0052: Role of kinins in diabetic wound healing

The diabetic foot is associated with pain, decrease in patient's quality of life, considerable costs, and amputation. In this study, we determined the role of KKS, via activation of bradykinin receptors (B1R or B2R), in a mouse model of diabetic wound healing. Diabetic or nondiabetic mice are wounded with an 8-mm punch biopsy and then are treated or not with specific B1R or B2R agonists (720nmol/kg.d-1) and/or B2R antagonist (Icatibant, 500μg/kg.dg/d-1). The wound-healing surface was daily followed up. At 11 days, the scar were analysed by histology (Masson's trichrome staining) and B1R and B2R expression were assessed (RT-qPCR). Effects of the agonists on cells (fibroblasts and keratinocytes) migration and proliferation were also analysed. In diabetic condition, mRNA of B1R and B2R was increased in skin (p<0.01). B1R activation had no effect on wound closure in our model. In contrast, B2R activation dramatically delayed wound healing in diabetic (p<0.001) or nondiabetic (p<0.01) mice. Histological analysis of scar showed significant skin disorganization and epidermis thickening with B2R agonist (p<0.05). In vitro, B2R agonist induced an increase of keratinocyte proliferation (+46% after 48h, p<0.01) and a stimulation of keratinocyte migration (+30% after 24h, p<0.05). These effects was associated with ERK phosphorylation which occurs downstream of EGFR activation (p<0.05). B2R agonist had no effect on fibroblast migration but decreased fibroblast proliferation (–33% after 48h, p<0.05). Co-treatment with Icatibant abrogated in vivo and in vitro effects observed with B2R agonist. Moreover, Icatibant alone hastened wound healing and decrease the epidermis thickening induced by diabetes. In conclusion, KKS, through the B2R but not the B1R, plays a critical role in proliferation and remodelling phases of skin wound healing in mice. While more studies are needed, Icatibant could be used to correct the diabetic wound healing defect

Rôle de la réaction inflammatoire dans le processus de néovascularisation

Dans certaines conditions physiologiques ou pathologiques, le développement de nouveaux vaisseaux en réponse à l'ischémie est diminué. Ainsi, la compréhension des mécanismes qui régulent la néovascularisation post-ischémique est un enjeu majeur pour le développement de nouvelles stratégies thérapeutiques. Nous nous sommes intéressés dans ce travail au rôle de la réaction inflammatoire dans le processus de néovascularisation. Nous avons mis en évidence le rôle des chimiokines dans le recrutement des cellules inflammatoires et des cellules progénitrices au niveau du territoire ischémique. Le récepteur aux chimiokines CXCR3 est impliqué dans le recrutement des leucocytes lors d'une ischémie des membres inférieurs. Les leucocytes s'infiltrent alors dans le tissu et participent à la néovascularisation en sécrétant des facteurs de croissance et en dégradant la matrice extracellulaire. Les chimiokines sont aussi impliquées dans le recrutement des cellules progénitrices issues de la moelle (BMCs) au niveau du territoire ischémique. Lors d'une ischémie, la chimiokine SDF-1 permet l'adhésion des BMCs au niveau de l'endothélium activé induisant une sécrétion de NO indispensable à la néovascularisation. Nous nous sommes également intéressés à l'utilisation d'un modulateurs de la réaction inflammatoire comme stratégie thérapeutique. L'emploi d'un peptide appelé AcSDKP a pour effet d'augmenter la réaction inflammatoire et la néovascularisation post-ischémique chez des souris. Ces résultats présentent donc la réaction inflammatoire et ses acteurs comme une cible thérapeutique potentielle qui pourrait être modulée pour le développement de traitements des maladies occlusives vasculaires.PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Re-epithelialization of pathological cutaneous wounds is improved by local mineralocorticoid receptor antagonism

International audienceImpaired cutaneous wound healing is a social burden. It occurs as a consequence of glucocorticoid treatment and in several pathologies. Glucocorticoids (GC) bind not only to the glucocorticoid receptor (GR), but also to the mineralocorticoid receptor (MR), both expressed by keratinocytes. In addition to its beneficial effects through the GR, GC exposure may lead to inappropriate MR occupancy. We hypothesized that dermatological use of MR antagonists (MRA) may be beneficial by overcoming the negative impact of GC treatment on pathological wounds. The potent GC clobetasol, applied as an ointment to mouse skin, or added to cultured human skin explants, induced delayed wound closure and outgrowth of epidermis with reduced proliferation of keratinocytes. Delayed wound re-epithelialization was rescued by local MRA application. Normal skin was unaffected by MRA. The benefit of MR blockade is explained by the increased expression of MR in clobetasol-treated mouse skin. Blockade of the epithelial sodium channel by phenamil also rescued cultured human skin explants from GC- impaired growth of the epidermis. MRA application over post-biopsy wounds of clobetasol-treated skin zones of healthy volunteers (from the SPIREPI clinical trial) also accelerated wound closure. In conclusion, we propose repositioning MRA for cutaneous application to improve delayed wound closure occurring in pathology

Vascular dysfunctions in the isolated aorta of double-transgenic hypertensive mice developing aortic aneurysm.

Angiotensin-II and oxidative stress are involved in the genesis of aortic aneurysms, a phenomenon exacerbated by endothelial nitric oxide synthase (eNOS) deletion or uncoupling. The purpose of this work was to study the endothelial function in wild-type C57BL/6 (BL) and transgenic mice expressing the h-angiotensinogen and h-renin genes (AR) subjected to either a control, or a high-salt diet plus a treatment with a NO-synthase inhibitor, N-ω-nitro-L-arginine-methyl-ester (L-NAME; BLSL and ARSL). BLSL showed a moderate increase in blood pressure, while ARSL became severely hypertensive. Seventy-five percent of ARSL developed aortic aneurysms, characterized by major histo-morphological changes and associated with an increase in NADP(H) oxidase-2 (NOX2) expression. Contractile responses (KCl, norepinephrine, U-46619) were similar in the four groups of mice, and relaxations were not affected in BLSL and AR. However, in ARSL, endothelium-dependent relaxations (acetylcholine, UK-14304) were significantly reduced, and this dysfunction was similar in aortae without or with aneurysms. The endothelial impairment was unaffected by catalase, superoxide-dismutase mimetic, radical scavengers, cyclooxygenase inhibition, or TP-receptor blockade and could not be attributed to sGC oxidation. Thus, ARSL is a severe hypertension model developing aortic aneurysm. A vascular dysfunction, involving both endothelial (reduced role of NO) and smooth muscle cells, precedes aneurysms formation and, paradoxically, does not appear to involve oxidative stress

The platelet receptor for type III collagen (TIIICBP) is present in platelet membrane lipid microdomains (rafts)

International audiencePlatelet interactions with collagen are orchestrated by the presence or the migration of platelet receptor(s) for collagen into lipid rafts, which are specialized lipid microdomains from the platelet plasma membrane enriched in signalling proteins. Electron microscopy shows that in resting platelets, TIIICBP, a receptor specific for type III collagen, is present on the platelet membrane and associated with the open cana-licular system, and redistributes to the platelet membrane upon platelet activation. After platelet lysis by 1% Triton X-100 and the separation of lipid rafts on a dis-continuous sucrose gradient, TIIICBP is recovered in lipid raft-containing fractions and Triton X-100 insoluble fractions enriched in cytoskeleton proteins. Platelet aggregation, induced by type III collagen, was inhibited after disruption of the lipid rafts by cholesterol depletion , whereas platelet adhesion under static conditions did not require lipid raft integrity. These results indicate that TIIICBP, a platelet receptor involved in platelet interaction with type III collagen, is localized within platelet lipid rafts where it could interact with other platelet receptors for collagen (GP VI and a2b1 integrin) for efficient platelet activation

Regulatory T cells modulate postischemic neovascularization

CD4+ and CD8+ T lymphocytes are key regulators of postischemic neovascularization. T-cell activation is promoted by 2 major costimulatory signalings, the B7/CD28 and CD40-CD40 ligand pathways. Interestingly, CD28 interactions with the structurally related ligands B7-1 and B7-2 are also required for the generation and homeostasis of CD4+CD25+ regulatory T cells (Treg cells), which play a critical role in the suppression of immune responses and the control of T-cell homeostasis. We hypothesized that Treg cell activation may modulate the immunoinflammatory response to ischemic injury, leading to alteration of postischemic vessel growth. Ischemia was induced by right femoral artery ligation in CD28-, B7-1/2-, or CD40-deficient mice (n=10 per group). CD40 deficiency led to a significant reduction in the postischemic inflammatory response and vessel growth. In contrast, at day 21 after ischemia, angiographic score, foot perfusion, and capillary density were increased by 2.0-, 1.2-, and 1.8-fold, respectively, in CD28-deficient mice, which showed a profound reduction in the number of Treg cells compared with controls. Similarly, disruption of B7-1/2 signaling or anti-CD25 treatment and subsequent Treg deletion significantly enhanced postischemic neovascularization. These effects were associated with enhanced accumulation of CD3-positive T cells and Mac-3-positive macrophages in the ischemic leg. Conversely, treatment of CD28(-/-) mice with the nonmitogenic anti-CD3 monoclonal antibody enhanced the number of endogenous Treg cells and led to a significant reduction of the postischemic inflammatory response and neovascularization. Finally, coadministration of Treg cells and CD28(-/-) splenocytes in Rag1(-/-) mice with hindlimb ischemia abrogated the CD28(-/-) splenocyte-induced activation of the inflammatory response and neovascularization. Treg cell response modulates postischemic neovascularizatio