The relationship between serum uric acid level and concentration of proangiogenic mononuclear progenitor cells in patients with chronic heart failure

Correspondence: Alexander E. Berezin, Cardiology Unit, Internal Medicine Department, State Medical University, 26, Mayakovsky av., Zaporozhye, Postcode, Ukraine. Email: dr_berezin@mail.ru Received: 15.04.2014, Accepted: 24.12.2014 Copyright © JCEI / Journal of Clinical and Experimental Investigations 2014, All rights reserved JCEI / 2014; 5 (4): 511-520 Journal of Clinical and Experimental Investigations doi: 10.5799/ahinjs.01.2014.04.0449


Conclusion:
Circulated level of proangiogenic MPCs is declined progressively depended on quartiles of serum UA level in CHF subjects. We suggest that mild elevation of serum UA might be considered as a predictor of low proangiogenic MPCs in CHF patients. J Clin Exp Invest 2014; 5 (4): 511-520

INTRODUCTION
Chronic heart failure (CHF) has been remained a potential fatal complication of any cardiovascular diseases and it is characterized by a systemic inflammatory response that leads to end organ damage [1]. Serum uric acid (SUA) has been shown to be an independent predictor of outcome in the general population and in patients with CHF, the metabolic syndrome, type 2 diabetes mellitus (T2DM), and atherosclerosis, chronic kidney disease [2,3]. Although sustained hyperuricaemia is considered as independent adverse factor in CHF-related mortality [4], a causal role of SUA is not yet to be established [5]. It has been suggested that there is a relationship between SUA as a "phenotypical" marker of metabolic disorders and a tenderness of reparative processes affected vascular wall and contributed endothelial function. However, less is known about the association between SUA level and circulating mononuclear progenitor cells (MPCs), which have an effect on angiogenesis and tissue reparation [6]. Currently it is well established that MPCs might be recruited resulting in proinflammatory cytokines production that are suitable for CHF [7,8]. Substantially, many studies have demonstrated that level of MPCs is declined progressively in the peripheral circulation with increasing severity of symptomatic CHF [9][10][11]. However, we have been postulated that depletion numerous and functional disability of MPCs in circulation may link SUA with inflammatory response and outcomes in CHF. Therefore, CD34+ MPC populations are not related to cardiovascular remodeling or clinical outcome in CHF patients [11,12]. Recent evidence suggests circulating proangiogenic CD14+CD309+ and CD14+CD309+Tie2+ MPCs levels are decreased in patients with stable CHF [13], but an association of MPCs level with SUA irrespective to clinically significant hyperuricemia is still not investigated.
The objective of this study was to evaluate a relationship between serum uric acid level and circulating proangiogenic MPCs in patients with ischemic mild-to-severe CHF.

Study population
The study population was structured retrospectively after determining the coronary artery disease (CAD) by contrast-enhanced spiral computed tomography angiography in 126 subjects with symptomatic ischemic mild-to-severe CHF. Chronic heart failure (CHF) was diagnosed according to current clinical guidelines [14]. All patients were Caucasians, have given their written informed consent for participation in the study and met the following inclusion criteria: Q-wave and non-Q-wave MI within 3 months prior to study enrolment; severe kidney and liver diseases that may affect clinical outcomes; malignancy; creatinin plasma level above 440 μmol/L; estimated glomerular filtration rate (GFR) < 35 ml/ min/м 2 ; brain injury within 3 months prior to study enrolment; pulmonary edema; tachyarrhythmia; valvular heart disease; thyrotoxicosis; ischemic stroke; intracranial hemorrhage; acute infections; surgery; trauma; all the ischemic events within 3 previous months; inflammations within a previous month; neoplasm; pregnancy; implanted pacemaker, any disorder that, according to investigators, might discontinue patient's participation in the study; and patient's refusal to participate in the study or to give his consent for it.
The study was approved by an institutional review committee. The investigators followed strictly all the requirements to clinical trials in conformity with the World Medical Association Declaration of Helsinki, 1964, Good Clinical Practice provided by International Conference on Harmonization (GCP-ICH), Council of Europe Convention for the Protection of. Human Rights and Dignity of the Human Being in view of using achievements in biology and medicine, Convention on Human Rights and Biomedicine, including Additional Protocol to the Convention on Human Rights and Biomedicine, concerning Biomedical Research, and legislation of Ukraine.

Methods for visualization of coronary arteries
Multispiral computed tomography angiography (n=74) and/or angiographic study (n=52) have been carried out to verify the ischemic origin of CHF and have been performed for all patients prior to their inclusion in the study. When atherosclerotic lesions of the coronary arteries were verified, patients were subjected to conventional angiographic examination provided indications for revascularization were available. CAD was considered to be diagnosed upon availability of previous angiographic examinations carried out not later than 6 months ago provided no new cardiovascular events occurred for this period, and the procedure are available for assay. The coronary artery wall structure was measured by means of contrast spiral computed tomography angiography [15] on Somatom Volum Zoom scanner (Siemens, Erlangen, Germany) with two detector rows when holding patient's breathe at the end of breathing in. After preliminary native scanning, nonionic contrast Omnipak (Amersham Health, Ireland) was administered for the optimal image of the coronary arteries. To reconstruct the image, 0.6-mmwidth axial tomographic slices were used.

Echocardiography examination
Transthoracic ultrasonic echocardiography was performed according to a conventional procedure on ACUSON apparatus, SIEMENS, Germany, in В-mode regimen and tissue Doppler echocardiography regimen from parasternal, subcostal, and apical positions over the short and long axis with probe Р of 5 МHz. Left ventricular end-diastolic and endsystolic volumes were measured by modified Simpson's planimetric method. Left ventricular ejection fraction (LVEF) was assessed in compliance with the requirements of American Society of Echocardiography [16]. Tissue Doppler echocardiography was carried out in 4-, 3-and 2-chamber projections in each of 16 segments of the left ventricle and in 4 spots of the mitral annulus: at the base of posterior septal, lateral, inferior, and anterior left ventricular walls [17].

Calculation of glomerular filtration rate
Calculation of glomerular filtration rate (GFR) was carried out using MDRD-6 formula [18].

Blood sampling and biomarker measurements
Venous blood samples were drawn in the fasting state in the morning (at 7-8 a.m.) at baseline into cooled silicone test tubes to detect serum uric acid, N-terminal pro-brain natriuretic peptide (NTpro-BNP), total cholesterol and cholesterol fractions, any biochemical parameters. Samples were processed according to the recommendations of the manufacturer of the analytical technique used. They were centrifuged upon permanent cooling at 6,000 rpm for 3 minutes. Then, plasma was refrigerated immediately to be stored at a temperature not higher than -35°С.

Serum uric acid level measurement
Serum uric acid level was measured by enzymatic methods using chemical analyzer Beckman Synchron LX20. Analytical Range average for serum uric acid was 0.5-82 mmol / L.

NT-pro-Brain Natriuretic Peptide level measurement
NT-pro-BNP level was measured by immunoelectrochemiluminesence method using sets by R&D Systems (USA) on Elecsys 1010 analyzer (Roche, Mannheim, Germany). Calibration of the assay was performed according to the manufacturer's recommendations and values were normalized to a standard curve.

Cholesterol level measurement
Concentrations of total cholesterol (TC) and high density lipoprotein (HDL) cholesterol were determined with Dimension Clinical Chemistry System (Dade Behring Inc, Newark, NJ). Low density lipoprotein (LDL) cholesterol was calculated using Friedewald formula [19].

Circulating EPCs
The flow cytometric technique (FCT) was used for predictable distinguish circulating cells subsets, which depend on expression of CD45, CD34, CD14, Tie-2, and VEGFR2, using High-Definition Fluorescence Activated Cell Sorter (HD-FACS) methodology [20]. Accordingly, the cells in question were phenotyped on the basis of their forward scatter characteristic (FSC) and side scatter characteristic (SSC) profiles. The cells were directly stained and analyzed for the phenotypic expression of surface proteins using anti-human monoclonal antibodies, including anti-CD45 FITS (BD Biosciences, USA), anti-CD34 FITS (BD Biosciences, USA), anti-VEG-FR-2 known as anti-CD309 (BD Biosciences, USA), anti-Tie2 (BD Biosciences, USA) and anti-CD14 (BD Biosciences, USA). The fluorescence minus one technique was used to provide negative controls and establish positive stain boundaries. After lysis of erythrocytes with UTILIZE wash solution, the samples were centrifuged at 200 g for 15 min; then they were washed twice with PBS and fixed immediately.
Double-or triple-positive events were determined using Boolean principles ('and', 'not', 'or', etc.). Circulating EPCs are defined as CD34 / VEGFR2 positive cells in lack of CD45 expression. 500,000 events were analyzed from each tube. For CD14+ populations, coexpression with Tie-2-and/ or VEGFR-2-was determined using quadrant analysis. Standardized cell counts were presented as a percentage of total white blood cells count, which were identified as the total number of all CD45+ cells.

Statistical analysis
Statistical analysis of the results obtained was carried out in SPSS system for Windows, Version 20  Baseline angiographic and treatment characteristics of patients with CHF are presented in Table  2. Coronary arteries with plaques were determined in 36.5%; 33.3%; and 20.2% for 1 vessel, 2 vessels, 3 and more vessels respectively. All the CHF patients were informed about coronary angiography, and they were treated according to current clinical guidelines with diet, lifestyle modification, and drug therapy that included ACE inhibitors / ARBs, betablockers, mineralocorticoid antagonists, aspirin or other antiagregants, ivabradin, diuretics, as well as statins and metformin if needed. No significant difference between patients related to coronary arteries with plaques determined depending SUA quartiles were found. ACEI/ARBs and aspirin were given for all patients across SUA quartiles in similar proportions. Compared with QI SUA cohort, patients with QII-IV SUA cohorts had a higher prescribing rate of beta-blockers, mineralocorticoid antagonists diuretics (P<0.05), but lower prescribing rate of i/f channel blocker ivabradin, statins (P<0.05). Circulating MPCs level in the study patient population Table 3 shows the incidence of various phenotypes of circulating CD34+ MPCs. There was a significant change in level of circulating MPCs depended on quartiles of SUA. Subjects with higher SUA quartile had significantly lower MPCs counts when compared with patient with low quartiles.  HbA1c, % 6.8 (95% CI=4.1-9.5) 6.8 (95% CI=3.9-8.9) 6.9 (95% CI=3.5-9.6) 6.8 (95% CI=3.7-8.9) 6.9 (95% CI=3. 8

Association between SUA level and biomarkers
The    The predictive value of SUA level with respect to the MPCs with phenotypes CD14+CD309+ and CD14+CD309+Tie2+ in the patients with CHF was performed using ROC-analysis, the results of which are presented in Fig. 1. The findings suggest a high predictive power of SUA in the both models for declining of CD14+CD309+ and CD14+CD309+Tie2+ MPCs in CHF patients. The estimated AUCs (area under curves) were 0.631 (sensitivity = 63.9%; specificity = 56.2%) and 0.687 (sensitivity = 72.2%; specificity = 52.9%) respectively. In this case, the cut-off point for the SUA level that had the best prognostic potential on the risk of decreasing MPCs in both models was 31.5 mmol / L. Thus, these data suggest that for the CHF patient elevation of SUA might be considered as a predictor of lowed proangiogenic MPCs.  The test result variables: CD14 + CD309 + , CD14 + CD309 + Tie2 + has at least one tie between the positive actual state group and the negative actual state group. a. Under the nonparametric assumption b. Null hypothesis: true area = 0.5

DISCUSSION
Previously reports have been predominantly elucidated a relationship between cardiovascular outcomes and documented hyperuricemia in patients with acute and chronic heart failure [3,21,22]. The effects of SUA on all-cause mortality at different SUA cut-offs in CHF patient population was evaluated using meta-regression. There was a linear association between SUA after 7 mg/dL and mortality [23]. Arguing against a pure protective role of SUA in cardiovascular disease [24], we found that levels of SUA remained independently associated with lowed proangiogenic MPCs after adjusting for parameters with known impact on concentrations of MPCs. Moreover, even tendency to increase of SUA in CHF patient population associated with pro-gressively declining proangiogenic MPCs, which have a tremendous tissue repair capacity. Probably, these findings might be taken into consideration to be explaining controversial role of SUA in CHF evolution and outcomes. Really, significant association between high SUA level and BMI, diuretic use, some biomarkers, such as NT-pro-BNP, as wells as with hemodynamic performances (E/Ea and LVEF) even beyond declining eGFR was frequently noted in recent investigations [23,25]. Amin et al. [26] reported that mild elevated SUA levels in patients with systolic CHF is associated with impaired clinical and hemodynamic profile and might be used as a noninvasive indicator of elevated left ventricular filling pressures. Misra et al. have been evaluated the independent impact of CHF status (compensation or decompensation) on SUA levels among men with high cardiovascular risk profile [27]. Investigators found that mild elevated SUA associated with increased risk of CHF decompensation (OR = 1.67; 95% CI 1.21 to 2.32).
Although hyperuricemia predominantly affects men, in our study we have not received a confirmation of differences in SUA between men and women with CHF. Noted, that there was not a documented hyperuricemia (SUA ≥6 mg/dL for women and ≥8 mg/dL for men) especially required treatment in patients enrolled in the study. However, the interpretation of SUA levels for individual CHF patients may be confusing, but even small increased SUA levels at symptomatic CHF might be discussed as a marker of endothelial dysfunction and, probably, as an indicator of tissue repair disorders. Current evidence suggests that SUA could be a marker of oxidative damage in several settings distinguished CHF, such as overweight, obesity, diuretic use. We confirmed a slight linear association SUA with BMI and diuretic use, but direct effect of BMI and diuretic use on number of circulating MPCs was not found. It has predisposed that SUA may realize their capacity for modulating tissue damage through other mechanisms irrespective SUA clearance. Therefore, it is not clear whether this increase in SUA levels may be a counter-regulatory process or a pathophysiological detrimental factor. Because SUA is a product of xanthine oxidase (XO), apoptosis and tissue hypoxia that are suitable for CHF lead to increased purine catabolism, which, in turn, increases XO activity and subsequently SUA levels [28]. Indeed, has a significant association with poor outcomes in CHF patients without CKD but not in those with CKD [28][29][30], suggesting that hyperuricemia may predict poor outcomes when it is primarily a marker of increased XO activity, but not when it is primarily due to impaired renal excretion of uric acid [29]. In controversy of data presented by Filippatos et al (2011) [28], no association between SUA and BMI was found in our study. Diuretics, widely used to treat CHF, increase SUA by stimulating the reabsorption of sodium and urate in the proximal tubule. Although we obtained an association between SUA and diuretics administration, the direct effect of diuretics in depletion of MPCs in patient study population was not determined. Additionally, increased SUA might also associated with coronary artery disease and with its risk factors, such as obesity, hypertension, hypertriglyceridemia, dyslipidemia and T2DM, and worsen renal function. Multivariable linear regression analyses that was performed for CD34+ phenotypes of MPCs with adjustment for eGFR, LVEF, NYHA, diuretics, and T2DM, has been showed an independent impact of SUA on counts of CD14+CD309+ MPCs and CD14+CD309+Tie2+ MPCs. We suggest that tissue ischemia determines an increase in XO, which leads to an increase in SUA levels, and mediates suppression of recruitment, mobbing, differentiation and functional status of MPCs through Akt / STAT /MAP-kinase mechanisms, that is reflection of chronic inflammatory, oxidative stress and, probably, catabolic state suitable for CHF [31,32].

Study Restrictions
This study has some restrictions. The authors believe that a greater cohort is to be desirable to improve the power of the study. There is a variation in the definition of EPCs, the number of existing cardiovascular risk factors in various patients, and in the interaction between EPCs and other hematopoietic progenitor, inflammatory cells or platelets. The authors suppose that these restrictions might have no significant impact on the study data interpretation.
Finally, significant confounder impacting SUA levels on population of proangiogenic MPCs with involving several pathogenetic mechanisms are predisposed. It is possible to address to new investigations whether relationships between SUA and proangiogenic MPCs are multidimensional, or if they can be associated with clinical outcomes.
In conclusion, circulating level of proangiogenic MPCs is declined progressively depended on quartiles of SUA level in CHF subjects. We suggest that mild elevation of SUA (>31.5 mmol / L) might be considered as a predictor of lowed proangiogenic MPCs in CHF patient population.