Early IKKβ-dependent anabolic signature governs vascular smooth muscle cells fate and abdominal aortic aneurysm development

Abstract

ABSTRACTBackgroundAbdominal aortic aneurysm (AAA) is a detrimental disease with no effective pharmacological therapy. While inflammation is recognized as one of the key regulators of AAA, targeting inflammatory pathways once the disease is established does not impact the outcomes. However, understanding the earliest molecular indicators could shed light on the precise biological targets and prognostic markers for AAA.MethodsUsing apolipoprotein E (ApoE)-deficient mice fed with a standard diet and infused with Angiotensin II (Ang II), we conducted bulk RNA-sequencing (RNA-Seq) analysis on suprarenal (SRA) regions obtained from both unchallenged and challenged WT mice, specifically examining responses 24 hours after Ang II infusion to capture the initial phases of aortic stress response. We further created a unique model of hyperlipidemic mice in which the expression of the inhibitor of nuclear factor kappa B kinase subunit beta (IKKβ) can be conditionally (via tamoxifen injection) suppressed in vascular smooth muscle cells (VSMC). The development of AAA was evaluated using in situ examination and quantified using RT-qPCR, immunohistochemistry and fluorescence microscopy. Cultured VSMC were exposed to the selective IKKβ inhibitor MLN120b and the expression levels of phenotypic markers kruppel like factor 4 (KLF4), β-Catenin and cellular communication network factor 2 (CCN2) were addressed using cellular extracts and immunoblot analysis.ResultsRNA-Seq data support the presence of early anabolic events in SRA regions detailing activation of the mammalian target of rapamycin complex 1 (mTORC1) pathway, which paralleled cellular anabolic processes including mitochondria, ribosome and sterol biosynthesis, the Unfolded Protein Response (UPR) and fibrogenesis. Conditional deletion of theIkbkbgene in VSMC significantly reduces the incidence of SRA lesions as well as the rate of aneurysm ruptures in mice exposed to Ang II.In situanalysis further demonstrated that the protection conferred by the lack of IKKβ expression in VSMC is associated with reduced inflammatory response, the preservation of the contractile over the degradative VSMC phenotypes, and the absence of an anabolic signature.ConclusionOur results not only reinforce the major roles played by VSMC in the rapid adaptation leading to the deleterious remodeling of the vascular wall and aortic lesions but also support a paradigm aiming at repositioning the efforts focusing on anabolic rather than inflammatory events.