Single-Cell Multimodal Profiling Highlights Persistent Aortic Smooth Muscle Cell Changes in Diabetic Mice Despite Glycemic Control

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Abstract
BACKGROUND: Type 2 diabetes is associated with accelerated vascular complications such as hypertension and atherosclerosis. Phenotypic switching of vascular smooth muscle cells (SMCs), a major driver of these complications, is enhanced in diabetes. Despite adequate glycemic control, SMC dysfunction can persist due to metabolic memory of prior hyperglycemia. However, the mechanisms are unclear. Here, leveraging single-cell multiomics, we examined the effect of glucose normalization on transcriptomic and epigenomic changes associated with SMC phenotypic transition in type 2 diabetes mice. METHODS: Type 2 diabetes db/db mice were treated with the antidiabetic drug dapagliflozin (DAPA) or vehicle and nondiabetic control db/+ mice with vehicle for 6 weeks. Dissected aortas were subjected to single-cell RNA sequencing, single-cell assay for transposase-accessible chromatin with sequencing, and spatial transcriptomics (Xenium) to determine single-cell changes in gene expression and chromatin accessibility. RESULTS: DAPA conferred effective glycemic control in db/db mice, with significant reductions in blood glucose and hemoglobinA1c. scRNA and single-cell assay for transposase-accessible chromatin with sequencing analysis of aortas identified SMC, fibroblasts, endothelial, and immune cells. SMCs were further clustered into 9 subtypes, including contractile and fibromyocyte-like cells. Interestingly, SMC contractile phenotype–associated pathways decreased in diabetes and remained decreased despite DAPA treatment. Fibrosis and inflammation-associated pathways in SMC and fibroblasts, and dysfunction markers in endothelial cells, increased in diabetes and were partly reversed by DAPA. Pseudotime trajectory analysis of SMC revealed increased activities of fibromyocyte-enriched TFs (transcription factors) during the contractile to fibromyocyte transition. Pairwise analysis for differentially accessible regions revealed diabetes-associated differentially accessible regions, enrichment of TF motifs, and related disease-associated biological processes. However, no differentially accessible regions were identified between db/db and db/dbDAPA groups. Spatial transcriptomics mapped aortic cell types within intact aortas and validated single-cell sequencing data. CONCLUSIONS: Type 2 diabetes induces gene expression and chromatin accessibility changes associated with profound SMC phenotypic switching. These changes are not efficiently reversed by a widely used antidiabetic drug, DAPA, underscoring the need for more effective therapies targeting hyperglycemic memory.
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