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

MacMillan CJ, Doucette CD, Warford J, Furlong SJ, Hoskin DW, Easton AS - PLoS ONE (2014)

Bottom Line: B20-4.1.1 reduced spinal cord vascular permeability while K(1-3) had no effect.B20-4.1.1 reduced peripheral T cell proliferation while K(1-3) had no effect.We conclude that these angiogenesis inhibitors mitigate EAE by both suppressing spinal cord angiogenesis and reducing peripheral T cell activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Dalhousie University, Halifax, Nova Scotia, Canada.

ABSTRACT
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.

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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.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 (MOG35–55) 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.
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pone-0089770-g005: 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.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 (MOG35–55) 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.

Mentions: To assess the effect of B20-4.1.1 and K(1-3) on peripheral immune responses, lymph nodes were harvested at day 21 to coincide with peak disease scores. Compared to healthy (wild type) controls, there was a significant increase in lymphoid cell numbers in mice with untreated EAE (P<0.01). A similar increase was noted in mice treated with K(1-3) (P<0.001), but numbers were not significantly increased in mice treated with B20.4.1.1 or combined B20.4.1.1 and K(1-3) (fig. 5A). Flow cytometry was used to assess the proportions of different cell types among the lymphoid cells based on surface markers. Mice with untreated EAE showed a significant reduction (P<0.05) in the proportion of CD4+ TcRβ+ cells compared to controls but this was not significantly different in the treated animals (fig. 5B). All of the EAE groups (untreated or treated) had a significant reduction (P<0.001) in the proportion of non-CD4+ T cells (CD4− TcRβ+, fig. 5C) with significant compensatory increases (P<0.01–0.001) in the proportion of non-T cells (TcRβ−, fig. 5D). Next, the fraction of proliferating CD4+ T cells was assessed by Oregon Green labeling. As cells proliferate, each generation contains a reduced amount of Oregon Green so that fluorescence shifts to the left. The proportion of CD4+ T cells in this group was used to calculate a proliferation fraction. The right panel in fig. 5E shows representative flow cytometry plots used for this calculation. The x-axis shows Oregon Green fluorescence, while the y-axis shows CD4 fluorescence. The proliferating fraction has a high expression of CD4 and contains cells whose Oregon Green fluorescence has shifted to the left upon stimulation (outlined in purple on the flow cytometry plots). The proliferation fraction (%) is the number of proliferating CD4+ T cells divided by the total number of CD4+ T cells and then multiplied by 100 to give a percent. The proliferation fraction increased when cells were treated with the inciting antigen (MOG35–55) and significantly blunted (P<0.001) in cells taken from mice treated with B20.4.1.1, while K(1-3) or the combination of B20.4.1.1 and K(1-3) had no effect. Proliferation was enhanced by stimulation with CD3/CD28 coated microbeads, but there were no differences between the groups (fig. 5E).


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

MacMillan CJ, Doucette CD, Warford J, Furlong SJ, Hoskin DW, Easton AS - PLoS ONE (2014)

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.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 (MOG35–55) 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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3935931&req=5

pone-0089770-g005: 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.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 (MOG35–55) 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.
Mentions: To assess the effect of B20-4.1.1 and K(1-3) on peripheral immune responses, lymph nodes were harvested at day 21 to coincide with peak disease scores. Compared to healthy (wild type) controls, there was a significant increase in lymphoid cell numbers in mice with untreated EAE (P<0.01). A similar increase was noted in mice treated with K(1-3) (P<0.001), but numbers were not significantly increased in mice treated with B20.4.1.1 or combined B20.4.1.1 and K(1-3) (fig. 5A). Flow cytometry was used to assess the proportions of different cell types among the lymphoid cells based on surface markers. Mice with untreated EAE showed a significant reduction (P<0.05) in the proportion of CD4+ TcRβ+ cells compared to controls but this was not significantly different in the treated animals (fig. 5B). All of the EAE groups (untreated or treated) had a significant reduction (P<0.001) in the proportion of non-CD4+ T cells (CD4− TcRβ+, fig. 5C) with significant compensatory increases (P<0.01–0.001) in the proportion of non-T cells (TcRβ−, fig. 5D). Next, the fraction of proliferating CD4+ T cells was assessed by Oregon Green labeling. As cells proliferate, each generation contains a reduced amount of Oregon Green so that fluorescence shifts to the left. The proportion of CD4+ T cells in this group was used to calculate a proliferation fraction. The right panel in fig. 5E shows representative flow cytometry plots used for this calculation. The x-axis shows Oregon Green fluorescence, while the y-axis shows CD4 fluorescence. The proliferating fraction has a high expression of CD4 and contains cells whose Oregon Green fluorescence has shifted to the left upon stimulation (outlined in purple on the flow cytometry plots). The proliferation fraction (%) is the number of proliferating CD4+ T cells divided by the total number of CD4+ T cells and then multiplied by 100 to give a percent. The proliferation fraction increased when cells were treated with the inciting antigen (MOG35–55) and significantly blunted (P<0.001) in cells taken from mice treated with B20.4.1.1, while K(1-3) or the combination of B20.4.1.1 and K(1-3) had no effect. Proliferation was enhanced by stimulation with CD3/CD28 coated microbeads, but there were no differences between the groups (fig. 5E).

Bottom Line: B20-4.1.1 reduced spinal cord vascular permeability while K(1-3) had no effect.B20-4.1.1 reduced peripheral T cell proliferation while K(1-3) had no effect.We conclude that these angiogenesis inhibitors mitigate EAE by both suppressing spinal cord angiogenesis and reducing peripheral T cell activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Dalhousie University, Halifax, Nova Scotia, Canada.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus