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NDT Advance Access originally published online on January 10, 2008
Nephrology Dialysis Transplantation 2008 23(7):2265-2273; doi:10.1093/ndt/gfm943
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© The Author [2008]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.For Permissions, please e-mail: journals.permissions@oxfordjournals.org


Association between endothelial progenitor cell depletion in blood and mild-to-moderate renal insufficiency in stable angina

Andrzej Surdacki1, Ewa Marewicz2, Ewa Wieteska1, Grzegorz Szastak1, Tomasz Rakowski1, Ewa Wieczorek-Surdacka3, Dariusz Dudek1, Juliusz Pryjma2 and Jacek S. Dubiel1

1 2nd Department of Cardiology 2 Department of Immunology 3 Department of Nephrology, Jagiellonian University, Cracow, Poland

Correspondence and offprint requests to: Andrzej Surdacki, 2nd Department of Cardiology, Jagiellonian University, 17 Kopernika Street, 31-501 Cracow, Poland. Tel/Fax: +48-12-636-8039; E-mail: surdacki.andreas{at}gmx.net



   Abstract
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 Abstract
Summary
 Introduction
 Subjects and methods
 Results
 Discussion
 Supplementary data
 References
 
Background. Low blood counts of CD34/kinase-insert domain receptor double-positive cells (CD34+/KDR+ cells)—a leukocytes subpopulation enriched for bone marrow-derived endothelial progenitor cells (EPC)— predict adverse outcomes in coronary artery disease (CAD). The dependence of EPC numbers on the glomerular filtration rate (GFR), another prognostic factor, has not been reported in CAD yet. Our aim was to assess CD34+/KDR+ cell counts versus GFR in stable angina.

Methods. We studied 102 stable angina men with severe angiographic CAD and normal left-ventricular systolic function. CD34+/KDR+ cells were enumerated by flow cytometry.

Results. With lowering GFR, CD34+/KDR+ cell numbers (% of lymphocytes, median and interquartile range) decreased: 0.04 (0.03–0.06), 0.03 (0.02–0.05) and 0.02 (0.01–0.03)% for GFR ≥90, 60–89 and 30–59 ml/min/1.73 m2, respectively (P < 0.001 for trend). CD34+/KDR+ cell counts correlated with GFR (r = 0.25, P = 0.01), CAD extension score (r = –0.20, P = 0.04), soluble form of vascular cell adhesion molecule-1 (sVCAM-1) (r = –0.22, P = 0.03) and homocysteine (r = –0.20, P = 0.04) levels. A GFR <90 ml/min/1.73 m2 was associated with insignificantly higher plasma erythropoietin concentrations (r = –0.22, P = 0.09 for trend) that correlated with haemoglobin levels (r = –0.33, P = 0.01, n = 59). The GFR–CD34+/KDR+ cells relation was attenuated, yet maintained (β = 0.19 ± 0.09, P = 0.04) on adjustment for the remaining multivariate determinants of CD34+/KDR+ cell numbers: sVCAM-1 (β = –0.20 ± 0.09, P = 0.03) and haemoglobin (β = 0.18 ± 0.09, P = 0.05).

Conclusions. Mild-to-moderate renal dysfunction accompanying stable angina is associated with CD34+/KDR+ cell depletion, which partially depends on concomitant endothelial dysfunction and a tendency to anaemia (despite insignificantly higher erythropoietin) irrespective of an angiographic CAD extent. This may exacerbate an imbalance between endothelial injury and EPC-mediated repair, thus contributing to high cardiovascular risk in CAD coexisting with renal insufficiency.

Keywords: coronary artery disease; endothelial progenitor cells; renal insufficiency



   Summary
Top
 Abstract
 Summary
Introduction
 Subjects and methods
 Results
 Discussion
 Supplementary data
 References
 
We described an independent positive correlation between estimated GFR and blood counts of CD34/kinase-insert domain receptor double-positive cells (CD34+/KDR+ cells)—a leukocyte subpopulation enriched for bone marrow-derived endothelial progenitor cells (EPC)—in a relatively homogenous group of 102 subjects with stable angina, angiographically significant coronary artery disease (CAD), normal left-ventricular systolic function and GFR ≥ 30 ml/min/1.73 m2. The relationship was attenuated after adjustment for levels of soluble form of vascular cell adhesion molecule-1 (sVCAM-1) and haemoglobin. This was found despite the fact that a lower GFR was associated with insignificantly higher erythropoietin levels that correlated inversely with haemoglobin. Irrespective of mechanisms involved, CD34+/KDR+ cell depletion in mild-to-moderate renal dysfunction accompanying stable angina may exacerbate an imbalance between endothelial injury and EPC-mediated repair, thus contributing to excessive cardiovascular risk in CAD coexisting with renal insufficiency.



   Introduction
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 Abstract
 Summary
 Introduction
Subjects and methods
 Results
 Discussion
 Supplementary data
 References
 
Chronic kidney disease (CKD) is associated with excessive cardiovascular morbidity [1] and increases cardiovascular event risk independently of and additively to preexistent cardiovascular disease [2]. The estimated glomerular filtration rate (GFR) <60 ml/min/1.73 m2 was a strong predictor of cardiovascular events in 1 120 295 adults, being only slightly attenuated after adjustment for a plethora of coexistent diseases including coronary artery disease (CAD) [3]. There is growing evidence that excessive cardiovascular risk appears already in subjects with even mildly depressed GFR over the above CKD-defining threshold in high-risk populations, especially in acute coronary syndromes (ACS) [4]. The prognostic relevance of mild renal derangement in stable CAD has recently been confirmed in 7311 stable angina patients as serum creatinine contributed to a combined risk score beginning from a cut-off value of 1.15 mg/dl [5], which is equivalent to a respective GFR cut-off value higher than 60 ml/min/1.73 m2 [6].

Low numbers of circulating bone marrow-derived circulating endothelial progenitor cells (EPC) [7]—depressed in the presence of classical atherosclerotic risk factors [8,9] and endothelial dysfunction [9]—independently predict adverse outcomes in CAD [10,11]. It is assumed that a disequilibrium between ongoing endothelial injury and repair—which partially depends on EPC [12,13]—may be responsible for these associations. As clustering of various risk factors impairing nitric oxide (NO) bioavailability is pronounced in CKD [1] and the mobilization of EPC from the bone marrow into the blood is controlled by NO [14], we hypothesized that mild-to-moderate renal insufficiency can negatively affect EPC number in CAD. Negative correlations between mildly-to-moderately depressed GFR and EPC counts were reported exclusively in some [15,16] but not other [17] studies dealing with kidney transplant recipients. Additionally, in advanced CKD EPC numbers were depressed in non-dialyzed subjects [18], whereas either decreased [19–21], increased [22] or unchanged [23] in patients on maintenance haemodialysis. Although no correlation between serum creatinine and EPC count in CAD has been reported [10,11,24–26], GFR values were not provided in these studies and the computation of GFR is superior to creatinine for renal function assessment [6] and cardiovascular risk prediction [27,28], especially at a creatinine level close to the upper limit of the normal range.

Additionally, no clear consensus protocols have been agreed upon with regard to optimal EPC quantification [29]. Moreover, available studies provided inconsistent data on the relation between EPC counts and angiographic CAD extent [8,10,11,24–26] and on the effects of ACS on EPC mobilization [10,11,25,30–33]. ACS subjects frequently demonstrate unstable haemodynamics and in the presence of rapidly fluctuating renal function changes in GFR estimates lag behind changes in real GFR [6].

Our aim was to investigate the relationship between estimated GFR and CD34/kinase-insert domain receptor double-positive cells (CD34+/KDR+ cells) in a relatively homogenous group of subjects with stable angina, angiographically significant CAD and normal left-ventricular systolic function. Decreased counts of circulating CD34+/KDR+ cells—a leukocyte subpopulation enriched for EPC—were associated with adverse outcomes in CAD [10,11] and preclinical atherosclerosis in healthy subjects [34]. It is noteworthy that the only studies which have reported a depressed CD34+/KDR+ cell number in renal dysfunction were dealing with patients on maintenance haemodialysis [21,23]. Surprisingly, after successful renal transplantation Herbrig et al. [23] have shown increases in the EPC count by the in vitro EPC culture assay with no changes in CD34+/KDR+ cell numbers.



   Subjects and methods
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 Abstract
 Summary
 Introduction
 Subjects and methods
Results
 Discussion
 Supplementary data
 References
 
Patients
We studied 102 men hospitalized in our centre for a planned coronary angiography. Inclusion criteria were stable angina (angina class II–III according to the Canadian Cardiovascular Society), a standard therapy (low-dose aspirin, angiotensin-converting enzyme inhibitors (ACEI) and statins) for ≥3 preceding months and angiographically significant CAD defined as the presence of a diameter stenosis of ≥70% of at least one major epicardial artery segment according to the American College of Cardiology/American Heart Association guidelines on stable angina [35].

A wide set of exclusion criteria and requirements concerning therapy was adopted in order to limit the previously reported effects of statins [36], ACEI [37], ACS [10,25,30–33], heart failure [38] and depressed left-ventricular ejection fraction (EF) [11,38] on EPC. Out of 358 eligible patients 256 had been eliminated by the exclusion criteria that included the following: age >75 years, ACS within past 3 months, percutaneous revascularization in past 1 month, any surgery within past 6 months, GFR <30 ml/min/1.73 m2 (calculated according to the simplified Modification of Diet in Renal Disease study equation [6]), significant valvular heart disease, congenital heart disease, EF <50%, heart failure, arterial hypertension uncontrolled adequately by drugs, significant extracoronary atherosclerosis, haemoglobin <11 g/dl, thrombocytopenia (<105/µl), thyroid or liver function abnormalities, malignant or inflammatory diseases, infections within past 2 months and any chronic non-cardiovascular medication.

Blood samples for fluorescence-activated cell sorter analysis and extended biochemical assays were taken after an overnight fast from an antecubital vein on the occasion of routine blood sampling 0–2 days prior to the planned angiography. The Bioethical Committee of our university approved the protocol and the patients gave informed consent.

Quantification of CD34+/KDR+ cells in blood
As described previously [8,10], 100 µl of blood was incubated in the dark (<60 min after venopuncture) with mouse monoclonal antibodies against human vascular endothelial growth factor (VEGF) receptor type-2 (KDR) (Sigma, St Louis, MO, USA) followed by rabbit fluoroscein isothiocyanate (FITC)-labelled secondary antibodies (Dako, Denmark) and phycoerythrin (PE)-conjugated mouse monoclonal antibodies against human CD34 (Becton Dickinson, Franklin Lakes, NJ, USA). Control blood samples were incubated with mouse isotype-matched antibodies (IgG1-FITC and IgG2a-PE, {gamma}1/{gamma}2a Simultest, Becton Dickinson). Following lysis of erythrocytes, data acquisition was performed on a flow cytometer (FACScan, Becton Dickinson GmbH, Heidelberg, Germany) including 100 000 cytometric events. The CD34+/KDR+ cell count was expressed as a percentage of peripheral blood mononuclear cells (PBMC) in the lymphocyte gate (Supplementary Figures 1 and 2) [8,10,11]. As a test of reproducibility, in 10 subjects we determined CD34+/KDR+ cell counts twice on Days 0 and 7 under similar conditions, which revealed a close correlation (r = 0.89, P < 0.001). Our laboratory personnel were unaware of patients’ clinical background.

Angiographic CAD quantification
Coronary angiogram (Philips Integris HM 3000) was assessed by a cardiologist blinded to the clinical data and CD34+/KDR+ cell count. In addition to the number of main coronary vessels with luminal diameter stenoses of ≥70% and the maximal percent diameter stenosis, the CAD extension score (a proportion of the visible coronary arterial tree with angiographically detectable atheroma) was computed following a method proposed by Sullivan et al. [39].

Biochemical assays
In addition to routine measurements, high-sensitivity C-reactive protein (hs-CRP) and homocysteine concentrations were assessed using commercially available chemiluminescent immunoassay systems (Immulite 1000 and Immulite 2000, DPC, Flanders, NJ, USA). Plasma levels of VEGF and soluble form of vascular cell adhesion molecule-1 (sVCAM-1) were measured by enzyme-linked immunoadsorbent assays (R&D Systems, Minneapolis, MN, USA). The lower detection limits for VEGF and sVCAM-1 were 5 pg/ml and 0.6 µg/l, respectively, and intra-assay and inter-assay coefficients of variation 6.7 and 8.8% (VEGF) and 2.3 and 7.8% (sVCAM-1), respectively. In a subgroup of 59 patients, plasma concentrations of erythropoietin were determined by an automated chemiluminescence-based immunoassay (detection limit 1.0 mIU/ml; normal range 3.7–29.5 mIU/ml) (DPC, Flanders, NJ, USA).

Statistical analysis
Data are presented as means ± SD for continuous variables with normal distribution, medians and interquartile ranges (25th to 75th percentile) for not normally distributed parameters (CD34+/KDR+ cell counts, levels of hs-CRP, homocysteine, sVCAM-1 and erythropoietin), and counts (n) (proportions) for categorical variables. The subjects were divided into three groups on the basis of the GFR value according to the National Kidney Foundation [1]. Trend effects across the GFR categories were analysed by Spearman's rank-order correlation coefficients. Intergroup differences in CD34+/KDR+ cell numbers were also assessed by nonparametric tests. For bivariate correlations, Spearman's or Pearson's correlation coefficients (r) were computed. Multivariate determinants of the CD34+/KDR+ cell count were identified by backward stepwise multiple linear regression with logarithmic derivatives of CD34+/KDR+ cell numbers as a dependent variable. Only the variables presenting the P-value of ≤0.20 in the univariate analysis were taken into account in the multiple regression. The F-to-remove value was set at 4.0, which is an approximate variance ratio (F) (variability due to regression divided by variability about the regression line) for >30 degrees of freedom at a P-value of 0.05. As in four patients the cell counts equalled 0, the logarithmic transformation to obtain a normal distribution consisted in the computation of log [raw value (%) + 0.01]. A P-value ≤0.05 was considered significant.



   Results
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 Abstract
 Summary
 Introduction
 Subjects and methods
 Results
Discussion
 Supplementary data
 References
 
Patient characteristics in relation to GFR
Characteristics of the study group stratified by GFR categories are shown in Tables 1 and 2. Across decreasing GFR categories we observed a gradual increase in CAD extension score, the number of major coronary vessels with severe stenoses (Table 1) as well as in levels of sVCAM-1, homocysteine and hs-CRP (Table 2). Haemoglobin was insignificantly lower in those with GFR 30–59 ml/min, whereas erythropoietin at a GFR of ≥90 ml/min (Table 2). As a continuous variable, GFR correlated with CAD extension score (r = –0.28, P = 0.004) and levels of homocysteine (r = –0.26, P = 0.008) and sVCAM-1 (r = –0.23, P = 0.02).


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  		Table 1 Clinical and angiographic data of the patients stratified by GFR categories

 

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  		Table 2 Biochemical parameters of the patients stratified by GFR categories

 
Univariate correlates of CD34+/KDR+ cell counts
CD34+/KDR+ cell counts were correlated with GFR (r = 0.25, P = 0.01) (Figure 1), sVCAM-1 level (r = –0.22, P = 0.03), CAD extension score (r = –0.20, P = 0.04) and homocysteine concentration (r = –0.20, P = 0.04) (Table 3). The relationship between renal function and CD34+/KDR+ cell numbers was also confirmed across the three GFR categories: 0.04 (0.03–0.06), 0.03 (0.02–0.05) and 0.02 (0.01–0.03)% for GFR ≥90, 60–89 and 30–59 ml/min/1.73 m2, respectively (Figure 2). Intergroup differences reached the significance by Spearman's r (P < 0.001 for trend), Kruskal–Wallis ANOVA (P = 0.015) and Mann–Whitney U-tests (Figure 2). Additionally, insignificant tendencies towards correlations between CD34+/KDR+ cell counts and levels of creatinine (r = –0.17, P = 0.08), haemoglobin (r = 0.16, P = 0.11) and erythropoietin (r = –0.19, P = 0.14, n = 59) were found (Table 3). Plasma erythropoietin correlated inversely with haemoglobin concentrations (r = –0.33, P = 0.01, n = 59).


Figure 1
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  		Fig. 1 A significant positive correlation between CD34+/KDR+ cell counts and estimated glomerular filtration rate (GFR) in stable angina. r, Spearman's rank-order correlation coefficient; PBMC, peripheral blood mononuclear cells.

 

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  		Table 3 Univariate correlates of CD34+/KDR+ cell counts

 

Figure 2
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  		Fig. 2 A gradual fall in CD34+/KDR+ cell counts with decreasing categories of the estimated glomerular filtration rate (GFR) in stable angina. Medians, interquartile ranges (25th to 75th percentile) and P-values by Mann–Whitney U-tests have been shown. PBMC, peripheral blood mononuclear cells.

 
Neither white blood cells and lymphocyte counts nor the numbers of CD34+ cells correlated with GFR, CAD extension score, sVCAM-1, homocysteine or haemoglobin levels (r < 0.13, P > 0.2).

Multivariate determinants of CD34+/KDR+ cell counts
By stepwise multivariate linear regression GFR, log-transformed sVCAM-1 levels and haemoglobin concentrations entered the final regression equation describing logarithmic derivatives of CD34+/KDR+ cell counts (Table 4). After removal of sVCAM-1 and haemoglobin levels from the equation, the strength of the GFR–CD34+/KDR+ cells relationship increased (β = 0.25 ± 0.09, P = 0.007), which suggests that the adjustment for sVCAM-1 and haemoglobin concentrations attenuated the association.


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  		Table 4 Multivariate determinants of log-transformed CD34+/KDR+ cell counts (for the final regression model: adjusted R2 = 0.22, P < 0.001)

 


   Discussion
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 Abstract
 Summary
 Introduction
 Subjects and methods
 Results
 Discussion
Supplementary data
 References
 
Our principal finding is that deficiency of CD34+/KDR+ cells in CAD men with stable angina and angiographically proven CAD was proportional to the degree of renal insufficiency, which was in part mediated by factors affecting endothelial function and haemoglobin levels irrespective of an angiographic CAD extent, inflammatory activation or homocysteine accumulation. Although EPC deficiency in proportion to GFR reduction had already been demonstrated in kidney transplant recipients [15,16], this has never been shown in non-dialyzed patients for CD34+/KDR+ cell count, a negative correlate of adverse outcome in CAD [10,11]. To the best of our knowledge, for the first time we have described a depressed CD34+/KDR+ cell number in parallel to the degree of mild-to-moderate renal dysfunction in CAD.

Intergroup differences in patient characteristics as an unlikely cause of the GFR–CD34+/KDR+ cell count relationship
In concordance with previous studies [4,27], our patients with decreased GFR exhibited a significantly wider angiographic CAD extent, elevated homocysteine and hs-CRP as well as insignificant differences, i.e. older age and higher incidence of diabetes and hypertension and lower prevalence of smoking habit (Table 1). Therefore, it might be suggested that the observed relationship could have rather reflected these intergroup differences than be a consequence of renal impairment by itself.

Nevertheless, CD34+/KDR+ cell counts were unrelated to traditional risk factors and hs-CRP, and the significance of effects of CAD extension score and homocysteine disappeared on multivariate adjustment. The observed lack of the hs-CRP–CD34+/KDR+ cell count association is consistent with other studies [10,26] and a negative effect of homocysteine was reported at intergroup differences in homocysteine much higher compared to ours [40].

Comparison with other studies relating EPC to angiographic CAD parameters
Among studies which defined significant coronary narrowings similar to the present work, i.e. diameter stenoses of ≥70–75% [25,26], Kunz et al. [25] observed a lower EPC count—quantified as the number of colony-forming units of endothelial cells (CFU) on Day 7 of culture—in subjects with multivessel CAD. However they were not compared with pure one-vessel CAD (as in our study), but with a pooled group of 51 patients with zero-vessel and 24 patients with one-vessel CAD. Güven et al. [26] found higher CFU numbers on Days 14–28 of culture in stable angina subjects with one severe (≥70%) stenosis with reference to those with less advanced narrowings, whereas a further rise in the number of such stenoses did not affect EPC count, similar to the present study. Following the concept set forth by Güven et al. [26] that severe coronary stenoses (associated with impaired coronary flow reserve [41]) might provoke EPC mobilization via repetitive episodes of coronary ischaemia [42], this factor, if present, was likely to comparably affect all our patients. Indeed, maximal percent coronary stenosis, angina class and VEGF levels were similar across three GFR categories, whereas close correlations between ischaemia-dependent rises in VEGF and subsequent increases in CD34+/KDR+ cell numbers (for 24– 48 h) had been reported after a single episode of exercise-induced myocardial ischaemia in stable angina [42]. Moreover, an extension of this hypothesis [26] may also explain apparent inconsistencies between studies relating EPC to angiographic CAD [8,10,11,24–26]. Accordingly, the non-necrotic tissue ischaemia-driven EPC mobilization [31,42] could be superimposed on chronically depressed EPC number associated with atherogenesis with a variable net EPC count depending on the relative proportion of these contradictory effects in a given patient group. In agreement with this concept, a decreased CD34+/KDR+ cell count was associated with preclinical carotid atherosclerosis in healthy subjects [34], i.e. without evidence of tissue ischaemia.

As to papers which used a 50% cut-off value for significant coronary stenoses [10,11,24], Thum et al. [24] have observed a fall in both CD34+/CD133+ cell counts and CFU numbers after 3 days of culture with an increasing CAD extent. However, Werner et al. [11] have found no such relationship for CD34+/KDR+ cell counts and CFU numbers on Day 7 of culture, whereas in the study by Schmidt-Lucke et al. [10] the relation between the CAD extent and CD34+/KDR+ cells has lost the significance on the multivariate approach, as in our data.

Proposed mechanisms of the positive GFR–CD34+/KDR+ cell count relationship
In the present study the correlation between GFR and CD34+/KDR+ cell count—previously shown to be inversely related to the risk of future cardiovascular events in CAD [10,11]—was not due to different characteristics of patients with decreased GFR, except for a tendency to anaemia. Interestingly, multivariate adjustment that included a similar set of variables (in particular the CAD extent, homocysteine and hs-CRP) only slightly affected the relationship between moderate renal insufficiency and the risk of death or nonfatal myocardial infarction in 1484 consecutive catheterized patients, out of whom 72% presented as stable angina [27]. Accordingly, we hypothesized that both a lower CD34+/KDR+ cell count and excessive cardiovascular risk accompanying renal insufficiency might be due to common factors other than the CAD extent, inflammatory activation or homocysteine accumulation.

In search of these determinants, abnormalities affecting endothelial function and haemoglobin levels are to be considered as adjustment for a marker of endothelial dysfunction (sVCAM-1) and haemoglobin, considerably attenuated the GFR–CD34+/KDR+ relationship. Similarly, in a sample of the Hoorn study, the association between mild-to-moderate renal insufficiency and cardiovascular mortality risk has been markedly weakened on adjustment for biomarkers of endothelial dysfunction including sVCAM-1 [43], which originates mainly from activated endothelial cells on atherosclerotic plaques and reflects leukocyte–endothelial interactions [44]. Additionally, in the ARIC study a prognostic effect of a mildly-to-moderately depressed GFR on the risk of CAD events was much stronger in patients with anaemia, defined as haemoglobin <13.5 g/dl in men [45], i.e. about the mean value in our patients with moderate renal insufficiency.

Among non-traditional risk factors that might precipitate endothelial dysfunction in renal insufficiency, accumulation of dimethylated L-arginine analogues and oxidative stress—factors impairing NO bioavailability—should be mentioned [1]. As NO governs the mobilization of EPC from the bone marrow into the blood [14], derangement of the L-arginine–NO pathway can explain the association of decreased GFR with both lower CD34+/KDR+ cell count and excessive cardiovascular risk. Among dimethylated L-arginine analogues, accumulation of an endogenous NO generation inhibitor, asymmetric dimethyl-L-arginine (ADMA), appears a plausible possibility as this compound is linked to atherosclerotic risk factors, endothelial dysfunction and elevated cardiovascular risk [46,47]. Moreover, Thum et al. [24] have observed an inverse correlation between plasma ADMA and EPC counts in stable angina, whereas ADMA and sVCAM-1 levels were independently correlated in mild-to-moderate renal insufficiency [48]. Additionally, a stereoisomer of ADMA, symmetric dimethyl-L-arginine (SDMA), whose circulating levels increase at earlier stages of renal dysfunction compared to ADMA [49], also inhibits NO synthesis, presumably by the interference with L-arginine uptake by NO-synthesizing cells [50]. Unfortunately, we have not measured ADMA and SDMA, whereas the strength of the GFR–CD34+/KDR+ association, although significant, was very modest.

As to haemoglobin, a tendency towards decreased CD34+/KDR+ cell counts in the presence of lower haemoglobin levels could have reflected relative erythropoietin deficiency, which had previously been demonstrated already at moderate renal dysfunction in 395 CAD patients [51]. Erythropoietin enhances both late erythrocyte development and EPC mobilization [52,53] and CD34+/KDR+ [53] or CD34+/CD133+/KDR+ [54] cell numbers were positively related to erythropoietin levels in CAD. However, relative erythropoietin deficiency is equivalent to the inadequacy of erythropoietin levels for the degree of anaemia (presumably due to a lowered set-point for erythropoietin synthesis), whereas a decrease in absolute erythropoietin concentrations was not found even in severe renal failure [51]. Moreover, with the reference to those with a normal renal function, Radtke et al. [55] reported increased erythropoietin levels irrespective of the stage of renal dysfunction. Similar to the above cited studies [51,55], we observed a tendency to an elevated erythropoietin level in parallel to a lower haemoglobin at a depressed GFR, which presumably reflected an intact regulatory feedback between haemoglobin and erythropoietin synthesis in the study patients, i.e. with a GFR of ≥30 ml/min/1.73 m2 and haemoglobin ≥11 g/dl. Accordingly, lower CD34+/KDR+ cell counts were found despite higher erythropoietin levels. That EPC mobilization from the bone marrow could be more resistant to erythropoietin than erythroid lineage was also suggested by Prunier et al. [56], who observed EPC elevations at a two-fold higher dose of darbepoietin than that necessary to increase haematocrit in rats after experimental myocardial infarction. Interestingly, Thum et al. [24] have reported nearly significantly lower haemoglobin and higher creatinine concentrations (versus controls) in patients with three-vessel CAD who had also the lowest EPC count. That a tendency of anaemia and progenitor cells deficiency could be due to a common mechanism was also suggested by Andreotti et al. [57], who described positive correlations of haemoglobin with GFR and CD34+ progenitor cell numbers in 52 consecutive CAD patients. This indicates that in some clinical settings the association between erythropoietin and EPC mobilization might be obscured by as yet unidentified mediators which interfere with erythropoietin signalling in the bone marrow and probably accumulate at renal dysfunction.

It cannot be excluded that CD34+/KDR+ cell depletion might have been a contributory cause rather than a pure consequence of renal insufficiency, as EPC take part in the maintenance of endothelial integrity in the kidney. Bone marrow-derived cells participated in glomerular endothelial repair after severe damage [58] and intrarenal infusion of culture-modified bone marrow-derived progenitor cells diminished endothelial injury and mesangial activation in experimental glomerulonephritis [59]. Additionally, endothelial chimerism was demonstrated in male-to-female transplanted kidneys irrespective of rejection [60].

Potential clinical relevance of the positive GFR–CD34+/KDR+ cell count relationship
EPC are supposed to participate in ongoing renewal of the endothelial layer. This concept is supported by chimerism of coronary endothelia within the female-to-male transplanted hearts [12], incorporation of labelled EPC into regenerating endothelial layers [13] and the ability of in vitro-differentiated EPC from wild-type animals to persistently correct endothelial function in apolipoprotein E-deficient mice [61]. Therefore EPC deficiency might facilitate the destabilization of atherosclerotic plaques [10,62], the basis for ACS.

Additionally, as circulating EPC participate in the neovascularization of ischaemic tissues [7] and their count was positively related to collateral flow index in one-vessel CAD [63], EPC depletion may contribute to impaired development of coronary collaterals already at GFR <80 ml/min [64], which can also augment cardiovascular risk. The importance of circulating progenitor cells for neovascularization has recently been supported by improvements in coronary flow reserve [65], EF and prognosis [66] after intracoronary administration of heterogeneous bone marrow-derived progenitor cells in acute myocardial infarction.

That the ability of bone marrow to mobilize EPC in response to erythropoietin may translate into beneficial effects can be concluded from experimental studies. Urao et al. [67] observed augmented incorporation of bone marrow-derived EPC into the regenerated endothelium with consequent accelerated reendothelialization after transluminal endothelial injury of the femoral artery in erythropoietin-treated mice. Similarly, in rats receiving erythropoietin after myocardial infarction, Westenbrink et al. [68] described increased homing of EPC to coronary microvessels in the peri-infarction zone with subsequently potentiated neovascularization and improved ventricular function.

In summary, CD34+/KDR+ cell deficiency may exacerbate the disequilibrium between endothelial injury and EPC-mediated repair, thus adding to mechanisms of excessive cardiovascular morbidity in the presence of renal dysfunction and further supporting a need for aggressive prevention in CAD associated with even mildly depressed GFR.

Limitations of the study
First, due to methodological limitations we have not studied functional properties of cultured EPC—their migratory capacity, adhesive properties and differentiation—all of which may be impaired in CKD [18,20,22,23]. Age-related endothelial dysfunction was demonstrated to be linked to the depressed capacity of culture-enriched EPC to migrate and proliferate despite comparable CD34+/KDR+ cell counts in young and elderly subjects [69], which points to the relevance of EPC activity independently of their number.

Second, we have enumerated CD34+/KDR+ cells, whereas the CFU assay and the EPC culture assay are generally preferable for EPC quantification. Additionally, it has been suggested that CD133-positivity, typical for less mature EPC, should be included when defining EPC by flow cytometry [70]. Nevertheless, no clear consensus protocols have been agreed upon with regard to optimal EPC quantification [70]. In healthy subjects, George et al. [29] have observed the lack of correlation between the number of endothelial cell colonies enumerated after 7 days of culture and flow cytometric analysis (CD34+/KDR+ or CD34+/CD133+/KDR+ cell count), which implied that the former approach integrates the number of true EPC with their functional properties. However, even within the CFU assay methodological inconsistencies exist. Indeed, in contrast to papers where the enumeration had been performed on Day 7 or 9 of culture [9,11,25,29], longer periods of culture have been proposed to identify true EPC [26,71,72]. As to the EPC culture assay (based on the ability of adherent cells cultured for 4 days on fibronectin-coated plates to subsequently uptake acetylated low-density lipoprotein and bind to Ulex europaeus agglutinin I), Rehman et al. [73] have provided evidence that the majority of cells identified by this method do not proliferate, yet secrete proangiogenic growth factors, being in fact cells derived of monocyte/macrophages which may be more appropriately referred to as circulating angiogenic cells.

On the other hand, there is evidence that the CD34+/KDR+ cell count is clinically relevant. Indeed, similar conclusions were reached irrespective of the EPC-defining criterion—CD34/KDR-double positivity, CD133-positivity or the CFU assay—with regard to prognosis in CAD [11]. Similarly, effects of statins [36] and exercise-induced myocardial ischaemia [42] on EPC were comparable for CD34+/KDR+ cells and the EPC culture assay. Moreover, out of six subpopulations of EPC-related cells defined by flow cytometry, exclusively the CD34+/KDR+ cell numbers correlated inversely with carotid intima-media thickness in 137 healthy subjects [34].



   Supplementary data
Top
 Abstract
 Summary
 Introduction
 Subjects and methods
 Results
 Discussion
 Supplementary data
References
 
Supplementary data is available online at http://ndt.oxfordjournals.org



   Acknowledgments
 
This work was supported by State Committee for Scientific Research grants 2PO5A01227 and CR/109/L.

Conflict of interest statement. None declared.



   References
Top
 Abstract
 Summary
 Introduction
 Subjects and methods
 Results
 Discussion
 Supplementary data
 References
 

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Received for publication: 14. 9.07
Accepted in revised form: 19.12.07


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