Blandine Dizier

Thrombospondin-1 Is a Plasmatic Marker of Peripheral Arterial Disease That Modulates Endothelial Progenitor Cell Angiogenic Properties

Objective—We examined whether plasma levels of angiogenic factors are altered in plasma of patients with peripheral arterial disease (PAD) and whether these factors affect endothelial progenitor cell–induced angiogenesis. Methods and Results—Plasma was collected from 184 patients with PAD and 330 age-matched healthy controls. Vascular endothelial growth factor and placental growth factor concentrations did not differ between the groups, whereas we found a linear correlation between PAD disease and thrombospondin (TSP)-1 plasma level. TSP-1 was expressed in newly formed vessels in PAD patients having received local injections of bone marrow mononuclear cells. To analyze the functional role of TSP-1 during neoangiogenesis, we used a Matrigel-plug assay and showed that vascularization of implanted Matrigel-plugs was increased in TSP-1−/− mice. Moreover, injections of TSP-1 in C57Bl6/J mice after hindlimb ischemia induced a significant decrease of blood flow recovery. To investigate the effects of TSP-1 on human endothelial colony-forming cell (ECFC) angiogenic potential, recombinant human TSP-1 and a small interfering RNA were used. In vitro, TSP-1 N-terminal part significantly enhanced ECFC adhesion, whereas recombinant human TSP-1 had a negative effect on ECFC angiogenic potential. This effect, mediated by CD47 binding, modulated stromal cell–derived factor 1/CXC chemokine receptor 4 pathway. Conclusion—TSP-1 is a potential biomarker of PAD and ECFC-induced angiogenesis, suggesting that TSP-1 modulation might improve local tissue ischemia in this setting. (Clinical trial registration: NCT00377897.)

A switch toward angiostatic gene expression impairs the angiogenic properties of endothelial progenitor cells in low birth weight preterm infants.

Low birth weight (LBW) is associated with increased risk of cardiovascular diseases at adulthood. Nevertheless, the impact of LBW on the endothelium is not clearly established. We investigate whether LBW alters the angiogenic properties of cord blood endothelial colony forming cells (LBW-ECFCs) in 25 preterm neonates compared with 25 term neonates (CT-ECFCs). We observed that LBW decreased the number of colonies formed by ECFCs and delayed the time of appearance of their clonal progeny. LBW dramatically reduced LBW-ECFC capacity to form sprouts and tubes, to migrate and to proliferate in vitro. The angiogenic defect of LBW-ECFCs was confirmed in vivo by their inability to form robust capillary networks in Matrigel plugs injected in nu/nu mice. Gene profile analysis of LBW-ECFCs demonstrated an increased expression of antiangiogenic genes. Among them, thrombospondin 1 (THBS1) was highly expressed at RNA and protein levels in LBW-ECFCs. Silencing THBS1 restored the angiogenic properties of LBW-ECFCs by increasing AKT phosphorylation. The imbalance toward an angiostatic state provide a mechanistic link between LBW and the impaired angiogenic properties of ECFCs and allows the identification of THBS1 as a novel player in LBW-ECFC defect, opening new perspectives for novel deprogramming agents.

The Wnt Antagonist Dickkopf-1 Increases Endothelial Progenitor Cell Angiogenic Potential

Objective—To determine the role of Wnt antagonist Dickkopf (DKK) 1 in human endothelial colony-forming cells (ECFCs) in view of the emerging importance of Wnt pathways in vascular biology. Methods and Results—Endothelial progenitor cells have been proposed to be crucial in tumor neovascularization. Recombinant DKK1 has been tested in ECFC angiogenic properties in vitro. DKK1 enhanced ECFC proliferation and the capacity of ECFCs to form pseudotubes in Matrigel. These effects have been attributed to enhancement of vascular endothelial growth factor receptor 2, SDF-1, and CXCR4. DKK1 gene silencing has been realized on ECFCs and mesenchymal stem cells, and we found that DKK1 silencing in the 2 cell types decreased their angiogenic potential. We then examined the possible role of DKK1 in tumor neovasculogenesis and found that blood vessels of breast cancer tissues expressed DKK1 far more strongly in human breast tumors than in normal breast tissues. By studying 62 human breast tumors, we found a significant positive correlation between DKK1 expression and von Willebrand factor. In vivo, DKK1 strongly enhanced the vascularization of Matrigel plugs and increased tumor size in a xenograft model of human breast carcinoma in nude mice. Conclusion—DKK1 enhances angiogenic properties of ECFCs in vitro and is required for ECFC and mesenchymal stem cell angiogenic phenotypes in vivo. DKK1 also increases tumoral angiogenesis. Thus, we demonstrated a major role of DKK1 in angiogenic processes.

Interleukin-34 promotes tumor progression and metastatic process in osteosarcoma through induction of angiogenesis and macrophage recruitment.

Interleukin‐34 (IL‐34) was recently characterized as the M‐CSF “twin” cytokine, regulating the proliferation/differentiation/survival of myeloid cells. The implication of M‐CSF in oncology was initially suspected by the reduced metastatic dissemination in knock‐out mice, due to angiogenesis impairment. Based on this observation, our work studied the involvement of IL‐34 in the pathogenesis of osteosarcoma. The in vivo effects of IL‐34 were assessed on tissue vasculature and macrophage infiltration in a murine preclinical model based on a paratibial inoculation of human osteosarcoma cells overexpressing or not IL‐34 or M‐CSF. In vitro investigations using endothelial cell precursors and mature HUVEC cells were performed to analyse the involvement of IL‐34 in angiogenesis and myeloid cell adhesion. The data revealed that IL‐34 overexpression was associated with the progression of osteosarcoma (tumor growth, lung metastases) and an increase of neo‐angiogenesis. In vitro analyses demonstrated that IL‐34 stimulated endothelial cell proliferation and vascular cord formation. Pre‐treatment of endothelial cells by chondroitinases/heparinases reduced the formation of vascular tubes and abolished the associated cell signalling. In addition, IL‐34 increased the in vivo recruitment of M2 tumor‐associated macrophages into the tumor tissue. IL‐34 increased in vitro monocyte/CD34+ cell adhesion to activated HUVEC monolayers under physiological shear stress conditions. This work also demonstrates that IL‐34 is expressed by osteosarcoma cells, is regulated by TNF‐α, IL‐1β, and contributes to osteosarcoma growth by increasing the neo‐angiogenesis and the recruitment of M2 macrophages. By promoting new vessel formation and extravasation of immune cells, IL‐34 may play a key role in tumor development and inflammatory diseases.

α6-Integrin Subunit Plays a Major Role in the Proangiogenic Properties of Endothelial Progenitor Cells

Objective—Alpha6 integrin subunit (&agr;6) expression is increased by proangiogenic growth factors such as vascular endothelial growth factor (VEGF) and fibroblast growth factor. This increase correlates with enhanced in vitro tube formation by endothelial cells and their progenitors called Endothelial Colony-Forming Cells (ECFCs). We thus studied the role of &agr;6 in vasculogenesis induced by human ECFCs, in a mouse model of hindlimb ischemia. Methods and Results—We used small interfering RNA (siRNA) to inhibit &agr;6 expression on the surface of ECFCs. For in vivo studies, human ECFCs were injected intravenously into a nude mouse model of unilateral hind limb ischemia. Transfection with siRNA &agr;6 abrogated neovessel formation and reperfusion of the ischemic hind limb induced by ECFCs (P<0.01 and P<0.001, respectively). It also inhibited ECFC incorporation into the vasculature of the ischemic muscle (P<0.001). In vitro, siRNA &agr;6 inhibited ECFC adhesion (P<0.01), pseudotube formation on Matrigel, migration, and AKT phosphorylation (P<0.0001), with no effect on cell proliferation or apoptosis. Conclusion—&agr;6 Expression is required for ECFC migration, adhesion, recruitment at the site of ischemia, and the promotion of the postischemic vascular repair. Thus, we have demonstrated a major role of &agr;6 in the proangiogenic properties of ECFCs.

Osteoprotegerin, a new actor in vasculogenesis, stimulates endothelial colony-forming cells properties.

Summary.u2002 Background:u2002Osteoprotegerin (OPG), a soluble receptor of the tumour necrosis factor family, and its ligand, the receptor activator of nuclear factor‐κB ligand (RANKL), are emerging as important regulators of vascular pathophysiology. Objectives:u2002We evaluated their effects on vasculogenesis induced by endothelial colony‐forming cells (ECFC) and on neovessel formation in vivo. Methods:u2002Effects of OPG and RANKL on in vitro angiogenesis were evaluated after ECFC incubation with OPG or RANKL (0–50u2003ngu2003mL−1). Effects on microvessel formation were evaluated with an in vivo murin Matrigel plug assay. Vascularization was evaluated by measuring plug hemoglobin and vascular endothelial growth factor (VEGF)‐R2 content 14u2003days after implantation. Results:u2002We found that ECFC expressed OPG and RANK but not RANKL mRNA. Treatment of ECFC with VEGF or stromal cell‐derived factor‐1 (SDF‐1) upregulated OPG mRNA expression. OPG stimulated ECFC migration (Pu2003<u20030.05), chemotaxis (Pu2003<u20030.05) and vascular cord formation on Matrigel® (Pu2003< 0.01). These effects were correlated with SDF‐1 mRNA overexpression, which was 30‐fold higher after 4u2003h of OPG stimulation (Pu2003<u20030.01). OPG‐mediated angiogenesis involved the MAPK signaling pathway as well as Akt or mTOR cascades. RANKL also showed pro‐vasculogenic effects in vitro. OPG combined with FGF‐2 promoted neovessel formation in vivo, whereas RANKL had no effect. Conclusions:u2002OPG induces ECFC activation and is a positive regulator of microvessel formation in vivo. Our results suggest that the OPG/RANK/RANKL axis may be involved in vasculogenesis and strongly support a modulatory role in tissue revascularization.

Therapeutic effect of fucoidan-stimulated endothelial colony-forming cells in peripheral ischemia

Summary.u2002 Background:u2002Fucoidan, an antithrombotic polysaccharide, can induce endothelial colony‐forming cells (ECFC) to adopt an angiogenic phenotype in vitro. Objectives: We evaluated the effect of fucoidan on vasculogenesis induced by ECFC in vivo. Methods:u2002We used a murine hindlimb ischemia model to probe the synergic role of fucoidan‐treatment and ECFC infusion during tissue repair. Results:u2002We found that exposure of ECFC to fucoidan prior to their intravenous injection improved residual muscle blood flow and increased collateral vessel formation. Necrosis of ischemic tissue was significantly reduced on day 14, to 12.1% of the gastronecmius cross‐sectional surface area compared with 40.1% in animals injected with untreated‐ECFC. ECFC stimulation with fucoidan caused a rapid increase in cell adhesion to activated endothelium in flow conditions, and enhanced transendothelial extravasation. Fucoidan‐stimulated ECFC were resistant to shear stresses of up to 21u2003dynu2003cm−2. Direct binding assays showed strong interaction of fucoidan with displaceable binding sites on the ECFC membrane. Bolus intramuscular administration of fucoidan 1u2003day after surgery reduces rhabdomyolysis. Mice injected with fucoidan (15u2003mgu2003kg−1) had significantly lower mean serum creatine phosphokinase (CPK) activity than control animals. This CPK reduction was correlated with muscle preservation against necrosis (Pu2003<u20030.001). Conclusions:u2002Fucoidan greatly increases ECFC‐mediated angiogenesis in vivo. Its angiogenic effect would be due in part to its transportation to the ischemic site and its release after displacement by proteoglycans present in the extracellular matrix. The use of ECFC and fucoidan together, will be an efficient angiogenesis strategy to provide therapeutic neovascularization.

Impaired platelet activation and cAMP homeostasis in MRP4-deficient mice

Molecules that reduce the level of cyclic adenosine 5-monophosphate (cAMP) in the platelet cytosol, such as adenosine 5-diphosphate (ADP) secreted from dense granules, trigger platelet activation. Therefore, any change in the distribution and/or availability of cyclic nucleotides or ADP may interfere with platelet reactivity. In this study, we evaluated the role of multidrug resistance protein 4 (MRP4, or ABCC4), a nucleotide transporter, in platelet functions in vivo and in vitro by investigating MRP4-deficient mice. MRP4 deletion resulted in a slight increase in platelet count but had no impact on platelet ultrastructure. In MRP4-deficient mice, the arterial occlusion was delayed and the tail bleeding time was prolonged. In a model of platelet depletion and transfusion mimicking a platelet-specific knockout, mice injected with MRP4(-/-) platelets also showed a significant increase in blood loss compared with mice injected with wild-type platelets. Defective thrombus formation and platelet activation were confirmed in vitro by studying platelet adhesion to collagen in flow conditions, integrin αIIbβ3 activation, washed platelet secretion, and aggregation induced by low concentrations of proteinase-activated receptor 4-activating peptide, U46619, or ADP. We found no role of MRP4 in ADP dense-granule storage, but MRP4 redistributed cAMP from the cytosol to dense granules, as confirmed by increased vasodilator-stimulated phosphoprotein phosphorylation in MRP4-deficient platelets. These data suggest that MRP4 promotes platelet aggregation by modulating the cAMP-protein kinase A signaling pathway, suggesting that MRP4 might serve as a target for novel antiplatelet agents.

Efficacy of prothrombin complex concentrate to reverse the anticoagulant effect of the pentasaccharide fondaparinux in a rabbit model

As a potent anticoagulant agent, fondaparinux exposes a risk of bleeding. An effective way to reverse its effects is needed. It was the objective to study efficacy and safety of prothrombin complex concentrate (PCC) to reverse the anticoagulant effect of fondaparinux in a rabbit model of bleeding and thrombosis. In anaesthetised and ventilated rabbits, the Folts model was applied: a stenosis (75%) and an injury were carried out on the carotid artery, inducing thrombosis. Blood flow decreased as thrombus size increased until the pressure gradient was such that the thrombus was released and local blood flow was suddenly restored. This is known as a cyclic flow reduction (CFR). After the first CFR, rabbits were randomised into three groups: control (saline and saline after 1 minute), fondaparinux (fondaparinux [3 mg.kg-1] and saline), PCC (fondaparinux and PCC [40 UI.kg-1]). Then CFRs were recorded over 20 minutes. The following were measured: ear immersion bleeding time (BT), haemoglobin blood level (Hb1) and thrombelastometric parameters (ROTEM®). Finally, a hepatosplenic section was performed; 15 minutes later, the amount of blood loss was recorded as primary endpoint and Hb2 was measured. Blood loss was increased with fondaparinux and normalised with PCC. Regarding ROTEM® INTEM, fondaparinux increased clotting time and clotting formation time. PCC normalised these parameters. EXTEM and FIBTEM tests were not modified. Regarding safety, PCC did not increase CFRs. PCC reduced bleeding without increasing thrombosis and was effective to reverse the haemorrhagic effect of fondaparinux in this rabbit model.

Tie2-dependent knockout of α6 integrin subunit in mice reduces post-ischaemic angiogenesis

AIMSnIntegrins α6β1 and α6β4 are receptors for laminins, the main components of the basement membrane underlying the endothelial cells. In vitro, α6 integrin subunit (α6) expression at the surface of endothelial cells and their progenitors (EPCs) is up-regulated by pro-angiogenic growth factors and is crucial for adhesion, migration, and pseudotube formation. We investigated the role for α6 in post-ischaemic vascular repair in vivo.nnnMETHODS AND RESULTSnWe used the cre-lox system to generate a mouse line with specific α6 gene deletion in Tie2-lineage cells. In a model of hind-limb ischaemia, Tie2-dependent α6 deletion reduced neovessel formation and reperfusion of the ischaemic limb. Concerning the role for α6 in post-ischaemic vasculogenesis, we showed previously that α6 was required for EPC recruitment at the site of ischaemia. Here, we found that α6 deletion also reduced EPC mobilization from the bone marrow after ischaemia. Examination of the ischaemic muscles showed that Tie2-dependent α6 deletion decreased the recruitment of pro-angiogenic Tie2-expressing macrophages. In the Matrigel plug assay, fibroblast growth factor-2-induced vascularization was diminished in mice lacking endothelial α6. To specifically investigate the role for α6 in angiogenesis, aortic rings were embedded in Matrigel or collagen and cultured ex vivo. In Matrigel, neovessel outgrowth from rings lacking α6 was strongly diminished, whereas no genotype-dependent difference occurred for rings in collagen.nnnCONCLUSIONnα6 plays a major role in both post-ischaemic angiogenesis and vasculogenesis.