Recovery from hind limb ischemia is less effective in type 2 than in type 1 diabetic mice: Roles of endothelial nitric oxide synthase and endothelial.

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Date of download: 7/7/2016 Copyright © The American College of Cardiology. All rights reserved. From: Sustained Release of Erythropoietin Using Biodegradable.

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Recovery from hind limb ischemia is less effective in type 2 than in type 1 diabetic mice: Roles of endothelial nitric oxide synthase and endothelial progenitor cells Jinglian Yan, PhD, Guodong Tie, PhD, Brian Park, MD, Yagai Yan, PhD, Philip T. Nowicki, MD, Louis M. Messina, MD Journal of Vascular Surgery Volume 50, Issue 6, Pages 1412-1422 (December 2009) DOI: 10.1016/j.jvs.2009.08.007 Copyright © 2009 Society for Vascular Surgery Terms and Conditions

Fig 1 Dermal blood flow in the (top) foot and (bottom) calf was measured in the same animal at multiple times before and after induction of ischemia (n = 5 animals in each group). Flow data represent the ratio of the ischemic to nonischemic hind limb. Mean data are presented with the standard deviation (error bars). Pre, Preoperative; Post, immediately postoperative, POD, postoperative day. #P < .05 for type 1 or type 2 diabetic mice vs control mice and *P < .05 for type 2 vs type 1 diabetic mice. Journal of Vascular Surgery 2009 50, 1412-1422DOI: (10.1016/j.jvs.2009.08.007) Copyright © 2009 Society for Vascular Surgery Terms and Conditions

Fig 2 Effect of ischemia on collateral arteries was studied 28 days after the induction of ischemia (n = 5 animals in each group). A, Mean data for collateral artery diameter in thigh muscles are expressed in micrometers. The error bars indicate the standard deviation. #P < .05 for type 1 or type 2 diabetic mice vs control mice, *P < .05 for type 2 vs type 1 diabetic mice, and +P < .05 for ischemic vs nonischemic hind limb. B, In representative photomicrographs (original magnification ×200), CD31 is stained red, smooth muscle actin is stained green, and nuclei are stained blue. C, Data for angioscores represent the mean number of collateral arteries crossing fixed points on a grid superimposed on the radiograph (n = 5 animals in each group). The error bars indicate the standard deviation. *P < .05 for type 1 or type 2 diabetic mice vs control mice. D, Representative radiographs are shown for control, type 1, and type 2 diabetic mice. Films were obtained 28 days after induction of hind limb ischemia. The radiographs depict the distal one third of the mouse. Journal of Vascular Surgery 2009 50, 1412-1422DOI: (10.1016/j.jvs.2009.08.007) Copyright © 2009 Society for Vascular Surgery Terms and Conditions

Fig 3 Effect of ischemia on capillary density was studied 28 days after the induction of ischemia (n = 5 animals in each group). A, Data for capillary density in gastrocnemius muscle are expressed as the mean number of C31+ cells per myofiber, with error bars showing the standard deviation. #P < .05 for type 1 or type 2 diabetic mice vs control mice, *P < .05 for type 2 vs type 1 diabetic mice, and +P < .05 for ischemic vs nonischemic hind limb. B, In these representative photomicrographs (original magnification ×200), CD31+ cells are stained brown. Journal of Vascular Surgery 2009 50, 1412-1422DOI: (10.1016/j.jvs.2009.08.007) Copyright © 2009 Society for Vascular Surgery Terms and Conditions

Fig 4 Photomicrographs (original magnification ×200) show representative Oil Red O staining of hind limb muscles harvested 28 days after induction of ischemia, in which fat is stained red; counterstained with hematoxylin. These photomicrographs are representative of observations made in 5 animals from each group. Journal of Vascular Surgery 2009 50, 1412-1422DOI: (10.1016/j.jvs.2009.08.007) Copyright © 2009 Society for Vascular Surgery Terms and Conditions

Fig 5 Endothelial nitric oxide synthase (eNOS) and nitrotyrosine expression was measured 28 days after induction of ischemia in hind limb muscle (n = 3 mice in each group). Mean data are presented with the standard deviation (error bars). #P < .05 for type 1 or type 2 diabetic mice vs control mice, *P < .05 for type 2 vs type 1 diabetic mice, and +P < .05 for ischemic vs nonischemic hind limb. Insets, Representative Western blots; lane designations from left to right: control, nonischemic hind limb; control, ischemic hind limb; type 1 diabetes, ischemic hind limb; type 1 diabetes, nonischemic hind limb; type 2 diabetes, nonischemic hind limb; type 2 diabetes, ischemic hind limb. Expression of eNOS is shown in the (A) gastrocnemius and (B) thigh muscles. Nitrotyrosine expression is shown in the (C) gastrocnemius and (D) thigh muscles. Journal of Vascular Surgery 2009 50, 1412-1422DOI: (10.1016/j.jvs.2009.08.007) Copyright © 2009 Society for Vascular Surgery Terms and Conditions

Fig 6 Expression of (A) endothelial nitric oxide synthase (eNOS) and (B) nitrotyrosine in bone marrow-derived epithelial progenitor cells (EPCs) was measured. EPCs were harvested from the bone marrow derived from the femur and tibia from ischemic and nonischemic hind limbs 7 days after induction of ischemia (n = 3 animals in each group). Mean data are presented with the standard deviation (error bars). #P < .05 type 1 or type 2 diabetic mice vs control mice, *P < .05 type 2 vs type 1 diabetic mice, and +P < .05 ischemic vs nonischemic hind limb. Insets, Representative Western blots; lane designations from left to right: control, nonischemic hind limb; control, ischemic hind limb; type 1 diabetes, ischemic hind limb; type 1 diabetes, nonischemic hind limb; type 2 diabetes, nonischemic hind limb; type 2 diabetes, ischemic hind limb. Journal of Vascular Surgery 2009 50, 1412-1422DOI: (10.1016/j.jvs.2009.08.007) Copyright © 2009 Society for Vascular Surgery Terms and Conditions

Fig 7 The percentage of endothelial progenitor cells (EPCs) was measured in bone marrow and peripheral blood samples harvested from the femur and tibia of the nonischemic or ischemic hind limbs 7 days after induction of ischemia (n = 6 animals per group). EPCs were characterized as CD34+, Flk-1+, CD133,+ and cell quantitation was by fluorescence-activated cell-sorting analysis. Mean data are shown with the standard deviation (error bars). #P < .05 for type 1 or type 2 diabetic mice vs control mice, *P < .05 for type 2 vs type 1 diabetic mice, and +P < .05 for ischemic vs nonischemic hind limb. Journal of Vascular Surgery 2009 50, 1412-1422DOI: (10.1016/j.jvs.2009.08.007) Copyright © 2009 Society for Vascular Surgery Terms and Conditions

Fig 8 In vitro functional assays of endothelial progenitor cells (EPCs) harvested from the femur and tibia of ischemic and nonischemic hind limbs 7 days after the induction of ischemia and grown in culture for 3 days (n = 6 animals in each group). A, EPC migration assay. The white bars represent migration in response to medium alone (baseline), and the black bars represent migration in response to vascular endothelial growth factor (VEGF). Mean data are presented with the standard deviation (error bars). +P < .05 for migration in response to VEGF vs migration in response to medium, #P < .05 for type 1 or type 2 diabetic mice vs control mice. B, Representative photomicrographs (original magnification ×200) of migration assay. UE lectin is stained green, Dil-Ac-LDL is stained red, and nuclei are stained blue. C, Incorporation of EPCs into tubular structures (n = 6 animals in each group). Mean data are presented with the standard deviation (error bars). #P < .05 for type 1 or type 2 diabetic mice vs control mice. D, Representative photomicrographs (original magnification ×200) of tubular incorporation studies. The upper panels are bright field images. The lower panels are fluorescent images. EPCs are stained red. Journal of Vascular Surgery 2009 50, 1412-1422DOI: (10.1016/j.jvs.2009.08.007) Copyright © 2009 Society for Vascular Surgery Terms and Conditions