Objectives: Marfan syndrome (MFS) is a genetic disorder caused by mutations in the fibrillin-1 gene leading to aortic medial degeneration, aneurysm formation, and dissection. MFS is known to affect men more severely than women. Neprilysin (NEP) is a membrane metallo-endopeptidase expressed inter alia in vascular cells, cleaving and thereby inactivating a variety of physiologically relevant peptides. NEP can be released into the circulation by ectodomain shedding, resulting in a soluble form (sNEP) shown to be related to cardiovascular diseases. Maximal sNEP enzymatic activity is much lower in plasma, suggesting enzymatic activity is predominantly cell-based. Recently, NEP/angiotensin II receptor blockers sacubitril/valsartan have been shown to attenuate aortic remodeling in a mouse MFS model. This study aims to determine if the aortic phenotype of MFS correlates with an increased NEP expression in the aortic media and sNEP plasma content in a fibrillin-1 hypomorphic Marfan mouse model and patients with MFS, respectively.

Methods: As part of survival experiments, the ascending aorta (AAo) of male and female Fbn1^tm2Rmz (mgR/mgR) mice were routinely evaluated by echocardiographic examination starting at 8 weeks of age. NEP/sNEP protein content was detected in the plasma, AAo, and descending aorta (DAo) of Marfan mice, wildtype littermates (10 weeks old), and MFS patients or control individuals (age 19-64 years) by using immunofluorescence microscopy and enzyme-linked immunosorbent assay (ELISA).

Results: Female mgR/mgR mice have a significantly prolonged median survival than males (females 24 weeks, males 14 weeks; p<0.0001). Male mgR/mgR mice reached a mean AAo diameter of 2.8±1.3 mm (n=6) at 14 weeks, whereas females had a comparable diameter of 2.8±0.5 mm (n=5) after 20 weeks. Wildtype siblings of comparable age usually reach diameters ranging from 1.2-1.6 mm. Immunofluorescent staining of AAo and DAo showed a gender-independent significantly higher NEP expression in the AAo (+44%, p=0.0009) and DAo (+48%, p=<0.0001) of mgR/mgR mice compared to wildtype animals. The correlation of NEP expression and postmortem AAo diameter measurements revealed a significant positive relationship (Pearson’s r=0,54, p=0,045). sNEP plasma levels were 44% lower in mgR/mgR mice compared to wildtype mice (p=0.0025), particularly in male mgR/mgR mice (-66%, p=0.0003). Exemplary NEP immunofluorescent staining of aortic tissue derived from MFS patients (n=2) also revealed a 47% higher NEP expression in the AAo media compared to control specimens (n=2). However, in MFS patients sNEP plasma levels were 78% higher (p=0.004, n=24-29), independent of gender, age, aortic diameter, and clinical data such as pre-/ post-surgery.

Conclusion: The significantly increased NEP expression in the aortic medial layer and the correlation of NEP expression with the AAo diameter allows the hypothesis that NEP may play a role in aneurysm development in mgR/mgR mice and MFS patients. The observed lower sNEP levels in mgR/mgR male mice point to a lower cleavage of NEP from cells in the medial layer, which may predispose male mgR/mgR mice to increased aneurysm formation. However, credible explanations for the increased sNEP plasma levels in MFS patients should be sought by further investigations. Adapting medical therapy for young MFS patients by inhibiting NEP activity may have beneficial effects on aneurysm progression or even prevent the need for surgical intervention at young age.