First steps in unveiling drug effects on phenotypic switching of vascular smooth muscle cells in atherosclerosis

Abstract

Introduction: Cardiovascular disease remains the leading cause of death worldwide, mainly due to ischemic heart disease (IHD) and stroke, with atherosclerosis as the primary underlying mechanism. Atherosclerosis is a chronic inflammatory disease involving the accumulation of lipids, vascular smooth muscle cells (VSMCs), immune cells, and necrotic debris within arteries, leading to plaque formation and vessel occlusion. The phenotypic switching of VSMCs from contractile to synthetic cells can contribute to plaque formation and progression and, finally, to plaque instability. Two main drugs are used to treat atherosclerosis: statins and colchicine. Statins are used to lower plasma LDL cholesterol and can have pleiotropic effects. Colchicine is an anti-inflammatory agent to increase the immune response in atherosclerosis. The impact of both drugs on phenotypic switching in atherosclerosis remains poorly understood and may affect patient outcomes. Aim: This study aims to investigate the effects of atorvastatin and colchicine on the gene expression of genes associated with VSMC phenotypic switching on both non-plaque VSMCs and CEA plaque VSMCs using quantitative PCR. Additionally, we investigate the COX-2 inhibitors celecoxib and rofecoxib, which, despite being banned due to severe cardiovascular risks, are included to examine their impact on VSMC phenotypic switching-related gene expression. By comparing these drugs, this study aims to reveal how both therapeutic and risk-inducing treatments alter gene expression involved in VSMC behavior to improve treatment strategies for atherosclerosis. Results: An extensive literature review identified and narrowed down a list of genes associated with VSMC phenotypic switching to those involved in transitioning from contractile to non-contractile phenotype. Analysis of DESeq2 data from treated non-plaque VSMCs with atorvastatin, colchicine, and celecoxib revealed differential expression in these genes, leading to the selection of six key genes—KLF4, Il1β, SRF, CNN1, PTEN, and ACTA2—for further investigation. In non-plaque VSMCs, colchicine promoted contractility by upregulating contractile genes and downregulating IL1β, while celecoxib did the opposite, promoting a de-differentiated state. Atorvastatin showed mixed effects, upregulating both KLF4 and CNN1. In plaque VSMCs, atorvastatin had inconsistent effects on contractile genes, colchicine had no significant impact, and rofecoxib upregulated IL1β. Non-plaque and plaque VSMCs exhibited similar KLF4 responses but varied IL1β, SRF, CNN1, PTEN, and ACTA2 trends. Gene expression changes often showed dose-dependent responses, especially for atorvastatin. Higher drug concentrations generally led to greater expression changes. Conclusion: This study highlights the complex, dose-dependent effects of atorvastatin, colchicine, and celecoxib/rofecoxib on phenotypic switching in VSMCs. Colchicine enhances contractility by upregulating contractile genes and downregulating inflammatory markers, suggesting plaque stabilization. Conversely, celecoxib/rofecoxib inhibits contractility, stimulates immune response, and promotes a more de-differentiated VSMC phenotype, potentially contributing to plaque instability. Atorvastatin's effects are more nuanced, showing both upregulation of contractile and non-contractile genes, indicating mixed impacts on the VSMC phenotypic state. The study highlights the need for further research on KLF4's role and the dose-dependent effects of these drugs to understand their implications for atherosclerosis progression better.