Figure 5.

Phospho-SMAD3 signaling is reduced, in endothelial cells of integrin β8 mutants. A, Top, Representative thin sections from P9 retinas illustrating phospho-SMAD3 (pSMAD3, red) colocalization with endothelial cell-specific-CD31 (green) in control and itgb8 complete knock-out retinas. Bottom are rainbow spectrum intensity maps of phospho-SMAD3 staining delimited by CD31 staining from boxed regions in panels above (red, most intense staining; blue, least intense staining). Whereas most endothelial cells in the superficial and deep vascular plexuses of control mice are strongly positive (arrows) and few weakly positive (arrowheads) for pSMAD3 staining, relatively few endothelial cells are strongly positive and most are weakly positive for pSMAD3 in mutants. Note the thickened superficial vascular plexus (SVP) and lack of a deep vascular plexus (DVP) in the mutant versus control sections. B, Quantification of vascular nuclei per 100 μm length of the vasculature documents a significant increase in the density of vasculature-associated endothelial cells in the mutant. The intensity of phospho-SMAD3 labeling of each endothelial nucleus was quantified in B in arbitrary units. The intensity of labeling in individual nuclei as well as the mean leveling densities presented in this graph documents a significant reduction in endothelial cell-specific phospho-SMAD3 levels in mutants compared with controls. p-values from Student's t test: *p = 0.019, ***p < 0.0001; N = 3 (controls), N = 3 (β8icr^−/− mutants). Error bars in all graphs represent SEM. Scale bars: (A), 100 μm; (C), 50 μm.