Aldehyde dehydrogenase activity allows reliable EPC enumeration in stored peripheral blood samples

Background Interest in the biology of endogenous progenitor cells (EPCs) continues to grow as evidence of their role in vascular repair mounts. EPC enumeration requires specialized laboratory techniques and is performed immediately after sample acquisition, limiting the clinical contexts in which EPC enumeration can be performed and the ability to increase sample sizes through multi-center participation. Methods We compared the numbers of EPCs enumerated in samples processed immediately after acquisition (n = 36) with EPCs enumerated in specimens stored for 24 hours or after cryopreservation of mononuclear cells (MNC) using two EPC identification strategies: cell surface marker expression (CD133/CD34) and aldehyde dehydrogenase activity (ALDH^br cells). Results EPCs assessed in fresh samples correlated with EPCs enumerated after whole blood storage (r = 0.699 for CD133⁺CD34⁺ cells, r = 0.880 for ALDH^br cells, P < 0.005 and P < 0.0001, respectively) or mononuclear cryopreservation (r = 0.590 for CD133⁺CD34⁺ cells, r = 0.894 for ALDH^br cells, P < 0.0001 for each); however, correlation based on assessment of ALDH^br cells was higher (P < 0.0003 for comparison of correlation coefficients). Initial results from a multi-site clinical trial suggest that EPC enumeration after mononuclear cell cryopreservation is feasible. Conclusion EPC analysis based on ALDH activity is reproducible, even after extended whole blood storage or MNC cryopreservation.     

In acute kidney injury, indoxyl sulfate impairs human endothelial progenitor cells: modulation by statin

Renal ischemia rapidly mobilizes endothelial progenitor cells (EPCs), which provides renoprotection in acute kidney injury (AKI). Indoxyl sulfate (IS) is a protein-binding uremic toxin with a potential role in endothelial injury. In this study, we examined the effects and mechanisms of action of IS on EPCs in AKI. Forty-one consecutive patients (26 male; age, 70.1 ± 14.1 years) diagnosed with AKI according to the AKIN criteria were enrolled. The AKI patients had higher serum IS levels than patients with normal kidney function (1.35 ± 0.94 × 10^?4M vs. 0.02 ± 0.02 × 10^?4M, P < 0.01). IS levels were negatively correlated to the number of double-labeled (CD34⁺KDR⁺) circulating EPCs (P < 0.001). After IS stimulation, the cells displayed decreased expression of phosphorylated endothelial nitric oxide (NO) synthase, vascular cell adhesion molecule-1, increased reactive oxygen species, decreased proliferative capacity, increased senescence and autophagy, as well as decreased migration and angiogenesis. These effects of IS on EPCs were reversed by atorvastatin. Further, exogenous administration of IS significantly reduced EPC number in Tie2-GFP transgenic mice and attenuated NO signaling in aortic and kidney arteriolar endothelium after kidney ischemia–reperfusion injury in mice, and these effects were restored by atorvastatin. Our results are the first to demonstrate that circulating IS is elevated in AKI and has direct effects on EPCs via NO-dependent mechanisms both in vitro and in vivo. Targeting the IS-mediated pathways by NO-releasing statins such as atorvastatin may preempt disordered vascular wall pathology, and represent a novel EPC-rescued approach to impaired neovascularization after AKI.     

Circulating Endothelial Progenitor Cells as Markers for Severity of Ischemic Chronic Heart Failure

IntroductionDespite a high potential of endothelial progenitor cells (EPCs) for diagnostic purposes, the EPC role in developing ischemic chronic heart failure (CHF) has not been determined obviously.The objective of this study was to assess the counts of CD45⁺CD34⁺, CD45⁻CD34⁺, CD14⁺CD309⁺, and CD14⁺CD309⁺Tie2⁺ phenotyped circulating EPCs of various subpopulations in patients with ischemic CHF.Methods and ResultsThe study involved 153 patients (86 male), aged 48–62 years, with angiographically proven coronary artery disease (CAD) and 25 healthy volunteers. CHF was diagnosed in 109 patients (71.2%). Mononuclear cell populations were phenotyped by flow cytofluorimetry. Cardiovascular risk factors, such as type 2 diabetes mellitus, hyperlipidemia, arterial hypertension, and adherence to smoking, may have a negative effect on circulating EPC counts in CAD patients regardless of the presence of CHF. The depletion of the CD14⁺CD309⁺- and CD14⁺CD309⁺Tie2⁺-phenotyped circulating EPC counts is associated with the severity of left ventricular dysfunction, whereas the CD45⁺CD34⁺- and CD45⁻CD34⁺-mononuclear cell counts are more representative of the severity of atherosclerotic coronary artery lesions.ConclusionThe authors found that New York Heart Association functional class of CHF, left ventricular ejection fraction <42%, the N-terminal pro–B-type natriuretic peptide level >554 pg/mL, and Е/Еm ratio >15 U had the highest predictive value for the depletion of the EPC count in CAD patients.     

Effects of myocardial postconditioning on the recruitment of endothelial progenitor cells

Background: Ischemic postconditioning (PostC), brief repetitive cycles of ischemia and reperfusion during early reperfusion, is suggested to protect the myocardium in patients with stent thrombosis‐elevation myocardial infarction (STEMI) by improved endothelial dysfunction and alteration of cytokine release. These mechanisms are also of importance for the recruitment of endothelial progenitor cells (EPC), an endogenous repair mechanism for re‐endothelialization and neoangiogenesis. The aim of this study was to investigate the effect of PostC on recruitment of EPC. Methods: EPC were analyzed in 20 patients with STEMI randomized to receive four cycles of PostC following percutaneous coronary intervention (PCI) or conventional PCI. Different subpopulations of EPC were quantified immediately and on day 4 using flow cytometry. Myocardium at risk, and infarct size was determined by cardiovascular magnetic resonance. Results: There was no influence of PostC on the number of different EPC (CD34⁺, CD133⁺, CD34⁺CD133⁺, CD34⁺KDR⁺, CD34^?CD133⁺KDR⁺, CD34⁺CD133⁺KDR⁺). Left ventricular ejection fraction, myocardium at risk, and infarct size did not correlate to the mobilization of EPC. There was an inverse correlation between the symptom‐to‐balloon time and the mobilization of progenitor precursor cells (CD34⁺ cells: R =?0.527, P = 0.02; CD133⁺ cells: R =?0.624, P = 0.004; CD34⁺CD133⁺ cells: R =?0.466, P = 0.04). Discussion: Ischemic PostC did not result in improved mobilization of EPC in STEMI patients. The recruitment of progenitor cells seems to be related to the duration of ischemia rather than the size of the ischemic myocardial area. More effort is needed to understand the changes of endothelial surface markers by PostC and their role in EPC recruitment and homing. (J Interven Cardiol 2012;25:103–110)     

Circulating Bone Marrow-Derived CD45−/CD34+/CD133+/VEGF+ Endothelial Progenitor Cells in Adults with Crohn’s Disease

Background

Circulating endothelial progenitor cells (EPCs) are bone marrow-derived stem cells able to migrate to sites of damaged endothelium and differentiate into endothelial cells. Altered EPC level and function have been described in various inflammatory diseases and have been shown to augment vasculogenesis in murine models. Previous studies of EPC in the context of Crohn’s disease (CD) have yielded conflicting results.

Aim

To determine whether the circulating levels of EPCs are changed in the context of CD.

Methods

CD patients and healthy controls were recruited. Disease activity was assessed by CDAI. Peripheral blood mononuclear cells were isolated and EPC numbers evaluated by FACS analysis using anti-CD34, anti-VEGF receptor-2, anti-CD133, and anti-CD45 markers.

Results

Eighty-three subjects, including 32 CD patients and 51 controls were recruited, including 19 (59.4 %) and 23 (45 %) males (p = 0.26), aged 34.8 ± 14.9 and 43.3 ± 18.5 years (p = 0.64), in cases and controls, respectively. Mean CDAI was 147 ± 97, disease duration was 12.7 ± 11.1 years, and 28 (87.5 %) were receiving biologics for a mean duration of 21.7 ± 16.8 months. The mean level of peripheral EPCs in CD patients was 0.050 ± 0.086 percent and 0.007 ± 0.013 % in controls (p < 0.01). There was no significant correlation between EPC levels and age (r = ?0.13, p = 0.47), CDAI (r = ?0.26, p = 0.15), disease duration (r = ?0.04, p = 0.84), or duration of treatment with biologics (r = 0.004, p = 0.99).

Conclusion

EPCs are elevated in patients with CD. Further studies are needed to examine the function of EPCs and their possible role as a marker of disease severity or therapeutic response.

     

The association between circulating endothelial progenitor cells and coronary collateral formation

Aim

We investigated the relationship between coronary collateral formation and circulating endothelial progenitor cells (EPC) in patients undergoing coronary angiography.

Methods and results

Circulating CD133⁺/34⁺ and CD34⁺/KDR⁺ EPCs were determined in 68 patients (normal coronary vessels in 24 patients and coronary artery disease (CAD) in 44 patients) (age: 58.7 ± 10.1, 64.7% male). Circulating EPCs were higher among patients with normal coronary vessels compared to patients with CAD for CD133⁺/34⁺ (p < 0.05) and CD34⁺/KDR⁺ cells (p < 0.05). The number of EPCs were significantly greater in patients with good coronary collateral formation (p < 0.05). EPC count was independent predictor for coronary collateral formation after adjustment for other cardiovascular risk factors and extent of CAD (p = 0.037).

Conclusion

In patients with severe coronary stenosis, those with increased circulating EPCs had better collateral formation compared to those with lower EPC counts. Our findings implicate that in addition to presence of critical stenosis, intact response of bone marrow is necessary for collateral formation in CAD.     

Effects of rosuvastatin and allopurinol on circulating endothelial progenitor cells in patients with congestive heart failure: the impact of inflammatory process and oxidative stress

ObjectiveEndothelial progenitor cells (EPCs) contribute to the maintenance of endothelial integrity and function. We investigated the effects of rosuvastatin and allopurinol on the number of EPCs in patients with heart failure and aimed to provide insight into the molecular inflammatory and oxidative mechanisms that could be responsible for the alterations in EPC levels after treatment.MethodsSixty patients with systolic heart failure were randomized to receive rosuvastatin 10 mg/d, allopurinol 300 mg/d or placebo and followed up for 1 month. The number of CD34⁺/KDR⁺ and CD34⁺/CD133⁺/KDR⁺ EPCs in blood was evaluated by flow cytometry. Endothelial function was assessed by brachial artery flow-mediated dilation. Levels of markers of inflammation and oxidative stress were also determined.ResultsCirculating EPCs were significantly increased after rosuvastatin treatment (from 230 (170–380) and 10 (8–24) to 390 (230–520) and 19 (8–33) cells/10⁶ lymphomonocytes, respectively, p = 0.004 and p = 0.008), whereas they remained unchanged in the other groups. The increase in EPC levels was not associated with the changes in the levels of the measured inflammatory and oxidative markers.ConclusionShort-term treatment with rosuvastatin, but not allopurinol, significantly increases the number of circulating EPCs in patients with heart failure providing further insights into its role in these individuals. The impact of rosuvastatin on EPCs is not mediated by changes in inflammatory and oxidative status.     

Prolongation of PR interval is associated with endothelial dysfunction and activation of vascular repair in high-risk cardiovascular patients

Purpose

Epidemiological studies showed that PR prolongation is associated with increased risk of adverse cardiovascular outcomes. We investigated the relations of PR interval with indices of vascular function and endothelial repair as the underlying mechanisms.

Methods

The study comprised 348 high-risk patients with prior coronary artery disease, ischemic stroke, and/or diabetes mellitus recruited from medical outpatient clinics and 150 healthy subjects without such a history. PR interval was considered prolonged if >200 ms, as determined from resting 12-lead electrocardiogram. Vascular function was assessed by brachial flow-meditated dilatation (FMD) using high-resolution ultrasound. Circulating CD133⁺/KDR⁺ endothelial progenitor cell (EPC) levels were measured by flow cytometry.

Results

Among healthy subjects, PR interval was inversely associated with FMD (R?=??0.20, P?=?0.015), but not with the level of circulating CD133⁺/KDR⁺ EPC (R?=?0.05, P?=?0.58). Among high-risk cardiovascular patients, PR prolongation >200 ms was more common compared with healthy subjects (45/348 (13 %) versus 4/150 (3 %), P?<?0.001). PR interval was associated inversely with FMD (R?=??0.14, P?=?0.01) and positively with circulating CD133⁺/KDR⁺ EPC level (R?=?+0.14, P?=?0.009). Circulating CD133⁺/KDR⁺ EPC level was significantly increased in patients with PR prolongation >200 ms (0.87?±?0.37 versus 0.68?±?0.42 (log, ×10^?3/ml), P?=?0.005). Adjusted for potential confounders, increased PR interval remained independently associated with increased CD133⁺/KDR⁺ EPC by +0.002 (95 % confidence interval (CI) 0.000 to 0.004 (log, ×10^?3/ml), P?=?0.011) and depressed FMD (B?=??0.014 %, 95 % CI ?0.027 to ?0.002, P?=?0.026).

Conclusions

PR prolongation is associated with endothelial dysfunction and evidence of endothelial repair activation in patients with high cardiovascular risk.     

Increased circulating CD3+/CD31+ T cells in patients with acute coronary syndrome

The number of circulating endothelial progenitor cells (EPCs) is considered to be a surrogate marker for coronary artery disease (CAD). Recent studies have identified a novel T-cell subset labeled with CD3⁺/CD31⁺, which is necessary for EPC colony formation and constitutes the central cluster. However, the clinical relevance of the CD3⁺/CD31⁺ T cells in CAD remains unclear. We sought to clarify whether circulating CD3⁺/CD31⁺ T cells are increased in patients with acute coronary syndrome (ACS). Circulating CD3⁺/CD31⁺ T cells were determined in 16 ACS patients undergoing emergency percutaneous coronary intervention (PCI) and in 16 control subjects with angiographically normal coronary arteries. Although no differences between the groups were found in baseline patient characteristics, the ratio of circulating CD3⁺/CD31⁺ T cells before PCI was higher in ACS patients as compared with that in control subjects (51.8 % ± 7.8 % vs 31.8 % ± 9.6 %, respectively; P < 0.001). The increased ratio of CD3⁺/CD31⁺ T cells in ACS patients was not altered 24 h after PCI, but became comparable with that in control subjects within 6 months after PCI. These results suggest that mobilization of CD3⁺/CD31⁺ T cells occurs in ACS, but is no longer detectable at 6 months after PCI.     

Reduced numbers of regulatory B cells are negatively correlated with disease activity in patients with new-onset rheumatoid arthritis

This study is aimed at determining the numbers of circulating Treg and Breg cells in patients with new-onset rheumatoid arthritis and during subsequent drug therapies. Patients were treated orally with 10 mg methotrexate weekly, and 20 mg leflunomide and 60 mg common threewingnut root daily (Lei Gong Teng) for 12 weeks, but received no steroid therapy. Basal measurements were performed of serum C-reactive protein, anticyclic citrullinated peptide antibody, and erythrocyte sedimentation rate, and the numbers of cluster of differentiation CD4⁺CD25⁺Foxp3⁺ T cells, interleukin 10 (IL10)-expressing on CD5⁺CD1d⁺ and TIM1⁺ B cells. Compared with the healthy controls, patients exhibited significantly less numbers of circulating CD19⁺TIM1⁺IL10⁺, CD19⁺CD5⁺CD1d⁺IL10⁺ B cells and CD4⁺CD25⁺Foxp3⁺ T cells (P?<?0.001, all). Drug therapy modulated the balance of different subsets of Breg and Treg cells. The numbers of CD19⁺TIM1⁺IL10⁺ and CD19⁺CD5⁺CD1d⁺IL10⁺ B cells correlated positively with the numbers of CD4⁺CD25⁺Foxp3⁺ T cells in these patients (r?=?0.707, P?=?0.001; r?=?0.481, P?=?0.007, respectively). The values of DAS28 were negatively correlated with the numbers of CD19⁺TIM1⁺IL10⁺ and CD19⁺CD5⁺CD1d⁺IL10⁺ B cells, and CD4⁺CD25⁺Foxp3⁺ T cells (r?=??0.533, P?=?0.023; r?=??0.442, P?=?0.016; and r?=??0.444, P?=?0.014, respectively). Of note, TIM1⁺ B cells identified more circulating IL10⁺ B cells than CD5⁺CD1d⁺ B cells. Our data indicate that Breg and Treg cells have a potentially crucial role in controlling disease activity in rheumatoid arthritis patients, and TIM1⁺ Breg cells may be a viable therapeutic target for these patients.     

Clonogenic assay of endothelial progenitor cells

In stem cell biology, CD34⁺ or CD133⁺ hematopoietic stem cells (HSCs) give rise to two types of endothelial progenitor cell (EPC) colonies: primitive and definitive EPC-colony forming units (primitive EPC-CFU and definitive EPC-CFU), which can be morphologically defined. Based on their morphology, an evaluation of the number or the ratio of each EPC colony constitutes the Endothelial Progenitor Cell Clonogenic Forming Assay (EPC-CFA), a novel assay to quantify the differentiation of colony forming EPCs. This assay system allows us to practically evaluate the vasculogenic potential of primary or cultured stem cell populations, i.e., mononuclear cells or fractionated stem cells (CD34⁺ or CD133⁺ cells) in peripheral blood, bone marrow, or umbilical cord blood. EPC-CFA can be used not only for basic research in vascular biology but also for evaluating the vascular reparative activity of patients with cardiovascular diseases. This review summarizes the underlying concepts and significance of the EPC-CFA in vascular biology.     

Endothelial progenitor cells are associated with response to chemotherapy in human non-small-cell lung cancer

Purpose

Bone marrow-derived endothelial progenitor cells (EPCs) play an important role in angiogenesis and tumor growth. However, the clinical relevance of EPCs in non-small-cell lung cancer (NSCLC) remains unclear. Recently, some reports suggested that EPCs correlate with clinical behavior of cancer patients. We assessed the hypothesis that EPCs correlate with efficient of therapy, prognosis, and clinicopathological factors, and EPCs may offer a possible biomarker for treatment outcome in NSCLC.

Methods

EPCs labeled with CD34, CD133, and vascular endothelial growth factor receptor-2 (VEGFR-2) antibodies were counted by flow cytometry in the peripheral blood of 31 NSCLC patients. We categorized two groups of NSCLC patients according to circulating EPC numbers. We examined age, pathological stage, histological type, Fluoro-d-glucose Positron emission tomography (FDG-PET), response to therapy, progression-free survival, and tumor size of NSCLC patients and investigated whether these factors correlate with EPC counts.

Results

Circulating EPC numbers before antitumor therapy were increased in NSCLC patients compared with healthy controls (P?P?P?Conclusion Peripheral blood levels of bone marrow-derived EPCs are significantly increased in patients with NSCLC and correlate with response to chemotherapy. EPCs may offer a possible biomarker for efficient of treatment and prognosis.     

Population alterations of l-arginase- and inducible nitric oxide synthase-expressed CD11b+/CD14−/CD15+/CD33+ myeloid-derived suppressor cells and CD8+ T lymphocytes in patients with advanced-stage non-small cell lung cancer

Background

Immune aberrations have been demonstrated in tumorogenesis, and myeloid-derived suppressor cells (MDSC) have shown to play a pivotal role in mediating immune suppression in animal models of human tumors. In the present study, we explored the clinical relevance of CD11b⁺/CD14^?/CD15⁺/CD33⁺ MDSCs and the association of MDSCs with CD8⁺ cytotoxic T lymphocytes in patients with non-small-cell lung cancer (NSCLC).

Patients and methods

The population of CD11b⁺/CD14^? cells in peripheral blood mononuclear cells (PBMNC) was determined in 173 patients with NSCLC and 42 control subjects. The expression of CD15, CD33, IL-4R, INF-γR, iNOS and l-arginase were analyzed. Cocultures with CD8⁺ T lymphocytes and Jurkat cells were developed to determine the impact of MDSCs on the expression of CD3ζ of CD8⁺ T lymphocytes.

Results

Patients with treatment-naïve, advanced-stage NSCLC (n = 87) had an increased subpopulation of CD11b⁺/CD14^?/CD15⁺/CD33⁺ cells in the PBMNCs with characteristics of MDSCs (P < 0.0001). The CD11b⁺/CD14^? cells in PBMNC also express IL-4R and INF-γR and can suppress CD3ζ expression in CD8⁺ T lymphocytes. The subpopulation of CD11b⁺/CD14^? cells in PBMNC was decreased in the advanced-stage NSCLC patients who had responsiveness to chemotherapy (n = 41, P < 0.0001) and in the early-stage NSCLC patients after removal of tumor (n = 8, P = 0.0391). Notably, a negative association existed between the population of CD11b⁺/CD14^? cells in PBMNC and the frequency of CD8⁺ T lymphocytes (n = 48, r = ?0.3141, P = 0.0297).

Conclusions

Our study provided evidence of an increased pool of CD11b⁺/CD14^?/CD15⁺/CD33⁺ MDSCs in the peripheral blood of NSCLC patients. For the suppressive effect of the cells on CD8⁺ T lymphocytes, these findings suggest the important role of the CD11b⁺/CD14^?/CD15⁺/CD33⁺ MDSCs in mediating immunosuppression in NSCLC.     

Advanced glycation end products, carotid atherosclerosis, and circulating endothelial progenitor cells in patients with end-stage renal disease

Numbers of endothelial progenitor cells (EPCs) have been shown to be decreased in subjects with end-stage renal disease (ESRD), the mechanism of which remained poorly understood. In this study, mutual association among circulating EPC levels, carotid atherosclerosis, serum pentosidine, and skin autofluorescence, a recently established noninvasive measure of advanced glycation end products accumulation, was examined in 212 ESRD subjects undergoing hemodialysis. Numbers of circulating EPCs were measured as CD34⁺ CD133⁺ CD45^low VEGFR2⁺ cells and progenitor cells as CD34⁺ CD133⁺ CD45^low fraction by flow cytometry. Skin autofluorescence was assessed by the autofluorescence reader; and serum pentosidine, by enzyme-linked immunosorbent assay. Carotid atherosclerosis was determined as intimal-medial thickness (IMT) measured by ultrasound. Circulating EPCs were significantly and inversely correlated with skin autofluorescence in ESRD subjects (R = −0.216, P = .002), but not with serum pentosidine (R = −0.079, P = .25). Circulating EPCs tended to be inversely associated with IMT (R = −0.125, P = .069). Intimal-medial thickness was also tended to be correlated positively with skin autofluorescence (R = 0.133, P = .054) and significantly with serum pentosidine (R = 0.159, P = .019). Stepwise multiple regression analyses reveal that skin autofluorescence, but not serum pentosidine and IMT, was independently associated with low circulating EPCs. Of note, skin autofluorescence was also inversely and independently associated with circulating progenitor cells. Thus, tissue accumulated, but not circulating, advanced glycation end products may be a determinant of a decrease in circulating EPCs in ESRD subjects.     

Vascular endothelial growth factor polymorphism (–460 T/C) is related to hypertension-associated chronic kidney disease

Our aim was to characterize the endothelial progenitor cells (EPCs) in normotensive controls and treated hypertensive individuals within the vascular endothelial growth factor (VEGF) –460 C/T polymorphism as well as to investigate whether this polymorphism predisposes to hypertension-related chronic kidney disease. The hypertensive patients bearing the TT genotype had the highest levels of immature EPC with the following phenotypes: CD34⁺, CD34⁺CD45^dim, CD34⁺CD133⁺CD45^dim. The study showed the estimated glomerular filtration rate values significantly lower and creatinine and BUN parameters higher among the TT hypertensive patients. We presume that the highest mobilization of EPCs from bone marrow may signalize more severe renal hypertension-related complications in the VEGF –460 TT genotype.     

The comparison of EPC count and function in the situation of vascular repair at early and late stage

To investigate the relationship between the changes in the number and function of the late endothelial progenitor cells (EPC) in peripheral blood and the carotid artery stenosis. 60 cases were selected and were divided into the carotid artery stenosis group of 40 cases (mild stenosis in 20 cases, moderate/severe stenosis in 20 cases), normal control group of 20 cases with the global cerebral angiography. Extracted the blood of femoral artery from the patients during the global cerebral angiography, mononuclear cells were isolated from peripheral blood by density-gradient centrifugation and were cultured to 21 days when they were identified as late endothelial progenitor cells, counted the colony numbers of late EPC. Then the proliferation, migration and adherentce ability of late EPC were determined by the MTT assay, modified Boyden and the HFN culturing plates. The amount of the late EPC colonies(34.30 ± 4.90, 25.38 ± 6.33) were significantly reduced in the patients with carotid artery stenosis compared with the control group (46.00 ± 5.64) (P < 0.05); the function of proliferation, migration, adhesion of the late EPC in patiens with cerebral artery stenosis were significantly lower (P < 0.05), the number and function of late EPC decreased with the worsening of vascular stenosis (P < 0.05). The number of EPC in peripheral blood of patients with the carotid artery stenosis decreased, the function was impaired, and number and function changes of late EPC were negatively correlated with the degree of carotid artery stenosis.     

Circulating endothelial progenitor cell numbers are not associated with donor organ age or allograft vasculopathy in cardiac transplant recipients

IntroductionIncreasing age is associated with reduced numbers of circulating endothelial progenitor cells (EPCs). It is unclear whether this relates to depletion or impairment of bone marrow progenitors, or to deficient mobilization signals from aging tissues. In cardiac transplant patients, one previous study has reported an association between circulating EPCs and the risk of cardiac allograft vasculopathy (CAV). We investigated whether increased donor heart age, a strong risk factor for CAV, was associated with reduced circulating EPC numbers in a group of cardiac transplant recipients matched for factors which influence EPC numbers, but with maximally discordant donor heart ages.MethodsWe identified 32 patient pairs, matched for factors known to influence EPC numbers, but who had discordant donor heart ages by at least 20 years. EPCs were quantified using flow cytometry for absolute counts of cells expressing all the combinations of CD45, CD34, CD133 and the kinase domain receptor (KDR).ResultsThere were no significant differences in the numbers of circulating EPCs between patients with old or young donor heart age. There was no association between the presence of CAV and circulating EPC numbers.ConclusionsWe suggest that the increased susceptibility to CAV of older donor hearts is not mediated via circulating EPCs. Our results are consistent with the theory that the normal age-related decline in EPC numbers relates to bone marrow aging rather than failure of target tissues to induce EPC mobilization.     

Imbalance of circulating endothelial cells and progenitors in idiopathic pulmonary fibrosis

Background

Fibrogenesis during idiopathic pulmonary fibrosis (IPF) is strongly associated with abnormal vascular remodeling. Respective abundance of circulating endothelial cells (CEC) and endothelial progenitor cells (EPC) might reflect the balance between vascular injury and repair and potentially serve as biomarkers of the disease.

Objectives and Methods

We postulated that CEC and EPC subtypes might be differently modulated in IPF. Sixty-four consecutive patients with newly diagnosed IPF were prospectively enrolled and compared to thirteen healthy volunteers. CEC were counted with immunomagnetic CD146-coated beads; progenitors CD34+45^dim/CD34+133+/CD34+KDR+were assessed through flow cytometry and EPC (colony-forming-units-Endothelial Cells, CFU-EC, and endothelial colonies forming cells, ECFC) were quantified by cell culture assays.

Results

IPF patients were characterized by a marked increase in CEC associated to an EPC defect: both CD34⁺KDR⁺ cells and CFU-EC were decreased versus controls. Moreover, in IPF subjects with a low diffusing capacity of the lung for carbon monoxide (DL_CO) < 40 %, CFU-EC and ECFC were higher compared to those with DL_CO > 40 %. Finally, ECFC were negatively correlated with DL_CO. During an 18 month follow up, CEC levels increased in patients with exacerbation, including those who died during follow up. Finally, ECFC from patients with exacerbation proliferative potential was strongly increased.

Conclusion

IPF is basically associated with both a vascular injury and a repair defect. This study highlights an adaptative process of EPC mobilization in the most severe forms of IPF, that could reflect enhanced homing to the pulmonary vasculature, which clinical consequences remain to be determined.     

An association of serum vistafin level and number of circulating endothelial progenitor cells in type 2 diabetes mellitus patients

BackgroundThe decreased number and impaired functions of endothelial progenitor cells (EPCs) may associate with cardiovascular disease (CV) including atherosclerosis. However, the role of vistafin in regulation of angiogenic EPC subset maturation in T2DM patients without known atherosclerosis is still not fully understood.The aim of the studyTo investigate an association of serum vistafin level and number of circulating EPCs in T2DM patients beyond known CV disease.MethodsThis case–control observational investigation was evolved 54 subjects with T2DM and 35 healthy volunteers. The flow cytometry was used for predictably distinguishing cell subsets, which depend on expression of CD45, CD34, CD14, Tie-2, and VEGFR2. Biomarkers were measured at baseline of the study.ResultsAll T2DM patients were divided depending median of vistafin level (5.88 ng/mL) in to two cohorts with low vistafin level (<5.88 ng/mL; n = 29) and high vistafin level (≥5.88 ng/mL; n = 25) respectively. Logistic regression analysis has shown that visfatin, hs-CRP, age and BMI were the best variables in the prediction of EPC number labeled as CD14⁺CD309⁺ and CD14⁺CD309⁺Tie²⁺ cells. After adjustment of the model to age and BMI elevated visfatin level remained the best predictor for both CD14⁺CD309⁺ and CD14⁺CD309⁺Tie²⁺ EPCs (OR 0.92, 95% CI: 0.88–0.95; P = 0.001 and OR 0.90, 95% CI: 0.87–0.96; P = 0.001 respectively).ConclusionWe found that elevated level of vistafin was an independent predictor for declined numerous of non-classical EPCs labeled as CD14⁺CD309⁺ and CD14⁺CD309⁺Tie²⁺, whereas CD34⁺ subsets of EPCs did not associate with vistafin level in T2DM individuals.     

Impaired endothelial progenitor cell function predicts age-dependent carotid intimal thickening

Objectives  We investigated whether qualitative or quantitative alterations of the endothelial progenitor cell (EPC) pool predict age-related structural vessel wall changes. Background  We have previously shown that age-related endothelial dysfunction is accompanied by qualitative rather than quantitative changes of EPCs. Animal studies suggest that impaired EPC functions lead to accelerated arterial intimal thickening. Methods  Intima-media thickness (IMT) was measured in the common carotid artery in our previously published groups of younger (25 ± 1 years, n = 20) and older (61 ± 2 years, n = 20) healthy non-smoking volunteers without arterial hypertension, hypercholesterolemia, and diabetes mellitus. Endothelial progenitor cells (EPCs, KDR⁺/CD34⁺ and KDR⁺/CD133⁺) were counted in peripheral blood using flow cytometry. In ex vivo expanded EPCs, the function was determined as chemotaxis to VEGF, proliferation, and survival. Results  We observed thicker IMT in older as compared to younger subjects (0.68 ± 0.03 mm Vs. 0.48 ± 0.02 mm, P < 0.001). Importantly, there were significant inverse univariate correlations between IMT, EPC chemotaxis, and survival (r = −0.466 P < 0.05; r = −0.463, P < 0.01). No correlation was observed with numbers of circulating EPCs. Multivariate regression analysis revealed that age, mean arterial pressure and migration of EPCs were independent predictors of IMT (R ² = 0.58). Conclusion  Impaired EPC function may lead to accelerated vascular remodeling due to chronic impairment of endothelial maintenance. Returned for 1. Revision: 13 December 2007 1. Revision received: 16 June 2008 Returned for 2. Revision: 20 June 2008 2. Revision received: 17 July 2008