AFM-based nanoindentation indicates an impaired cortical stiffness in the AAV-PCSK9DY atherosclerosis mouse model

Abstract

Investigating atherosclerosis and endothelial dysfunction has mainly become established in genetically modified ApoE−/− or LDL-R−/− mice transgenic models. A new AAV-PCSK9DYDY mouse model with no genetic modification has now been reported as an alternative atherosclerosis model. Here, we aimed to employ this AAV-PCSK9DY mouse model to quantify the mechanical stiffness of the endothelial surface, an accepted hallmark for endothelial dysfunction and forerunner for atherosclerosis. Ten-week-old male C57BL/6 N mice were injected with AAV-PCSK9DY (0.5, 1 or 5 × 1011 VG) or saline as controls and fed with Western diet (1.25% cholesterol) for 3 months. Total cholesterol (TC) and triglycerides (TG) were measured after 6 and 12 weeks. Aortic sections were used for atomic force microscopy (AFM) measurements or histological analysis using Oil-Red-O staining. Mechanical properties of in situ endothelial cells derived from ex vivo aorta preparations were quantified using AFM-based nanoindentation. Compared to controls, an increase in plasma TC and TG and extent of atherosclerosis was demonstrated in all groups of mice in a viral load-dependent manner. Cortical stiffness of controls was 1.305 pN/nm and increased (10%) in response to viral load (≥ 0.5 × 1011 VG) and positively correlated with the aortic plaque content and plasma TC and TG. For the first time, we show changes in the mechanical properties of the endothelial surface and thus the development of endothelial dysfunction in the AAV-PCSK9DY mouse model. Our results demonstrate that this model is highly suitable and represents a good alternative to the commonly used transgenic mouse models for studying atherosclerosis and other vascular pathologies.

Incidence of microvascular dysfunction is increased in hyperlipidemic mice, reducing cerebral blood flow and impairing remote memory

“…Afterwards, AAV-PCSK9 DY -injected mice were fed a WD (WD, EF TD88137 mod. +1.25% cholesterol, Sniff Spezialdiäten GmbH, Metabolizable Energy 19.1 MJ/kg, 42kJ% fat, 15kJ% protein, 43kJ% carbohydrates; Crude Nutrients [%], crude protein (N x 6.25) 17.3, crude fat 21.1, crude fibre 5.0, crude ash 4.2, starch 13.4, sugar 34.1, N free extracts 48.7, cholesterol 12,950 mg/kg) for 3 months, while the saline-treated controls received standard maintenance chow ( 12 ). Body weight was monitored and body composition was determined at week 15 using the nuclear magnetic resonance (NMR) method (Minispec BCA analyzer, LF-110, Bruker) 2 h after the mice were transferred to the NMR room to acclimatize to the environment.…”

“…Body weight was monitored and body composition was determined at week 15 using the nuclear magnetic resonance (NMR) method (Minispec BCA analyzer, LF-110, Bruker) 2 h after the mice were transferred to the NMR room to acclimatize to the environment. For the measurements, mice were put into a restrainer, which was then placed in the analyzer ( 12 , 17 ). Cognition of mice was determined from week 11 to week 14 by open-field (OF), object place recognition (OPR), elevated plus maze (EPM), as well as Barnes maze (BM) tests as previously described ( 12 , 17 ).…”

“…For the measurements, mice were put into a restrainer, which was then placed in the analyzer ( 12 , 17 ). Cognition of mice was determined from week 11 to week 14 by open-field (OF), object place recognition (OPR), elevated plus maze (EPM), as well as Barnes maze (BM) tests as previously described ( 12 , 17 ). At the end of the study, CBF was determined by ASL-MRI at week 16 ( 17 ).…”

“…PCSK9 inhibition is, therefore, a rational therapeutic target in treating patients suffering from atherosclerosis ( 9 ). In contrast, the single injection of the PCSK9 DY mutant results in hypercholesterolemia and accelerated atherosclerosis formation ( 10 , 11 ) accompanied by the development of endothelial dysfunction of the aorta ( 12 ), while it is not yet known whether the integrity and functionality of the cerebral vessels in particular is also deteriorated. We deliberately used the PCSK9 DY /WD based atherosclerosis model in contrast to the ApoE or LepR ko-models, as this model does not require any breeding effort and low (cost) effort in terms of the 3R rule and we intend to continue this approach in follow-up (therapy) studies by also using transgenic mouse lines.…”

Modulation of Arterial Intima Stiffness by Disturbed Blood Flow

“…[11][12][13] By surgically inducing d-flow in a previously healthy and straight arterial segment, the development of atherosclerotic lesions can be rapidly stimulated in hyperlipidemic mice. 11,14,15 In the presence of physiological levels of cholesterol and triglycerides, however, prolonged exposure (~two weeks) to PCL-induced d-flow leads to arterial wall remodeling and stiffening, similar to the effects of aging. 16 This model offers, therefore, a unique opportunity to study how mechanical forces contribute to abnormal molecular patterns and cellular phenotypes that drive the progression of atherosclerosis.…”