EPC mobilization after erythropoietin treatment in acute ST-elevation myocardial infarction: the REVEAL EPC substudy

Erythropoietin (EPO) was hypothesized to mitigate reperfusion injury, in part via mobilization of endothelial progenitor cells (EPCs). The REVEAL trial found no reduction in infarct size with a single dose of EPO (60,000 U) in patients with ST-segment elevation myocardial infarction. In a substudy, we aimed to determine the feasibility of cryopreserving and centrally analyzing EPC levels to assess the relationship between EPC numbers, EPO administration, and infarct size. As a prespecified substudy, mononuclear cells were locally cryopreserved before as well as 24 and 48–72 h after primary percutaneous coronary intervention. EPC samples were collected in 163 of 222 enrolled patients. At least one sample was obtained from 125 patients, and all three time points were available in 83 patients. There were no significant differences in the absolute EPC numbers over time or between EPO- and placebo-treated patients; however, there was a trend toward a greater increase in EPC levels from 24 to 48–72 h postintervention in patients receiving ≥30,000 U of EPO (P = 0.099 for CD133⁺ cells, 0.049 for CD34⁺ cells, 0.099 for ALDH^br cells). EPC numbers at baseline were inversely related to infarct size (P = 0.03 for CD133⁺ cells, 0.006 for CD34⁺ cells). Local whole cell cryopreservation and central EPC analysis in the context of a multicenter randomized trial is feasible but challenging. High-dose (≥30,000 U) EPO may mobilize EPCs at 48–72 h, and baseline EPC levels may be inversely associated with infarct size.     

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     

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.     

Intradialytic Hypertension and its Association with Endothelial Cell Dysfunction

Summary

Background and objectives

Intradialytic hypertension is associated with adverse outcomes, yet the mechanism is uncertain. Patients with intradialytic hypertension exhibit imbalances in endothelial-derived vasoregulators nitric oxide and endothelin-1, indirectly suggesting endothelial cell dysfunction. We hypothesized that intradialytic hypertension is associated in vivo with endothelial cell dysfunction, a novel predictor of adverse cardiovascular outcomes.

Design, settings, participants, & measurements

We performed a case-control cohort study including 25 hemodialysis (HD) subjects without (controls) and 25 with intradialytic hypertension (an increase in systolic BP pre- to postdialysis ≥10 mmHg ≥4/6 consecutive HD sessions). The primary outcome was peripheral blood endothelial progenitor cells (EPCs) assessed by aldehyde dehydrogenase activity (ALDH^br) and cell surface marker expression (CD34⁺CD133⁺). We also assessed endothelial function by ultrasonographic measurement of brachial artery flow-mediated vasodilation (FMD) normalized for shear stress. Parametric and nonparametric t tests were used to compare EPCs, FMD, and BP.

Results

Baseline characteristics and comorbidities were similar between groups. Compared with controls, 2-week average predialysis systolic BP was lower among subjects with intradialytic hypertension (144.0 versus 155.5 mmHg), but postdialysis systolic BP was significantly higher (159.0 versus 128.1 mmHg). Endothelial cell function was impaired among subjects with intradialytic hypertension as measured by decreased median ALDH^br cells and decreased CD34⁺CD133⁺ cells (ALDH^br, 0.034% versus 0.053%; CD34⁺CD133⁺, 0.033% versus 0.059%). FMD was lower among subjects with intradialytic hypertension (1.03% versus 1.67%).

Conclusions

Intradialytic hypertension is associated with endothelial cell dysfunction. We propose that endothelial cell dysfunction may partially explain the higher event rates observed in these patients.     

Circulating endothelial progenitor cells are reduced in SLE in the absence of coronary artery calcification

Circulating endothelial progenitor cells (EPCs) are reduced in patients with systemic lupus erythematosus (SLE). A reduced number of EPCs are associated with the presence of atherosclerosis in other populations. We sought to determine whether the reduction in EPC numbers in SLE is dependent on the presence of advanced coronary artery calcification (CAC). Patients with SLE had previous coronary calcium scores which placed them in either the >75th percentile or <25th percentile for their age. Seventeen patients with SLE and 13 healthy controls (HC) were included in the study. White blood cells were stained for EPC and progenitor cell markers including CD34, CD133, and VEGFR and analyzed by flow cytometry. SLE patients had repeated coronary imaging as well as carotid ultrasound. There was no difference in age between groups. SLE patients with advanced CAC were more likely to be hypertensive, to be smokers, and to have longer disease duration than SLE patients without CAC. SLE patients without evidence of CAC had a significantly lower number of EPCs (CD34+/CD133+/VEGFR+) compared to HC (median (IQR)) 0 (0, 6.7) vs. 10.2 (5.8, 12.3) (P = 0.02). Total numbers of PCs (CD133+/CD34+) were not significantly decreased in patients with SLE ((mean ± SEM) 1,007 ± 154 vs. 824 ± 170 (P = 0.20)). No significant difference was seen in EPC number between SLE patients without CAC and those with advanced CAC. Increased carotid intima-media thickness did not correlate with CAC or EPC number in SLE patients. Reduced numbers of EPCs in SLE patients may be observed compared to HC even in the absence of CAC. Differences in measured risk factor profiles and depletion of total circulating PCs do not fully explain this finding.     

Reduced circulating endothelial progenitor cell number in healthy young adult hyperinsulinemic men

Background and aims

The number of Endothelial Progenitor Cells (EPCs) is considered a novel marker of cardiovascular (CV) disease. It is not clear which are the main determinants of EPC number in apparently healthy subjects in the absence of overt clinical CV or metabolic abnormalities. We evaluated the main clinical determinants of EPC levels in a population of healthy subjects with normal glucose tolerance.

Methods and results

EPC number was determined in 122 healthy subjects (73M/49F;36.6 ± 8yrs). Blood samples were collected to test biochemical variables. OGTT was performed and insulin resistance/compensatory hyperinsulinemia was defined according to fasting plasma insulin (FPI) levels. EPCs were identified as cells co-expressing CD133/CD34/KDR antigens by flow-cytometry. CD133⁺/KDR⁺ count inversely correlated with BMI (rho=−0.18;p < 0.05), waist circumference (−0.2;<0.05), diastolic (−0.23;<0.01) and systolic blood pressure (−0.21;<0.05), uric acid (−0.24;<0.005), PAI-1 (−0.197; <0.05) and FPI (−0.2;<0.05) and directly correlated with HDL cholesterol (0.182;<0.05). CD34⁺/CD133⁺/KDR⁺ count inversely correlated with uric acid (−0.28;<0.005) and FPI (−0.2;<0.05). EPC number was lower in males (p < 0.05) and gender was the only independent predictor of EPC count (p < 0.05). By dividing the population in four subgroups based on gender and insulin resistance, CD133⁺/KDR⁺ levels were lower in insulin resistant compared to insulin sensitive males (p < 0.05) with no differences in females.

Conclusion

The male gender is an independent predictor of low EPC levels in healthy subjects. This might contribute to explaining the higher CV risk in males compared to pre-menopausal age-matched females. In this study a reduced EPC number seems to be associated with insulin resistance in male subjects.     

Exposure to platelets promotes functional properties of endothelial progenitor cells

Recent evidence indicates that endothelial progenitor cells (EPCs) have an important role in the process of repair following vascular injury, and that platelets mediate their recruitment to sites of injury. Platelets and EPCs can interact and bind directly. However, there is limited information on the effect of platelets on EPC function following this interaction. We, therefore, aimed to assess the in vitro effect of platelets on functional properties of EPCs. Human EPCs were isolated from donated Buffy coats and purified on a magnetic separation column specific for CD133. They were incubated either on fibronectin matrix, or co-incubated with washed platelets (isolated from healthy volunteers), for 7 days. Number of EPC colony forming units (CFU) was quantified, and endothelial cell lineage confirmed by immunostaining. Functional properties of the cultured cells were evaluated by MTT—proliferation assay and migration assay using the Boyden chamber. Co-incubation of EPCs with platelets compared to incubation of EPCs alone (on fibronectin matrix) resulted in higher number of CFUs after 7 days (6.5 ± 1.3 vs. 3.5 ± 0.5 CFUs/well, respectively, P = 0.005). In addition, co-incubation of EPCs with platelets versus EPCs alone was associated with higher proportion of living cells, by the MTT assay (0.2 ± 0.01 vs. 0.12 ± 0.04 MTT 570 nm respectively, P = 0.003), and higher number of migrated EPCs, assessed by the migration assay (1400 ± 212 vs. 580 ± 180 migrated cells/2000 cells, respectively, P < 0.0001). In vitro exposure to platelets promotes the capacity of EPCs to form colonies, proliferate and migrate. Therefore, the interaction with platelets appears to augment EPC functional properties.     

Depletion of circulating progenitor cells precedes overt diabetes: A substudy from the VA enhanced fitness trial

BackgroundOne theory of aging and disease development is that chronic injury (pathology) results in activation of regenerative processes and initial repair, with overt disease arising only after exhaustion of reparative capability leads to inadequate repair. While depletion of circulating progenitor cells (CPCs) has been noted in diabetes, the degree to which CPC depletion predates and is associated with propensity to develop overt disease is unclear.MethodsThe Enhanced Fitness trial enrolled overweight/obese (body mass index > 25) sedentary patients with glucose intolerance but without overt diabetes. Baseline CPCs were measured in 129 patients based on the cell surface markers CD34, CD133, and aldehyde dehydrogenase (ALDH) activity. HgbA_1C, fasting insulin and glucose levels, and HOMA calculations were ascertained.ResultsLower counts of early angiogenic CPCs identified as CD34⁺, CD34⁺CD133⁺, and ALDH-bright (ALDH^br) cells were associated with impairments in glucose homeostasis as reflected by HgbA_1C, but not fasting insulin, glucose, or HOMA-IR. These associations remained when corrected for age and cardiovascular risk factors.Conclusions/InterpretationThe numbers of CD34⁺ and ALDH^br CPCs were significantly lower in patients with impaired glucose tolerance. Depletion of reparative capacity as reflected by loss of CPCs may presage overt disease as exemplified in this pre-diabetes model.     

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.     

Circulating endothelial progenitor cells, endothelial function, carotid intima–media thickness and circulating markers of endothelial dysfunction in people with type 1 diabetes without macrovascular disease or microalbuminuria

Aims/hypothesis  Type 1 diabetes is associated with premature arterial disease. Bone-marrow derived, circulating endothelial progenitor cells (EPCs) are believed to contribute to endothelial repair. The hypothesis tested was that circulating EPCs are reduced in young people with type 1 diabetes without vascular injury and that this is associated with impaired endothelial function and increased carotid intima–media thickness (CIMT). Methods  We compared 74 people with type 1 diabetes with 80 healthy controls. CD34, CD133, vascular endothelial (VE) growth factor receptor-2 (VEGFR-2) and VE-cadherin antibodies were used to quantify EPCs and progenitor cell subtypes using flow-cytometry. Ultrasound assessment of endothelial function by brachial artery flow-mediated dilatation (FMD) and CIMT was made. Circulating endothelial markers, inflammatory markers and plasma plasminogen activator inhibitor-1 (PAI-1) levels were measured. Results  CD34+VE-cadherin+, CD133+VE-cadherin+ and CD133+VEGFR-2+ EPC counts were significantly lower in people with diabetes (46–69%; p = 0.004–0.043). In people with type 1 diabetes, FMD was reduced by 45% (p < 0.001) and CIMT increased by 25% (p < 0.001), these being correlated (r = −0.25, p = 0.033). There was a significant relationship between FMD and CD34+VE-cadherin+ (r = 0.39, p = 0.001), CD133+VEGFR-2+ (r = 0.25, p = 0.037) and CD34+ (r = 0.34, p = 0.003) counts. Circulating high-sensitivity C-reactive protein, PAI-1, interleukin-6 and E-selectin were significantly higher in the diabetes group (p < 0.001 to p = 0.049), the last two of these correlating with FMD (r = −0.27, p = 0.028 and r = −0.24, p = 0.048, respectively). Conclusions/interpretation  These findings suggest that abnormalities of endothelial function in addition to pro-inflammatory and pro-thrombotic states are already common in people with type 1 diabetes before development of clinically evident arterial damage. Low EPC counts confirm risk of macrovascular complications and may account for impaired endothelial function and predict future cardiovascular events. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorised users.     

Increased levels of IL‐21 responses are associated with the severity of liver injury in patients with chronic active hepatitis B

Interleukin‐21 (IL‐21) participates in tissue damage in various immune‐mediated diseases. Its role in the pathogenesis of chronic active hepatitis B (CAHB) has not been clarified. The frequency of circulating IL‐21⁺ T cells and the levels of serum and intrahepatic IL‐21 have been characterized in 70 CAHB patients, 32 inactive carrier (IC), 18 chronic hepatitis C (CHC) and 20 healthy controls (HC). Their potential association with liver injury was analysed. The percentages of IL‐21⁺CD3⁺CD8⁻ and IL‐21⁺CD3⁺CD8⁺ T cells and the levels of serum IL‐21 in CAHB patients were significantly higher than that in the IC, CHC patients and HC (P < 0.001) and were correlated positively with the levels of serum alanine aminotransferase (ALT, r = 0.424, P < 0.001; r = 0.392, P = 0.001) and aspartate aminotransferase (AST, r = 0.388, P = 0.001; r = 0.329, P = 0.005) in CAHB patients, respectively. The levels of IL‐21 expression in the liver tissues were associated significantly with increased degrees of inflammation and fibrosis in CAHB patients (P < 0.01 or P < 0.05). Our findings suggest that aberrant IL‐21 responses may be associated with the progression of CHB.     

Abnormal Levels of Circulating Endothelial Progenitor Cells During Exacerbations of COPD

Cardiovascular morbidity and mortality is increased in patients with chronic obstructive pulmonary disease (COPD). Reduced levels of circulating endothelial progenitor cells (EPCs) are associated with increased risk of death in patients with stable coronary artery disease (CAD). Likewise, during acute events of CAD, the number of circulating EPCs increases under the influence of vascular endothelial growth factor (VEGF) and systemic inflammation. Abnormal levels of circulating EPCs have been reported in patients with COPD. However, the response of EPCs to episodes of exacerbation of the disease (ECOPD) has not been investigated yet. We hypothesized that similar to what occurs during acute events of CAD, levels of circulating EPCs would increase during ECOPD. We compared levels of circulating EPCs (assessed by the % of CD34⁺KDR⁺ cells determined by flow cytometry) in patients hospitalized because of ECOPD (n = 35; 65 ± 9 years [mean ± SD]; FEV₁ = 46 ± 15% predicted), patients with stable COPD (n = 44; 68 ± 8 years; FEV₁ = 49 ± 17% predicted), smokers with normal lung function (n = 10; 60 ± 9 years), and healthy never smokers (n = 10; 62 ± 4 years). To investigate potential mechanisms of EPC regulation, we assessed both VEGF and high-sensitivity C-reactive protein (hsC-RP) in plasma. Our results show that EPC levels were higher (p < 0.05) in patients with ECOPD (1.46 ± 1.63%) than in those with stable disease (0.68 ± 0.83%), healthy smokers (0.65 ± 1.11%), and healthy never smokers (1.05 ± 1.36%). The percentage of circulating EPCs was positively related to VEGF plasma levels during ECOPD (r = 0.51, p = 0.003). In a subset of 12 patients who could be studied during both ECOPD and clinical stability, the EPCs levels increased during ECOPD. We conclude that EPC levels are increased during ECOPD, likely in relation to VEGF upregulation.     

Dynamics of endothelial progenitor cells in patients with advanced hepatocellular carcinoma

Background and aimsCirculating endothelial progenitor cells (EPC) predict tumor vascularization and disease progression, but limited information is available on their dynamics in hepatocellular carcinoma (HCC) undergoing systemic treatment.MethodsWe prospectively analyzed different populations of EPC in 16 patients with advanced HCC receiving sorafenib. Patients were studied before therapy (T0, n = 16) and after two (T2, n = 12) and eight weeks (T8, n = 8), using high-performance flow-cytometry. The tumor response at T8 was categorized as progressive disease (PD) or clinical benefit (CB, all other responses).ResultsAt T0, higher levels of CD34⁺CD133⁺KDR⁺ and CD34⁺KDR⁺ were observed in patients with alpha-fetoprotein ≥400 ng/ml or non-viral liver disease, whereas CD34⁺CD133⁺KDR⁺ cells were virtually absent in patients with vascular invasion. CD34⁺KDR⁺ and CD34⁺CD133⁺KDR⁺ were directly correlated with platelet count. Frequencies of all populations of EPC declined in patients receiving sorafenib. Levels of CD34⁺CD133⁺ were higher at T0 in patients with CB compared to patients with PD. In patients belonging to the CB group CD34⁺KDR⁺ cells at T0 were directly correlated to platelet count.ConclusionIn patients with advanced HCC, EPC are directly correlated with platelet count, suggesting a common activation of selected bone marrow pathways. Levels of a CD34⁺KDR⁺ are higher at baseline in patients responding to sorafenib.     

Exercise training improves function of circulating angiogenic cells in patients with chronic heart failure

Alterations in circulating angiogenic cells (CAC) and endothelial progenitor cells (EPC), known to contribute to endothelial repair, could explain the reversal of endothelial function in response to exercise training. Moreover, training-induced vascular remodeling might affect the acute response of EPC and CAC following a single exercise bout. We studied the impact of exercise training on CAC function and numbers of CD34⁺/KDR⁺ EPC in patients with chronic heart failure (CHF) and we assessed the effect of acute exercise on CAC and EPC in sedentary and trained patients. Twenty-one sedentary CHF patients underwent 6-month exercise training and were compared to a non-trained control group (n = 17) and 10 healthy age-matched subjects. At baseline and follow-up, flow-mediated dilation was assessed and graded exercise testing (GXT) was performed. Before and immediately after GXT, CAC migratory capacity was assessed in vitro and circulating CD34⁺/KDR⁺ EPC were quantified using flow cytometry. At baseline, CAC migration was significantly impaired in sedentary CHF patients but normalized acutely after GXT. Training corrected endothelial dysfunction, which coincided with a 77% increase in CAC migration (P = 0.0001). Moreover, the GXT-induced improvement detected at baseline was no longer observed after training. Numbers of CD34⁺/KDR⁺ EPC increased following 6-month exercise training (P = 0.021), but were not affected by GXT, either prior or post-training. In conclusion, the present findings demonstrate for the first time that exercise training in CHF reverses CAC dysfunction and increases numbers of CD34⁺/KDR⁺ EPC, which is accompanied by improvement of peripheral endothelial function. The acute exercise-induced changes in CAC function wane with exercise training, suggesting that repetitive exercise bouts progressively lead to functional endothelial repair.     

Effects of rituximab and dexamethasone on regulatory and proinflammatory B‐cell subsets in patients with primary immune thrombocytopenia

Objective

To investigate the cytokine production and surface marker composition of B cells in adult patients with newly diagnosed primary immune thrombocytopenia (ITP) before and 12 months after treatment with rituximab + dexamethasone (RTX+DXM) or dexamethasone (DXM).

Methods

Peripheral blood mononuclear cells were isolated from nine patients treated with RTX+DXM, seven patients treated with DXM, and seven healthy donors. Expression of the cell‐surface markers CD5, CD27, CD25, and CD19, and intracellular content of IL‐6 and IL‐10 were measured by flow cytometry.

Results

PBMCs from ITP patients at baseline contained a lower proportion of IL‐10⁺ B cells (P < .01) and IL‐6⁺ B cells (P < .01) than healthy controls. All patients responded to therapy and levels were normalized at 12 months. The proportion of CD5⁺ B cells increased (P < .01) and CD27⁺ memory B cells decreased (P < .05) 12 months after treatment with RTX+DXM compared to baseline, with an inverse correlation between platelet numbers and the proportion of CD27⁺ B cells (R = ?0.71; P < .05).

Conclusion

Both treatment regimens normalized the frequencies of cytokine‐producing B cells. The additional increase in CD5⁺ B cells after RTX+DXM is compatible with induction of Bregs.     

Effect of smoking cessation on the number and adhesive properties of early outgrowth endothelial progenitor cells

Background

Endothelial progenitor cells participate in angiogenesis and vascular repair, and cardiovascular risk factors may reduce their numbers or impair their functional properties. Cigarette smoking is a leading cause of preventable cardiovascular death, however, the functional properties of these cells before and after discontinuation of tobacco use have not been systematically analyzed.

Methods

We examined changes in the number and function of early outgrowth endothelial progenitor cells (EPC), isolated from individuals (n = 144; mean age, 47.8 ± 12.0 years; 43% males; more than 50% with additional cardiovascular risk factors or disease) who successfully completed a 5-week smoking cessation (SC) programme.

Results

SC significantly reduced total white blood cell count (WBC; P < 0.0001), plasma LDL cholesterol (P = 0.0002) and fibrinogen (P < 0.0001) levels, but did not alter the number of circulating CD34⁺, VEGFR2⁺ or CD34⁺, CD133⁺ cells (P = 0.14 and 0.57, respectively). Fewer acLDL⁺, lectin⁺ cells could be expanded from peripheral blood mononuclear cells in comparison to baseline (P < 0.001). Furthermore, SC was associated with reduced EPC adhesion to fibronectin (P < 0.001) or TNFα-activated endothelial cells (P = 0.003), and a diminished incorporation of EPC into endothelial cell networks (P = 0.035). Mechanistically, significantly reduced β1- and β2-integrin expression (P < 0.001 and 0.007) and lower contents of intracellular reactive oxygen species (P < 0.007) were detected in EPC following SC, in addition to reduced plasma asymmetric dimethyl-L-arginine (ADMA) levels (P = 0.0003).

Conclusions

Our findings suggest that the oxidative and inflammatory stress reduction associated with smoking cessation impair the adhesiveness of monocyte-derived EPC.     

Irradiation induces homing of donor endothelial progenitor cells in allogeneic hematopoietic stem cell transplantation

Functional abnormalities of the endothelial system may be caused by allogeneic hematopoietic stem cell transplantation (HSCT). The aim of this study is to explore the possibility that endothelial progenitor cells (EPCs) can be used in endothelial repair post-HSCT. EPCs were isolated from mouse bone marrow by density centrifugation and differential adherence. Numbers of endothelial cells (ECs) (CD31⁺CD133⁻CD45⁻), EPCs (CD31⁺CD133⁺–CD45^low/−) and carboxyfluorescein succinimidyl ester (CFSE)-positive cells in peripheral blood, spleen and bone marrow were determined at various time points by flow cytometry. The distribution of labeled EPCs was observed by fluorescence microscopy; morphological alterations of tissues were assessed by light microscopy and transmission electron microscopy. In the irradiated group, the numbers of circulating ECs and EPCs were elevated after pre-conditioning, reaching peaks at days 3 and 5; the counts remained high for about 5 days. In addition, CFSE-labeled cells were visualized in tissue and bone marrow. In conclusion, these results suggest the following: (a) the EPCs derived from mouse bone marrow mononuclear cells express phenotypes characteristic of normal EPCs, (b) irradiation during preconditioning damaged the endothelium, which initiated mobilization of EPCs, and (c) injury to the endothelium also caused extrinsic EPCs home to the damaged tissue.     

Bone marrow characterization in sickle cell disease: inflammation and stress erythropoiesis lead to suboptimal CD34 recovery

Stress erythropoiesis and chronic inflammation in subjects with sickle cell disease (SCD) may have an impact on the bone marrow (BM) haematopoietic stem and progenitor cell (HSPC) quality and yield necessary for effective autologous, ex vivo HSPC gene therapy. BM from 19 subjects with SCD and five volunteers without SCD (non-SCD) was collected in different anticoagulants and processed immediately (day 0) or the following day (day 1). Inflammatory, contamination and aggregation markers within the mononuclear layer, and CD34, CD45 and Glycophorin-A (GPA) expression on HSPCs after CD34⁺ selection were analysed by conventional and imaging flow cytometry. Compared to non-SCD BM, multiple markers of inflammation, contamination (red cells, P < 0·01; platelets, P < 0·01) and aggregates (platelet/granulocytes, P < 0·01; mononuclear/red cells, P < 0·01) were higher in SCD BM. Total CD34⁺ cell count was lower in SCD BM (P < 0·05), however CD34⁺ count was higher in SCD BM when collected in acid citrate dextrose-A (ACDA) versus heparin (P < 0·05). Greater than 50% of CD34⁺ HSPCs from SCD BM are CD34^dim due to higher erythroid lineage expression (P < 0·01) as single cell CD34⁺CD45⁺GPA⁺ (P < 0·01) and CD34⁺CD45⁻GPA⁺ (P < 0·01) HSPCs. SCD BM is characterized by increased inflammation, aggregation and contamination contributing to significant differences in HSPC quality and yield compared to non-SCD BM.     

Inverse Correlation Between Circulating Endothelial Progenitor Cells With CD34+CD133+ and the Severity of Coronary Atherosclerosis Assessed by Syntax Score

BackgroundThe number of endothelial progenitor cells (EPCs) descends when atherosclerosis developed. The objective was to compare the number of CD34 + CD133 + cells with the severity of atherosclerosis assessed by Syntax score.MethodsThe study included 80 patients with stable angina undergoing coronary angiography. Patients were classified into single-vessel group, multiple-vessel group and normal group according to angiography. The percentage of CD34 + CD133 + cells in the mononuclear cells isolated from peripheral blood of different groups by flow cytometric analysis was compared. The quantity of CD34 + CD133 + EPCs was log transformed to improve normality (lgEPC). Syntax score was used in this study to assess the extent of coronary artery disease.Results:The level of lgEPC was lower in the single-vessel group than that in the normal group (− 3.42 ± 0.44 versus − 3.17 ± 0.39, P < 0.05), and the level of lgEPC was lower in the multiple-vessel group than that in the single vessel group (− 3.63 ± 0.31 versus − 3.42 ± 0.44, P < 0.05). An inverse correlation between lgEPC and Syntax score analyzed by linear regression.ConclusionsEPC level probably serves as a predictor of the development and severity of atherosclerosis on a cellular level. EPC, a relatively more important risk factor, perhaps protects against coronary artery disease.     

Potential Link Between C3a,C3b and Endothelial Progenitor Cells in Resistant Hypertension

IntroductionEndothelial progenitor cells (EPC) and complement C3 are involved in the pathophysiology of arterial hypertension. C3a is the negative regulator of progenitor cells egress during their mobilization from bone marrow. Previously, higher plasma concentration of C3 was observed in resistant arterial hypertension (RAH) than in controlled arterial hypertension (CAH). Thus, we hypothesized that RAH would be associated with complement C3 activation and reduced number of circulating EPCs.ObjectiveTo compare C3a, C3b and their correlation with circulating EPC in subjects with RAH and CAH.MethodsBlood pressure was measured by electronic sphygmomanometer. EPCs were identified as CD34+/CD133+/KDR+ cells by flow cytometry. C3a and C3b were determined using enzyme-linked immunosorbent assay (Quidel, CA).ResultsRAH group (n = 20) and CAH group (n = 20) and 17 healthy individuals (control group) were recruited. In the RAH group, C3a (858.1 ± 70.6 μg/dL) was higher than in the CAH group (816.1 ± 123.3 μg/dL; P < 0.001), and in the control group (751.3 ± 98.8; P < 0.001), C3b (564.1 ± 54.7 μg/dL) was higher than in the CAH group (490.2 ± 58.5 μg/dL; P < 0.001). In control group (456.3 ± 98.8; P < 0.001), statistically significant negative correlation was observed between C3a and blood levels of EPC (r = ?0.523, P = 0.018); statistically significant positive correlation was observed between systolic blood pressure and blood levels of C3a (r = 0.52, P = 0.02) and between systolic blood pressure and blood levels of C3b (r = 0.57, P = 0.009).ConclusionRAH is characterized by higher levels of C3 component fragments and a negative correlation between circulating C3a and EPCs.