At 8 days post-CN injury, apoptosis was still suppressed in the presence of SHH protein (ratio of apoptotic cells to all cells within ~330 im of SHH protein-treated CN8 = 0.24 6 0.07 and control heat-inactivated SHH protein-treated CN8 = 0.71 6 0.06 penes, P-value = 9.03E-05). CN injury-induced apoptosis was not suppressed in control penes.
The amount of apoptosis suppression was dependent on the distance from the Affi-Gel beads at ~95 im (ratio of apoptotic cells to all cells for control heat-inactivated 2X SHH protein CN4 — 0.80 6 0.03 and 2X SHH protein CN4— 0.05 6 0.03, P-value — 4.32E-05), ~215 im (ratio of apoptotic cells to all cells for control heat-inactivated 2X SHH protein CN4 — 0.78 6 0.05 and 2X SHH protein CN4 — 0.38 6 0.14, P-value — 0.02), and at ~330 im (ratio of apoptotic cells to all cells for control heat-inactivated 2X SHH protein CN4 — 0.91 6 0.04 and 2X SHH protein CN4 — 0.57 6 0.07, P-value — 0.008) from the beads.
SHH Induction of VEGFA
Adult Sprague Dawley rat penes were treated with either 5E1 SHH inhibitor or SHH protein via Affi-Gel beads, and IHC was performed on penis tissue assaying for VEGFA. VEGFA protein was upregulated in response to SHH protein treatment and was reduced in the presence of SHH inhibitor (Fig. 8).
FIG. 8. IHC analysis of VEGFA localization in control (top), SHH-protein-treated (middle), and SHH-inhibited (bottom) adult Sprague Dawley rat penes. VEGFA protein is normally abundant within the corpus cavernosal sinusoidal tissue (original magnification X400). VEGFA protein was upregulated after SHH protein treatment and downregulated after SHH inhibition in the corpora cavernosa (original magnification X250), suggesting that SHH may be an upstream regulator of VEGFA. Arrows indicate VEGFA protein;b, Affi-Gel bead.