Background: Marfan syndrome (MFS) is the most prevalent inherited connective tissue disorder characterized by aortic root aneurysm formation with subsequent aortic dissection contributing to a shortened lifespan. MFS is caused by mutations of Fibrillin-1, which encodes for Fibrillin-1, the main component of microfibrils. As such Fibrillin-1 serves as a scaffold anchor and supports the structure of the arterial wall. Its deficiency impairs the integrity of the extracellular matrix (ECM) and leads to aneurysm formation. The molecular mechanisms involved in thoracic aortic aneurysm (TAA) formation in MFS are not fully understood. Increased TGFβ-signaling is involved in the pathogenesis, but the exact role and contribution of the TGFβ signaling pathway in TAA formation remains controversial. Multiple studies suggested that inflammation contributes to MFS progression. Neutrophils (PMN) are key mediators of inflammation and potent producers of heme enzyme myeloperoxidase (MPO). MPO enters the subendothelial space where it oxidizes nitric oxide (NO) and thereby impairs the humoral integrity of the vessel wall. Recently it was shown that the uncoupled eNOS is a central mediator of TAA formation in a MFS mouse model. Furthermore, MPO influences the smooth muscle cell phenotype by reactive oxygen species that stimulate the secretion of proteases and matrix-modulating cytokines such as TGF-β. MPO can also directly modify multiple ECM proteins with consecutive fibrotic remodeling. We hypothesize that MPO adversely affects vascular signal transduction and thereby exacerbates aortic aneurysm formation in MFS.

Methods and Results: Thoracic aortas from heterozygous knock-in Fbn1^C1039G/+ (Fbn1) mice, which develop MFS, had an increased MPO concentration compared to wildtype mice as assayed by ELISA. We next crossed MPO-deficient (Mpo^-/-) mice with heterozygous Fbn1 mice (Fbn1-DKO). As expected, histological analyses (Van-Giesson staining) of the aorta showed increased fragmentation of elastic fibers in Fbn1- vs. WT mice, which was significantly reduced in Fbn1-DKO mice. Staining of aortic sections for reactive oxygen species (ROS) with dihydroethidium (DHE) showed a significantly higher amount of ROS in Fbn1 compared to wildtype or Fbn1-DKO mice. Immunofluorescence staining of human smooth aortic muscle cells treated with MPO and H₂O₂ demonstrated a colocalization of 3-nitrotyrosine and fibrillin-1. Aortic segments from Fbn1 mice showed higher staining of 3-nitrotyrosine compared to wildtype and Fbn1-DKO, indicating increased activity of MPO  in diseased aortic walls and underpinning the role of NO-dependent posttranslational modification. Furthermore, we performed in situ zymography on aortic sections and detected elevated activity of the matrix metalloproteases (MMP) 2 & 9 in Fbn1 vs. WT and a lower activity in Fbn1-DKO mice compared to Fbn1 mice. Finally, ultrasound analyses of aortic root and ascending aorta in Fbn1 animals showed an increased aortic diameter compared to wildtype mice. No structural alterations were observed in Fbn1-DKO mice.

Conclusion: Here we show that MPO, a pro-inflammatory regulator of oxidative stress, is a key contributor to MFS. MPO influences NO-dependent production of reactive species and leads to the activation of matrix-degrading enzymes thus promoting TAA formation in a mouse model of MFS.