Endothelial specific deletion of HMGB1 increases blood pressure and retards ischemia recovery through eNOS and ROS pathway in mice

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  Fig. 1. HMGB1 mediates important roles in HUVECs. A, Immunofluorescent staining (HMGB1, red; DAPI, blue; scale bars, 25 μm) for HUVECs exposed to hypoxia (1%) for 24 h. Photomicrographs are representative of three independent experiments. B, Representative capillary-like networks (tube formation, scale bars, 50 μm) of HUVECs with or without purified recombinant human HMGB1 treatment. C and D, Representative Western blot (C) and quantitative analysis (D) of HMGB1 in the cell media of HUVECs exposed to hypoxia. E, Total tube length from each of four randomly chosen fields was quantified using the image analysis software image. F and H, HMGB1 silencing of HUVECs. F, representative blots for HMGB1 silencing of HUVECs. H, HMGB1 siRNA silenced 60% of HUVEC HMGB1 protein expression. G, J, I, Tube formation and proliferation were tested after HMGB1 siRNA transfection. G, Representative capillary-like networks of HUVECs after HMGB1 silencing. (scale bars, 100 μm). and J, Quantifications of EC network formation in HUVECs with HMGB1 siRNA. I, the proliferation of HUVECs transfected with HMGB1 siRNA was decreased by 24%. Data are the average of triplicates from single experiments that were…

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  Fig. 2. Generation and characterization of HMGB1ECKO mice. A, Schematic diagram of the transgenic mice used to generate HMGB1ECKO mice. B, PCR analysis for HMGB1ECKO mice genotype. C, IF stain of HMGB1 (red) and CD31 (white) in aortas from HMGB1ECKO and control mice. Scale bar = 50 μm. D. Characterization of MLECs. Subconfluent MLECs (third passage) grown on glass cover slips were stained with endothelial surface markers, vWF, using anti-vWF antibody (primary) and a FITC-labeled secondary antibody. eNOS antibody (primary) and a Cys 3 secondary antibody. Scale bar = 20 μm. E. Representative Western Blot of HMGB1 expression in MLEC from control and HMGB1ECKO mice. Data are the average of triplicates from single experiments that were independently repeated 3 times. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

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  Fig. 3. Impaired EDR in HMGB1ECKO mice. A, Blood pressure including systolic, mean, and diastolic blood pressures from male control and HMGB1ECKO mice at three months was measured using the noninvasive computerized tail cuff system (n = 10). *P < 0.05 vs. control. B, Response to sodium nitroprusside in mice aortas. Vessels were precontracted with phenylephrine. All values are mean ± SD. C, Relaxation response to acetylcholine of aortas obtained from control and HMGB1ECKO mice with or without l-NAME. D, response to phenylephrine of aortas from control and HMGB1ECKO mice. E, representative response curve of aortas from control and HMGB1ECKO mice. F, Relaxation response to acetylcholine of aortas from HMGB1ECKO mice with ad-HMGB1 transfection or ad-Vector transfection. G, I, Representative Western Blot and quantitative analysis of HMGB1 expression in aortas from ad-HMGB1 or ad-Vector group. H, IF stain of HMGB1 in aortas transfected with or without ad-HMGB1 from HMGB1ECKO mice. (HMGB1, red; DAPI, blue; CD31, white; scale bars, 50 μm, 100 μm for enlarged window). Data are the average of triplicates from single experiments that were independently repeated 3 times. Comparisons were performed…

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  Fig. 4. Ischemia-initiated blood flow recovery and angiogenesis were impaired in HMGB1ECKO mice. A, Representative Western Blot of HMGB1 expression in ischemic muscles on day 0, 1, 7 and 28. B, Quantitative densitometry analysis of HMGB1 expression in ischemic muscle on day 1 and 7 and 28. C, HMGB1ECKO mice developed necrotic toes at 7 days–14 days after femoral artery resection while WT littermate mice did not. Representative images for necrotic toes after surgery were shown. D, Representative images of Laser Doppler blood flow on day 0, 1, 7, 14, 21 and 28 post ischemia. E, Blood flow in ischemic hind limb was measured. Results were expressed as a ratio of the right (ischemic) to left (control, nonischemic) limb perfusion. F. Proliferated endothelial cells were marked with IF stain of CD31 and ki67. DAPI, blue; CD31, red; ki67, green; scale bars, 100 μm. G. Quantification of capillary density, calculated as the number of endothelial cells positive with both CD31 and Ki67 per field. H and I, Microvessel sprouting in aortic ring assay. Representative micrographs and statistic results of sprouting microvessels from aortic ring grown in the EGM-2 medium after 4 days were shown. vs control.…

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  Fig. 5. NO decreased and ROS increased after loss of HMGB1. A, NO was measured in serum from WT and HMGB1ECKO mice. B, ROS production in HUVECs after HMGB1 siRNA transfection. C, DHE staining of aortas from WT and HMGB1ECKO mice. D, Representative Western Blot of p-eNOS, eNOS, p-AKT and AKT in aortas from WT and HMGB1ECKO mice. E and F, Quantitative densitometry analysis showed p-AKT/AKT and p-eNOS/eNOS ratio were decreased significantly in aortas from HMGB1ECKO mice. G and H, Representative Western Blot of p-eNOS and NOS in MLEC. p-eNOS level was decreased in MLEC from HMGB1ECKO mice. I, Representative Western Blot of p-eNOS, eNOS, p-AKT and AKT in HUVECs transfected with HMGB1 siRNA. J, K and L, Quantitative densitometry analysis showed p-AKT/AKT and p-eNOS/eNOS expression were decreased significantly. M, Representative Western Blot of HMGB1, p-eNOS, eNOS, p-AKT and AKT in HUVECs after transfected with ad-HMGB1. N, O and P, Quantitative densitometry analysis showed HMGB1 expression increased in HUVECs after ad-HMGB1 transfection. p-AKT/AKT and p-eNOS/eNOS ratio were rescued significantly. Band densities were normalized with that of β-actin or GAPDH. Comparisons were performed by using…

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  Fig. 6. SOD rescued endothelial dependent EDR and angiogenesis. A, EDR was tested in aortas from HMGB1ECKO mice with or without pre-incubation of PEG-SOD. B, HMGB1ECKO mice were injected with Ad-GFP or Ad-SOD2 and SOD2 levels examined in the adductor muscle group by Western Blot after 4 days of infection (C). Ad-SOD2 improved the time course of blood flow recovery (D). HUVECs transfected with HMGB1 siRNA were treated with or without PEG-SOD (100 μg/ml), then proliferation(G) and tube formation were tested. (E, F) Representative images were shown. Comparisons were performed by using two-tailed unpaired Student's t-tests for C, one-way ANOVA for F, G and two-way ANOVA for A, D. (n = 4–6, *P < 0.05).

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  Fig. 7. Illustration of effect and possible pathway after HMGB1 loss in endothelial cells.