Endothelial cell CD36 deficiency prevents normal angiogenesis and vascular repair

Endothelial cells (ECs) maintain vascular integrity and mediate vascular repair and angiogenesis, by which new blood vessels are formed from pre-existing blood vessels. Hyperglycemia has been shown to increase EC angiogenic potential. However, few studies have investigated effects of fatty acids (FAs) on EC angiogenesis. Cluster of differentiation 36 (CD36) is a FA transporter expressed by ECs, but its role in EC proliferation, migration, and angiogenesis is unknown. We sought to determine if circulating FAs regulate angiogenic function in a CD36-dependent manner. CD36-dependent effects of FAs on EC proliferation and migration of mouse heart ECs (MHECs) and lung ECs (MLECs) were studied. We used both silencing RNA and antisense oligonucleotides to reduce CD36 expression. Oleic acid (OA) did not affect EC proliferation, but significantly increased migration of ECs in wound healing experiments. CD36 knockdown prevented OA-induced increases in wound healing potential. In EC transwell migration experiments, OA increased recruitment and migration of ECs, an effect abolished by CD36 knockdown. Phospho-AMP-activated protein kinase (AMPK) increased in MHECs exposed to OA in a CD36-dependent manner. To test whethe

Rôle de l’axe CD40L/CD40 dans les cellules endothéliales progénitrices

Les cellules endothéliales progénitrices («Endothelial Progenitor Cells», EPCs) sont des précurseurs endothéliaux qui possèdent un potentiel considérable dans la réparation et la régénération vasculaire. Dans le contexte des maladies cardiovasculaires, la compréhension du rôle des EPCs dans la régulation de la thrombogenèse et la réparation endothéliale est pertinente et nécessaire pour comprendre leur potentiel thérapeutique. Nous avons rapporté que les EPCs interagissent avec les plaquettes via la P-sélectine et inhibent l’adhésion, l’activation et l’agrégation des plaquettes ainsi que la formation de thrombus. Plus récemment, nous avons démontré que les EPCs expriment le récepteur inflammatoire CD40 et il est bien connu que les plaquettes constituent la source principale de la forme soluble de son agoniste le CD40L («soluble CD40 Ligand», sCD40L). Ainsi, nous avons émis l’hypothèse principale que l’axe CD40L/CD40 dans les EPCs influence leurs fonctions anti-thrombotique et pro-angiogénique. Pour vérifier cette hypothèse, nous avons réussi à générer des «early» et «late» EPCs à partir de cellules mononucléaires du sang périphérique («Peripheral Blood Mononuclear Cells», PBMCs) en culture. Nous avons mis en évidence l’existence de l’axe CD40L/CD40 dans ces EPCs en démontrant l’expression des protéines adaptatrices, nommées les facteurs associés au récepteur du facteur de nécrose tumorale («TNF Receptor Associated Factors», TRAFs). Dans une première étude, nous avons investigué l’effet du sCD40L sur la fonction des «early» EPCs dans l’agrégation plaquettaire. En effet, nous avons démontré que le sCD40L renverse leur effet inhibiteur sur l’agrégation plaquettaire, et ce sans avoir un effet significatif sur la sécrétion de prostacycline (PGI2) et d’oxyde nitrique («Nitric Oxide», NO) par ces cellules. De plus, aucun effet du sCD40L n’a été noté sur l’apoptose et la viabilité de ces cellules. Par contre, nous avons noté une augmentation importante du stress oxydatif dans les «early» EPCs suite à leur stimulation avec le sCD40L. L’inhibition du stress oxydatif renverse l’effet du sCD40L sur les «early» EPCs dans l’agrégation plaquettaire. Ces résultats pourraient expliquer, en partie, la fonction réduite des EPCs chez les individus présentant des niveaux élevés de sCD40L en circulation. Dans une deuxième étude, nous avons étudié l’effet de sCD40L dans la fonction des «early» EPCs en relation avec l’angiogenèse. Nous avons identifié, dans un premier temps,les métalloprotéinases de la matrice («Matrix Metalloproteinases», MMPs) qui sont sécrétées par ces cellules. Nous avons trouvé que les «early» EPCs relâchent principalement la MMP-9 et que cette relâche est augmentée par le sCD40L. Le sCD40L induit aussi la phosphorylation de la p38 MAPK qui contribue à augmenter la sécrétion de MMP-9. Des études fonctionnelles ont démontré que le prétraitement des «early» EPCs au sCD40L potentialise la réparation endothéliale des HUVECs. En conclusion, l’ensemble de nos travaux, dans le cadre de ce projet de doctorat, nous a permis d’élucider les mécanismes responsables de l’action du sCD40L sur les effets inhibiteur et angiogénique des «early» EPCs dans l’agrégation plaquettaire et l’angiogenèse, respectivement. Ces résultats ajoutent de nouvelles connaissances sur le rôle des EPCs et pourront constituer la base pour des études futures permettant de corréler les niveaux élevés du sCD40L circulant et l’incidence des maladies cardiovasculaires, particulièrement l’athérothrombose.Endothelial progenitor cells (EPCs) are endothelial precursors which possess a considerable therapeutic potential in vascular repair and regeneration. In the context of cardiovascular diseases, the understanding of the role of EPCs in the regulation of thrombogenesis and endothelial repair is relevant and necessary to the understanding of their therapeutic potential. We have shown that EPCs interact with platelets via P-selectin and inhibit the adhesion, activation and aggregation of platelets as well as thrombus formation. Recently, we have shown that EPCs express the inflammatory receptor CD40 and it is well known that platelets are the main source of the soluble form of its agonist CD40L («soluble CD40 ligand», sCD40L). Hence, we have hypothesized that the CD40L/CD40 axis in EPCs influences the anti-thrombotic and pro-angiogenic functions of EPCs. To verify this hypothesis, we have successfully generated early and late EPCs from peripheral blood mononuclear cells in culture. We have demonstrated the existence of the CD40L/CD40 axis in EPCs by showing the expression of adaptor proteins, named tumor necrosis factor associated factors (TRAFs). In our first study, we investigated the effect of sCD40L on the function of early EPCs in platelet aggregation. Indeed, we have shown that sCD40L reverses their inhibitory effect on platelet aggregation without having an effect on prostacyclin (PGI2) and nitric oxide (NO) secretion by these cells. Moreover, no effect of sCD40L has been noted on the apoptosis and viability of these cells. However, we have shown a significant increase in oxidative stress in early EPCs following sCD40L stimulation. The inhibition of oxidative stress reverses the effect of sCD40L on early EPCs in platelet aggregation. These results could partially explain the decreased function of EPCs in individuals displaying higher levels of sCD40L in circulation. In our second study, we have studied the effect of sCD40L on the function of early EPCs in relation to angiogenesis. First, we have identified the matrix metalloproteinases (MMPs) which are secreted by these cells. We have found that early EPCs mainly release MMP-9 and that this release is increased by sCD40L. The sCD40L also induces the phosphorylation of p38 MAPK which contributes to increase the secretion of MMP-9. In functional studies, we have shown that pretreatment of early EPCs with sCD40L can potentialize HUVEC endothelial repair. In conclusion, our work in the context of this doctoral research project has allowed us to study the mechanisms involved in the role of sCD40L in the inhibitory and angiogenic function of early EPCs in platelet aggregation and angiogenesis, respectively. These results add new insights to the role of EPCs and could constitute the basis for future studies allowing for the correlation between high levels of sCD40L and the incidence of cardiovascular disease, particularly atherothrombosis

Soluble CD40 ligand stimulates the pro-angiogenic function of peripheral blood angiogenic outgrowth cells via increased release of matrix metalloproteinase-9.

The role of endothelial progenitor cells in vascular repair is related to their incorporation at sites of vascular lesions, differentiation into endothelial cells, and release of various angiogenic factors specifically by a subset of early outgrowth endothelial progenitor cells (EOCs). It has been shown that patients suffering from cardiovascular disease exhibit increased levels of circulating and soluble CD40 ligand (sCD40L), which may influence the function of EOCs. We have previously shown that the inflammatory receptor CD40 is expressed on EOCs and its ligation with sCD40L impairs the anti-platelet function of EOCs. In the present study, we aimed at investigating the effect of sCD40L on the function of EOCs in endothelial repair. Human peripheral blood mononuclear cell-derived EOCs express CD40 and its adaptor proteins, the tumor necrosis factor receptor-associated factors; TRAF1, TRAF2 and TRAF3. Stimulation of EOCs with sCD40L increased the expression of TRAF1, binding of TRAF2 to CD40 and phosphorylation of p38 mitogen activated protein kinase (MAPK). In an in vitro wound healing assay, stimulation of EOCs with sCD40L increased the release of matrix metalloproteinase 9 (MMP-9) in a concentration-dependent manner and significantly enhanced the angiogenic potential of cultured human umbilical vein endothelial cells (HUVECs). Inhibition of p38 MAPK reversed sCD40L-induced MMP-9 release by EOCs, whereas inhibition of MMP-9 reversed their pro-angiogenic effect on HUVECs. This study reveals the existence of a CD40L/CD40/TRAF axis in EOCs and shows that sCD40L increases the pro-angiogenic function of EOCs on cultured HUVECs by inducing a significant increase in MMP-9 release via, at least, the p38 MAPK signaling pathway

sCD40L increases MMP-9 release by EOCs.

<p>A) Representative gelatin zymography gel of MMP-2 and MMP-9 release by HUVECs and EOCs. Histogram represents the mean of data ± SEM of optical density. n ≥ 4, **P<0.01 <i>vs</i>. HUVECs. B) Representative gelatin zymography gel showing the release of MMP-9 by control EOCs and the effects of increasing concentration of sCD40L (0.1, 0.5 and 1.0 μg/mL) following 24 hours stimulation of EOCs. Histogram represents the mean of data ± SEM of optical density ratio. n ≥ 3,*P<0.05 <i>vs</i>. control. </p

Enhancement of MMP-9 release in sCD40L-stimulated EOCs is p38 MAPK-dependent.

<p>Representative gelatin zymography gel of supernatants of EOCs and sCD40L (1 μg/mL)-, p38 inhibitor SB203580 (10 μM)-, and double sCD40L- and SB203580-treated EOCs. Histogram represents the mean of data ± SEM of fold increase in optical density over control EOCs. n ≥ 3, *P<0.05 <i>vs</i>. other groups. </p

CD40 and TRAF expression and binding to CD40 in EOCs.

<p>A) Comparative CD40 and TRAF expression profiles in PBMCs and EOCs, as determined by Western Blots of cell lysates analyzed for CD40, TRAF1, 2, 3, 5 and 6 expressions; the loading control β-actin is representative of the proteins loaded for each TRAF (blots are representative of n ≥ 4). B) Representative blots showing the effect of sCD40L on TRAF association with CD40. EOCs were stimulated with sCD40L (1 μg/mL) for 15 or 30 minutes or left untreated (baseline) and CD40 was immunoprecipitated from lysates using an anti-CD40 monoclonal antibody. Immunoprecipitates were then assayed for TRAF1, 2 and 3 by SDS-PAGE. Histogram represents the mean of data ± SEM of fold increase in optical density over baseline. n ≥ 4, **P<0.01 <i>vs</i>. baseline. C) Representative blots showing the effect of sCD40L on TRAF expression in EOCs following 24 hour stimulation with sCD40L (1 μg/mL); the loading control β-actin is representative of the proteins loaded for each TRAF. Histogram represents the mean of data ± SEM of fold increase in optical density over control. n=3, *P<0.05. </p

An USH2A founder mutation is the major cause of Usher syndrome type 2 in Canadians of French origin and confirms common roots of Quebecois and Acadians

Congenital hearing loss affects approximately one child in 1000. About 10% of the deaf population have Usher syndrome (USH). In USH, hearing loss is complicated by retinal degeneration with onset in the first (USH1) or second (USH2) decade. In most populations, diagnostic testing is hampered by a multitude of mutations in nine genes. We have recently shown that in French Canadians from Quebec, USH1 largely results from a single USH1C founder mutation, c.216G>A (‘Acadian allele'). The genetic basis of USH2 in Canadians of French descent, however, has remained elusive. Here, we have investigated nine USH2 families from Quebec and New Brunswick (the former Acadia) by haplotype analyses of the USH2A locus and sequencing of the three known USH2 genes. Seven USH2A mutations were identified in eight patients. One of them, c.4338_4339delCT, accounts for 10 out of 18 disease alleles (55.6%). This mutation has previously been reported in an Acadian USH2 family, and it was found in homozygous state in the three Acadians of our sample. As in the case of c.216G>A (USH1C), a common haplotype is associated with c.4338_4339delCT. With a limited number of molecular tests, it will now be possible in these populations to estimate whether children with congenital hearing impairment of different degrees will develop retinal disease – with important clinical and therapeutic implications. USH2 is the second example that reveals a significant genetic overlap between Quebecois and Acadians: in contrast to current understanding, other genetic disorders present in both populations are likely based on common founder mutations as well