A Model of Disturbed Flow-Induced Atherosclerosis in Mouse Carotid Artery by Partial Ligation and a Simple Method of RNA Isolation from Carotid Endothelium

Abstract

Despite the well-known close association, direct evidence linking disturbed flow to atherogenesis has been lacking. We have recently used a modified version of carotid partial ligation methods to show that it acutely induces low and oscillatory flow conditions, two key characteristics of disturbed flow, in the mouse common carotid artery. Using this model, we have provided direct evidence that disturbed flow indeed leads to rapid and robust atherosclerosis development in Apolipoprotein E knockout mouse. We also developed a method of endothelial RNA preparation with high purity from the mouse carotid intima. Using this mouse model and method, we found that partial ligation causes endothelial dysfunction in a week, followed by robust and rapid atheroma formation in two weeks in a hyperlipidemic mouse model along with features of complex lesion formation such as intraplaque neovascularization by four weeks. This rapid in vivo model and the endothelial RNA preparation method could be used to determine molecular mechanisms underlying flow-dependent regulation of vascular biology and diseases. Also, it could be used to test various therapeutic interventions targeting endothelial dysfunction and atherosclerosis in considerably reduced study duration.

Alternative Splicing of Endothelial Fibronectin Is Induced by Disturbed Hemodynamics and Protects Against Hemorrhage of the Vessel Wall

“…This severely reduces mean blood velocity in the ligated carotid artery (by ∼75%, Figure 1 & 4) and results in the pro-inflammatory activation of the endothelium 23 . We collected RNA from the intima of these mice by a Trizol flush following published methods, yielding a 3000-fold enrichment of endothelial mRNA Pecam1 (CD31) relative to smooth muscle mRNA acta2 ( alpha-smooth muscle actin) in the intimal preparation relative to the remainder of the carotid artery (Figure IA online) 23, 24 . Purified intimal RNA was pooled from three groups of 4-5 vessels for each flow regime: low-flow (ligated) or high-flow (contralateral to the ligated artery).…”

Role of Flow-Sensitive microRNAs in Endothelial Dysfunction and Atherosclerosis

“…It is interesting that miR-21 was upregulated by both LS and OS when compared to static conditions, suggesting that the mechano-sensitivity of miR-21 is a transient adaptive response to imposed shear. Interestingly, miR-21 was increased in arterial endothelium exposed to d-flow as compared to that of s-flow in the mouse PCL model 4, 39 . Whether miR-21 plays a pro- or anti-atherogenic role still remains to be determined.…”

“…In this model, 3 out of 4 of the caudal branches of the left common carotid artery (LCA) are ligated, thus inducing d-flow along the entire length of the straight section of the artery. As the contralateral right common carotid artery (RCA) is left undisturbed, it continues to be exposed to s-flow 4, 39 . This model directly demonstrates the causal relationship between d-flow and atherosclerosis, as the LCA develops atherosclerosis within two weeks of surgery, while the RCA remains plaque-free.…”

Pro-atherogenic role of smooth muscle Nox4-based NADPH oxidase

“…Endothelial dysfunction and leukocyte adhesion initiate the development of atherosclerotic lesions. Partial left carotid artery (LCA) ligation is an established model of acutely induced, disturbed blood flow that leads to endothelial dysfunction and atherosclerosis [2–4]. The importance of smooth muscle cells (SMC) in development of atherosclerosis remains underappreciated.…”