MVL-PLA2, a Snake Venom Phospholipase A2, Inhibits Angiogenesis through an Increase in Microtubule Dynamics and Disorganization of Focal Adhesions

Integrins are essential protagonists of the complex multi-step process of angiogenesis that has now become a major target for the development of anticancer therapies. We recently reported and characterized that MVL-PLA2, a novel phospholipase A2 from Macrovipera lebetina venom, exhibited anti-integrin activity. In this study, we show that MVL-PLA2 also displays potent anti-angiogenic properties. This phospholipase A2 inhibited adhesion and migration of human microvascular-endothelial cells (HMEC-1) in a dose-dependent manner without being cytotoxic. Using Matrigel™ and chick chorioallantoic membrane assays, we demonstrated that MVL-PLA2, as well as its catalytically inactivated form, significantly inhibited angiogenesis both in vitro and in vivo. We have also found that the actin cytoskeleton and the distribution of αvβ3 integrin, a critical regulator of angiogenesis and a major component of focal adhesions, were disturbed after MVL-PLA2 treatment. In order to further investigate the mechanism of action of this protein on endothelial cells, we analyzed the dynamic instability behavior of microtubules in living endothelial cells. Interestingly, we showed that MVL-PLA2 significantly increased microtubule dynamicity in HMEC-1 cells by 40%. We propose that the enhancement of microtubule dynamics may explain the alterations in the formation of focal adhesions, leading to inhibition of cell adhesion and migration

Snake venomics: Comparative analysis of the venom proteomes of the Tunisian snakes Cerastes cerastes, Cerastes vipera and Macrovipera lebetina

International audienceThe protein composition of the crude venoms of the three most important vipers of Tunisia was analyzed by RP-HPLC, N-terminal sequence analysis, MALDI-TOF mass determination, and in-gel tryptic digestion followed by PMF and CID-MS/MS of selected peptide ions in a quadrupole-linear IT instrument. Our results show that the venom proteomes of Cerastes cerastes, Cerastes vipera, and Macrovipera lebetina are composed of proteins belonging to a few protein families. However, each venom showed distinct degree of protein composition complexity. The three venoms shared a number of protein classes though the relative occurrence of these toxins was different in each snake species. On the other hand, the venoms of the Cerastes species and Macrovipera lebetina each contained unique components. The comparative proteomic analysis of Tunisian snake venoms provides a comprehensible catalogue of secreted proteins, which may contribute to a deeper understanding of the biological effects of the venoms, and may also serve as a starting point for studying structure-functio

Antitumoral Potential of Tunisian Snake Venoms Secreted Phospholipases A2

Phospholipases type A2 (PLA2s) are the most abundant proteins found in Viperidae snake venom. They are quite fascinating from both a biological and structural point of view. Despite similarity in their structures and common catalytic properties, they exhibit a wide spectrum of pharmacological activities. Besides being hydrolases, secreted phospholipases A2 (sPLA2) are an important group of toxins, whose action at the molecular level is still a matter of debate. These proteins can display toxic effects by different mechanisms. In addition to neurotoxicity, myotoxicity, hemolytic activity, antibacterial, anticoagulant, and antiplatelet effects, some venom PLA2s show antitumor and antiangiogenic activities by mechanisms independent of their enzymatic activity. This paper aims to discuss original finding against anti-tumor and anti-angiogenic activities of sPLA2 isolated from Tunisian vipers: Cerastes cerastes and Macrovipera lebetina, representing new tools to target specific integrins, mainly, and integrins

A Novel Single-Domain Antibody Against von Willebrand Factor A1 Domain Resolves Leukocyte Recruitment and Vascular Leakage During Inflammation—Brief Report

International audienceObjective—von Willebrand factor (VWF) is crucial to hemostasis, but also plays a role in inflammatory processes. Unfortunately, no proper monoclonal antibodies to study VWF function in mice are currently available. We therefore aimed to generate single-domain antibodies (sdAbs) recognizing murine VWF and blocking its function in vivo.Approach and Results—Llama-derived sdAbs recognizing both human and murine VWF were isolated via phage display technology. One of them (designated KB-VWF-006) recognized the VWF A1 domain with picomolar affinity. This sdAb avidity was strongly enhanced via dimerization using a triple Ala linker (KB-VWF-006bi). When administered in vivo to wild-type mice, KB-VWF-006bi dose dependently induced bleeding in a tail clip model. In 2 distinct models of inflammation, KB-VWF-006bi efficiently interfered with leukocyte recruitment and vascular leakage.Conclusions—KB-VWF-006bi is an sdAb recognizing the A1 domain of human VWF and murine VWF that interferes with VWF–platelet interactions in vivo. By using this sdAb, we now also show that the A1 domain is pertinent to the participation of VWF in the inflammatory response

Les sélectines : acteurs de l'adhérence cellulaire et potentiel cible thérapeutique

International audienceSelectins belong to the family of adhesion molecules that recognize sugars as ligands through their Carbohydrate Recognition Domain (CRD). There are three types of selectin: the L-selectin (CD62L), which is constitutively expressed by most leukocyte populations, the P-selectin (CD62P) is found on activated platelets and endothelial cells, and the E-selectin (CD62E) expressed by activated endothelial cells. These three molecules exhibit high homology in their structures. Selectin-ligand interactions are among the most studied protein-glycan interactions in biology. The selectins and theirs ligands are involved in regulating inflammatory and immunological events that occur at the interface of the bloodstream and vessel walls. Their molecular partners are surface glycoconjugates harboring groups of the sialyl-Lewis antigens. This review presents an inventory of our current knowledge on the structures and functions of selectins and their ligands. We also provide an update on their involvement in pathophysiological processes, especially during inflammation and tumor development.Les sélectines font partie de la famille des molécules d’adhérence et ont la particularité de posséder un domaine de liaison aux sucres, le CRD (Carbohydrate Recognition Domain). On distingue trois types de sélectine: la L-sélectine (CD62L) qui est exprimée constitutivement par la plupart des populations leucocytaires, la P-sélectine (CD62P) qui est retrouvée sur le s plaquettes et les cellules endothéliales et la E-sélectine (CD62E) exprimée par les cellules endothéliales activées. Ces trois molécules possèdent une forte homologie au niveau de leur séquence primaire et de leurs structures. Les interactions sélectine-ligand sont les plus étudiées parmi les interactions protéine-glycanne connues en biologie. Elles sont impliquées dans la régulation des évènements inflammatoires et immunologiques à l’interface paroi vasculaire/ circulation sanguine. Leurs partenaires moléculaires sont des glyco conjugués de surface exprimant des groupements de la famille des sialyl-Lewis. Cette revue présente l’état des lieux des connaissances sur la structure et l’expression des sélectines et de leurs ligands. Elle fait aussi le point sur leur implication en physiopathologie, principalement lors de l’inflammation et du développement tumora

PIVL, a snake venom Kunitz-type serine protease inhibitor, inhibits in vitro and in vivo angiogenesis.

International audience: Development and homeostasis of the vascular system requires integrin-promoting endothelial cell adhesion, migration and survival. Nowadays, integrins represent potential targets for pharmacological agents and open new avenues for the control of metastatic spread in the treatment of tumor malignancies. We have already reported that PIVL, a serine protease inhibitor isolated from Macrovipera lebetina venom, displays an anti-tumor effect through the interference with integrin receptor function. Here, we report that PIVL inhibits human vascular endothelial cell adhesion and migration onto fibrinogen and fibronectin in a dose-dependent manner without any cytotoxicity. Furthermore, we show that PIVL increases microtubule dynamic instability in HMEC-1 transfected with EGFP-tagged α-tubulin. Using Matrigel™ and chick chorioallantoic membrane assays, we demonstrate that PIVL exhibits a strong anti-angiogenic effect both in vitro and in vivo. Interestingly, results herein reveal that the potent anti-angiogenic properties of PIVL are mediated by its RGD-like motif ((41)RGN(43))

Expression of a functional recombinant C-type lectin-like protein lebecetin in the human embryonic kidney (HEK) cells.

International audienceLebecetin is an anticoagulant C-type lectin-like protein (CLPs) that was previously isolated from Macrovipera lebetinavenom and described to consist of two subunits (alpha and beta). It was reported to potently prevent platelet aggregation by binding to glycoprotein Ib (GPIb) and to exhibit a broad spectrum of inhibitory activities on various integrin-mediated functions of tumour cells, including adhesion, proliferation, and cell migration. The present study aimed to investigate the structure-function of lebecetin. Accordingly, the cDNA of each subunit was cloned and separately or jointly expressed in the human embryonic kidney cells (HEK)using two vectors with different selectable tags. The immunofluorescence analysis of transfected cells revealed significant expression levels and co-localization of the two lebecetin subunits. The recombinant proteins were efficiently secreted and purified using metal-chelating affinity chromatography. We found that the Lebecetin alpha and beta subunits were produced as a mixture of homodimers and heterodimers and that the heterodimerization represent a prerequisite for functioning

Complex formation with pentraxin-2 regulates factor X plasma levels and macrophage interactions

International audienceRecently, we have identified scavenger receptor class A member I (SR-AI) as a receptor for coagulation factor X (FX), mediating the formation of an FX reservoir at the macrophage surface. Here, we demonstrate that the FX/SR-AI-complex comprises a third protein, pentraxin-2 (PTX2). The presence of PTX2 is essential to prevent internalization of FX by SR-AI, and the presence of FX is needed to interfere with internalization of PTX2. Binding studies showed that FX, SR-AI, and PTX2 independently bind to each other (KD,app: 0.2-0.7 μM). Surprisingly, immunoprecipitation experiments revealed that FX and PTX2 circulate as a complex in plasma, and complex formation involves the FX activation peptide. No binding of PTX2 to other vitamin K–dependent proteins was observed. Short hairpin RNA–mediated inhibition of PTX2 levels in mice resulted not only in reduced levels of PTX2, but also in similarly reduced FX levels. Moreover, PTX2 and FX levels were correspondingly reduced in SR-AI–deficient mice. Analysis of 71 human plasma samples uncovered a strong correlation between FX and PTX2 plasma levels. Furthermore, plasma samples of patients with reduced FX levels (congenital/acquired FX deficiency or after anti–vitamin K treatment) were characterized by concomitantly decreased PTX2 levels. In conclusion, we identified PTX2 as a novel partner for FX, and both proteins cooperate to prevent their SR-AI–mediated uptake by macrophages. Interestingly, their respective plasma levels are interdependent. These findings seem of relevance in perspective of ongoing clinical trials, in which plasma depletion of PTX2 is used as a therapeutical approach in the management of systemic amyloidosis

Two purified and characterized phospholipases A2 from Cerastes cerastes venom, that inhibit cancerous cell adhesion and migration.

International audienceTwo non-toxic PLA2s were purified to homogeneity from Cerastes cerastes Tunisian snake venom. The purification process employed gel filtration on Sephadex G-75 followed by C18 reverse phase high-pressure liquid chromatography. These two acidic enzymes, namely CC-PLA2-1 and CC-PLA2-2, have a molecular weight of 13,737.52 and 13,705.63 Da, respectively. These two PLA2 are the first reported glycosylated phospholipases A2 purified from snake venom. The rates of glycosylation are 2.5% and 0.5% (w/w), respectively. Specific activities of 1800 U/mg and 2400 U/mg for CC-PLA2-1 and CC-PLA2-2, respectively, were measured at optimal conditions. CC-PLA2-1 and CC-PLA2-2 strongly inhibited coagulation. They also exhibited a marked dose-dependent inhibitory effect on platelet aggregation induced by ADP and arachidonic acid in platelet-rich plasma. Interestingly, CC-PLA2-1 and CC-PLA2-2 inhibited in a dose-dependent manner adhesion of IGR39 melanoma and HT1080 fibrosarcoma cells to fibrinogen and fibronectin. Furthermore, both CC-PLA2-1 and CC-PLA2-2 abolished HT1080 cell migration towards fibrinogen and fibronectin. This activity is reported for the first time for PLA2 enzymes