Multiplex Imaging for Cell Phenotyping of Early Human Atherosclerosis

Abstract

Background Previous studies using animal models and cultured cells suggest that vascular smooth muscle cells (SMCs) and inflammatory cytokines are important players in atherogenesis. Validating these findings in human disease is critical to designing therapeutics that target these components. Multiplex imaging is a powerful tool for characterizing cell phenotypes and microenvironments using biobanked human tissue sections. However, this technology has not been applied to human atherosclerotic lesions and needs to first be customized and validated. Methods and Results For validation, we created an 8‐plex imaging panel to distinguish foam cells from SMC and leukocyte origins on tissue sections of early human atherosclerotic lesions (n=9). The spatial distribution and characteristics of these foam cells were further analyzed to test the association between SMC phenotypes and inflammation. Consistent with previous reports using human lesions, multiplex imaging showed that foam cells of SMC origin outnumbered those of leukocyte origin and were enriched in the deep intima, where the lipids accumulate in early atherogenesis. This new technology also found that apoptosis or the expression of pro‐inflammatory cytokines were not more associated with foam cells than with nonfoam cells in early human lesions. More CD68+ SMCs were present among SMCs that highly expressed interleukin‐1β. Highly inflamed SMCs showed a trend of increased apoptosis, whereas leukocytes expressing similar levels of cytokines were enriched in regions of extracellular matrix remodeling. Conclusions The multiplex imaging method can be applied to biobanked human tissue sections to enable proof‐of‐concept studies and validate theories based on animal models and cultured cells.

Keywords

• atherosclerosis
• inflammatory cytokines
• multiplex imaging
• smooth muscle cells