Spatial proteomics reveals the dynamic phenotypic landscape of vascular smooth muscle cells (VSMCs) in atherosclerosis

Atherosclerosis, the primary cause of cardiovascular diseases, is marked by plaque accumulation in arteries, which consist of fatty and fibrous material. Identifying high-risk plaques and effective therapies remains difficult due to limited understanding of underlying mechanisms. Vascular smooth muscle cells (VSMCs) are critical in atherosclerosis progression and plaque stability, exhibiting remarkable plasticity with either atheroprotective or atherogenic roles. To explore the mechanisms behind these roles in plaque formation, we employed in vitro and spatial in situ proteomics.

We initially treated primary carotid SMCs with various stimuli, inducing diverse phenotypes including contractile, synthetic, osteogenic, macrophage-like and inflammatory states. Subsequently, we applied an integrative proteomics approach, encompassing cellular, secreted and phospho proteomics, to generate an extensive quantitative profile of VSMC phenotypes in vitro. This quantitatively profiled more than 9,000 proteins, including regulatory members of TGFβ, JAK-STAT, NFκB and TNFα signaling pathways. Our findings reveal distinct shifts in protein expression patterns corresponding to VSMC phenotypes, revealing links between plasticity, key signaling pathways and extracellular matrix (ECM) remodeling.

To extend our findings to clinically relevant samples, we analyzed formalin-fixed paraffin-embedded (FFPE) human carotid plaques by Deep Visual Proteomics (DVP). Our approach integrates immunofluorescence imaging, automated cellular segmentation and spatial clustering with laser microdissection and ultra-high sensitive quantitative mass spectrometry. It allowed us to individually dissect and in situ analyze the spatial distribution of VSMC phenotypes in plaque microtomes. For each in vivo VSMC population, we achieved a median protein depth of 4,000 protein groups, and hallmark VSMC proteins such as smooth muscle actin (ACTA2) and transgelin (TAGLN) were highly abundant, confirming proteomic specificity. Principal component analysis (PCA) revealed that dissected cellular populations cluster according to their spatial location in the plaque. Remarkably, unsupervised clustering identified two clusters, one associated with upregulation of smooth muscle contraction and ECM organization, and the other with inflammatory and lipid processes. This opens up for direct study of various VSMC phenotypes, such as contractile and inflammatory states in situ. Finally, we used phenotype-specific signatures of the in vitro experiments to assign proteomes to the in situ populations.

In summary, our study leverages advanced proteomics to enhance understanding of VSMC biology in atherosclerosis, identifying novel proteins associated with VSMC phenotypic changes, potentially serving as therapeutic targets.