1. VEGF-A produced by chronically inflamed tissue induces lymphangiogenesis in draining lymph nodes
by Cornelia Halin, Nadja E. Tobler, Benjamin Vigl, Lawrence F. Brown, and Michael Detmar
Blood
Volume 110(9):
November 1, 2007
©2007 by American Society of Hematology

2. The number of LECs increases in inflamed ears of VEGF-A Tg mice.
The number of LECs increases in inflamed ears of VEGF-A Tg mice. A DTH response to oxazolone was induced in the ears of VEGF-A Tg mice. Ear tissue was analyzed 9 days after challenge. (A) At this time point, the inflamed ears were markedly increased in thickness compared with ears from control (ctr) animals. (B) FACS analysis was performed on cells derived from enzymatically digested ears (example shown: inflamed ears). Cells were stained for CD45, CD31, and podoplanin to differentiate between leukocytes (CD45+CD31−podoplanin−), BECs (CD45+CD31+podoplanin−), and LECs (CD45+CD31+podoplanin+). (C-E) Quantitative FACS analysis revealed that total numbers of leukocytes (C), BECs (D), and LECs (E) were increased in cell suspensions of inflamed ears compared with those of control ears. *P < .05; ***P < .001 (compared with control). (F) Immunofluorescence analysis of LYVE-1 (green) and MECA-32 (red) expression confirmed that vascularization was increased in inflamed compared with control ears. Scale bars represent 50 μm. Error bars are SE.
Cornelia Halin et al. Blood 2007;110:
©2007 by American Society of Hematology

3. The number of LECs increases in auricular LNs that drain the inflamed ears of VEGF-A Tg mice.
The number of LECs increases in auricular LNs that drain the inflamed ears of VEGF-A Tg mice. Auricular LNs were analyzed 9 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) were markedly increased in LNs draining inflamed ears compared with those draining control (ctr) ears. (C) FACS analysis of cell suspensions of auricular LN was used to differentiate between leukocytes, BECs, and LECs. (D,E) Quantitative FACS analysis revealed that the total number of LECs (D) was increased in inflamed compared with control LNs, whereas the total number BECs (E) remained unchanged. ***P < .001 (compared with control). (F) Immunofluorescence analysis of LYVE-1 (green) and MECA-32 (red) expression confirmed that lymphatic structures were markedly expanded in LNs draining inflamed compared with control ears. Scale bars represent 100 μm. Error bars are SE.
Cornelia Halin et al. Blood 2007;110:
©2007 by American Society of Hematology

4. The number of LECs increases in auricular LNs that drain inflamed ears of WT mice subjected to repeated DTH challenges.
The number of LECs increases in auricular LNs that drain inflamed ears of WT mice subjected to repeated DTH challenges. A DTH response to oxazolone was induced in the ears of WT mice and maintained by repeatedly applying oxazolone on the ears for 9 days. (A-C) After 9 days, the total numbers of leukocytes (A) and of LECs (B) and BECs (C) were significantly increased in inflamed ears over control (ctr) ears, as determined by quantitative FACS analysis. (D) Immunofluorescence analysis of LYVE-1 (green) and MECA-32 (red) expression confirmed that vascularization was increased in inflamed compared with control ears. Scale bars represent 50 μm. (E,F) Analysis of ear draining auricular LNs revealed that LN weight (E) and cellularity (F) was markedly increased in inflamed compared with control animals. (G,H) Quantitative FACS analysis detected elevated numbers of LECs (G) in LNs draining inflamed compared with control ears, but no change in BEC (H) numbers. (I) Immunofluorescence analysis of LYVE-1 (green) and MECA-32 (red) expression confirmed that lymphatic structures were markedly expanded in LNs draining inflamed compared with control ears. Scale bars represent 100 μm. (J) Differential immunofluorescence staining for LYVE-1 (green) and Ki67 (red) revealed the presence of proliferating LECs (white arrows) in inflamed LNs. Scale bars represent 25 μm. *P < .05; ***P < .001 (compared with control). Error bars are SE.
Cornelia Halin et al. Blood 2007;110:
©2007 by American Society of Hematology

5. Blockade of VEGF-A, but not of VEGF-C, inhibits inflammation-induced LN lymphangiogenesis.
Blockade of VEGF-A, but not of VEGF-C, inhibits inflammation-induced LN lymphangiogenesis. A DTH response to oxazolone was induced in the ears of WT mice and maintained by repeatedly applying oxazolone for 9 days. Mice with inflamed ears were exposed to anti–VEGF-A Ab (A-F), VEGFR3-Fc (G-L), or control IgG (A-L), and the effects were compared with those observed in mice in which inflammation had not been induced (ctr). (A-F) Effect of anti–VEGF-A: Weight (A) and cellularity (B) of the auricular LN was significantly increased in mice with inflamed ears, exposed to anti–VEGF-A or IgG. This effect was less pronounced in inflamed LNs of mice that had been administered anti–VEGF-A compared with IgG. (C) Exposure to anti–VEGF-A potently blocked the increase in LEC numbers in LNs draining inflamed ears, whereas exposure to IgG had no effect. (D) Inflammation led to a significant increase in leukocyte numbers in the ears of both anti–VEGF-A or IgG treated mice, compared with uninflamed the ears of control animals. (E, F) Administration of anti–VEGF-A potently blocked the increase in LEC (E) and BEC (F) numbers in inflamed ears of mice. (G-L) Effect of VEGFR3-Fc: Weight (G) and cellularity (H) of the auricular LN was similarly increased in mice with inflamed ears exposed to VEGFR3-Fc or IgG. (I) Administration of VEGFR3-Fc did not block the increase in LEC numbers in LNs draining inflamed ears. (J) Leukocyte numbers were strongly increased in inflamed ears of mice exposed to IgG. Administration of VEGFR3-Fc led to a significant reduction in the numbers of leukocytes in inflamed ears. Administration of VEGFR3-Fc had no effect on the numbers of LECs (K) or BECs (L) in inflamed ears of mice. *P < .05, **P < .01, ***P < .001 (compared with control [ctr]); #P < .05, ##P < .01, ###P < .001 (anti–VEGF-A or VEGFR3-Fc, compared with IgG). Error bars are SE.
Cornelia Halin et al. Blood 2007;110:
©2007 by American Society of Hematology

6. VEGF-A is expressed in the inflamed ears but not in the draining LNs
VEGF-A is expressed in the inflamed ears but not in the draining LNs. A DTH response was induced and maintained in the ears of WT or of VEGF-A Tg mice, and VEGF-A mRNA and protein levels in ears and draining auricular LN were determined on study days 2 and 9.
VEGF-A is expressed in the inflamed ears but not in the draining LNs. A DTH response was induced and maintained in the ears of WT or of VEGF-A Tg mice, and VEGF-A mRNA and protein levels in ears and draining auricular LN were determined on study days 2 and 9. (A) In situ hybridization on study day 9 revealed that VEGF-A mRNA was up-regulated in inflamed ears of both WT mice (WT-infl) and of VEGF-A Tg (TG-infl) mice compared with WT and VEGF-A Tg control mice (WT-ctr and TG-ctr, respectively). (B) Virtually no VEGF-A mRNA expression was detected in LN sections from inflamed or control VEGF-A Tg (TG-infl and TG-ctr) and WT mice (WT-infl and WT-ctr). (C,D) Quantitative RT-PCR was performed on RNA extracted from auricular LNs of WT (C) or of VEGF-A Tg (D) mice. No increase in VEGF-A mRNA expression in inflamed over control LNs could be detected, neither when analyzing RNA extracted on study day 2 (D2) nor on study day 9 (D9). VEGF-A mRNA levels in salivary glands were assayed as positive control. (E,F) A VEGF-A ELISA was performed on tissue homogenates of ears (E) and draining LNs (F) on study day 9. (E) VEGF-A protein concentration was significantly elevated in homogenates of inflamed ears, compared with controls. (F) The amount of VEGF-A protein was significantly higher in homogenates from inflamed LNs compared with control LNs. *P < .05; ***P < .001 (compared with control). Error bars are SE.
Cornelia Halin et al. Blood 2007;110:
©2007 by American Society of Hematology

7. Lymphangiogenesis in LNs that drain inflamed ears occurs in the absence of nodal B cells.
Lymphangiogenesis in LNs that drain inflamed ears occurs in the absence of nodal B cells. A DTH response to oxazolone was induced in the ears of B cell-deficient JHT mice and maintained by repeatedly challenging the ears of the mice with oxazolone over 9 days. Ears and draining auricular LNs were analyzed on study day 9. (A-C) Quantitative FACS analysis detected a significant increase in the numbers of leukocytes (A) and of LECs (B) and BECs (C) in cell suspensions of inflamed compared with control (ctr) ears. (D) Immunofluorescence analysis of LYVE-1 (green) and MECA-32 (red) expression confirmed that vascularization was increased in inflamed compared with control ears. Scale bars represent 50 μm. (E,F) Analysis of ear draining auricular LNs revealed that LN weight (E) and cellularity (F) was significantly increased in LNs of inflamed animals compared with LNs of control animals. (G,H) Quantitative FACS analysis detected elevated numbers of LECs (G) in LNs draining inflamed compared with LNs draining control ears, but no change in BEC numbers (H). (I) Immunofluorescence analysis of LYVE-1 (green) and MECA-32 (red) expression confirmed that lymphatic structures were markedly expanded in LNs draining inflamed compared with control ears. Scale bars represent 100 μm. **P < .01; ***P < .001 (compared with control). Error bars are SE.
Cornelia Halin et al. Blood 2007;110:
©2007 by American Society of Hematology