Figure 7
Figure 7. Slit3 promotes angiogenesis in vivo and Slit3−/− mice display disrupted angiogenesis in developing diaphragm. (A-D) CAM assay. Gelfoam sponges soaked with Slit3 (4.0 nmol/L), FGF-2 (18 nmol/L), or BSA (6.5 nmol/L) were applied to chorioallantoic membrane. After 72 hours, the membranes around sponges were fixed and dissected out, and the number of vessels sprouting into sponges were counted and represented as mean ± SD (n = 8 for each group). The asterisk indicates a statistically significant increase compared with BSA treatment (**P < .01 by Student t test). (E-N) Mouse corneal micropocket assay. Hydron pellets containing Slit3 (1.3 nmol/L) or FGF-2 (4.8 nmol/L) were implanted into micropockets of the C57BL/6 mouse cornea. The pellets with only balanced buffers were used as a negative control. On day 5, the corneal neovascularization response to test factors was photographed and quantified. To reduce experimental variation, every mouse was implanted into both eyes with either test-factor or control. n = 8 for each group. Asterisks indicate statistically significant difference in the vessel area of the Slit3-inplanted cornea compared with the control group by Student t test (H,**P < .01; ***P < .001). The mouse corneas were further examined for ECs and pericytes/VSMCs by immunostaining with anti-PECAM-1 (red) and anti-PDGFR (green) antibodies, respectively. The control corneas (I-K) did not show any PECAM-1 position cells, showing that neovascularization was not induced, whereas the Slit3-implanted corneas (L-N) exhibited PECAM-1–positive cells and were covered by PDGFR-positive pericytes/VSMCs (indicated by the arrows), confirming that Slit3 potently induced angiogenesis in cornea. Scale bare = 50 μm. (O-T). Whole-mount staining of embryonic day 15.5 diaphragms of Slit3+/− and Slit3−/− mice with anti-mouse PECAM-1 antibody. Quantification of the vascular parameter showed that, compared with the phenotypically normal littermate control Slit3+/− diaphragm, the Slit3−/− diaphragm showed reduced vascular density and branch points. n = 4-6 for each group. Asterisks indicate statistically significant difference compared with Slit3+/− control by Student t test (***P < .001). Red scale bar = 250 μm; black scale bar = 100 μm.

Slit3 promotes angiogenesis in vivo and Slit3−/−mice display disrupted angiogenesis in developing diaphragm. (A-D) CAM assay. Gelfoam sponges soaked with Slit3 (4.0 nmol/L), FGF-2 (18 nmol/L), or BSA (6.5 nmol/L) were applied to chorioallantoic membrane. After 72 hours, the membranes around sponges were fixed and dissected out, and the number of vessels sprouting into sponges were counted and represented as mean ± SD (n = 8 for each group). The asterisk indicates a statistically significant increase compared with BSA treatment (**P < .01 by Student t test). (E-N) Mouse corneal micropocket assay. Hydron pellets containing Slit3 (1.3 nmol/L) or FGF-2 (4.8 nmol/L) were implanted into micropockets of the C57BL/6 mouse cornea. The pellets with only balanced buffers were used as a negative control. On day 5, the corneal neovascularization response to test factors was photographed and quantified. To reduce experimental variation, every mouse was implanted into both eyes with either test-factor or control. n = 8 for each group. Asterisks indicate statistically significant difference in the vessel area of the Slit3-inplanted cornea compared with the control group by Student t test (H,**P < .01; ***P < .001). The mouse corneas were further examined for ECs and pericytes/VSMCs by immunostaining with anti-PECAM-1 (red) and anti-PDGFR (green) antibodies, respectively. The control corneas (I-K) did not show any PECAM-1 position cells, showing that neovascularization was not induced, whereas the Slit3-implanted corneas (L-N) exhibited PECAM-1–positive cells and were covered by PDGFR-positive pericytes/VSMCs (indicated by the arrows), confirming that Slit3 potently induced angiogenesis in cornea. Scale bare = 50 μm. (O-T). Whole-mount staining of embryonic day 15.5 diaphragms of Slit3+/− and Slit3−/− mice with anti-mouse PECAM-1 antibody. Quantification of the vascular parameter showed that, compared with the phenotypically normal littermate control Slit3+/− diaphragm, the Slit3−/− diaphragm showed reduced vascular density and branch points. n = 4-6 for each group. Asterisks indicate statistically significant difference compared with Slit3+/− control by Student t test (***P < .001). Red scale bar = 250 μm; black scale bar = 100 μm.

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