A wild-type mouse-based model for the regression of inflammation in atherosclerosis

Abstract

Atherosclerosis can be induced by the injection of a gain-of-function mutant of proprotein convertase subtilisin/kexin type 9 (PCSK9)-encoding adeno-associated viral vector (AAVmPCSK9), avoiding the need for knockout mice models, such as low-density lipoprotein receptor deficient mice. As regression of atherosclerosis is a crucial therapeutic goal, we aimed to establish a regression model based on AAVmPCSK9, which will eliminate the need for germ-line genetic modifications. C57BL6/J mice were injected with AAVmPCSK9 and were fed with Western diet for 16 weeks, followed by reversal of hyperlipidemia by a diet switch to chow and treatment with a microsomal triglyceride transfer protein inhibitor (MTPi). Sixteen weeks following AAVmPCSK9 injection, mice had advanced atherosclerotic lesions in the aortic root. Surprisingly, diet switch to chow alone reversed hyperlipidemia to near normal levels, and the addition of MTPi completely normalized hyperlipidemia. A six week reversal of hyperlipidemia, either by diet switch alone or by diet switch and MTPi treatment, was accompanied by regression of atherosclerosis as defined by a significant decrease of macrophages in the atherosclerotic plaques, compared to baseline. Thus, we have established an atherosclerosis regression model that is independent of the genetic background.

Fig 1. Hypercholesterolemia induced by recombinant Adeno-Associated Virus (rAAV)8-D377Y-murine Proprotein Convertase Subtilisin/Kexin type 9 (mPCSK9) in chow diet-fed mice.

A. Serum levels of murine PCSK9 protein in mice injected with AAVmPCSK9 virus, AAVnull–injected or saline-injected control mice. **P<0.01. B. Liver LDL receptor protein levels analyzed by western blot and normalized to α-tubulin (n = 3). C. Plasma total cholesterol of chow diet—fed mice. **P<0.01 compared with control, ***P<0.01 compared with control and AAVmPCSK9. D. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured at the same time point and determined as described in Materials and Methods. Values are mean ± SEM; n = 3.

Fig 2. Hypercholesterolemia in AAVmPcsk9-injected, Western diet-fed mice can be reversed by a diet switch and MTPi treatment.

A. Experimental design: C57BL/6J mice were i.p. injected once with AAVmPcsk9 and were placed on a Western diet for 16 weeks. At 16 weeks, the baseline group of mice was sacrificed and the remaining were continued with Western diet (progression group) or switched to chow (chow regression group) or MTPi diet (MTPi regression group), for another 6 weeks. B. Plasma levels (mg/dL) of total cholesterol in baseline, progression, chow regression and MTPi regression groups. ***P<0.001 when compared with base-line, # p<0.01 when compared with chow regression group. Values are mean±SEM; n = 9–11. C. Fast protein liquid chromatography (FPLC) profile of cholesterol (Chol) and triglyceride (TG) in pooled samples at the end of the experiment. CM/VLDL, chylomicron and very low-density lipoprotein; HDL, high-density lipoprotein; IDL/LDL, intermediate density lipoprotein.

Fig 3. Regression of atherosclerosis induced by recombinant Adeno-Associated Virus (rAAV) 8-D377Y-murine Proprotein Convertase Subtilisin/Kexin type 9 (mPCSK9).

A. Plaques of aortic root sections stained for macrophages (CD68) and (B.) Oil Red O from baseline, progression, chow regression and MTPi regression groups. (magnification ×10). C. Quantitative analysis of atherosclerotic lesion size in CD68–stained aortic sections, *p < 0.05 compared with progression group. D. Quantitative analysis of CD68-positive area in aortic sections. E. Quantitative analysis of CD68-positive area as percentage of plaque area, in aortic sections. *p < 0.05 compared with base-line group and progression group. Values are mean ± SEM; n = 5–9 in each group.

Fig 4. A. AAVmPCSK9 and MTPi do not contribute to liver toxicity induced by Western diet in mice.

A. Representative H&E stained mouse liver sections of baseline, progression, chow regression and MTPi regression groups. “Western only” mice were saline-injected and Western diet-fed, serving as control for hepatotoxicity induced by Western diet alone. B. Plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST), were measured at the same time point and determined as described in Materials and Methods. *** p-value<0.001 compared with ALT values of baseline, progression and Western diet-only groups. Values are mean ± SEM; (n = 5).