Murine experimental autoimmune encephalomyelitis is diminished by treatment with the angiogenesis inhibitors B20-4.1.1 and angiostatin (K1-3).

Angiogenesis is the formation of new blood vessels form pre-existing vasculature whose contribution to inflammatory conditions of the Central Nervous System is being studied in order to generate novel therapeutic targets. This study is the first to investigate the impact of two particular angiogenesis inhibitors on murine Experimental Autoimmune Encephalomyelitis (EAE), an inflammatory disease that mimics aspects of the human disease Multiple Sclerosis. The inhibitors were chosen to reduce angiogenesis by complimentary means. Extrinsic factors were targeted with B20-4.1.1 through its ability to bind to murine Vascular Endothelial Growth Factor (VEGF). Vascular processes connected to angiogenesis were targeted directly with K(1-3), the first three kringle domains of angiostatin. Mice treated with B20-4.1.1 and K(1-3) from onset of signs had reduced clinical scores 18-21 days after EAE induction. Both agents suppressed spinal cord angiogenesis without effect on local VEGF expression. B20-4.1.1 reduced spinal cord vascular permeability while K(1-3) had no effect. T cell infiltration into the spinal cord at day 21 was unaffected by either treatment. B20-4.1.1 reduced peripheral T cell proliferation while K(1-3) had no effect. Lymphoid cells from treated mice produced reduced levels of the T helper-17 (Th-17) cell cytokine interleukin (IL)-17 with no effect on the Th-1 cytokine interferon (IFN)-γ or Th-2 cytokine IL-4. However, when both drugs were added in vitro to naive T cells or to antigen stimulated T cells from mice with untreated EAE they had no effect on proliferation or levels of IL-17 or IFN-γ. We conclude that these angiogenesis inhibitors mitigate EAE by both suppressing spinal cord angiogenesis and reducing peripheral T cell activation

B20-4.1.1 and K(1-3) Have no Effect on Proliferation and Cytokine Secretion by Isolated T cells.

<p>A,C,E naive CD4⁺ T cells co-stimulated with T cell expander beads (CD3/CD28). B,D,F. T cells from mice with untreated EAE restimulated with MOG_35–55 added to bone marrow derived dendritic cells. Data is normalized to untreated CD3/CD28 or MOG stimulated cells and shown as mean ± SEM, n = 3 per group; each data point pooled from 2 mice. IgG = immunoglobulin G control.</p

B20.4.1.1 but not K(1-3) Reduces Permeability-Surface Area Product (PS) Values During EAE.

<p>A. Upper panels show representative permeability maps for indicated treatment groups at day 21. Arrows indicate regions of increased PS within the spinal cord. Scale bar = 200 µm. Lower bar chart shows PS values at day 14 and 21 for EAE, EAE+B20.4.1.1 and EAE+K(1-3). PS values per mouse in each group (EAE only group: n = 6; B20.4.1.1 treated group, n = 6, K(1-3) treated group, n = 8); data are shown as mean ± SEM, significance compares EAE with each treatment (***; P<0.001). B, C. Regression plots for values from individual mice from day 14 (filled circles) and day 21 (open circles), comparing PS to vessel counts for mice treated with B20.4.1.1 (B) or K(1-3) (C). Regression lines through each data set are shown with the regression coefficient (r²).</p

Reduced IL-17 Production in Lymphoid Cells From Mice Treated With B20-4.1.1 and K(1-3) During EAE.

<p>Data is also shown for stimulation with MOG_35–55 as well as T cell expander beads (CD3/CD28). Supernatants were analyzed by ELISA. Bars indicate release for wild type controls, untreated EAE, EAE+B20.4.1.1, EAE+K(1-3) and EAE+treatments combined. Data are shown as mean ± SEM, each sample was pooled from 2 mice, n = 3–9 samples for each group; significance compares untreated EAE to treated groups (*–**, P<0.05–0.01). A. IL-17 release. B. IFN-γ release. C. IL-4 release.</p

B20-4.1.1 and K(1-3) Reduce Clinical Scores in EAE.

<p>A. EAE treated with B20-4.1.1. B. Treatment with K(1-3). C. Treatment with IgG. Arrows indicate treatment days. Data shown are mean ± standard error of the mean (SEM). Indicated significance is either relative to control (*–***; P<0.05–0.001) or else compares untreated EAE with treated EAE (#,###; P<0.05, 0.001). Number of mice (n): B20-4.1.1 group: EAE only, n = 3, treated group, n = 24 up to day 14, 12 after day 14, B20-4.1.1 only, n = 3. K(1-3) group: EAE only: n = 6, treated group, n = 16 up to day 14, 8 after day 14, K(1-3) only, n = 4; IgG group: EAE only: n = 4, IgG treated, n = 12 up to day 14, 6 after day 14, IgG only, n = 2.</p

B20.4.1.1 Treatment During EAE Minimizes the Increase in Cell Numbers in Peripheral Lymph Nodes and Reduces Proliferative Responses in CD4⁺ T cells.

<p>Data are shown as mean ± SEM for wild type controls, untreated EAE, EAE+B20.4.1.1, EAE+K(1-3) and EAE+agents combined. Significance is relative to untreated EAE (ns = not significant, ***, P<0.05–0.001). A. Effect on total cell numbers (n = 3–5 samples for each group, each sample obtained by pooling lymph nodes from 2 mice). B. % of lymphoid cells co-expressing CD4 and TcRβ (n = 3–4). C. % CD4⁻TcRβ⁺ cells (n = 3–4). D. % cells lacking TcRβ (n = 3–4). E. Proliferation assay in vitro for culture medium (negative control), the inciting antigen (MOG_35–55) and co-stimulation with T cell expander beads (CD3/CD28); n = 3–4 samples (each sample pooled from 2 mice) for each group. Representative flow cytometry plots are shown in right side panels for indicated groups. X-axis shows Oregon Green fluorescence, y-axis shows CD4 labeling. Gating for CD4⁺ cells is outlined in blue, while gating for proliferating cells is outlined in purple.</p

Treatment With B20.4.1.1 or K(1-3) has no Effect on VEGF Expression During EAE.

<p>A,B. Bar charts show per mouse data on VEGF expression, using the % area of positive staining in the dorsal columns. Control mice were disease-free. EAE alone is compared to mice treated during EAE with B20-4.1.1 (A) or K(1-3) (B). Day 21 data is further subdivided into high and low expression (n = 6 mice in EAE alone, n = 5 at day 21, high expression group n = 3/5, low n = 2/5; n = 9 mice for B20-4.1.1 treated mice, n = 5/9 for high expression group, low n = 4/9; n = 10 mice for K(1-3) treated mice, n = 6/10 for high expression group, low n = 4/10; data is mean ± SEM, significance relative to untreated controls, *–***, P<0.05–0.001). C–E. Representative images of dorsal column expression of VEGF for untreated controls (C), high expression B20.4.1.1 treated EAE day 21 (D) and low expression K(1-3) treated EAE day 21 (E). Scale bar = 100 µm.</p

B20-4.1.1 and K(1-3) Inhibit Spinal Cord Angiogenesis During EAE.

<p>Left sided panels show vessel counts as a function of disease duration. Filled circles indicate data for untreated EAE, open circles for EAE treated with B20-4.1.1 (upper left) or K(1-3) (lower left). Data are shown as mean ± SEM obtained from per mouse values from 6-8 mice. Significance is relative to control or between points linked by cross bars (*, ***; P<0.05, 0.001). Right sided images show CD31 positive vessels in spinal cord for indicated groups. Arrows point to examples of individual blood vessels. Scale bar = 50 µm.</p

Treatment With B20-4.1.1 and K(1-3) During EAE has Little Impact on Spinal Cord Infiltration of T cells.

<p>A. Effect on total mononuclear cells isolated from spinal cord (samples pooled from 2 mice, n = 3 samples for each data set). B–D. Proportion of surface marker defined cell populations in samples from untreated EAE and treated EAE. CD4 and TcRβ status as indicated in each panel (each sample pooled from 2 mice, n = 3 samples for each data set). Data are shown as mean ± SEM, significance relative to untreated EAE (*, P<0.05).</p

Marie-Claire / dir. Jean Prouvost

15 janvier 19441944/01/15 (A0,N304)-1944/01/15.Appartient à l’ensemble documentaire : UnivJeun