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Blood, Vol. 110, Issue 9, 3158-3167, November 1, 2007
VEGF-A produced by chronically inflamed tissue induces lymphangiogenesis in draining lymph nodes
Blood Halin et al.
110: 3158
Supplemental materials for: Halin et al
Files in this Data Supplement:
- Figure S1. Analysis of LNs of VEGF-A Tg mice 2 days after the induction of a DTH response in the ear (PDF, 59.7 KB) -
Auricular LNs were analyzed 2 days after induction of a DTH response to oxazolone in the ears of VEGF A Tg mice. (A&B) At this time point, LN weight (A) and cellularity (B) was markedly increased in LNs draining inflamed ears as compared to those draining control (ctr) ears. (C&D) Quantitative FACS analysis detected no difference in the total number of LECs (C) and BECs (D) present in LNs draining inflamed or control ears at this early time point of analysis. ** p < 0.01; *** p < 0.001 (versus control). (E) Immunofluorescence analysis of LYVE-1 (green) and MECA-32 (red) expression revealed that lymphatic structures were already slightly expanded in LNs draining inflamed ears as compared to LNs draining control ears. Scale bars = 100 µm.
- Figure S2. Analysis of LNs of WT mice 2 days after the induction of a DTH response in the ear (PDF, 67.9 KB) -
Auricular LNs were analyzed 2 days after induction of a DTH response to oxazolone in the ears of WT mice. (A&B) At this time point, LN weight (A) and cellularity (B) was markedly increased in LNs draining inflamed ears as compared to those draining control (ctr) ears. (C&D) Quantitative FACS analysis detected no difference in the total number of LECs (C) and BECs (D) present in LNs draining inflamed or control ears at this early time point of analysis. ** p < 0.01; *** p < 0.001 (versus control). (E) Immunofluorescence analysis of LYVE-1 (green) and MECA-32 (red) expression revealed that lymphatic structures were already slightly expanded in LNs draining inflamed ears as compared to LNs draining control ears. Scale bars = 100 µm. (F) High magnification immunofluorescence, staining for LYVE-1 (green) and Ki67 (red), revealed the presence of proliferating LECs (white arrows) in inflamed LNs. Scale bars = 25 µm. * p < 0.05; *** p < 0.001 (compared to control).
- Figure S3. VEGF-C mRNA is weakly upregulated in inflamed ears as compared to control ears (PDF, 371 KB) -
A DTH response to oxazolone was induced in the ears of WT or of VEGF-A Tg mice and maintained over 9 days. At this time point, animals were sacrificed and sections from ears and draining auricular LNs were analyzed by in situ hybridization with a VEGF-C specific probe. (A) VEGF-C mRNA was weakly upregulated in inflamed ears of both WT mice (WT-infl) and of VEGF-A Tg (TG-infl) mice, as compared to WT and VEGF-A Tg control mice (WT-ctr and TG-ctr, respectively). (B) Virtually no VEGF-C mRNA expression was detected in LN sections from inflamed or control VEGF-A Tg (TG-infl and TG-ctr) or WT mice (WT-infl and WT-ctr).
- Figure S4. Analysis of VEGF-A protein levels in ears and draining LNs on day 2 after the induction of a DTH response (PDF, 35 KB) -
A DTH response was induced in the ears of WT or of VEGF-A Tg mice. 2 days after challenge, animals were sacrificed and tissue homogenates of ears and draining LNs were analyzed by ELISA. (A) VEGF A protein concentration was significantly elevated in homogenates of inflamed ears, as compared to homogenates of control ears. (B) The amount of VEGF A protein was significantly higher in homogenates from inflamed LNs as compared to homogenates of control LNs. Groups: WT ctr (control WT mice); WT infl (DTH-challenged WT mice), TG ctr (control VEGF-A TG mice); TG infl (DTH-challenged VEGF-A TG mice). * p < 0.05; ** p < 0.01; *** p < 0.001 (compared to control).
- Figure S5. SCID/beige mice repeatedly challenged with oxazolone develop inflamed ears and LN lymphangiogenesis (PDF, 49.7 KB) -
A DTH response to oxazolone was induced in the ears of SCID/beige mice and maintained by repeatedly applying oxazolone on the ears for 9 days. (A) At this time point, the treated ears were markedly increased in thickness as compared to ears from control (ctr) animals. (B) Immunofluorescence analysis of LYVE-1 (green) and MECA-32 (red) expression showed that vascularization was increased in inflamed as compared to control ears. Scale bars = 50 µm. (C) The weight of LNs draining inflamed ears was markedly increased compared to the one of LNs draining control (ctr) ears. (D) Immunofluorescence analysis of LYVE-1 (green) and MECA-32 (red) expression revealed that lymphatic structures were markedly expanded in LNs draining inflamed as compared to control ears. Scale bars = 100 µm. *** p < 0.001 (compared to control).
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