Background:
Necroptosis is a type of regulated cell death and is induced by receptor-interacting protein kinases 1 (RIPK1) and its downstream substrate mixed lineage kinase-like (MLKL). Necroptosis causes the release of damage associated molecular patterns (DAMPs), thereby necroptosis is considered a pro-inflammatory process compared to apoptosis. The role of necroptosis in abdominal aortic aneurysm (AAA) formation remains to be clarified, as inflammation and regulated cell death of SMCs are established hallmarks  of AAA  progression.

Hypotheses:
We hypothesize that necroptotic cell death of smooth muscle cells (SMC) within the tunica media causes the release of DAMPs, which in turn leads to increased inflammatory activation and recruitment of polymorphonuclear neutrophils (PMN). These activated PMN then further induce SMC death thereby establishing a ‘vicious circle’ of AAA formation.
Methods and Results:
We performed in vivo studies in MLKL-deficient- (Mlkl^-/-) and RIPK1 inactivated (Ripk1^D138N/D138N) animals while wild-type (WT-C57BL/6) mice served as control. We induced abdominal aneurysm in these mice using two separate models: a) the angiotensin II (AngII) model and b) the porcine pancreatic elastase perfusion (PPE) model. To investigate the progression of aortic aneurysm formation, echocardiographic analyses were performed after 7 and 14 days of subcutaneous AngII application by osmotic mini-pumps and 3, 7, 14, 21, and 28 days following PPE surgery. Aortic sections were further analyzed using histological staining. Analyses of the aortic diameter indicate that Mlkl^-/-- as well as Ripk1^D138N/D138N- mice were protected from vascular dilatation upon AngII infusion at day 14 (increase of aortic diameter: WT: 61.4 ± 9.8 % vs. Ripk1^D138N/D138N: 28.0 ± 5.9 % vs. Mlkl^-/-: 23.2 ± 6.5 %; n=5/5/5; P (WT vs. Ripk1^D138N/D138N, WT vs. Mlkl^-/-) < 0.05) and after PPE surgery at day 21 (increase of aortic diameter: WT:  138.8 ± 29.4 % vs. Ripk1^D138N/D138N:  52.8 ± 9.1 vs. Mlkl^-/-: 60.3 ± 14.6 %; n=6/3/9; P (WT vs. Ripk1^D138N/D138N, WT vs. Mlkl^-/-) < 0.05). Puls wave velocity was increased in AAA formation, but attenuated with Necroptosis deficiency after PPE.
Aortic hematoxylin/eosin staining revealed increased tunica media thickness in untreated Mlkl^-/- mice as compared to WT animals. Furthermore, collagen deposition within the tunica media of Mlkl^-/-- animals was significantly elevated as compared to WT animals after AngII and  PPE AAA induction, indicating a role of necroptosis in vascular remodeling during AAA formation. Necroptosis deficiency stabilized elastin fibers, as the number of elastin strand breaks significantly decrease in Ripk1^D138N/D138N  and Mlkl^-/- animals after AngII and PPE treatment.
To further assess the influence of inflammatory stimuli on necroptosis induction we treated human primary aortic SMC with tumor necrosis factor (TNF) resulting in increased expression of MLKL (rel. Mlkl mRNA expression: control: 85.5 ± 30.3 % vs. TNF: 241.1 ± 53.5 %; n=5/6; P (control vs. TNF) < 0.05). Furthermore, TNF-alpha induction of primary SMCs of Ripk1^D138N/D138N and Mlkl^-/- animals showed decreased IL-6 expression with Necroptosis deficiency. 

Conclusion:
This study has revealed a crucial role of MLKL-induced necroptosis in the development and progression of AAA. Data from this study also confirmed that RIPK1 inactivity and MLKL deficiency protect from AAA development and pointing towards a potential new treatment strategy in this disease.