artery (upper-arm occlusion), was inflated after the baseline phase to 50 mmhg above the subject s systolic blood pressure (or until no blood flow was observed in the brachial artery by Doppler probe), and held for 5 minutes. Increased flow ( hyperemic state ) was subsequently induced by sudden cuff deflation, followed by a continuous scan at deflation, 60 and 90 seconds, with frozen and Doppler measurements recorded at similar intervals from baseline. Nitroglycerin (NTG)-induced (non-endothelium-dependent) vasodilation A second 2-minute baseline resting scan was recorded to confirm vessel recovery 13 minutes after cuff deflation. Scanning was performed continuously for 5 minutes following administration of a sublingual NTG tablet (Nitrostat, 0.4 mg; Park-Davis). Data analysis Ultrasound images were recorded on an S-VHS videotape with an SLV-RS7 videocassette recorder (SONY). The brachial artery diameter was measured from the anterior to the posterior interface between the media and adventitia ( m line ) at a fixed distance. The mean diameter was calculated from four cardiac cycles synchronized with the R-wave peaks on the ECG. All measurements were calculated at end

3 Beigel R et al. 301 diastole to avoid possible errors resulting from variable arterial compliance. The internal diameter was calculated with PC Prosound software (USC; Los Angeles, CA, USA) using a Horita Data Translation Image Processing board (DT Hz; Mission Viejo, CA, USA). 15 The diameter percent change caused by endothelium-dependent FMD (%FMD), and endothelium-independent percent change from baseline in NTG-mediated vasodilation (%NTG) were expressed as the percent change relative to that at the initial resting scan. %FMD and %NTG were computed from the formula [(maximum diameter baseline diameter)/baseline diameter 100]. %FMD, measured 1:30 minutes after cuff deflation, was used as an index of endothelium-dependent dilation; percent dilation obtained 5 minutes after the administration of nitroglycerin represented %NTG. FMD and NTG vasodilatory response measurements were carried out in a blind fashion. The intra-observer correlation coefficients for baseline and deflation diameters were The absolute error between measurements ranged from 0 to 0.12 mm (for brachial artery diameter) and 0.02% to 2.98% (for FMD). The determination of endothelial function was performed in accordance with published guidelines. 5 Blood pressure measurements Following a 5-minute quiet rest period, readings of clinical blood pressure were obtained from the left arm of the supine patients with a mercury sphygmomanometer. Systolic and diastolic blood pressure were recorded at the first appearance (phase I) and disappearance (phase V) of Korotkoff sounds. Each blood pressure was measured three times, 5 minutes apart, and the values provided are the mean of the three measurements. Echocardiographic assessment All patients underwent 2-dimensional echocardiographic assessment within 30 days of the endothelial function assessment by the same senior cardiologist (M.S.F.) who was blinded to the patients clinical status and endothelial function results. Echocardiographic data included left ventricular dimensions and volumes, left atrial dimension and volume (biplane modified Simpson s), left ventricular ejection fraction (LVEF) (biplane modified Simpson s), global and regional wall motion, and valve anatomy and function. Statistical analysis Group data are expressed as mean ± SD. Differences between clinical characteristics and brachial artery vasodilator responses were evaluated and analyzed between the two study groups ( and > the median pulse pressure of 50 mmhg) by independent t-tests. Comparison of biochemical measurements was performed using independent Student s t-test and the Wilcoxon signed-rank test. All the clinical and laboratory data were analyzed using SPSS software (version 17). The chi-square test was used to test the significance of differences between the groups in categorical variables. The effects of independent predictors on %FMD were evaluated by univariate analyses and, successively, by a stepwise multiple linear regression with forward selection. Initially, we tested a baseline model by using the following variables: BMI, age, sex, serum glucose, serum cholesterol, serum triglycerides, family history of CAD, concomitant medications, and smoking. Subsequently, the model was improved by entering, one at a time, the resting heart rate, systolic blood pressure, diastolic blood pressure, mean blood pressure, and PP. Statistical significance was determined by a p-value of < Results The study population comprised 525 middle-aged volunteers [mean age of 52 ± 11 years, of whom 323 (61%) were men], without known CAD at relatively low CAD risk with an estimated Framingham 10-year risk of 7.8 ± 2.1%. Thirty percent of the study cohort had hypertension (25% received anti-hypertension therapy), 38% hypercholesterolemia, 40% a family history of CAD, 8% type 2 diabetes mellitus and 13% were current smokers. The mean BMI was 26 ± 4 kg/m 2, LVEF 60 ± 3%, mean systolic, diastolic, PP and mean arterial blood pressures were 130 ± 20, 80 ± 40, 58 ± 13 and 99 ± 10 mmhg, respectively. Lipoprotein values of all participants were within the NCEP ATP III guidelines. Endothelial function and pulse pressure The mean %FMD of the study population was 12.2 ± 3.2% and NTG was 15.5 ± 3.5%. Univariate analysis used to assess the effects of various covariates on %FMD revealed a significant inverse correlation between %FMD and PP (r = 0.65, p < 0.01) (Figure 1), systolic blood pressure (r = 0.52, p < 0.01) and age (r = 0.21, p < 0.05). Following adjustment in a multivariate stepwise linear regression model, PP was the best predictor of %FMD (R 2 = 0.26, p < 0.008), followed by systolic blood pressure (R 2 = 0.18, p < 0.02) and age (R 2 = 0.12, p < 0.04). PP remained the strongest independent predictor of % FMD r = 0.65; p < 0.01 n = Pulse Pressure (mmhg) Figure 1. Correlation between pulse pressure and flowmediated dilation (%FMD) of the study cohort (n = 525). It should be noted that there is a strong digital bias regarding the blood pressure measurement.

4 302 Vascular Medicine 15(4) %FMD, even when the number of data points above a PP of 80 mmhg was excluded, or when hypertensive patients were excluded (R 2 = 0.10, p = 0.02). The study cohort median PP was 50 mmhg. On the basis of these findings, subjects were divided into two groups: group A (n = 290) equal or below, and group B (n = 235) above the median PP of 50 mmhg. Baseline characteristics of the two groups are presented in Tables 1 and 2. Male sex, hypertension, type 2 diabetes mellitus, BMI, resting heart rate, and the use of aspirin, long-acting nitrates, calcium channel blockers, angiotensin-converting enzyme inhibitors/angiotensin receptor blockers and beta blockers, were significantly more common in group B compared with group A. Brachial artery %FMD was significantly higher in Table 1. Coronary artery disease risk factors and medications by median pulse pressure Parameter MPP 50 mmhg n = 290 MPP > 50 mmhg n = 235 p-value Age (years) 55 ± ± Males 215 (74%) 108 (46%) < Hypertension 52 (18%) 120 (51%) < Hyperlipidemia 110 (38%) 106 (45%) Current smokers 46 (16%) 28 (12%) Type 2 diabetes mellitus 12 (4%) 26 (11%) Family history 133 (46%) 94 (40%) Body mass index (kg/m 2 ) 26 ± 4 28 ± Aspirin 49 (17%) 80 (34%) < Statins 58 (20%) 59 (25%) Long-acting nitrates 2 (0.7%) 12 (5%) Calcium channel blockers 15 (5%) 49 (21%) < Furosemide 6 (2%) 16 (7%) Spironolactone 0 (0%) 2 (1%) Angiotensin-converting enzyme inhibitors/ 17 (6%) 52 (22%) < angiotensin-receptor blockers Beta blockers 23 (8%) 52 (22%) < Multi-vitamins 17 (6%) 28 (12%) MPP, median pulse pressure. Values are mean ± SD. Table 2. Lipoproteins, blood pressure and endothelial function by median pulse pressure Parameter MPP 50 mmhg n = 290 MPP > 50 mmhg n = 235 p-value Fasting blood glucose (mg/dl) 89 ± 9 95 ± Total cholesterol (mg/dl) 200 ± ± Low-density lipoprotein cholesterol (mg/dl) 123 ± ± Triglycerides (mg/dl) 131 ± ± High-density lipoprotein cholesterol (mg/dl) 53 ± ± Homocysteine (mmol/l) 13 ± 3 13 ± Systolic blood pressure (mmhg) 123 ± ± 18 < Diastolic blood pressure (mmhg) 79 ± 7 82 ± 9 < Resting heart rate (beats/min) 65 ± ± 10 < 0.01 Pulse pressure (mmhg) 44 ± 6 69 ± 14 < Mean arterial pressure (mmhg) 93.3 ± ± 11.7 < High-sensitivity C-reactive protein (mg/l) 4.2 ± ± Framingham risk score (% risk/10 years) 7.3 ± ± Baseline brachial artery diameter (mm) 5.59 ± ± % Flow-mediated dilation 14.9 ± ± 8.8 < % Nitroglycerin-induced dilation 16.1 ± ± MPP, median pulse pressure. Values are mean ± SD.

5 Beigel R et al. 303 % p < p = 0.38 % FMD % NTG Figure 2. Bar graphs showing brachial artery flow-mediated dilation (%FMD) and endothelium-independent nitroglycerinmediated vasodilation (%NTG) in subjects with pulse pressure equal or below (solid bars, n = 290) and above (open bars, n = 235) the median pulse pressure of 50 mmhg. Data are expressed as mean ± SD. group A compared with group B, while %NTG and brachial artery diameter did not differ significantly between the two groups (Figure 2). Furthermore, the two study groups were comparable regarding the Framingham 10-year CAD risk, lipoproteins and hs-crp as a marker of inflammation. Discussion Our current study demonstrates that PP is inversely associated with brachial artery FMD in a large sample of middle-aged subjects (30% with hypertension, 25% on anti-hypertension therapy) with no apparent heart disease. This relationship persists after adjustment for the significant influence of other covariates. Furthermore, the reciprocal relationship between PP and endothelial function is supported by the observation that more patients with higher than median PP received antihypertensive (such as calcium channel blockers, beta blockers, angiotensin-converting enzyme inhibitors and/or angiotensin receptor blockers) or other medications (such as aspirin, multi-vitamins) which could potentially improve endothelial function. PP rises as a consequence of the episodic nature of cardiac contractions and arterial circulation properties. In its simplest two-element forms, PP is the ratio between stroke volume and compliance of arterial circulation. 3 While the rise in PP with aging can be related to loss of arterial elasticity and a fall in arterial circulation, 22 its elevation in young people is mainly related to a rise in stroke volume. 23 Although there is substantial evidence linking elevated PP to adverse cardiovascular outcomes, studies are scarce regarding possible mechanisms that link PP to cardiovascular pathology. Compared with laminar flow, oscillatory and pulsatile flow patterns are associated with an increase in endothelial oxidative stress, resulting in impairment of endothelial function. 24 Thus, the pulsatile blood pressure component can affect endothelium function independently from other blood pressure components. Endothelial dysfunction is also related to poor outcome. Many studies have shown that endothelial dysfunction is an independent predictor for future cardiovascular events in healthy volunteers without apparent CAD 25 and in patients with overt cardiovascular disease, where invasive and non-invasive methods are used for evaluating endothelial function. Only very few studies have shown a relationship between PP and endothelial dysfunction. In one study, PP was shown to be associated with endothelial dysfunction as assessed by acetylcholine reactivity in small vessels, 12 while another showed that PP is inversely associated with endothelial dysfunction in naïve hypertensive subjects. 13 Although it is currently premature to identify the reduction of PP as a therapeutic target, there is growing evidence for an association between it and cardiovascular outcomes. 40 While the mechanisms underlying this association are still at best unclear, we have shown one connection by which PP might play a role in the pathogenesis of the atherosclerotic process. Whether endothelial dysfunction develops as a consequence of elevated PP chronic hemodynamics, or is involved in the pathogenesis of elevating PP itself, remains unclear. As outlined by prior studies, this link might relate to a decrease in nitric oxide bioavailability, which results in both endothelial function impairment and increased arterial stiffness. 3,41 While our current study demonstrates an association between PP and endothelial function, it did not establish a cause and effect relationship. Whereas our current study implies that abnormalities in PP contribute to endothelial dysfunction, the opposite is as likely to be the case. Physiologic studies have suggested that endothelial function contributes to increased vascular stiffness and thereby would likely raise PP. Kinlay et al. 41 and Wilkinson et al. 42 have shown in humans and sheep, respectively, that decreased nitric oxide bioavailability increases vascular stiffness. Ceravolo et al. 13 found that PP is inversely correlated with endothelium-dependent vadodilation assessed by acetylcholine-stimulated forearm blood flow in hypertensive patients, while Scuteri et al. 43 found that salt loading in normotensive postmenopausal women increased PP and this was inversely correlated with the changes in FMD. In contrast, however, Sharman et al. 44 found that the nitric oxide synthase antagonist L-NMMA increased brachial diastolic blood pressure significantly but not systolic or PP. Study limitations It should be mentioned that subjects participating in the current study were usually from a high socio-economic background (those without private health insurance were required to pay for the test), as well as being more educated (awareness of primary care facilities), thereby generating a biased study cohort. Although stress tests were not performed on all patients, the exclusion of CAD was based upon clinical examination, electrocardiography and echocardiography which were performed on all participants.

6 304 Vascular Medicine 15(4) The observational nature of this study prohibits drawing conclusions regarding a causal link between endothelial dysfunction and PP. Endothelial dysfunction could be a cause of elevated PP rather than the other way around, since unmeasured confounders may have contributed to the observed associations. We did not measure other potential causes for enhanced endothelial function, such as changes in free fatty acids and inflammatory cytokines, nitric oxide synthase expression and endothelin. Since we did not want to harm those subjects (25% of the study cohort were on chronic anti-hypertension therapy) who were on chronic treatment/medication and who were stabilized on those concomitant medications, we thought that at least an overnight fasting of 12 hours would be sufficient, although we were aware that some of the long-acting medications have some effect on FMD and NTG beyond 12 hours and therefore may have affected endothelial function, thereby possibly influencing our findings. However, most subjects stopped their concomitant medications > 24 hours from the study testing. Nevertheless, the use of concomitant medications was equally distributed in the two study groups (< and > the median FMD). Conclusions PP is inversely associated with endothelial function assessed by brachial artery FMD in middle-aged subjects without apparent heart disease, suggesting a potential mechanism whereby elevated PP contributes to cardiovascular disease. 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