Partial carotid ligation is a model of acutely induced disturbed flow, leading to rapid endothelial dysfunction and atherosclerosis

Abstract

Atherosclerosis is closely associated with disturbed flow characterized by low and oscillatory shear stress, but studies directly linking disturbed flow to atherogenesis is lacking. The major reason for this has been a lack of an animal model in which disturbed flow can be acutely induced and cause atherosclerosis. Here, we characterize partial carotid ligation as a model of disturbed flow with characteristics of low and oscillatory wall shear stress. We also describe a method of isolating intimal RNA in sufficient quantity from mouse carotid arteries. Using this model and method, we found that partial ligation causes upregulation of proatherogenic genes, downregulation of antiatherogenic genes, endothelial dysfunction, and rapid atherosclerosis in 2 wk in a p47(phox)-dependent manner and advanced lesions by 4 wk. We found that partial ligation results in endothelial dysfunction, rapid atherosclerosis, and advanced lesion development in a physiologically relevant model of disturbed flow. It also allows for easy and rapid intimal RNA isolation. This novel model and method could be used for genome-wide studies to determine molecular mechanisms underlying flow-dependent regulation of vascular biology and diseases.

Fig. 1.

Partial ligation of left common carotid artery (LCA) causes low and oscillatory flow. A: three branches of the LCA [external carotid artery (ECA), internal carotid artery (ICA), and occipital artery (OA)] were ligated in the LCA, while leaving the superior thyroid artery (STA) open. B: ultrasound showing flow velocity profiles and revealing that partial ligation induces flow reversal (indicated by arrows) in LCA during diastole. Flow in the right common carotid artery (RCA) remains unchanged after ligation. Images shown were obtained from an ApoE KO mouse and are representative of at least 20 mice deficient in apolipoprotein E (ApoE KO). Partial ligation in C57BL/6 mice results in similar flow reversal profiles (data not shown). C: partial ligation reduces blood flow through the LCA, without significantly raising flow in RCA. The dotted line indicates the preligation flow level. Shown are means ± SE, n = 4 experiments.

Fig. 2.

Computational fluid dynamics (CFD) study. Partial ligation results in low and oscillatory shear stress. A and B: CFD was carried out using the values shown in Fig. 1 (ligated ApoE KO mice). A: wall shear stress (WSS) over a cardiac cycle. B: pseudocolor images of time-averaged WSS levels over the cardiac cycle in carotid arteries before and 1 day after partial ligation.

Fig. 3.

A–C: method of intimal RNA preparation. Intimal RNA and medial + adventitial (m + a) RNA were obtained from sham-operated RCA and LCA in C57BL/6 mice. RNAs were analyzed by semiquantitative RT-PCR (A) and quantitative real-time PCR (qPCR) (B and C) for platelet endothelial cell adhesion molecule-1 (PECAM-1) and α-smooth muscle actin (α-SMA) using 18s as an internal control. Bar graphs are means ± SE, n = 3. D: partial ligation results in decreases in kruppel-like factor 2 (KLF2) and endothelial nitric oxide synthase (eNOS) while increasing bone morphogenic protein 4 (BMP4), intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1). Intimal RNA from sham and partially ligated C57BL/6 mice were collected from LCA and RCA, respectively, 2 days after surgery and analyzed by qPCR using 18S as an internal control. Data are shown as a ratio of mRNA expressed in LCA over RCA of sham and partially ligated mice. Means ± SE are shown, n = 3 sham 5 ligated. E and F: partial ligation increases ICAM-1 and VCAM-1 protein expression in LCA. C57BL/6 mice underwent partial ligation, and LCA and RCA were collected 2 days postligation. Frozen sections were stained for ICAM-1, VCAM-1, and PECAM-1 (red). Nuclei were counterstained with Hoechst (blue). Images are representative of n = 4 (E). Average staining intensity per stained area for each was quantified and shown as the mean ± SE (F).

Fig. 4.

Partial ligation induces endothelial dysfunction. Arterial rings were obtained from LCA and RCA that were partially ligated as in Fig. 1 and fed high-fat diet for 2 and 7 days in ApoE KO mice. Rings preconstricted with PGF2α were dilated with increasing concentrations of acetylcholine (A) or sodium nitroprusside (SNP; B) for endothelial-independent relaxation. Shown are means ± SE, n = 2 for 2 days, and 6 for 7 days.

Fig. 5.

Partial ligation and high-fat diet rapidly induce atherosclerosis in LCA of ApoE KO mice, and this atherogenesis is delayed in mice deficient in p47^phox in ApoE KO background (p47^phox_ApoE DKO). ApoE KO and p47^phox_ ApoE DKO mice were partially ligated and fed the high-fat diet for 1–3 wk. Shown are representative images of at least n = 6 (A). Frozen sections from LCA were stained with Oil red O. Intimal lesion was quantified for each and shown as the mean ± SE (B).

Fig. 6.

Partial ligation and high-fat diet induce features of advanced atherosclerosis in LCA. ApoE KO mice were partially ligated and fed high-fat diet for 4 wk. Paraffin sections obtained from LCA and RCA were stained with pentachrome. Note needle-shaped cholesterol clefts (*) and intraplaque neovessels (arrows) containing red blood cells.

Fig. 7.

Partial ligation in ApoE KO mice increases superoxide production in LCA in a p47^phox NADPH oxidase-dependent manner. ApoE KO and p47^phox_ApoE DKO mice underwent partial ligation and were fed high-fat diet for 2 days. Frozen sections were stained with dihydroethidium (DHE; red), and autofluorescence of elastic laminas is shown in green (A). Shown in A are representative images of n = 3–5 mice. DHE staining intensity was quantified and shown as the mean ± SE (B).