Upregulation Of Receptor Interacting Protein Kinase-3 Augments Abdominal Aortic Aneurysms

Objective: Aortic smooth muscle cell (SMC) depletion is a major pathological feature of abdominal aortic aneurysm (AAA). Our lab has previously reported elevated levels of Receptor Interacting Protein Kinase-3 (RIPK3), a major mediator of necroptosis, in human AAA tissues. Pharmacologic inhibition of RIPK3 or deletion of the Ripk3 gene prevents aortic SMC death and attenuates aneurysm growth in murine models. In the current study, we tested the hypothesis that manipulation of Ripk3 gene expression would alter AAA development. Methods: We manipulated Ripk3 gene expression by mutating a conserved putative transcription enhancer site using CRISPR-Cas9-mediated gene editing. AAA was induced in 8-week old male CRISPR mutated (CM) and wild-type (WT) mice by perivascular application of CaCl 2 or NaCl (sham). Aortic tissues were harvested on days 4 or 28 post-surgery and analyzed by immunohistochemistry (IHC). Primary aortic SMCs isolated from both groups were stimulated with inflammation-related cytokines and Ripk3 mRNA was examined by RT-PCR. Results: Both CM and WT mice developed AAA in response to CaCl 2 . Change in aortic diameter was greater in CM mice than WT at 28 days (66.81±4.512 vs 44.87±6.685%, P =0.0108). IHC showed CM aneurysm tissue to exhibit fewer SMCs and a greater number of non-SMC cells in the tunica media, presumably infiltrated immune cells. Furthermore, mutant tissues contained higher levels of RIPK3 positivity than WT aortas. Consistently, CM aortic SMCs expressed more Ripk3 mRNA than WT SMCs, both with vehicle and following stimulation with IL-1β, IL-25, and IL-33. Conclusions: Our data show a positive correlation between Ripk3 expression and aneurysm size. We are currently analyzing data from experiments aimed to prove the causal relationship between Ripk3 gene editing, SMC necroptosis, inflammation, and aneurysm growth. This study further illustrates the potential of RIPK3 as a therapeutic target of AAA.