PROJECT SUMMARY Ascending thoracic aortic aneurysms and dissections (ATAAD) are extremely lethal diseases. The contribution of endothelial dysfunction in ATAAD has received limited attention and thus remains poorly understood. Com- pelling evidence from our preliminary studies suggests a critical role for endothelial cell (EC) injury and endo- thelial hyperpermeability in aortic degeneration and ATAAD development. [scRNA-seq analysis of aortas from sporadic ATAAD patients revealed significant EC dysfunction with upregulation of RIP3 and GSDMD that trig- ger necroptosis and pyroptosis, respectively. In a sporadic ATAAD mouse model, aortic challenge with high- fat diet and angiotensin II infusion induced RIP3 and GSDMD in ECs, caused endothelial hyperpermeability, nanoparticle infiltration, interlaminar space expansion, elastic fiber delamination, and microdissection. EC-Rip3- /- or EC-Gsdmd-/- showed markedly reduced nanoparticle infiltration and aortic degeneration.] Further investiga- tion with scATAC-seq showed that aortic challenge increased chromatin accessibility at promoters/enhancers of the pro-inflammatory/pro-death genes, likely by activating pro-inflammatory transcription factors (TFs) such as IRF3. In human ECs, cytosolic DNA and STING-TBK1 signaling was a potent upstream trigger for the acti- vation of IRF3 and BRG1 and induction of GSDMD expression. The objective of this application is to test the central hypothesis that aortic stress activates inflammatory signaling within ECs that triggers epigenetic in- duction of a pro-inflammatory and pro-death program, leading to EC necroptosis/pyroptosis, barrier dysfunc- tion, blood component infiltration, aortic wall degeneration, and ATAAD formation. [Aim 1 will test the hypothe- sis that RIP3-mediated EC necroptosis and GSDMD-mediated EC pyroptosis compromise EC barrier function, thereby facilitating the infiltration of inflammatory blood components and promoting aortic degeneration and ATAAD formation. In our sporadic ATAAD model, we will compare aortic degeneration and ATAAD formation in control mice, EC-Rip3-/- mice, EC-Gsdmd-/- mice, and WT mice treated with a necroptosis/pyroptosis inhibi- tor.] Aim 2 will test the hypothesis that aortic stress activates inflammatory signaling (e.g., STING pathway) within ECs that triggers epigenetic induction of a pro-inflammatory and pro-death program. We will perform scRNA-seq and scATAC-seq analyses of ECs in aortas from unchallenged and challenged WT mice and EC- specific Sting KO mice. Aim 3 will test the hypothesis that stress signals (e.g., cytosolic DNA) trigger sensing- signaling pathways (e.g., STING-TBK) that directly activates chromatin remodeling complexes (e.g., BRG1/SWI/SNF) and TFs (e.g., IRF3) that induce GSDMD expression and promote EC pyroptosis. We will test the hypothesis in cultured human ECs (Aim 3A) and [confirm the mechanistic links in human ATAAD aortas (Aim 3B)]. We expect that the proposed studies will provide novel insigh...