The Role of Necroptosis in Marfan Syndrome-associated Thoracic Aortic Aneurysm Disease

Background:
Marfan Syndrome (MFS) is an autosomal dominant connective tissue disorder caused by mutations in the Fibrillin-1 (Fbn1) gene. Increased morbidity and early mortality in this patient population are primarily due to aortic complications, such as thoracic aortic aneurysms (TAA) and dissections. Despite extensive research, targeted treatment options are still not available, and surgical aortic replacement is often required. MFS-associated TAA is accompanied by increased vascular inflammation and structural degradation, suggesting that necroptosis, a regulated form of cell death, may play a significant role. Necroptosis is promoted by inflammatory stimuli leading to the phosphorylation of the executor protein mixed lineage kinase like protein (MLKL), resulting in loss of cell membrane integrity and cell death. Previous research has demonstrated that the loss of MLKL prevents necroptosis and protects mice from developing abdominal aortic aneurysms. However, the role of MLKL in TAA remains unknown.
 
Methods and Results:
To investigate necroptosis in MFS-associated TAA, we crossbred MFS mice (Fbn1C1041G/+ / Mlkl+/+ = MFS) with mice carrying a phosphorylation site mutation in MLKL (Fbn1+/+ / MlklS345/7A/S345/7A = pmMLKL), rendering MLKL inactive, thereby creating necroptosis-deficient MFS mice (Fbn1C1041G/+ / MlklS345/7A/S345/7A = pmDKO). Echocardiographic analyses have revealed a significantly enlarged aortic bulb diameter in pmMLKL compared to Wildtype (WT), and even more so in pmDKO compared to MFS mice. Proteomic analyses of aortic tissue from pmMLKL and pmDKO mice suggest modifications in extracellular matrix (ECM) organization, mitochondrial metabolism, and regulation of endocytosis. These alterations indicate a switch from a contractile smooth muscle cell (SMC) phenotype to a mixed synthetic/contractile phenotype. Primary murine SMCs were isolated, and qPCR was performed to investigate the expression of genes related to ECM structure and integrity. We found an upregulation of Lysyl-oxidase, Collagen type 1 and 3 and Matrix-metalloproteinases 2 and 13 in pmMLKL compared to WT and in pmDKO compared to MFS mice. Aligned with the qPCR data, picrosirius red staining revealed numerically higher aortic collagen deposition in pmMLKL and a significantly higher deposition in pmDKO mice compared to the respective controls. Transmission electron microscopy of aortic tissue showed pronounced structural degradation and altered ECM composition in pmMLKL and pmDKO mice compared to WT and MFS mice, respectively. When subjected to an accelerated aortic disease model using high-dose Angiotensin II (3125 ng/kg/min), pmDKO mice exhibited aggravated aortic bulb dilation and higher mortality rates compared to MFS mice.
 
Conclusion:
We show that MLKL deficiency in MFS mice exacerbates the TAA phenotype and increases mortality, highlighting the critical role of necroptosis in maintaining aortic integrity in Marfan Syndrome.