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Abdominal aortic aneurysms (AAA) are the third cause of cardiovascular mortality in industrialized countries. AAA consist in progressive dilatation of the aorta which can lead to spontaneous rupture. Aneurysm evolution depends on the balance between degradation and repair of elastin and collagens in the aortic extracellular matrix (ECM), due to inflammatory cells infiltration in parallel with loss of vascular smooth muscle cells (VSMC). The cross-linking enzymes, lysyl oxidase (LOX), lysyl oxidase-like protein 2 (LOXL2) and transglutaminase 2 (TG2) are involved in the maturation of ECM components and arterial remodeling, by covalently bonding elastin and collagens. These crosslinking enzymes are therefore good candidates for AAA stabilisation and prevention of rupture, through ECM remodeling. We show that TG2 and LOXL2 are expressed in endothelial cells and VSMC, in addition to LOX, as they were detected in the intima and the media of healthy mouse aortic wall by immunofluorescence. In addition, these enzymes are all present in the ECM Triton-resistant fraction. We used a murine CaCl2-induced aneurysm model, in which AAA progression is associated with a decrease in VSMC density, as demonstrated by smooth muscle alpha-actin immunostaining. Whereas LOX and TG2 expression was strongly decreased in parallel with the loss of VSMC, LOXL2 expression was less severely affected. The involvement of lysyl oxidases in AAA was investigated using the irreversible inhibitor, beta-aminopropionitrile (BAPN). After induction of AAA by CaCl2, BAPN-treated mice showed a greater increase of the internal aortic diameter in comparison with untreated controls, suggesting that activity from remaining lysyl oxidase partially prevents AAA progression. In parallel, fibrillar collagens content is determined by Sirius red staining and second harmonic generation imaging; and elastin fibers organisation by Orcein-staining. These ongoing experiments will characterize the involvement of crosslinking enzymes in ECM repair during the evolution of AAA. Altogether, this study will allow to determine whether LOXL2 and/or TG2 could be therapeutic targets for stabilizing AAA progression through ECM repair. [Copyright &y& Elsevier]

Hypoxia stimulates angiogenesis during development, as well as in the course of ischemic cardiovascular diseases and tumor growth. In a hypoxic environment, endothelial cells (EC) are key players of the angiogenic response. Their activation leads to remodeling of the extracellular matrix (ECM): a degradation step generates a provisional ECM supporting EC proliferation and migration; assembly of a new basal lamina leads to pericyte recruitment and neovessel maturation. In order to identify endothelial ECM proteins regulated by hypoxia, 2D gel electrophoresis was performed on ECM samples prepared from cultured EC of micro or large vessels (HDMEC or HUVEC respectively). Mass spectrometry identified lysyl oxidase-like protein 2 (LOXL2) as a highly hypoxia-induced protein. Lysyl oxidases are secreted enzymes involved in ECM maturation through covalent cross-linking of its major components, collagens and elastin. The induction of LOXL2 expression was detected both at the mRNA and protein levels. Using siRNA, we demonstrated that LOXL2 is responsible for 65 % of lysyl oxidase total activity in hypoxic EC. In addition, LOXL2 protein was colocalised with type IV collagen in endothelial ECM. We further investigated the expression of LOXL2 in vivo. The enzyme was expressed in rat EC from retina during postnatal vascular development, and from adult skeletal muscle. In a murine model of hindlimb ischemia, LOXL2 was upregulated at the protein and mRNA levels. In situ hybridization revealed its expression in both EC and macrophages. Involvement of LOXL2 at different steps of the angiogenic process was further studied in vitro. We demonstrated that LOXL2 increases EC migration on fibronectin, as well as migration and tube formation in fibrin 3D gels. In addition, LOXL2 knock-down inhibited type IV collagen deposition in the ECM, suggesting a major role for LOXL2 in the organisation of endothelial basal lamina. Finally, whereas the efficiency of endothelial ECM for recruiting VSMC was increased by hypoxia, this effect was reduced upon knocking down endothelial LOXL2 expression. Altogether, these data suggest that LOXL2 plays a major role in EC migration, vascular basal lamina deposition and neovessel stabilisation during hypoxia-induced angiogenesis. [Copyright &y& Elsevier]