Rationale: Depletion of medial smooth muscle cell (SMC) Receptor-Interacting Protein Kinase 3 Contributes to Abdominal Aortic Aneurysms via Smooth Muscle Cell Necrosis and Inflammation

is a major pathological characteristic of abdominal aortic aneurysm (AAA), although the mechanism by which these cells are eliminated remains incompletely understood. We reasoned that necroptosis, a recently described form of necrosis mediated by receptor-interacting protein kinase 3 (RIP3), may contribute to AAA pathology through the induction of SMC death and the significant production of inflammatory cytokines. Objective: To test the hypothesis that RIP3-mediated necroptosis is actively involved in aneurysm pathogenesis. Methods and Results: RIP3 and RIP1 levels were found to be elevated in human AAAs, most noticeably in SMCs. Elevations of RIP3 and SMC necrosis were also observed in the elastase-induced mouse model of AAAs. Deletion of one or both copies of Rip3 prevented AAA formation. By transplanting Rip3 +/ − aortae to Rip3 +/+ mice, we demonstrated that reduced Rip3 expression in arterial wall was the primary cause of aneurysm resistance. In vitro, adenoviral overexpression of RIP3 was sufficient to trigger SMC necroptosis. Protein kinase C-delta contributed to tumor necrosis factor- α –induced SMC necroptosis by regulating Rip3 expression. Furthermore, Rip3 deficiency impaired tumor necrosis factor- α –induced inflammatory gene expression in aortic SMCs, which was at least in part because of attenuation of p65 Ser536 phosphorylation. In vivo, the lack of RIP3 diminished activation of p65 in SMCs, implicating a necrosis independent function of RIP3 in aneurysms. Conclusions: Enhanced RIP3 signaling in aneurysmal tissues contributes to AAA progression by causing SMC necroptosis, as well as stimulating vascular inflammation, and therefore may serve as a novel therapeutic target for AAA treatment. ( Circ Res . 2015;116:600-611. DOI: nonparametric test for skewed data were used to compare ≥ 3 means. Differences with P <0.05 considered statistically The mouse was then placed supine on the operative field. A long midline abdominal incision was made and the abdominal aorta, extending from where the left renal vein crosses the aorta to the iliac bifurcation, was isolated. The external diameter of the largest portion of abdominal aorta was measured with a digital caliber. All of the aortic branches between the renal arteries and bifurcation were ligated with 9-0 sutures. After placing temporary 6-0 silk ligatures around the proximal and distal aorta, an aortostomy was created with a 30G needle at the bifurcation. A heat-tapered segment of PE-10 polyethylene tubing (Baxter Healthcare Corp., Deerfield, IL) was introduced through the aortostomy and secured with a silk ligature. 0.45 U/mL type I porcine pancreatic elastase (Sigma, St. Louis, MO) was administrated to the infrarenal aorta through the tubing and allowed to incubate for 5 min at a constant pressure of 100 mm Hg. The 100mm Hg was calibrated using a saline bag hung at a height of 136cm. As a control, a separate group of mice were treated with equal concentration of heat-inactivated (100°C for 15 min) elastase for 5 min at a constant pressure of 100 mm Hg. The heat-tapered PE-10 polyethylene tubing was then removed and the aortotomy was closed with a 10-0 suture. The distal ligature was removed, followed by the removal of the proximal ligature. The abdominal incision was closed in two layers with 6-0 nylon sutures. Lidocaine 1% drops were placed on the wound. The mouse was kept on a warming pad until fully recovered from anesthesia. After the operation, mice were monitored each hour for the first 4 hours and then once daily. The external diameter of the largest portion of an abdominal aorta was measured and recorded at sacrifice, as it had been prior to elastase perfusion.