Targeting Modulated Vascular Smooth Muscle Cells in Atherosclerosis via FAP-Directed Immunotherapy [Human_CITEseq]
Vascular smooth muscle cell (VSMCs) cell diversification drives atherosclerotic coronary artery disease (CAD). Mechanisms governing these cell state transitions remain unclear. We applied multi-omic single-cell profiling, epitope mapping, and spatial transcriptomics across 27 human coronary arteries, identifying fibroblast activation protein (FAP) as a marker of modulated VSMCs. Lineage tracing in mice indicated that FAP? cells originate from Myh11? VSMCs, and FAP PET imaging in CAD patients showed plaque uptake. FAP? cells states resided in the macrophage-rich neo-intima. Therapeutically, we developed an anti-FAP bispecific T-cell engager, which reduced plaque burden and remodeled the stromal–immune microenvironment through T-cell clonal expansion. Our study delivers a single-cell and spatial atlas of human CAD, establishes FAP as a marker of modulated VSMCs, and highlights immunotherapy for lipid-independent targets. Overall design: Ethical Approval for Human Specimens The study is compliant with all relevant ethical regulations and has been approved by the Washington University School of Medicine Institutional Review Board (IRB #201104172). Informed consent was obtained from each patient prior to tissue collection by Washington University School of Medicine, and no compensation was provided in exchange for subject participation in the study. All demographic and clinical data have been de-identified and provided in Supplementary Table 1. Patients included in this study span diverse races, ages, and sexes to provide an inclusive trans-ethnic study population.  Inclusion Criteria Prior to tissue collection, specific inclusion criteria were applied to ensure well-controlled study groups. Any patients with HIV or hepatitis and known genetic cardiomyopathies were excluded from this study. The left anterior descending coronary artery was isolated and flash frozen from donor hearts: patients with stable ejection fractions and no known history of cardiac disease who experienced a non-cardiac cause of death/transplant and patients with chronic heart failure. For all samples, the proximal left anterior descending coronary artery was used.  Human single cell isolation for CITE-seq Fresh human coronary arteries from explanted hearts at the time of transplantation or donors declared dead after cardiac death (DCD) or brain death (DBD) at Washington University School of Medicine and Mid America Transplant Service were harvested, perfused with cardioplegia, and transported on ice. Starting at the coronary sinus, the left main, left anterior descending, and left circumflex arteries were dissected removing any epicardial fat while preserving the adventitia. Sections were partitioned for histopathology, spatial transcriptomics, single cell isolation, and flash frozen. Cells were isolated as before (17). Briefly: Coronaries were minced using a razor blade on ice and transferred to a 15 mL conical tube containing 3 mL DMEM with 170 µL Collagenase IV (250 U mL-1 final concentration), 35 µL DNAse1 (60 U mL-1), and 75 µL Hyaluronidase (60 U mL-1, all purchased from Sigma Aldrich) and incubated at 37 °C for 45 min with agitation. After 45 min, the digestion reaction was quenched with 6 mL of HBB buffer (2% FBS and 0.2% BSA in HBSS), filtered through 40 µm filters into a 50 mL conical tube and transferred back into a 15 mL conical tube to obtain tighter pellets. Samples were then centrifuged at 4°C, 1,200 rpm for 5 min, and the supernatant was discarded. The pellets were resuspended in 1 mL ACK Lysis buffer (#A10492-01, Gibco) and incubated at room temperature for 5 min followed by the addition of 9 mL DMEM and centrifugation at 4°C, 1,200 rpm for  5 min. Supernatant was discarded and the pellet was resuspended in 2 mL FACS buffer (2% FBS and 2 mM EDTA in calcium/magnesium free PBS), and centrifugation was repeated in above conditions and supernatant aspirated. The TotalSeq A 277 panel (BioLegend) antibody cocktail was resuspended in 100 µL of FACS buffer and 1 µL each of custom oligo-labeled FAP (Amgen) and LRRC15 (Amgen) were added.  The combined 102 µL were used to resuspend the pellet with the addition of 1 µL of DRAQ5 (#564907, Thermo Fisher Scientific) and incubated on ice for 30 min. Solution was washed with FACS buffer three times following same centrifugation as above and then resuspended in 500 µL of FACS buffer and 1 µL DAPI (#564907, BD Biosciences), then filtered into filter-top FACS tubes. First singlets were gated and subsequent DRAQ5+/DAPI- events were collected in 300 µL cell resuspension buffer (0.04% BSA in PBS). The collected cells were centrifuged as above and resuspended in collection buffer to a target concentration of 1,000 cells µL-1. Cells were counted on a hemocytometer before proceeding with the 10x protocol.