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Exploring soluble urokinase plasminogen activator receptor and its relationship with arterial stiffness in a bi-ethnic population: the SAfrEIC-study
Thrombosis Research 130 (2012) 273–277
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Regular Article
Exploring soluble urokinase plasminogen activator receptor and its relationship with arterial stiffness in a bi-ethnic population: the SAfrEIC-study Aletta E. Schutte a,⁎, Anélda Myburgh a, Michael H. Olsen b, Jesper Eugen-Olsen c, Rudolph Schutte a a b c
Hypertension in Africa Research Team (HART), North-West University, Potchefstroom Campus, South Africa Research Centre for Prevention and Health, Copenhagen University Hospital, Glostrup Clinical Research Centre, Copenhagen University, Hvidovre Hospital, Hvidovre
a r t i c l e
i n f o
Article history: Received 10 August 2011 Received in revised form 3 October 2011 Accepted 30 October 2011 Available online 21 November 2011 Keywords: suPAR cardiovascular African Caucasian ethnicity gender
a b s t r a c t Introduction: Elevated soluble urokinase-type plasminogen activator receptor (suPAR) indicates an inflammatory state caused by conditions such as HIV and cancer. Recently suPAR was identified as an indicator of cardiovascular disease (CVD). CVD is highly prevalent in black South Africans, but the potential role of suPAR is unknown. We investigated suPAR as a possible marker of arterial stiffness in Africans and Caucasians. Methods: This study involved 207 Africans and 314 Caucasians (aged 20–70 yrs). C-reactive protein (CRP) and suPAR were determined in fasting blood samples. We measured blood pressure, pulse wave velocity (PWV) and Windkessel arterial compliance (Cwk). Results: Africans displayed higher suPAR, CRP, PWV and lower Cwk (pb 0.001) compared to Caucasians. SuPAR was elevated in Africans irrespective of gender and smoking. We found strong relationships between PWV and suPAR (r= 0.27; p b 0.001) and Cwk and suPAR (r= −0.39; p b 0.001) in the whole group, but found no independent relationship of any arterial stiffness measure and suPAR in Africans after adjustment for confounders. Caucasian men indicated a weak significant independent association between Cwk and suPAR (β = −0.09; p = 0.028). Conclusion: Africans had higher levels of suPAR and arterial stiffness than Caucasians (pb 0.001), but there was no independent relationship between arterial stiffness and suPAR in the Africans. It is speculated that due to the inflammatory role of suPAR, it will have stronger relationships with atherosclerosis, which has not yet manifested in this relatively young population group. SuPAR may therefore not be an ideal early marker of cardiovascular dysfunction, but may rather indicate established CVD. © 2011 Elsevier Ltd. All rights reserved.
Introduction Cardiovascular disease is globally recognized as a major health concern [1]. It is known that African populations have a higher risk of developing hypertension than Caucasians [2] and is believed to be due, at least in part, to increasing urbanization, and associated obesity and diabetes. This increases the risk of cardiovascular morbidity and mortality dramatically [3], which is currently second to infectious diseases in Africa. Soluble urokinase plasminogen activator receptor (suPAR) is a soluble form of the urokinase plasminogen activator receptor (uPAR) and is found in various body fluids [4]. SuPAR reflects the immune and proinflammatory status of patients [5], mainly caused by human immunodeficiency virus (HIV)-infection [6,7], tuberculosis [8,9], as well as ⁎ Corresponding author at: Hypertension in Africa Research Team (HART), NorthWest University, Private Bag X6001, Potchefstroom, 2520, South Africa. Tel.: + 27 182992444; fax: + 27 182991053. E-mail address: [email protected] (A.E. Schutte). 0049-3848/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2011.10.034
several cancers [10]. Apart from the clear link between suPAR and infectious diseases, recent studies point to its association with atherosclerosis [11] and the development of cardiovascular disease [12], especially in young individuals. In uremic patients, circulating suPAR concentrations were independently associated with carotid intima-media thickness [13,14], a marker of atherosclerotic disease. SuPAR reflects different aspects of inflammation as opposed to C-reactive protein (CRP). SuPAR is less related to anthropometric indices, and therefore characterizes a dysmetabolic phenotype [12]. Most black urban South Africans present multiple risk factors for cardiovascular disease [15]. It is therefore important to identify markers that may explain their increased risk and may contribute to the early detection and prevention of cardio-metabolic abnormalities. To date no evidence is available whether ethnic differences exist regarding suPAR levels and if suPAR relates to cardiovascular function in black South Africans. The aims of this study were therefore, to compare suPAR levels of Africans and Caucasians, as well as to evaluate the associations of suPAR with arterial stiffness measures in an African and Caucasian population.
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Materials and methods Study design The SAfrEIC (South African study on the influence of Sex, Age and Ethnicity on Insulin sensitivity and Cardiovascular function) study was a cross-sectional study, including 750 participants. Inclusion criteria were men and women aged 20–70 years, while pregnant and lactating women were excluded. For the present study a subsample of 207 African and 314 Caucasian participants were included. A total of 229 participants were excluded due to the following reasons: nonfasting (n = 46); use of oral contraception (n = 48); missing data (n = 29); hyperglycemic (n = 6); or previous diagnosis of HIV infection (n = 98) or diabetes (n = 2). All participants signed informed consent forms and participated voluntarily. The study complied with the Declaration of Helsinki 1975 (as revised in 2008) for investigation of human subjects. The Ethics Committee of the North-West University approved the study.
percentile intervals. We compared means, adjusted means and proportions by using independent t-tests, analysis of co-variance and the chi-square test, respectively. Interactions of gender, ethnicity and smoking were tested for the relationships between arterial stiffness measures (pulse wave velocity and arterial compliance) and inflammatory markers (suPAR and CRP). We plotted quartiles of suPAR (log) against age, separately for gender and ethnicity. The following covariates were considered by means of backward stepwise regression analyses for inclusion into the final regression models: age, body mass index, mean arterial pressure, high-density lipoprotein cholesterol (HDL-C), fasting glucose, γ-glutamyl transferase, cotinine, CRP, suPAR, physical activity, use of anti-inflammatory and antihypertensive medication. Forward stepwise regression analyses were performed with either PWV or Cwk as dependent variable within each ethnic and gender group, and the final list of independent variables included: age, body mass index, mean arterial pressure, cotinine, γ-glutamyl transferase, glucose, HDL-C and suPAR.
Questionnaires
Results
Demographic, lifestyle and physical activity questionnaires were used to assess alcohol use, smoking habit and physical activity level.
The Africans and Caucasians were of similar age, however all anthropometric measures were higher in the Caucasians (Table 1). Despite this, the Africans displayed significantly higher blood pressure, pulse wave velocity and lower Windkessel compliance. Caucasian participants showed an unfavorable lipid profile (p b 0.001), whereas Africans used significantly more tobacco products (p b 0.001) and had higher γ-glutamyl transferase (p b 0.001). In addition to hs-CRP levels, suPAR levels were also significantly higher in Africans (P b 0.001). When stratifying the participants according to gender (Table 2), suPAR was significantly higher in African men and women compared to their Caucasian counterparts. This was confirmed after adjustments for various confounders – as shown in Table 2. When dividing our population into smokers and non-smokers (Table 2), suPAR levels were again significantly higher in the African smokers and non-smokers when compared to Caucasians, also after adjusting for cotinine only (p b 0.001), and after adjusting for various confounders. When viewing suPAR (log) according to quartiles of age (Fig. 1), adjusted for body mass index, suPAR of both African men (P b 0.001) and Caucasian men (P b 0.001) and women (P = 0.020) increased significantly with age. Moreover, the suPAR levels of African men and women were significantly higher than those of the Caucasians within each age quartile. We performed single regression analyses between cardiovascular measurements (SBP, DBP, PWV and Cwk) and inflammatory markers (suPAR and CRP) within the whole population group, and found significant correlations between all variables (p b 0.05). Overall the strongest relationships were found with suPAR, namely between Cwk and suPAR (r = −0.39; p b 0.001) and PWV and suPAR (r = 0.27; p b 0.001). Similar relationships with CRP were weaker, namely Cwk and CRP (r = −0.10; p = 0.03) and PWV and suPAR (r = 0.14; p = 0.001). We found a significant interaction of ethnicity regarding the relationship between Cwk and CRP (p = 0.002), and with gender with regards to the relationship between Cwk and suPAR (p b 0.001). Subsequent multivariate analyses were therefore performed separately in gender and ethnic groups. Forward stepwise regression analyses indicated no significant independent relationships between PWV or Cwk with inflammatory markers in African men or women (PWV, African men: R 2 = 0.50, β = 0.05, p = 0.77; African women: R2 = 0.63, β = 0.09, p = 0.22; Cwk African men: R2 = 0.83, β = 0.04, p = 0.38; African women: R 2 = 0.83, β = −0.02, p = 0.09). In Caucasian men and women these relationships were also non-significant, but suPAR contributed significantly towards the variance in Cwk (p= 0.028) in the Caucasian men.
Anthropometric measurements Anthropometric measurements were done according to standard methods described by Marfell-Jones et al. [16], and included height (Invicta, IP 1465, Leicester, UK), weight (Precision Health Scale, A&D Company, Japan) and waist circumference. Cardiovascular measurements Each participant rested for 10 minutes prior to blood pressure measurements. Blood pressure was measured using the OMRON HEM-757 (Omron, Kyoto, Japan) apparatus. Two measurements were taken, with a 5-min rest interval between measurements. Thereafter, the Finometer device (Finapres Measurement Systems, Amsterdam, The Netherlands) [17,18] was connected and a 5-min continuous measurement of Windkessel arterial compliance cardiovascular variables was taken [19]. The carotid dorsalis-pedis pulse wave velocity (PWV) was measured by the same two observers, using the Complior SP (Artech Medical, Pantin, France) on the left side of each subject in the supine position and made use of the subtraction method when calculating the distances. Biochemical analyses Plasma and serum samples were prepared according to standard methods and stored at −80 °C until analyses were performed. Serum C-reactive protein (CRP), lipids, liver enzymes and creatinine were determined with the Konelab 20i™ auto-analyser (Thermo Fisher Scientific, Oy, Vantaa, Finland). Creatinine clearance was estimated using the Cockroft-Gault formula. Plasma (EDTA) suPAR levels were determined using the suPARnostic® ELISA kit (ViroGates, Copenhagen, Denmark). Cotinine analyses were performed with the use of the IMMULITE 2000 Nicotine Metabolite assay (Siemens Medical Solutions Diagnostics Ltd., Los Angeles, CA, USA), a solid-phase competitive chemiluminescent immunoassay (Catalog Number L2KNM6). Statistical analysis We used used Statistica version 10 (Statsoft, Inc., Tulsa, OK, 2010) for statistical analyses. Variables with a non-Gaussian distribution were logarithmically transformed and the central tendency and spread represented by the geometric mean and the 5th and 95th
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Table 1 Physical and metabolic characteristics of the African and Caucasian participants.
Number of participants Gender (men/women) Age (years) Anthropometric measurements Height (m) Weight (kg) Body mass index (kg/m²) Waist circumference (cm) Cardiovascular measurements Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Pulse pressure (mmHg) Windkessel compliance (mL/mmHg) Pulse wave velocity (m/s) Lipid profile High-density lipoprotein cholesterol (mmol/L) Low-density lipoprotein cholesterol (mmol/L) Triglycerides (mmol/L) Estimated creatinine clearance rate (mL/min) Biochemical variables Glucose (mmol/L) γ-glutamyl transferase (U/L) C-reactive protein (mg/L) suPAR (ng/mL) Lifestyle Physical activity index Cotinine (ng/mL) Smoking, n (%) Alcohol use, n (%) Hypertensive, n (%) Medication Anti-hypertensive,n (%) Anti-inflammatory, n (%)
African
Caucasian
P-value
207 116/91 42.5 ± 13.1
314 158/156 41.2 ± 13.0
0.20 0.24
1.64 ± 0.09 61.8 ± 15.7 23.2 ± 6.33 77.8 ± 12.5
1.73 ± 0.09 84.2 ± 19.5 28.0 ± 5.92 88.9 ± 14.9
b b b b
128 ± 21.8 85.3 ± 13.5 42.6 ± 13.1 1.57 ± 0.50 8.32 ± 1.60
120 ± 16.3 78.5 ± 9.98 41.7 ± 10.7 2.14 ± 0.59 7.88 ± 1.17
b 0.001 b 0.001 0.37 b 0.001 b 0.001
1.60 ± 0.59 2.35 ± 0.88 1.02 [0.95;1.09] 108 ± 31.9
1.36 ± 0.42 3.75 ± 1.12 1.31 [1.23;1.39] 134 ± 33.0
b b b b
0.001 0.001 0.001 0.001
5.06 ± 0.76 68.9 [19.3; 432] 2.21 [1.74;2.81] 3.01 [2.86;3.17]
5.53 ± 0.86 32.0 [14.3; 101] 1.23 [1.00;1.50] 2.27 [2.20;2.34]
b b b b
0.001 0.001 0.001 0.001
7.90 ± 1.35 93.0 [74.9;116] 137 (66.5) 152 (73.4) 77 (37.2)
7.57 ± 1.40 15.1 [13.2;17.3] 50 (16) 206 (65.6) 45 (14.3)
0.009 b 0.001 b 0.001 0.059 b 0.001
0 (0) 1 (0.5)
67 (21.4) 22 (7.03)
b 0.001 b 0.001
0.001 0.001 0.001 0.001
Data are arithmetic mean ± SD or geometric mean (5th and 95th percentile intervals) for logarithmically transformed variables. SuPAR, soluble urokinase plasminogen activator receptor. Table 2 Ethnic differences in inflammatory and cardiovascular measurements stratified by gender and smoking status. Men
N SBP (mmHg) DBP (mmHg) PWV (m/s) Cwk (ml/mmHg) suPAR (ng/mL) suPAR (ng/mL)* CRP (mg/L) CRP (mg/L)*
Women
African
Caucasian
116 131 ± 20.4 84.5 ± 13.6 8.58 ± 1.52 1.67 ± 0.52 2.91 [1.66; 4.94] 2.76 [2.59; 2.96] 1.61 [0.01; 21.4] 2.00 [1.34; 2.99]
158 125 ± 13.1 79.7 ± 9.00 8.06 ± 1.14 2.40 ± 0.59 2.15 [1.38; 3.46] 2.31 [2.17; 2.45] 1.23 [0.02; 9.53] 1.01 [0.70; 1.45]
P 0.004 b 0.001 0.001 b 0.001 b 0.001 b 0.001 0.23 0.22
Smokers
N SBP (mmHg) DBP (mmHg) PWV (m/s) Cwk (ml/mmHg) suPAR (ng/mL) suPAR (ng/mL)† CRP (mg/L) CRP (mg/L)†
African
Caucasian
91 124 ± 22.9 86.2 ± 13.2 7.98 ± 1.64 1.44 ± 0.45 3.08 [1.88; 5.72] 2.81 [2.63; 2.99] 3.29 [0.48; 25.3] 2.89 [2.08; 4.04]
156 115 ± 17.7 77.3 ± 10.8 7.69 ± 1.17 1.87 ± 0.46 2.40 [1.66; 3.58] 2.61 [2.48; 2.76] 1.23 [0.02; 15.6] 1.37 [1.04; 1.80]
P b 0.001 b 0.001 0.11 b 0.001 b 0.001 b 0.001 b 0.001 b 0.001
Non-Smokers
African
Caucasian
137 128 ± 18.2 85.4 ± 11.8 8.60 ± 1.50 1.50 ± 0.49 3.25 [3.06; 3.46] 2.93 [2.75; 3.11] 2.34 [0.02; 26.9] 2.67 [1.83; 3.89]
50 119 ± 14.6 78.5 ± 10.5 7.86 ± 1.09 2.33 ± 0.58 2.54 [2.29; 2.81] 2.77 [2.53; 3.03] 1.78 [0.01; 15.9] 1.54 [0.90; 2.66]
Ps 0.002 b 0.001 0.002 b 0.001 b 0.001 b 0.001 0.38 0.38
African
Caucasian
69 127 ± 27.7 85.3 ± 16.4 7.74 ± 1.65 1.68 ± 0.50 2.60 [2.43; 2.78] 2.63 [2.46; 2.80] 2.01 [0.20; 17.4] 2.27 [1.56; 3.25]
263 120 ± 16.6 78.5 ± 9.87 7.88 ± 1.18 2.10 ± 0.58 2.22 [2.15; 2.30] 2.19 [2.12; 2.28] 1.15 [0.05; 11.7] 1.01 [0.82; 1.26]
P 0.02 b 0.001 0.42 b 0.001 b 0.001 b 0.001 0.015 0.004
Data are mean ± SD or geometric mean (5th and 95th percentile intervals) for logarithmically transformed variables. SBP, systolic blood pressure; DBP, diastolic blood pressure, Cwk, Windkessel arterial compliance; suPAR, soluble urokinase plasminogen activator receptor; CRP, high-sensitivity C-reactive protein. *Adjusted for age, body mass index, high-density lipoprotein cholesterol, fasting glucose, γ-glutamyl transferase and cotinine. † Adjusted for age, gender, body mass index, high-density lipoprotein cholesterol, fasting glucose, γ-glutamyl transferase and cotinine.
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0.60
# #
p trend < 0.001*
Log suPAR (ng/ml)
0.55
# #
#
0.50
p trend = 0.163 # #
0.45 #
p trend = 0.020*
p trend < 0.001*
0.40 0.35 0.30
African women Caucasian women
African men Caucasian men
0.25 20
30
40
50
60
20
30
40
50
60
Age (years) Fig. 1. Soluble urokinase plasminogen activator receptor (suPAR) according to age quartiles of African and Caucasian men and women adjusted for body mass index.* Significant differences between quartile 1 and 4 (P b 0.05); # Significant ethnic differences between suPAR levels within a specific quartile (P b 0.05). Bars indicate standard error of mean.
Discussion This is the first study, to our knowledge, to investigate suPAR as a marker of arterial stiffness in an African population. We found that although Africans had significantly higher levels of suPAR and arterial stiffness compared to Caucasians (pb 0.001), there was no independent relationship between arterial stiffness and suPAR in the Africans. Contrarily we found a significant independent relationship between Windkessel compliance and suPAR in Caucasian men (p= 0.028). In 2010 Eugen-Olsen et al. [12] was the first to publish positive results showing a significant independent relationship of plasma suPAR concentration with the development of cancer, cardiovascular disease, type 2 diabetes and mortality in a population from Copenhagen. Additive to their results, Sehestedt et al. [20] confirmed that suPAR is associated with atherosclerotic plaques and cardiovascular events in a population from Copenhagen. These studies highlight the potential importance of suPAR in the development of cardiovascular disease, but were clearly performed in a specific Caucasian population. This urged Eugen-Olsen et al. [12] to state that further validation of their results are needed in other ethnic populations. Our results clearly showed that black South Africans have significantly elevated suPAR concentrations compared to Caucasians when viewing the population as a whole, also irrespective of gender and smoking status. We also adjusted our results for additional confounders such as age, body mass index and fasting glucose, but the result was robust and remained highly significant (pb 0.001). The higher suPAR levels observed along with elevated CRP levels in Africans are not surprising since previous studies have shown that Africans have higher levels of inflammatory markers compared to Caucasians [21,22]. This study therefore confirms this but also adds to current knowledge by showing elevated suPAR levels in Africans. Furthermore, our results correspond well with previous findings that found suPAR to be associated with increasing age [23] with Africans showing higher suPAR in all different age quartiles, independent of body mass index. Eugen-Olsen et al. [12] found that both suPAR concentrations and cardiovascular disease incidence increased with age. In our study this was confirmed. As a possible measure of their risk for cardiovascular disease and type 2 diabetes, the elevated suPAR levels are aligned with South African studies [15] confirming the heavy burden of non-communicable diseases in the urban populations. The African population from the present study originates from an area of low socio-economic status, which may explain their increased risk further due to unemployment, stress and
increased use of tobacco and alcohol products. Also, none of our black study participants used anti-hypertensive medication despite the fact that 37% suffered from hypertension. In an attempt to elucidate a possible mechanism for the development of hypertension in Africans, our second aim was to explore the association of suPAR with arterial stiffness measurements. We hypothesized that due to the elevated cardiovascular risk and arterial stiffness profile observed in Africans, suPAR may contribute towards this model. Despite Africans having significantly elevated suPAR, we did not find any independent relationships between PWV or Windkessel compliance with either inflammatory marker in the African population. Although unadjusted linear regression analyses showed significant associations of PWV and Windkessel compliance with suPAR, these associations were heavily confounded by age, blood pressure and γ-glutamyl transferase. In hemodialysis patients, Pawlak et al. [14] indicated that suPAR was significantly related to the prevalence of cardiovascular disease, due to observed uPAR upregulation at sites of vascular pathologies such as atherosclerotic plaque. This is also supported by Eugen-Olsen et al. [12] and Sehestedt et al. [20] who found an association between elevated suPAR levels and cardiovascular disease (atherosclerotic plaques) in a Caucasian population. But similar to our African population, Sehestedt et al. [20] did not find a significant relationship between PWV and suPAR in their population from Copenhagen. Interestingly, in our group of 158 Caucasian men, we did find a relatively weak, but independent relationship between Windkessel compliance and suPAR (p= 0.028). It is known that the link with cardiovascular measures are stronger in younger individuals, which may explain this relationship found in our study (aged 40 ± 13.0 yrs) compared to the population of Sehestedt et al. [20] who had age ranges between 48 and 56 years. The inconsistency of this relationship in the other subgroups of the present study (and previous studies) may indicate that suPAR is closer linked to the inflammatory processes in the later stages of the development of atherosclerosis, and not as much to arterial stiffness as a measure of the hemodynamic load. The potential implications of these findings are that suPAR may be considered as a marker of cardiovascular disease where disease progression has already taken place, but it may not be considered as an early marker of cardiovascular dysfunction per se. This needs to be explored further in especially African studies where measures of sub-clinical atherosclerosis, such as carotid intima-media thickness, and cardiovascular morbidity and mortality data are available. This study has to be interpreted within the context of its limitations and strengths. This was a cross-sectional study design with relatively
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young and apparently healthy participants. Older participants are needed to investigate suPAR levels with regard to disease development. Although our results are consistent after multiple adjustments, we cannot exclude possible residual confounding. Participants without having tuberculosis or cancer could not be guaranteed due to asymptomaticity and therefore, could have influenced the suPAR levels. The difference in socioeconomic status between the African and Caucasian individuals is another limitation, where Africans had a much lower income than the Caucasians. Due to distinct differences in tobacco use between African and Caucasian participants, it may have confounded the results, although adjustments for serum cotinine levels were made. Thus, the confounding effects of smoking could perhaps not have been adequately adjusted for. Smoking reduces the bioavailability of endothelium-derived nitric oxide [24], augmenting inflammation of the arterial wall [25] and related arterial stiffness [26]. Instead of carotid-femoral PWV, we measured carotid-dorsalis pedis PWV due to participant sensitivity. As with carotid-femoral PWV, carotid-dorsalis pedis PWV is also an indication of the arterial stiffness of the central conduit arteries (as well as the more muscular arteries), and shows a strong correlation with carotid-femoral PWV. Overall, this was the first well-designed study investigating suPAR in an African population, implemented under highly controlled conditions. Plasma suPAR samples are known to be very stable throughout the day [27] and few variations are present in the suPAR levels of HIVinfected patients using antiretroviral therapy [28]. There is evidence that suPAR levels of plasma samples are also not affected by repeated freeze-thaw procedures [29]. These properties make suPAR an attractive measure of cardiovascular risk in African settings, and may contribute to establish disease progression more effectively. To conclude, although African men and women from different age quartiles presented significantly higher blood pressure, arterial stiffness and suPAR concentrations than age-matched Caucasians, we did not find an independent relationship between arterial stiffness and suPAR in the African group. Due to the relatively young age of our study population atherosclerosis has possibly not yet manifested itself, resulting in the absence of a link between inflammatory conditions and measures of arterial stiffness. This could indicate that suPAR may not be an ideal early marker of vascular dysfunction, but may be a more effective marker of established cardiovascular disease. Conflict of interest statement Jesper Eugen-Olsen is a founder, shareholder and board member of ViroGates A/S, Denmark, the company that produces the suPARnostic® assay. Jesper Eugen-Olsen is an inventor on a patent on suPAR and risk. Copenhagen University Hospital Hvidovre, Denmark, owns the patent, which is licensed to ViroGates A/S. Acknowledgements We thank the participants, as well as all supporting staff and postgraduate students for their involvement in this project. We are also grateful to our sources of support: the South African National Research Foundation (GUN 2073040), the Medical Research Council, and the Africa Unit for Transdisciplinary Health Research (AUTHeR) of the North-West University (Potchefstroom Campus), South Africa. References [1] Writing Group MembersLloyd-Jones D, Adams R, Carnethon M, De Simone G, Ferguson TB, et al. Heart disease and stroke statistics - 2009 update: A report from the American Heart Association Statistics Committee and Stroke Statistics Subcommittee. Circulation 2009;119:e21–181. [2] Xue JL, Ma JZ, Louis TA, Collins AJ. Forecast of the number of patients with end-stage renal disease in the United States to the year 2010. J Am Soc Nephrol 2001;12:2753–8.
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Regular Article
Exploring soluble urokinase plasminogen activator receptor and its relationship with arterial stiffness in a bi-ethnic population: the SAfrEIC-study Aletta E. Schutte a,⁎, Anélda Myburgh a, Michael H. Olsen b, Jesper Eugen-Olsen c, Rudolph Schutte a a b c
Hypertension in Africa Research Team (HART), North-West University, Potchefstroom Campus, South Africa Research Centre for Prevention and Health, Copenhagen University Hospital, Glostrup Clinical Research Centre, Copenhagen University, Hvidovre Hospital, Hvidovre
a r t i c l e
i n f o
Article history: Received 10 August 2011 Received in revised form 3 October 2011 Accepted 30 October 2011 Available online 21 November 2011 Keywords: suPAR cardiovascular African Caucasian ethnicity gender
a b s t r a c t Introduction: Elevated soluble urokinase-type plasminogen activator receptor (suPAR) indicates an inflammatory state caused by conditions such as HIV and cancer. Recently suPAR was identified as an indicator of cardiovascular disease (CVD). CVD is highly prevalent in black South Africans, but the potential role of suPAR is unknown. We investigated suPAR as a possible marker of arterial stiffness in Africans and Caucasians. Methods: This study involved 207 Africans and 314 Caucasians (aged 20–70 yrs). C-reactive protein (CRP) and suPAR were determined in fasting blood samples. We measured blood pressure, pulse wave velocity (PWV) and Windkessel arterial compliance (Cwk). Results: Africans displayed higher suPAR, CRP, PWV and lower Cwk (pb 0.001) compared to Caucasians. SuPAR was elevated in Africans irrespective of gender and smoking. We found strong relationships between PWV and suPAR (r= 0.27; p b 0.001) and Cwk and suPAR (r= −0.39; p b 0.001) in the whole group, but found no independent relationship of any arterial stiffness measure and suPAR in Africans after adjustment for confounders. Caucasian men indicated a weak significant independent association between Cwk and suPAR (β = −0.09; p = 0.028). Conclusion: Africans had higher levels of suPAR and arterial stiffness than Caucasians (pb 0.001), but there was no independent relationship between arterial stiffness and suPAR in the Africans. It is speculated that due to the inflammatory role of suPAR, it will have stronger relationships with atherosclerosis, which has not yet manifested in this relatively young population group. SuPAR may therefore not be an ideal early marker of cardiovascular dysfunction, but may rather indicate established CVD. © 2011 Elsevier Ltd. All rights reserved.
Introduction Cardiovascular disease is globally recognized as a major health concern [1]. It is known that African populations have a higher risk of developing hypertension than Caucasians [2] and is believed to be due, at least in part, to increasing urbanization, and associated obesity and diabetes. This increases the risk of cardiovascular morbidity and mortality dramatically [3], which is currently second to infectious diseases in Africa. Soluble urokinase plasminogen activator receptor (suPAR) is a soluble form of the urokinase plasminogen activator receptor (uPAR) and is found in various body fluids [4]. SuPAR reflects the immune and proinflammatory status of patients [5], mainly caused by human immunodeficiency virus (HIV)-infection [6,7], tuberculosis [8,9], as well as ⁎ Corresponding author at: Hypertension in Africa Research Team (HART), NorthWest University, Private Bag X6001, Potchefstroom, 2520, South Africa. Tel.: + 27 182992444; fax: + 27 182991053. E-mail address: [email protected] (A.E. Schutte). 0049-3848/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2011.10.034
several cancers [10]. Apart from the clear link between suPAR and infectious diseases, recent studies point to its association with atherosclerosis [11] and the development of cardiovascular disease [12], especially in young individuals. In uremic patients, circulating suPAR concentrations were independently associated with carotid intima-media thickness [13,14], a marker of atherosclerotic disease. SuPAR reflects different aspects of inflammation as opposed to C-reactive protein (CRP). SuPAR is less related to anthropometric indices, and therefore characterizes a dysmetabolic phenotype [12]. Most black urban South Africans present multiple risk factors for cardiovascular disease [15]. It is therefore important to identify markers that may explain their increased risk and may contribute to the early detection and prevention of cardio-metabolic abnormalities. To date no evidence is available whether ethnic differences exist regarding suPAR levels and if suPAR relates to cardiovascular function in black South Africans. The aims of this study were therefore, to compare suPAR levels of Africans and Caucasians, as well as to evaluate the associations of suPAR with arterial stiffness measures in an African and Caucasian population.
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Materials and methods Study design The SAfrEIC (South African study on the influence of Sex, Age and Ethnicity on Insulin sensitivity and Cardiovascular function) study was a cross-sectional study, including 750 participants. Inclusion criteria were men and women aged 20–70 years, while pregnant and lactating women were excluded. For the present study a subsample of 207 African and 314 Caucasian participants were included. A total of 229 participants were excluded due to the following reasons: nonfasting (n = 46); use of oral contraception (n = 48); missing data (n = 29); hyperglycemic (n = 6); or previous diagnosis of HIV infection (n = 98) or diabetes (n = 2). All participants signed informed consent forms and participated voluntarily. The study complied with the Declaration of Helsinki 1975 (as revised in 2008) for investigation of human subjects. The Ethics Committee of the North-West University approved the study.
percentile intervals. We compared means, adjusted means and proportions by using independent t-tests, analysis of co-variance and the chi-square test, respectively. Interactions of gender, ethnicity and smoking were tested for the relationships between arterial stiffness measures (pulse wave velocity and arterial compliance) and inflammatory markers (suPAR and CRP). We plotted quartiles of suPAR (log) against age, separately for gender and ethnicity. The following covariates were considered by means of backward stepwise regression analyses for inclusion into the final regression models: age, body mass index, mean arterial pressure, high-density lipoprotein cholesterol (HDL-C), fasting glucose, γ-glutamyl transferase, cotinine, CRP, suPAR, physical activity, use of anti-inflammatory and antihypertensive medication. Forward stepwise regression analyses were performed with either PWV or Cwk as dependent variable within each ethnic and gender group, and the final list of independent variables included: age, body mass index, mean arterial pressure, cotinine, γ-glutamyl transferase, glucose, HDL-C and suPAR.
Questionnaires
Results
Demographic, lifestyle and physical activity questionnaires were used to assess alcohol use, smoking habit and physical activity level.
The Africans and Caucasians were of similar age, however all anthropometric measures were higher in the Caucasians (Table 1). Despite this, the Africans displayed significantly higher blood pressure, pulse wave velocity and lower Windkessel compliance. Caucasian participants showed an unfavorable lipid profile (p b 0.001), whereas Africans used significantly more tobacco products (p b 0.001) and had higher γ-glutamyl transferase (p b 0.001). In addition to hs-CRP levels, suPAR levels were also significantly higher in Africans (P b 0.001). When stratifying the participants according to gender (Table 2), suPAR was significantly higher in African men and women compared to their Caucasian counterparts. This was confirmed after adjustments for various confounders – as shown in Table 2. When dividing our population into smokers and non-smokers (Table 2), suPAR levels were again significantly higher in the African smokers and non-smokers when compared to Caucasians, also after adjusting for cotinine only (p b 0.001), and after adjusting for various confounders. When viewing suPAR (log) according to quartiles of age (Fig. 1), adjusted for body mass index, suPAR of both African men (P b 0.001) and Caucasian men (P b 0.001) and women (P = 0.020) increased significantly with age. Moreover, the suPAR levels of African men and women were significantly higher than those of the Caucasians within each age quartile. We performed single regression analyses between cardiovascular measurements (SBP, DBP, PWV and Cwk) and inflammatory markers (suPAR and CRP) within the whole population group, and found significant correlations between all variables (p b 0.05). Overall the strongest relationships were found with suPAR, namely between Cwk and suPAR (r = −0.39; p b 0.001) and PWV and suPAR (r = 0.27; p b 0.001). Similar relationships with CRP were weaker, namely Cwk and CRP (r = −0.10; p = 0.03) and PWV and suPAR (r = 0.14; p = 0.001). We found a significant interaction of ethnicity regarding the relationship between Cwk and CRP (p = 0.002), and with gender with regards to the relationship between Cwk and suPAR (p b 0.001). Subsequent multivariate analyses were therefore performed separately in gender and ethnic groups. Forward stepwise regression analyses indicated no significant independent relationships between PWV or Cwk with inflammatory markers in African men or women (PWV, African men: R 2 = 0.50, β = 0.05, p = 0.77; African women: R2 = 0.63, β = 0.09, p = 0.22; Cwk African men: R2 = 0.83, β = 0.04, p = 0.38; African women: R 2 = 0.83, β = −0.02, p = 0.09). In Caucasian men and women these relationships were also non-significant, but suPAR contributed significantly towards the variance in Cwk (p= 0.028) in the Caucasian men.
Anthropometric measurements Anthropometric measurements were done according to standard methods described by Marfell-Jones et al. [16], and included height (Invicta, IP 1465, Leicester, UK), weight (Precision Health Scale, A&D Company, Japan) and waist circumference. Cardiovascular measurements Each participant rested for 10 minutes prior to blood pressure measurements. Blood pressure was measured using the OMRON HEM-757 (Omron, Kyoto, Japan) apparatus. Two measurements were taken, with a 5-min rest interval between measurements. Thereafter, the Finometer device (Finapres Measurement Systems, Amsterdam, The Netherlands) [17,18] was connected and a 5-min continuous measurement of Windkessel arterial compliance cardiovascular variables was taken [19]. The carotid dorsalis-pedis pulse wave velocity (PWV) was measured by the same two observers, using the Complior SP (Artech Medical, Pantin, France) on the left side of each subject in the supine position and made use of the subtraction method when calculating the distances. Biochemical analyses Plasma and serum samples were prepared according to standard methods and stored at −80 °C until analyses were performed. Serum C-reactive protein (CRP), lipids, liver enzymes and creatinine were determined with the Konelab 20i™ auto-analyser (Thermo Fisher Scientific, Oy, Vantaa, Finland). Creatinine clearance was estimated using the Cockroft-Gault formula. Plasma (EDTA) suPAR levels were determined using the suPARnostic® ELISA kit (ViroGates, Copenhagen, Denmark). Cotinine analyses were performed with the use of the IMMULITE 2000 Nicotine Metabolite assay (Siemens Medical Solutions Diagnostics Ltd., Los Angeles, CA, USA), a solid-phase competitive chemiluminescent immunoassay (Catalog Number L2KNM6). Statistical analysis We used used Statistica version 10 (Statsoft, Inc., Tulsa, OK, 2010) for statistical analyses. Variables with a non-Gaussian distribution were logarithmically transformed and the central tendency and spread represented by the geometric mean and the 5th and 95th
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Table 1 Physical and metabolic characteristics of the African and Caucasian participants.
Number of participants Gender (men/women) Age (years) Anthropometric measurements Height (m) Weight (kg) Body mass index (kg/m²) Waist circumference (cm) Cardiovascular measurements Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Pulse pressure (mmHg) Windkessel compliance (mL/mmHg) Pulse wave velocity (m/s) Lipid profile High-density lipoprotein cholesterol (mmol/L) Low-density lipoprotein cholesterol (mmol/L) Triglycerides (mmol/L) Estimated creatinine clearance rate (mL/min) Biochemical variables Glucose (mmol/L) γ-glutamyl transferase (U/L) C-reactive protein (mg/L) suPAR (ng/mL) Lifestyle Physical activity index Cotinine (ng/mL) Smoking, n (%) Alcohol use, n (%) Hypertensive, n (%) Medication Anti-hypertensive,n (%) Anti-inflammatory, n (%)
African
Caucasian
P-value
207 116/91 42.5 ± 13.1
314 158/156 41.2 ± 13.0
0.20 0.24
1.64 ± 0.09 61.8 ± 15.7 23.2 ± 6.33 77.8 ± 12.5
1.73 ± 0.09 84.2 ± 19.5 28.0 ± 5.92 88.9 ± 14.9
b b b b
128 ± 21.8 85.3 ± 13.5 42.6 ± 13.1 1.57 ± 0.50 8.32 ± 1.60
120 ± 16.3 78.5 ± 9.98 41.7 ± 10.7 2.14 ± 0.59 7.88 ± 1.17
b 0.001 b 0.001 0.37 b 0.001 b 0.001
1.60 ± 0.59 2.35 ± 0.88 1.02 [0.95;1.09] 108 ± 31.9
1.36 ± 0.42 3.75 ± 1.12 1.31 [1.23;1.39] 134 ± 33.0
b b b b
0.001 0.001 0.001 0.001
5.06 ± 0.76 68.9 [19.3; 432] 2.21 [1.74;2.81] 3.01 [2.86;3.17]
5.53 ± 0.86 32.0 [14.3; 101] 1.23 [1.00;1.50] 2.27 [2.20;2.34]
b b b b
0.001 0.001 0.001 0.001
7.90 ± 1.35 93.0 [74.9;116] 137 (66.5) 152 (73.4) 77 (37.2)
7.57 ± 1.40 15.1 [13.2;17.3] 50 (16) 206 (65.6) 45 (14.3)
0.009 b 0.001 b 0.001 0.059 b 0.001
0 (0) 1 (0.5)
67 (21.4) 22 (7.03)
b 0.001 b 0.001
0.001 0.001 0.001 0.001
Data are arithmetic mean ± SD or geometric mean (5th and 95th percentile intervals) for logarithmically transformed variables. SuPAR, soluble urokinase plasminogen activator receptor. Table 2 Ethnic differences in inflammatory and cardiovascular measurements stratified by gender and smoking status. Men
N SBP (mmHg) DBP (mmHg) PWV (m/s) Cwk (ml/mmHg) suPAR (ng/mL) suPAR (ng/mL)* CRP (mg/L) CRP (mg/L)*
Women
African
Caucasian
116 131 ± 20.4 84.5 ± 13.6 8.58 ± 1.52 1.67 ± 0.52 2.91 [1.66; 4.94] 2.76 [2.59; 2.96] 1.61 [0.01; 21.4] 2.00 [1.34; 2.99]
158 125 ± 13.1 79.7 ± 9.00 8.06 ± 1.14 2.40 ± 0.59 2.15 [1.38; 3.46] 2.31 [2.17; 2.45] 1.23 [0.02; 9.53] 1.01 [0.70; 1.45]
P 0.004 b 0.001 0.001 b 0.001 b 0.001 b 0.001 0.23 0.22
Smokers
N SBP (mmHg) DBP (mmHg) PWV (m/s) Cwk (ml/mmHg) suPAR (ng/mL) suPAR (ng/mL)† CRP (mg/L) CRP (mg/L)†
African
Caucasian
91 124 ± 22.9 86.2 ± 13.2 7.98 ± 1.64 1.44 ± 0.45 3.08 [1.88; 5.72] 2.81 [2.63; 2.99] 3.29 [0.48; 25.3] 2.89 [2.08; 4.04]
156 115 ± 17.7 77.3 ± 10.8 7.69 ± 1.17 1.87 ± 0.46 2.40 [1.66; 3.58] 2.61 [2.48; 2.76] 1.23 [0.02; 15.6] 1.37 [1.04; 1.80]
P b 0.001 b 0.001 0.11 b 0.001 b 0.001 b 0.001 b 0.001 b 0.001
Non-Smokers
African
Caucasian
137 128 ± 18.2 85.4 ± 11.8 8.60 ± 1.50 1.50 ± 0.49 3.25 [3.06; 3.46] 2.93 [2.75; 3.11] 2.34 [0.02; 26.9] 2.67 [1.83; 3.89]
50 119 ± 14.6 78.5 ± 10.5 7.86 ± 1.09 2.33 ± 0.58 2.54 [2.29; 2.81] 2.77 [2.53; 3.03] 1.78 [0.01; 15.9] 1.54 [0.90; 2.66]
Ps 0.002 b 0.001 0.002 b 0.001 b 0.001 b 0.001 0.38 0.38
African
Caucasian
69 127 ± 27.7 85.3 ± 16.4 7.74 ± 1.65 1.68 ± 0.50 2.60 [2.43; 2.78] 2.63 [2.46; 2.80] 2.01 [0.20; 17.4] 2.27 [1.56; 3.25]
263 120 ± 16.6 78.5 ± 9.87 7.88 ± 1.18 2.10 ± 0.58 2.22 [2.15; 2.30] 2.19 [2.12; 2.28] 1.15 [0.05; 11.7] 1.01 [0.82; 1.26]
P 0.02 b 0.001 0.42 b 0.001 b 0.001 b 0.001 0.015 0.004
Data are mean ± SD or geometric mean (5th and 95th percentile intervals) for logarithmically transformed variables. SBP, systolic blood pressure; DBP, diastolic blood pressure, Cwk, Windkessel arterial compliance; suPAR, soluble urokinase plasminogen activator receptor; CRP, high-sensitivity C-reactive protein. *Adjusted for age, body mass index, high-density lipoprotein cholesterol, fasting glucose, γ-glutamyl transferase and cotinine. † Adjusted for age, gender, body mass index, high-density lipoprotein cholesterol, fasting glucose, γ-glutamyl transferase and cotinine.
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0.60
# #
p trend < 0.001*
Log suPAR (ng/ml)
0.55
# #
#
0.50
p trend = 0.163 # #
0.45 #
p trend = 0.020*
p trend < 0.001*
0.40 0.35 0.30
African women Caucasian women
African men Caucasian men
0.25 20
30
40
50
60
20
30
40
50
60
Age (years) Fig. 1. Soluble urokinase plasminogen activator receptor (suPAR) according to age quartiles of African and Caucasian men and women adjusted for body mass index.* Significant differences between quartile 1 and 4 (P b 0.05); # Significant ethnic differences between suPAR levels within a specific quartile (P b 0.05). Bars indicate standard error of mean.
Discussion This is the first study, to our knowledge, to investigate suPAR as a marker of arterial stiffness in an African population. We found that although Africans had significantly higher levels of suPAR and arterial stiffness compared to Caucasians (pb 0.001), there was no independent relationship between arterial stiffness and suPAR in the Africans. Contrarily we found a significant independent relationship between Windkessel compliance and suPAR in Caucasian men (p= 0.028). In 2010 Eugen-Olsen et al. [12] was the first to publish positive results showing a significant independent relationship of plasma suPAR concentration with the development of cancer, cardiovascular disease, type 2 diabetes and mortality in a population from Copenhagen. Additive to their results, Sehestedt et al. [20] confirmed that suPAR is associated with atherosclerotic plaques and cardiovascular events in a population from Copenhagen. These studies highlight the potential importance of suPAR in the development of cardiovascular disease, but were clearly performed in a specific Caucasian population. This urged Eugen-Olsen et al. [12] to state that further validation of their results are needed in other ethnic populations. Our results clearly showed that black South Africans have significantly elevated suPAR concentrations compared to Caucasians when viewing the population as a whole, also irrespective of gender and smoking status. We also adjusted our results for additional confounders such as age, body mass index and fasting glucose, but the result was robust and remained highly significant (pb 0.001). The higher suPAR levels observed along with elevated CRP levels in Africans are not surprising since previous studies have shown that Africans have higher levels of inflammatory markers compared to Caucasians [21,22]. This study therefore confirms this but also adds to current knowledge by showing elevated suPAR levels in Africans. Furthermore, our results correspond well with previous findings that found suPAR to be associated with increasing age [23] with Africans showing higher suPAR in all different age quartiles, independent of body mass index. Eugen-Olsen et al. [12] found that both suPAR concentrations and cardiovascular disease incidence increased with age. In our study this was confirmed. As a possible measure of their risk for cardiovascular disease and type 2 diabetes, the elevated suPAR levels are aligned with South African studies [15] confirming the heavy burden of non-communicable diseases in the urban populations. The African population from the present study originates from an area of low socio-economic status, which may explain their increased risk further due to unemployment, stress and
increased use of tobacco and alcohol products. Also, none of our black study participants used anti-hypertensive medication despite the fact that 37% suffered from hypertension. In an attempt to elucidate a possible mechanism for the development of hypertension in Africans, our second aim was to explore the association of suPAR with arterial stiffness measurements. We hypothesized that due to the elevated cardiovascular risk and arterial stiffness profile observed in Africans, suPAR may contribute towards this model. Despite Africans having significantly elevated suPAR, we did not find any independent relationships between PWV or Windkessel compliance with either inflammatory marker in the African population. Although unadjusted linear regression analyses showed significant associations of PWV and Windkessel compliance with suPAR, these associations were heavily confounded by age, blood pressure and γ-glutamyl transferase. In hemodialysis patients, Pawlak et al. [14] indicated that suPAR was significantly related to the prevalence of cardiovascular disease, due to observed uPAR upregulation at sites of vascular pathologies such as atherosclerotic plaque. This is also supported by Eugen-Olsen et al. [12] and Sehestedt et al. [20] who found an association between elevated suPAR levels and cardiovascular disease (atherosclerotic plaques) in a Caucasian population. But similar to our African population, Sehestedt et al. [20] did not find a significant relationship between PWV and suPAR in their population from Copenhagen. Interestingly, in our group of 158 Caucasian men, we did find a relatively weak, but independent relationship between Windkessel compliance and suPAR (p= 0.028). It is known that the link with cardiovascular measures are stronger in younger individuals, which may explain this relationship found in our study (aged 40 ± 13.0 yrs) compared to the population of Sehestedt et al. [20] who had age ranges between 48 and 56 years. The inconsistency of this relationship in the other subgroups of the present study (and previous studies) may indicate that suPAR is closer linked to the inflammatory processes in the later stages of the development of atherosclerosis, and not as much to arterial stiffness as a measure of the hemodynamic load. The potential implications of these findings are that suPAR may be considered as a marker of cardiovascular disease where disease progression has already taken place, but it may not be considered as an early marker of cardiovascular dysfunction per se. This needs to be explored further in especially African studies where measures of sub-clinical atherosclerosis, such as carotid intima-media thickness, and cardiovascular morbidity and mortality data are available. This study has to be interpreted within the context of its limitations and strengths. This was a cross-sectional study design with relatively
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young and apparently healthy participants. Older participants are needed to investigate suPAR levels with regard to disease development. Although our results are consistent after multiple adjustments, we cannot exclude possible residual confounding. Participants without having tuberculosis or cancer could not be guaranteed due to asymptomaticity and therefore, could have influenced the suPAR levels. The difference in socioeconomic status between the African and Caucasian individuals is another limitation, where Africans had a much lower income than the Caucasians. Due to distinct differences in tobacco use between African and Caucasian participants, it may have confounded the results, although adjustments for serum cotinine levels were made. Thus, the confounding effects of smoking could perhaps not have been adequately adjusted for. Smoking reduces the bioavailability of endothelium-derived nitric oxide [24], augmenting inflammation of the arterial wall [25] and related arterial stiffness [26]. Instead of carotid-femoral PWV, we measured carotid-dorsalis pedis PWV due to participant sensitivity. As with carotid-femoral PWV, carotid-dorsalis pedis PWV is also an indication of the arterial stiffness of the central conduit arteries (as well as the more muscular arteries), and shows a strong correlation with carotid-femoral PWV. Overall, this was the first well-designed study investigating suPAR in an African population, implemented under highly controlled conditions. Plasma suPAR samples are known to be very stable throughout the day [27] and few variations are present in the suPAR levels of HIVinfected patients using antiretroviral therapy [28]. There is evidence that suPAR levels of plasma samples are also not affected by repeated freeze-thaw procedures [29]. These properties make suPAR an attractive measure of cardiovascular risk in African settings, and may contribute to establish disease progression more effectively. To conclude, although African men and women from different age quartiles presented significantly higher blood pressure, arterial stiffness and suPAR concentrations than age-matched Caucasians, we did not find an independent relationship between arterial stiffness and suPAR in the African group. Due to the relatively young age of our study population atherosclerosis has possibly not yet manifested itself, resulting in the absence of a link between inflammatory conditions and measures of arterial stiffness. This could indicate that suPAR may not be an ideal early marker of vascular dysfunction, but may be a more effective marker of established cardiovascular disease. Conflict of interest statement Jesper Eugen-Olsen is a founder, shareholder and board member of ViroGates A/S, Denmark, the company that produces the suPARnostic® assay. Jesper Eugen-Olsen is an inventor on a patent on suPAR and risk. Copenhagen University Hospital Hvidovre, Denmark, owns the patent, which is licensed to ViroGates A/S. Acknowledgements We thank the participants, as well as all supporting staff and postgraduate students for their involvement in this project. We are also grateful to our sources of support: the South African National Research Foundation (GUN 2073040), the Medical Research Council, and the Africa Unit for Transdisciplinary Health Research (AUTHeR) of the North-West University (Potchefstroom Campus), South Africa. References [1] Writing Group MembersLloyd-Jones D, Adams R, Carnethon M, De Simone G, Ferguson TB, et al. Heart disease and stroke statistics - 2009 update: A report from the American Heart Association Statistics Committee and Stroke Statistics Subcommittee. Circulation 2009;119:e21–181. [2] Xue JL, Ma JZ, Louis TA, Collins AJ. Forecast of the number of patients with end-stage renal disease in the United States to the year 2010. J Am Soc Nephrol 2001;12:2753–8.
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