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racial differences in circulating markers of angiogenesis and their association with cardiovascular risk factors and cardiovascular disease
International Journal of Cardiology 167 (2013) 1247–1250
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International Journal of Cardiology journal homepage: www.elsevier.com/locate/ijcard
Ethnic/racial differences in circulating markers of angiogenesis and their association with cardiovascular risk factors and cardiovascular disease Philip C. Bennett a, b, Paramjit S. Gill c, 1, Stanley Silverman b, Andrew D. Blann a, Balu Balakrishnan a, Gregory Y.H. Lip a,⁎, 1 a b c
University of Birmingham Centre for Cardiovascular Sciences, City Hospital, Birmingham, B18 7QH, United Kingdom Department of Vascular Surgery, City Hospital, Birmingham, B18 7QH, United Kingdom Primary Care Clinical Sciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, England, United Kingdom
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Article history: Received 12 January 2012 Accepted 17 March 2012 Available online 9 April 2012 Keywords: Ethnic differences Angoipoietin-1 Angiopoietin-2 Soluble Tie-2 receptor Angiogenin Cardiovascular risk factors
a b s t r a c t Objective: To determine (a) whether ethnic/racial differences exist in circulating markers of angiogenesis (Angiopoietin-1 (Ang-1), Angiopoietin-2 (Ang-2), soluble Tie-2 receptor (sTie-2) and Angiogenin) between South Asian (SA; from India, Pakistan and Bangladesh); Black African-Caribbean and White (W) ethnic groups, and (b) associations between these markers in stable cardiovascular disease (CVD) and its risk factors. Patients and methods: We recruited 243 subjects (82 SA, 84 Black and 77 W) with symptomatic and clinically confirmed CVD (n = 108), risk factor controls (with ≥ 1 cardiovascular risk factor, e.g. smoking, diabetes mellitus, dyslipidaemia, hypertension) and with ankle brachial pressure index > 1) (n = 64) and healthy controls free of CVD and risk factors (n = 56). Angiogenic markers were measured by enzyme linked immunoassay. Results: In healthy controls, angiogenin was higher in SA and Black subjects, compared to Whites (p b 0.05). sTie-2 correlated inversely with angiogenin (p = 0.001), was higher in women (p = 0.029) and was lower in smokers (p = 0.007). Overall, age (p = 0.001) was the only independent factor associated with angiopoietin-1. Angiogenin (p = 0.01) and SBP (p = 0.014) were both independently higher in the Black group compared to the White group. Conclusions: Ethnic, racial, and demographic differences are evident in certain circulating markers of angiogenesis. With the exception of an effect of smoking on sTie-2, these differences are not influenced by the presence of other risk factors, nor the presence of stable cardiovascular disease. © 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Cardiovascular disease, manifesting as peripheral arterial disease, coronary artery disease or cerebrovascular disease, is an important consequence of atherosclerosis. Angiogenesis, the formation of new blood vessels, is evident in these conditions and is closely related to atherogenesis, thrombogenesis and the development of intra-plaque vasa vasorum [1,2]. Furthermore, angiogenesis is a feature of disease development and progression [3,4], and is especially frequent in advanced cardiovascular disease and may characterise atherosclerotic lesions at high risk of haemorrhage or rupture [4–6]. Vascular endothelial growth factor (VEGF), acting on the endothelium, is one of the major growth factors driving angiogenesis, and raised plasma levels in diabetes, peripheral artery disease and coronary artery disease is evidence of this increased angiogenic activity [7].
⁎ Corresponding author. Tel.: + 44 121507 5080; fax: + 44 121 5544083. E-mail address: [email protected] (G.Y.H. Lip). 1 These authors contributed equally to this work. 0167-5273/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijcard.2012.03.158
In addition to VEGF, the angiopoietins (Angiopoietin-1 (Ang-1) and Angiopoietin-2 (Ang-2) are additional angiogenic growth factors that are specific for endothelium. Ang-1 and Ang-2 play modulatory roles by binding a common receptor, the endothelial cell-specific tyrosine kinase receptor Tie-2 [8,9], a soluble form of which (i.e. sTie-2) can be detected in plasma. Together, the angiopoietins regulate endothelial cell differentiation: Ang-1 accelerates the maturation of the blood vessel, whilst Ang-2, an antagonist of Ang-1, destabilises the vessel and degrades the basal lamina [10,11]. Plasma levels of Ang-1 and Ang-2 levels are abnormal in, and are associated with, cardiovascular risk in patients with cardiovascular disease [12] and also in other manifestations of atherosclerosis, including heart failure, acute coronary syndromes and hypertension [8,13–17]. Angiogenin is a small polypeptide implicated in angiogenesis normally found in the vasculature, but also in some physiological and pathological conditions, including peripheral and coronary atherosclerosis [18–20]. Elevated angiogenin levels have been found to be higher in those with severe peripheral atherosclerosis, compared to mild and moderate disease [19], and raised levels in acute coronary syndromes predict a poor outcome at six months [20]. Thus, angiogenin
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could potentially be an indicator of endothelial activity related to progression of vascular disease. Given improvements in biochemical techniques there has been an increasing interest in value of biomarkers related to the development of atherosclerosis and its symptomatic complications. Despite ethnic and racial differences having been documented in the prevalence of atherosclerosis [21], much of the research on pathophysiology has been carried out on predominantly White populations [19,20] and limited research has been directed on these biomarkers in relation to ethnic and/or racial susceptibility to this disease [22]. We hypothesised the following: (1) that there would be differences in levels of circulating markers of angiogenesis between South Asian (SA i.e. people originating from India, Pakistan and Bangladesh]), Black African-Caribbean (AC), and White ethic groups, and (2) that Ang-1, Ang-2, soluble Tie-2 receptor (sTie-2) and angiogenin levels would be higher in those with risk factor compared to healthy controls, with a further increase in those with clear cardiovascular disease. 2. Methods Participants were recruited from among those attending cardiovascular disease outpatient clinics (cardiology and peripheral artery disease) and from hypertension clinics. Coronary artery disease (CAD) was defined as myocardial infarction, coronary artery bypass graft, or coronary atherosclerosis on angioplasty. Peripheral artery disease (PAD) was defined as radiologically confirmed disease or peripheral artery surgery (e.g. distal bypass procedures and major limb amputations). Cerebrovascular disease was defined as stroke, transient ischaemia attack, or carotid artery surgery. All patients had stable disease. Risk factor controls and healthy controls were recruited from patients attending hypertension clinics, from relatives of patients attending outpatient clinics, from subjects participating in a community-based screening survey for heart disease [23] and from hospital and university staff. Risk factor controls had at least one cardiovascular risk factor, that is, smoking [current, or ceased with the past year], hypertension [defined as blood pressure >140/90 mm Hg or on treatment], diabetes mellitus [documented or on treatment], and dyslipidaemia [documented or on treatment] but not PAD, defined by an ankle brachial pressure Index (ABPI) > 1.0. Healthy controls were free of all these risk factors. In all subjects blood pressure was measured three times, and the final measurement reported. Height and weight were obtained to provide body mass index as the weight (kg) divided by the square of the height (m). Exclusion criteria for all subjects were infectious diseases, rheumatoid arthritis and other chronic inflammatory disorders, sepsis, malignancy, haemodynamically significant valvular heart disease, atrial fibrillation, renal failure, immunity-modulating drugs and hormone replacement therapy, recurrent venous thromboembolism, congestive cardiac failure, multiple sclerosis and anaemia. The study was approved by Birmingham East, North & Solihull Local Research Ethnics Committee and written informed consent was obtained from all study participants.
the CVD group, we recruited in excess in each group for extra confidence. Statistical analyses were carried out using Minitab version 15 (State Coll, PA, USA).
3. Results We recruited 243 subjects with a mean age of 64.3 (SD 11.9) years. Diabetes was present in 25.9% and 55.1% were male. Breakdown by age, sex, blood pressure, body mass index, clinical grouping, risk factors, cardiovascular territory and use of aspirin according to racial/ethnic group is presented in Table 1. The three groups were matched for age, sex and proportion of subjects who were healthy volunteers, risk factor controls, and those who had CVD or actual risk factors (smoking, hypertension, dyslipidaemia, and diabetes), cardiovascular disease territory and use of aspirin. SBP and DBP were lower in White Europeans compared to the other two groups, whilst BMI was lower in SA compared to the AC group. There were fewer diabetics in the White group compared to the other two groups. The risk factor group consisted of 42 people with a single risk factor, 25 with two risk factors and one person with all four risk factors. Of those with cardiovascular disease, 80 had disease in one territory, 33 had disease in two and 4 patients had disease in all three. Univariate analysis of the angiogenic markers are shown in Table 2. Angiogenin was lower in White compared to SA and to AC groups. Compared to healthy controls, angiopoietin-2 was higher in the risk factor and the cardiovascular disease group. In the 56 healthy controls, only angiogenin varied between the ethnic groups: 6.35 (3.2–8.6) ng/mL in 18 SA, 5.3 (3.7–7.5) ng/mL in 18 AC, and 3.3 (1.7–6.0) in the 20 White (p = 0.043 overall, p b 0.05 SA v White). sTie-2 correlated inversely with angiogenin (r = −0.44, p = 0.001, Fig. 1). sTie-2 was higher in the women (124 (83–170) ng/mL) compared to the men (86 (75–133) ng/mL (p = 0.029). In those with one or more risk factors, none of the research indices were associated with ethnic/racial group. Indeed, the effect of ethic/ racial group on angiogenin seen in the absence of risk factors became not significant (p = 0.087). sTie2 was lower in 16 smokers (87 (61–130) ng/mL) compared to 48 non-smokers (136 (100–166) ng/ mL, p = 0.007), and the effect of sex on sTie2 seen in those free of Table 1 Clinical and demographic details of study participants. South Asian (n = 82)
Africa/Black Caribbean (n = 84)
White European (n = 77)
P-value
Age (years) Male sex (%) SBP (mm Hg) DBP (mm Hg) BMI (kg/m2)
62 [10] 60.7 142 [27] 81 [13] 26.7 [4.4]
66 [13] 48.3 144 [21] 80 [12] 28.9 [5.5]
63 [11] 54.4 134 [18] 76 [9] 27.8 [5.2]
0.146 0.254 0.024 0.020 0.018
Clinical grouping Healthy Controls (%) Risk factor controls (%) Cardiovascular disease (%)
21.9 18.3 59.7
22.6 33.3 44.0
26.0 32.5 41.4
0.102
Risk factors Current smoking (%) Diabetes (%) Hypertension (%) Dyslipidaemia (%)
12.2 31.7 63.4 55.1
21.4 30.9 66.7 46.1
24.7 14.3 57.1 49.4
0.115 0.019 0.450 0.879
Cardiovascular disease territory PAD (%) 30.5 CAD (%) 35.4 CeVD (%) 11.0 Use of aspirin (%) 50.0
34.5 14.3 7.1 46.6
36.4 20.8 6.5 49.3
2.1. Laboratory Venous blood was taken from the antecubital fossa into sodium citrate. Blood was stored on ice for a maximum of 2 hours before being centrifuged at 1500 × g; the plasma supernatant was then frozen at − 70 °C for future batch analysis. Samples were tested for angiogenic markers using commercially available ELISA kits (R&D systems, Abingdon. UK). Intra-assay and inter-assay variances of all assays were b 5% and b 10% respectively. Lower limits of detection were 3.8 ng/ml for Ang-1, 0.1 ng/ml Ang-2, 0.1 ng/ml Angiogenin and 1.0 ng/mL for sTie 2.
2.2. Sample size justification and statistical analysis Data are presented as mean with standard deviation (SD) or median with interquartile range (IQR) for continuous parameters, as distribution defined. Chi-squared test was used for categorical data. One way ANOVA or the Kruskal–Wallis test was used to assess differences between three groups (SA, AC, White, and Healthy controls, risk factors, cardiovascular disease) for continuous variables, with Tukey's post-hoc analysis performed for inter-group differences. Determinants of the four research indices were sought using a general linear model, inputting ethnic group and sex as nominal categories, disease progression state as an ordinal category. When necessary, data was logged for normality. A p-value b 0.02 was deemed significant. Two-sided Spearman correlations were sought with a 1-beta power of 0.85: a correlation coefficient of >0.4 was considered large enough to minimise false positives [24]. We hypothesised a 25%–50% difference in one of the research indices in one of the three nominal groups of SA, AC and Whites, and of a similar increase across the spectrum of health (modelling 100[90–140] U/mL), to risk factors (125[115–165] U/mL) to cardiovascular disease (150[149–190] U/mL). To achieve this at p b 0.001, a sample size of 50 per group is required. However, with four research indices, and expecting greater heterogeneity in
0.154 0.887
Data are % or mean (standard deviation); SBP: systolic blood pressure; DBP: diastolic blood pressure; BMI: body mass index; PAD: peripheral arterial disease; CAD: coronary artery disease; CeVD: cerebrovascular disease. Age, SBP, DBP, BMI analysed by ANOVA, all others by chi-squared.
P.C. Bennett et al. / International Journal of Cardiology 167 (2013) 1247–1250 Table 2 Univariate comparisons of angiogenic markers. (a) Analysis by racial/ethnic group
South Asian (n = 82)
African/Black Caribbean (n = 84)
White European (n = 77)
P-value
Angiopoitein-1 Angiopoietin-2 sTie2 Angiogenin
16.8 1.3 103 240
12.4 [6.0–32.5] 1.2 [0.69–2.65] 119 [80–168] 261 [187–367]
9.4 1.35 127 169
0.246 0.301 0.191 0.01*
(b) Analysis by disease process
Healthy controls (n = 56)
Risk factors (n = 64)
Cardiovascular disease (n = 108)
Angiopoitein-1 Angiopoietin-2 sTie2 Angiogenin
14.8 0.88 117 260
12.4 1.32 126 221
12.3 1.52 114 238
[4.9–33.2] [0.85–1.78] [79–146] [135–386]
[5.2–33.0] [0.69–1.71] [79–158] [137–372]
[5.2–35.8] [0.81–3.1] [87–153] [114–314]
[5.1–21.0] [0.87–3.3] [83–166] [95–319]
[5.3–26.8] [0.9–2.4] [82–162] [135–371]
0.950 0.006** 0.851 0.347
Units for all growth factors, ng/mL. Data analysed by the Kruskall–Wallis Test for nonparametric testing between 3 groups. *Lower in White Europeans compared to South Asians and to Africans/Black Caribbeans (p b 0.05). Multivariate analysis showed this relationship to be present only in those free of risk factors or cardiovascular disease. **Higher in risk factors and cardiovascular disease (p b 0.05). However, the effect of the disease spectrum on angiopoietin-2 disappeared in multivariate analysis.
risk factors also became non-significant. There were no correlations where the coefficient exceeded 0.4. In the 108 cases with stable cardiovascular disease, there was no effect of ethnicity, or of the number of symptomatic cardiovascular territories on the research indices, and no effect of smoking or sex on levels of sTie2. There were no correlations where the coefficient exceeded 0.4. In multivariate analysis, general linear models identified age (p = 0.001) to be the only independent factor associated with angiopoietin-1, whilst ethnic group (p = 0.018) and SBP (p = 0.01) were the only factors associated with angiogenin. In nominal regression analysis, SBP (p = 0.014) and levels of angiogenin (p = 0.01) both remained higher in the AC group compared to the White group. 4. Discussion We report for the first time an analysis of circulating markers of angiogenesis in three different ethnic groups and the effect of risk factors and in stable atherosclerotic disease. We have also investigated the inter-relationships between Ang-1, Ang-2, sTie-2 and angiogenin in patients with CVD, risk factors and healthy subjects. We found angiogenin levels to be significantly lower in healthy White than in both SA and AC groups. This effect disappeared in the presence of risk factors, and in stable cardiovascular disease, indicating some effect of the latter two processes. We, and others, have reported
Scatterplot of sTie-2 (ng/mL) vs Angiogenin (ng/mL)
sTie-2 (ng/mL)
400
300
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raised angiogenin in acute coronary syndromes [19,20,25], but not in stable coronary artery disease [25] in predominantly White populations. We now extend previous work by reporting no change in angiogenin in SA and AC with stable cardiovascular disease. Angiogenin has been thought to be an indicator of endothelial damage related to progression of vascular disease, it binds actin on the surface of endothelial cells, and its plasma levels are dependent on the presence of an angiogenic stimulus [26]. Its levels may therefore be expected to be associated with related molecules, and the correlation with sTie-2 as found in this study may reflects this. Several studies have suggested that angiogenin, and other angiogenic factors, could promote atherosclerosis and potentially de-stabilise plaques by promoting intra-lesional angiogenesis [4,27]. If so, our data suggest that plasma levels do not reflect this process. The higher levels in healthy asymptomatic middle aged (mean age 56 years) SAs and ACs may in part explain the apparently higher cardiovascular disease burden in these ethnic groups when compared to the general population [22,28]. It is also possible that the higher angiogenin levels could in part be attributable to higher blood pressure and/or higher prevalence of diabetes amongst our SA and AC with respect to the White groups. Of note, the relationship between angiogenin and race/ethnicity present in health was only just non-significant in the presence of risk factors (p =0.087), but markedly so in stable cardiovascular disease (p= 0.595), imply an effect to the latter two aspects on the growth factor. Such an effect may be effect of pathophysiology and/or drug therapy on the endothelium. sTie-2 was higher in our middle aged healthy women than in health men, precisely the reverse of a much larger community-based study, although the latter subjects were younger (and the women more likely to be pre-menopausal [mean age 40 years]) and had more disease [29], although in our study this effect disappeared in risk factors and in frank disease. Some of this may be due to the effect of smoking. Notably, this finding parallels that of reduced plasma levels of the receptor for VEGF, Flt-1 in smokers [30]. Curiously, this may have a real cardiovascular effect as smoking, and so lower sFlt-1, protects against pre-eclampsia [31]. Failure to find altered sTie-2 in stable cardiovascular disease contrasts with others who reported raised levels in acute coronary syndromes [32], suggesting that alterations in angiogenic markers are the result of the acute changes. Cleavage of the extra-cellular domain of Tie-2 on endothelial cells occurs in response to inflammation and results in circulating sTie-2, a process which is reduced by hypoxia [9]. In acute inflammatory states, sTie-2 in the plasma is bound by Ang-1 preferentially over Ang-2 due to its higher affinity for the receptor, leading to greater binding of Ang-2 to endothelial Tie-2 and increasing endothelial permeability [9,33]. In hypoxic states, the relatively lower endothelial release of sTie-2, leads to less Ang-1 binding within the plasma and greater endothelial binding, which accelerates vessel maturation [2]. Whether or not these effects which are prominent in cell biology, have any role in the pathophysiology of cardiovascular disease we cannot say. Our only significant correlation was inversely between sTie2 and angiogenin. As there seems to be no clear physiological or pathological relationship between the two, it could be argued that this is spurious. However, the association was far from weak (r = 0.4) so a genuine association may be present.
200
4.1. Limitations 100
0 0
100
200
300
400
500
600
Angiogenin (ng/mL) Fig. 1. Correlation between sTie-2 and angiogenin in the healthy controls.
This study is limited by its cross-sectional design, but it still represents the first study to investigate ethnic differences in circulating markers of angiogenesis and their association with cardiovascular risk factors and frank, but stable, cardiovascular disease. Although we have adjusted for various know risk factors, as well as age and gender, in our multivariate analysis, residual confounding may still be present.
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5. Conclusion Ethnic differences are evident in circulating markers of angiogenesis, which may reflect susceptibility of particular groups to a greater predilection to cardiovascular disease. However, once cardiovascular disease or its risk factors are present, the value of these plasma markers diminishes. Acknowledgements We are grateful to all the subjects and the Department of Vascular Surgery, Sandwell & West Birmingham Hospitals NHS Trust. We thank the British Heart Foundation for part-funding of this project (PG/05/036). The authors of this manuscript have certified that they comply with the Principles of Ethical Publishing in the International Journal of Cardiology. References [1] Sluimer JC, Daemen MJ. Novel concepts in atherogenesis: angiogenesis and hypoxia in atherosclerosis. J Pathol 2009;218:7–29. [2] Post S, Peeters W, Busser E, et al. Balance between angiopoietin-1 and angiopoietin-2 is in favour of angiopoietin-2 in atherosclerotic plaques with high microvessel density. J Vasc Res 2008;45:244–50. [3] Moreno PR, Purushothaman KR, Fuster V, et al. Plaque neovascularisation is increased in ruptures atherosclerotic lesions of human aorta: implications for plaque vulnerability. Circulation 2004;110:2032–8. [4] Kolodgie FD, Gold HK, Burke AP, et al. Intraplaque haemorrhage and progression of coronary atheroma. New Engl Med J 2003;349:2316–25. [5] McCarthy MJ, Loftus IM, Thimpson MM, et al. Angiogenesis and the atherosclerotic carotid plaque: an association between symptomatology and plaque morphology. J Vasc Surg 1999;30:261–8. [6] Mofidi R, Crotty TB, McCarthy P, Sheehan SJ, Mehigan D, Keaveny TV. Association between plaque instability, angiogenesis and symptomatic carotid occlusive disease. Br J Surg 2001;88:945–50. [7] Blann AD, Belgore FM, McCollum CN, Silverman S, Lip PL, Lip GY. Vascular endothelial growth factor and its receptor, Flt-1, in the plasma of patients with coronary or peripheral atherosclerosis, or Type II diabetes. Clin Sci 2002;102:187–94. [8] Chong AY, Caine GJ, Freestone B, Blann AD, Lip GY. Plasma angiopoietin-1, angiopoietin-2, and angiopoietin receptor tie-2 levels in congestive heart failure. J Am Coll Cardiol 2004;43:423–8. [9] Van der Heijen M, Van Nieuw Amerongen GP, Van Hinsbergh VWM, Groeneveld ABJ. The interaction of soluble Tie2 with angiopoietins and pulmonary vascular permeability in septic and nonseptic critically ill patients. Shock 2010;33:263–8. [10] Felmelden DC, Blann AD, Lip GYH. Angiogenesis: basic pathophysiology and implications for disease. Eur Heart J 2003;24:586–603. [11] Stetler-Stevenson WG. Matrix metalloproteinases in angiogenesis: moving target for therapeutic intervention. J Clin Invest 1999;103:1237–41. [12] Findley CM, Mitchell RG, Discha BD, Annex BH, Kontos CD. Plasma levels of soluble Tie2 and VEGF distinguish critical limb ischaemia from intermittent claudication in patients with peripheral arterial disease. J Am Coll Cardiol 2008;52:387–93. [13] Felmelden DC, Spencer CG, Belgore FM, Blann AD, Beevers DG, Lip GYH. Endothelial damage and angiogenesis in hypertensive patients: relationship to cardiovascular risk factors and risk factor management. Am J Hypertens 2003;16:11–20.
[14] Felmeden DC, Spencer CGC, Chung N, et al. Relation of thrombogenesis in systemic hypertension to angiogenesis and endothelial damage/dysfunction (a substudy of the Anglo-Scandinavian Cardiac Outcomes Trial [ASCOT]). Am J Cardiol 2003;92: 400–5. [15] Nadar SK, Blann A, Beevers DG, Lip GY. Abnormal angiopoietins 1&2, angiopoietin receptor Tie-2 and vascular endothelial growth factor levels in hypertension: relationship to target organ damage [a sub-study of the Anglo-Scandinavian Cardiac Outcomes Trial (ASCOT)]. J Intern Med 2005;258:336–43. [16] Lim HS, Blann AD, Chong AY, Freestone B, Lip GY. Plasma vascular endothelial growth factor, angiopoietin-1, and angiopoietin-2 in diabetes: implications for cardiovascular risk and effects of multifactorial intervention. Diabetes Care 2004;27:2918–24. [17] Lee KW, Lip GY, Blann AD. Plasma angiopoietin-1, angiopoietin-2, angiopoietin receptor tie-2, and vascular endothelial growth factor levels in acute coronary syndromes. Circulation 2004;110:2355–60. [18] Bond MD, Valee BL. Isolation and sequencing of mouse angiogenin DNA. Biochem Biophys Res Commun 1990;171:988–95. [19] Burgmann H, Hollenstein U, Maca T, et al. Increased serum laminin and angiogenin concentrations in patients with peripheral arterial occlusive disease. J Clin Pathol 1996;49:508–10. [20] Tello-Montoliu A, Marín F, Patel J, et al. Plasma angiogenin levels in acute coronary syndromes: implications for prognosis. Eur Heart J 2007;28:3006–11. [21] Bennett PC, Silverman SH, Gill PS, Lip GYH. Ethnicity and peripheral artery disease. QJM 2009;102:3–16. [22] Allison MA, Criqui MH, McClelland RL, et al. The effect of novel cardiovascular risk factors on the ethnic-specific odds for peripheral arterial disease in the Multi-Ethnic Study of Atherosclerosis (MESA). J Am Coll Cardiol 2006;48: 1190–7. [23] Gill PS, Davis R, Davies, Freemantle N, Lip GYH. Rationale and study design of a cross sectional study documenting the prevalence of Heart Failure amongst the minority ethnic communities in the UK: the E-ECHOES Study (Ethnic-Echocardiographic Heart of England Screening Study). BMC Cardiovasc Disord 2009;9:47. [24] Machin D, Campbell M. Statistical Tables for the Design of Clinical Trials. Oxford: Blackwell Scientific; 1987. [25] Idriss NK, Lip GY, Balakrishnan B, Jaumdally R, Boos CJ, Blann AD. Plasma haemoxygenase-1 in coronary artery disease. A comparison with angiogenin, MMP-9, TIMP-1 and vascular endothelial growth factor. Thromb Haemost 2010;104:1029–37. [26] Tello-Montoliu A, Patel JV, Lip GY. Angiogenesis; a review of the pathophysiology and potential clinical applications. J Thromb Haemost 2006;4:1864–74. [27] Khurana R, Simons M, Martin JF, Zachary IC. Role of angiogenesis in cardiovascular disease: a critical appraisal. Circulation 2005;112:1813–24. [28] Gill PS, Kai J, Bhopal RS, Wild S; Health Care Needs Assessment: Black and Minority Ethnic Groups. In: Raftery J, Stevens A, Mant J, editors. Health care needs assessment. The Epidemiologically Based Needs Assessment Reviews. Third SeriesAbingdon: Radcliffe Publishing Ltd; 2007. [29] Lieb W, Zachariah JP, Xanthakis V, et al. Clinical and genetic correlates of circulating angiopoietin-2 and soluble Tie-2 in the community. Circ Cardiovasc Genet 2010;3: 300–6. [30] Belgore FM, Lip GY, Blann AD. Vascular endothelial growth factor and its receptor, Flt-1, in smokers and non-smokers. Br J Biomed Sci 2000;57:207–13. [31] Jeyabalan A, Powers RW, Durica AR, Harger GF, Roberts JM, Ness RB. Cigarette smoke exposure and angiogenic factors in pregnancy and preeclampsia. Am J Hypertens 2008;21:943–7. [32] Wang C, Fu P, Li H, Gao R, Xiu R. Soluble angiopoietin receptor Tie-2 in patients with acute myocardial infarction and its effects on angiogenesis. Clin Hemorheol Microcirc 2005;33:1–10. [33] Jones N, Iljin K, Dumont DJ, Alitalo K. Tie receptors: new modulators of angiogenic and lymphangiogenic responses. Nat Rev Mol Cell Biol 2001;2:257–67.
Contents lists available at SciVerse ScienceDirect
International Journal of Cardiology journal homepage: www.elsevier.com/locate/ijcard
Ethnic/racial differences in circulating markers of angiogenesis and their association with cardiovascular risk factors and cardiovascular disease Philip C. Bennett a, b, Paramjit S. Gill c, 1, Stanley Silverman b, Andrew D. Blann a, Balu Balakrishnan a, Gregory Y.H. Lip a,⁎, 1 a b c
University of Birmingham Centre for Cardiovascular Sciences, City Hospital, Birmingham, B18 7QH, United Kingdom Department of Vascular Surgery, City Hospital, Birmingham, B18 7QH, United Kingdom Primary Care Clinical Sciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, England, United Kingdom
a r t i c l e
i n f o
Article history: Received 12 January 2012 Accepted 17 March 2012 Available online 9 April 2012 Keywords: Ethnic differences Angoipoietin-1 Angiopoietin-2 Soluble Tie-2 receptor Angiogenin Cardiovascular risk factors
a b s t r a c t Objective: To determine (a) whether ethnic/racial differences exist in circulating markers of angiogenesis (Angiopoietin-1 (Ang-1), Angiopoietin-2 (Ang-2), soluble Tie-2 receptor (sTie-2) and Angiogenin) between South Asian (SA; from India, Pakistan and Bangladesh); Black African-Caribbean and White (W) ethnic groups, and (b) associations between these markers in stable cardiovascular disease (CVD) and its risk factors. Patients and methods: We recruited 243 subjects (82 SA, 84 Black and 77 W) with symptomatic and clinically confirmed CVD (n = 108), risk factor controls (with ≥ 1 cardiovascular risk factor, e.g. smoking, diabetes mellitus, dyslipidaemia, hypertension) and with ankle brachial pressure index > 1) (n = 64) and healthy controls free of CVD and risk factors (n = 56). Angiogenic markers were measured by enzyme linked immunoassay. Results: In healthy controls, angiogenin was higher in SA and Black subjects, compared to Whites (p b 0.05). sTie-2 correlated inversely with angiogenin (p = 0.001), was higher in women (p = 0.029) and was lower in smokers (p = 0.007). Overall, age (p = 0.001) was the only independent factor associated with angiopoietin-1. Angiogenin (p = 0.01) and SBP (p = 0.014) were both independently higher in the Black group compared to the White group. Conclusions: Ethnic, racial, and demographic differences are evident in certain circulating markers of angiogenesis. With the exception of an effect of smoking on sTie-2, these differences are not influenced by the presence of other risk factors, nor the presence of stable cardiovascular disease. © 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Cardiovascular disease, manifesting as peripheral arterial disease, coronary artery disease or cerebrovascular disease, is an important consequence of atherosclerosis. Angiogenesis, the formation of new blood vessels, is evident in these conditions and is closely related to atherogenesis, thrombogenesis and the development of intra-plaque vasa vasorum [1,2]. Furthermore, angiogenesis is a feature of disease development and progression [3,4], and is especially frequent in advanced cardiovascular disease and may characterise atherosclerotic lesions at high risk of haemorrhage or rupture [4–6]. Vascular endothelial growth factor (VEGF), acting on the endothelium, is one of the major growth factors driving angiogenesis, and raised plasma levels in diabetes, peripheral artery disease and coronary artery disease is evidence of this increased angiogenic activity [7].
⁎ Corresponding author. Tel.: + 44 121507 5080; fax: + 44 121 5544083. E-mail address: [email protected] (G.Y.H. Lip). 1 These authors contributed equally to this work. 0167-5273/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijcard.2012.03.158
In addition to VEGF, the angiopoietins (Angiopoietin-1 (Ang-1) and Angiopoietin-2 (Ang-2) are additional angiogenic growth factors that are specific for endothelium. Ang-1 and Ang-2 play modulatory roles by binding a common receptor, the endothelial cell-specific tyrosine kinase receptor Tie-2 [8,9], a soluble form of which (i.e. sTie-2) can be detected in plasma. Together, the angiopoietins regulate endothelial cell differentiation: Ang-1 accelerates the maturation of the blood vessel, whilst Ang-2, an antagonist of Ang-1, destabilises the vessel and degrades the basal lamina [10,11]. Plasma levels of Ang-1 and Ang-2 levels are abnormal in, and are associated with, cardiovascular risk in patients with cardiovascular disease [12] and also in other manifestations of atherosclerosis, including heart failure, acute coronary syndromes and hypertension [8,13–17]. Angiogenin is a small polypeptide implicated in angiogenesis normally found in the vasculature, but also in some physiological and pathological conditions, including peripheral and coronary atherosclerosis [18–20]. Elevated angiogenin levels have been found to be higher in those with severe peripheral atherosclerosis, compared to mild and moderate disease [19], and raised levels in acute coronary syndromes predict a poor outcome at six months [20]. Thus, angiogenin
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could potentially be an indicator of endothelial activity related to progression of vascular disease. Given improvements in biochemical techniques there has been an increasing interest in value of biomarkers related to the development of atherosclerosis and its symptomatic complications. Despite ethnic and racial differences having been documented in the prevalence of atherosclerosis [21], much of the research on pathophysiology has been carried out on predominantly White populations [19,20] and limited research has been directed on these biomarkers in relation to ethnic and/or racial susceptibility to this disease [22]. We hypothesised the following: (1) that there would be differences in levels of circulating markers of angiogenesis between South Asian (SA i.e. people originating from India, Pakistan and Bangladesh]), Black African-Caribbean (AC), and White ethic groups, and (2) that Ang-1, Ang-2, soluble Tie-2 receptor (sTie-2) and angiogenin levels would be higher in those with risk factor compared to healthy controls, with a further increase in those with clear cardiovascular disease. 2. Methods Participants were recruited from among those attending cardiovascular disease outpatient clinics (cardiology and peripheral artery disease) and from hypertension clinics. Coronary artery disease (CAD) was defined as myocardial infarction, coronary artery bypass graft, or coronary atherosclerosis on angioplasty. Peripheral artery disease (PAD) was defined as radiologically confirmed disease or peripheral artery surgery (e.g. distal bypass procedures and major limb amputations). Cerebrovascular disease was defined as stroke, transient ischaemia attack, or carotid artery surgery. All patients had stable disease. Risk factor controls and healthy controls were recruited from patients attending hypertension clinics, from relatives of patients attending outpatient clinics, from subjects participating in a community-based screening survey for heart disease [23] and from hospital and university staff. Risk factor controls had at least one cardiovascular risk factor, that is, smoking [current, or ceased with the past year], hypertension [defined as blood pressure >140/90 mm Hg or on treatment], diabetes mellitus [documented or on treatment], and dyslipidaemia [documented or on treatment] but not PAD, defined by an ankle brachial pressure Index (ABPI) > 1.0. Healthy controls were free of all these risk factors. In all subjects blood pressure was measured three times, and the final measurement reported. Height and weight were obtained to provide body mass index as the weight (kg) divided by the square of the height (m). Exclusion criteria for all subjects were infectious diseases, rheumatoid arthritis and other chronic inflammatory disorders, sepsis, malignancy, haemodynamically significant valvular heart disease, atrial fibrillation, renal failure, immunity-modulating drugs and hormone replacement therapy, recurrent venous thromboembolism, congestive cardiac failure, multiple sclerosis and anaemia. The study was approved by Birmingham East, North & Solihull Local Research Ethnics Committee and written informed consent was obtained from all study participants.
the CVD group, we recruited in excess in each group for extra confidence. Statistical analyses were carried out using Minitab version 15 (State Coll, PA, USA).
3. Results We recruited 243 subjects with a mean age of 64.3 (SD 11.9) years. Diabetes was present in 25.9% and 55.1% were male. Breakdown by age, sex, blood pressure, body mass index, clinical grouping, risk factors, cardiovascular territory and use of aspirin according to racial/ethnic group is presented in Table 1. The three groups were matched for age, sex and proportion of subjects who were healthy volunteers, risk factor controls, and those who had CVD or actual risk factors (smoking, hypertension, dyslipidaemia, and diabetes), cardiovascular disease territory and use of aspirin. SBP and DBP were lower in White Europeans compared to the other two groups, whilst BMI was lower in SA compared to the AC group. There were fewer diabetics in the White group compared to the other two groups. The risk factor group consisted of 42 people with a single risk factor, 25 with two risk factors and one person with all four risk factors. Of those with cardiovascular disease, 80 had disease in one territory, 33 had disease in two and 4 patients had disease in all three. Univariate analysis of the angiogenic markers are shown in Table 2. Angiogenin was lower in White compared to SA and to AC groups. Compared to healthy controls, angiopoietin-2 was higher in the risk factor and the cardiovascular disease group. In the 56 healthy controls, only angiogenin varied between the ethnic groups: 6.35 (3.2–8.6) ng/mL in 18 SA, 5.3 (3.7–7.5) ng/mL in 18 AC, and 3.3 (1.7–6.0) in the 20 White (p = 0.043 overall, p b 0.05 SA v White). sTie-2 correlated inversely with angiogenin (r = −0.44, p = 0.001, Fig. 1). sTie-2 was higher in the women (124 (83–170) ng/mL) compared to the men (86 (75–133) ng/mL (p = 0.029). In those with one or more risk factors, none of the research indices were associated with ethnic/racial group. Indeed, the effect of ethic/ racial group on angiogenin seen in the absence of risk factors became not significant (p = 0.087). sTie2 was lower in 16 smokers (87 (61–130) ng/mL) compared to 48 non-smokers (136 (100–166) ng/ mL, p = 0.007), and the effect of sex on sTie2 seen in those free of Table 1 Clinical and demographic details of study participants. South Asian (n = 82)
Africa/Black Caribbean (n = 84)
White European (n = 77)
P-value
Age (years) Male sex (%) SBP (mm Hg) DBP (mm Hg) BMI (kg/m2)
62 [10] 60.7 142 [27] 81 [13] 26.7 [4.4]
66 [13] 48.3 144 [21] 80 [12] 28.9 [5.5]
63 [11] 54.4 134 [18] 76 [9] 27.8 [5.2]
0.146 0.254 0.024 0.020 0.018
Clinical grouping Healthy Controls (%) Risk factor controls (%) Cardiovascular disease (%)
21.9 18.3 59.7
22.6 33.3 44.0
26.0 32.5 41.4
0.102
Risk factors Current smoking (%) Diabetes (%) Hypertension (%) Dyslipidaemia (%)
12.2 31.7 63.4 55.1
21.4 30.9 66.7 46.1
24.7 14.3 57.1 49.4
0.115 0.019 0.450 0.879
Cardiovascular disease territory PAD (%) 30.5 CAD (%) 35.4 CeVD (%) 11.0 Use of aspirin (%) 50.0
34.5 14.3 7.1 46.6
36.4 20.8 6.5 49.3
2.1. Laboratory Venous blood was taken from the antecubital fossa into sodium citrate. Blood was stored on ice for a maximum of 2 hours before being centrifuged at 1500 × g; the plasma supernatant was then frozen at − 70 °C for future batch analysis. Samples were tested for angiogenic markers using commercially available ELISA kits (R&D systems, Abingdon. UK). Intra-assay and inter-assay variances of all assays were b 5% and b 10% respectively. Lower limits of detection were 3.8 ng/ml for Ang-1, 0.1 ng/ml Ang-2, 0.1 ng/ml Angiogenin and 1.0 ng/mL for sTie 2.
2.2. Sample size justification and statistical analysis Data are presented as mean with standard deviation (SD) or median with interquartile range (IQR) for continuous parameters, as distribution defined. Chi-squared test was used for categorical data. One way ANOVA or the Kruskal–Wallis test was used to assess differences between three groups (SA, AC, White, and Healthy controls, risk factors, cardiovascular disease) for continuous variables, with Tukey's post-hoc analysis performed for inter-group differences. Determinants of the four research indices were sought using a general linear model, inputting ethnic group and sex as nominal categories, disease progression state as an ordinal category. When necessary, data was logged for normality. A p-value b 0.02 was deemed significant. Two-sided Spearman correlations were sought with a 1-beta power of 0.85: a correlation coefficient of >0.4 was considered large enough to minimise false positives [24]. We hypothesised a 25%–50% difference in one of the research indices in one of the three nominal groups of SA, AC and Whites, and of a similar increase across the spectrum of health (modelling 100[90–140] U/mL), to risk factors (125[115–165] U/mL) to cardiovascular disease (150[149–190] U/mL). To achieve this at p b 0.001, a sample size of 50 per group is required. However, with four research indices, and expecting greater heterogeneity in
0.154 0.887
Data are % or mean (standard deviation); SBP: systolic blood pressure; DBP: diastolic blood pressure; BMI: body mass index; PAD: peripheral arterial disease; CAD: coronary artery disease; CeVD: cerebrovascular disease. Age, SBP, DBP, BMI analysed by ANOVA, all others by chi-squared.
P.C. Bennett et al. / International Journal of Cardiology 167 (2013) 1247–1250 Table 2 Univariate comparisons of angiogenic markers. (a) Analysis by racial/ethnic group
South Asian (n = 82)
African/Black Caribbean (n = 84)
White European (n = 77)
P-value
Angiopoitein-1 Angiopoietin-2 sTie2 Angiogenin
16.8 1.3 103 240
12.4 [6.0–32.5] 1.2 [0.69–2.65] 119 [80–168] 261 [187–367]
9.4 1.35 127 169
0.246 0.301 0.191 0.01*
(b) Analysis by disease process
Healthy controls (n = 56)
Risk factors (n = 64)
Cardiovascular disease (n = 108)
Angiopoitein-1 Angiopoietin-2 sTie2 Angiogenin
14.8 0.88 117 260
12.4 1.32 126 221
12.3 1.52 114 238
[4.9–33.2] [0.85–1.78] [79–146] [135–386]
[5.2–33.0] [0.69–1.71] [79–158] [137–372]
[5.2–35.8] [0.81–3.1] [87–153] [114–314]
[5.1–21.0] [0.87–3.3] [83–166] [95–319]
[5.3–26.8] [0.9–2.4] [82–162] [135–371]
0.950 0.006** 0.851 0.347
Units for all growth factors, ng/mL. Data analysed by the Kruskall–Wallis Test for nonparametric testing between 3 groups. *Lower in White Europeans compared to South Asians and to Africans/Black Caribbeans (p b 0.05). Multivariate analysis showed this relationship to be present only in those free of risk factors or cardiovascular disease. **Higher in risk factors and cardiovascular disease (p b 0.05). However, the effect of the disease spectrum on angiopoietin-2 disappeared in multivariate analysis.
risk factors also became non-significant. There were no correlations where the coefficient exceeded 0.4. In the 108 cases with stable cardiovascular disease, there was no effect of ethnicity, or of the number of symptomatic cardiovascular territories on the research indices, and no effect of smoking or sex on levels of sTie2. There were no correlations where the coefficient exceeded 0.4. In multivariate analysis, general linear models identified age (p = 0.001) to be the only independent factor associated with angiopoietin-1, whilst ethnic group (p = 0.018) and SBP (p = 0.01) were the only factors associated with angiogenin. In nominal regression analysis, SBP (p = 0.014) and levels of angiogenin (p = 0.01) both remained higher in the AC group compared to the White group. 4. Discussion We report for the first time an analysis of circulating markers of angiogenesis in three different ethnic groups and the effect of risk factors and in stable atherosclerotic disease. We have also investigated the inter-relationships between Ang-1, Ang-2, sTie-2 and angiogenin in patients with CVD, risk factors and healthy subjects. We found angiogenin levels to be significantly lower in healthy White than in both SA and AC groups. This effect disappeared in the presence of risk factors, and in stable cardiovascular disease, indicating some effect of the latter two processes. We, and others, have reported
Scatterplot of sTie-2 (ng/mL) vs Angiogenin (ng/mL)
sTie-2 (ng/mL)
400
300
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raised angiogenin in acute coronary syndromes [19,20,25], but not in stable coronary artery disease [25] in predominantly White populations. We now extend previous work by reporting no change in angiogenin in SA and AC with stable cardiovascular disease. Angiogenin has been thought to be an indicator of endothelial damage related to progression of vascular disease, it binds actin on the surface of endothelial cells, and its plasma levels are dependent on the presence of an angiogenic stimulus [26]. Its levels may therefore be expected to be associated with related molecules, and the correlation with sTie-2 as found in this study may reflects this. Several studies have suggested that angiogenin, and other angiogenic factors, could promote atherosclerosis and potentially de-stabilise plaques by promoting intra-lesional angiogenesis [4,27]. If so, our data suggest that plasma levels do not reflect this process. The higher levels in healthy asymptomatic middle aged (mean age 56 years) SAs and ACs may in part explain the apparently higher cardiovascular disease burden in these ethnic groups when compared to the general population [22,28]. It is also possible that the higher angiogenin levels could in part be attributable to higher blood pressure and/or higher prevalence of diabetes amongst our SA and AC with respect to the White groups. Of note, the relationship between angiogenin and race/ethnicity present in health was only just non-significant in the presence of risk factors (p =0.087), but markedly so in stable cardiovascular disease (p= 0.595), imply an effect to the latter two aspects on the growth factor. Such an effect may be effect of pathophysiology and/or drug therapy on the endothelium. sTie-2 was higher in our middle aged healthy women than in health men, precisely the reverse of a much larger community-based study, although the latter subjects were younger (and the women more likely to be pre-menopausal [mean age 40 years]) and had more disease [29], although in our study this effect disappeared in risk factors and in frank disease. Some of this may be due to the effect of smoking. Notably, this finding parallels that of reduced plasma levels of the receptor for VEGF, Flt-1 in smokers [30]. Curiously, this may have a real cardiovascular effect as smoking, and so lower sFlt-1, protects against pre-eclampsia [31]. Failure to find altered sTie-2 in stable cardiovascular disease contrasts with others who reported raised levels in acute coronary syndromes [32], suggesting that alterations in angiogenic markers are the result of the acute changes. Cleavage of the extra-cellular domain of Tie-2 on endothelial cells occurs in response to inflammation and results in circulating sTie-2, a process which is reduced by hypoxia [9]. In acute inflammatory states, sTie-2 in the plasma is bound by Ang-1 preferentially over Ang-2 due to its higher affinity for the receptor, leading to greater binding of Ang-2 to endothelial Tie-2 and increasing endothelial permeability [9,33]. In hypoxic states, the relatively lower endothelial release of sTie-2, leads to less Ang-1 binding within the plasma and greater endothelial binding, which accelerates vessel maturation [2]. Whether or not these effects which are prominent in cell biology, have any role in the pathophysiology of cardiovascular disease we cannot say. Our only significant correlation was inversely between sTie2 and angiogenin. As there seems to be no clear physiological or pathological relationship between the two, it could be argued that this is spurious. However, the association was far from weak (r = 0.4) so a genuine association may be present.
200
4.1. Limitations 100
0 0
100
200
300
400
500
600
Angiogenin (ng/mL) Fig. 1. Correlation between sTie-2 and angiogenin in the healthy controls.
This study is limited by its cross-sectional design, but it still represents the first study to investigate ethnic differences in circulating markers of angiogenesis and their association with cardiovascular risk factors and frank, but stable, cardiovascular disease. Although we have adjusted for various know risk factors, as well as age and gender, in our multivariate analysis, residual confounding may still be present.
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5. Conclusion Ethnic differences are evident in circulating markers of angiogenesis, which may reflect susceptibility of particular groups to a greater predilection to cardiovascular disease. However, once cardiovascular disease or its risk factors are present, the value of these plasma markers diminishes. Acknowledgements We are grateful to all the subjects and the Department of Vascular Surgery, Sandwell & West Birmingham Hospitals NHS Trust. We thank the British Heart Foundation for part-funding of this project (PG/05/036). The authors of this manuscript have certified that they comply with the Principles of Ethical Publishing in the International Journal of Cardiology. References [1] Sluimer JC, Daemen MJ. Novel concepts in atherogenesis: angiogenesis and hypoxia in atherosclerosis. J Pathol 2009;218:7–29. [2] Post S, Peeters W, Busser E, et al. Balance between angiopoietin-1 and angiopoietin-2 is in favour of angiopoietin-2 in atherosclerotic plaques with high microvessel density. J Vasc Res 2008;45:244–50. [3] Moreno PR, Purushothaman KR, Fuster V, et al. Plaque neovascularisation is increased in ruptures atherosclerotic lesions of human aorta: implications for plaque vulnerability. Circulation 2004;110:2032–8. [4] Kolodgie FD, Gold HK, Burke AP, et al. Intraplaque haemorrhage and progression of coronary atheroma. New Engl Med J 2003;349:2316–25. [5] McCarthy MJ, Loftus IM, Thimpson MM, et al. Angiogenesis and the atherosclerotic carotid plaque: an association between symptomatology and plaque morphology. J Vasc Surg 1999;30:261–8. [6] Mofidi R, Crotty TB, McCarthy P, Sheehan SJ, Mehigan D, Keaveny TV. Association between plaque instability, angiogenesis and symptomatic carotid occlusive disease. Br J Surg 2001;88:945–50. [7] Blann AD, Belgore FM, McCollum CN, Silverman S, Lip PL, Lip GY. Vascular endothelial growth factor and its receptor, Flt-1, in the plasma of patients with coronary or peripheral atherosclerosis, or Type II diabetes. Clin Sci 2002;102:187–94. [8] Chong AY, Caine GJ, Freestone B, Blann AD, Lip GY. Plasma angiopoietin-1, angiopoietin-2, and angiopoietin receptor tie-2 levels in congestive heart failure. J Am Coll Cardiol 2004;43:423–8. [9] Van der Heijen M, Van Nieuw Amerongen GP, Van Hinsbergh VWM, Groeneveld ABJ. The interaction of soluble Tie2 with angiopoietins and pulmonary vascular permeability in septic and nonseptic critically ill patients. Shock 2010;33:263–8. [10] Felmelden DC, Blann AD, Lip GYH. Angiogenesis: basic pathophysiology and implications for disease. Eur Heart J 2003;24:586–603. [11] Stetler-Stevenson WG. Matrix metalloproteinases in angiogenesis: moving target for therapeutic intervention. J Clin Invest 1999;103:1237–41. [12] Findley CM, Mitchell RG, Discha BD, Annex BH, Kontos CD. Plasma levels of soluble Tie2 and VEGF distinguish critical limb ischaemia from intermittent claudication in patients with peripheral arterial disease. J Am Coll Cardiol 2008;52:387–93. [13] Felmelden DC, Spencer CG, Belgore FM, Blann AD, Beevers DG, Lip GYH. Endothelial damage and angiogenesis in hypertensive patients: relationship to cardiovascular risk factors and risk factor management. Am J Hypertens 2003;16:11–20.
[14] Felmeden DC, Spencer CGC, Chung N, et al. Relation of thrombogenesis in systemic hypertension to angiogenesis and endothelial damage/dysfunction (a substudy of the Anglo-Scandinavian Cardiac Outcomes Trial [ASCOT]). Am J Cardiol 2003;92: 400–5. [15] Nadar SK, Blann A, Beevers DG, Lip GY. Abnormal angiopoietins 1&2, angiopoietin receptor Tie-2 and vascular endothelial growth factor levels in hypertension: relationship to target organ damage [a sub-study of the Anglo-Scandinavian Cardiac Outcomes Trial (ASCOT)]. J Intern Med 2005;258:336–43. [16] Lim HS, Blann AD, Chong AY, Freestone B, Lip GY. Plasma vascular endothelial growth factor, angiopoietin-1, and angiopoietin-2 in diabetes: implications for cardiovascular risk and effects of multifactorial intervention. Diabetes Care 2004;27:2918–24. [17] Lee KW, Lip GY, Blann AD. Plasma angiopoietin-1, angiopoietin-2, angiopoietin receptor tie-2, and vascular endothelial growth factor levels in acute coronary syndromes. Circulation 2004;110:2355–60. [18] Bond MD, Valee BL. Isolation and sequencing of mouse angiogenin DNA. Biochem Biophys Res Commun 1990;171:988–95. [19] Burgmann H, Hollenstein U, Maca T, et al. Increased serum laminin and angiogenin concentrations in patients with peripheral arterial occlusive disease. J Clin Pathol 1996;49:508–10. [20] Tello-Montoliu A, Marín F, Patel J, et al. Plasma angiogenin levels in acute coronary syndromes: implications for prognosis. Eur Heart J 2007;28:3006–11. [21] Bennett PC, Silverman SH, Gill PS, Lip GYH. Ethnicity and peripheral artery disease. QJM 2009;102:3–16. [22] Allison MA, Criqui MH, McClelland RL, et al. The effect of novel cardiovascular risk factors on the ethnic-specific odds for peripheral arterial disease in the Multi-Ethnic Study of Atherosclerosis (MESA). J Am Coll Cardiol 2006;48: 1190–7. [23] Gill PS, Davis R, Davies, Freemantle N, Lip GYH. Rationale and study design of a cross sectional study documenting the prevalence of Heart Failure amongst the minority ethnic communities in the UK: the E-ECHOES Study (Ethnic-Echocardiographic Heart of England Screening Study). BMC Cardiovasc Disord 2009;9:47. [24] Machin D, Campbell M. Statistical Tables for the Design of Clinical Trials. Oxford: Blackwell Scientific; 1987. [25] Idriss NK, Lip GY, Balakrishnan B, Jaumdally R, Boos CJ, Blann AD. Plasma haemoxygenase-1 in coronary artery disease. A comparison with angiogenin, MMP-9, TIMP-1 and vascular endothelial growth factor. Thromb Haemost 2010;104:1029–37. [26] Tello-Montoliu A, Patel JV, Lip GY. Angiogenesis; a review of the pathophysiology and potential clinical applications. J Thromb Haemost 2006;4:1864–74. [27] Khurana R, Simons M, Martin JF, Zachary IC. Role of angiogenesis in cardiovascular disease: a critical appraisal. Circulation 2005;112:1813–24. [28] Gill PS, Kai J, Bhopal RS, Wild S; Health Care Needs Assessment: Black and Minority Ethnic Groups. In: Raftery J, Stevens A, Mant J, editors. Health care needs assessment. The Epidemiologically Based Needs Assessment Reviews. Third SeriesAbingdon: Radcliffe Publishing Ltd; 2007. [29] Lieb W, Zachariah JP, Xanthakis V, et al. Clinical and genetic correlates of circulating angiopoietin-2 and soluble Tie-2 in the community. Circ Cardiovasc Genet 2010;3: 300–6. [30] Belgore FM, Lip GY, Blann AD. Vascular endothelial growth factor and its receptor, Flt-1, in smokers and non-smokers. Br J Biomed Sci 2000;57:207–13. [31] Jeyabalan A, Powers RW, Durica AR, Harger GF, Roberts JM, Ness RB. Cigarette smoke exposure and angiogenic factors in pregnancy and preeclampsia. Am J Hypertens 2008;21:943–7. [32] Wang C, Fu P, Li H, Gao R, Xiu R. Soluble angiopoietin receptor Tie-2 in patients with acute myocardial infarction and its effects on angiogenesis. Clin Hemorheol Microcirc 2005;33:1–10. [33] Jones N, Iljin K, Dumont DJ, Alitalo K. Tie receptors: new modulators of angiogenic and lymphangiogenic responses. Nat Rev Mol Cell Biol 2001;2:257–67.