A spatial transcriptomic approach to understanding coronary atherosclerotic plaque stability

Description

ABSTRACT Background Coronary atherosclerotic plaques susceptible to acute coronary syndrome (ACS) have traditionally been characterized by their surrounding cellular architecture. However, with the advent of intravascular imaging, novel mechanisms of coronary thrombosis have emerged, challenging our contemporary understanding of ACS. These intriguing findings underscore the necessity for a precise molecular definition of plaque stability. Considering this, our study aimed to investigate the vascular microenvironment in patients with stable and unstable plaques using spatial transcriptomics. Methods Autopsy-derived coronary arteries were preserved and categorized by plaque stability (N = 5 patients / group). We utilized the GeoMx digital spatial profiling platform and Whole Transcriptome Atlas to link crucial histologic morphology markers in coronary lesions with differential gene expression in specific regions of interest, thereby mapping the vascular transcriptome. This innovative approach allowed us to conduct cell morphologic and spatially resolved transcriptional profiling of atherosclerotic plaques while preserving crucial intercellular signaling pathways. Results We observed intriguing spatial and cell-specific transcriptional patterns in stable and unstable atherosclerotic plaques, showcasing regional variations within the tunica intima and media. These regions exhibited differential expression of pro-inflammatory molecules (e.g., IFN-gamma, MHC Class II, pro-inflammatory cytokines) and pro-thrombotic signaling pathways. By employing lineage-tracing through spatial deconvolution of intimal CD68+ cells, we characterized unique, intraplaque subpopulations originating from endothelial, smooth muscle, and myeloid lineages with distinct regional locations associated with plaque instability. In addition, unique transcriptional signatures were observed in vascular smooth muscle and CD68+ cells among plaques exhibiting coronary calcification. Conclusion Our study sheds light on the distinct cell-specific and regional transcriptional alterations present in unstable arterial plaques. Furthermore, we characterize the first spatially-resolved, in situ evidence supporting cellular transdifferentiation and intra-plaque plasticity as significant contributors to plaque instability in human coronary atherosclerosis. Our results provide a powerful resource for the identification of novel mediators of ACS, opening new avenues for preventative and therapeutic treatments.

Steps to reproduce

Human subjects. Post-mortem samples of coronary conduit artery tissues were collected from deceased patients, reviewed by an independent cardiovascular anatomic pathologist (Silvio Litovsky, MD, American Board of Pathology Certified in Anatomic and Clinical Pathology), and grouped by atherosclerotic plaque stability (N = 5 stable and unstable patients per group, respectively). Plaques were characterized as “unstable” by the presence of a large necrotic core, intraplaque hemorrhage, intimal neovascularization and/or vaso vasorum proliferation, thin fibrous cap, and increased density of macrophages9,21. Coronary plaques featuring small lipid cores, minimal monocyte infiltration, absent necrotic centers, and thick fibrous caps were classified as "stable". Histology and Immunofluorescence. Formalin-fixed, paraffin-embedded (FFPE) coronary arterial tissues were selected prior to GeoMx digital spatial profiling by hematoxylin and eosin (H&E) and immunofluorescence staining (see Supplemental Methods for detailed protocol). Briefly, Alexa Fluor® 594-conjugated anti-CD68 (Santa Cruz, sc20060AF594, 1:200) and Alexa Fluor® 647-conjugated anti-smooth muscle myosin heavy chain 11 (MYH11; Abcam, ab196982, 1:100) antibodies were used as fluorescent morphologic markers (see GeoMx digital spatial profiling below). Nuclei were stained with SYTO13 (ThermoFisher, S7575, 0.5 μM in Buffer W) found within the GeoMx RNA slide preparation kit (NanoString). Images were acquired using the GeoMx epifluorescence microscope included in the digital spatial platform. GeoMx digital spatial profiling. FFPE tissues were processed for digital spatial profiling as previously described18,19. Briefly, fluorescently labeled anti-CD68 and anti-MYH11 antibodies were used as morphologic markers and combined with the GeoMx Human Whole Transcriptome Atlas gene set. Regions of interest (ROIs) were then selected based on immunofluorescent staining and/or histopathologic architecture consistent with coronary arterial tissue. In particular, ROIs were selected from the tunica media, tunica intima, and atheroma. SYTO13 stained nuclei were used to ensure that at least 50 nuclei were included per ROI analyzed. All experimental comparisons (e.g. stable vs unstable plaques) were analyzed per ROI replicate from each patient as unique samples (See Statistical Analysis below). Each figure illustrates representative ROI replicates for a given patient group. Quantification. Libraries of the oligo tags collected with the GeoMx platform were quantified as previously described18,19. Sequencing through the GeoMx platform is performed on the RNA probe tag and not on the transcript itself to ensure an accurate transcript count with less sequencing bias. RNA probe counts were selected following sequencing quality control (QC) and probe QC according to NanoString protocols. Please see Supplemental Methods for detailed experimental protocol.

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