American Society of Hematology

Figure 1.

$Figure 1. Angiogenesis in the collagen CAM onplants is accompanied by infiltration of chMMP-9–positive heterophils and accumulation of chMMP-9 protein. Collagen onplants were supplemented with buffer alone (control onplants: panels A, C, E) or angiogenic growth factors (bFGF/VEGF: panels B, D, F, H). Collagen onplants with the underlying CAM were harvested at 24 hours (A-B), 48 hours (C-D), or 66 hours (E-F, H), embedded in OCT compound, and frozen. Cryosections were immunostained for chMMP-9 with an affinity-purified rabbit polyclonal antibody (red). Tissue sections were counterstained with YO-PRO-1 iodide resulting in green-stained cell nuclei. The blank circular areas represent areas previously occupied by the 2-tiered nylon meshes, which are frequently displaced during tissue processing. At low magnification, scattered chMMP-9 staining appears to be associated with individual cells or small clusters of cells (arrowheads). The insets in panels A and B illustrate at higher magnification heterophils with distinctively shaped nuclei (green) and cytoplasmic chMMP-9–positive granules (red). * indicates specific staining of the extracellular fibrils for chMMP-9 in panel F. lu denotes the lumens of large blood vessels in panels B, C, and E; #, the bright ribbonlike staining at the upper border of the collagen-air interface in panel A. In panel H, a 66-hour growth factor–containing onplant is chMMP-9 negative after the chMMP-9 antibody was immunodepleted by preincubation with purified chMMP-9, confirming the specificity of the staining in the other panels. Bar represents 200 μm. The scatter graph (G) illustrates the levels of angiogenesis in control and growth factor–containing (bFGF/VEGF) onplants scored at 66 hours and presented as a fraction of grids with newly developed blood vessels (mean ± SEM). *P < .001. (I) Expression of chMMP-9 protein in the collagen CAM onplants. At 3 hours (lanes 2-3), 17 hours (lanes 4-5), 26 hours (lanes 6-7), 50 hours (lanes 8-9), and 66 hours (lanes 10-11) the collagen onplants with (+) or without (-) angiogenic growth factors (bFGF/VEGF) were excised from the CAM, pooled, extracted, and analyzed by SDS-PAGE and Western blotting with chMMP-9 antibody. As a positive control, the chMMP-9 proenzyme produced by PMA-treated chicken monocytic cells (HD11) was run in lane 1 (2 μL of cell lysate). The position of 75-kDa chMMP-9 is indicated on the right.$

Angiogenesis in the collagen CAM onplants is accompanied by infiltration of chMMP-9–positive heterophils and accumulation of chMMP-9 protein. Collagen onplants were supplemented with buffer alone (control onplants: panels A, C, E) or angiogenic growth factors (bFGF/VEGF: panels B, D, F, H). Collagen onplants with the underlying CAM were harvested at 24 hours (A-B), 48 hours (C-D), or 66 hours (E-F, H), embedded in OCT compound, and frozen. Cryosections were immunostained for chMMP-9 with an affinity-purified rabbit polyclonal antibody (red). Tissue sections were counterstained with YO-PRO-1 iodide resulting in green-stained cell nuclei. The blank circular areas represent areas previously occupied by the 2-tiered nylon meshes, which are frequently displaced during tissue processing. At low magnification, scattered chMMP-9 staining appears to be associated with individual cells or small clusters of cells (arrowheads). The insets in panels A and B illustrate at higher magnification heterophils with distinctively shaped nuclei (green) and cytoplasmic chMMP-9–positive granules (red). ^* indicates specific staining of the extracellular fibrils for chMMP-9 in panel F. lu denotes the lumens of large blood vessels in panels B, C, and E; #, the bright ribbonlike staining at the upper border of the collagen-air interface in panel A. In panel H, a 66-hour growth factor–containing onplant is chMMP-9 negative after the chMMP-9 antibody was immunodepleted by preincubation with purified chMMP-9, confirming the specificity of the staining in the other panels. Bar represents 200 μm. The scatter graph (G) illustrates the levels of angiogenesis in control and growth factor–containing (bFGF/VEGF) onplants scored at 66 hours and presented as a fraction of grids with newly developed blood vessels (mean ± SEM). ^*P < .001. (I) Expression of chMMP-9 protein in the collagen CAM onplants. At 3 hours (lanes 2-3), 17 hours (lanes 4-5), 26 hours (lanes 6-7), 50 hours (lanes 8-9), and 66 hours (lanes 10-11) the collagen onplants with (+) or without (-) angiogenic growth factors (bFGF/VEGF) were excised from the CAM, pooled, extracted, and analyzed by SDS-PAGE and Western blotting with chMMP-9 antibody. As a positive control, the chMMP-9 proenzyme produced by PMA-treated chicken monocytic cells (HD11) was run in lane 1 (2 μL of cell lysate). The position of 75-kDa chMMP-9 is indicated on the right.

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