Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Fondazione Gigi & Pupa Ferrari ONLUS Background Calcific aortic stenosis (AS) affects 3% of older adults and lacks medical treatment. The well-known deacetylase Sirtuin1 (SIRT1) could be involved in many pathways linked to AS progression. Sodium-glucose co-transporter 2 inhibitors (SGLT2i), a class of antihyperglycemic agents, have been shown to reduce cardiovascular events and hospitalization for heart failure (possibly via SIRT1), but their possible benefits in AS are not yet known. Purpose Our study aims to uncover if SIRT1 plays a key role in AS progression and if SGLT2i can potentially slow the valve fibro-calcification processes, potentially via SIRT1 modulation. Methods RNA sequencing of control (n=27) and AS (n=39) aortic valves was performed. SIRT1 knockdown (SIRT1-) and overexpressing (SIRT1+) valve interstitial cells (VIC) were generated by CRISPR/Cas9. A calcification assay with or without dapagliflozin (dapa 1uM as SGLT2i) was employed to evaluate valve endothelial cells (VEC) and VIC crass-talk on calcification potential. Patient data were collected from the Lombardy regional healthcare utilization database and diabetic patients who were taking sulphonylureas (sul; not affecting SIRT1) and SGLT2i (acting on SIRT1) were selected. Patients were matched 1:1 by age, sex, and multisource comorbidity score. Kaplan-Maier analysis was used to generate time-to-event curves for aortic valve replacement (AVR) cumulative incidence at 2 years and Cox regression analysis was used to account for confounding effects. Results Whole tissue RNA-seq showed that SIRT1 could be a master regulator of multiple pathways linked to AS. Functional analysis and validations on mutant VIC revealed that inflammation, extracellular-matrix remodeling, and calcification mechanisms are directly regulated by SIRT1. Calcium assays unveiled an increment of calcification in SIRT1- VICs (+50.8%, p<0.01), while SIRT1+ VICs deposited less calcium (-23.6%; p<0.01) compared to wild-type ones. VIC exposed to dapa-treated VEC medium showed reduced calcification (-35.2%, p<0.01). In the dapa-treated VEC medium, we found an increase in NO levels (+19.9%, p<0.01) and VIC treated with an NO donor (detanonate 50uM) showed reduced calcification (-23.2%, p<0.05), while VIC treated with a SIRT1 inhibitor (EX527 25uM) showed an increased calcification (+68.1%, p<0.01). To corroborate these findings, we retrospectively enrolled 233,546 diabetic patients, and after matching, we obtained two groups (sul and SGLT2i) with 37,081 patients each. Adjusted Cox regression analysis revealed that patients taking SGLT2i had a lower AVR incidence than patients taking sulp with a hazard ratio of 0.65 (95%CI 0.47-0.89) resulting in a 35% risk reduction. Conclusion Our data highlights SIRT1 as a key regulator of fibro-calcific processes involved in AS and SGLT2i exhibit potential in slowing AS progression, via SIRT1 modulation. This study unveils promising molecular insights and suggests SGLT2i as a potential therapeutic avenue for AS.

中文翻译：

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Sirt1 聚焦：揭示 SGLT2i 抑制剂治疗钙化性主动脉瓣狭窄的分子见解和治疗前景


资助致谢 资助来源类型：基金会。主要资金来源：Fondazione Gigi 和 Pupa Ferrari ONLUS 背景 钙化性主动脉瓣狭窄 (AS) 影响 3% 的老年人，且缺乏治疗。众所周知的去乙酰酶 Sirtuin1 (SIRT1) 可能参与与 AS 进展相关的许多途径。钠-葡萄糖协同转运蛋白 2 抑制剂 (SGLT2i) 是一类降血糖药，已被证明可以减少心血管事件和心力衰竭住院（可能通过 SIRT1），但其在 AS 中可能的益处尚不清楚。目的 我们的研究旨在揭示 SIRT1 是否在 AS 进展中发挥关键作用，以及 SGLT2i 是否可以通过 SIRT1 调节来减缓瓣膜纤维钙化过程。方法 对对照 (n=27) 和 AS (n=39) 主动脉瓣进行 RNA 测序。 SIRT1 敲低 (SIRT1-) 和过表达 (SIRT1+) 瓣膜间质细胞 (VIC) 通过 CRISPR/Cas9 生成。采用含或不含达格列净（dapa 1uM，SGLT2i）的钙化测定来评估瓣膜内皮细胞（VEC）和 VIC 对钙化潜力的影响。从伦巴第地区医疗保健利用数据库收集患者数据，并选择服用磺脲类药物（sul；不影响 SIRT1）和 SGLT2i（作用于 SIRT1）的糖尿病患者。患者按年龄、性别和多源合并症评分进行 1:1 匹配。 Kaplan-Maier 分析用于生成主动脉瓣置换术 (AVR) 2 年累积发生率的时间-事件曲线，并使用 Cox 回归分析来解释混杂效应。结果全组织 RNA-seq 显示 SIRT1 可能是与 AS 相关的多个途径的主要调节因子。 对突变 VIC 的功能分析和验证表明，炎症、细胞外基质重塑和钙化机制直接受 SIRT1 调节。钙测定显示，与野生型相比，SIRT1- VIC 中的钙化增加（+50.8%，p<0.01），而 SIRT1+ VIC 沉积的钙较少（-23.6%；p<0.01）。暴露于 dapa 处理的 VEC 培养基的 VIC 显示钙化减少（-35.2%，p<0.01）。在 dapa 处理的 VEC 培养基中，我们发现 NO 水平增加（+19.9%，p<0.01），并且用 NO 供体（去腺酸 50uM）处理的 VIC 显示钙化减少（-23.2%，p<0.05），而 VIC用 SIRT1 抑制剂 (EX527 25uM) 治疗显示钙化增加 (+68.1%, p<0.01)。为了证实这些发现，我们回顾性纳入了 233,546 名糖尿病患者，经过匹配后，我们获得了两组（sul 和 SGLT2i），每组 37,081 名患者。调整后的 Cox 回归分析显示，服用 SGLT2i 的患者的 AVR 发生率低于服用 sulp 的患者，风险比为 0.65 (95%CI 0.47-0.89)，导致风险降低 35%。结论 我们的数据强调 SIRT1 作为参与 AS 的纤维钙化过程的关键调节因子，并且 SGLT2i 通过 SIRT1 调节表现出减缓 AS 进展的潜力。这项研究揭示了有希望的分子见解，并表明 SGLT2i 作为 AS 的潜在治疗途径。