Increased Ratio of CD31 + /CD42 - Microparticles to Endothelial Progenitors as a Novel Marker of Atherosclerosis in Hypercholesterolemia

【摘要】  Objectives- Atherosclerosis may be caused by increased endothelial damage and by a consumptive loss of endothelial repair capacity by endothelial progenitors. Arterial stiffness is a reliable marker of atherosclerosis and a positive correlate of endothelial damage. We investigated whether an increased ratio of CD31 + /CD42 - microparticles to endothelial progenitors, a possible indicator of endothelial damage and impaired endothelium reparation, may contribute to aortic stiffness in hypercholesterolemia. We also studied the in vitro effect of microparticles from hypercholesterolemic patients on endothelial progenitor survival.

Methods and Results- Circulating CD31 + /CD42 - microparticles, endothelial progenitors, and aortic pulse wave velocity (aPWV), a measure of aortic stiffness, were measured in 50 patients with never-treated hypercholesterolemia and 50 normocholesterolemic controls. Hypercholesterolemic patients had more circulating CD31 + /CD42 - microparticles, less endothelial progenitors, and a stiffer aorta than controls. aPWV was associated with CD31 + /CD42 - microparticles ( r =0.61; P <0.001), endothelial progenitors ( r =-0.45, P <0.001), and with cholesterol levels ( r =0.51; P <0.001). High plasma cholesterol and a high ratio of CD31 + /CD42 - microparticles to endothelial progenitors independently predicted an increased aPWV. Microparticles from hypercholesterolemic patients caused a significant endothelial progenitor loss in vitro.

Conclusions- Hypercholesterolemia-related aortic stiffness is promoted by plasma cholesterol directly, increased endothelial damage, and reduced endothelium repair capacity by endothelial progenitors.

Atherosclerosis may be caused by increased endothelial damage and loss of endothelial repair by circulating progenitors. We found that hypercholesterolemic patients have a stiffer aorta, more CD31 + /CD42 - microparticles, and less endothelial progenitors than normocholesterolemic subjects. An increased ratio of CD31 + /CD42 - microparticles to endothelial progenitors independently predicted increased aortic stiffness.

【关键词】  arterial stiffness endothelial progenitor cells hypercholesterolemia microparticles

Introduction

A few studies have reported conflicting data on the influence of high plasma cholesterol levels on reduced arterial elasticity. 1-6 Some initial studies reported an increased aortic distensibility in subjects with heterozygous familial hypercholesterolemia 4 and an inverse association between cholesterol and aortic stiffness; 5 more recently, it has been found that patients with hypercholesterolemia have stiffer blood vessels than matched controls 2,3 and that cholesterol reduction may have a role in reducing large artery stiffness. 7,8 It is also debated whether in patients with hypercholesterolemia the high plasma cholesterol levels alone contribute to the arterial stiffening, or whether there are other factors that also reduce the arterial distensibility. 3,8,9 Hypercholesterolemia decreases the release of nitric oxide from the endothelium of blood vessels, 10 and the bioavailability of nitric oxide is considered critical in maintaining arterial distensibility. 11,12 Given that the bioavailability of nitric oxide relies on endothelial integrity, 13 endothelial damage and reduced endothelial renovation might impair arterial distensibility.

Recently there has been considerable interest in a novel surrogate marker of endothelial injury, endothelial microparticles (EMPs). 14,15 Elevated levels of EMPs, mostly defined as CD31 + /CD42 - MPs, 14,15 are encountered in patients with a variety of diseases involving the vascular system such as acute coronary syndromes, peripheral arterial disease, and diabetes mellitus. 15,16 In these conditions EMPs were initially believed as a sign of vascular damage. 15,16 However, a recent study showed that circulating microparticles of endothelial origin are closely associated with endothelial dysfunction and arterial stiffness in end-stage kidney failure; 17 interestingly, in these patients EMPs also decreased the release of nitric oxide by endothelial cells, 17 thus suggesting that microparticles may play also a pro-atherogenic role other than being a consequence of vascular damage.

Endothelial progenitor cells (EPCs) may contribute to the maintenance of the endothelium by replacing injured mature endothelial cells. 18-20 The number of EPCs has been shown to be reduced in patients with cardiovascular disease, 21,22 leading to the speculation that atherosclerosis is caused by a consumptive loss of endothelial repairing capacity. 23

It is still unknown whether in hypercholesterolemia aortic stiffness is associated with an increased ratio of CD31 + /CD42 - MPs to EPCs. If a relationship were found, this could establish a novel marker of early atherosclerosis and cardiovascular risk. To clarify this issue, we performed a clinical study in patients with a wide range of cholesterol levels, from normocholesterolemia to frank hypercholesterolemia, in whom we assessed aortic pulse wave velocity and the degree of cell injury and replacement by circulating endothelial progenitors. Moreover, to further investigate the relationship between MPs and EPCs, we performed an "in vitro" substudy testing whether MPs isolated from hypercholesterolemic patients have an influence on EPCs apoptosis and colony forming capacity.

Methods

Study Subjects

The study population consisted of 50 consecutive outpatients with newly diagnosed, never-treated primary hypercholesterolemia, and 50 age- and sex-matched normocholesterolemic controls selected among the staff working at the hospital of Perugia (Italy). Diagnosis of hypercholesterolemia was based on the mean of 2 measurements of serum low-density lipoprotein (LDL) cholesterol 160 mg/dL (4.1 mmol/L). Hypercholesterolemic patients were enrolled if they had never been treated with lipid-lowering drugs before the study starting. Additional exclusion criteria were secondary hyperlipidemia caused by diabetes mellitus, renal, liver or thyroid diseases, current smoking, and alcohol consumption 40 g/d. Subjects with history of cardiovascular disease, hypertension, ultrasonographic evidence of atherosclerotic plaques of the carotid arteries, or with any clinical or laboratory evidence of inflammation over a period of 1 month before the beginning of the study were excluded. None of the participants was receiving drug treatment with anti-platelet, anti-inflammatory, hypolipidemic agents, or hormone replacement therapy. Among 100 subjects enrolled for the study, 10 untreated hypercholesterolemic patients and 10 age- and sex-matched normocholesterolemic controls were selected for the in vitro substudy (isolation of plasma microparticles). The study was approved by the local Ethics Committee and all participants gave their informed consent.

Clinical Evaluation

All the determinations were made at the medical center at 8:00 AM, with a room temperature between 21°C and 23°C, after a 14-hour overnight fast. Weight, height, and waist circumference were measured and body mass index (BMI) was calculated. Brachial blood pressure was measured by a physician with a mercury sphygmomanometer after patients sat for 10 minutes or longer. The average of 3 measurements was considered for the analysis.

Measurement of Aortic Pulse Wave Velocity

Aortic pulse wave velocity (aPWV) was determined using an automatic device, the SphygmoCor Vx system (AtCor, Sydney, Australia), as previously described. 24 It uses a single-lead ECG and a high-fidelity applanation tonometer to measure the pressure pulse waveform sequentially in 2 peripheral artery sites, one at the base of the neck for the common carotid artery and the other over the femoral artery. aPWV is calculated from measurements of pulse transit time and the distance between the 2 sites, according to the following formula: PWV (m/s)=distance (m)/transit time (s).

The numerator is the distance between the suprasternal notch and the femoral artery minus the distance between the carotid sampling site and the suprasternal notch; the denominator is the time interval between the systolic R wave and the femoral systolic up-stroke minus the time interval between the systolic R wave and the carotid systolic up-stroke. The distance between the 2 sites is measured using a standard compass system, thus avoiding having the measurement influenced by thoracic and abdominal profiles. An average of 10 different cardiac cycles on each site was used for the analysis. All measurements were performed by the same observer, who was blinded to whether participants were hypercholesterolemic or normocholesterolemic. The intra-observer variability measured in 50 healthy young volunteers was 5.1%.

Assay of Lipid Profile

Total cholesterol, triglycerides, and high-density lipoprotein (HDL) cholesterol were determined by enzymatic-colorimetric method (Dimension Autoanalyzer; DADE Inc, Newark, NJ); LDL cholesterol was calculated by the Friedewald equation in all participants 400 mg/dL.

Assay of CD31 + /CD42 - MPs

Detection of circulating CD31 + /CD42 - MPs, which are mostly considered of endothelial origin, was performed as previously described by Jimenez et al. 25,26 Blood samples were drawn into citrated Vacutainer tubes (5 mL) and were centrifuged for 10 minutes at 160 g to prepare platelet-rich plasma. The platelet-rich plasma was then centrifuged for 6 minutes at 1000 g to prepare platelet-poor plasma. MPs were assayed immediately after venipuncture. Fifty µL platelet-poor plasma in 12 x 75 mm polypropylene tube was incubated with 4 µL of anti-CD31-PE (BD Biosciences, San Jose, Calif) plus 4 µL anti-CD42 fluorescein isothiocyanate (FITC) (BD Biosciences) for 20 minutes with gentle (100 rpm) orbital shaking. Then 1 mL of phosphate-buffered saline (PBS) was added, and the sample was ready for flow cytometry on a BD FACSCanto (BD Biosciences Immunocytometry Systems). MPs were defined as CD31 + /CD42 - particles with a diameter <1.5 µm, being MP size calibrated with flow cytometry size calibrations beads (Molecular Probes; Invitrogen, Eugene, Ore). We tested in a sample of 18 healthy volunteers at what extent leukocyte MPs expressing the surface pan-leukocyte CD45 antigen might contaminate the CD31 + /CD42 - population; we found <5% of CD31 + MPs coexpressed CD45, in agreement to what observed by other authors. 25,27-29 The number of CD31 + /CD42 - microparticles per microliter of platelet-poor plasma was calculated by dividing the number of CD31 + /CD42 - events in the final volume of the sample (50 µL of platelet-poor plasma, 8 µL of antibodies suspension, 1000 µL PBS) by the volume of platelet-poor plasma tested (50 µL). To assess the reproducibility of CD31 + /CD42 - MPs measurements, circulating MPs were measured twice from the same subjects in a subsample of 20 participants; in these subjects, CD31 + /CD42 - MPs were measured in 2 separate blood samples, getting a very close correlation between the measurements ( r =0.81; P <0.001).

Assay of EPCs

Mononuclear cells were isolated from peripheral venous blood by density centrifugation (Lymphoprep; Axis-Shield PoC AS, Oslo, Norway). Freshly isolated mononuclear cells were incubated for 30 minutes at 4°C in the dark with PE-conjugated antibodies against human KDR (R&D Systems, Minneapolis, Minn) and with FITC-conjugated antibodies against human CD34 (BD Biosciences). Isotype-identical antibodies served as controls (BD Biosciences). After incubation, quantitative analysis was performed on a BD FACSCanto (BD Bioscences Immunocytometry Systems) measuring 1 000 000 cells per sample. EPCs were defined as CD34 + /KDR + cells. The number of EPCs was calculated by multiplying the frequency of CD34 + /KDR + events in the gate of lymphocytes by the total lymphocyte count. EPCs count in 2 separate blood samples for each participant (subsample of 20 subjects) was highly reproducible ( r =0.88; P <0.001).

Isolation of MPs and Assay of Cultured EPCs Apoptosis

Isolation of circulating MPs was performed in 10 cardiovascular disease free patients with never-treated primary hypercholesterolemia and 10 normocholesterolemic age- and sex-matched controls, as previously described by Boulanger et al. 30 After citrated fasting venous blood samples were collected, platelet-free plasma (obtained after centrifugation at 11 000 g for 2 minutes) was subjected to centrifugation at 13 000 g for 45 minutes. The microparticle pellets were resuspended in 1 mL of PBS and the approximate number of microparticles was defined cytofluorimetrically in the gate of the events <1.5 µm. For each hypercholesterolemic patient and normocholesterolemic control aliquots containing approximately the same number of MPs (1.5 x 10 6 MPs) were prepared to be added in the EPCs cultures. The number of MPs we used in the in vitro experiments (1.5 x 10 6 /mL of platelet-free plasma) is the lowest we found in vivo among 10 hypercholesterolemic patients; this number was higher than the mean level of MPs/mL measured in 10 healthy subjects. The number of MPs we measured is likely to be overestimated because flow cytometry events in the gate <1.5 µm may also include background noise, and although subtraction of PBS-related noise was performed in all samples, the concentration of MPs (1.5 x 10 6 /mL of platelet free plasma) in the aliquots may be approximate. Equal volumes of MPs suspensions were also prepared for each subjects and used in the in vitro experiments (results not shown because comparable to that obtained with the use of 1.5 x 10 6 MPs aliquots). After mononuclear cells were isolated from 10 buffy coats from healthy volunteers, 2.5 x 10 7 cells were plated on 60-mm culture dishes coated with human fibronectin (BD Biosciences, San Jose, Calif) and maintained in endothelial cell basal medium-2 (EBM-2) (Clonetics, San Diego, Calif) with supplements. After 48 hours in culture, nonadherent cells were removed by washing with PBS, and transferred with fresh media into new fibronectin-coated 60-mm dishes as well as into 24-well fibronectin-coated plates. Cell transfer into fibronectin-coated 60 mm dishes and 24-well plates was performed with or without adding to the culture medium aliquots of previously isolated MPs (1 aliquot/mL of medium) from either hypercholesterolemic patients or normocholesterolemic controls. After 7 days, EPCs apoptosis was evaluated after adherent cells were detached nonenzymatically (Cambrex; Clonetics) and stained with PE-conjugated antibodies against human KDR (R&D Systems, Minneapolis, Minn), FITC-conjugated antibodies against human CD34 (BD Biosciences), and allophycocyanin (APC)-conjugated antibodies against annexin-V (BD Biosciences). Staining with propidium iodide (Sigma Chemical Co, St. Louis, Miss) was also performed in separate samples to exclude dead cells (propidium iodide-positive and annexin-V-negative). Apoptotic EPCs were defined by the expression of CD34, KDR, and annexin-V; 24-well plates were also analyzed for the count of the number of EPCs colonies as described by Hill et al. 31 The samples were analyzed independently by 2 observers who were unaware of whether EPCs cultures were exposed to microparticles and of the source of microparticles (from hypercholesterolemic or normocholesterolemic participants).

Statistical Analysis

SPSS statistical package, release 10.0 (SPSS Inc, Chicago, Ill) was used for all statistical analyses. Values are expressed as the mean±SEM. Independent sample t test was used to compare the study variables between hypercholesterolemic patients and control subjects. Correlation analyses were performed using the Pearson coefficient of correlation. Multivariate linear regression analysis was used to estimate prediction of aPWV by including all the following independent variables in the model: age, gender, BMI, waist circumference, systolic blood pressure, heart rate, plasma lipids and glucose, and CD31 + /CD42 - MPs to EPCs ratio. Standardized coefficients were calculated as a measure for the relative predictive value. ANOVA was used to compare the number EPCs colonies and the frequency of apoptotic EPCs between cultures added with MPs from either hypercholesterolemic patients, normocholesterolemic controls, or added with the vehicle only (no MPs). Statistical significance was assumed if a null hypothesis could be rejected at P =0.05.

Results

The characteristics of 100 participants, 50 with newly diagnosed never-treated hypercholesterolemia and 50 age- and sex-matched normocholesterolemic subjects, are summarized in Table 1. Hypercholesterolemic patients differed from normocholesterolemic subjects with respect to plasma total and LDL cholesterol levels and aortic pulse wave velocity (8.9±0.17 m/sec versus 7.6±0.14 m/sec), which were all significantly higher among hypercholesterolemic patients ( P <0.001). Figure 1 illustrates the distribution (median, quartiles, and extreme values within each category) of the number of circulating peripheral CD31 + /CD42 - MPs, EPCs, and CD31 + /CD42 - MPs to EPCs ratio in hypercholesterolemic patients and controls; hypercholesterolemic patients had a significantly higher ratio of CD31 + /CD42 - MPs to EPCs compared with normocholesterolemic subjects (mean±SEM, 2.18±0.13 versus 0.99±0.05; P <0.001), as a result of both an increased number/µL of circulating CD31 + /CD42 - MPs (mean±SEM, 817±39 versus 540±21; P <0.001) and a reduced number/mL of peripheral EPCs (mean±SEM, 403±16 versus 574±17; P <0.001). The number of CD31 + /CD42 - MPs and that of EPCs were negatively associated with each other ( r =-0.33, P =0.001). The number of CD31 + /CD42 + MPs, generally considered of platelet origin, was significantly higher in hypercholesterolemic than in controls (mean±SEM, 1698±48 versus 1520±42; P =0.026), albeit this difference was less striking than that of CD31 + /CD42 - MPs.

TABLE 1. Clinical and Biological Characteristics of 50 Hypercholesterolemic Patients (HC) and 50 Age- and Sex-Matched Normocholesterolemic Subjects (NC)

Figure 1. Box-and-whisker plots illustrating the distribution of the number of circulating peripheral CD31 + /CD42 - MPs (A), EPCs (B), and CD31 + /CD42 - MPs to EPCs ratio (C) in 50 hypercholesterolemic patients (HC) and 50 normocholesterolemic (NC) age- and sex-matched controls. Each box shows the median, quartiles, and extreme values within a category. MPs indicates microparticles; EPCs, endothelial progenitor cells.

CD31 + /CD42 - MPs to EPCs Ratio and Cardiovascular Risk Factors

Figure 2 A shows the direct correlation of CD31 + /CD42 - MPs to EPCs ratio with LDL cholesterol ( r =0.61; P <0.001). The ratio was also significantly associated with age ( r =0.23; P =0.02), BMI ( r =0.33; P =0.001), waist circumference ( r =0.35; P =0.001), and systolic blood pressure ( r =0.29; P =0.01).

Figure 2. Correlation of CD31 + /CD42 - MPs to EPCs ratio with LDL cholesterol (A) and aPWV (B). LDL indicates low-density lipoprotein; aPWV, aortic pulse wave velocity.

Univariate and Multivariate Correlates of aPWV

Figure 2 B shows the correlation of CD31 + /CD42 - MPs to EPCs ratio with aPWV ( r =0.68; P <0.001). aPWV was associated with CD31 + /CD42 - MPs ( r =0.61; P <0.001), EPCs ( r =-0.48; P <0.001), and plasma LDL cholesterol levels ( r =0.53; P <0.001). Finally, aPWV showed a significant correlation with the number of CD31 + /CD42 + MPs ( r =0.31, P <0.05).

To identify independent predictors for aPWV, we performed a multivariate linear regression analysis including age, gender, BMI, waist circumference, systolic blood pressure, plasma lipids, glucose, and the ratio of CD31 + /CD42 - MPs to EPCs as independent variables. Both plasma LDL cholesterol levels (ß=0.21; P =0.017) and CD31 + /CD42 - MPs to EPCs ratio (ß=0.45; P <0.001) were independently associated with aPWV. Age, waist circumference, LDL cholesterol, and CD31 + /CD42 - MPs to 50% of the total variability of aPWV (adjusted R 2 =0.508; P <0.001). No sex-based differences were observed in the analyses.

In Vitro Effect of MPs on EPCs Survival

We tested the effect of isolated MPs on cultured EPCs ( Table 2 ). We found that 7 days of exposure of cultured EPCs to MPs from never-treated hypercholesterolemic patients caused a significant EPCs loss; in fact, the mean percentage of apoptotic (annexin-V + /propidium iodide - ) cultured EPCs was 60% higher in cultures exposed to hypercholesterolemic patients MPs than that in cultures not exposed to MPs ( P <0.05). The percentage of late apoptotic EPCs (annexin-V + /propidium iodide + ) was negligible (<1%) in cultures exposed and not exposed to MPs (results not shown). Moreover, the mean percentage of EPCs colonies/well ranged from 32±4 without MPs exposure to 19±3 after exposure to MPs from hypercholesterolemic patients ( P <0.05). EPCs exposure to MPs from normocholesterolemic controls caused a nonsignificant EPCs loss in vitro ( Table 2 ).

TABLE 2. Effect of Microparticles (MPs) Isolated From Hypercholesterolemic Patients (HC) and Normocholesterolemic Controls (NC) on Buffy Coat-Derived EPCs Cultures

Discussion

Conventional wisdom holds that large artery stiffness mainly arises from the detrimental influence of major cardiovascular risk factors on endothelium integrity and function. 27,32 In this respect, the present and previous observations 1,2,3,7,8 of a positive association between hypercholesterolemia and arterial stiffness may be not too surprising, given that high plasma cholesterol levels contribute to endothelial cells injury and dysfunction, 33,34 which in turn has been positively associated with arterial stiffness. 35,36

To elucidate the mechanisms underlying the association between plasma cholesterol levels, endothelial injury and arterial stiffness we recruited subjects with a wide range of plasma cholesterol levels, from normocholesterolemia to frank hypercholesterolemia; in these subjects, we measured plasma lipid levels, aPWV, CD31 + /CD42 - MPs, which have been shown to derive mostly from the endothelium, 25,27-29 EPCs, and the ratio of circulating CD31 + /CD42 - MPs to EPCs, as an index of endothelium injury and repair ability.

The main findings of the present study are that hypercholesterolemic patients have higher circulating CD31 + /CD42 - MPs and lower EPCs than normocholesterolemic subjects and also that the ratio of CD31 + /CD42 - MPs to EPCs is directly associated with aPWV. To our knowledge this is the first observation that hypercholesterolemia may contribute to large artery stiffness both by increasing the release of microparticles that are mainly of endothelial origin and by reducing the number of circulating endothelial progenitors.

Microparticles, formed during cell activation and apoptosis, 14 may be more than just a marker of endothelial exposure to an unfavorable plasma milieu; 15 they may also actively contribute to the pathogenesis of cardiovascular disease because of their pro-inflammatory effects, their ability to promote thrombosis, and endothelial dysfunction. 15,37 In this respect, it has been shown that isolated microparticles may diminish endothelium-dependent relaxation; 30 moreover, Amabile et al found that high circulating endothelial MPs levels are associated with reduced flow-mediated dilation and increased aortic stiffness in patients with end-stage kidney failure; 17 in the same study, 17 exposure of cultured endothelial cells to EMPs reduced endothelial nitric oxide release by almost 60%. To further corroborate this observation we found that microparticles, besides injuring mature endothelial cells, 17 may also play a role in reducing the vitality of EPCs; in fact, we observed that microparticles from hypercholesterolemic patients caused a significant in vitro EPCs apoptosis and reduced their colony forming capacity. Moreover, a negative correlation was found between CD31 + /CD42 - MPs, CD31 + /CD42 + MPs, and the number of circulating EPCs. Hence, MP-induced endothelial dysfunction and subsequent reduction in nitric oxide bioavailability 17,30 and MP-induced EPCs loss might represent possible mechanisms of cholesterol-induced endothelial damage and impaired vascular reparation, which in turn may contribute to increase vascular tone and arterial stiffness.

Our finding of a lower number of EPCs in hypercholesterolemic patients compared with normocholesterolemic subjects, and the inverse association between the number of circulating EPCs and aPWV, further suggests that, also in the case of hypercholesterolemia, the integrity of the endothelium appears essential for the preservation of a proper aortic distensibility. Although simply quantifying peripheral EPCs does not explain the reason and the mechanism of their reduced number in hypercholesterolemia, our "in vitro" finding of an increased EPCs apoptosis and of a reduced EPCs colony forming capacity after exposure to MPs might suggest a novel mechanism of human EPCs incompetence. The demonstration of low plasma EPCs levels is therefore important because it reflects an impaired potential of EPCs to participate in the repair of injured endothelium. 19-23 As a consequence of their reduced repairing capacity, low EPCs levels have been found to be associated with endothelial dysfunction. 31 In conclusion, our finding in hypercholesterolemia of a strong positive correlation between CD31 + /CD42 - MPs to EPCs ratio and aPWV suggests that both increased endothelium injury and impaired endothelial repair may contribute to reduce aortic distensibility.

Limitations of our study have to be acknowledged. First, we assumed CD31 + /CD42 - MPs are mainly of endothelial origin, although also leukocytes express CD31; assessment of CD45 expression in all samples of the present research would have ruled out the possible contamination of endothelial MPs with leukocyte MPs. Alternatively, CD105 might be used to more specifically detect EMPs. However, our and previous observations 25,27-29 of a negligible number of CD31 + /CD45 + MPs (<5% of all CD31 + MPs) contaminating the whole CD31 + MPs population might have lessened this limit. Second, we did not rule out that CD31 + /CD42 - MPs population may also contain EPCs-derived MPs; it is an intriguing hypothesis because it would establish a novel mechanism of EPCs loss; to what extent MPs production from a rare circulating population of EPCs may contribute to CD31 + MPs is, however, still unknown. Finally, we did not distinguish between MPs formed during cell activation or apoptosis, or whether MPs had a different phenotype in hypercholesterolemic compared with normocholesterolemic subjects; it would have given further information on the possible mechanisms of MPs formation and the differential influence of such MPs on vascular function.

The balance between endothelium injury and repair is critical for the maintenance of the vascular homeostasis. A further understanding of the mechanisms underlying hypercholesterolemia-related endothelium damage and of the role of EPCs in the regeneration of a functional endothelium is of extreme importance for the development of more effective therapies aimed toward restoring a physiological vascular function.

Acknowledgments

The authors thank Dr Iliana Lega for her assistance in the preparation of the manuscript.

Disclosures

None.

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作者单位：Unit of Internal Medicine, Angiology, and Arteriosclerosis (M.P., G.S., R.P., F.B., C.M., M.R.M., E.M.) and the Division of Hematology and Clinical Immunology (M.C., A.V.), Department of Clinical and Experimental Medicine, University of Perugia, Italy.