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High mobility group box 1 promotes endothelial cell angiogenic behavior in vitro and improves muscle perfusion in vivo in response to ischemic injury 

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Presentation on theme: "High mobility group box 1 promotes endothelial cell angiogenic behavior in vitro and improves muscle perfusion in vivo in response to ischemic injury "— Presentation transcript:

1 High mobility group box 1 promotes endothelial cell angiogenic behavior in vitro and improves muscle perfusion in vivo in response to ischemic injury  Ulka Sachdev, MD, Xiangdong Cui, MD, Guiying Hong, MD, Seung Namkoong, PhD, Jenny M. Karlsson, MS, Catherine J. Baty, DVM, PhD, Edith Tzeng, MD  Journal of Vascular Surgery  Volume 55, Issue 1, Pages (January 2012) DOI: /j.jvs Copyright © Terms and Conditions

2 Fig 1 Effect of hypoxia and serum deprivation is shown on nuclear high mobility group box 1 (HMGB1) release. A, Human dermal microvascular endothelial cells (HDMVECs) were cultured in normoxia or 1% hypoxia and fixed at 0 (time 0) or 6 hours for immunohistochemistry for HMGB1 (red, Cy3), nuclei (blue, 4',6-diamidino-2-phenylindole), and actin (green). Representative photomicrographs are shown at original magnification ×60. Experiments were performed in duplicate and repeated 3 times. FBS, Fetal bovine serum. B, Western blotting for HMGB1 and β-actin performed on cytosolic fractions from HDMVECs treated in normoxia or hypoxia in Dulbecco's modified Eagle medium with 1% or 10% FBS for 6 hours. Graph demonstrates cytosolic level of HMGB1 protein by densitometry, expressed as fold-increase above 10% serum-treated, normoxic cells. C, Western blotting for HMGB1 and β-actin performed on nuclear fractions from HDMVECs cultured in normoxia or 1% hypoxia in 1% FBS for 3 and 24 hours. Graph demonstrates nuclear levels of HMGB1 protein by densitometry, expressed as fold increase above T0 cells. The error bars show the standard error of the mean. *P < .01. Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © Terms and Conditions

3 Fig 2 Effect of exogenous high mobility group box 1 (HMGB1) and neutralizing anti-HMGB1 antibody on endothelial tube formation in vitro. A, Representative photomicrographs of endothelial cell (EC) tubing in growth-factor reduced Matrigel at 24 hours in the presence of 1 μg/mL HMGB1 or control buffer under normoxia or 1% hypoxia (arrow, tubules; star, boxes; original magnification ×40). B, EC tube formation with polyclonal anti-HMGB1 antibody. Images represent findings after 6 hours of incubation in hypoxia (original magnification ×20). C, Time course of EC tubing from 0 to 6 hours with and without 2g7 monoclonal anti-HMGB1 antibody (select images from time-lapse imaging, original magnification ×10). IgG, Immunoglobulin G. Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © Terms and Conditions

4 Fig 3 Effect of high mobility group box 1 (HMGB1) on LC3II formation. A, Serum-depleted HDMVECs were treated with increasing doses of HMGB1 and 2g7 or immunoglobulin (Ig) G (20 μg/mL). Western blotting of cell lysates was performed for light-chain 3I (LC3I), LC3II, and β-actin. B, Effect of 2g7, nonspecific IgG, and the autophagy inhibitor 3-methyladenine (3MA) on LC3II production was examined by Western blotting. Blots are representative of 3 separate experiments. Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © Terms and Conditions

5 Fig 4 The role of autophagy in endothelial tube formation in vitro. A, Endothelial cell tube formation in growth-factor reduced Matrigel was examined in cells cultured in 1% serum (to induce autophagy) in the presence of increasing doses of the autophagy inhibitors 3-methyladenine (3MA) and chloroquine (CQ). Images were obtained after 18 hours of culture (original magnification ×10, representative of 3 experiments). B, Quantification of tubing in the presence of increasing doses of 3MA or CQ was performed by counting box formation. The error bars show the standard error of the mean for experiments repeated 3 times. *P < .001 for CQ, 3MA. Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © Terms and Conditions

6 Fig 5 Effect of rapamycin, an autophagy inducer, on endothelial tubing in vitro. Human dermal microvascular endothelial cells were seeded on growth-factor reduced Matrigel in normoxic, serum-replete conditions with increasing doses of rapamycin to induce autophagy. Tubing was assessed at 4 hours of culture. Representative images are shown. Experiments were performed in duplicate and repeated. Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © Terms and Conditions

7 Fig 6 Regulation of high mobility group box 1 (HMGB1) release by autophagy. The relationship between autophagy and HMGB1 release was examined by immunohistochemistry (IHC). A, IHC was performed on human dermal microvascular endothelial cells (HDMVECs) cultured under pro- and anti-autophagy conditions to examine the cellular localization of HMGB1. HDMVECs were cultured in 1% serum and hypoxia to stimulate autophagy and 3-methyladenine (3MA) was added to block autophagy. HDMVECs were cultured in normoxia and 10% serum and were treated with rapamycin to induce autophagy. Cells were stained after fixation at 6 hours for HMGB1 (red, Cy3), nuclei (blue, 4',6-diamidino-2-phenylindole), and actin cytoskeleton (green, phalloidin). Representative photomicrographs are shown at original magnification ×60). Pink, Overlap of nuclear protein with HMGB1. B, Western blotting for HMGB1 in cytosolic fractions isolated from HDMVEC treated with hypoxia, serum deprivation, and rapamycin or (C) 3MA (representative blots of 3 separate experiments). Graphs demonstrate cytosolic level of HMGB1 protein by densitometry, expressed as fold-increase above 10% serum-treated, normoxic cells. FBS, Fetal bovine serum. The error bars show the standard error of the mean. *P < .05. Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © Terms and Conditions

8 Fig 7 Role of high mobility group box 1 (HMGB1) release in ischemic mouse hind limbs. A, Representative photomicrographs demonstrate the effect of ischemia on HMGB1 release in skeletal muscle. Mouse calf muscles were harvested 4 hours after femoral artery ligation and stained for HMGB1 (red staining; n = 3 mice/group, P < .001). B, Mice were injected with recombinant HMGB1 (20 μg) or buffer into the right hind limb, followed by right femoral artery ligation. Hind limb perfusion was assessed by laser Doppler perfusion imaging (LDPI) after 14 days. Representative LDPI images from control (n = 3) and HMGB1 (n = 4) injected animals are shown. C, The ratio of ischemic to contralateral, nonischemic limb perfusion 14 days after femoral artery ligation is demonstrated. D, Photomicrographs show vascular density by CD31 staining of ischemic hind limbs after injection with control buffer or 20 μg HMGB1 (original magnification ×40). Muscle tissues shown were harvested 14 days after the initiation of ischemia. Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © Terms and Conditions

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