Purpose: Atherosclerosis is the main driver of cardiovascular disease, with serious events such as myocardial infarction and stroke. The main risk factors for atherosclerosis, such as obesity, diabetes and lipid metabolism disorders, accumulate in metabolic syndrome, which leads to accelerated atherosclerosis development. In this study, we investigated the effects of metabolic syndrome on monocytes, a key player in atherosclerosis progression, using a mouse model.
Methods: Metabolic syndrome was induced by crossing mice with a dysfunctional leptin receptor, which leads to type II diabetes mellitus, with ApoE knockout mice, which provokes pronounced dyslipidaemia. Bone marrow of these db/dbApoE-/- mice was obtained to isolate monocytes via negative immunological magnetic isolation. Cell biology methods such as qPCR and flow cytometry were used to study molecules of interest. Migration experiments were performed using Transwell assays. CX3CL1, the cytokine ligand for the adhesion molecule CX3CR1, was immobilised on murine endothelial cells (b.End.5) to study adhesion. SHP099 was used to inhibit SHP2 tyrosine phosphatase.
Results: The db/dbApoE-/- mice showed a clinical phenotype of metabolic syndrome with dramatic increases in weight and blood glucose levels compared to the control group which was not influenced by SHP099 treatment. Diabetic conditions led to enhanced expression of CX3CR1 on monocytes, which is essential for migration into atherosclerotic plaques. db/dbApoE-/- mice monocytes exhibited elevated chemokinesis compared to wild type, reflecting increased activation. In addition, augmented chemotaxis towards the chemoattractant CX3CL1 was observed in monocytes exposed to metabolic syndrome. The same behaviour was documented in response to the important pro-atherosclerotic chemokine monocyte chemoattractant protein-1 (MCP-1). Moreover, db/dbApoE-/- monocytes adhere more intensively to immobilised CX3CL1 compared to wild-type monocytes, reflecting their pro-adhesive phenotype. Furthermore, we demonstrated that increased expression and activation of SHP2 in diabetic monocytes led to augmented expression of the CX3CR1 adhesion molecule. Treatment of mice with SHP099 reversed the pro-migratory phenotype as chemotaxis to CX3CL1 and MCP-1 was reduced to levels comparable to the levels of wildtype group. Moverover, significantly reduced adhesion of db/dbApoE-/- monocytes to CX3CL1 was observed when mice were pretreated with SHP099.
Conclusions: The presented data provide evidence for the pro-adhesive and pro-migratory phenotype caused by upregulation of the CX3CR1-CX3CL1 signalling pathway in a mouse model of metabolic syndrome. Treatment with SHP099 reversed the pro-atherosclerotic phenotype of db/dbApoE-/- monocytes, representing a novel and potential therapeutic approach to prevent the accelerated development of atherosclerosis induced by metabolic syndrome.
