Name: murine aortic crush injury: an efficient in vivo model of smooth muscle cell proliferation and endothelial function
Uploaded: 2017-06-11T14:00:00
Description: Watch this Scientific Journal Video about Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function at JoVE.com

We thank Hsia Ru-ching PhD, from the Electron Microscopy Core Facility of University of Maryland School of Medicine, for her technical support in processing the scanning electronic microscopy samples.

Ethics Statement: The protocols for animal handling were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Maryland (protocol number 0416009) and conducted according to AAALAC-International standards.
1. Surgical Procedure
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We have characterized the effects of a murine aortic injury model that results in medial hyperplasia and endothelial barrier dysfunction. Partial EC detachment along the aorta intima accompanied the loss of cell-cell contact and enhancement of cell protrusions. Correspondingly, endothelial barrier function was significantly impaired, which stimulated the mitogen-sensitive signaling pathways, leading to proliferation of VSMCs and thickening of the vessel wall. The strengths of this model is that it is technically easier t...

Restenosis following cardiovascular procedures (bypass surgery, angioplasty, or stenting) is a significant problem reducing the durability of these procedures. All revascularization procedures are plagued by restenosis. Present strategies to prevent restenosis (drug-eluting stents and drug-coated balloons) inhibit both vascular smooth muscle cell (VSMC) and endothelial cell proliferation (EC). Consequently, these interventions prevent VSMC mediated restenosis, but also prevent the regeneration of the endothelium. Without an intact endothelium, patients are required to be on potent antiplatelet agents to decrease the risk of in situ thrombosis at the risk of bleeding complications. An ideal therapy would inhibit VSMC proliferation while promoting regeneration of the endothelium. Thus, there is a need to simultaneously study VSMC proliferation and endothelial barrier function in vivo.
Presently, there are several mouse models of restenosis 1. These models include carotid ligation and femoral artery wire injury 2. Aortic models include stent placement 3, balloon injury 4, and aortic allograft 5. All of the present models are limited. Carotid ligation generates a flow-mediated neointimal lesion and does not have endothelial injury. Additionally, both carotid and femoral arteries have many fold fewer cell layers than human vessels, limiting their translational value. The mouse aorta which is approximately 1.3 mm in diameter, is the only vessel that approximates a clinically relevant (coronary) human artery (3).
Despite the translational potential of murine aortic models of disease, current models have limitations. These models require advanced microsurgical skills and specialized equipment such as angioplasty balloons and stents. Herein, we present a novel, reproducible technique to simultaneously induce VSMC proliferation and disrupt endothelial barrier function.

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murine aortic crush injury: an efficient in vivo model of smooth muscle cell proliferation and endothelial function

Arterial reconstruction, whether angioplasty or bypass surgery, involves iatrogenic trauma causing endothelial disruption and vascular smooth muscle cell (VSMC) proliferation. Common murine models study small vessels such as the carotid and femoral arteries. Herein we describe an in vivo system in which both VSMC proliferation and endothelial barrier function can be simultaneously assessed in a large vessel. We studied the infrarenal aortic response to injury in C57BL/6 mice. The aorta was injured from the left renal vein to the aortic bifurcation by 30 transmural crushes of 5-seconds duration with a cotton-tipped applicator. Morphological changes were assessed with conventional histology. Aorta wall thickness was measured from the luminal surface to the adventitia. EdU integration and counter staining with DAPI and alpha-actin was used to demonstrate VSMC proliferation. Activation of ERK1/2, a known moderator of intimal hyperplasia formation, was determined by Western Blot analysis. The effect of inflammation was determined by immunohistochemistry for B-cells, T-cells, and macrophages. En face sections of endothelium were visualized with scanning electron microscopy (SEM). Endothelial barrier function was determined with Evans Blue staining. Transmural injury resulted in aortic wall thickening. This injury induced VSMC proliferation, most prominently at 3 days after injury, and early activation of ERK1/2 and decreased p27kip1 expression. Injury did not result in increased B-cells, T-cells, or macrophages infiltration in the vessel wall. Injury caused partial endothelial cell denudation and loss of cell-cell contact. Injury resulted in a significant loss of endothelial barrier function, which returned to baseline after seven days. The murine transmural blunt aortic injury model provides an efficient system to simultaneously study both VSMC proliferation and endothelial barrier function in a large vessel.

Transverse sections aorta embedded in OCT were sectioned, and stained with hematoxylin and eosin then counter stained with Verhoeff-Van Gieson (VVG) stain to identify the internal and external elastic lamina 7. Crush injury induced aortic wall thickening compared to the aortas of animals treated with a sham procedure (laparotomy and small bowel mobilization alone). Wall thickness, as assessed by the distance from adventitia to the lumen, was greatest three days aft...

Watch this Scientific Journal Video about Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function at JoVE.com

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

Restenosis following cardiovascular procedures (bypass surgery, angioplasty, or stenting) is a significant problem reducing the durability of these procedures. An ideal therapy would inhibit smooth muscle cell (VSMC) proliferation while promoting regeneration of the endothelium. We describe a model for simultaneous assessment of VSMC proliferation and endothelial function in vivo.

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

Arterial reconstruction, whether angioplasty or bypass surgery, involves iatrogenic trauma causing endothelial disruption and vascular smooth muscle cell ...

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