Christiaan J. Vrints

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.

High-Dose Folic Acid Pretreatment Blunts Cardiac Dysfunction During Ischemia Coupled to Maintenance of High-Energy Phosphates and Reduces Postreperfusion Injury

Background— The B vitamin folic acid (FA) is important to mitochondrial protein and nucleic acid synthesis, is an antioxidant, and enhances nitric oxide synthase activity. Here, we tested whether FA reduces myocardial ischemic dysfunction and postreperfusion injury. Methods and Results— Wistar rats were pretreated with either FA (10 mg/d) or placebo for 1 week and then underwent in vivo transient left coronary artery occlusion for 30 minutes with or without 90 minutes of reperfusion (total n=131; subgroups used for various analyses). FA (4.5×10−6 mol/L IC) pretreatment and global ischemia/reperfusion (30 minutes/30 minutes) also were performed in vitro (n=28). After 30 minutes of ischemia, global function declined more in controls than in FA-pretreated rats (&Dgr;dP/dtmax, −878±586 versus −1956±351 mm Hg/s placebo; P=0.03), and regional thickening was better preserved (37.3±5.3% versus 5.1±0.6% placebo; P=0.004). Anterior wall perfusion fell similarly (−78.4±9.3% versus −71.2±13.8% placebo at 30 minutes), yet myocardial high-energy phosphates ATP and ADP reduced by ischemia in controls were better preserved by FA pretreatment (ATP: control, 2740±58 nmol/g; ischemia, 947±55 nmol/g; ischemia plus FA, 1332±101 nmol/g; P=0.02). Basal oxypurines (xanthine, hypoxanthine, and urate) rose with FA pretreatment but increased less during ischemia than in controls. Ischemic superoxide generation declined (3124±280 cpm/mg FA versus 5898±474 cpm/mg placebo; P=0.001). After reperfusion, FA-treated hearts had smaller infarcts (3.8±1.2% versus 60.3±4.1% placebo area at risk; P<0.002) and less contraction band necrosis, terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling positivity, superoxide, and nitric oxide synthase uncoupling. Infarct size declined similarly with 1 mg/d FA. Conclusions— FA pretreatment blunts myocardial dysfunction during ischemia and ameliorates postreperfusion injury. This is coupled to preservation of high-energy phosphates, reducing subsequent reactive oxygen species generation, eNOS-uncoupling, and postreperfusion cell death.

Mechanisms and potential therapeutic targets for folic acid in cardiovascular disease

Folic acid (FA) is a member of the B-vitamin family with cardiovascular roles in homocysteine regulation and endothelial nitric oxide synthase (eNOS) activity. Its interaction with eNOS is thought to be due to the enhancement of tetrahydrobiopterin bioavailability, helping maintain eNOS in its coupled state to favor the generation of nitric oxide rather than oxygen free radicals. FA also plays a role in the prevention of several cardiac and noncardiac malformations, has potent direct antioxidant and antithrombotic effects, and can interfere with the production of the endothelial-derived hyperpolarizing factor. These multiple mechanisms of action have led to studies regarding the therapeutic potential of FA in cardiovascular disease. To date, studies have demonstrated that FA ameliorates endothelial dysfunction and nitrate tolerance and can improve pathological features of atherosclerosis. These effects appear to be homocysteine independent but rather related to their role in eNOS function. Given the growing evidence that nitric oxide synthase uncoupling plays a major role in many cardiovascular disorders, the potential of exogenous FA as an inexpensive and safe oral therapy is intriguing and is stimulating ongoing investigations.

Impaired Fibrillin-1 Function Promotes Features of Plaque Instability in Apolipoprotein E–Deficient Mice

Background— Arterial stiffness has been associated with an increased cardiovascular risk. The aim of this study was to investigate the interaction between arterial stiffness and atherosclerosis. Methods and Results— Mice with a mutation (C1039G+/−) in the fibrillin-1 gene leading to fragmentation of the elastic fibers were crossbred with apolipoprotein E–deficient (ApoE−/−) mice. Subsequently, ApoE−/− and ApoE−/−C1039G+/− mice were fed a Western-type diet for 10 or 20 weeks. Our results show that the interaction between arterial stiffness and atherosclerosis is bidirectional. On the one hand, arterial stiffness in ApoE−/−C1039G+/− mice increased more rapidly in the presence of atherosclerotic plaques. On the other hand, arterial stiffness promoted the development of larger and more unstable plaques in ApoE−/−C1039G+/− mice. The plaque area at the aortic root was increased 1.5- and 2.1-fold in ApoE−/−C1039G+/− mice after 10 and 20 weeks of Western-type diet, respectively. After 10 weeks of Western-type diet, plaques of ApoE−/−C1039G+/− mice showed increased apoptosis of smooth muscle cells, which was associated with a decrease in collagen content, an enlargement of the necrotic core, and an increase in macrophages. After 20 weeks of Western-type diet, the number of buried fibrous caps was increased in atherosclerotic lesions of ApoE−/−C1039G+/− mice, not only at the level of the aortic valves but also in the brachiocephalic artery and in the upper, middle, and lower thoracic aorta. Furthermore, acute plaque rupture was observed. Conclusion— These results indicate that fragmentation of the elastic fibers leads to increased vascular stiffness, which promotes features of multifocal plaque instability.

Performant Mutation Identification Using Targeted Next‐Generation Sequencing of 14 Thoracic Aortic Aneurysm Genes

At least 14 causative genes have been identified for both syndromic and nonsyndromic forms of thoracic aortic aneurysm/dissection (TAA), an important cause of death in the industrialized world. Molecular confirmation of the diagnosis is increasingly important for gene‐tailored patient management but consecutive, conventional molecular TAA gene screening is expensive and labor‐intensive. To circumvent these problems, we developed a TAA gene panel for next‐generation sequencing of 14 TAA genes. After validation, we applied the assay to 100 Marfan patients. We identified 90 FBN1 mutations, 44 of which were novel. In addition, Multiplex ligation‐dependent probe amplification identified large deletions in six of the remaining samples, whereas false‐negative results were excluded by Sanger sequencing of FBN1, TGFBR1, and TGFBR2 in the last four samples. Subsequently, we screened 55 syndromic and nonsyndromic TAA patients. We identified causal mutations in 15 patients (27%), one in each of the six following genes: ACTA2, COL3A1, TGFBR1, MYLK, SMAD3, SLC2A10 (homozygous), two in NOTCH1, and seven in FBN1. We conclude that our approach for TAA genetic testing overcomes the intrinsic hurdles of consecutive Sanger sequencing of all candidate genes and provides a powerful tool for the elaboration of clinical phenotypes assigned to different genes.

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.

Euro Heart Survey 2009 Snapshot: Regional variations in presentation and management of patients with AMI in 47 countries

Aims: Detailed data on patients admitted for acute myocardial infarction (AMI) on a European-wide basis are lacking. The Euro Heart Survey 2009 Snapshot was designed to assess characteristics, management, and hospital outcomes of AMI patients throughout European Society of Cardiology (ESC) member countries in a contemporary ‘real-world’ setting, using a methodology designed to improve the representativeness of the survey. Methods: Member countries of the ESC were invited to participate in a 1-week survey of all patients admitted for documented AMI in December 2009. Data on baseline characteristics, type of AMI, management, and complications were recorded using a dedicated electronic form. In addition, we used data collected during the same time period in national registries in Sweden, England, and Wales. Data were centralized at the European Heart House. Results: Overall, 4236 patients (mean age 66±13 years; 31% women) were included in the study in 47 countries. Sixty per cent of patients had ST-segment elevation myocardial infarction, with 50% having primary percutaneous coronary intervention and 21% fibrinolysis. Aspirin and thienopyridines were used in >90%. Unfractionated and low-molecular-weight heparins were the most commonly used anticoagulants. Statins, beta-blockers, and angiotensin-converting enzyme inhibitors were used in >80% of the patients. In-hospital mortality was 6.2%. Regional differences were observed, both in terms of population characteristics, management, and outcomes. Conclusions: In-hospital mortality of patients admitted for AMI in Europe is low. Although regional variations exist in their presentation and management, differences are limited and have only moderate impact on early outcomes.

Unraveling new mechanisms of exercise intolerance in chronic heart failure. Role of exercise training

Despite remarkable progress in the therapeutic approach of patients with chronic heart failure (CHF), exercise intolerance remains one of the hallmarks of the disease. During the past two decades, evidence has accumulated to underscore the key role of both endothelial dysfunction and skeletal muscle wasting in the process that gradually leads to physical incapacity. Whereas reverse ventricular remodeling has been attributed to aerobic exercise training, the vast majority of studies conducted in this specific patient population emphasize the reversal of peripheral abnormalities. In this review, we provide a general overview on underlying pathophysiological mechanisms. In addition, emphasis is put on recently identified pathways, which contribute to a deeper understanding of the main causes of exercise tolerance and the potential for reversal through exercise training. Recently, deficient bone marrow-related endothelial repair mechanisms have received considerable attention. Both acute exercise bouts, as well as exercise training, affect the mobilization of endothelial progenitor cells and their function. The observed changes following exercise training are believed to significantly contribute to improvement of peripheral endothelial function, as well as exercise capacity. With regard to skeletal muscle dysfunction and energy deprivation, adiponectin has been suggested to play a significant role. The demonstration of local skeletal muscle adiponectin resistance may provide an interesting and new link between the insulin resistant state and skeletal muscle wasting in CHF patients.

Immunohistochemical characterisation of dendritic cells in human atherosclerotic lesions: possible pitfalls

Background: Previously we demonstrated decreased blood myeloid (m) and plasmacytoid (p) dendritic cell (DC) counts in atherosclerotic patients. Therefore, we examined whether DCs, in particular DC precursors, accumulate in human plaques. Methods: Blood DC antigen (BDCA)-1, CD11c (mDCs), BDCA-2, CD123 (pDCs), langerin, fascin, S-100 (immature/mature DCs), and CD1a and CD83 (mature DCs) were investigated by immunohistochemistry of carotid arteries obtained by endarterectomy (EAS, frozen n = 11, fixed n = 11) or autopsy (fixed, n = 87). Results: Fascin and S-100 required formaldehyde fixation, other markers needed cryo-preservation. BDCA-1, BDCA-2, langerin, and S-100 appeared specific for intimal DCs, unlike CD123 and fascin (staining endothelial cells), CD11c and CD1a (staining monocytes, foam cells) or CD83 (staining lymphocytes). BDCA-1+ and BDCA-2+ cells were detected in EAS, preferentially near microvessels. S-100+ cells increased successively from intimal thickening, via pathological intimal thickening, fibrous cap atheroma and finally complicated plaques. Fascin+ cells followed the same pattern, but were more abundant. However, in lesions containing microvessels (complicated plaques, plaque shoulders and most EAS) this was partly explained by fascin positive endothelial cells. Even complicated plaques contained relatively few mature CD83+ DCs. Conclusions: Accumulation of BDCA-1 and BDCA-2 around neovessels showed that mDCs and pDCs are recruited to advanced plaques, which is in line with the previously described decline of circulating blood DCs in patients with coronary artery disease. Unexpectedly, several DC markers yielded false positive signals. Hence, some accounts on numbers, trafficking and activation of DCs in atherosclerotic plaques may require re-evaluation.

Human C-Reactive Protein Activates Monocyte-Derived Dendritic Cells and Induces Dendritic Cell-Mediated T-Cell Activation

Objective—Recent studies proposed a pathogenic role for C-reactive protein (CRP), an independent predictor of cardiovascular disease (CVD), in atherosclerosis. Therefore, we tested whether CRP may modulate dendritic cell (DC) function, because these professional antigen-presenting cells have been implicated in atherogenesis. Methods and Results—Human monocyte-derived immature DCs were cultured with human CRP (0 to 60 &mgr;g/mL) for 24 hours. Thereafter, activation markers were measured by flow-cytometry and DCs were cocultured with CFSE-labeled lymphocytes to measure T-cell proliferation and interferon (IFN)-γ secretion after 8 days. Exposure to 60 &mgr;g/mL CRP (n=5) induced an activated cell morphology and significant (CD40 increase MFI 5.23±0.28, P<0.01 paired t test; CD80 6.18±0.51, P<0.01) to modest (CD83 1.38±0.17, P<0.05, CCR7 1.60±0.29, P=0.05) upregulation of DC activation markers. The expression of CD86 and HLA-DR was high, but not affected. T-lymphocytes incubated with CRP-pulsed DCs displayed increased IFN-γ secretion and proliferation (P<0.001). DC activation was concentration-dependent and detected from 2 &mgr;g/mL CRP; the maximum effect was equivalent to that seen with 0.1 &mgr;g/mL lipopolysaccharide (LPS). Polymyxin B abolished the LPS response, without influencing CRP effects. Finally, immunohistochemistry could demonstrate DC/CRP colocalization in human atherosclerotic lesions. Conclusions—These findings suggest that CRP in plaques or found circulating in CVD patients can influence DC function during atherogenesis.