Genetically Engineered Clostridium Prevents Perivascular Adipose Tissue and Endothelial Senescence and Dysfunction in Aging

Abstract Introduction (Peri)vascular senescence is a bona fide risk for several aging-associated diseases that threatens healthspan. Gut microbiome may be one of triggers that promotes vascular diseases as host ages. However, the interplay between vascular senescence and gut microbiome is largely unknown. Our recent studies revealed an increase in gut-derived phenylacetic acid (PAA) in the aging population (TwinsUK Aging Cohort, n=7,303) and aged mice. Our preliminary shotgun metagenomics also showed that ppfor-harboring Clostridium sp. ASF356 age-dependently generates PAA, which induces perivascular adipose tissue (PVAT) and endothelial cell (EC) senescence and dysfunction in old mice. Yet, it remains unclear whether and how genetic engineering of Clostridium sp. ASF356 (by depleting ppfor gene) can prevent aortic PVAT-EC senescence and restore vascular function in aging. Methods For bacterial genetic engineering, we depleted ppfor gene in Clostridium ASF356 by ClosTron technique and further mono-colonized aged mice (24-months old) and germ-free mice, as control, to reduce plasma PAA and thereby prevent aortic PVAT-EC senescence and improve endothelial dysfunction. Results As a proof of the concept, our studies exhibited that mono-colonization of germ-free mice with wild-type Clostridium sp. ASF356 markedly elevates plasma PAA levels. It was accompanied by hallmarks of aortic PVAT-EC senescence, including increased DNA damage (↑ γ-H2A.X phosphorylation), heightened SASP (↑ IL-6 and VCAM1), and proliferative arrest (↑ p16INK4a and p21WAF1/Cip1). Additionally, our findings revealed upreglation of Notch1 in PVAT, contributing to reduced energy supply (↓ UCP1) and augmented senescence-messaging secretome (↑ IL-6) towards ECs. Our further studies revealed that mono-colonization of aged mice (pre-treated with antibiotics) with Δppfor Clostridium ASF356 mutants intriguingly resulted in decreased plasma PAA levels. This intervention led to the rescues of cellular senescence in both aortic PVAT and ECs, consequently improving vascular function. Specifically, mice subjected to this treatment exhibited a significant reduction in SA-β-galactosidase+ cells, as well as decreased levels of γ-H2A.X phosphorylation, p16INK4a, p21WAF1/Cip1, and IL-6 in their aortic PVAT and ECs compared to wild-type aged mice. Conclusions The current study suggests that genetically engineered Δppfor Clostridium ASF356 mutants reverse cellular senescence and restore function in aortic PVAT and endothelial cells, highlighting the potential for targeted gut microbiome manipulation as an innovative senotherapy to combat vascular aging.