Modulation of Arterial Intima Stiffness by Disturbed Blood Flow

Abstract Background The intima, comprising the endothelium and the subendothelial matrix, plays a crucial role in the development of atherosclerotic plaques, especially in bifurcations and curved segments of arteries. The mechanical stress arising from disturbed blood flow (d-flow) and the stiffening of the arterial wall contributes to endothelial dysfunction. However, the specific impacts of these physical forces on the mechanical environment of the intima remain undetermined. To address this gap in knowledge, we investigated whether inhibiting collagen crosslinking could ameliorate the detrimental effects of persistent d-flow on the mechanical properties of the intima. Methods To explore this hypothesis, we performed partial ligation (PCL) of the left carotid artery (LCA) in male and female C57BL/6J mice, inducing d-flow. The right carotid artery (RCA) served as an internal control. Carotids were collected two days and two weeks after PCL to study acute and chronic effects of d-flow on the mechanical phenotype of the intima. To decouple the chronic effects of d-flow from the ensuing arterial wall stiffening, we used subcutaneous implants delivering either phosphate-buffered saline (Saline) or 150 mg/kg/day of β-aminopropionitrile (BAPN), an inhibitor of elastin and collagen crosslinking lysyl oxidase (LOX) and LOX-like (LOXL) enzymes. Atomic force microscopy (AFM) measurements allowed us to determine stiffness of the endothelium and the denuded subendothelial matrix in en face carotid preparations. In addition, we determined the stiffness of human aortic endothelial cells (HAEC) cultured on soft and stiff hydrogels. Results Acute exposure to d-flow caused a slight decrease in endothelial stiffness in male mice but had no effect on the stiffness of the subendothelial matrix in either sex. Regardless of sex, the intact endothelium was softer than the subendothelial matrix. In contrast, exposure to chronic d-flow led to a substantial increase in the endothelial and subendothelial stiffness in both sexes. The effects of chronic d-flow were largely prevented by concurrent BAPN administration. Notably, the subendothelial matrix of ligated, BAPN-treated arteries was softer than that of unligated, saline-treated counterparts. Furthermore, HAEC displayed reduced stiffness when cultured on soft vs. stiff hydrogels. Conclusions Exposure to chronic d-flow results in marked stiffening of arterial intima, which can be effectively prevented by pharmacological inhibition of LOX/LOXL enzymes. Highlights Acute exposure to d-flow slightly softens the endothelium in males. Chronic exposure to d-flow causes stiffening of the arterial intima. Inhibition of LOX/LOXL enzymes prevents intimal stiffening arising from chronic d-flow.