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Blood, Vol. 110, Issue 7, 2457-2465, October 1, 2007
Extracellular RNA mediates endothelial-cell permeability via vascular endothelial growth factor
Blood Fischer et al.
110: 2457
Supplemental materials for Fischer et al, Vol 110, Issue 7, 2457-2465
Files in this Data Supplement:
- Figure S1. Change of RNA-induced endothelial cell permeability by preincubation with RNAse (JPG, 19.3 KB)
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RNA and RNase were preincubated together for different time periods as indicated and added simultanously with RNA or 1 hour later to BMECs. Permeability changes were quantitated 3 hours after RNA addition. Control flux determined after 3 hours was set to 100%, and values represent the mean (± SEM, n = 12) for each condition; *P<.05 compared to untreated cells.
- Figure S2. Influence of extracellular RNA on endothelial cell electrical resistance (JPG, 53.3 KB)
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(A) Following addition of different concentrations of RNA to confluent HUVEC monolayers, on-line measurements of transendothelial resistance were performed as detailed in Document 1, “Supplemental methods, Measurement of electrical resistance across HUVEC monolayers.” As a positive control, transient thrombin-induced decrease of endothelial resistance is shown. Data are presented as the change in resistive portions of the resistance normalized to its value at time zero. Values represent the mean (± SEM, n = 3). (B) Analysis of RNase activity was measured in 24-hour conditioned media of HUVEC or BMEC cultures as well as in the corresponding cell lysates. Values represent the mean (± SEM, n = 5).
- Figure S3. Expression of tight junction proteins and VE-cadherin in nucleic acid-treated BMEC (JPG, 49.2 KB)
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BMEC were incubated for 3 hours with RNA (50 µg/mL), poly-I:C (25 µg/mL), heparin (10 µg/mL), DNA (25 µg/mL), or ssRNA (0.25 µg/mL) followed by isolation of the whole cell lysate, the cytosolic, and the nucleic fractions. After SDS-gel electrophoresis and transfer of proteins, western blot analysis was completed with antibodies against ZO-1, ZO-2, occludin, claudin-5, VE-cadherin, and, as loading control, anti- -actin as indicated.
- Figure S4. Expression of VEGF-receptors in BMEC (JPG, 11.7 KB)
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Western blot analysis for VEGF-R2 or VEGF-R1 of lysates prepared from untreated BMEC (control) or cells after preincubation for 2 days with antisense oligonucleotides against VEGF-R2 or VEGF-R1 (each 2 µM) is shown. As loading control, membranes were stripped and probed for -actin expression.
- Figure S5. RNA-induced release of VEGF from cell cultures (JPG, 39.7 KB)
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(A) Confluent BMEC were cultured in the absence (control, 100%) or presence of RNA (50 µg/mL), poly-I:C (25 µg/mL), heparin (10 µg/mL), DNA (25 µg/mL), or ssRNA (0.25 µg/mL) for 24 hours and the amount of VEGF released into the culture medium was determined. Data represent the mean (±SEM, n = 9); *P<.05 as compared to control. (B) For the detection of mRNA expression of different VEGF isoforms, BMEC were treated with the same doses of RNA, poly-I:C, heparin, DNA, or ssRNA for 3 hours as in panel A. Semiquantitative PCR was carried out using specific primers for amplification of VEGF and -actin as indicated.
- Figure S6. RNA-mediated signal transduction in endothelial cells (JPG, 53.9 KB)
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(A) After culturing confluent BMEC in the absence (control) or presence of RNA (50 µg/mL), poly-I:C (25 µg/mL), heparin (10 µg/mL), DNA (25 µg/mL), or ssRNA (0.25 µg/mL) for 3 hours, lysates were prepared and analyzed by Western blot analysis for phospho-p42/44, or p42/44 and for phospho-SAPK/JNK or SAPK/JNK expression. (B) BMEC monolayers left untreated (control) or pretreated with PD98059 (20 µM), SB203580 (10 µM), or SP600125 (25 µM) for 30 minutes, were exposed to RNA (50 µg/mL), poly-I:C (25 µg/mL), or ssRNA (0.25 µg/mL) as indicated, followed by analysis of permeability changes. (C) BMEC monolayers left untreated (control) or pretreated with NMMA (100 nM), wortmannin, (WM; 100 nM), or 2-aminopurine (AP; 10 µM) for 30 minutes, were exposed to RNA, poly-I:C, or ssRNA at the same doses as indicated in panel B, followed by analysis of permeability changes. Flux determined after 3 hours in the absence of added compounds was set to 100%, and values represent the mean (± SEM, n = 24) for each condition; *P<.05 compared to control.
- Figure S7. Influence of anti-VEGF antibody and FXIIa-inhibitor pretreatment on venous thrombosis in rats (JPG, 31.9 KB)
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Vessel occlusion was induced by FeCl3-exposure to the Sinus sagittalis superior. Thirty minutes prior to the start of the experiment, animals were pretreated with either saline (Control), anti-VEGF antibody, or FXIIa-inhibitor (both at the dose of 10 mg/kg body weight). Haematoxylin-eosin staining of paraffin-embedded brain sections prepared from each group was performed. Note the occluded vessel (*) and massive edema formation (arrows) in the control and FXIIa-inhibitor-pretreated group, whereas in the anti-VEGF antibody treatment group, both vessel occlusion (*) and edema (arrows) were largely reduced, to a similar extent as with RNase treatment. Bar = 20 µm. Slides were inspected with a Leica microscope (Leica, Bensheim, Germany) under a magnification of 40/0.75 (NA objective). Images were taken using a Wisitron Systems GmbH (Puchheim, Germany) and analysed by image-acquisition with MetaMorph software, version 7 (Molecular Devices Corp., Berkshire, United Kingdom).
- Figure S8. Influence of RNase on claudin-5 labeling in infarcted brain tissue (JPG, 46.2 KB)
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Tissue cryosections of rat brains after middle cerebral artery occlusion were prepared from infarcted brain pretreated with either saline (A = Control), heparin (C), RNase (D), or DNase (E) or from noninfarcted brain areas (B). All sections were stained with a polyclonal antibody against claudin-5 (green fluorescence) and with a monoclonal antibody against alpha-smooth muscle actin (red fluorescence). Staining of the nuclei in the corresponding area is shown in the right corner of each panel. Claudin-5 staining is dramatically reduced in infarcted brain areas (A), which is partly restored with RNase pretreatment (D). Specimens were prepared as described in “Materials and Methods, Immunolabeling and confocal microscopy,” and slides were inspected with a DMRB Leica microscope, magnification 40× /1.00-0.5 (NA objective). Images were taken using a digital camera DFC300FX and image-acquisition software IM500 from Leica.
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