Accelerated atherosclerosis development in C57Bl6 mice by overexpressing AAV-mediated PCSK9 and partial carotid ligation

Abstract

Studying the role of a particular gene in atherosclerosis typically requires a time-consuming and often difficult process of generating double-knockouts or transgenics on ApoE−/− or LDL receptor−/− background. Recently, it was reported that adeno-associated-virus-8 (AAV8) mediated overexpression of PCSK9 (AAV8-PCSK9) rapidly induced hyperlipidemia. However, using this method in C57BL6 wild-type (C57) mice, it took approximately 3 months to develop atherosclerosis. Our partial carotid ligation model is used to rapidly develop atherosclerosis by inducing disturbed flow in the left common carotid artery within 2 weeks in ApoE−/− or LDLR−/− mice. Here, we combined these two approaches to develop an accelerated model of atherosclerosis in C57 mice. C57 mice were injected with AAV9-PCSK9 or AAV9-Luciferase (control) and high-fat diet was initiated. A week later, partial ligation was performed. Compared to the control, AAV-PCSK9 led to elevated serum PCSK9, hypercholesterolemia, and rapid atherosclerosis development within 3 weeks as determined by gross plaque imaging, and staining with Oil-Red-O, Movat’s pentachrome and CD45 antibody. These plaque lesions were comparable to the atherosclerotic lesions that have been previously observed in ApoE−/− or LDLR−/− mice that were subjected to partial carotid ligation and high-fat diet. Next, we tested whether our method can be utilized to rapidly determine the role of a particular gene in atherosclerosis. Using eNOS−/− and NOX1−/y mice on C57 background, we found that the eNOS−/− mice developed more advanced lesions, while the NOX1−/y mice developed less atherosclerotic lesions as compared to the C57 controls. These results are consistent with the previous findings using double knockouts (eNOS−/−_ApoE−/− and NOX1−/y_ApoE−/−). AAV9-PCSK9 injection followed by partial carotid ligation is an effective and time-saving approach to rapidly induce atherosclerosis. This accelerated model is well-suited to quickly determine the role of gene(s) interest without generating double- or triple-knockouts.

NOTCH1 is a mechanosensor in adult arteries

“…To investigate the role of endothelial Notch1 in a pathophysiological setting, we utilized an atherosclerosis mouse model (Fig. 9a ) in which hypercholesterolemia was induced via PCSK9-AAV 38 . In brief, 2 weeks post tamoxifen, control and Notch1 ECKO animals were injected with PCSK9-AAV or control AAV and subjected to a high-fat diet for 3 months.…”

Assessment of ICAM-1 N-glycoforms in mouse and human models of endothelial dysfunction

“…ICAM-1 N-glycoforms in endothelial dysfunction [20] by a cardiovascular pathologist using the defining characteristics of fibrosis, calcification, and fatty deposits. All procedures were performed per Institutional Review Board approved protocols.…”

“…Carotid arteries were also collected from male ApoE-/-mice fed a control diet. In the second model, [8][9][10] week old, male ApoE-/-mice underwent partial ligation of the left carotid artery to induce disturbed flow as described previously [19,20]. After induction of anesthesia (5% isoflurane, followed by 2% isoflurane for maintenance of anesthesia), the external, internal, and occipital arteries were ligated with a 7-0 silk suture leaving the superior thyroid artery patent, and the resulting disturbed flow pattern was verified by ultrasound measurements using a VisualSonics VEVO3100 system.…”

Endothelial reprogramming by disturbed flow revealed by single-cell RNA and chromatin accessibility study

“…To understand how flow regulates expression of endothelial genes at the transcript and epigenetic levels in vivo, we and others used a bulk RNAs or DNAs obtained from the pooled ECs exposed to either s-flow or d-flow using the mouse partial carotid ligation (PCL) model or pig arteries (Dunn et al, 2014;Jiang et al, 2015;Ni et al, 2010;Son et al, 2013). We developed the PCL model and showed that d-flow rapidly induces, whereas s-flow prevents, robust atherosclerosis development within two weeks in hypercholesterolemic mice (Kumar et al, 2017;Nam et al, 2010;Son et al, 2013 ). The PCL model involves the ligation of 3 of 4 distal branches of the left common carotid artery (LCA) to induce d-flow, while using the contralateral right common carotid artery (RCA) that continues to be exposed to s-flow as an internal control.…”