Background: Marfan syndrome (MFS) is the most prevalent inherited connective tissue disorder (fibrillin‐1 gene mutation) characterized by aortic aneurysm formation with subsequent dissection. Pharmacological therapy is limited to ß-blockers, which do not halt abnormal aortic growth or prevent dissection or death. MFS is accompanied by impaired endothelial function, caused by yet unknown molecular mechanisms. Immunohistochemical analysis of aortic sections from MFS patients and mice confirmed leukocyte infiltration in the aortic tunica media and aorta, respectively. Myeloperoxidase (MPO) is one of the principal proteins released by neutrophils, monocytes and macrophages. Under inflammatory conditions the MPO/H2O2 system may serve as a catalytic sink for NO, limiting its bioavailability and function. MPO has been shown to upregulate inducible nitric oxide synthase, potentially further aggravating its pathological role in MFS. The role of the innate immune system and its mediators is - in striking contrast to many other types of cardiovascular diseases - in MFS poorly understood. Given the fatal course of the disease and the limited pharmacological treatment options, characterization of the contributing mechanisms and the innate immune system is needed. Here, we aim to unravel the role of MPO in the progression of MFS to identify potential novel treatment strategies for MFS patients.

Methods and Results: MPO plasma levels of MFS (N=53) patients were significantly increased compared to healthy controls (N=18, p < 0.032). In accordance, circulating and thoracic aorta MPO levels were increased in a common MFS model, heterozygous fibrillin-1^C1041G/+ knock-in mice (MFS), vs. WT mice. To further study the function of MPO on MFS, MPO-deficient mice were crossbred with MFS mice to obtain MFSxMpo^-/-. Histological analysis showed increased elastic fiber breaks in aortic root sections in MFS- vs. WT mice whereas MFSxMpo^-/- showed less fragmentation. In-gel and in-situ zymography revealed elevated MMP-activity in the aorta of MFS mice. Immunofluorescence staining of the aortic root showed an increase of 3-nitrotyrosine in MFS vs. WT and MFSxMpo^-/- mice demonstrating NO-dependent posttranslational modifications. In silico analysis of a single-cell RNA-sequencing data set revealed two endothelial cell clusters in MFS mice that showed enrichment of inflammatory gene sets. In comparison, bulk RNA-Seq data from isolated endothelial cells by the "modified Häutchen method" showed less upregulation of inflammatory pathways in MFSxMpo-/--derived cells compared with MFS. ICAM-1, a leukocyte adhesion molecule, was increased in the aortic endothelium of Fbn1 mice but not in MFSxMpo^-/- mice. Intravital microscopy of the cremaster arteries revealed pronounced leukocyte adhesion and rolling in MFS mice, which was not observable in WT and MFSxMpo^-/-. To investigate the pharmaceutical inhibition of MPO, we decided to feed 6-week-old MFS mice, harboring a modest aortic phenotype, a diet containing the specific MPO inhibitor AZM198 for 6 to 12 weeks. AZM198 significantly reduced the formation of aortic aneurysms and elastic strand breaks.

Conclusion: Here, we show that MFS mice exhibit pathologically activated endothelium, leading to enhanced leukocyte-endothelial interactions that might result in release of MPO. Subsequent activation of matrix-degrading enzymes promotes the formation of TAA. These insights coincide with elevated MPO plasma levels in human MFS patients.