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European Heart Journal Advance Access originally published online on January 12, 2008
European Heart Journal 2008 29(3):332-338; doi:10.1093/eurheartj/ehm602
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Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2008. For permissions please email: journals.permissions@oxfordjournals.org

Evidence for genetic regulation of endothelial progenitor cells and their role as biological markers of atherosclerotic susceptibility

Andrew Whittaker, Jasbir S. Moore, Mariuca Vasa-Nicotera, Suzanne Stevens and Nilesh J. Samani*

Department of Cardiovascular Sciences, University of Leicester, Clinical Sciences Wing, Glenfield Hospital, Groby Road, Leicester LE3 9QP, UK

Received 23 April 2007; revised 28 November 2007; accepted 6 December 2007; online publish-ahead-of-print 2 January 2008.

*Corresponding author. Tel: + 44 116 256 3021, Fax: + 44 116 287 5792, Email: njs{at}le.ac.uk


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 Funding
 References
 
Aims: Endothelial progenitor cells (EPCs) are found in the peripheral circulation and are capable of endothelial repair and neovascularization. EPC number and function are reduced in subjects with cardiovascular risk factors or proven coronary artery disease (CAD). We hypothesized that EPC number and/or function may be genetically regulated and may vary in healthy adult offspring depending on parental history of CAD.

Methods and results: We studied 102 subjects comprising 24 healthy parent–healthy offspring pairs and 27 CAD parent–healthy offspring pairs. We measured the number of circulating CD34+VEGFR-2+ and AC133+VEGFR-2+ EPCs, the number of EPCs grown in culture, and the migration capacity of cultured EPCs towards vascular endothelial growth factor. There was significant correlation in the number of cultured EPCs between healthy parents and their offspring (R = 0.492, P = 0.015) and CAD parents and their offspring (R = 0.751, P < 0.001). Offspring of subjects with CAD had significantly higher numbers of circulating CD34+VEGFR-2+ and AC133+VEGFR-2+ cells (P = 0.018 and P < 0.001, respectively). There was no difference in migration capacity between groups.

Conclusion: Our results suggest that EPC number is, at least in part, genetically regulated. Circulating EPCs may represent biological markers of occult vascular damage in offspring with hereditary risk of CAD.

Key Words: Endothelial progenitor cells • Coronary artery disease • Heritability • Biological markers • Atherosclerosis


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 Funding
 References
 
The intricate molecular relationships between the traditional cardiovascular risk factors, such as diabetes mellitus, dyslipidaemia, hypertension, and smoking, and the development of atherosclerosis and coronary artery disease (CAD) are becoming better understood.1,2 There is a significant genetic contribution to the aetiology of CAD3 though the mechanisms underlying this have yet to be fully elucidated. One of the earliest demonstrable features of atherosclerosis is endothelial dysfunction, characterized by impairment of endothelial nitric oxide production and activation of the endothelial cell monolayer.1,2 This in turn leads to homing and adhesion of circulating blood monocytes and macrophages at the site of endothelial injury,1,2 which subsequently migrate through the vessel wall, adopt a scavenger phenotype, and trigger the atherosclerotic process.1,2 Efficient repair of the endothelial cell monolayer is therefore paramount in maintaining normal endothelial function and raises the hypothesis that exhaustion of endothelial cell reparative capacity may predispose an individual to increased atherogenesis.4 Mature endothelial cells have a low regenerative capacity, which makes them unlikely candidates for endothelial repair.5 A circulating pool of bone marrow-derived angiogenic precursor cells, which are thought to be progeny of the common haemangioblast, were described in 1997 by Asahara et al.6 These cells, which were shown to express CD34, AC133, and VEGFR-2 surface receptors, are capable of differentiation into mature endothelial cells in vitro and of vascular repair and neoangiogenesis in vivo.6 Further studies have confirmed the existence of endothelial progenitor cells (EPCs), which can be characterized by their co-expression of progenitor (CD34 or AC133) and endothelial lineage (VEGFR-2) surface markers, typical in vitro appearance and uptake of acetylated-LDL and lectin,6–8 and their stemness characteristics.7 Both CD34+ and AC133+ cells have been shown to differentiate into endothelial cell phenotype in appropriate culture conditions in vitro and contribute to re-endothelialization and ischaemia-induced neovascularization in animal models.6,9,10 CD34+/AC133+/VEGFR-2+cells are commonly seen in the bone marrow; however, only a small proportion are represented in the circulating leukocyte population.11 Although CD34 and VEGFR-2 receptors remain present on EPCs through to maturity, AC133+ is lost quickly on release into the peripheral circulation.10

In previous studies, EPC number and function have been shown to be reduced both in individuals with risk factors for CAD and those with overt CAD, compared with healthy controls.8,12,13 EPC number and function are also impaired in subjects with either type 1 or type 2 diabetes mellitus.14,15 In addition, EPC numbers have been shown to correlate with cardiovascular outcomes, including cardiovascular death, in subjects with angiographically proven CAD.16 We hypothesized that the number and/or function of EPCs may be genetically regulated and could contribute to the mechanisms by which CAD is inherited. We investigated the potential genetic regulation of EPCs by comparing EPC number and function in parents and their offspring. We also investigated whether EPC number and/or function differ between healthy adult offspring of subjects with CAD and those without such a history.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 Funding
 References
 
Subjects
Coronary artery disease subjects and their offspring
Subjects with severe premature CAD (<65 years of age) were identified from individuals undergoing elective coronary angiography at Glenfield Hospital, Leicester. From these, 27 consecutive non-diabetic subjects with triple-vessel CAD (>50% stenosis in all three vessels), who also had a healthy adult offspring willing to be studied, were recruited. All the CAD patients were stable and none had experienced an acute coronary syndrome in the previous 2 months.

Healthy adult subjects and their offspring
Separately, 76 randomly chosen families (out of 500 families) who had previously participated in a family-based study17 of subjects representative of the general population were invited to participate in the present study. Out of 26 families who responded, 24 were willing to participate. From these, one healthy parent and one healthy offspring were recruited per family. Decision regarding which family members would participate was based on willingness and availability to attend. For the offspring in this healthy group, neither parent had a history of CAD.

Demographic information including cardiovascular risk factor status was recorded at the time of individual recruitment from both parents and offspring. Hypertension and hypercholesterolaemia were defined by patient self-reporting. In addition, fasting serum total cholesterol and HDL cholesterol levels were available for the healthy parents and their offspring.17 Smoking history was obtained by subject interview and recorded as current, ex-, and never smoker. Exclusion criteria for all subjects were diabetes mellitus, malignancy, concomitant inflammatory conditions (including asthma), and renal impairment (serum creatinine >150 µmol/L). The local research Ethics Committee approved the project. All subjects provided informed consent prior to participation. The investigation conforms with the principles outlined in the Declaration of Helsinki.

Flow cytometric quantification of circulating endothelial progenitor cells
This was carried out as described previously.8 Briefly, duplicate samples of 100 µL peripheral blood were incubated with 4 µL of fluorescein isothiocyanate (FITC)-conjugated human anti-CD34 antibody, APC-conjugated human anti-AC133 antibody, or isotype control for 15 min in the dark at room temperature. This was followed by incubation with either 4 µL of PE-conjugated anti-human VEGFR-2 antibody or isotype control for a further 15 min. Red cells were lysed with 1x BD FACS Lysis solution (BD Pharmingen, UK). Leukocytes were washed twice and suspended in 500 µL of D-PBS without CaCl2, MgCl2. Samples were analysed on a BD FACSCalibur flow cytometer with CellSystems® software (Becton Dickinson, UK). Lymphocytes were identified by their light scatter characteristics and were gated for dual-colour analysis. A total of 100 000 events per tube for CD34+VEGFR-2+ cells and 200 000 events per tube for AC133+VEGFR-2+ cells were counted. EPCs were recorded as a percentage of the total gated lymphocyte population.

Isolation, culture and characterization of endothelial progenitor cells in vitro
Peripheral blood mononuclear cells were separated from whole blood by density gradient centrifugation with Biocoll (Autogen Bioclear, UK), washed in phosphate buffered saline (PBS) (Invitrogen, UK), and resuspended in endothelial cell basal medium (EBM) supplemented endothelial growth medium SingleQuots® and 20% fetal calf serum (Cambrex BioWhittaker, UK) at a cell concentration of 8 x 106 cells/mL, as described previously.8 Cells were plated at a density of 2.1 x 106 cells/cm2 into fibronectin-coated wells (10 µg/mL, Sigma-Aldrich, UK) and incubated at 37°C/5% CO2 for 4 days. Non-adherent cells were removed on day 3 and fresh media was applied. On day 4, adherent cells were stained with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine-labelled acetylated low-density lipoprotein (DiL-ac-LDL, 2.4 µg/mL, Biogenesis Ltd, UK), fixed with 2% formaldehyde, and counter-stained with FITC-labelled Ulex europaeus agglutinin I (10 mg/mL, Sigma-Aldrich, UK). Adherent, dual-staining cells were judged to represent EPCs8 and were counted in six random high-powered fields (hpf) per subject.

Vascular endothelial growth factor migration assay
This was performed as described previously.8 Briefly, from separate plates on culture day 4, adherent cells were detached using 1 mM EDTA pH 7.4, counted, and re-suspended in EBM supplemented as described previously (maximum 2 x 105 cells/mL). An equal number of EPCs (maximum 100 000) were placed into the upper chamber of two transwell inserts (Becton Dickinson). The transwells were placed in a 24-well culture dish containing EBM with recombinant human vascular endothelial growth factor (VEGF) (50 ng/mL, Sigma, UK) in one well only. After 24 h incubation at 37°C/5% CO2, the lower filter was washed with PBS. Cells were then stained with DiL-ac-LDL (2.4 µg/mL) and fixed with 2% formaldehyde. Cells migrating onto the lower aspect of the filter were counted in five random high-powered microscopic fields. The number of cells that had migrated towards VEGF compared with those that had migrated towards media alone represents the migration ratio. A migration ratio >1.00 reflects greater migration towards VEGF than control media.

Serum vascular endothelial growth factor and stromal cell-derived factor-1{alpha} concentrations
Quantitative sandwich ELISAs for VEGF and stromal cell-derived factor (SDF)-1{alpha} were performed in duplicate on each subject as per the manufacturer's instructions (R&D Systems, UK). Optical density was read at 450 nm using a BIO-TEK ELx-800 plate reader and KC Junior software (BioTek Instruments, Inc., VT, USA). Cytokine concentration was determined by linear regression from standard curves generated using serial dilutions of the standard in each kit. Mean regression coefficients for standard curves were 0.998 for VEGF and 0.996 for SDF-1{alpha}.

Plasma von Willebrand factor activity
Quantitative ELISA was performed in duplicate on each offspring subject as per the manufacturer's instructions (Axis-Shield Diagnostics, UK). Optical density was read as before. von Willebrand factor (vWF) activity was determined by linear regression from a standard curve generated from serial dilutions using the standard provided in each kit. Mean regression coefficient for standard curves was 0.997.

Statistical analysis
Continuous data are presented as mean ± standard deviation. The means of continuous variables were compared using a generalized linear model for normally distributed data. For non-normal data, a Mann–Whitney U test compared the medians. Categorical data were investigated using a {chi}2 test of association or Fisher's exact test, where appropriate. Adjustment for parental age was carried out using a generalized linear model for continuous variables and logistic regression for categorical variables. Correlation was measured using Pearson's coefficient for normally distributed data and Spearman's rank for non-normally distributed data. Linear regression analysis was used to investigate the relationship between cultured EPC numbers in offspring and parents, accounting for potential factors that may affect EPC numbers including age, gender, body mass index (BMI), and smoking status in both generations. Two-sided tests were used with statistical significance deemed present at a level of P < 0.05. No formal adjustments were made for multiple testing but all P-values are interpreted with caution. Analysis were performed using SPSS v14 and SAS version 9.1 (SAS Institute).


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 Funding
 References
 
Demographic data of subjects
Characteristics of the subjects are given in Table 1. The two offspring groups were well matched apart from a trend towards a greater number of male subjects and a slightly higher BMI in offspring of CAD patients. It should be emphasized that for the healthy offspring cohort, neither the recruited parent nor the other parent had a known history of CAD. The two parent groups are substantially different in the majority of characteristics, reflecting the recruitment strategy (see Methods), but this is not of direct relevance as there were no plans to compare the two parent groups directly.


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  		Table 1 Demographic data of study subjects

 
Circulating endothelial progenitor cell numbers
Consistent with previous studies, circulating levels of AC133+VEGFR-2+ and CD34+VEGFR-2+ cells, expressed as a percentage of the total gated lymphocyte population, were low in both parent and offspring subjects and in a significant proportion of subjects in each group; there was no detectable level of either cell (Table 2 and Figure 1). Taking this into consideration, healthy offspring of subjects with CAD had significantly higher levels of circulating CD34+VEGFR-2+ and AC133+VEGFR-2+ cells than offspring of healthy subjects (P = 0.018 and P < 0.001, respectively) (Figure 1). There was a significant inverse correlation in circulating AC133+VEGFR-2+ cells between healthy parent subjects and their offspring (R = –0.449, P = 0.028). In CAD subjects, there was a significant positive correlation between parents and offspring in circulating AC133+VEGFR-2+ cells (R = 0.390, P = 0.044). There was no correlation in either CAD subjects or healthy subjects and their respective offspring in circulating CD34+VEGFR-2+ cell numbers (CAD subjects: R = –0.212, P = 0.289; healthy subjects: R = –0.119, P = 0.580).


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  		Table 2 Circulating and cultured endothelial progenitor cell number, migratory function, serum cytokine levels, and von Willebrand factor activity in study subjects

 

Figure 1
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  		Figure 1 Dot plots showing distribution of circulating CD34+VEGFR-2+ and AC133+VEGFR-2+ endothelial progenitor cells in offspring subjects. (A) Data for CD34+VEGFR-2+ cells. (B) Data for AC133+VEGFR-2+ cells. Bars represent median endothelial progenitor cell number. For offspring of healthy subjects, the median was 0.000% for both cell types.

 
Cultured endothelial progenitor cell numbers
There was wide variation in the number of cultured EPCs in both parent and offspring subjects (nine-fold in healthy parent and offspring subjects, 44-fold in parent CAD subjects, and 17-fold in offspring of CAD subjects) (Table 2). There was a positive correlation in the number of cultured EPCs between healthy parents and their offspring (R = 0.492, P = 0.015) and between CAD parents and their offspring (R = 0.751, P < 0.001) (Figure 2). There was no significant correlation between unrelated subjects (data not shown). For both healthy parents and their offspring and CAD parents and their offspring, the relationship in EPC numbers remained significant after adjustment for age, gender, BMI, and smoking status in both generations (P = 0.003 and P < 0.001, respectively). In CAD subjects, for every one unit increase in EPC number in parents, the EPC number in offspring increases by 0.93 (SE 0.16). Similarly for healthy subjects, for every one unit increase in EPC number in parents, the EPC number in offspring increases by 0.90 (SE 0.24). There was no significant difference in the number of cultured EPCs between offspring of subjects with CAD and offspring of healthy subjects (Table 2). Furthermore, in all groups, there was no correlation between the number of cultured EPCs and the number of either type of circulating EPC (data not shown).


Figure 2
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  		Figure 2 Correlation in cultured endothelial progenitor cell number between healthy parents and their offspring. Pearson's correlation coefficient between parent and offspring values and significance level are shown. (A) Data for healthy parent–offspring pairs (n = 24). (B) Data for CAD parent–offspring pairs (n = 27). EPC, endothelial progenitor cells; hpf, high-power field.

 
Endothelial progenitor cell migratory function
There was no significant correlation in migration ratio to VEGF between parents and offspring (CAD group: R = 0.092, P = 0.663; healthy group: R = –0.008, P = 0.970). No significant difference in migratory capacity was seen between the offspring of subjects with CAD and offspring of healthy subjects (Table 2). Migratory capacity of EPCs in the offspring was not associated with any demographic variable.

Serum vascular endothelial growth factor and stromal cell-derived factor-1{alpha} concentrations
Serum VEGF was undetectable in one offspring of CAD subjects and two offspring of healthy subjects. There was no difference in the serum VEGF levels between the two offspring groups (P = 0.395) (Table 2). SDF-1{alpha} level was undetectable in 23 (85%) parent CAD subjects, 14 (61%) healthy parent subjects, 11 (46%) offspring of healthy parents, and 20 (74%) offspring of CAD parents. Marginally more offspring of healthy subjects had detectable levels of serum SDF-1{alpha} than offspring of CAD subjects (P = 0.049). There was no correlation in serum VEGF level between parents and offspring (R = 0.036, P = 0.876). There was no correlation between serum VEGF concentration and circulating EPC numbers, cultured EPCs numbers or migration ratio in offspring subjects.

Plasma von Willebrand factor activity
The mean vWF activity was similar in offspring of subjects with CAD and offspring of healthy parents (P = 0.382, Table 2). There was no correlation between vWF activity and circulating EPC numbers, cultured EPCs, or migration ratio in the offspring.


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
Funding
 References
 
The primary object of this study was to ascertain whether EPC number and/or function display heritability. We present novel evidence of a significant correlation in cultured EPC number between healthy parents and their healthy adult offspring, as well as between subjects with CAD and their offspring. The parental cohorts were not well matched including for the use of medication such as statins, which have been shown to influence EPC numbers.18–20 Despite these differences in the parental groups, consistent correlations between parental and offspring EPC numbers were seen in both comparisons, adding to the robustness of the association. Although we cannot exclude an effect of ‘shared environment’ on EPC number and/or function, we feel this is unlikely to be significant, as most of the offspring in our study were living separately from their parents at the time of participation. Therefore, our results suggest that EPC numbers are, at least partially, genetically regulated.

Our findings further suggest that any genetic regulation is specific. Thus, we observed correlation between parents and offspring for the number of cultured EPCs but, for example, did not see any correlation for migration sensitivity to VEGF. Moreover, with regard to circulating EPC numbers, our findings were mixed. There was a significant positive correlation between parents with premature CAD and their offspring in the level of circulating AC133+VEGFR-2+ cells, but a negative correlation between healthy subjects and their offspring. For CD34+VEGFR-2+ cells, there was no correlation in either group. These findings require more cautious interpretation, as, unlike the cultured cells, for a significant proportion of both parents and offspring cohorts, levels of circulating EPCs were undetectable or very low, increasing the margin of any errors for a correlation analysis.

The other interesting finding in our study was that offspring of subjects with CAD had significantly higher levels of both types of circulating EPCs than offspring of healthy parents. Although at first this may appear paradoxical, as CAD has been associated with reduced EPCs,8,12,13 it may suggest that although the offspring of CAD had no clinically apparent coronary disease, they could have occult vascular damage and the raised EPCs could reflect a necessary repair response. Indeed, healthy, young adult subjects with a family history of CAD have previously been shown to exhibit endothelial dysfunction, as measured by brachial artery activity.21 If this is the case, then our results suggest that elevated EPCs, particularly of the immature AC133+VEGFR-2+ type, may represent a biological marker of future risk of CAD in healthy young adults. Increased serum vWF activity has been reported to be a marker of endothelial activation.22 We did not find any difference in vWF activity between the two offspring groups, but by itself, this does not exclude the presence of endothelial damage, which could be stimulating an EPC response. The lack of difference between the offspring groups in the number of cultured EPCs suggests that either the inherent ability of the offspring's cells to adhere and differentiate is not impaired by a family history of premature CAD, or that different cell(s) are measured by culture methods (discussed below).

An important observation is that we did not see any correlation between the level of either type of circulating EPC and the number of EPCs grown in vitro in any of the groups. This was the case even when we excluded subjects with no detectable levels of circulating EPCs. We used an established flow cytometric protocol to identify circulating EPCs,8 which form a very small fraction of the lymphocyte population, and our finding of undetectable levels in a proportion of subjects has been observed previously.8,23 The lack of correlation between the level of circulating EPCs and the number of EPCs grown in vitro suggests that these two methods may not be identifying the same cell or group of cells and are therefore not interchangeable. Our results are consistent with another small study, whose authors also found no correlation between the number of adherent cultured cells and either CD34+VEGFR-2+ or CD34+AC133+VEGFR-2+ cells.24 Antibody-guided labelling of cells by surface marker expression is a specific method of cell identification, whereas in vitro culture of any precursor cell, exhibiting plasticity, in endothelial conditions could potentially result in an endothelial phenotype. Since findings from either approach are currently being widely used as an index of EPC numbers, our results emphasize that care needs to be taken in the interpretation of data, using the two methods.

VEGF and SDF-1 are important regulators of EPC mobilization and homing.25–27 We therefore examined whether potentially heritable differences in these factors could explain the parent–offspring correlation in cultured EPC numbers. We saw no relationship between VEGF concentrations and cultured EPC numbers in either parent or offspring cohorts. SDF-1 levels could only be classified as detectable or not and there was no difference between the cohorts.

Despite the differences between the methods of EPC identification, there does appear to be a genetic contribution to the number of EPCs and/or the differentiation capacity of a subpopulation of MNCs to endothelial lineage cells, between parents and their offspring. Further work is needed to determine the true identity of functionally active EPCs, which are capable of re-endothelialization and neovascularization shown in animal studies.6,9,10 This is of paramount importance for the future of EPC research and for studies examining the therapeutic use of EPCs in human randomized controlled trials. Once this is more clearly understood, the genetic contribution to EPC biology can be further delineated.

The parental cohorts were not well matched for demographic variables, especially gender or use of cardiovascular medications such as HMG-CoA reductase inhibitors that have been shown to affect EPC numbers.18–20 Since the primary objective of the study was to investigate parent–offspring correlations of EPC numbers and to examine EPC number and function in healthy subjects with contrasting familial risk of CAD, this does not affect the interpretation of our principle findings. However, because of the differences in demographics and the inability to account for variables such as treatment effect, it is not appropriate to compare the parental groups.

Other limitations of our study include the relatively small number of subjects. For the type of primary hypotheses we were testing, the cohorts were of a reasonable size. Nonetheless, the findings require replication, and for comparisons of healthy offspring with contrasting family histories of CAD, need to include more sophisticated methods to measure endothelial (dys)function and early atherosclerosis. Moreover, prospective larger scale studies are required to determine whether measurement of EPC numbers could be useful in identifying those at higher risk of future events. In conclusion, our data suggest that a degree of the heritability seen in CAD could be explained through the genetic regulation of EPCs. In addition, circulating EPCs may represent a biological marker of subclinical atherosclerosis in healthy young adults.


   Funding
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Funding
References
 
A.W. was supported by the Edith Murphy Foundation. J.S.M. is funded by the British Heart Foundation. N.J.S. holds a British Heart Foundation Chair of Cardiology.


   Acknowledgments
 
Conflict of interest: none declared.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Funding
 References
 

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