Decreased Number and Impaired Angiogenic Function of Endothelial Progenitor Cells in Patients With Chronic Renal Failure
http://www.100md.com
《动脉硬化血栓血管生物学》
From the Department of Medicine, Samsung Medical Center, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Seoul, Korea.
ABSTRACT
Objective— Increased risk of cardiovascular disease in patients with chronic renal failure (CRF) has been explained by accelerated atherosclerosis and impaired angiogenesis, in which endothelial progenitor cells (EPCs) may play key roles. We hypothesized that altered EPC biology may contribute to the pathophysiology of CRF.
Methods and Results— EPCs were isolated from CRF patients on maintenance hemodialysis (n=44) and from a normal control group (n=30). CRF patients showed markedly decreased numbers of EPC (44.6%) and colonies (75.3%) when compared with the controls (P<0.001). These findings were corroborated by 30.5% decrease in EPC migratory function in response to vascular endothelial growth factor (VEGF) (P=0.040) and 48.8% decrease in EPC incorporation into human umbilical vein endothelial cells (HUVEC) (P<0.001). In addition, Framingham’s risk factor score of both CRF (r=–0.461, P=0.010) and normal group (r=–0.367, P=0.016) significantly correlated with the numbers of EPC. Indeed, the number of circulating EPC was significantly lower in CRF patients than in normal group under the same burden of risk factors (P<0.001). A significant correlation was also observed between dialysis dose (Kt/V) and EPC incorporation into HUVEC (r=0.427, P=0.004).
Conclusions— EPC biology, which is critical for neovascularization and the maintenance of vascular function, is altered in CRF. Our data strongly suggest that dysfunction of circulating EPC has a role in the progression of cardiovascular disease in patients with CRF.
Our study shows that EPC is numerically and functionally impaired in CRF. This may contribute to the accelerated atherosclerosis and impaired angiogenesis observed in patients with CRF. Altered biology of EPC may therefore account for the increased cardiovascular disease risk in CRF.
Key Words: endothelial progenitor cell ? risk factors ? vascular biology ? renal physiology
Introduction
The lifespan of patients with chronic renal failure (CRF) is reduced, and coronary artery disease is the most important cause of morbidity and mortality in these patients.1,2 Even the results of therapeutic strategies such as percutaneous coronary intervention and bypass surgery have shown poor procedural success rates and dismal long-term event-free survival in CRF patients.3,4
Most of the increased cardiovascular morbidity and mortality in CRF has been accounted for by the rapid progression of atherosclerosis, which is clinically shown to be accelerated in CRF.5,6 Experimental studies have also shown that even mild renal dysfunction causes a dramatic acceleration of atherosclerosis.7 Angiogenesis, which is an essential compensation for myocardial ischemia, is also impaired in CRF.8 However the mechanism underlying the acceleration of atherosclerosis and impaired angiogenesis by CRF has not been examined closely. Although the phenomenon has been partially explained by the higher prevalence of established risk factors in CRF, such as hypertension, abnormal carbohydrate metabolism, and increased low density lipoprotein (LDL) cholesterol, the extent and severity of cardiovascular disease is clearly disproportionately high relative to the underlying risk factor profile.9,10
Recent studies have identified that normal adults have a small amount of circulating endothelial progenitor cell (EPC) in the peripheral blood. In response to cytokine stimulation and ischemic insult, these cells are mobilized from bone marrow, home to the ischemic tissue, and contribute to neovascularization and angiogenesis.11–14 Moreover, EPC is regarded to have a key role in the maintenance of vascular integrity and to act as "repair" cells in response to the endothelial injury,15,16 which has been regarded as an initial step in atherosclerosis and a result of the actions of various cardiovascular risk factors.17 Current data suggest that decrease in circulating EPC contributes not only to impaired angiogenesis but also to the progression of atherosclerosis,18 and patients at risk for coronary artery disease have a decreased number of circulating EPC with impaired activity.19–22
Therefore, we reasoned that EPC, which is critical for neovascularization and the maintenance of vascular integrity, might be numerically or functionally impaired in CRF. This would contribute to the accelerated atherosclerosis and impairment of angiogenesis, which may account for the increased cardiovascular risk and poor clinical outcomes observed in CRF. We investigated the number and angiogenic function of EPC obtained from patients on maintenance hemodialysis and compared them with the corresponding parameters of healthy volunteers.
Methods
Study Subjects
We studied 44 male CRF patients on maintenance hemodialysis and 30 healthy male volunteers who were older than age 21 years. Chronic stable patients on maintenance hemodialysis were selected to exclude potential factors that may affect biology of circulating EPC in the setting of recent aggravation of renal function. Patients with clinical evidence of symptomatic coronary artery disease, peripheral vascular disease, cerebrovascular disease, or carotid artery stenosis were excluded. Subjects with any condition, such as neoplasm, wounds, or significant retinopathy, which might involve neovascularization, were also excluded.14 Medications, including statins, glucose-controlling drugs, and antihypertensive drugs, were continued. No new medications, including vitamins, were used for at least 2 weeks before the study. Laboratory data of each subject were obtained within 2 weeks from the date of blood sampling. All enrolled subjects underwent a detailed assessment of cardiovascular risk after signing an informed consent document approved by the Institutional Review Board of Samsung Medical Center.
Isolation of EPC
Peripheral blood (30 mL) was drawn by venipuncture using a heparin-coated syringe. Samples of CRF patients were drawn just before the beginning of hemodialysis to exclude any possible influence of dialyzer on the cell. Mononuclear cells were isolated by density gradient method using Ficoll-Paque Plus (Amersham, Buckinghamshire, UK),and resuspended in EGM-2 MV Singlequot medium (Cambrex, East Rutherford, NJ), which contains multiple growth factors including human vascular endothelial growth factor (VEGF) A, human fibroblast growth factor-2, human endothelial growth factor, insulin-like growth factor-1, and ascorbic acid. Cells were plated on 6-well plates (Becton Dickinson, Franklin Lakes, NJ) coated with 2% gelatin (Sigma, St. Louis, Mo). The initial seeding density was standardized at 4x106 cells per well. After 3 days of culture, nonadherent cells were removed and the media were changed. The culture was maintained through day 7. After the numbers of EPC and EPC colony were counted, cells were assayed or harvested for further study.
EPC Characterization
Endothelial cell (EC) phenotype of EPC was investigated at day 7. Adherent cells were incubated with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)-labeled acetylated LDL (Molecular Probes, Eugene, Ore) at 37°C for 3 hours, and then with fluorescein-isothiocyanate-conjugated Ulex europaeus agglutinin (UEA)-1 lectin (10 μg/mL, Sigma) for 4 hours. Samples were examined with an inverted fluorescence microscope (Zeiss) and only cells exhibiting double fluorescence were identified as EPC.21,23 Cultured human umbilical vein endothelial cells (HUVEC) and NIH 3T3 cells were stained simultaneously and served as the positive and negative controls, respectively. To confirm endothelial cell phenotype of EPC, cells were also simultaneously stained with primary antibodies against the VEGF receptor KDR (Sigma) and von Willebrand factor (vWF) (BD Pharmingen, San Diego, Calif). Fluorescein-isothiocyanate and rhodamine-linked secondary monoclonal antibodies were used.
To confirm that the EPCs of our study population were capable of intracellular nitric oxide (NO) synthesis, we examined the EPCs of normal subjects with a membrane NO-specific fluorescence indicator, diamino-fluorescein-2 diacetate (DAF-2 DA; Daiichi, Japan). EPCs were gently washed twice with calcium-free phosphate-buffered saline (PBS) and bathed in Krebs-Henseleit buffer containing L-arginine (1 mmol/L) and DAF-2 DA (10 μmol/L) for 15 minutes. The cells were then incubated at 37°C for an additional 15 minutes, and 50 ng/mL of recombinant human VEGF165 (R&D Systems, Minneapolis, Minn) was added to the wells. The samples were then examined with fluorescence microscopy.
Cell surface antigens were investigated with fluorescence-activated cell sorter (FACS) analysis.11,23 Cultured cells were detached by brief incubation with trypsin/1 mmol/L EDTA followed by forceful pipetting, and cells (1x105 cells each) were used for FACS analysis. Antibodies to the VEGF receptor KDR (Sigma), vWF (BD Pharmingen), and VE-cadherin (BD Pharmingen) were used as primary antibodies. Peripheral blood mononuclear cells served as controls.
EPC Count
The numbers of EPC and EPC colony were determined by counting 12 random high-power (x100) microscope fields per subject and were expressed as cells or colonies per mm2. Spindle-shaped cells and colonies consisting of multiple thin, flat cells emanating from a central cluster of rounded cells were counted.
EPC Migration Assay
EPC migratory function, which is essential for angiogenesis, was examined using a modified Boyden chamber technique. A 24-well Transwell apparatus (Coster) was used, with each well containing a 6.5-mm polycarbonate membrane with 8-μm pores, coated with type I collagen (Sigma). EPCs (4x104) were placed on the membrane, and the chamber was immersed in a 24-well plate, which was filled with growth factor-free EBM-2 culture media or EBM-2 with 50 ng/mL of human VEGF165. After incubation for 24 hours, the membrane was washed briefly with PBS and the upper side of membrane was wiped gently with a cotton ball. The membrane was then removed and stained using Giemsa solution. The magnitude of EPC migration was evaluated by counting the migrated cells in 4 random high-power (x100) microscope fields.
Matrigel Tube Formation Assay
A Matrigel tube formation assay was performed to assess the ability of EPC to incorporate into endothelial cell vascular structures, which is believed to be important in new vessel formation.16 Matrigel (Becton Dickinson) was spread onto 4-well chamber slides (Nalge Nunc). EPCs were marked with DiI (Molecular Probes) to distinguish them from HUVEC. DiI-labeled EPC (1x104) and HUVEC (4x104) were plated together and incubated at 37°C for 24 hours with EGM-2 culture medium. Incorporated cells were counted from 4 random high-power (x100) microscope fields per each subject. The numbers and line lengths of the circle formed by cells were also calculated.
Statistical Analysis
All data are presented as means±SEM. Continuous variables were evaluated by nonparametric Mann–Whitney test or Wilcoxon signed rank test. Categorical variables were evaluated by Fischer exact test. Univariate correlations were made with Spearman correlation coefficient. Multiple linear regression for the analysis of covariance was used to identify predictors of changes in EPC counts in a multivariate setting. SPSS release 11.0 (SPSS Inc) was used, and differences were considered statistically significant at 2-tailed P<0.05.
Results
Patients Characteristics
Clinical data of the study subjects are summarized in the Table. Patients in the CRF group were 4.6 years older than those in the normal group and had a lower body mass index. Total cholesterol and high-density lipoprotein cholesterol were significantly lower in the CRF group. All CRF patients were treated with erythropoietin, and most of them (97.7%, n=43) were using antihypertensive medication. Plasma VEGF levels measured with an enzyme-linked immunosorbent assay kit (R&D Systems) showed no difference between CRF and normal groups. There was no difference in total leukocyte counts between 2 groups. The underlying cause of CRF was diabetic nephropathy (n=22) or other causes (n=22).
Characteristics of Study Subjects
EPC Characterization
Culture of total peripheral blood mononuclear cell resulted in the emergence of characteristic spindle-shaped cells and colonies consisting of peripheral spindle-shaped cells emanating from round central cells within 72 hours of culture (Figure 1A and 1B). These cells could be shown to uptake acetylated LDL and bind UEA-1, consistent with endothelial lineage cells (Figure 1C through 1H). Coexpression of 2 endothelial lineage-specific markers, vWF and KDR (Figure IA through IC, available online at http://atvb.ahajournals.org) and specific NO synthesis in these cells were also confirmed (Figure ID through IF, available online at http://atvb.ahajournals.org). FACS analysis showed that the percentage of cells positive for endothelial lineage markers, KDR, vWF, and VE-cadherin were 23.9%, 39.1%, and 53.3%, respectively (Figure IG, available online at http://atvb.ahajournals.org).
Figure 1. Identification of the endothelial lineage phenotype of cultured EPC. Culture of peripheral blood mononuclear cells resulted in the emergence of characteristic spindle-shaped EPCs (A) and EPC colonies characterized by a central cluster of rounded cells surrounded by radiating spindle-shaped cells (B). EPCs (C through E) and EPC colonies (F to H) were shown to uptake acetylated LDL (C, F) and bind UEA-1 (D, G). Merged images show that most cells are dual-positive (E, H).
EPC Count
Significantly fewer EPC colonies were formed from mononuclear cell culture of CRF patients compared with normal controls at day 7 (133.7±21.6 versus 542.5±64.1 per 103 mm2; P<0.001) (Figure 2A through 2C). Significantly fewer EPCs were also identified from CRF patients (10.0±1.2x103 versus 17.8±2.0x103 per 103 mm2; P<0.001) (Figure 2D through 2F). Despite differences in age, body mass index, total cholesterol, high-density lipoprotein cholesterol, hypertension, and usage of statin, which might affect EPC biology (Table),15,18,24 the statistically significant differences were maintained after adjusting for these factors (EPC colonies of CRF versus normal; P<0.001, EPC of CRF versus normal; P=0.005) (Figure IIA and IIB, available online at http://atvb.ahajournals.org). There was no difference in the numbers of EPCs or EPC colonies between diabetic and nondiabetic CRF patients.
Figure 2. The number of EPCs and EPC colonies counted after 1 week of culture is reduced in CRF. Representative photos of EPC colony culture from the normal (A) and CRF group (B), demonstrating fewer colonies in the CRF group. C, EPC colonies were significantly reduced (by 75.3%) in the CRF group compared with the normal group, and this was independent of other clinical factors (P<0.001) (Figure IIA). Representative photos of EPC cultures from the normal (D) and CRF group (E), demonstrating fewer EPCs in the CRF group. F, EPCs were also significantly reduced (by 44.6%) in the CRF group. *Statistical significance (P<0.05).
EPC Migration Assay
The migratory function of EPC in response to VEGF, which is believed to be important during neovascularization,19 was evaluated using a modified Boyden chamber. The basal EPC migratory functions of CRF patients and normal controls were not significantly different (P>0.05). After supplementation with 50 ng/mL of VEGF, the migratory function of EPC in both groups was significantly augmented (baseline versus VEGF-augmented migration, CRF patients; 14.9±3.3 versus 22.4±3.7 per x100 high-power field; P=0.001, normal group; 19.1±3.2 versus 32.9±5.3 per x100 high-power field; P=0.016) (Figure 3A through 3E). However, the VEGF-induced augmentation of EPC migration was relatively impaired in CRF. VEGF-induced augmentation of EPC migration was reduced by 52.8% in CRF patients relative to that of the normal group (7.5±1.4 versus 15.8±3.5 per x100 high-power field, P=0.036) (Figure 3F), and the total number of migrated EPC was also lower in CRF patients independently of clinical factors described (22.4±3.7 versus 32.9±5.3 per x100 high-power field, P=0.017) (Figure IIC, available online at http://atvb.ahajournals.org).
Figure 3. EPC migratory function is impaired in CRF. A modified Boyden chamber assay was used with growth factor-free medium or VEGF supplemented medium. Baseline (A) and VEGF-stimulated (B) EPC migration in the normal group. Baseline (C) and VEGF-stimulated (D) EPC migration in the CRF group. Representative photos are shown. The small dots are holes of the Boyden chamber membrane. E, Baseline migratory function of EPC did not differ between 2 groups (P>0.05). Supplementation with VEGF led to significantly augmented migration of EPCs in both groups (normal group; P=0.001, CRF group; P=0.016). However, EPC of the CRF group showed a reduced increase in the number of migrated cells (50.2% increase) relative to that of the normal group (72.5% increase). F, The reduced increase in EPC migration in CRF was statistically significant. *P<0.05.
Matrigel Tube Formation Assay
EPC incorporation into the tubular networks formed by HUVEC was evaluated in culture using Matrigel, which is used to evaluate EC differentiation.25 Significantly fewer EPCs of CRF patients were incorporated into tubules compared with the EPCs from normal controls (8.2±0.5 per mm2 versus 16.0±1.1 per mm2, P<0.001) (Figure 4A through 4C), independently of clinical factors described (P<0.001) (Figure IID, available online at http://atvb.ahajournals.org). Significantly fewer circles and shorter line lengths of the tubules were also identified (Figure IIIA through IIID, available online at http://atvb.ahajournals.org). There was no difference in EPC incorporation or tubule formation between diabetic and nondiabetic CRF.
Figure 4. EPC incorporation into EC and stimulation of EC tube formation is impaired in CRF. EPCs were labeled with DiI and cocultured with HUVEC in Matrigel-coated 4-well chamber slide for 24 hours. Fluorescence and light images of identical fields were merged. Significantly fewer EPCs from CRF patients were incorporated into the tubules formed by HUVEC (A) than those of the normal controls (B). EPC incorporation in the CRF group was reduced by 51.9% (C). *P<0.05.
Risk of Coronary Artery Disease and the Level of EPC
We next investigated the relationship between the level of circulating EPC and the risk of coronary artery disease, which is a clinical consequence of accelerated atherosclerosis and impaired angiogenesis in CRF.5–8 Because none of our study subjects showed any clinical evidence of coronary artery disease, the 10-year coronary artery disease risk was estimated from the total burden of risk factors according to the Framingham risk score.20,26 The EPC number was significantly inversely correlated with the estimated 10-year coronary artery disease risk in CRF patients (r=–0.367, P=0.016) and in the normal group (r=–0.461, P=0.010) (Figure 5A and 5B), suggesting that a lower level of EPC is associated with a higher risk of coronary artery disease in CRF as well as in normal renal function group.20
Figure 5. The level of circulating EPC is inversely correlated with the risk factor score of coronary artery disease, and the EPC incorporation function is correlated with the dose of dialysis (Kt/V). The correlation between the number of EPC and the 10-year coronary artery disease risk estimated from the Framingham risk score was evident in the normal group (A) and CRF group (B). C, The age-adjusted relative risk of coronary artery disease was calculated to compare the numbers of EPC under the same risk burden. The correlation was maintained in the normal (r=–0.399, P=0.029) and CRF group (r=–0.422, P=0.005), and there were significantly fewer EPCs in the CRF group than in the normal group after correction for risk burden (P<0.001). D, The number of EPC was significantly reduced not only in the total CRF group but also in the low-risk (P=0.009) and high-risk subgroup (P<0.001). The EPC incorporation function was significantly different between lower doses and higher doses of dialysis (P=0.015) (E) and correlated significantly with the dose of dialysis (r=0.427, P=0.004) (F).
To compare the EPC numbers in both groups under the same risk burden, the age-adjusted relative risk of each subject was estimated.26 EPC numbers were also significantly inversely correlated with the age-adjusted relative risk, both in CRF patients (r=–0.422, P=0.005) and in the normal group (r=–0.399, P=0.029). The numbers of EPC were significantly lower in CRF patients after adjustment for age-adjusted relative risk (P<0.001) (Figure 5C). We next divided the study subjects into a low-risk group (for whom the 10-year coronary artery disease risk was <2.0-times the optimal risk profile) and a high-risk group (2.0-times). The number of EPC in CRF patients was significantly lower than that in the normal group independently of whether they were in the low-risk or high-risk group (low-risk group; CRF versus normal; 11.7±1.6x104 versus 20.3±2.8x104 per 103 mm2; P=0.009, high-risk group; 8.2±1.8x104 versus 15.3±2.8x104 per 103 mm2; P<0.001) (Figure 5D). From these observations, it would appear that the level of EPC is reduced in CRF irrespective of the risk burden.
Impact of Dialysis Dose on the EPC Migratory Function
As shown, a lower number of EPC was correlated with a higher future coronary artery disease risk. We presumed that the number or function of EPC might also be related to the dose of dialysis calculated by Kt/V, which is known to be related to mortality risk in CRF patients.27 Because a Kt/V of 1.3 is regarded as the minimal standard for adequate dialysis,2,27 we compared the EPC incorporation function of patients receiving a lower dialysis dose (Kt/V <1.3) with that of patients receiving a higher dialysis dose (Kt/V 1.3). The EPCs of patients receiving lower doses of dialysis showed significantly impaired incorporation into EC compared with those of patients receiving higher doses of dialysis (5.3±1.1x103 versus 8.6±0.5x103 EPC per 103 mm2, P=0.015) (Figure 5E). Furthermore, the dose of dialysis was significantly correlated with the degree of incorporation of EPC into EC (r=0.427, P=0.004) (Figure 5F). This interesting finding suggests that higher doses of dialysis may be related to improved angiogenic function of EPC. However, other parameters such as the number of EPC, the number of EPC colonies, and migratory functions were not correlated with the dose of dialysis.
Discussion
In this study, we demonstrate that the number of circulating EPC is decreased and the angiogenic activity of EPC is impaired in CRF, a disease in which endothelial dysfunction and impaired angiogenesis have been described.8,28 There is growing evidence that bone marrow-derived EPC participates in the repair of endothelial dysfunction.15,16,20 This process can be divided into 3 stages: mobilization from bone marrow, homing into the sites of vascular injury, and incorporation into the endothelium of the injured or newly formed blood vessels.29 In our study, the number of EPC is decreased in CRF despite no difference in plasma VEGF levels. This implies that there may be a fundamental impairment in the production, mobilization, or half-life of EPC in CRF. The exhaustion of a presumed finite supply of EPC by continuous endothelial damage and consumption of EPC for vascular endothelial repair might also contribute to the decreased EPC numbers.18–20
Current data suggest that EPC may be incorporated into damaged endothelium and may work in concert with existing endothelial cells to form blood vessels rather than forming entirely new vessels.18–20,30 It is interesting that not only EPC migration and incorporation into EC was reduced but that EC network formation was also reduced when EC was cocultured with the EPC of CRF patients. These observations suggest that EPC incorporation into damaged endothelium or neovascularization foci may be impaired in CRF, and that the differentiation of normal EC may also be affected when EPC is functionally impaired. These phenomena could contribute to the deterioration in the repair of damaged endothelium or angiogenesis in CRF.8,28
In our study, the number of EPC was much lower in the CRF group than in the normal group, even when both groups had the same burden of risk factors. Furthermore, the number of EPC was inversely correlated with the estimated future coronary artery disease risk in both groups. These findings are consistent with the clinical observation that coronary artery disease risk in CRF is disproportionately high relative to the underlying risk factor profile.9,10 Our study suggests that EPC mobilization and incorporation into damaged endothelium or neovascularization foci may be impaired in CRF, as it is in other traditional risk factors such as aging, diabetes, and hypercholesterolemia.18–22,31
The dose of dialysis is an index of the removal of uremic toxins and is a key determinant of prognosis for end-stage CRF patients.1,27,32,33 Our data show that the dose of dialysis is inversely related to the capacity for EPC incorporation, which is a principal step in tissue vascularization by bone marrow-derived progenitor cells.16 This result may indicate the mechanism by which the greater removal of uremic toxins by higher doses of dialysis can improve the angiogenic function of EPC and the prognosis of CRF patients.
Possible mechanisms for the altered biology of EPC observed in CRF are presented here. Excessive oxidative stress, which is known to be related to increased cardiovascular risk in CRF, may inhibit the differentiation of EPC into the mature EC and contribute to the impaired repair of injured vascular endothelium.34,35 Another candidate is the deficient NO production in CRF.36,37 NO synthase, which has an essential role in mobilization of EPC,38 is blocked by endogenous inhibitory actions of guanidine compounds, which are major components of uremic toxins.39,40 Therefore, deficient NO production might lead to decreased mobilization of EPC from bone marrow. Finally, the function of EPC may also be impaired by uremic toxins, similar to the way activation of T and B lymphocyte is impaired in CRF.41 In our study, the level of blood urea nitrogen, which represents an approximate burden of uremic toxin burden, was weakly correlated to the number of EPC (P=0.06, data not shown). However, considering the fact that the blood urea nitrogen varies widely between measurements and is not an accurate measure of uremic toxin burden, the correlation between the level of uremic toxin and the number of EPC may be suggestive and could hardly conclusive.
Recently a new therapeutic strategy involving the administration of autologous EPC to increase neovascularization has generated great interest.23,42–44 The results of the present study suggest that if such approach is to reach clinical fruition, the function of the transplanted cells must also be considered. The effects of therapeutic angiogenesis using autologous cell transplantation may not be satisfactory in patients with EPC dysfunction, such as diabetic, aged, or those with CRF. However, our study shows that higher dose of dialysis, which may eliminate the cause of EPC dysfunction, is related to better EPC function. If the angiogenic function of EPC can be improved by modification of the pathophysiology of EPC dysfunction, not only the therapeutic outcome of EPC transplantation but also the future coronary artery disease risk may be improved.
In conclusion, our data demonstrate that EPC is numerically and functionally impaired in CRF, and that this is inversely related to the risk factor score of coronary artery disease. The results of our study may account for the acceleration of atherosclerosis, impairment of angiogenesis, and increased coronary artery disease risk observed in CRF patients.
Acknowledgments
This work was supported by National Research Laboratory Grants from Korea Institute of Science and Technology Evaluation and Planning (M1-0203-00-0048) to D. K. Kim.
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ABSTRACT
Objective— Increased risk of cardiovascular disease in patients with chronic renal failure (CRF) has been explained by accelerated atherosclerosis and impaired angiogenesis, in which endothelial progenitor cells (EPCs) may play key roles. We hypothesized that altered EPC biology may contribute to the pathophysiology of CRF.
Methods and Results— EPCs were isolated from CRF patients on maintenance hemodialysis (n=44) and from a normal control group (n=30). CRF patients showed markedly decreased numbers of EPC (44.6%) and colonies (75.3%) when compared with the controls (P<0.001). These findings were corroborated by 30.5% decrease in EPC migratory function in response to vascular endothelial growth factor (VEGF) (P=0.040) and 48.8% decrease in EPC incorporation into human umbilical vein endothelial cells (HUVEC) (P<0.001). In addition, Framingham’s risk factor score of both CRF (r=–0.461, P=0.010) and normal group (r=–0.367, P=0.016) significantly correlated with the numbers of EPC. Indeed, the number of circulating EPC was significantly lower in CRF patients than in normal group under the same burden of risk factors (P<0.001). A significant correlation was also observed between dialysis dose (Kt/V) and EPC incorporation into HUVEC (r=0.427, P=0.004).
Conclusions— EPC biology, which is critical for neovascularization and the maintenance of vascular function, is altered in CRF. Our data strongly suggest that dysfunction of circulating EPC has a role in the progression of cardiovascular disease in patients with CRF.
Our study shows that EPC is numerically and functionally impaired in CRF. This may contribute to the accelerated atherosclerosis and impaired angiogenesis observed in patients with CRF. Altered biology of EPC may therefore account for the increased cardiovascular disease risk in CRF.
Key Words: endothelial progenitor cell ? risk factors ? vascular biology ? renal physiology
Introduction
The lifespan of patients with chronic renal failure (CRF) is reduced, and coronary artery disease is the most important cause of morbidity and mortality in these patients.1,2 Even the results of therapeutic strategies such as percutaneous coronary intervention and bypass surgery have shown poor procedural success rates and dismal long-term event-free survival in CRF patients.3,4
Most of the increased cardiovascular morbidity and mortality in CRF has been accounted for by the rapid progression of atherosclerosis, which is clinically shown to be accelerated in CRF.5,6 Experimental studies have also shown that even mild renal dysfunction causes a dramatic acceleration of atherosclerosis.7 Angiogenesis, which is an essential compensation for myocardial ischemia, is also impaired in CRF.8 However the mechanism underlying the acceleration of atherosclerosis and impaired angiogenesis by CRF has not been examined closely. Although the phenomenon has been partially explained by the higher prevalence of established risk factors in CRF, such as hypertension, abnormal carbohydrate metabolism, and increased low density lipoprotein (LDL) cholesterol, the extent and severity of cardiovascular disease is clearly disproportionately high relative to the underlying risk factor profile.9,10
Recent studies have identified that normal adults have a small amount of circulating endothelial progenitor cell (EPC) in the peripheral blood. In response to cytokine stimulation and ischemic insult, these cells are mobilized from bone marrow, home to the ischemic tissue, and contribute to neovascularization and angiogenesis.11–14 Moreover, EPC is regarded to have a key role in the maintenance of vascular integrity and to act as "repair" cells in response to the endothelial injury,15,16 which has been regarded as an initial step in atherosclerosis and a result of the actions of various cardiovascular risk factors.17 Current data suggest that decrease in circulating EPC contributes not only to impaired angiogenesis but also to the progression of atherosclerosis,18 and patients at risk for coronary artery disease have a decreased number of circulating EPC with impaired activity.19–22
Therefore, we reasoned that EPC, which is critical for neovascularization and the maintenance of vascular integrity, might be numerically or functionally impaired in CRF. This would contribute to the accelerated atherosclerosis and impairment of angiogenesis, which may account for the increased cardiovascular risk and poor clinical outcomes observed in CRF. We investigated the number and angiogenic function of EPC obtained from patients on maintenance hemodialysis and compared them with the corresponding parameters of healthy volunteers.
Methods
Study Subjects
We studied 44 male CRF patients on maintenance hemodialysis and 30 healthy male volunteers who were older than age 21 years. Chronic stable patients on maintenance hemodialysis were selected to exclude potential factors that may affect biology of circulating EPC in the setting of recent aggravation of renal function. Patients with clinical evidence of symptomatic coronary artery disease, peripheral vascular disease, cerebrovascular disease, or carotid artery stenosis were excluded. Subjects with any condition, such as neoplasm, wounds, or significant retinopathy, which might involve neovascularization, were also excluded.14 Medications, including statins, glucose-controlling drugs, and antihypertensive drugs, were continued. No new medications, including vitamins, were used for at least 2 weeks before the study. Laboratory data of each subject were obtained within 2 weeks from the date of blood sampling. All enrolled subjects underwent a detailed assessment of cardiovascular risk after signing an informed consent document approved by the Institutional Review Board of Samsung Medical Center.
Isolation of EPC
Peripheral blood (30 mL) was drawn by venipuncture using a heparin-coated syringe. Samples of CRF patients were drawn just before the beginning of hemodialysis to exclude any possible influence of dialyzer on the cell. Mononuclear cells were isolated by density gradient method using Ficoll-Paque Plus (Amersham, Buckinghamshire, UK),and resuspended in EGM-2 MV Singlequot medium (Cambrex, East Rutherford, NJ), which contains multiple growth factors including human vascular endothelial growth factor (VEGF) A, human fibroblast growth factor-2, human endothelial growth factor, insulin-like growth factor-1, and ascorbic acid. Cells were plated on 6-well plates (Becton Dickinson, Franklin Lakes, NJ) coated with 2% gelatin (Sigma, St. Louis, Mo). The initial seeding density was standardized at 4x106 cells per well. After 3 days of culture, nonadherent cells were removed and the media were changed. The culture was maintained through day 7. After the numbers of EPC and EPC colony were counted, cells were assayed or harvested for further study.
EPC Characterization
Endothelial cell (EC) phenotype of EPC was investigated at day 7. Adherent cells were incubated with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)-labeled acetylated LDL (Molecular Probes, Eugene, Ore) at 37°C for 3 hours, and then with fluorescein-isothiocyanate-conjugated Ulex europaeus agglutinin (UEA)-1 lectin (10 μg/mL, Sigma) for 4 hours. Samples were examined with an inverted fluorescence microscope (Zeiss) and only cells exhibiting double fluorescence were identified as EPC.21,23 Cultured human umbilical vein endothelial cells (HUVEC) and NIH 3T3 cells were stained simultaneously and served as the positive and negative controls, respectively. To confirm endothelial cell phenotype of EPC, cells were also simultaneously stained with primary antibodies against the VEGF receptor KDR (Sigma) and von Willebrand factor (vWF) (BD Pharmingen, San Diego, Calif). Fluorescein-isothiocyanate and rhodamine-linked secondary monoclonal antibodies were used.
To confirm that the EPCs of our study population were capable of intracellular nitric oxide (NO) synthesis, we examined the EPCs of normal subjects with a membrane NO-specific fluorescence indicator, diamino-fluorescein-2 diacetate (DAF-2 DA; Daiichi, Japan). EPCs were gently washed twice with calcium-free phosphate-buffered saline (PBS) and bathed in Krebs-Henseleit buffer containing L-arginine (1 mmol/L) and DAF-2 DA (10 μmol/L) for 15 minutes. The cells were then incubated at 37°C for an additional 15 minutes, and 50 ng/mL of recombinant human VEGF165 (R&D Systems, Minneapolis, Minn) was added to the wells. The samples were then examined with fluorescence microscopy.
Cell surface antigens were investigated with fluorescence-activated cell sorter (FACS) analysis.11,23 Cultured cells were detached by brief incubation with trypsin/1 mmol/L EDTA followed by forceful pipetting, and cells (1x105 cells each) were used for FACS analysis. Antibodies to the VEGF receptor KDR (Sigma), vWF (BD Pharmingen), and VE-cadherin (BD Pharmingen) were used as primary antibodies. Peripheral blood mononuclear cells served as controls.
EPC Count
The numbers of EPC and EPC colony were determined by counting 12 random high-power (x100) microscope fields per subject and were expressed as cells or colonies per mm2. Spindle-shaped cells and colonies consisting of multiple thin, flat cells emanating from a central cluster of rounded cells were counted.
EPC Migration Assay
EPC migratory function, which is essential for angiogenesis, was examined using a modified Boyden chamber technique. A 24-well Transwell apparatus (Coster) was used, with each well containing a 6.5-mm polycarbonate membrane with 8-μm pores, coated with type I collagen (Sigma). EPCs (4x104) were placed on the membrane, and the chamber was immersed in a 24-well plate, which was filled with growth factor-free EBM-2 culture media or EBM-2 with 50 ng/mL of human VEGF165. After incubation for 24 hours, the membrane was washed briefly with PBS and the upper side of membrane was wiped gently with a cotton ball. The membrane was then removed and stained using Giemsa solution. The magnitude of EPC migration was evaluated by counting the migrated cells in 4 random high-power (x100) microscope fields.
Matrigel Tube Formation Assay
A Matrigel tube formation assay was performed to assess the ability of EPC to incorporate into endothelial cell vascular structures, which is believed to be important in new vessel formation.16 Matrigel (Becton Dickinson) was spread onto 4-well chamber slides (Nalge Nunc). EPCs were marked with DiI (Molecular Probes) to distinguish them from HUVEC. DiI-labeled EPC (1x104) and HUVEC (4x104) were plated together and incubated at 37°C for 24 hours with EGM-2 culture medium. Incorporated cells were counted from 4 random high-power (x100) microscope fields per each subject. The numbers and line lengths of the circle formed by cells were also calculated.
Statistical Analysis
All data are presented as means±SEM. Continuous variables were evaluated by nonparametric Mann–Whitney test or Wilcoxon signed rank test. Categorical variables were evaluated by Fischer exact test. Univariate correlations were made with Spearman correlation coefficient. Multiple linear regression for the analysis of covariance was used to identify predictors of changes in EPC counts in a multivariate setting. SPSS release 11.0 (SPSS Inc) was used, and differences were considered statistically significant at 2-tailed P<0.05.
Results
Patients Characteristics
Clinical data of the study subjects are summarized in the Table. Patients in the CRF group were 4.6 years older than those in the normal group and had a lower body mass index. Total cholesterol and high-density lipoprotein cholesterol were significantly lower in the CRF group. All CRF patients were treated with erythropoietin, and most of them (97.7%, n=43) were using antihypertensive medication. Plasma VEGF levels measured with an enzyme-linked immunosorbent assay kit (R&D Systems) showed no difference between CRF and normal groups. There was no difference in total leukocyte counts between 2 groups. The underlying cause of CRF was diabetic nephropathy (n=22) or other causes (n=22).
Characteristics of Study Subjects
EPC Characterization
Culture of total peripheral blood mononuclear cell resulted in the emergence of characteristic spindle-shaped cells and colonies consisting of peripheral spindle-shaped cells emanating from round central cells within 72 hours of culture (Figure 1A and 1B). These cells could be shown to uptake acetylated LDL and bind UEA-1, consistent with endothelial lineage cells (Figure 1C through 1H). Coexpression of 2 endothelial lineage-specific markers, vWF and KDR (Figure IA through IC, available online at http://atvb.ahajournals.org) and specific NO synthesis in these cells were also confirmed (Figure ID through IF, available online at http://atvb.ahajournals.org). FACS analysis showed that the percentage of cells positive for endothelial lineage markers, KDR, vWF, and VE-cadherin were 23.9%, 39.1%, and 53.3%, respectively (Figure IG, available online at http://atvb.ahajournals.org).
Figure 1. Identification of the endothelial lineage phenotype of cultured EPC. Culture of peripheral blood mononuclear cells resulted in the emergence of characteristic spindle-shaped EPCs (A) and EPC colonies characterized by a central cluster of rounded cells surrounded by radiating spindle-shaped cells (B). EPCs (C through E) and EPC colonies (F to H) were shown to uptake acetylated LDL (C, F) and bind UEA-1 (D, G). Merged images show that most cells are dual-positive (E, H).
EPC Count
Significantly fewer EPC colonies were formed from mononuclear cell culture of CRF patients compared with normal controls at day 7 (133.7±21.6 versus 542.5±64.1 per 103 mm2; P<0.001) (Figure 2A through 2C). Significantly fewer EPCs were also identified from CRF patients (10.0±1.2x103 versus 17.8±2.0x103 per 103 mm2; P<0.001) (Figure 2D through 2F). Despite differences in age, body mass index, total cholesterol, high-density lipoprotein cholesterol, hypertension, and usage of statin, which might affect EPC biology (Table),15,18,24 the statistically significant differences were maintained after adjusting for these factors (EPC colonies of CRF versus normal; P<0.001, EPC of CRF versus normal; P=0.005) (Figure IIA and IIB, available online at http://atvb.ahajournals.org). There was no difference in the numbers of EPCs or EPC colonies between diabetic and nondiabetic CRF patients.
Figure 2. The number of EPCs and EPC colonies counted after 1 week of culture is reduced in CRF. Representative photos of EPC colony culture from the normal (A) and CRF group (B), demonstrating fewer colonies in the CRF group. C, EPC colonies were significantly reduced (by 75.3%) in the CRF group compared with the normal group, and this was independent of other clinical factors (P<0.001) (Figure IIA). Representative photos of EPC cultures from the normal (D) and CRF group (E), demonstrating fewer EPCs in the CRF group. F, EPCs were also significantly reduced (by 44.6%) in the CRF group. *Statistical significance (P<0.05).
EPC Migration Assay
The migratory function of EPC in response to VEGF, which is believed to be important during neovascularization,19 was evaluated using a modified Boyden chamber. The basal EPC migratory functions of CRF patients and normal controls were not significantly different (P>0.05). After supplementation with 50 ng/mL of VEGF, the migratory function of EPC in both groups was significantly augmented (baseline versus VEGF-augmented migration, CRF patients; 14.9±3.3 versus 22.4±3.7 per x100 high-power field; P=0.001, normal group; 19.1±3.2 versus 32.9±5.3 per x100 high-power field; P=0.016) (Figure 3A through 3E). However, the VEGF-induced augmentation of EPC migration was relatively impaired in CRF. VEGF-induced augmentation of EPC migration was reduced by 52.8% in CRF patients relative to that of the normal group (7.5±1.4 versus 15.8±3.5 per x100 high-power field, P=0.036) (Figure 3F), and the total number of migrated EPC was also lower in CRF patients independently of clinical factors described (22.4±3.7 versus 32.9±5.3 per x100 high-power field, P=0.017) (Figure IIC, available online at http://atvb.ahajournals.org).
Figure 3. EPC migratory function is impaired in CRF. A modified Boyden chamber assay was used with growth factor-free medium or VEGF supplemented medium. Baseline (A) and VEGF-stimulated (B) EPC migration in the normal group. Baseline (C) and VEGF-stimulated (D) EPC migration in the CRF group. Representative photos are shown. The small dots are holes of the Boyden chamber membrane. E, Baseline migratory function of EPC did not differ between 2 groups (P>0.05). Supplementation with VEGF led to significantly augmented migration of EPCs in both groups (normal group; P=0.001, CRF group; P=0.016). However, EPC of the CRF group showed a reduced increase in the number of migrated cells (50.2% increase) relative to that of the normal group (72.5% increase). F, The reduced increase in EPC migration in CRF was statistically significant. *P<0.05.
Matrigel Tube Formation Assay
EPC incorporation into the tubular networks formed by HUVEC was evaluated in culture using Matrigel, which is used to evaluate EC differentiation.25 Significantly fewer EPCs of CRF patients were incorporated into tubules compared with the EPCs from normal controls (8.2±0.5 per mm2 versus 16.0±1.1 per mm2, P<0.001) (Figure 4A through 4C), independently of clinical factors described (P<0.001) (Figure IID, available online at http://atvb.ahajournals.org). Significantly fewer circles and shorter line lengths of the tubules were also identified (Figure IIIA through IIID, available online at http://atvb.ahajournals.org). There was no difference in EPC incorporation or tubule formation between diabetic and nondiabetic CRF.
Figure 4. EPC incorporation into EC and stimulation of EC tube formation is impaired in CRF. EPCs were labeled with DiI and cocultured with HUVEC in Matrigel-coated 4-well chamber slide for 24 hours. Fluorescence and light images of identical fields were merged. Significantly fewer EPCs from CRF patients were incorporated into the tubules formed by HUVEC (A) than those of the normal controls (B). EPC incorporation in the CRF group was reduced by 51.9% (C). *P<0.05.
Risk of Coronary Artery Disease and the Level of EPC
We next investigated the relationship between the level of circulating EPC and the risk of coronary artery disease, which is a clinical consequence of accelerated atherosclerosis and impaired angiogenesis in CRF.5–8 Because none of our study subjects showed any clinical evidence of coronary artery disease, the 10-year coronary artery disease risk was estimated from the total burden of risk factors according to the Framingham risk score.20,26 The EPC number was significantly inversely correlated with the estimated 10-year coronary artery disease risk in CRF patients (r=–0.367, P=0.016) and in the normal group (r=–0.461, P=0.010) (Figure 5A and 5B), suggesting that a lower level of EPC is associated with a higher risk of coronary artery disease in CRF as well as in normal renal function group.20
Figure 5. The level of circulating EPC is inversely correlated with the risk factor score of coronary artery disease, and the EPC incorporation function is correlated with the dose of dialysis (Kt/V). The correlation between the number of EPC and the 10-year coronary artery disease risk estimated from the Framingham risk score was evident in the normal group (A) and CRF group (B). C, The age-adjusted relative risk of coronary artery disease was calculated to compare the numbers of EPC under the same risk burden. The correlation was maintained in the normal (r=–0.399, P=0.029) and CRF group (r=–0.422, P=0.005), and there were significantly fewer EPCs in the CRF group than in the normal group after correction for risk burden (P<0.001). D, The number of EPC was significantly reduced not only in the total CRF group but also in the low-risk (P=0.009) and high-risk subgroup (P<0.001). The EPC incorporation function was significantly different between lower doses and higher doses of dialysis (P=0.015) (E) and correlated significantly with the dose of dialysis (r=0.427, P=0.004) (F).
To compare the EPC numbers in both groups under the same risk burden, the age-adjusted relative risk of each subject was estimated.26 EPC numbers were also significantly inversely correlated with the age-adjusted relative risk, both in CRF patients (r=–0.422, P=0.005) and in the normal group (r=–0.399, P=0.029). The numbers of EPC were significantly lower in CRF patients after adjustment for age-adjusted relative risk (P<0.001) (Figure 5C). We next divided the study subjects into a low-risk group (for whom the 10-year coronary artery disease risk was <2.0-times the optimal risk profile) and a high-risk group (2.0-times). The number of EPC in CRF patients was significantly lower than that in the normal group independently of whether they were in the low-risk or high-risk group (low-risk group; CRF versus normal; 11.7±1.6x104 versus 20.3±2.8x104 per 103 mm2; P=0.009, high-risk group; 8.2±1.8x104 versus 15.3±2.8x104 per 103 mm2; P<0.001) (Figure 5D). From these observations, it would appear that the level of EPC is reduced in CRF irrespective of the risk burden.
Impact of Dialysis Dose on the EPC Migratory Function
As shown, a lower number of EPC was correlated with a higher future coronary artery disease risk. We presumed that the number or function of EPC might also be related to the dose of dialysis calculated by Kt/V, which is known to be related to mortality risk in CRF patients.27 Because a Kt/V of 1.3 is regarded as the minimal standard for adequate dialysis,2,27 we compared the EPC incorporation function of patients receiving a lower dialysis dose (Kt/V <1.3) with that of patients receiving a higher dialysis dose (Kt/V 1.3). The EPCs of patients receiving lower doses of dialysis showed significantly impaired incorporation into EC compared with those of patients receiving higher doses of dialysis (5.3±1.1x103 versus 8.6±0.5x103 EPC per 103 mm2, P=0.015) (Figure 5E). Furthermore, the dose of dialysis was significantly correlated with the degree of incorporation of EPC into EC (r=0.427, P=0.004) (Figure 5F). This interesting finding suggests that higher doses of dialysis may be related to improved angiogenic function of EPC. However, other parameters such as the number of EPC, the number of EPC colonies, and migratory functions were not correlated with the dose of dialysis.
Discussion
In this study, we demonstrate that the number of circulating EPC is decreased and the angiogenic activity of EPC is impaired in CRF, a disease in which endothelial dysfunction and impaired angiogenesis have been described.8,28 There is growing evidence that bone marrow-derived EPC participates in the repair of endothelial dysfunction.15,16,20 This process can be divided into 3 stages: mobilization from bone marrow, homing into the sites of vascular injury, and incorporation into the endothelium of the injured or newly formed blood vessels.29 In our study, the number of EPC is decreased in CRF despite no difference in plasma VEGF levels. This implies that there may be a fundamental impairment in the production, mobilization, or half-life of EPC in CRF. The exhaustion of a presumed finite supply of EPC by continuous endothelial damage and consumption of EPC for vascular endothelial repair might also contribute to the decreased EPC numbers.18–20
Current data suggest that EPC may be incorporated into damaged endothelium and may work in concert with existing endothelial cells to form blood vessels rather than forming entirely new vessels.18–20,30 It is interesting that not only EPC migration and incorporation into EC was reduced but that EC network formation was also reduced when EC was cocultured with the EPC of CRF patients. These observations suggest that EPC incorporation into damaged endothelium or neovascularization foci may be impaired in CRF, and that the differentiation of normal EC may also be affected when EPC is functionally impaired. These phenomena could contribute to the deterioration in the repair of damaged endothelium or angiogenesis in CRF.8,28
In our study, the number of EPC was much lower in the CRF group than in the normal group, even when both groups had the same burden of risk factors. Furthermore, the number of EPC was inversely correlated with the estimated future coronary artery disease risk in both groups. These findings are consistent with the clinical observation that coronary artery disease risk in CRF is disproportionately high relative to the underlying risk factor profile.9,10 Our study suggests that EPC mobilization and incorporation into damaged endothelium or neovascularization foci may be impaired in CRF, as it is in other traditional risk factors such as aging, diabetes, and hypercholesterolemia.18–22,31
The dose of dialysis is an index of the removal of uremic toxins and is a key determinant of prognosis for end-stage CRF patients.1,27,32,33 Our data show that the dose of dialysis is inversely related to the capacity for EPC incorporation, which is a principal step in tissue vascularization by bone marrow-derived progenitor cells.16 This result may indicate the mechanism by which the greater removal of uremic toxins by higher doses of dialysis can improve the angiogenic function of EPC and the prognosis of CRF patients.
Possible mechanisms for the altered biology of EPC observed in CRF are presented here. Excessive oxidative stress, which is known to be related to increased cardiovascular risk in CRF, may inhibit the differentiation of EPC into the mature EC and contribute to the impaired repair of injured vascular endothelium.34,35 Another candidate is the deficient NO production in CRF.36,37 NO synthase, which has an essential role in mobilization of EPC,38 is blocked by endogenous inhibitory actions of guanidine compounds, which are major components of uremic toxins.39,40 Therefore, deficient NO production might lead to decreased mobilization of EPC from bone marrow. Finally, the function of EPC may also be impaired by uremic toxins, similar to the way activation of T and B lymphocyte is impaired in CRF.41 In our study, the level of blood urea nitrogen, which represents an approximate burden of uremic toxin burden, was weakly correlated to the number of EPC (P=0.06, data not shown). However, considering the fact that the blood urea nitrogen varies widely between measurements and is not an accurate measure of uremic toxin burden, the correlation between the level of uremic toxin and the number of EPC may be suggestive and could hardly conclusive.
Recently a new therapeutic strategy involving the administration of autologous EPC to increase neovascularization has generated great interest.23,42–44 The results of the present study suggest that if such approach is to reach clinical fruition, the function of the transplanted cells must also be considered. The effects of therapeutic angiogenesis using autologous cell transplantation may not be satisfactory in patients with EPC dysfunction, such as diabetic, aged, or those with CRF. However, our study shows that higher dose of dialysis, which may eliminate the cause of EPC dysfunction, is related to better EPC function. If the angiogenic function of EPC can be improved by modification of the pathophysiology of EPC dysfunction, not only the therapeutic outcome of EPC transplantation but also the future coronary artery disease risk may be improved.
In conclusion, our data demonstrate that EPC is numerically and functionally impaired in CRF, and that this is inversely related to the risk factor score of coronary artery disease. The results of our study may account for the acceleration of atherosclerosis, impairment of angiogenesis, and increased coronary artery disease risk observed in CRF patients.
Acknowledgments
This work was supported by National Research Laboratory Grants from Korea Institute of Science and Technology Evaluation and Planning (M1-0203-00-0048) to D. K. Kim.
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