Single-cell and spatial transcriptomics uncovers the role of senescent vascular cells in pathological arterial remodeling during murine atherosclerosis
Studying cardiovascular senescence is crucial in understanding the diverse manifestations of disease-related changes in the cardiovascular system and their implications for overall health and disease.  To systematically investigate the heterogeneity of senescent vascular cells in atherosclerosis, we employed the senescence reporter mouse p16TdTomato+/-, injected with an adeno-associated virus expressing PCSK9 to induce atherosclerosis when fed a high-fat diet (HFD).  We then treated this mouse with the senolytic drug ABT-737 or vehicle.  Whole-aorta single-cell RNA-sequencing (scRNA-seq) and Gene Set Enrichment Analysis (GSEA) using the SenMayo panel, uncovered 10 cell clusters displaying increased senescent features that were reduced by treatment with ABT-737.  Interestingly, these clusters do not express 'classical' senescence markers such as p16 and p21, but were senescent based on SenMayo GSEA, indicating that the senescence transcriptomic signature in this niche requires deeper characterization.  Using unbiased subclustering of the top four potentially senescent clusters, we identified subsets of cells increasing by HFD and reduced by ABT-737 treatment.  Accordingly, unique transcripts associated with each subpopulation, such as Spp1, Ctsb, and Tnfrsf11b, implicated these cells in senescence-related signaling pathways.  We then validated these findings by spatial transcriptomic profiling, locating senescent cells in the cap and core of brachiocephalic arteries from atherosclerotic mice.  Our results uncover a new vascular-specific transcriptomic signature that may be exploited for therapeutic targeting in age-related vascular diseases. Overall design: Mice of both sexes were entered in the study at 8 weeks of age, when all animals received 3x1010 viral genomes of PCSK9 AAV8 via retro-orbital injection.  Two weeks later, normal chow was maintained or replaced with an atherogenic diet (42% Fat, 0.2% cholesterol, Harlan atherogenic diet TD.88137).  After 16 weeks, a subset of mice on atherogenic diet were administered ABT-737 via intra peritoneal (IP) injection twice per week for an additional 8 weeks while maintaining the atherogenic diet.  All mouse experiments, including the import, housing, experimental procedures, and euthanasia, were performed strictly under an Animal Study Proposal (ASP #474-LGG-2023) and associated amendments, reviewed and approved by the Animal Care and Use Committee (ACUC) of National Institute on Aging (NIA).