Senohemostatic role for DPP4 in atherosclerosis

We performed scRNA sequencing to understand the composition and differential gene expression changes in aortic cells from LDLR-/- mice fed a normal diet (ND), high-fat diet (HFD), or HFD + DPP4i. We combined this technique with CITE-sequencing to measure the cell surface protein expression (DPP4) and transcriptomic changes associated with our experimental conditions as well as changes associated with DPP4 cell surface expression in all aortic cells. Overall design: LDL receptor knock out (Ldlr-/-) mice on the C57BL/6 background purchased from Jackson labs (Catalog #002207) were bred to obtain homozygous Ldlr-/- knock out mice. Mice of both sexes were entered in the study at 8 weeks of age when normal chow was replaced with an atherogenic diet (42% Fat, 0.2% cholesterol, Harlan atherogenic diet TD.88137). For the treatment groups, mice were gavaged with 10 mg/kg/day of Vildagliptin from SelleckChem or an equal volume of DMSO, 5 days per week for 16 weeks. Mice were anesthetized and blood was extracted using cardiac puncture. The whole aorta was collected after left ventricular perfusion with 10?mL of PBS and quickly transferred to cold PBS. To prepare a single-cell suspension, perivascular adipose tissue was removed and whole aortas from three mice were cut into ~1-mm pieces and digested with an enzyme solution consisting of 10 mg/ml Collagenase II (Sigma) and 1 mg/ml elastase (Worthington) for 15?min at 37?°C. The cell suspension was strained through a 40-µm filter and centrifuged at 500 x g for 5 min. Cells were resuspended in 100 µl Cell Staining Buffer with 15% FBS and 5 µl of Human TruStain FcX™ Fc Blocking reagent (or 0.5 µl of TruStain FcX™ PLUS (anti-mouse CD 16/32) blocking reagent. Cells were incubated for 10 minutes at 4°C, followed by the addition of 1 µg of DPP4 (CD26)-APC antibody and incubated for 30 minutes at 4°C. Cells were incubated with TotalSeq™ APC secondary antibody for 30 minutes at 4C. Next, cells were washed with 1 ml of Cell Staining Buffer, spun at 400g for 5 minutes, and resuspended in 1 mL of PBS + 1X EDTA (0.5M = 100x). Cells were stained with viability dye as described in the Flow Cytometry method and single, viable cells were collected for 10X library preparation.