Abstract 4145115: Chemogenetic hydrogen sulfide donor rescues aortic perivascular adipose tissue and endothelial cell senescence in aging

Introduction: Hydrogen sulfide (H 2 S) is increasingly recognized as an important signaling small molecule that mitigates vascular aging and extends lifespan. Reduced H 2 S production in aged vasculature underscores the unmet need for effective strategies to controllably generate cell-specific H 2 S in vascular cells predisposed to cellular senescence in aging. Here, we develop a chemogenetic technology to offer a new class of H 2 S-donors to rescue perivascular adipose tissue (PVAT)-endothelial cell (EC) senescence in aging. Methods: To characterize our innovative (peri)vascular senotherapy, we employed a chemogenetic approach combined with biosensor-based real-time fluorescence imaging, and senescence and cell-cell interaction analyses. Results: We employed a recombinant AAV2/8-Adipo construct expressing a yeast D-amino acid oxidase (DAAO), which oxidizes D-Cysteine to yield 3-mercaptopyruvate (3MP), converting to H 2 S and pyruvate by mercaptopyruvate sulfurtransferase (MPST) in PVAT. We cloned DAAO as a fusion protein with the H 2 S biosensor hsGFP, enabling both generation (DAAO) and detection (hsGFP) of H 2 S in the cell mitochondria. Old mice (>24 months) exhibiting senescence in their aortic PVAT and ECs (indicated by ROS-induced oxidative stress, mitochondrial dysfunction, increased SASP component IL6, and proliferative arrest marker p16 INK4a ), were infected with AAV2/8-Adipo-DAAO-hsGFP-mito constructs. Fluorescence imaging documented robust in vivo H 2 S production in the distinct subcellular organelle after treatment with D-Cysteine (10 mM). Interestingly, these mice showed restored mitochondrial function and adipocyte marker UCP1, alongside downregulated IL6 in aortic PVAT. Our old PVAT-EC co-culture studies revealed that chemogenetic H2S, by reducing IL6 release towards ECs, rescues senescence and dysfunction in ECs (decreased SA-β-gal activity, DNA damage response (↓ γ-H2A.X), and proliferative arrest (↓ p16 INK4a &p19 INK4d )). The rescued ECs also exhibited restored angiogenic capacity, demonstrated by increased tube formation. Conclusions: Our recent advances in chemogenetic technology provide a remote-control (Turn ON/OFF) and cell-specific tool for intracellular H 2 S-mediated rescue of PVAT-EC senescence in aging. This innovative approach offers promising avenues for developing a new class of H 2 S-based senotherapies with significant potential for clinical applications.