N. De Freitas Caires

Endocan (endothelial cell-specific molecule-1) as a pertinent biomarker of endothelial dysfunction in sepsis

One of the main players in the severity of sepsis is the endothelium integrity. Endocan, also called endothelial cell-specific molecule-1 (ESM-1), was shown to be preferentially expressed in lung vasculature. Structurally, endocan/ESM-1 is a 50 kDa proteoglycan that can interact with ICAM-1 and LFA-1 integrins and consequently prevents inflammatory events. In an experimental rat endotoxemic shock model, we previously showed that a decrease in the leukocyte-endothelial cell contacts (induced by drugs) is clearly linked to an increase of blood endocan levels. Blood levels of endocan/ESM-1 were also shown to be associated with the severity and evolution of septic states in preliminary studies.

Identification of cathepsin G in the generation of elastase-resistant fragment of vascular endocan: involvement in the regulation of LFA-1-dependent cascade

The migration of polymorphonuclear neutrophils (PMN) into inflamed tissue requires fine interactions with the endothelial cell surface. The PMN serine proteases cathepsin G (CG), neutrophil elastase (NE) and proteinase 3 (PR3) were originally thought to play a role by the cleavage of endothelial cell proteins that control the PMN firm adhesion and the transendothelial cell migration. However, how these proteases participate in leukocyte adhesion and transmigration remains controversial. Vascular endocan, also called esm1, is a restricted endothelial cell-secreted proteoglycan constituted by a protein core of 20 kDa and by a unique glycosaminoglycan chain of dermatan sulphate (DS). Endocan is preferentially expressed in lung and kidney tissues. Endocan binds to its leukocytic receptor, the LFA-1 integrin, with an affinity of 18 nM, and inhibits the LFA-1-ICAM-1 interactions. Its expression is upregulated by the proinflammatory mediators TNF, IL-1, and lipopolysaccharide (LPS). In human sepsis, the serum endocan increases from fivefold to 30-fold the normal value and correlates with bad prognosis. Here, we examined the role of PMN-derived proteases in the degradation of endocan.

Differential kinetics of endothelial cell activation biomarkers E-selectin and endocan during nonlethal endotoxemia in 129Sv mice: a role for PMN-derived serine proteases in the transient decrease of circulating endocan levels

Severe septic syndrome remains one of the most frequent causes of death in ICUs. One of the main players in this pathology is the endothelium integrity. Our laboratory has demonstrated in preliminary clinical studies among the various biomarkers of endothelial dysfunction that blood levels of endocan (ESM-1), a pulmonary vascular endothelial cell-specific molecule participating in the control of endothelial-leukocyte interactions, are associated with the severity and evolution of septic states. On the other hand, we showed in vivo that antiprotease therapy is associated with a decrease of the leukocyte rolling and firm leukocyte adhesion to endothelium in sepsis. This decrease in the leukocyte-endothelial cell contacts was associated with an increase of blood endocan levels, suggesting a linkage between leukocyte proteases, endocan, and inflammation during sepsis.

012 Étude du catabolisme d’endocan : rôle des protéases du polynucléaire neutrophile

Introduction Endocan est un proteoglycane secrete specifiquement par la cellule endotheliale pulmonaire. Il est detecte au niveau serique chez des individus sains et retrouve surexprime chez des patients atteints de sepsis ou de cancer bronchique. En plus d’etre correlee au mauvais pronostic de ces deux maladies, la surexpression d’endocan est impliquee dans leur physiopathologie. Ainsi, pour pouvoir explorer endocan comme cible therapeutique potentielle, il apparait interessant de controler son expression, notamment en jouant sur son catabolisme. Cependant, le mecanisme de degradation d’endocan est encore a ce jour totalement inconnu. Endocan etant retrouve pour l’essentiel dans le sang, interagissant avec les leucocytes, nous nous sommes orientes sur l’etude de la degradation d’endocan par les leucocytes et plus precisement par les polynucleaires neutrophiles (PNNs). Methodes Des PNNs sont isoles a partir de poches de sang et resuspendus dans un milieu HBSS a 10 6 cellules/ml pour etre stimules par 10 nM de Phorbol Myristate Acetate, pendant 3 heures a 37°C. Le surnageant de PNNs, contenant les proteines secretees, est ensuite recueilli et teste pour son activite de degradation vis-a-vis d’endocan en le mettant en contact avec de l’endocan recombinant pendant 2 a 72 heures a 37 °C. La degradation d’endocan est ensuite evaluee par dosage ELISA et visualisee par Western blot. L’activite proteasique du surnageant de PNNs sur endocan est determinee par utilisation d’un cocktail inhibiteur de proteases. Resultats Les resultats montrent que les PNNs secretent des proteases clivant endocan en differents sites. Si l’extremite N-termi-nale d’endocan n’est pas degradee, les proteases coupent le corps proteique entre les acides amines 107 et 136 pour former des fragments de 15 et 14 kDa, et entre les acides amines 81 et 106 pour donner un peptide de 10 kDa. Un point important concerne le fragment de 14 kDa, qui constitue le metabolite majoritaire. Les donnees cinetiques montrent qu’il apparait tres precocement et persiste au moins pendant 3 jours en presence de proteases neutrophiles. Conclusion On peut penser qu’au cours d’etats infectieux severes, les PNNs actives sont capables de degrader plus rapidement endocan et modifient ainsi les activites biologiques de cette molecule.