Early Adventitial Activation and Proliferation in a Mouse Model of Arteriovenous Stenosis: Opportunities for Intervention

Abstract

Background: Arteriovenous fistula (AVF) stenosis remains an important cause of AVF maturation failure, for which there are currently no effective therapies. We examined the pattern and phenotype of cellular proliferation at different timepoints in a mouse model characterized by a peri-anastomotic AVF stenosis.

Methods: Standard immunohistochemical analyses for cellular proliferation and macrophage infiltration were performed at 2, 7 and 14 d on our validated mouse model of AVF stenosis to study the temporal profile, geographical location and cellular phenotype of proliferating and infiltrating cells in this model.

Results: Adventitial proliferation and macrophage infiltration (into the adventitia) began at 2 d, peaked at 7 d and then declined over time. Surprisingly, there was minimal macrophage infiltration or proliferation in the neointimal region at either 7 or 14 d, although endothelial cell proliferation increased rapidly between 2 d and 7 d, and peaked at 14 d.

Conclusions: Early and rapid macrophage infiltration and cellular proliferation within the adventitia could play an important role in the downstream pathways of both neointimal hyperplasia and inward or outward remodelling.

Keywords: adventitia; arteriovenous fistula; macrophage; proliferation.

Figure 1

Mouse AVF Model Histology: (a–d) Note the rapid increase in neointimal hyperplasia from 2 d to 7 d to 14 d. Double-headed arrows in white and black indicate the extent of the neointimal hyperplasia; SMA = smooth muscle alpha actin.

Figure 2

Semi-quantitative scoring for cellular proliferation and macrophage infiltration.

Figure 3

Cellular proliferation in the mouse AVF stenosis model: (a–c) show the immunohistochemistry for Ki-67 at 2 d, 7 d and 14 d, respectively. Note the early (2 d) and persistent (7 d and 14 d) presence of cellular proliferation in our mouse AVF stenosis model. (d,e) show our semi-quantitative scoring system for Ki-67 across different time points and different regions of the AV anastomosis. Small black arrows indicate proliferating cells; Endo = luminal side; Adv = adventitial side.

Figure 4

Macrophage infiltration in the mouse AVF stenosis model: (a–c) show the immunohistochemistry for Mac-2 at 2 d, 7 d and 14 d, respectively. Note the early (2 d adventitia) and persistent (7 d and 14 d) presence of macrophage infiltration in our mouse AVF stenosis model, which is predominantly located in the adventitia but also increases in the neointimal region by 14 d. (d,e) show our semi-quantitative scoring system for Mac-2 across different timepoints and different regions of the AV anastomosis. Small black arrows indicate macrophages; Endo = luminal side; Adv = adventitial side.

Figure 5

Mouse AVF surgery and histology techniques: (a) describes the final AVF between the side of the carotid artery (red arrow) and the end of the jugular vein (blue arrow). (b) (lower panel) documents the technique that we will use to section the anastomosis. Black arrows indicate direction of blood flow; green double-headed arrows indicate how we sectioned the anastomotic region. Heart and Head labels help to indicate the directionality of flow in both panels.