Ulka Sachdev, MD, Xiangdong Cui, MS, Edith Tzeng, MD

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HMGB1 and TLR4 mediate skeletal muscle recovery in a murine model of hindlimb ischemia Ulka Sachdev, MD, Xiangdong Cui, MS, Edith Tzeng, MD Journal of Vascular Surgery Volume 58, Issue 2, Pages 460-469 (August 2013) DOI: 10.1016/j.jvs.2012.11.071 Copyright © 2013 Terms and Conditions

Fig 1 C57BL6 (A) and C3H/HeOuJ (B) mice underwent femoral artery ligation on the right hindlimb and exposure of the femoral vessels without ligation on the left. Anterior tibialis muscles were harvested from both sides after sacrifice at 24 hours after femoral ligation. Paraffin sections were stained for high-mobility group box 1 (HMGB1) (arrow). C, Quantification of HMGB1 staining nuclei (arrow) was expressed as % total nuclei (*P < .001; †P < .05; n = 4-5; scale bar = 50 μm). Western blots for HMGB1 were performed on nuclear (D) and cytoplasmic (E) fractions of skeletal muscle from C3H/HeOuJ mice. Animal number refers to individual mice within the experiment. Journal of Vascular Surgery 2013 58, 460-469DOI: (10.1016/j.jvs.2012.11.071) Copyright © 2013 Terms and Conditions

Fig 2 A, C3H/HeOuJ mice were treated with intraperitoneal injection of polyclonal anti-high-mobility group box 1 (HMGB1) antibody (600 mg; n = 9) or IgG (n = 6) 30 minutes before femoral artery ligation. The anterior muscle compartments of both limbs were harvested at day 14 for histologic analysis (asterisk = necrotic muscle; scale bar = 50 μm. B, Scatter plot of the % muscle necrosis identified in each mouse (error bars = standard error of the mean; *P = .02). Journal of Vascular Surgery 2013 58, 460-469DOI: (10.1016/j.jvs.2012.11.071) Copyright © 2013 Terms and Conditions

Fig 3 A, Calf muscles from ischemic hindlimbs of HeJ and HeOuJ mice obtained at day 14 were paraffin embedded and stained for von Willebrand factor (vWF) (red). Note areas of fat replacement (arrow), and muscle necrosis (asterisk). Scale bar = 50 μm. B, Quantification of vascular density (vessels per high power field [HPF]), % area muscle regeneration, necrosis, and fat replacement in HeOuJ and HeJ mice. Values are obtained from four to five images per section, three sections per animal; n = 3-4 animals/group. (*P = .03; †P = .04). Journal of Vascular Surgery 2013 58, 460-469DOI: (10.1016/j.jvs.2012.11.071) Copyright © 2013 Terms and Conditions

Fig 4 A, Calf muscle from control C57B6 and receptor for advanced glycation end-products (RAGE) knockout (KO) mice were harvested 14 days after femoral ligation and stained for von Willebrand factor (vWF) (red) to evaluate vascular density (scale bar = 50 μm; n = 4 each). B, Quantification of number of vWF positive structures per high power field (HPF). Values are obtained from four to five images per section, three sections per animal. Journal of Vascular Surgery 2013 58, 460-469DOI: (10.1016/j.jvs.2012.11.071) Copyright © 2013 Terms and Conditions

Fig 5 Laser Doppler perfusion imaging was performed 14 days after femoral artery ligation in (A) control and receptor for advanced glycation end-products (RAGE) knockout (KO) mice (n = 4 each), as well as (B) HeOuJ and HeJ mice (n = 6 each). Perfusion recovery is shown as ratio between ischemic and nonischemic limb. *P < .05. Journal of Vascular Surgery 2013 58, 460-469DOI: (10.1016/j.jvs.2012.11.071) Copyright © 2013 Terms and Conditions

Fig 6 A, Human dermal microvascular endothelial cells (HDMVECs) were exposed to 1% hypoxia for 6 or 24 hours, and probed for expression of either Toll-like receptor 4 (TLR4) or (B) receptor for advanced glycation end-products (RAGE). Protein expression was determined using mean fluorescence intensity normalized to actin. Percent change in protein expression with hypoxia compared with air is depicted (C) (*P < .03; n = 4/group). Journal of Vascular Surgery 2013 58, 460-469DOI: (10.1016/j.jvs.2012.11.071) Copyright © 2013 Terms and Conditions

Fig 7 A, Phosphorylated extracellular signal-regulated kinase (P-ERK) and total ERK expression in human dermal microvascular endothelial cells (HDMVECs) exposed to high-mobility group box 1 (HMGB1) over 1 hour. B-C, Percent change in P-ERK/total ERK ratio with exposure to IgG, anti-Toll-like receptor (TLR)2, and anti-TLR4 after 30 minutes; *P < .03; n = 3. Journal of Vascular Surgery 2013 58, 460-469DOI: (10.1016/j.jvs.2012.11.071) Copyright © 2013 Terms and Conditions