Figure 3.

AXT107 inhibits TNFα-mediated upregulation of leukocyte adhesion molecules and leukostasis in eye vessels. (A,B) Left—Histograms for HUVECs pre-treated with DMSO or 100 μM AXT107 followed by 10 ng/mL TNFα for varying durations as indicated that were stained with PE-labeled antibodies for VCAM-1 (A) or ICAM-1 (B) in absence of permeabilization. Right—Quantification of the number of VCAM-1 (A) or ICAM-1 (B) molecules per cell determined by comparing sample geometric means relative to the DMSO sample at 0 h. n = 4 (3 at 24 h), two-way ANOVA with Bonferroni posttest, ** indicates significance relative to the corresponding DMSO control, p < 0.05, 0.01, and 0.001, respectively; df = 1; F = 14.88 (VCAM-1) or F = 11.32 (ICAM-1). (C,D) Representative immunofluorescence images of vessels in retina flat mounts from mouse eyes pre-treated with intraocular injections of PBS (C) or 1 μg AXT107 (D) followed by 50 ng TNFα that were flushed with PBS and stained for adherent leukocytes with fluorescein isothiocyanate (FITC)-conjugated conconavalin A. Higher-magnification images are shown on the right. Scale bars are 200 μm (left) and 100 μm (right). © Quantification of total leukocytes adherent in the blood vessels of isolated mouse retinas. n = 12, *** indicate significance relative to PBS control by Student’s t-test (two-tailed), p < 0.001.