RIPK1 and RIPK3dependent cell death has been identified as a crucial mediator of abdominal aortic aneurysm (AAA) development. RIPKs mediate phosphorylation of MLKL thereby inducing its oligomerization and translocation to the cell membrane. Given the dual role of RIPKs being involved in necroptosis as well as in apoptosis induction, the specific role of MLKL-induced necroptotic cell death in AAA remains unclear.

Methods:

We monitored elastase-perfusion (PPE) induced progression of AAA in C57BL/6N (WT), RIPK1 kinase deficient (Ripk1^D138N/D138N), MLKL phosphodeficient (Mlkl^S345/7A) and MLKL knockout (Mlkl^-/-) mice by ultrasound measurements, histological analyses and bulk mRNAseq techniques to assess structural and molecular aortic changes. Bone marrow transplantation studies in WT and Mlkl^S345/7A mice were utilized to dissect the role of MLKL in smooth muscle cells (SMCs) and myeloid cells in AAA development. MLKL expressing human SMCs were generated to investigate necroptosis-induced proinflammatory cytokine secretion and subsequent polymorphonuclear neutrophil (PMN) migration and activation in vitro. Of note, treatment with a novel MLKL inhibitor, MBA-m1, prevented adverse ECM remodeling, leukocyte infiltration, and protected mice from AAA development. 

Results:

Ultrasound analysis showed that ~70% of the WT animals developed PPE induced-AAA with significant aortic structural alterations and enhanced myeloid cell infiltration. In contrast, Ripk1^D138N/D138N (45%), Mlkl^S345/7A (40%), and Mlkl^-/- (20%) mice were protected from AAA. This protection was associated with reduced adverse extracellular matrix (ECM) remodeling and leukocyte infiltration. Bone marrow transplantation studies show that the lack of MLKL in SMCs is the main driver of AAA protection.  MLKL deficiency was associated with a significant downregulation of genes involved in fibrinolysis, anti-inflammatory response, immune response and complement activation in aortic tissue in AAA. Proinflammatory cytokine secretion was elevated in necroptosis induced SMCs and resulted in a significant accumulation and activation of PMN.

Conclusions:

Overall, these findings indicate that MLKL-induced necroptotic SMC death and subsequent proinflammatory leukocyte activation plays a causative role in AAA development and suggests that pharmacological inhibition of MLKL may represent a promising treatment strategy for AAA disease.