Y. Castier, S. Lehoux, Y. Hu, G. Fonteinos, A. Tedgui, Q. Xu 

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Characterization of neointima lesions associated with arteriovenous fistulas in a mouse model  Y. Castier, S. Lehoux, Y. Hu, G. Fonteinos, A. Tedgui, Q. Xu  Kidney International  Volume 70, Issue 2, Pages 315-320 (July 2006) DOI: 10.1038/sj.ki.5001569 Copyright © 2006 International Society of Nephrology Terms and Conditions

Figure 1 Schematic representation of AVF-associated lesion formation in mice. A window was cut on the side of the jugular vein, and the carotid artery was transected distally and sutured to the vein. (a) Arrows represent direction of blood flow in the artery (A) and vein (V). (b) Lesion formation (gray shading) occurred at the site of anastomosis and on the vein wall opposite to the arterial opening. (c) Lesion size progressed with time, encroaching on the lumen, and protruding into the vein. Panel (d) shows a photograph of an AVF after performing anastomosis, with black arrows indicating the direction of blood flow. Note that the head of the mouse is at the left and the heart at the right. Kidney International 2006 70, 315-320DOI: (10.1038/sj.ki.5001569) Copyright © 2006 International Society of Nephrology Terms and Conditions

Figure 2 Hematoxylin and eosin-stained sections of mouse artery and vein at the site of AVF anastomosis. At 3 weeks after opening of the AVF, vascular segments were fixed in situ in 4% phosphate-buffered formaldehyde (pH 7.2), embedded in paraffin, sectioned, and stained with hematoxylin–eosin. A series of sections were cut in the artery at different levels approaching (a–e) the anastomosis and (f–h) in the vein. Note that the lesion size increases near (d) the anastomosis. (f) Shows a normal vein vessel wall before surgery, whereas (g and h) represent vein sections 3 weeks after AVF. Bar=50 μm. Kidney International 2006 70, 315-320DOI: (10.1038/sj.ki.5001569) Copyright © 2006 International Society of Nephrology Terms and Conditions

Figure 3 Neointimal quantification in the AVF anastomosis. Hematoxylin and eosin-stained sections were prepared as described in Figure 2. Lesion area was measured microscopically as described in the Materials and Methods. (a–d) Representative pictures of anastomoses obtained 12 h, 1 week, 2 weeks, and 3 weeks after AVF creation. Panels (e–h) are photographs at higher magnification corresponding to panels (a–d). Bar=50 μm. The graph shows mean±s.d. (n=6/group). *P<0.05 and **P<0.01 versus unoperated controls. Kidney International 2006 70, 315-320DOI: (10.1038/sj.ki.5001569) Copyright © 2006 International Society of Nephrology Terms and Conditions

Figure 4 Immunohistochemical staining demonstrates the presence of MAC-1+ mononuclear cells and SMCs in neointimal lesions 3 weeks post-AVF. Frozen sections derived from the site of anastomosis in arteries at (a and b) 1, (c) 2, and (d) 3 weeks post-AVF were probed with normal rat immunoglobulin or with a rat monoclonal antibody (CD11b/18; b–d) against MAC-1+ leukocytes. For SMC staining, sections of arteries obtained (e and f) 1, (g) 2, and 3 weeks post-AVF were probed with (e) normal mouse serum or with (f–h) a mouse monoclonal anti-α-actin antibody. A counterstaining (blue) with hematoxylin was performed. Arrows indicate typical positive cells. Bar=10 μm. The graph shows mean±s.d. (n=6/group) (i). Kidney International 2006 70, 315-320DOI: (10.1038/sj.ki.5001569) Copyright © 2006 International Society of Nephrology Terms and Conditions

Figure 5 Non-bone marrow origin of SMCs in neointimal lesions. Bone marrow cells of SM-LacZ mice were transplanted into irradiated wild-type mice 3 weeks before AVF surgery. Frozen sections were stained for (a–c) X-gal or for both X-gal and (d) α-actin. (a and b) Carotid arteries from SM-LacZ and wild-type mice, respectively. (c and d) Sections from AVF artery anatomoses. Arrows indicate the luminal surface of the vessels. Bar=50 μm. Kidney International 2006 70, 315-320DOI: (10.1038/sj.ki.5001569) Copyright © 2006 International Society of Nephrology Terms and Conditions