Induction of downstream targets S100A8 and S100A9 but normalized keratinocyte proliferation after G6–31 treatment. Immunohistochemistry of S100A9 (A–D) and S100A8 (E–H), target genes of Jun proteins, showed an up-regulation in both IgG-treated mice (B, F) and anti-VEGF–treated DKO* mice (C, D, G, H) after the deletion of JunB and c-Jun. (I–L) Ki67 staining shows a significant reduction of proliferation in anti-VEGF–treated animals (K, L, Q; total skin: P < 2.21E-08; epidermis only: P < 0.00022) compared with DKO* IgG-injected mice (J). Moreover, staining for the histone H3 phosphorylation on serine 10 (P-H3-S10) revealed that the proliferation is more pronounced in the epidermis than in the dermis (M–P, R; epidermis control IgG-treated vs. DKO* IgG-treated: P < 5.7E-08; epidermis DKO* IgG-treated vs. DKO* anti-VEGF–treated: P < 9.7E-05; epidermis control IgG-treated vs. DKO* anti-VEGF–treated: P < 1.0E-05). Data represent mean ± SD. (Scale bars, 100 μm.) Quantification of Western blots of epidermal samples showed similar levels of S100A8 protein in DKO* IgG-treated and anti-VEGF–treated mice (S and T).

Inhibition of VEGF reduces macrophage and lymphocyte infiltration. Immunohistochemistry for leukocyte markers Gr-1 (A–D), F4/80 (E–H), and CD11b (I–L) revealed decreased dermal leukocyte infiltration (C, D, G, H, K, L) and decreased epidermal as well as dermal CD3⁺ and CD4⁺ T lymphocytes (O, P, S, T) in the skin of anti-VEGF–treated mice, as compared with the IgG-treated group (B, F, J, N, R). A normal Cre⁻ control mouse is shown for comparison (A, E, I, M, Q). (Scale bars, 100 μm in A–H and M–P and 50 μm in I–P and Q–T.)

Systemic inhibition of VEGF alleviates the psoriasis-like phenotype induced by epidermal deletion of JunB and c-Jun. (A) The schematic representation of the experimental procedure. (B–E) Ears of control IgG-treated DKO* mice showed prominent inflammatory, scaly skin lesions. Mice that responded only moderately to anti-VEGF antibody treatment (DKO* anti-VEGF^m; n = 6) displayed a mild psoriatic phenotype (D), whereas the ear skin appeared largely normal in mice with a strong response to the anti-VEGF treatment (DKO* anti-VEGF^s: n = 5) (E). A control IgG-injected Cre⁻ mouse is shown for comparison (n = 3). (F–I) H&E staining of control IgG-treated mice (F) and DKO* IgG-treated mice (G) reveals the typical signs of the psoriasis-like phenotype. (H, I) Anti-VEGF–treated mice showed significant improvement in these symptoms. (Scale bars, 100 μm.) (J–U) Normalized keratinocyte differentiation after G6–31 treatment. Immunofluorescence analyses showed strong induction of the hyperproliferation marker keratin 6 (K) and altered expression patterns of the epidermal differentiation-related proteins keratin 10 (O) and loricrin (S) in the isotype control IgG-treated DKO* mice, whereas after anti-VEGF treatment the expression patterns normalized showing reduced expression of keratin 6 (L, M) and restriction of the expression of keratin 10 (P, Q) and loricrin (T, U) to the suprabasal layers of the epidermis. A normal control mouse is shown for comparison (J, N, R). Blood vessels (MECA-32⁺) are shown in red; nuclei are stained in blue (Hoechst 33342 stain). (Scale bars, 50 μm.)

Reduced vascular abnormalities after G6–31 treatment. (A–D) Immunostaining for MECA-32 showed increased vascular density and enlargement of blood vessels in the control IgG-treated group (B), compared with anti-VEGF–treated mice (C, D) or normal Cre⁻ control mice (A). (E–H) Double immunofluorescence staining for CD31 (red) and for LYVE-1 (green) revealed enlarged lymphatic vessels in the skin of isotype control IgG-treated mice (F) compared with anti-VEGF–treated mice (G, H) or normal control mice (E). The positive staining of LYVE-1 in the stratum corneum in H is unspecific. (Scale bars, 100 μm.) (I–N) Computer-assisted measurements demonstrated that the ear thickness (I) and epidermal thickness (J) were significantly reduced after anti-VEGF mAb treatment (**, P < 0.01), as compared with the control treated group. (K–N) The average size of blood (L) and lymphatic (M) vessels and the number of blood vessels in the skin (K) were significantly reduced after systemic inhibition of VEGF (***, P < 0.001). No significant difference in the number of lymphatic vessels was found in the 2 groups (N). Data represent mean ± SD.

Expression analyses of genes involved in inflammation and epidermal differentiation. RT-PCR analysis of mRNA expression levels revealed strong expression of VEGF A, VEGFR1, and VEGFR2 in DKO* IgG-treated mice which was reduced in DKO* anti-VEGF–treated mice (A–C). S100A8 (D) and A9 (E) were increased in DKO* mice and decreased in anti-VEGF–injected mice, perhaps because of a posttranslational mechanism or a negative feedback loop. The expression levels of epidermal samples of the following genes (especially cytokines related to the IL-23/Th17 axis and the related receptors) reflect a normalization of the inflammatory process in anti-VEGF–injected mice: S100A7A (F), MMP3 (I), MMP9 (J), MMP2 (K), TNFα (L), IL-23 (M), IL-23R (N), IL-22 (O), and IL-12p40 (P). RNA expression levels of total skin for IL-1β (G) and CXCL2 (H) also are reduced back to control levels in anti-VEGF–injected mice. Controls are set to 1, and the y axis shows relative expression levels. Control IgG-treated: n = 3; DKO* IgG-treated: n = 3–4; DKO* anti-VEGF–treated: n = 4–5. D–F represent data (mean ± SD) from control IgG-treated: n = 3; DKO* IgG-treated: n = 5–6; DKO* anti-VEGF–treated: n = 10–12.