Edited by Gerald W. HartCollagens are the main structural component of the extracellular matrix and provide biomechanical properties to connective tissues. A critical step in collagen fibril formation is the proteolytic removal of N-and C-terminal propeptides from procollagens by metalloproteinases of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) and BMP1 (bone morphogenetic protein 1)/Tolloidlike families, respectively. BMP1 also cleaves and activates the lysyl oxidase (LOX) precursor, the enzyme catalyzing the initial step in the formation of covalent collagen crosslinks, an essential process for fibril stabilization. In this study, using murine skin fibroblasts and HEK293 cells, along with immunoprecipitation, LOX enzymatic activity, solidphase binding assays, and proteomics analyses, we report that the LOX precursor is proteolytically processed by the procollagen N-proteinases ADAMTS2 and ADAMTS14 between Asp-218 and Tyr-219, 50 amino acids downstream of the BMP1 cleavage site. We noted that the LOX sequence between the BMP1-and ADAMTS-processing sites contains several conserved tyrosine residues, of which some are posttranslationally modified by tyrosine O-sulfation and contribute to binding to collagen. Taken together, these findings unravel an additional level of regulation in the formation of collagen fibrils. They point to a mechanism that controls the binding of LOX to collagen and is based on differential BMP1-and ADAMTS2/14-mediated cleavage of a tyrosinesulfated domain.

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-β (TGF-β)- 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-β signaling pathways. The blockade of angiotensin II type I receptor with losartan restored TGF-β 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.