dysfunction (a Substudy of the Anglo-Scandinavian Cardiac Outcomes Trial [ASCOT])

Relation of Thrombogenesis in Systemic Hypertension to Angiogenesis and Endothelial Damage/Dysfunction (a Substudy of the Anglo-Scandinavian Cardiac Outcomes Trial [ASCOT]) Dirk C. Felmeden, MD, Charles G.C. Spencer, MRCP, Natali A.Y. Chung, MRCP, Funmi M. Belgore, PhD, Andrew D. Blann, PhD, D. Gareth Beevers, MD, and Gregory Y.H. Lip, MD Increasing evidence points toward a prothrombotic state in hypertension and atherosclerosis, conditions associated with thrombosis-related complications, such as myocardial infarction and stroke. We hypothesized that this increased risk of thrombogenesis may be related to endothelial damage/dysfunction and abnormal angiogenesis, and thus, an increased risk of future cardiovascular disease. Thrombogenesis, endothelial damage/ dysfunction, and angiogenesis can be assessed by measurement of tissue factor (TF), von Willebrand Factor (vWF), flow-mediated dilatation (FMD), and vascular endothelial growth factor (VEGF), respectively. To test this hypothesis, we measured TF, vWF, FMD, and VEGF in 76 patients with systemic hypertension (71 men; mean age 64; mean blood pressure 167/72 mm Hg), considered additional risk factors such as diabetes, and related them to the patient’s 10-year cardiovascular and cerebrovascular risk score using the Framingham equation. Patients were compared with 48 healthy normotensive controls. In these patients, the effects of 6 months of

intensified blood pressure and (where appropriate) lipid-lowering treatment were investigated. In our patients, TF, VEGF, and vWF levels were higher, but FMD was lower (all p <0.001) compared with the controls. All markers correlated with each other and with both cardiovascular and cerebrovascular risk scores (all p <0.001). After intensified blood pressure and hypercholesterolemia treatment, total cholesterol, blood pressure, TF, VEGF, and vWF levels all decreased, whereas FMD increased (all p <0.001). Thus, in subjects with hypertension and other risk factors, endothelial damage/dysfunction (and thus, atherogenesis), thrombogenesis, and angiogenesis are abnormal, correlate with overall cardiovascular risk, and importantly, can be related to each other in a “Birmingham Vascular Triangle.” Furthermore, these processes are beneficially affected by intensive blood pressure and lipid treatment. 䊚2003 by Excerpta Medica, Inc. (Am J Cardiol 2003;92:400 – 405)

e hypothesized that abnormal thrombogenesis (as assessed by tissue factor [TF]) is related to W endothelial damage/dysfunction (assessed by flow-

problem was hypertension, but who also had other risk factors for atherosclerosis such as diabetes and smoking.

mediated dilatation [FMD] and plasma von Willebrand factor [vWF] levels) and abnormal angiogenesis (assessed by plasma vascular endothelial growth factor [VEGF] levels). We also hypothesized that the measured indexes would also be of clinical relevance because they are related to the 10-year coronary heart disease and stroke risk as assessed by the Framingham equation.1 Finally, we hypothesized that the effects of 6 months of intensified blood pressure and (where applicable) hypercholesterolemia treatment on these indexes would be beneficial. Our hypotheses were tested in a group of subjects whose primary clinical From the Haemostasis, Thrombosis, and Vascular Biology Unit, University Department of Medicine, City Hospital, Birmingham, United Kingdom. Manuscript received February 14, 2003; revised manuscript received and accepted May 2, 2003. Address for reprints: Gregory Y.H. Lip, MD, Haemostasis, Thrombosis, and Vascular Biology Unit, University Department of Medicine, City Hospital, Birmingham B18 7QH, United Kingdom. E-mail: [email protected].

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©2003 by Excerpta Medica, Inc. All rights reserved. The American Journal of Cardiology Vol. 92 August 15, 2003

METHODS

Subjects: Patients were recruited from among those screened as part of the Anglo-Scandinavian Cardiac Outcome Trial (ASCOT) in Birmingham, England.2 In brief, the inclusion criteria were patients aged between 40 and 80 years with either newly diagnosed untreated hypertension with systolic blood pressure ⬎160 mm Hg and/or diastolic blood pressure ⬎100 mm Hg or treated hypertension with systolic blood pressure ⬎140 and/or diastolic blood pressure ⬎90 mm Hg. Blood pressure was measured after 10 minutes of rest in a quiet room. Three consecutive blood pressure readings were taken and the average of the last 2 readings was used. Patients with secondary hypertension or malignant-phase hypertension were excluded. Clinical and laboratory assessments also performed were: left ventricular hypertrophy according to the Cornell voltage duration product (⬎2440) or Sokolow-Lyon criteria (⬎38 mV); other electrocar0002-9149/03/$–see front matter doi:10.1016/S0002-9149(03)00657-X

antihypertensive treatment was adjusted to aim for a target blood presPatients With sure of ⬍140 mm Hg systolic and ⬍ Controls Hypertension 90 mm Hg diastolic for nondiabetic Variable (n ⫽ 48) (n ⫽ 76) p Value patients. In patients with diabetes Age (years) 62 (8.0) 64 (8.2) 0.280 mellitus, the goal was ⬍130 mm Hg Men/women 41/7 71/5 0.142 systolic and ⬍80 mm Hg diastolic. White/non-White 44/4 69/7 0.867 At the 6-month visit, further blood Medical history (N) samples were taken for the measureLeft ventricular hypertrophy on ECG 0 7 — Cerebrovascular event/transient ischaemic 0 11 — ment of lipoprotein, vWF, TF, and event/peripheral vascular disease VEGF levels. The control group conDiabetes mellitus 0 15 — sisted of 48 healthy normotensive Smoker 9 18 0.659 controls (41 men and 7 women, Systolic blood pressure (mm Hg) 134 (13) 167 (15) ⬍0.001 mean age 62 years, SD 8.0) recruited Diastolic blood pressure (mm Hg) 80 (8) 92 (11) ⬍0.001 Total cholesterol (mmol/L) 5.6 (1.0) 5.8 (1.1) 0.156 from healthy hospital staff, patients’ High-density lipoprotein cholesterol (mmol/L) 1.3 (0.5) 1.3 (0.4) 0.647 relatives, and those attending the Low-density lipoprotein cholesterol (mmol/L) 3.4 (0.8) 3.7 (1.0) 0.117 hospital for routine cataract surgery. Triglycerides (mmol/L) 1.7 (1.1) 1.8 (1.0) 0.634 2 The subjects had no clinical evidence 26.1 (3.6) 28.6 (4.0) ⬍0.001 Body mass index (kg/m ) Coronary heart disease risk (10-y; %) 9.8 (5.1–15.1) 21.4 (16.6–28.6) ⬍0.001 of vascular, metabolic, neoplastic, or Stroke risk (10-y; %) 1.9 (1.4–3.8) 7.5 (4.8–11.8) ⬍0.001 inflammatory disease (assessed by a FMD (%) 8.6 (2.2) 4.8 (1.3) ⬍0.001 careful history, examination, and Glyceryl trinitrate-mediated dilatation (%) 20.5 (2.3) 19.7 (5.7) 0.150 routine laboratory tests). These subTF (pg/ml) 10 (10–16) 83 (41–210) ⬍0.001 jects were normotensive and in sinus VEGF (pg/ml) 100 (46–200) 400 (210–1,450) ⬍0.001 vWF (IU/dl) 98 (19) 138 (28) ⬍0.001 rhythm and were not taking aspirin, warfarin, lipid-lowering or antihyValues are expressed as mean (SD) or median (interquartile range). pertensive drugs, nonsteroidal antiECG ⫽ electrocardiogram. inflammatory drugs, or antibiotics. Laboratory methods: Blood was diographic abnormalities (left ventricular strain pat- drawn after an 8-hour fasting period with minimal tern, abnormal Q waves, left bundle branch block, trauma from the antecubital vein. Samples were put on ST-T changes compatible with ischemic heart dis- ice for 5 minutes and then centrifuged at 2000 rpm for ease); diabetes mellitus according to World Health 20 minutes. The plasma was stored at ⫺70°C until Organization criteria; medical history of a cerebrovas- assayed. In the routine hospital pathology laboratory, cular event, including transient ischemic attack; male plasma was analyzed by standard techniques for total sex; age ⬎55 years; microalbuminuria/proteinuria; cholesterol, triglycerides, and high-density lipoprotein current smoking; increased plasma total cholesterol/ cholesterol. Low-density lipoprotein cholesterol was high-density lipoprotein cholesterol ratio; family his- calculated (where possible) using Friedewald’s equatory of coronary artery disease in a first-degree rela- tion. TF levels were analyzed by an enzyme-linked tive before the age of 55 (men) or 60 (women) years, immunosorbant assay method using commercially and peripheral vascular disease according to the Ed- available reagents (Sigma, Poole, Dorset, United inburgh Claudication Questionnaire.3 Using these Kingdom; and American Diagnostica Inc., Greendata, the Framingham risk calculations of coronary wich, Connecticut). The TF assay has a lower limit of heart disease and stroke risk score were derived.1 detection of 10 pg/ml, an intra-assay coefficient of These scores have recently been validated for other ⬍5%, and an interassay coefficient of ⬍10%. VEGF levels in citrated plasma were measured by enzymepopulations.4 The impact of cardiovascular risk management linked immunosorbant assay using commercially with blood pressure and hypercholesterolemia treat- available reagents and recombinant standards (R&D ment was determined in 76 patients (Table 1,which Systems, Abingdon, United Kingdom). The VEGF also shows clinical, demographic, and laboratory data) assay has a minimum sensitivity of 0.5 IU/dl, an who fulfilled ⱖ3 of the previously mentioned criteria, intra-assay coefficient of 4.4%, and an interassay coone of which was hypertension. Treatment was a efficient of 9.1%.5 Levels of vWF were analyzed with “package of care” that included health education a sandwich enzyme-linked immunosorbant assay us(smoking cessation, regular exercise, decreases in ing commercially available reagents and standards weight and salt intake) and antihypertensive therapy (Dako Ltd., Ely, United Kingdom). The assay has a (with either amlodipine and/or perindopril, or atenolol minimum sensitivity of 0.5 IU/dl, an intra-assay coand/or bendroflumethiazide). If total cholesterol was efficient of ⬍5%, and an interassay coefficient of 4.0 to 6.5 mmol/L (155 to 250 mg/ml), patients were ⬍10%. All laboratory work was performed in blinded randomized to the lipid arm in a double-blind manner fashion with respect to the identity of the samples. to receive atorvastatin 10 mg or matching placebo. FMD: High-resolution ultrasound was used to asPatients with total cholesterol ⬎6.5 mmol/L (250 mg/ sess changes in brachial artery diameter as previously ml) were referred back to their general practitioners described.6,7 Measurements were taken in the morning for management of their hypercholesterolemia. The after patients had fasted and rested in a supine position TABLE 1 Cross-sectional Data of Hypertensive Patients and Healthy Controls

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for 20 minutes in a quiet room. High-quality images were obtained by a single dedicated operator using a 10-MHz vascular ultrasound probe (GE Vingmed Ultrasound, System V; Slough, Berkshire, United Kingdom). A longitudinal section of the brachial artery was scanned approximately 5 cm above the elbow. The transducer remained in a fixed position relative to the patients arm throughout the procedure. Vessel diameter was assessed at end-diastole, with a measurement of leading edge to leading edge. After a baseline scan, a pneumatic cuff was placed at the level of the mid forearm and inflated to 250 to 300 mm Hg for 4.5 minutes. The second scan was performed 30 to 90 seconds after cuff release (peak changes during reactive hyperemia), and 15 minutes were allowed for vessel recovery. Subsequent scans of the brachial artery were done before, and again at 3.5 minutes after, the administration of sublingual glyceryl trinitrate (400-␮g spray) to assess endothelium-independent vasodilatation. Scans were recorded on super-VHS videotape for later analysis by an independent investigator blinded to treatment assignment. Inter- and intraobserver variation was ⬍10%. Flow-mediated (endothelium-dependent) dilatation and glyceryl trinitrate-induced (endothelium-independent) dilatation were estimated as percent change in diameter relative to their respective baseline measurements. Power calculations: We hypothesized significant correlations between TF, vWF, FMD, VEGF, and 10-year cardiovascular risk in a group of subjects at risk for atherosclerosis; we considered the hypertensive group would most likely demonstrate poor FMD and increased vWF. Because we believed that a correlation coefficient of ⬎0.3 would be meaningful, we needed ⬎72 sets of data to achieve this at p ⬍0.05 and 1-␤ ⫽ 0.825. By testing the effect of intervention, this number of subjects provides a 1-␤ of 0.80 at p ⬍0.05 to detect a change of 0.3 of a SD in 1 of the normally distributed variables due to the cardiovascular risk factor treatments. Control data are provided merely to provide a perspective of normal values. No case or control analysis is hypothesized because differences in the relevant indexes (vWF, TF, FMD, VEGF) between patients and controls are already well-established.8 –11 Statistical analysis: Continuous data were subjected to the Ryan-Joiner test to assess distribution. Age, blood pressure, FMD, and vWF levels were normally distributed and are expressed as mean and SD. VEGF and TF levels are not normally distributed; therefore, they are shown as median and interquartile range. Correlations between cardiovascular risk scores and measured parameters were assessed according to Spearman’s method. Paired comparisons were made using the paired t test or paired Wilcoxon’s test, as appropriate. Data between patients and controls were analyzed using Student’s t or the Mann-Whitney U test. All statistical calculations were performed on a microcomputer using a commercially available statistical package (SPSS 10.0 for Windows; SPSS Inc., Chicago, Illinois). A p value ⬍0.05 was considered statistically significant. 402 THE AMERICAN JOURNAL OF CARDIOLOGY姞

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TABLE 2 Spearman Correlations Between Framingham Cardiovascular and Cerebrovascular Risk Scores, von Willebrand Factor (vWF), and Vascular Endothelial Growth Factor (VEGF) Coronary Heart Disease Risk r

p Value

Stroke Risk r

p Value

0.343 0.485 0.308

⬍0.001 ⬍0.001 ⬍0.001

0.456 0.582 0.327

⬍0.001 ⬍0.001 ⬍0.001

TF vWF VEGF

RESULTS

Cross-sectional data (Table 1): Hypertensive patients and controls were similar with regards to age, sex, and racial mix, but, as expected, the hypertensive patients had a worse risk factor profile. Patients had higher TF, VEGF, and vWF levels, but lower FMD, when compared with controls. There was no difference in endothelial-independent vasodilatation due to glyceryl trinitrate. All case and control comparative data are largely confirmatory.8 –11 Correlations (Tables 2 and 3): All indexes correlated significantly with cardiovascular and cerebrovascular risk scores, and all indexes correlated significantly with each other. All research indexes also correlated significantly with systolic blood pressure. Effects of treatment (Table 4): As expected, patients’ risk factor profiles improved with the treatment of hypertension and hypercholesterolemia.With the exception of glyceryl–trinitrate-mediated dilation (endothelial-independent), all the research indexes improved (at least p ⫽ 0.004). However, in comparison with healthy control data in Table 1, all indexes, except FMD, were still far from normal. Before treatment, mean patient FMD was 56% that of the healthy controls (i.e., 100%). After six months of treatment, patients’ FMD improved to become 85% of that of the controls. There were no significant correlations between changes in blood pressure and corresponding changes in TF, VEGF, or vWF levels or FMD (data not shown).

DISCUSSION The present study confirms abnormal thrombogenesis (defined by TF) endothelial damage/dysfunction (vWF and FMD) and abnormal angiogenesis (VEGF) in subjects with hypertension and other risk factors or atherosclerosis.8 –11 However, we are unaware of previous studies with a concurrent assessment of the processes of thrombogenesis, endothelial damage/dysfunction, and angiogenesis in a “high-risk” hypertensive population. In addition, our data also suggest that these processes are (1) related not only to each other but also to the subject’s 10-year coronary heart disease and stroke risk scores (according to the Framingham equation), and (2) can be beneficially improved with pharmacologic treatment and cardiovascular risk management. The Framingham equations integrate the age, gender, systolic and diastolic blood pressures, smoking status, history of diabetes mellitus, presence AUGUST 15, 2003

linked immunosorbant assay. Its relevance for atherosclerosis is that high levels carry a poor prognoTF VEGF Systolic Blood Pressure sis.14 –16 Furthermore, vWF levels r p Value r p Value r p Value correlate with a mathematically devWF 0.382 ⬍0.001 0.560 ⬍0.001 0.593 ⬍0.001 rived index of the risk of future carFMD ⫺0.578 ⬍0.001 ⫺0.513 ⬍0.001 ⫺0.672 ⬍0.001 diovascular and cerebrovascular TF — 0.640 ⬍0.001 0.477 ⬍0.001 events (i.e., the Framingham equaVEGF 0.640 ⬍0.001 — 0.411 ⬍0.001 tion).4 Therefore, the inverse relation between the 2 indexes fits the paradigm of endothelial damage/dysfunction very well. TF: We were not surprised to find TABLE 4 Effects of Intensified Blood Pressure, Hypercholesterolemia Treatment, increased TF in this group of patients and Cardiovascular Risk Management in 76 Patients With Hypertension with hypertension additional disAfter 6 Months’ ease.17,18 TF, as the initiator of the Baseline Treatment p Value extrinsic clotting cascade, is a critical Risk factors component of thrombotic response in Total cholesterol (mmol/L) 5.8 (1.0) 5.3 (1.3) ⬍0.001 vivo. Its importance in atheroscleroHigh-density lipoprotein cholesterol 1.3 (0.4) 1.3 (0.4) 0.316 sis has also been shown in a recent (mmol/L) study in which high TF levels were Low-density lipoprotein cholesterol 3.7 (1.0) 3.3 (1.2) 0.001 (mmol/L) present in lipid-rich atherosclerotic Triglycerides (mmol/L) 1.8 (1.0) 1.7 (0.9) 0.116 plaques, which would suggest that an Systolic blood pressure (mm Hg) 167 (15) 144 (13) ⬍0.001 increase of TF in such lesions may Diastolic blood pressure (mm Hg) 92 (11) 83 (10) ⬍0.001 be predictive of plaque rupture, the Research indexes initial event in acute coronary synFMD (%) 4.8 (1.3) 7.3 (1.7) ⬍0.001 Glyceryl–trinitrate-mediated 19.7 (5.7) 19.9 0.126 dromes, and therefore, thrombogedilatation (%) nicity.19,20 Certainly, our patients TF (pg/ml) 83 (41–210) 65 (25–130) ⬍0.001 had a high risk of cardiovascular disVEGF (pg/ml) 400 (210–1,450) 270 (180–1,050) ⬍0.001 ease, including a variable degree of vWF (IU/dl) 138 (28) 129 (33) 0.004 peripheral vascular disease, diabetes Values expressed as mean (SD) or median (interquartile range). mellitus, previous stroke, and smoking, all of which have been shown to increase TF levels.21–23 It follows of left ventricular hypertrophy, and total and high- that we would expect TF to also correlate with 10-year density lipoprotein cholesterol.1,4 This validated Framingham cardiovascular risk, as indeed it does. method for assessing cardiovascular and stroke risk Nevertheless, the decrease of TF after 6 months of (based on individual risk factors) is now widely used antihypertensive therapy and cardiovascular risk manfor risk stratification to target the appropriate patients agement has not been described, although it is known for lipid-lowering therapy or the initiation of antihy- that antihypertensive therapy reduces elevated TF levpertensive treatment in persons with high to normal els in certain groups.18,23 and mild degrees of hypertension. Our observations Angiogenesis: Because we have described inthat TF, vWF, and VEGF positively correlate with the creased VEGF (which is associated with abnormal Framingham risk scores may assist as alternative in- angiogenesis) in hypertension,11 hyperlipidemia,24 dexes to assess the future cardiovascular and cerebro- and atherosclerosis,25 we were not surprised to find vascular risk in patients with hypertension. These similar changes in the present cohort of patients, all of markers also correlate with systolic blood pressure, but there was no significant correlation with total whom had some burden of actual or potential disease. cholesterol, high-density lipoprotein cholesterol, or Evidence of angiogenesis in atherosclerosis includes gender. TF, vWF, and VEGF may assess other risk increased numbers of vasa vasorum in arteries burfactors not included in the equation as it stands, and dened with atheroma with26increased expression of they may provide additional information needed to angiogenic growth factors. Angiogenesis has also been proposed as an important mechanism in the refine overall cardiovascular risk. 27 Endothelial cell damage/dysfunction: The hypothe- pathogenesis of hypertension. If, as we suspect, insis of endothelial cell damage/dysfunction in hyper- creased plasma VEGF reflects ongoing angiogenesis, tension is supported by increased plasma levels of then a relation with vWF is not surprising. However, vWF and decreased FMD.8,9,12,13 Although the latter the mechanistic relation between VEGF and Framingmethod is very sensitive, it is difficult to apply to large ham-defined risk is unclear and so it may be spurious. epidemiologic studies because it requires specialized The present strong correlation between vWF and equipment and technical expertise. A more convenient VEGF contrasts with our previous failure to correlate marker for the assessment of endothelial damage/ these indexes in patients with full-blown atheroscledysfunction is vWF, easily measured by an enzyme- rosis,25 suggesting different relations between markers TABLE 3 Spearman Correlations Between Tissue Factor (TF), Vascular Endothelial Growth Factor (VEGF), and Systolic Blood Pressure

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Thrombosis, and Vascular Biology Unit. We would like to thank Sisters Ranjit Dhillon, RGN, Maggie Lal, RGN, Zoe¨ Townend, RGN, and Ruth Watson, RGN, for help with data collection. Other ASCOT Investigators are listed in reference 2. 1. Kannel WB, McGee D, Gordon T. A general cardio-

FIGURE 1. The Birmingham Vascular Triangle.

and the endothelium in different parts of the development of atherosclerosis. A hypothesis relating the endothelium, thrombogenesis, and angiogenesis: Because nitric oxide is an im-

portant regulator of vascular tone, it appears likely that the loss of nitric oxide results in impaired FMD,28 and it has been suggested that FMD endothelial dysfunction, like increased vWF, is an early marker of atherogenesis.29,30 Such impaired FMD may be a consequence of decreased nitric oxide synthase activity, as well as an enhanced breakdown of nitric oxide resulting in impaired vasodilatation. Dysregulated nitric oxide production and breakdown as part of impaired FMD has, like increased vWF, been implicated in the pathogenesis of a variety of vascular diseases, including atherosclerosis and hypertension, because nitric oxide synthesized by endothelial cells induces vasodilatation and inhibits platelet aggregation and, therefore, thrombosis.30 The intimate links between endothelial damage/dysfunction (and thus, atherogenesis) to thrombogenesis and angiogenesis forms the basis of the Birmingham Vascular Triangle (Figure 1). Limitations: The present analysis is limited by its cross-sectional comparison, but includes a longitudinal component. Furthermore, because this substudy is part of the main ASCOT clinical trial, the investigations and treatment regimens allowed are very much protocol-driven and the substudy was secondary to the main ASCOT. Further planned analyses from this substudy should occur at the end of 5 years (on completion of the main ASCOT study) when we would hope to present complete data from the Birmingham center on these research indexes in relation to the full duration of follow-up, study end points, grades of blood pressure decrease, effects of different drug regimens (including statins), an so on. Acknowledgment: We acknowledge the support of the Sandwell & West Birmingham Hospitals NHS Trust Research and Development Program for the Hemostasis, 404 THE AMERICAN JOURNAL OF CARDIOLOGY姞

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