Emeline M. Van Craenenbroeck

Flow cytometric detection of endothelial microparticles (EMP): effects of centrifugation and storage alter with the phenotype studied.

INTRODUCTION Endothelial microparticles (EMP) are released into the circulation in case of endothelial disturbance, and are therefore increasingly investigated as a biomarker reflecting disease activity. Numerous pre-analytic methods have been proposed for their flow cytometric enumeration, but standardization is still lacking. In this study we evaluated the influence of centrifugation and storage conditions on EMP quantification. MATERIALS AND METHODS Platelet-poor plasma (PPP) from 10 healthy volunteers was prepared by centrifugation at 1,550 g for 20 minutes twice. A first aliquot of PPP was analyzed immediately, a second after storage at 4 degrees C for 7 hours. A third and fourth aliquot were snap-frozen and stored at -80 degrees C for 7 and 28 days. A final aliquot was further centrifuged at 10,000g for 10 minutes and analyzed immediately. EMP were defined as CD31+CD42b-, CD62E+, CD144+ or CD144+CD105+ particles, smaller than 1.0 microm. RESULTS High speed centrifugation led to a significant loss of CD31+CD42b- EMP (p=0.004). A good correlation between PPP and high speed centrifuged PPP was only found for CD144+ EMP (Kendall tau b=0.611, p=0.025). Storage at 4 degrees C did not affect EMP quantification. However, freezing at -80 degrees C increased CD31+CD42b- and CD62E+ EMP counts, and lowered CD144+ EMP (p<0.05). Nevertheless, the agreement among the different storage conditions was relatively good (Kendall coefficient of concordance >0.487; p<0.05). CONCLUSION The flow cytometric detection of EMP varies with the centrifugation protocol and the storage method used, and these changes also depend on the phenotype studied. The results of this study caution against comparing study results gathered with different EMP laboratory protocols.

A maximal exercise bout increases the number of circulating CD34+/KDR+ endothelial progenitor cells in healthy subjects. Relation with lipid profile

Mobilization of bone marrow-derived endothelial progenitor cells (EPC) might explain exercise-induced improvement of endothelial function. We assessed whether a maximal exercise bout could alter the number of circulating EPC in healthy subjects and whether this effect is related to their cardiovascular risk profile. Additionally, we investigated possible mediators of this effect, namely nitric oxide (NO) bioavailability and vascular endothelial growth factor (VEGF) release. Healthy subjects (group 1, n = 11; group 2, n = 14) performed a symptom-limited cardiopulmonary exercise test on a bicycle ergometer. Numbers of CD34+/kinase insert domain receptor (KDR)+ cells were determined by flow-cytometric analysis, either after magnetic separation of CD34+ cells (group 1) or starting from whole blood (group 2). Serum concentrations of VEGF and NO metabolites were measured by using ELISA. Following exercise, EPC increased by 76% (15.4 +/- 10.7 cells/ml vs. 27.2 +/- 13.7 cells/ml; P = 0.01) in group 1 and by 69% in group 2 (30.9 +/- 14.6 cells/ml vs. 52.5 +/- 42.6 cells/ml; P = 0.03). The increase in EPC correlated positively with LDL and total cholesterol/HDL ratio and negatively with peak oxygen consumption and oxygen consumption at anaerobic threshold. VEGF levels increased with exercise, with a strong trend toward significance (P = 0.055). NO levels remained unchanged. The present study demonstrates that a maximal bout of exercise induces a significant shift in CD34+ cells toward CD34+/KDR+ cells. This response was larger in subjects with a less favorable lipid profile.

Mutations in a TGF-β Ligand, TGFB3, Cause Syndromic Aortic Aneurysms and Dissections

Background Aneurysms affecting the aorta are a common condition associated with high mortality as a result of aortic dissection or rupture. Investigations of the pathogenic mechanisms involved in syndromic types of thoracic aortic aneurysms, such as Marfan and Loeys-Dietz syndromes, have revealed an important contribution of disturbed transforming growth factor (TGF)-β signaling. Objectives This study sought to discover a novel gene causing syndromic aortic aneurysms in order to unravel the underlying pathogenesis. Methods We combined genome-wide linkage analysis, exome sequencing, and candidate gene Sanger sequencing in a total of 470 index cases with thoracic aortic aneurysms. Extensive cardiological examination, including physical examination, electrocardiography, and transthoracic echocardiography was performed. In adults, imaging of the entire aorta using computed tomography or magnetic resonance imaging was done. Results Here, we report on 43 patients from 11 families with syndromic presentations of aortic aneurysms caused by TGFB3 mutations. We demonstrate that TGFB3 mutations are associated with significant cardiovascular involvement, including thoracic/abdominal aortic aneurysm and dissection, and mitral valve disease. Other systemic features overlap clinically with Loeys-Dietz, Shprintzen-Goldberg, and Marfan syndromes, including cleft palate, bifid uvula, skeletal overgrowth, cervical spine instability and clubfoot deformity. In line with previous observations in aortic wall tissues of patients with mutations in effectors of TGF-β signaling (TGFBR1/2, SMAD3, and TGFB2), we confirm a paradoxical up-regulation of both canonical and noncanonical TGF-β signaling in association with up-regulation of the expression of TGF-β ligands. Conclusions Our findings emphasize the broad clinical variability associated with TGFB3 mutations and highlight the importance of early recognition of the disease because of high cardiovascular risk.

Aerobic interval training and continuous training equally improve aerobic exercise capacity in patients with coronary artery disease: the SAINTEX-CAD study.

BACKGROUND Exercise-based cardiac rehabilitation increases peak oxygen uptake (peak VO₂), which is an important predictor of mortality in cardiac patients. However, it remains unclear which exercise characteristics are most effective for improving peak VO₂ in coronary artery disease (CAD) patients. Proof of concept papers comparing Aerobic Interval Training (AIT) and Moderate Continuous Training (MCT) were conducted in small sample sizes and findings were inconsistent and heterogeneous. Therefore, we aimed to compare the effects of AIT and Aerobic Continuous Training (ACT) on peak VO₂, peripheral endothelial function, cardiovascular risk factors, quality of life and safety, in a large multicentre study. METHODS Two-hundred CAD patients (LVEF >40%, 90% men, mean age 58.4 ± 9.1 years) were randomized to a supervised 12-week cardiac rehabilitation programme of three weekly sessions of either AIT (90-95% of peak heart rate (HR)) or ACT (70-75% of peak HR) on a bicycle. Primary outcome was peak VO₂; secondary outcomes were peripheral endothelial function, cardiovascular risk factors, quality of life and safety. RESULTS Peak VO₂ (ml/kg/min) increased significantly in both groups (AIT 22.7 ± 17.6% versus ACT 20.3 ± 15.3%; p-time<0.001). In addition, flow-mediated dilation (AIT+34.1% (range -69.8 to 646%) versus ACT+7.14% (range -66.7 to 503%); p-time<0.001) quality of life and some other cardiovascular risk factors including resting diastolic blood pressure and HDL-C improved significantly after training. Improvements were equal for both training interventions. CONCLUSIONS Contrary to earlier smaller trials, we observed similar improvements in exercise capacity and peripheral endothelial function following AIT and ACT in a large population of CAD patients.

Quantification of circulating endothelial progenitor cells: A methodological comparison of six flow cytometric approaches

OBJECTIVES The validity of endothelial progenitor cells as biomarkers and their therapeutic potential depend on the accuracy of techniques used for enumeration. This study assessed the agreement between 6 flow cytometric methods and a CFU assay used for EPC quantification. METHODS Two blood samples were obtained from 30 healthy volunteers (60 samples). CD34+/VEGFR2+ cells were analyzed with flow cytometry, starting from whole blood (A-C) or PBMC (D-F), using different gating strategies: A: lymphocyte gating; B and D: exclusion of autofluorescent cells (CD3 negative selection); C and E: exclusion of autofluorescence and cell aggregates (pulse shape analysis by FSCarea/FSCpeak); F: exclusion of autofluorescence, cell aggregates and non-nucleated cells (Draq 5). PBMC were cultured under endothelial cell conditions to assess CFU numbers. RESULTS Moderate agreement was found between methods B-C and D-E (ICC 0.647 and 0.530). Comparison of methods B-D and C-E showed poor agreement (ICC 0.178 and 0.249). This was also the case for techniques that considerably differed with regard to gating strategies (A-B, A-F, B-F). CFU numbers did not correlate with flow cytometric quantification (all p>0.05). CONCLUSIONS Agreement between methods for EPC quantification is moderate to poor, which may explain apparent controversies in literature. Although each protocol is highly reproducible, this study cautions against comparing study results gathered with different enumeration techniques.

High-Intensity Interval Training in Patients with Heart Failure with Reduced Ejection Fraction

Background: Small studies have suggested that high-intensity interval training (HIIT) is superior to moderate continuous training (MCT) in reversing cardiac remodeling and increasing aerobic capacity in patients with heart failure with reduced ejection fraction. The present multicenter trial compared 12 weeks of supervised interventions of HIIT, MCT, or a recommendation of regular exercise (RRE). Methods: Two hundred sixty-one patients with left ventricular ejection fraction ⩽35% and New York Heart Association class II to III were randomly assigned to HIIT at 90% to 95% of maximal heart rate, MCT at 60% to 70% of maximal heart rate, or RRE. Thereafter, patients were encouraged to continue exercising on their own. Clinical assessments were performed at baseline, after the intervention, and at follow-up after 52 weeks. Primary end point was a between-group comparison of change in left ventricular end-diastolic diameter from baseline to 12 weeks. Results: Groups did not differ in age (median, 60 years), sex (19% women), ischemic pathogenesis (59%), or medication. Change in left ventricular end-diastolic diameter from baseline to 12 weeks was not different between HIIT and MCT (P=0.45); left ventricular end-diastolic diameter changes compared with RRE were −2.8 mm (−5.2 to −0.4 mm; P=0.02) in HIIT and −1.2 mm (−3.6 to 1.2 mm; P=0.34) in MCT. There was also no difference between HIIT and MCT in peak oxygen uptake (P=0.70), but both were superior to RRE. However, none of these changes was maintained at follow-up after 52 weeks. Serious adverse events were not statistically different during supervised intervention or at follow-up at 52 weeks (HIIT, 39%; MCT, 25%; RRE, 34%; P=0.16). Training records showed that 51% of patients exercised below prescribed target during supervised HIIT and 80% above target in MCT. Conclusions: HIIT was not superior to MCT in changing left ventricular remodeling or aerobic capacity, and its feasibility remains unresolved in patients with heart failure. Clinical Trial Registration: URL: http://www.clinicaltrials.gov. Unique identifier: NCT00917046.

Current perspective of pathophysiological and interventional effects on endothelial progenitor cell biology: Focus on Pi3K/AKT/eNOS pathway

For more than a decade, endothelial progenitor cells (EPCs) have been implicated in cardiovascular homeostasis. EPCs are believed to reside within the bone marrow in close contact with surrounding stromal cells, and, under stimulation of pro-inflammatory cytokines, EPCs are mobilized out of the bone marrow. Hereafter circulating EPCs home to peripheral tissues, undergoing further proliferation and differentiation. Under certain pathophysiologic conditions this process seems to be blunted, resulting in a reduced capacity of EPCs to engage in vasculogenesis at sites of endothelial injury or tissue ischemia. In this review, we focus on the effects of traditional cardiovascular risk factors on EPC biology and we explore whether pharmacological, dietary and lifestyle interventions can favorably restore EPC mobilization, differentiation, homing and angiogenic properties. Because the PI3K/Akt/eNOS pathway plays a pivotal role in the process of EPC mobilization, migration and homing, we specifically emphasize the involvement of PI3K, Akt and eNOS in EPC biology under these different (patho)physiologic conditions. (Pre)clinically used drugs or lifestyle interventions that have been shown to ameliorate EPC biology are reviewed. These treatment strategies remain attractive targets to restore the regenerative capacity of EPCs in cardiovascular diseases.

High Intensity Interval Training in Heart Failure Patients with Reduced Ejection Fraction

Background: Small studies have suggested that high-intensity interval training (HIIT) is superior to moderate continuous training (MCT) in reversing cardiac remodeling and increasing aerobic capacity in patients with heart failure with reduced ejection fraction. The present multicenter trial compared 12 weeks of supervised interventions of HIIT, MCT, or a recommendation of regular exercise (RRE). Methods: Two hundred sixty-one patients with left ventricular ejection fraction ⩽35% and New York Heart Association class II to III were randomly assigned to HIIT at 90% to 95% of maximal heart rate, MCT at 60% to 70% of maximal heart rate, or RRE. Thereafter, patients were encouraged to continue exercising on their own. Clinical assessments were performed at baseline, after the intervention, and at follow-up after 52 weeks. Primary end point was a between-group comparison of change in left ventricular end-diastolic diameter from baseline to 12 weeks. Results: Groups did not differ in age (median, 60 years), sex (19% women), ischemic pathogenesis (59%), or medication. Change in left ventricular end-diastolic diameter from baseline to 12 weeks was not different between HIIT and MCT (P=0.45); left ventricular end-diastolic diameter changes compared with RRE were −2.8 mm (−5.2 to −0.4 mm; P=0.02) in HIIT and −1.2 mm (−3.6 to 1.2 mm; P=0.34) in MCT. There was also no difference between HIIT and MCT in peak oxygen uptake (P=0.70), but both were superior to RRE. However, none of these changes was maintained at follow-up after 52 weeks. Serious adverse events were not statistically different during supervised intervention or at follow-up at 52 weeks (HIIT, 39%; MCT, 25%; RRE, 34%; P=0.16). Training records showed that 51% of patients exercised below prescribed target during supervised HIIT and 80% above target in MCT. Conclusions: HIIT was not superior to MCT in changing left ventricular remodeling or aerobic capacity, and its feasibility remains unresolved in patients with heart failure. Clinical Trial Registration: URL: http://www.clinicaltrials.gov. Unique identifier: NCT00917046.

Exercise acutely reverses dysfunction of circulating angiogenic cells in chronic heart failure.

AIMS Recruitment of endothelial progenitor cells (EPCs) and enhanced activity of circulating angiogenic cells (CACs) might explain the benefits of exercise training in reversing endothelial dysfunction in chronic heart failure (CHF) patients. We studied baseline EPC numbers and CAC function and the effect of a single exercise bout. METHODS AND RESULTS Forty-one CHF patients (mild, n = 22; severe, n = 19) and 13 healthy subjects were included. Migratory activity of CACs was evaluated in vitro and circulating CD34+ and CD34+/KDR+ (EPC) cells were quantified by flow cytometry before and after cardiopulmonary exercise testing (CPET). Circulating stromal cell-derived factor-1alpha (SDF-1alpha) and vascular endothelial growth factor (VEGF) concentrations were measured. Both CAC migration as well as CD34+ cell numbers were significantly reduced in CHF, whereas CD34+/KDR+ cells were not different from controls. Endothelial dysfunction was related to impaired CAC migration (r = 0.318, P = 0.023). Cardiopulmonary exercise testing improved CAC migration in severe (+52%, P < 0.005) and mild CHF (+31%, P < 0.005), restoring it to levels similar to controls. Following CPET, SDF-1alpha increased in healthy controls and mild CHF (P < 0.005). Vascular endothelial growth factor, CD34+, and CD34+/KDR+ cell numbers remained unchanged. CONCLUSION The present findings reveal a potent stimulus of acute exercise to reverse CAC dysfunction in CHF patients with endothelial dysfunction.

Quantification of circulating CD34+/KDR+/CD45dim endothelial progenitor cells: analytical considerations.

The discovery of peripheral circulating cells that contribute to vasculogenesis and endothelial repair was one of the most fascinating breakthroughs in the domain of vascular research during the last two decades. The population of vasculogenic cells however, is heterogeneous and can be analyzed using different approaches including in vitro culture and flow cytometry. Circulating CD34(+)/KDR(+)/CD45(dim) endothelial progenitor cells (EPC) have a great potential as biomarkers in various cardiovascular diseases. With the expanding interest in this field, the development of standardized protocols is critical. In this review we describe in detail the pre-analytical and analytical factors that should be taken into account when quantifying CD34(+)/KDR(+)/CD45(dim) EPC using flow cytometry. Moreover, technical suggestions in order to enhance accuracy and reproducibility of this enumeration are provided.