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A03

The innate immune sensor RIG-I in aortic inflammation and calcification

Calcified aortic valve stenosis (AS) develops at an anatomical site of massive and continuous mechanical stress. Mechanical stress is known to induce sterile inflammation in a variety of tissue types, and, at this vascular site, it may drive the calcification process and AS progression. However, the specific molecular mechanisms leading to sterile inflammation in this context are not well understood. This project is based on recent insights into the pathogenesis of a rare monogenetic disease, Singleton-Merten Syndrome (SMS).
SMS is a family of autosomal-dominant disorders characterized by diverse calcification abnormalities, including defects in dental and skeletal mineralization as well intramural calcification of the proximal aorta extending into the aortic valve. Gain-of function mutations in RIG-I-like receptors of the innate immune system are known to cause SMS-2 (MIM: 616298, atypical SMS), which present with severe aortic and valvular calcification. RIG-I is a ubiquitously expressed cytoplasmic helicase, which, upon sensing of viral RNA with certain structural characteristics (double strand with blunt end and a 5’-triphosphate), induces an antiviral of atypical SMS patients suggest that enhanced signaling of RIG-I drives the pathogenesis of the disease.
In previous collaborative studies, we found that the two RIG-I mutations causing atypical SMS allow ATP binding to the ATP binding site of RIG-I but prevent hydrolysis of ATP. The resulting ATPase-deficient RIG-I variants constitutively signal through binding of endogenous RNA, and we identified the large subunit of ribosomal RNA as the endogenous ligand inducing continuous RIG-I activation in this situation. Thus, atypical forms of SMS caused by gain-of-function RIG-I variants provide evidence that an increased responsiveness of RIG-I to endogenous RNA ligands in humans in vivo cause calcification of the aorta.

This has led us to hypothesize that an enhanced availability of activating endogenous RNA ligands could also activate non-SMS RIG-I specifically at vascular sites of high mechanical stress, such as the aortic valve and adjacent aorta, and that such dysregulated RIG-I activity may contribute to the development of aortic calcification.

Further support for an involvement of RIG-I comes from our previous collaborative work that demonstrated impaired endothelial function and enhanced atherosclerotic plaque formation in response to activation of RIG-I-like receptors, and from our observation of a significantly increased aortic valve elasticity in RIG-I-/- mice. Notably, a recent publication by another group showed that RIG-I co-activates osteogenic transcription factors thereby conveying calcifying signals in aortic smooth muscle cells. Collectively, the current literature and our own data provide a well-supported rationale for studying the functional implication of RIG-I as a co-factor in AS. In this proposal, our main goal is to dissect the pathophysiological functional implications of RIG-I in AS.

The project plan is structured along the following four aims: Aim 1: to identify the predominant cell type in aortic tissue in which RIG-I is activated during AS; Aim 2: to identify and characterize the endogenous RIG-I ligands present in RIG-I active cell types; Aim 3: to characterize the environmental factors such as mechanical stress or co-factors like metabolic, inflammatory or biochemical stressors that trigger and modulate the formation of endogenous RIG-I ligands; and Aim 4: to identify signals downstream of endogenous RIG-I activation that mediate enhanced osteogenic differentiation and calcification. We will benefit from the vast expertise of this consortium and its techniques, tools and models to study aortic disease, especially the highly standardized mouse models of AS (S01). Furthermore, RIG-I-/- mice and the newly established hRIG-I E373A Tg mouse (cooperation with Hiroki Kato, Bonn) will serve as RIG-I negative and positive controls and will instruct the experimental design for the RIG-I wild-type situation. Our extensive experience in CRISPR/Cas genome engineering and in polyclonal CRISPRi will help to precisely pinpoint the relevant molecular contributions in aortic pathogenesis. Since we expect RIG-I-driven immunopathology to act in concert with the pathogenetic mechanisms studied in the other projects, this consortium provides a unique research environment to therapeutically tackle the key drivers of AS and of aortic degeneration.

 

A03

 

A03 Hypothesis: Activation of retinoic-acid inducible gene I (RIG-I)-like receptors (RLR) induced by endogenous ligands formed at vascular sites exposed to high mechanical stress triggers proinflammatory pathways including type I interferon production (IFN) involved in the development of aortic disease.

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