Sirtuin-1 (SirT1) is a nicotinamide adenine dinucleotide (NAD+)-dependent lysine deacetylase expressed ubiquitously in the body. In the vasculature, SirT1 is present in endothelial and vascular smooth muscle cells (VSMCs), where it has been shown to promote anti-inflammatory and anti-oxidant effects. As a result, SirT1 is known to play a protective role in the vasculature wall from pathologies such as atherosclerosis, arterial stiffness, and aortic aneurysm. Hence, SirT1 is considered an attractive therapeutic target for vascular diseases and potentially, aging-related and metabolic diseases. However, currently available SirT1 activators have failed to reach the clinic. Thus, novel approaches to activate SirT1 are needed. In this study, we first sought to optimize a novel fluorescence-based SirT1 activity assay, with which to reliably assess intracellular SirT1 activity and the efficacy of SirT1 activators and inhibitors. We next sought to use the SirT1 activity assay to screen novel compounds identified by an in silico docking analysis and hypothesized to activate SirT1. Lastly, we generated adeno-associated viruses (AAV) overexpressing wildtype (WT) or a redox-resistant (3M) SirT1 to analyze the effects of overexpressing SirT1 in VSMCs, in normal and oxidative stress conditions. For the activity assay, our results showed that an optimal standard curve range was between 0 ng and 12 ng of substrate (acetylated-p53 peptide). After testing different commercially available human recombinant SirT1s, the Anaspec SirT1 of the highest concentration showed a decrease in measured fluorescence for acetylated-p53 peptide with higher SirT1 (ng), indicating the enzyme and the assay were functional. However, when novel small molecules (A4, B4, and G3) hypothesized to activate SirT1 were added to reactions, the total p53 peptide fluorescence values increased compared to the control, suggesting some interference of the molecules with the assay detection. After AAV infection in VSMCs, SirT1 expression, measured by HA-tag, increased for AAV WT (n=3, p=0.04) and similarly for AAV 3M SirT1, indicating that the AAVs efficiently infect VSMCs. SirT1 activity, measured by Western Blot as decreased acetylated-histone (H3), also appeared to increase for both AAV WT and AAV 3M. A similar trend was shown for VSMCs under oxidant stress conditions (n=2). In conclusion, we successfully established a standard curve range for a novel SirT1 activity assay. Further trials are needed to ensure activity assay reproducibility before testing the efficacy of SirT1 activators and inhibitors. Infection of AAV WT and 3M SirT1 led to an increase in the expression and activity of SirT1 in VSMCs. The expression of SirT1 by AAV may be a promising therapeutic option for in vivo prevention and treatment of vascular diseases. / 2026-03-01T00:00:00Z
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/48315 |
| Date | 01 March 2024 |
| Creators | McElhinney, Priscilla |
| Contributors | Seta, Francesca, Trinkaus-Randall, Vickery E. |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
| Rights | Attribution 4.0 International, http://creativecommons.org/licenses/by/4.0/ |
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