Chun-Yu Wong, MD, Margreet R. de Vries, BSc, Yang Wang, MD, Joost R

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Vascular remodeling and intimal hyperplasia in a novel murine model of arteriovenous fistula failure Chun-Yu Wong, MD, Margreet R. de Vries, BSc, Yang Wang, MD, Joost R. van der Vorst, MD, Alexander L. Vahrmeijer, MD, PhD, Anton Jan van Zonneveld, PhD, Prabir Roy-Chaudhury, MD, PhD, Ton J. Rabelink, MD, PhD, Paul H.A. Quax, PhD, Joris I. Rotmans, MD, PhD Journal of Vascular Surgery Volume 59, Issue 1, Pages 192-201.e1 (January 2014) DOI: 10.1016/j.jvs.2013.02.242 Copyright © 2014 Society for Vascular Surgery Terms and Conditions

Fig 1 Surgical procedure. A, Dissection of the carotid artery (a) and external jugular vein (v). B, After placement of vascular clamps and ligation of the vein, an incision was made in the artery, followed by the transection of the vein. C, After the front side of the anastomosis is completed with interrupted sutures, the backside is exposed using a suture wire and by simultaneously twisting the vascular clamps 180°. D, Blood flow direction (arrows) in the arteriovenous fistula (AVF). E, Photograph of completed AVF. The white arrow depicts blood flow in the artery, and the asterisk (*) designates the venous outflow tract. Journal of Vascular Surgery 2014 59, 192-201.e1DOI: (10.1016/j.jvs.2013.02.242) Copyright © 2014 Society for Vascular Surgery Terms and Conditions

Fig 2 Morphometric parameters and fragmentation of internal elastic lamina (IEL). Venous section in the arteriovenous fistula (AVF) stained with Weigert elastin. A, The IEL, traced in black, is used to quantify the circumference. Intimal hyperplasia (IH) and luminal (lumen) area are shown. B, High magnification photomicrograph shows fragmentation of the IEL, which is indicated by the black arrow (original magnification, ×100; scale bar: 100 μm). Journal of Vascular Surgery 2014 59, 192-201.e1DOI: (10.1016/j.jvs.2013.02.242) Copyright © 2014 Society for Vascular Surgery Terms and Conditions

Fig 3 Quantification of outward remodeling (OR) and intimal hyperplasia (IH). A, OR was present in the first 14 days after arteriovenous fistula (AVF) creation. B, IH increased progressively in time, whereas the luminal area only increased in the first 14 days after AVF placement due to a reduction in OR after this time point. Furthermore, the increase in IH between consecutive time points was significant between days 14 and 28. C, The venous outflow tract was divided in four equal segments of 300 μm (A-D) and shows that the maximum IH (shaded area) was located at the site closest to the anastomosis. OR at days (D) 7, (E) 14, and (F) 28 and luminal area at days (G) 7, (H) 14, and (I) 28 were not differently distributed in the vein. At days 14 and 28, IH was the highest in the first 600 μm. J, The total amount of collagen increased in time. K, However, the amount of collagen divided by the total vessel area remained the same between the control and arterialized vein. *P < .05 compared with control. **P < .05 compared with the previous time point. Mean data are presented with the standard error of the mean (error bars). Journal of Vascular Surgery 2014 59, 192-201.e1DOI: (10.1016/j.jvs.2013.02.242) Copyright © 2014 Society for Vascular Surgery Terms and Conditions

Fig 4 Visualization of the arteriovenous fistula (AVF) with near-infrared fluorescence imaging. The proximal carotid artery and venous outflow tract are depicted by a red and blue arrow, respectively. A, Intravenous administration of indocyanine green (white) that is detected at 800 nm using a fluorescence-assisted resection and exploration (FLARE) imaging system indicates AVF patency directly after completion of the surgical intervention. B, Merged image of color video and 800-nm near-infrared fluorescence channel. C, At 28 days after surgery, the luminal area is reduced in the perianastomotic area due to intimal hyperplasia (IH). Journal of Vascular Surgery 2014 59, 192-201.e1DOI: (10.1016/j.jvs.2013.02.242) Copyright © 2014 Society for Vascular Surgery Terms and Conditions

Fig 5 Outward remodeling (OR), intimal hyperplasia (IH), and collagen distribution are shown in time. Venous sections stained with hematoxylin, phloxine, and saffron (HPS) at days 7, 14 and 28 after creation of arteriovenous fistula (AVF). A control vein (A) before arterialization shows no IH and has a modest circumference compared with the veins (B-D) after AVF placement. OR was primarily observed at (B) day 7 and (C) day 14 and was less evident at (D) day 28. E-H, Higher magnification photomicrographs of panels (A-D) shows the development of neo-IH, which starts with a (F) thrombus that has been repopularized with cells as time progresses. I-L, Sirius red staining shows the collagen content in time. M-P, Movat pentachrome staining shows fibrin-rich areas (bright red) in the intimal lesion at the different time points. (A-D, I-P: original magnification, ×100; E-H: original magnification, ×400). Partially repopularized thrombus indicated by asterisk (*), and black arrow indicates fibrin-rich area. Scale bar: 100 μm. Journal of Vascular Surgery 2014 59, 192-201.e1DOI: (10.1016/j.jvs.2013.02.242) Copyright © 2014 Society for Vascular Surgery Terms and Conditions

Fig 6 Immunohistochemical stainings. A, Immunohistochemical assessment of cells positive for α-smooth muscle actin (SMA) revealed a small band in the control vein, compared with a (B-D) time-dependant increase of α-SMA-positive cells in the intimal hyperplasia (IH) after arteriovenous fistula (AVF) surgery. E, The presence of leukocytes, which was absent in the control vein, was located in the (F-H) media and adventitia of the AVF vein at all time points. H, However, leukocytes were also found in the intima at day 14 and 28 as depicted by the black arrow. J and K, The luminal lining with cluster of differentiation (CD)31-positive endothelial cells, was disrupted at all time points but showed re-endothelialization in a time dependant manner. L, Adventitial capillaries were present at all time points (black arrow). I, The control vein showed an intact lining of endothelial cells. (A-D: original magnification, ×100; E-L: original magnification, ×200). The asterisk (*) indicates the lumen. Scale bar: 100 μm. Journal of Vascular Surgery 2014 59, 192-201.e1DOI: (10.1016/j.jvs.2013.02.242) Copyright © 2014 Society for Vascular Surgery Terms and Conditions