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Blood, Vol. 109, Issue 4, 1495-1502, February 15, 2007
Receptor activator of nuclear factor (NF)– B ligand (RANKL) increases vascular permeability: impaired permeability and angiogenesis in eNOS-deficient mice
Blood Min et al.
109: 1495
Supplemental materials for: Min et al, Vol 109, Issue 4, 1495-1502
Files in this Data Supplement:
- Figure S1. RANKL-induced leukocyte extravasation was impaired in eNOS KO mice (JPG, 91.6 KB)
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(A) Leukocyte extravasation in WT and eNOS KO mice (n = 5 per group). Twenty-four hours after RANKL (3 µg) application as described in “Leukocyte infiltration,” mice (n = 5 per group) were perfused with the lectin L esculentum to visualize extravasated leukocytes. This experiment was performed twice. (B) Quantitative analysis of extravasated leukocytes. Data are means ± SDs; **P < 0.01 versus RANKL in WT.
- Figure S2. The effects of RANKL on phosphorylation of eNOS at various sites (JPG, 71.8 KB)
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HUVECs were stimulated with 1 µg/mL RANKL for the indicated times, and the phosphorylated forms of eNOS in whole-cell extracts were detected with anti–phospho-eNOS antibodies (S116, T495, S615, S633, and S1177). The membranes were then stripped and reprobed with antibody against eNOS.
- Figure S3. Suppression of RANKL-induced aortic endothelial cell sprouting by inhibitors of PI3K and NOS (JPG, 64.5 KB)
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(A) Aortic vessels were harvested from rats (n = 7 per group), segments of which were embedded in Matrigel and cultured for 5 days with RANKL (10 µg/mL) in the presence or absence of 100 nM Wortmannin (W) or 1 mM NMA (N). (a) Control, (b) RANKL only, (c) staining of endothelial cells sprouted from RANKL-treated aorta with vWF, (d) W plus RANKL, (e) N plus RANKL. (A) Endothelial-cell sprouts forming branching cords from the margins of vessel segments taken from rats were photographed under a phase microscope. (B) Sprouting scores classified from 0 (least positive) to 5 (most positive). Data are means ± SEs.
- Figure S4. RANKL-induced angiogenesis was impaired in eNOS KO mice (JPG, 65 KB)
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(A) Representative micrographs of mouse corneas 6 days after pellet implantation. Arrow indicates implanted pellet. (B) Quantification of corneal angiogenesis as numbers of microvessels. This experiment was performed twice (n = 7 per group). Data are means ± SDs; *P < 0.05 versus RANKL in WT.
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