A novel direct inducible nongenetic murine model of diabetes-aggravated atherosclerosis
Authors: Gaul S , Shahzad K , Medert R , Gadi I , Maeder C , Schumacher D , Wirth A , Fatima S , Boeckel J N , Khawaja H , Brune M , Nawroth P P , Isermann B , Laufs U , Freichel M 
Published: October 2022

Abstract

Background and aims
Atherosclerosis, the main pathology underlying cardiovascular diseases is accelerated in diabetic patients (1,2). Genetic mouse models require breeding efforts which are time-consuming and costly. Our aim was to establish a new nongenetic model of inducible metabolic risk factors that mimics hyperlipidemia, hyperglycemia, or both and allows the detection of phenotypic differences dependent on the metabolic stressor(s) on any genetic background.

Methods and results
Wild type mice were injected with gain-of-function PCSK9D377Y (proprotein convertase subtilisin/kexin type 9) mutant adeno-associated viral particles (AAV) and streptozotocin (STZ) and fed either a high-fat diet (HFD) or high-cholesterol/high fat-diet (Paigen diet, PD) for 12 and 20 weeks. LDLR KO mice were used as reference control. Combined hyperlipidemic and hyperglycemic mice (HGHCi), but not hyperlipidemia (HCi) alone, displayed characteristic features of aggravated atherosclerosis characterized by larger and less stable plaques (necrotic core area in HGHCi HFD: 24% vs HCi HFD: 13% vs LDLR KO HFD: 18% area, at 20 weeks p&lt;0.05; fibrous cap thickness in HGHCi: 13 μm vs HCi HFD: 23 μm vs LDLR KO HFD: 17 μm, at 20 weeks, p&lt;0.05) which contained more macrophages (MOMA-2 in HGHCi HFD: 27% vs HCi HFD: 19% vs LDLR KO HFD: 46% area/plaque, at 20 weeks, p&lt;0.05) and less smooth muscle cells (α-SMA in HGHCi HFD: 12% vs HCi HFD: 25% vs LDLR KO HFD: 18% area/plaque, at 20 weeks, p&lt;0.05), on both HFD or PD diet. Diabetic atherosclerotic mice (HGHCi) fed a HFD showed 37% plaque area (of total lumen) compared to 16% plaque area in non-diabetic mice (HCi HFD) and 17% in LDLR KO HFD after 12 weeks; and 43% (HGHCi HFD) vs. 29% (HCi HFD) vs 39% plaque area (LDLR KO HFD) after 20 weeks (Figure 1A, B). Differences between the diabetic HGHCi and non-diabetic HCi HFD mice were confirmed using RNA-seq analysis of aortic tissue, revealing that significantly more genes were dysregulated in mice with combined hyperlipidemia and hyperglycemia than in the hyperlipidemia-only group. The HGHCi-associated genes were related to pathways regulating inflammation (increased Cd68, iNos, and Tnfa expression) and extracellular matrix degradation (Adamts4 and Mmp14) (Figure 1C). When comparing HFD with PD, the PD aggravated atherosclerosis to a greater extent in mice and showed plaque formation after 8 weeks (HGHCi PD: 48% plaque area vs. HCi PD: 30% plaque area), therefore, representing a direct inducible hyperglycemic atherosclerosis model compared with HFD-fed mice, in which atherosclerosis is severe by 8 weeks.

Conclusion
We established a nongenetic direct inducible mouse model of diabetes-aggravated atherosclerosis allowing comparative analyses of atherosclerosis in diabetic and non-diabetic conditions and its modification by diet, allowing analyses of multiple metabolic hits in mice.

Funding Acknowledgement
Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft (DFG)