Adeno-Associated Virus-Mediated Gain-of-Function mPCSK9 Expression in the Mouse Induces Hypercholesterolemia, Monocytosis, Neutrophilia, and a Hypercoagulative State

Abstract

Hypercholesterolemia has previously been induced in the mouse by a single intravenous injection of adeno-associated virus (AAV)-based vector harboring gain-of-function pro-protein convertase subtilisin/kexin type 9. Despite the recent emergence of the PCSK9-AAV model, the profile of hematological and coagulation parameters associated with it has yet to be characterized. We injected 1.0 × 10¹¹ viral particles of mPCSK9-AAV or control AAV into juvenile male C57BL/6N mice and fed them with either a Western-type high-fat diet (HFD) or standard diet over the course of 3 weeks. mPCSK9-AAV mice on HFD exhibited greater plasma PCSK9 concentration and lower low-density lipoprotein levels, concomitant with increased total cholesterol and non-high-density lipoprotein (non-HDL)-cholesterol concentrations, and lower HDL-cholesterol concentrations than control mice. Furthermore, mPCSK9-AAV-injected mice on HFD exhibited no signs of atherosclerosis at 3 weeks after the AAV injection. Hypercholesterolemia was associated with a thromboinflammatory phenotype, as neutrophil levels, monocyte levels, and neutrophil-to-lymphocyte ratios were higher and activated partial thromboplastin times (aPTTs) was lower in HFD-fed mPCSK9-AAV mice. Therefore, the mPCSK9-AAV is a suitable model of hypercholesterolemia to examine the role of thromboinflammatory processes in the pathogenesis of cardiovascular and cerebrovascular diseases.

Keywords: PCSK9; coagulation; hypercholesterolemia; monocytes; mouse; neutrophils.

Figure 1

Verification of the mPCSK9-AAV mouse model of hypercholesterolemia. (A) Body weights of the four experimental groups: Control AAV + Standard diet (black circles, n = 12), Control AAV + HFD (black squares, n = 10), mPCSK9-AAV + Standard diet (gray upward triangles, n = 11), and mPCSK9-AAV + HFD (gray downward triangles, n = 10). Mean ± SD; repeated-measures ANOVA with Holm–Sidak post-hoc test; **p ≤ 0.01, ****p ≤ 0.0001. (B) Mouse PCSK9 plasma concentration in Control AAV + Standard diet (circles, n = 7), Control AAV + HFD (squares, n = 6), mPCSK9-AAV + Standard diet (upward triangles, n = 7), and mPCSK9-AAV + HFD (downward triangles, n = 7) groups. Data are the mean of three independent replicates. Mean ± SD; two-way ANOVA with Tukey's post-hoc test; ****p ≤ 0.0001. (C,D) Hepatic Ldl receptor (Ldlr) levels in control-injected (n = 6) and mPCSK9-AAV-injected mice (n = 6) on standard diet normalized to total protein. Data are the mean of three independent replicates. Two gels were loaded per replicate. Mean ± SD; unpaired t-test; ****p ≤ 0.0001. (E) Representative images of Oil-red O-stained thoracic aortae of mPCSK9-AAV and control AAV mice on HFD. Mice did not develop atherosclerotic lesions in the selected time frame. Scale bar, 2 mm. (F–H) Serum total cholesterol (F), non-HDL cholesterol (G), and HDL cholesterol (H) in the different groups: Control AAV + Standard diet (n = 6), Control AAV + HFD (n ≥ 8), mPCSK9-AAV + Standard diet (n ≥ 9), and mPCSK9-AAV + HFD (n ≥ 7). Mean ± SD; two-way ANOVA with Tukey's post-hoc test; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. AAV, adeno-associated virus; HFD, high-fat diet; HDL, high-density lipoprotein.

Figure 2

Hematological profiling of the mPCSK9-AAV mouse model of hypercholesterolemia. Leukocytes (A), neutrophils (B), monocytes (C), lymphocytes (D), neutrophil-to-lymphocyte ratio (E), reticulocyte (F), and erythrocyte (G) levels in EDTA-blood of the experimental groups: Control AAV + Standard diet (n = 15, circles), Control AAV + HFD (n = 13, squares), mPCSK9-AAV + Standard diet (n = 12, upward triangles), and mPCSK9-AAV + HFD (n = 11, downward triangles). (H) Platelet levels in EDTA-blood of experimental groups: Control AAV + Standard diet (n = 11, circles), Control AAV + HFD (n = 8, squares), mPCSK9-AAV + Standard diet (n = 6, upward triangles), and mPCSK9-AAV + HFD (n = 7, downward triangles). Mean ± SD; two-way ANOVA with Tukey's post-hoc test; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.0005, ****p ≤ 0.0001. AAV, adeno-associated virus; HFD, high-fat diet.

Figure 3

aPTT and fibrinogen levels in mice. (A) aPTT profiles of the four experimental groups: Control AAV + Standard diet (circles, n = 8), Control AAV + HFD (squares, n = 11), mPCSK9-AAV + Standard diet (upward triangles, n = 8), and mPCSK9-AAV + HFD (downward triangles, n = 10). (B) Fibrinogen levels in the four experimental groups: Control AAV + Standard diet (circles, n = 7), Control AAV + HFD (squares, n = 10), mPCSK9-AAV + Standard diet (upward triangles, n = 9), and mPCSK9-AAV + HFD (downward triangles, n = 8). Mean ± SD; two-way ANOVA with Tukey's post-hoc test; *p ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.0001. aPTT, activated partial thromboplastin time; AAV, adeno-associated virus; HFD, high-fat diet.