Elevated expression levels of lysyl oxidases protect against aortic aneurysm progression in Marfan syndrome.

Patients with Marfan syndrome (MFS) are at high risk of life-threatening aortic dissections. The condition is caused by mutations in the gene encoding fibrillin-1, an essential component in the formation of elastic fibers. While experimental findings in animal models of the disease have shown the involvement of transforming growth factor-ß (TGF-ß)- and angiotensin II-dependent pathways, alterations in the vascular extracellular matrix (ECM) may also play a role in the onset and progression of the aortic disease. Lysyl oxidases (LOX) are extracellular enzymes, which initiates the formation of covalent cross-linking of collagens and elastin, thereby contributing to the maturation of the ECM. Here we have explored the role of LOX in the formation of aortic aneurysms in MFS. We show that aortic tissue from MFS patients and MFS mouse model (Fbn1(C1039G/+)) displayed enhanced expression of the members of the LOX family, LOX and LOX-like 1 (LOXL1), and this is associated with the formation of mature collagen fibers. Administration of a LOX inhibitor for 8weeks blocked collagen accumulation and aggravated elastic fiber impairment, and these effects correlated with the induction of a strong and rapidly progressing aortic dilatation, and with premature death in the more severe MFS mouse model, Fbn1(mgR/mgR), without any significant effect on wild type animals. This detrimental effect occurred preferentially in the ascending portion of the aorta, with little or no involvement of the aortic root, and was associated to an overactivation of both canonical and non-canonical TGF-ß signaling pathways. The blockade of angiotensin II type I receptor with losartan restored TGF-ß signaling activation, normalized elastic fiber impairment and prevented the aortic dilatation induced by LOX inhibition in Fbn1(C1039G/+) mice. Our data indicate that LOX enzymes and LOX-mediated collagen accumulation play a critical protective role in aneurysm formation in MFS.

Enhancement of collagen deposition and cross-linking by coupling lysyl oxidase with bone morphogenetic protein-1 and its application in tissue engineering.

Cultured cell-derived extracellular matrices (ECM)-based biomaterials exploit the inherent capacity of cells to create highly sophisticated supramolecular assemblies. However, standard cell culture conditions are far from ideal given the fact that the diluted microenvironment does not favor the production of ECM components, a circumstance particularly relevant for collagen. An incomplete conversion of procollagen by C-proteinase/bone morphogenetic protein 1 (BMP1) has been proposed to severely limit in vitro collagen deposition. BMP1 also catalyzes the proteolytic activation of the precursor of the collagen cross-linking enzyme, lysyl oxidase (LOX) to yield the active form, suggesting a deficit in cross-linking activity under standard conditions. We hypothesized that the implementation of fibroblast cultures with LOX and BMP1 may be an effective way to increase collagen deposition. To test it, we have generated stable cell lines overexpressing LOX and BMP1 and studied the effect of supernatants enriched in LOX and BMP1 on collagen synthesis and deposition from fibroblasts. Herein, we demonstrate that the supplementation with LOX and BMP1 strongly increased the deposition of collagen onto the insoluble matrix at the expense of the soluble fraction in the extracellular medium. Using decellularization protocols, we also show that fibroblast-derived matrices regulate adipogenic and osteogenic differentiation of human mesenchymal stem cells (MSC), and this effect was modulated by LOX/BMP1. Collectively, these data demonstrate that we have developed a convenient protocol to enhance the capacity of in vitro cell cultures to deposit collagen in the ECM, representing this approach a promising technology for application in tissue engineering.