Etienne Larger

The Soluble VEGF Receptor sFlt1 Contributes to Endothelial Dysfunction in CKD

Endothelial dysfunction contributes to the increased cardiovascular risk that accompanies CKD. We hypothesized that the soluble VEGF receptor 1 (sFlt-1), a VEGF antagonist, plays a role in endothelial dysfunction and decreased angiogenesis in CKD. We enrolled 130 patients with CKD stages 3 to 5 and 56 age- and gender-matched control patients. Plasma sFlt-1 levels were higher in patients with CKD and, after multivariate regression analyses, exclusively associated with renal function and levels of vWF, a marker of endothelial dysfunction. Compared with serum from control patients, both recombinant sFlt-1 and serum from patients with CKD had antiangiogenic activity in the chick chorioallantoic membrane (CAM) assay, induced endothelial cell apoptosis in vitro, and decreased nitric oxide generation in two different endothelial cell lines. Pretreating the sera with an antibody against sFlt-1 abrogated all of these effects. Furthermore, we observed increased sFlt1 levels in 5/6-nephrectomized rats compared with sham-operated animals. Finally, using real-time PCR and ELISA, we identified monocytes as a possible source of increased sFlt-1 in patients with CKD. Our findings show that excess sFlt-1 associates with endothelial dysfunction in CKD and suggest that increased sFlt-1 may predict cardiovascular risk in CKD.

Elevated Soluble Flt1 Inhibits Endothelial Repair in PR3-ANCA–Associated Vasculitis

Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis exhibits endothelial damage, but the capacity for vessel repair in this disorder is not well understood. Here, we observed a marked increase in serum levels of soluble Flt1 (sFlt1), a potent inhibitor of vascular endothelial growth factor, in patients with active ANCA-associated vasculitis compared with patients during remission and other controls. Serum levels of sFlt1 correlated with C5a, an anaphylatoxin released after complement activation. Serum from patients with acute ANCA-associated vasculitis disrupted blood flow in the chicken chorioallantoic membrane assay, suggesting an antiangiogenic effect. Preincubation with excess human vascular endothelial growth factor prevented this effect. Anti-proteinase-3 (PR3) mAb and serum containing PR3-ANCA from patients with active vasculitis both induced a significant and sustained release of sFlt1 from monocytes, whereas anti-myeloperoxidase (MPO) mAb or polyclonal antibodies did not. However, the serum containing polyclonal PR3-ANCA did not induce release of sFlt1 from cultured human umbilical vein endothelial cells. In summary, these data suggest that anti-PR3 antibodies, and to a much lesser extent anti-MPO antibodies, increase sFlt1 during acute ANCA-associated vasculitis, leading to an antiangiogenic state that hinders endothelial repair.

Angiotensinogen Modulates Renal Vasculature Growth

We have shown previously that angiotensinogen, like other members of the serine protease inhibitor family, has antiangiogenic properties in vitro on cultured endothelial cells and in ovo in the chick chorioallantoic membrane assay. The aim of this study was to show the effects of angiotensinogen on vascular wall remodeling in vivo. We measured the vessel wall thickness (tunica media) stained with an antibody to α-actin. In the kidney, arterioles were 21.5% thinner in male transgenic mice overexpressing human angiotensinogen than in male control animals. In other vascular beds, the arterial wall thickness was not affected. By using in situ hybridization and Northern blot analysis, human angiotensinogen expression was detected at a high level in the male kidney and at a much lower level in other organs. There is a relationship between the effect of angiotensinogen on arterial wall thickness and the local expression level of angiotensinogen in this model of transgenic mice. Because human angiotensinogen is not cleaved to a significant extent by mouse renin, the reduction in kidney arterial wall thickness is because of angiotensinogen itself and not angiotensin II, and we show that the reduction was not because of hypoplasia or hypotropia. In contrast, a marked difference in the expression of platelet-derived growth factor receptor-β was observed in the kidney arterioles at day 5 when compared with controls. Altogether, these observations provide the first quantitative evidence that a high level of angiotensinogen expression can inhibit the growth of kidney artery walls in vivo.

Potential Role of IL-17-Producing iNKT Cells in Type 1 Diabetes

We explored in this study the status and potential role of IL-17-producing iNKT cells (iNKT17) in type 1 diabetes (T1D) by analyzing these cells in patients with T1D, and in NOD mice, a mouse model for T1D. Our analysis in mice showed an increase of iNKT17 cells in NOD vs control C57BL/6 mice, partly due to a better survival of these cells in the periphery. We also found a higher frequency of these cells in autoimmune-targeted organs with the occurrence of diabetes, suggesting their implication in the disease development. In humans, though absent in fresh PMBCs, iNKT17 cells are detected in vitro with a higher frequency in T1D patients compared to control subjects in the presence of the proinflammatory cytokine IL-1β, known to contribute to diabetes occurrence. These IL-1β-stimulated iNKT cells from T1D patients keep their potential to produce IFN-γ, a cytokine that drives islet β-cell destruction, but not IL-4, with a reverse picture observed in healthy volunteers. On the whole, our results argue in favour of a potential role of IL-17-producing iNKT cells in T1D and suggest that inflammation in T1D patients could induce a Th1/Th17 cytokine secretion profile in iNKT cells promoting disease development.

O57 Rôle de l’angiopoïétine 2 dans les altérations de l’angiogenèse en hyperglycémie

Introduction Au cours du diabete de type 2, nous suggerons qu’une alteration de la vascularisation pancreatique puisse participer au defaut d’augmentation compensatoire de la masse des cellules b. Nous avons precedemment mis en evidence que l’hyperglycemie provoque des anomalies de la vascularisation pancreatique, associees a une surexpression de l’angiopoietine 2 (Ang2). Pour determiner le role de cette augmentation d’Ang2, nous avons bloque son action en situation ou non d’hyperglycemie et etudie la vascularisation pancreatique. Materiels et Methodes Nous travaillons dans un modele de greffe interspecifique : du pancreas d’embryon de poulet E14 est greffe sous la capsule renale de souris SCID normoglycemiques (NG) ou de souris rendues hyperglycemiques par injection de streptozotocine (HG), puis est recupere apres 2 semaines. L’inhibition de l’Ang2 a ete obtenue par injection de L1-10 (2x/sem, s.c. ; proteine de fusion, don de AMGEN). Resultats En NG, l’expression d’Ang2 est quasi inexistante, bloquer son action n’altere pas l’architecture pancreatique, ni insulaire ni vasculaire. En HG, l’expression d’Ang2 est fortement augmentee (x2,5 p Conclusion Ainsi, nous mettons en evidence un role majeur de l’expression de l’Ang2 dans les alterations de la vascularisation du pancreas diabetique dans ce modele. En bloquant l’action de l’Ang2, nous avons corrige la desorganisation vasculaire pancreatique induite par l’hyperglycemie et augmente la densite de cellules b.

O7 Interactions cellules β-cellules endothéliales. Effet de l’hyperglycémie

Introduction Nous suggerons quune alteration du dialogue entre cellules endocrines et endotheliales insulaires participe a la perte de masse et de fonction des cellules β dans le DT2. Pour analyser ce dialogue, nous avons developpe un modele de differenciation pancreatique interspecifique dans lequel le pancreas dun embryon de poulet se developpe sous la capsule renale dune souris. Nous avons etudie le role de 2 facteurs angiogeniques, le VEGF et langiopoietine 2 (Ang2) en situation ou non dhyperglycemie. Materiels et methodes Un pancreas dembryon de poulet E14 est greffe sous la capsule renale de souris SCID normoglycemiques (NG) ou hyperglycemiques (HG, STZ) et est recupere apres 2 semaines. La surexpression de VEGF est obtenue par infection in vitro , avant greffe, du pancreas par un virus RCAS exprimant le VEGF, infection specifique des cellules aviaires. Resultats En NG, le greffon se developpe, taille multipliee par 4, (n=4, p Conclusion Ce modele de greffe interspecifique permet detudier leffet de la modulation de facteurs de croissance vasculaire sur la vascularisation et la masse endocrine. Lhyperglycemie provoque des anomalies de vascularisation associees a la surexpression dAng2. La surexpression de VEGF augmente la vascularisation et la croissance du pancreas.

O76 Étude des interactions entre cellules endothéliales et cellules B pancréatiques

Introduction Les ilots de Langerhans font partie des tissus les plus richement vascularises de l’organisme. Plusieurs modeles animaux decrivent une alteration de la vascularisation des ilots de Langerhans au cours du developpement du diabete de type 2. Pour mieux analyser la relation qui lie la perte de secretion de l’insuline et la rarefaction vasculaire au cours du diabete de type 2, nous avons etabli un modele de differentiation du pancreas ou le pancreas se developpe dans un animal hote d’une espece differente, nous permettant ainsi d’analyser separement ce qui vient du pancreas de ce qui vient du systeme vasculaire de l’hote. Materiels et methodes A 13,5 jours de vie embryonnaire (E), l’epithelium pancreatique Wistar est preleve et greffe dans le coelome d’un embryon de poulet E3. Dix jours apres greffe, le greffon est preleve puis fixe dans du paraformaldehyde 4 %. Resultats A E13,5, l’epithelium pancreatique natif de l’embryon de rat contient de rares cellules a insuline et a glucagon. Aucune cellule a amylase n’est observee. Apres 10 jours de greffe chez le poulet, le tissu pancreatique endocrine et exocrine s’est differencie. On observe de nombreuses cellules a insuline, regroupees en amas, avec a proximite de ces amas des cellules a glucagon. Le reste du tissu est majoritairement represente par des cellules a amylase. Le tissu pancreatique apparait richement vascularise et peuple de globules rouges nuclees, nucleation caracteristique du poulet, ce qui montre que le greffon et l’hote ont etabli des connexions vasculaires fonctionnelles. Le reseau microvasculaire du greffon est par contre constitue de cellules endotheliales de rat. Conclusion Dans ce modele de developpement ex vivo du bourgeon pancreatique de rat, la differenciation des cellules endocrines et exocrines est tres proche de celle observee in vivo . L’apparition d’une vascularisation de rat en l’absence de cellules endotheliales au moment de la greffe signe une vasculogenese pancreatique. En modulant la reponse angiogenique dans ce modele nous disposons d’un outil pour etudier les interactions entre cellules endotheliales et cellules endocrines en pathologie diabetique.