Retinoic acid-inducible gene-I (RIG-I) is a putative RNA helicase and recently identified as a cytosolic RNA receptor in mammalian cells. The role of RIG-I in the regulation of vascular function under physiological and pathological conditions is unknown. Recent studies have shown that the inflammasome serves as a crucial initiator of cytokine-mediated inflammation mediating the pathogenesis of cardiovascular disease. The present study tested whether RIG-I activation triggers inflammasome formation and subsequent cytokine-mediated inflammation in the endothelium of mice coronary arteries. Using both genetic and pharmacological interventions of the RIG-I inflammasome, we first characterized whether specific activation of RIG-I via 3pRNA transfection induced the formation of an ASC-containing inflammasome in mouse vascular endothelial cells (MVECs). We confirmed that 3pRNA dose-dependently increased RIG-I protein levels and release of of type I IFNβ and IL-1b (a prototype cytokine from inflammasome activation), confirming RIG-I activation. We found that MVECs transfected with 3pRNA exhibited increased colocalization of RIG-I with apoptosis-associated speck-like protein (ASC) or caspase-1 and elevated active caspase-1 and IL-1β levels, indicating the formation and activation of the RIG-I inflammasome. This RIG-I inflammasome activation was accompanied by endothelial barrier dysfunction. In the presence of 3pRNA, ZO-1 and ZO-1 and VE-Cadherin expression diminished, and inhibiting caspase-1 and silencing inflammasome components attenuated this effect. To test the functional role of RIG-I and its affect on the permeability of mouse ECs, we performed a transwell permeability assay. Results confirmed that 3pRNA induced increased permeabilization of these mouse ECs, which was attenuated when inhibiting and silencing inflammasome components. These data indicate that increased expression and activity of RIG-I activate IL-1b producing inflammasomes in ECs, which may represent an early molecular mechanism mediating vascular inflammation and endothelial dysfunction independent of Nlrp3. Furthermore, we investigated the role of anti-aging gene Klotho in the regulation of RIG-I inflammasome activation. The Klotho protein has been shown to directly interact with RIG-I in senescent cells to block RIG-I multimerization and downstream production of pro-inflammatory cytokines. Administration of D-saccharic acid 1,4-lactone (saccharolactone) in vitro, a pharmacological inhibitor of Klotho activity, substantially increased inflammasome activation. In addition, mice injected with saccharolactone exhibited increased RIG-I inflammasome formation and activation within the coronary artery endothelium. These results suggest that decreased Klotho activity may activate RIG-I and thereby increase inflammasome activity. Therefore, the present study defines a novel role for RIG-I inflammasome activation in vascular dysfunction.
| Identifer | oai:union.ndltd.org:vcu.edu/oai:scholarscompass.vcu.edu:etd-6116 |
| Date | 01 January 2017 |
| Creators | Pitzer, Ashley |
| Publisher | VCU Scholars Compass |
| Source Sets | Virginia Commonwealth University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Theses and Dissertations |
| Rights | © The Author |
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