Elucidating the role of Protein Kinase RNA-like ER Kinase (PERK)-mediated unfolded protein response in modulating smooth muscle cells during atherosclerosis

Abstract

Atherosclerosis is the main underlying cause of cardiovascular disease (CVD) and is characterized by the buildup of inflammatory lipid-rich deposits containing smooth muscle cells (SMCs), fibroblasts, endothelial cells, and various infiltrating immune cells including macrophages and T-cells in the arterial vessel. The size and cellular composition of these lesions impact their stability, with less stable lesions imposing increased CVD pathological risk. Atherosclerotic lesion development begins within the first two decades of life, and while existing lipid-lowering therapies can significantly reduce atherosclerotic burden, additional strategies to bias developing lesions towards stability will further reduce residual CVD vulnerability. During lesion development, SMCs de-differentiate and modulate to form non-classical SMC-derived cell types (SDCs) in a process termed phenotype switching. Together, SMCs and SDCs form the majority of a lesion’s cellular content and can therefore influence lesion stability. However, little is known about SDC function, the factors driving their modulation, and their effect on CVD outcomes. A recent paper from Chattopadhyay et al. linked the modulation of a subset of SDCs to the Perk unfolded protein response (UPR). The UPR describes a three-pronged approach to reinstating endoplasmic reticulum (ER) homeostasis in response to ER stress. Perk, an ER stress sensor and activator of one prong of the UPR pathway, was reported to be necessary in the formation and expansion of a subset of SDCs in atherogenic mice. The UPR has been studied previously in the context of other diseases, and Perk inhibition was reported to influence infiltrating leukocyte proportions and function in developing lesions. However, it is not clear what role a Perk-dependent SDC population plays in atherosclerotic lesions and if this has any impact on lesion stability. Using single cell analysis (scRNA-seq) and SMC lineage-traced atherogenic mice, we assayed the effect of Perk on SMC modulation. As pharmacological Perk inhibition resulted in adverse events that confounded our efforts to characterize Perk’s role in lesion development, we created a SMC-specific, inducible Perk knockout (KO) mouse on our atherogenic SMC lineage-traced background. Across multiple timepoints of lesion development, we determined that SMC modulation was unchanged by SMC Perk depletion. Perk WT and Perk KO mice developed lesions of comparable sizes, compositions, and features predicting lesion stability. We integrated our data with scRNA-seq data from Chattopadhyay et al. and determined that the SDC population previously reported as being Perk-dependent was not entirely SMC derived nor detectable in other comparable studies. Furthermore, analysis of human carotid lesion samples failed to identify a correlation between UPR activity at the transcriptional level and CVD outcomes. Our findings suggest that the Perk UPR pathway in SMCs is not significantly coupled to its modulation, and that furthermore, UPR does not sufficiently correlate with lesion stability in human lesions to be an ideal therapeutic target for addressing residual CVD risk