A20 (tnfaip3) Knockdown Protects From Abdominal Aortic Aneurysms By Limiting Extra-cellular Matrix Degradation And Maintaining Aortic Smooth Muscle Cell Mass

Background: Our lab has recently demonstrated that haploinsufficiency of A20/TNFAIP3, a potent anti-inflammatory/NFκB inhibitory gene, protects mice from abdominal aortic aneurysms (AAA) by enhancing TGFβ signaling and modulating smooth muscle cell (SMC) differentiation and apoptosis. Here, we explored the impact of A20 knockdown on TGFβ-mediated SMC proliferation and extra cellular matrix (ECM) degradation, both involved in AAA pathogenesis. Methods: AAA was induced by periadventitial elastase application to the infrarenal aorta of both A20 wild-type (WT) and A20 heterozygous (Het) 10-week-old mice who received 0.2% BAPN in their drinking water. Aortae were harvested 28 days after for immunohistochemistry (IHC) analysis of SMC proliferation and ECM markers. In vitro , human aortic SMC cells (HAoSMC) were transduced with rAd.ShRNA A20 (siA20), resulting in 50-70% A20 knockdown, or control rAd.ShRNA Scramble (siScr). HAoSMC were starved for 24 hours, then treated with TGFβ. Proliferation was analyzed by MTS assay, and cell extracts assayed by Western blot (WB) for cell-cycle markers. Results: A20 haplo-insufficiency protected from AAA formation by: 1) decreasing ECM degradation, as gauged by significantly lower MMP-9 expression (n=3, p<0.01), and 2) promoting vessel wall thickening by increasing SMC proliferation (ki67+ nuclei/vessel wall, n=4, p<0.01). Increased SMC proliferation in Het vs. WT aortae correlated with significantly lower expression of the cell-cycle brake, p21 (p21+ nuclei/vessel wall, n=3-7, p<0.05). Akin mouse aortae, A20 knockdown significantly increased TGFβ-mediated HAoSMC proliferation, assessed by the MTS assay (siA20 vs. siScr, n=5, p<0.05). This associated with significantly higher protein levels of the proliferation regulator phospho-ERK1/2 in siA20 vs. siScr-transduced HAoSMC (WB, n=4, p<0.001). Conclusion: Our findings qualify the protective effect of A20 knockdown against AAA development by preserving ECM integrity and promoting TGFβ-mediated SMC proliferation. These targets of A20 knockdown were validated in HAoSMC, further supporting our pursuit of A20-siRNA based therapies to prevent or limit progression of AAA.