Abstract Body: Background: Hyperhomocysteinemia (Hhcy), defined as plasma homocysteine concentrations >15μM, is an independent risk factor for early-onset atherosclerosis and ischemic strokes. We have previously demonstrated that complex phenotypic modulation of smooth muscle cells (SMCs) during atherogenesis is regulated in part by cholesterol-induced endoplasmic reticulum (ER) stress and PERK signaling. We also determined that activation of cytosolic stress and heat shock factor 1 (HSF1) leading to increased intracellular cholesterol biosynthesis from activation of HMG-CoA reductase (HMGCR) can augment PERK signaling (HSF1→HMGCR→PERK) and phenotypic modulation, which is responsible for early-onset atherosclerosis associated with missense variants in the gene ACTA2, that codes for SMC-specific α-actin. Here, we investigated whether HSF1→HMGCR→PERK signaling drives augmented atherosclerosis associated with Hhcy.
Methods: Wildtype (WT) and SMC-specific Perk-deficient (Perk^SMC-/-) HC mice were fed a high fat diet for 12 weeks, with and without Hcy, and atherosclerotic plaque formation was characterized using en face aortic Oil Red O staining and histopathology. Single cell transcriptomics (scRNA-seq) of aortic tissue from SMC lineage-traced mice was pursued, while SMCs explanted from ascending aortas of WT and Perk^SMC-/- mice were used to investigate atherosclerosis-associated SMC phenotypic modulation.
Results: Hcy supplementation increased atherosclerotic plaque burden in WT mice, and both inhibition of HMGCR by pravastatin, and genetic depletion of Perk from SMCs successfully reversed the augmented plaque burden associated with Hhcy. scRNA-seq indicated that Hcy exposure disrupted protein folding, activated HSF1, and augmented PERK-mediated SMC phenotypic modulation. Cellular studies confirmed that Hcy treatment activated HSF1→HMGCR→PERK signaling, and sensitized WT SMCs to undergo atherosclerosis-associated phenotypic modulation at lower concentrations of cholesterol, and this effect was completely reversed in Perk-deficient SMCs.
Conclusion: These data establish a novel mechanism by which high Hcy levels in SMCs predispose to early onset atherosclerosis. Targeting components of HSF1→HMGCR→PERK signaling in SMCs, including the use of statins – which is not currently used commonly in Hhcy patients - could prove effective against augmented atherosclerosis in these patients.