Paul-Eduard Neagoe

Vascular endothelial growth factor (VEGF)-A165-induced prostacyclin synthesis requires the activation of VEGF receptor -1 and -2 heterodimer

We previously reported that vascular endothelial growth factor (VEGF)-A165 inflammatory effect is mediated by acute platelet-activating factor synthesis from endothelial cells upon the activation of VEGF receptor-2 (VEGFR-2) and its coreceptor, neuropilin-1 (NRP-1). In addition, VEGF-A165 promotes the release of other endothelial mediators including nitric oxide and prostacyclin (PGI2). However, it is unknown whether VEGF-A165 is mediating PGI2 synthesis through VEGF receptor-1 (VEGFR-1) and/or VEGF receptor-2 (VEGFR-2) activation and whether the coreceptor NRP-1 potentiates VEGF-A165 activity. In this study, PGI2 synthesis in bovine aortic endothelial cells (BAEC) was assessed by quantifying its stable metabolite (6-keto prostaglandin F1α, 6-keto PGF1α) by enzyme-linked immunosorbent assay. Treatment of BAEC with VEGF analogs, VEGF-A165 (VEGFR-1, VEGFR-2 and NRP-1 agonist) and VEGF-A121 (VEGFR-1 and VEGFR-2 agonist) (up to 10–9 m), increased PGI2 synthesis by 70- and 40-fold within 15 min. Treatment with VEGFR-1 (placental growth factor and VEGF-B) or VEGFR-2 (VEGF-C) agonist did not increase PGI2 synthesis. The combination of VEGFR-1 and VEGFR-2 agonists did not increase PGI2 release. Pretreatment with a VEGFR-2 inhibitor abrogated PGI2 release mediated by VEGF-A165 and VEGF-A121, and pretreatment of BAEC with antisense oligomers targeting VEGFR-1 or VEGFR-2 mRNA reduced PGI2 synthesis mediated by VEGF-A165 and VEGF-A121 up to 79%. In summary, our data demonstrate that the activation of VEGFR-1 and VEGFR-2 heterodimer (VEGFR-1/R-2) is essential for PGI2 synthesis mediated by VEGF-A165 and VEGF-A121, which cannot be reproduced by the parallel activation of VEGFR-1 and VEGFR-2 homodimers with corresponding agonists. In addition, the binding of VEGF-A165 to NRP-1 potentiates its capacity to promote PGI2 synthesis.

Expression and release of angiopoietin-1 from human neutrophils: Intracellular mechanisms

We recently demonstrated that Tie2 receptor activation on human neutrophils by both angiopoietins (Ang1 and Ang2) promoted platelet-activating factor synthesis, β2-integrin activation, and cell migration. Herein, we wanted to assess if human neutrophils express angiopoietins and further delineate their mechanisms of release. Employing Reverse transcriptase-polymerase chain reaction, Real time quantitative transcriptase-polymerase chain reaction, FACScan analysis and ELISA approaches, we observed that neutrophils express Ang1 but not Ang2. For each condition, vascular endothelial growth factor (VEGF) detection was performed as positive control. Using nitrogen cavitation, we observed that Ang1 is localized in the cytosolic fraction whereas VEGF is found in β-granules. Treatment of neutrophils with phorbol myristate acetate (PMA), N-Formyl-Met-Leu-Phe (fMLP) and tumor necrosis factor-α (TNF-α) induced VEGF release. Maximal effect was observed with PMA (80 nM) stimulation inducing a complete release of VEGF content (565 ± 100 pg/ml; 6 × 106 neutrophils), corresponding to a 18.9-fold increase as compared to phosphate buffer saline (PBS) treated neutrophils. By contrast, only a treatment with PMA (80 nM) induced Ang1 release. PMA treatment induced also a complete release of Ang1 (661 ± 148 pg/ml; 6 × 106 neutrophils), corresponding to 2.8-fold increase as compared to PBS-treated neutrophils. In both cases, PMA-mediated release of VEGF and Ang1 was nearly maximal by 15 min. Finally, we observed that the induction of Ang1 release was calcium-independent whereas VEGF release was not. These data demonstrate the capacity of human neutrophils to synthesize Ang1, which is stored and released differently as compared to VEGF. These data suggest a different cascade of events regarding the distribution of selected growth factors during inflammation and angiogenesis.

Effect of high-dose dexamethasone on endothelial haemostatic gene expression and neutrophil adhesion

Glucocorticoid usage especially at high doses is complicated by adverse outcomes such as thrombotic events or acceleration of inflammatory response in conditions like myeloma and osteonecrosis. The mechanism(s) through which high-dose dexamethasone (HDDEXA) causes vascular injury remains unclear. We hypothesized that HDDEXA sensitizes endothelial cells (EC) to the effect of inflammatory mediators and modulates endothelial haemostatic gene expression and leukocyte adhesion. Human umbilical vein endothelial cells (HUVECs) were grown in the absence or presence of HDDEXA and were also tested in the presence or absence of tumor necrosis factor-alpha (TNF-alpha), lipopolysaccharide (LPS) or thrombin. mRNA and protein expression were measured and the functional consequences of HDDEXA preconditioning on cell adhesion molecules (CAM) were determined by agonist-mediated leukocyte adhesion assay. Treatment with HDDEXA resulted in an increased induction of CAM, tissue factor and von Willebrand factor, while down-regulating thrombomodulin and urokinase. HDDEXA alone had no effect on adhesion but resulted in enhanced TNF-alpha- and LPS-mediated adhesion of neutrophils. Together, these findings suggest that HDDEXA sensitizes HUVEC to the effect of inflammatory mediators and induces a pro-adhesive environment in primary EC. This finding is of importance when glucocorticoid usage is required at therapeutic high doses in patients with or without thrombotic risk factors.

Angiopoietin-mediated endothelial P-selectin translocation: cell signaling mechanisms

Recently identified, angiopoietin‐1 (Ang1) and ‐2 (Ang2) bind to the tyrosine kinase receptor Tie2 and contribute to orchestrate blood vessel formation during angiogenesis. Ang1 mediates vessel maturation and integrity by favoring the recruitment of pericytes and smooth muscle cells. Ang2, initially identified as a Tie2 antagonist, may under certain circumstances, induce Tie2 phosphorylation and biological activities. As inflammation exists in a mutually dependent association with angiogenesis, we sought to determine if Ang1 and/or Ang2 could modulate proinflammatory activities, namely P‐selectin translocation, in bovine aortic endothelial cells (EC) and dissect the mechanisms implicated. P‐selectin, an adhesion molecule found in the Weibel‐Palade bodies of EC, is translocated rapidly to the cell surface upon EC activation during inflammatory processes. Herein, we report that Ang1 and Ang2 (1 nM) are capable of mediating a rapid Tie2 phosphorylation as well as a rapid and transient endothelial P‐selectin translocation maximal within 7.5 min (125% and 100% increase, respectively, over control values). In addition, we demonstrate for the first time that angiopoietin‐mediated endothelial P‐selectin translocation is calcium‐dependent and regulated through phospholipase C‐γ activation.

Sphingosine 1‐phosphate effect on endothelial cell PAF synthesis: Role in cellular migration

Sphingosine 1‐phosphate (S1P) and vascular endothelial growth factor (VEGF) are two inflammatory mediators capable of promoting endothelial cell (EC) migration and angiogenesis. As VEGF inflammatory effect is mediated by the synthesis of endothelial platelet‐activating factor (PAF) which is also contributing to VEGF chemotactic activity, we wanted to assess if S1P can trigger PAF synthesis in EC and if S1P‐induced migration is PAF‐dependent. Treatment of bovine aortic EC (BAEC) with S1P (10−10–10−6 M) increased dose‐ and time‐dependently the synthesis of PAF by up to 3.3‐fold above the basal level, with a maximal amount of PAF detected at 20 min post‐stimulation. This biological response was attenuated by inhibiting p38 mitogen‐activated protein kinase (MAPK), cytosolic or secreted phospholipase A2 (cPLA2, sPLA2) activity, suggesting that p38 MAPK activation by S1P promotes the conversion of membrane phospholipids into PAF through the combined activation of cPLA2 and sPLA2. Interestingly, pretreatment of BAEC with extracellular PAF receptor antagonists (BN52021, 10−5 M and CV3988, 10−6 M) reduced by up to 42% the cellular migration induced by S1P (10−6 M). These data demonstrate the capacity of S1P to induce PAF synthesis, which contributes in part to S1P chemotactic activity.

Heart Failure With Anemia Novel Findings on the Roles of Renal Disease, Interleukins, and Specific Left Ventricular Remodeling Processes

Background— Anemia is a highly prevalent and strong independent prognostic marker in heart failure (HF), yet this association is not completely understood. Whether anemia is simply a marker of disease severity and concomitant chronic kidney disease or represents the activation of other detrimental pathways remains uncertain. We sought to determine which pathophysiological pathways are exacerbated in patients with HF, reduced ejection fraction (HFrEF) and anemia in comparison with those without anemia. Methods and Results— In a prospective study involving 151 patients, selected biomarkers were analyzed, each representing proposed contributive mechanisms in the pathophysiology of anemia in HF. We compared clinical, echocardiographic, and circulating biomarkers profiles among patients with HFrEF and anemia (group 1), HFrEF without anemia (group 2), and chronic kidney disease with preserved EF, without established HF (chronic kidney disease control group 3). We demonstrate here that many processes other than those related to chronic kidney disease are involved in the anemia–HF relationship. These are linked to the pathophysiological mechanisms pertaining to left ventricular systolic dysfunction and remodeling, systemic inflammation and volume overload. We found that levels of interleukin-6 and interleukin-10, specific markers of cardiac remodeling (procollagen type III N-terminal peptide, matrix metalloproteinase-2, tissue inhibitor of matrix metalloproteinase 1, left atrial volume), myocardial stretch (NT-proBNP [N-terminal probrain natriuretic peptide]), and myocyte death (troponin T) are related to anemia in HFrEF. Conclusions— Anemia is strongly associated not only with markers of more advanced and active heart disease but also with the level of renal dysfunction in HFrEF. Increased myocardial remodeling, inflammation, and volume overload are the hallmarks of patients with anemia and HF. Clinical Trial Registration— URL: . Unique identifier: [NCT00834691][1]. [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT00834691&atom=%2Fcirchf%2F7%2F5%2F773.atomBackground—Anemia is a highly prevalent and strong independent prognostic marker in heart failure (HF), yet this association is not completely understood. Whether anemia is simply a marker of disease severity and concomitant chronic kidney disease or represents the activation of other detrimental pathways remains uncertain. We sought to determine which pathophysiological pathways are exacerbated in patients with HF, reduced ejection fraction (HFrEF) and anemia in comparison with those without anemia. Methods and Results—In a prospective study involving 151 patients, selected biomarkers were analyzed, each representing proposed contributive mechanisms in the pathophysiology of anemia in HF. We compared clinical, echocardiographic, and circulating biomarkers profiles among patients with HFrEF and anemia (group 1), HFrEF without anemia (group 2), and chronic kidney disease with preserved EF, without established HF (chronic kidney disease control group 3). We demonstrate here that many processes other than those related to chronic kidney disease are involved in the anemia–HF relationship. These are linked to the pathophysiological mechanisms pertaining to left ventricular systolic dysfunction and remodeling, systemic inflammation and volume overload. We found that levels of interleukin-6 and interleukin-10, specific markers of cardiac remodeling (procollagen type III N-terminal peptide, matrix metalloproteinase-2, tissue inhibitor of matrix metalloproteinase 1, left atrial volume), myocardial stretch (NT-proBNP [N-terminal probrain natriuretic peptide]), and myocyte death (troponin T) are related to anemia in HFrEF. Conclusions—Anemia is strongly associated not only with markers of more advanced and active heart disease but also with the level of renal dysfunction in HFrEF. Increased myocardial remodeling, inflammation, and volume overload are the hallmarks of patients with anemia and HF. Clinical Trial Registration—URL: http://www.clinicaltrials.gov. Unique identifier: NCT00834691.

Differences in Biomarkers in Patients With Heart Failure With a Reduced vs a Preserved Left Ventricular Ejection Fraction

BACKGROUND The differences in concentrations of biomarkers between heart failure (HF) patients with a preserved left ventricular ejection fraction (LVEF), or HF-PEF, and patients with HF with reduced LVEF (HF-REF) have yet to be defined. The objectives of this study were to compare the concentrations and correlation of biomarkers of inflammation, extracellular matrix (ECM) turnover and neurohormonal activation between these populations. METHODS We performed a cross-sectional study of 29 subjects with symptomatic HF-REF (LVEF = 25.6 ± 5.1%) and 29 subjects with symptomatic HF-PEF (LVEF = 63.3 ± 5.3%). Concentrations of N-terminal proB-type natriuretic peptide (NT-proBNP), high sensitivity C-reactive protein (hsCRP), procollagen type III amino-terminal peptide (PIIINP), matrix metalloproteinase (MMP)-2, MMP-9, and tissue inhibitor of MMP (TIMP)-1 were measured. RESULTS Although NT-proBNP and PIIINP concentrations were higher in patients with HF-REF compared with patients with HF-PEF (both P < 0.05), the only significant difference between the groups remaining after adjusting for possible confounding variables was NT-proBNP (P = 0.02). In patients with HF-REF, NT-proBNP correlated with PIIINP (P < 0.05), TIMP-1 (P < 0.05), and MMP-2 (P = 0.002), while PIIINP correlated with TIMP-1 (P < 0.05) and MMP-2 (P < 0.0001). In patients with a HF-PEF, only high sensitivity C-reactive protein correlated significantly with MMP-2 (P = 0.002). CONCLUSIONS Patients with HF-REF or HF-PEF presenting similar symptoms and functional limitations exhibit similar concentrations of biomarkers of ECM and inflammation. However, patients with HF-REF exhibit significantly higher NT-proBNP concentrations than patients with HF-PEF. The differences in the correlations observed between the biomarkers between these 2 populations suggest some heterogeneity and differences in the mechanisms related to the release or clearance of biomarkers in HF-REF vs HF-PEF.

Effect of everolimus on the immunomodulation of the human neutrophil inflammatory response and activation

The primary cause of mortality at 5 years following a cardiac transplantation is the development of atherosclerosis, termed coronary allograft vasculopathy (CAV). This pathology is characterized by diffused intimal hyperplasia and emanates from coronary arterial injuries caused by immune inflammatory cells. Neutrophils play an important role in this inflammatory process; however, their potential participation in the pathogenesis of CAV is poorly understood. Despite their essential contribution to the prevention of graft rejection, immunosuppressive drugs could have detrimental effects owing to their pro-inflammatory activities. Thus, we investigated the impact of different immunosuppressive drugs on the inflammatory response of neutrophils isolated from the blood of healthy volunteers. Under basal conditions, mammalian target of rapamycin (mTOR) inhibitors (sirolimus and everolimus) had the most potent anti-inflammatory effect, decreasing both IL-8 release (≈−80%) and vascular endothelial growth factor (VEGF) release (≈−65%) and preserving the release of the anti-inflammatory cytokine interleukin-1 receptor antagonist (IL-1RA). In TNF-α-treated neutrophils, pre-incubation with everolimus provided the most potent effect, simultaneously reducing the release of both VEGF and IL-8 while doubling the release of IL-1RA. This latter effect of everolimus was maintained even when administered in combination with other immunosuppressive drugs. Sirolimus and everolimus decreased the tumor necrosis factor (TNF)-α-induced adhesion of neutrophils to human endothelial cells and human extracellular matrix. This effect was largely dependent on the ability of these compounds to alter β2-integrin/CD18 activation. Our results suggest a potential mechanism for the beneficial effect of everolimus in the prevention of CAV in heart transplant recipients.

Angiopoietin-1 but not angiopoietin-2 promotes neutrophil viability: Role of interleukin-8 and platelet-activating factor.

We previously reported the expression of angiopoietin receptor Tie2 on human neutrophils. Both angiopoietins (Ang1 and Ang2) induce platelet activating factor (PAF) synthesis from endothelial cells (ECs) and neutrophils. Both angiopoietins can also modulate EC viability and since PAF can promote pro-survival activity on neutrophils, we addressed whether Ang1 and/or Ang2 could modulate neutrophil viability. Neutrophils were isolated from venous blood of healthy volunteers and neutrophil viability was assessed by flow cytometry using apoptotic and necrotic markers (annexin-V and propidium iodide (P.I.), respectively). Basal neutrophil viability from 0 to 24 h post-isolation decreased from 98% to ≈45%. Treatment with anti-apoptotic mediators such as interleukin-8 (IL-8; 25 nM) and PAF (100 nM) increased neutrophil basal viability by 34 and 26% (raising it from 43 to 58 and 55%) respectively. Treatment with Ang1 (0.001-50 nM) increased neutrophil viability by up to 41%, while Ang2 had no significant effect. Combination of IL-8 (25 nM) or PAF (100 nM) with Ang1 (10 nM) further increased neutrophil viability by 56 and 60% respectively. We also observed that Ang1, but not Ang2 can promote IL-8 release and that a pretreatment of the neutrophils with blocking anti-IL-8 antibodies inhibited the anti-apoptotic effect of IL-8 and Ang1 by 92 and 81% respectively. Pretreatment with a selective PAF receptor antagonist (BN 52021), did abrogate PAF pro-survival activity, without affecting Ang1-induced neutrophil viability. Our data are the first ones to report Ang1 pro-survival activity on neutrophils, which is mainly driven through IL-8 release.

Synthesis of Human Neutrophil Extracellular Traps Contributes to Angiopoietin-Mediated In Vitro Proinflammatory and Proangiogenic Activities

Neutrophil extracellular traps (NETs) are composed of nuclear DNA in a web-like structure extruded from neutrophils in response to either bacterial infection or inflammation. We previously reported the expression of angiopoietin Tie2 receptor on human neutrophils and the capacity of both angiopoietins (Ang1 and Ang2) to induce proinflammatory activities, such as synthesis and release of platelet-activating factor, upregulation of β2 integrin complex (CD11/CD18), and neutrophil chemotaxis. In contrast, only Ang1 but not Ang2 is capable of promoting translational and transcriptional activities in neutrophils. In this article, we addressed whether Ang1 and/or Ang2 could modulate the release of NETs and if they contribute to angiopoietin-mediated proinflammatory activities. We observed that Ang1 and Ang2, alone or combined (10 nM, 3 h), increase NET synthesis and release by ≈2.5-fold as compared with PBS-treated neutrophils. The release of NETs is Tie2 dependent and requires downstream intracellular participation of PI3K, p38, and p42/44 MAPK pathways; reactive oxygen species production; intracellular calcium store depletion; and protein arginine deiminase 4 activation. These isolated NETs induced neutrophil and endothelial cell activation, leading to neutrophil adhesion onto human extracellular matrix and HUVEC and in vitro formation of capillary-like tubes by endothelial cells. Our study reports the capacity of Ang1 and Ang2 to promote the release of NETs and that these NETs contribute to angiopoietin-mediated in vitro proinflammatory and proangiogenic activities.