Inhibition of chronic and acute skin inflammation by treatment with a vascular endothelial growth factor receptor tyrosine kinase inhibitor

Abstract

Although vascular remodeling is a hallmark of many chronic inflammatory disorders, antivascular strategies to treat these conditions have received little attention to date. We investigated the effects of a newly identified vascular endothelial growth factor (VEGF) receptor tyrosine-kinase inhibitor, NVP-BAW2881, on endothelial cell function in vitro and its anti-inflammatory activity in different animal models. NVP-BAW2881 inhibited proliferation, migration, and tube formation by human umbilical vein endothelial cells and lymphatic endothelial cells in vitro. In a transgenic mouse model of psoriasis, NVP-BAW2881 reduced the number of blood and lymphatic vessels and infiltrating leukocytes in the skin, and normalized the epidermal architecture. NVP-BAW2881 also displayed strong anti-inflammatory effects in models of acute inflammation; pretreatment with topical NVP-BAW2881 significantly inhibited VEGF-A-induced vascular permeability in the skin of pigs and mice. Furthermore, topical application of NVP-BAW2881 reduced the inflammatory response elicited in pig skin by UV-B irradiation or by contact hypersensitivity reactions. These results demonstrate for the first time that VEGF receptor tyrosine-kinase inhibitors might be used to treat patients with inflammatory skin disorders such as psoriasis.

Figures

Figure 1

NVP-BAW2881 inhibits VEGF-induced proliferation and migration of HUVECs and LECs in vitro. A–C: The effect of increasing concentrations of NVP-BAW2881 on VEGF-A- or VEGF-C-induced proliferation of cultured HUVECs and LECs was assayed. A: Effect of NVP-BAW2881 on HUVEC proliferation induced by VEGF-A. B: Effect of NVP-BAW2881 on LEC proliferation induced by VEGF-A. C: Effect of NVP-BAW2881 on LEC proliferation induced by VEGF-C. D and E: The effect of either 10 nmol/L or 1 μmol/L NVP-BAW2881 on migration of HUVECs (D) or LECs (E) toward VEGF-A, as assayed in transwell plates. *P < 0.05; **P < 0.01; ***P < 0.001. In each panel, the results from one representative of 2 to 3 experiments are shown. NVP-BAW2881 is abbreviated as BAW2881.

Figure 2

NVP-BAW2881 inhibits VEGF-A-induced tube formation of HUVECs and LECs in vitro. Tube formation assays were performed by covering confluent monolayers of HUVECs or LECs with collagen type I containing no additives (ctr), containing VEGF-A alone (VEGF-A), or containing VEGF-A together with either 10 nmol/L or 1 μmol/L NVP-BAW2881. A and B: Representative pictures showing the effect of NVP-BAW2881 on tube formation in HUVECs (A) and LECs (B). C and D: Quantification of the effect of NVP-BAW2881 on tube formation in HUVECs (C) and LECs (D). For quantification, the total tube length per picture was measured and normalized to the tube length measured in the control picture. **P < 0.01; ***P < 0.001. Each panel shows the results from one representative out of 2 to 3 experiments. NVP-BAW2881 is abbreviated as BAW2881.

Figure 3

Oral or topical treatment with NVP-BAW2881 reduces symptoms of chronic ear inflammation in K14/VEGF-A mice. Heterozygous female K14/VEGF-A TG mice were sensitized with oxazolone on the belly and paws on study day −5. On study day 0, mice were challenged by application of oxazolone onto the ears. Treatment with NVP-BAW2881 was started on day 7 and repeated once (oral NVP-BAW2881) or twice (topical NVP-BAW2881) each day until the end of the study (day 21). Control (ctr) mice were given vehicle alone. A and B: Effect of oral (A) or topical (B) administration of NVP-BAW2881 on inflammation-induced ear swelling. C and D: The effect of oral (C) or topical (D) administration of NVP-BAW2881 on ear redness was qualitatively evaluated on day 21 with a scoring system ranging from 0 (normal ear color) to 3 (dark red). E and F: Mice were sacrificed on day 21 and the effect of oral (E) or topical (F) administration of NVP-BAW2881 on ear weight was determined. G and H: Effects of oral (G) or topical (H) administration of NVP-BAW2881 on the weight of the ear-draining auricular LN was determined (day 21). **P < 0.01; ***P < 0.001. NVP-BAW2881 is abbreviated as BAW2881.

Figure 4

Oral or topical administration of NVP-BAW2881 reduces leukocyte infiltration and vascular abnormalities in the inflamed skin. A CHS response to oxazolone was induced in the ears of K14/VEGF-A TG mice and treated by either oral or topical application of NVP-BAW2881 7 to 21 days after CHS induction. Histological analysis was performed on study day 21. A: H&E staining. B: Immunofluorescence for CD45 (leukocytes, red) and LYVE-1 (lymphatic vessels, green). C: Immunofluorescence for CD11b+ (red), in combination with the lymphatic vessel marker LYVE-1 (green). D: Immunofluorescence for MECA-32 (blood vessels, red) and LYVE-1 (lymphatic vessels, green). Scale bars = 50 μm. E–G: Changes in leukocyte infiltration and in vascular parameters were quantified by computer-assisted image analysis: E, Leukocyte infiltration, measured as the percentage of ear tissue that stained positively for the leukocyte marker CD45; F, average size of LYVE-1-positive lymphatic vessels; and G, percentage of tissue area in the upper dermis (area up to 120 μm below the epidermis) that stained positively for the blood vascular marker MECA-32. NVP-BAW2881 is abbreviated as BAW2881.

Figure 5

Oral or topical administration of NVP-BAW2881 reduces epidermal hyperproliferation in the inflamed skin. A CHS response to oxazolone was induced in the ears of K14/VEGF-A TG mice and treated by either oral or topical application of NVP-BAW2881 7 to 21 days after CHS induction. Histological analysis was performed on study day 21. A: Immunofluorescence for the epidermal marker loricrin (red), in combination with the nuclear stain Hoechst 33342 (blue). B and C: Immunofluorescence for the epidermal marker keratin 6 (B) and keratin 10 (C) (both in red), in combination with CD31 (green). Scale bars = 50 μm. NVP-BAW2881 is abbreviated as BAW2881.

Figure 6

Topical pretreatment with NVP-BAW2881 blocks VEGF-A-induced vascular permeability in mouse and pig skin. VEGF-A-induced extravasation of intravenously injected dye into the skin of mice or domestic pigs was measured spectrophotometrically. A: In mice, one pretreatment of the skin with NVP-BAW2881 for 2 hours before intradermal injection of VEGF-A, significantly reduced the extravasation of Evans Blue. B: Pretreatment with NVP-BAW2881 did not affect vascular permeability induced by intradermal injection of PAF or histamine into the skin of mice. C: In domestic pigs, three epicutaneous pretreatments with NVP-BAW2881 (at −30, −7, and −2 hours) led to a significant reduction in Evans Blue extravasation. ***P < 0.001. NVP-BAW2881 is abbreviated as BAW2881.

Figure 7

Signs of acute skin inflammation are significantly reduced after topical treatment with NVP-BAW2881. The anti-inflammatory effects of NVP-BAW2881 were tested in UVB-induced erythema and in acute contact dermatitis (CHS response) in the skin of domestic pigs. A–C: A phototoxic inflammatory response was induced by irradiation with UVB. Treatment with NVP-BAW2881 at 0, 3, and 6 hours after irradiation resulted in a significant reduction in clinical inflammatory symptoms (A), measured skin redness (reflectometry) (B), and microperfusion of the exposed skin (C), as compared with controls (ctr). D and E: A CHS response toward DNFB was elicited on the back skin domestic pigs. At 0.5 and 6 hours after challenge, the test sites were treated with 0.1% or 0.5% of NVP-BAW2881. At 24 hours after challenge, the inflammatory response was evaluated by clinical score (D) and reflectometry (E). Topical NVP-BAW2881 caused a dose-dependent reduction of inflammatory symptoms (erythema and induration). *P < 0.05; **P < 0.01; ***P < 0.001. NVP-BAW2881 is abbreviated as BAW2881.