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Regulation of Cavernous Nerve Injury-Induced Apoptosis: DISCUSSION(7)

VEGFA acts downstream of SHH in the adult penis, since SHH treatment induces PTCH1 and VEGFA and SHH inhibition downregulates these targets. SHH induction of VEGFA is supported by reports in the literature in cardiac tissue and in ischemic limb. Schwann cells have been shown to induce VEGF expression. Mutations that eliminate Schwann cells prevent proper arteriogenesis, and in mutant embryos containing disorganized nerves the trajectory of blood vessel branching is altered to follow the nerve. These data suggest that peripheral nerves provide a template that determines the organotypic pattern of blood vessel branching and arterial differentiation during development, via local secretion of VEGFA.

Regulation of Cavernous Nerve Injury-Induced Apoptosis: DISCUSSION(6)

DISCUSSION(6)

We have shown that SHH protein is decreased following CN injury and that SHH inhibition is able to upregulate BMP4 in the corpora cavernosa. The decrease in SHH protein, accompanied by increased apoptosis, suggests that the mechanism described above may be active in the penis following CN injury. Bmp4 expression was unaltered with CN injury. However, Bmp4 plays a role in and is regulated by several pathways that do not involve Shh, and the protein levels of BMP4 may be different than the RNA expression levels with CN injury, as is the case with SHH.

Regulation of Cavernous Nerve Injury-Induced Apoptosis: DISCUSSION(5)

We hypothesize that CN injury-induced apoptosis is caused by decreased SHH protein. A mechanism for SHH involvement in apoptosis induction in the penis is presented in detail below, based on what is known about the Shh signaling pathway in the penis and in other organs (Fig. 9). In the absence of SHH, caspase 3 activity is increased and the G1 to S transition is inhibited, which leads to apoptosis which can be prevented by SHH application. The cell death program is initiated by the proapoptotic dependence receptor PTCH1.

Regulation of Cavernous Nerve Injury-Induced Apoptosis: DISCUSSION(4)

DISCUSSION(4)

In this work, we continue our ongoing efforts to determine what role neuropathy plays in ED development and to better understand the molecular mechanisms that regulate maintenance of penile architecture. We have shown that SHH inhibition causes apoptosis and that SHH protein is significantly decreased following CN injury. The localization of apoptosis in the corpora cavernosa after CN injury and after SHH inhibition appears very similar, with apoptotic cells present in the sinusoid smooth muscle and endothelium (Fig. 7). These results suggest that decreased SHH protein may be a cause of the abundant apoptosis that occurs following CN injury.

Regulation of Cavernous Nerve Injury-Induced Apoptosis: DISCUSSION(3)

It is an interesting finding that the RNA expression and precursor form of SHH protein increase while the active form of SHH protein decreases following CN injury. This suggests that there is inhibited posttranslational processing of the SHH protein with nerve injury and feedback regulation on Shh RNA expression in order to replace the decreased protein. Feedback regulation of Shh by downstream targets, including Bmp4 and Ptc, have been reported in other systems in the literature.

Regulation of Cavernous Nerve Injury-Induced Apoptosis: DISCUSSION(2)

DISCUSSION(2)

This shows that SHH has significant potential for development as a therapy for morphological and physiological changes in the penis that cause ED.

The reversibility of SHH inhibition has wide implications. Mutations in the Shh signaling pathway that target Ptchl, Smo, and Glil are associated with certain forms of cancer, including prostate, skin, and esophageal.

Regulation of Cavernous Nerve Injury-Induced Apoptosis: DISCUSSION(1)

Genes involved in cell fate decisions during development also play key roles in continuous cell fate decisions made by adult organs. The Shh signaling pathway is critical for establishing the sinusoid morphology of the corpora cavernosa, and Shh continues to regulate and maintain penile morphology in the adult organ. In two rat models of ED, the BB/WoR diabetic rat and in the CN-injured Sprague Dawley rat, SHH protein is significantly decreased. In these same models there are significant morphological changes in the corpora cavernosa, including increased apoptosis and decreased smooth muscle and endothelial staining.

Regulation of Cavernous Nerve Injury-Induced Apoptosis: RESULTS(7)

RESULTS(7)

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.

Regulation of Cavernous Nerve Injury-Induced Apoptosis: RESULTS(6)

Even sinusoidal tissue distant from the Affi-Gel bead vehicles showed abundant apoptosis after SHH inhibitor treatment. Apoptosis was not induced in control rats (Fig. 7) or in SHH protein-treated rats (SHH protein-treated = 1.6 6 0.7).

SHH Protein Treatment at the Time of CN Injury Prevents Post-CN Injury-induced Apoptosis

Affi-Gel beads soaked in SHH protein were injected into the corpora cavernosa at the time of bilateral CN injury in adult Sprague Dawley rats.

Regulation of Cavernous Nerve Injury-Induced Apoptosis: RESULTS(5)

RESULTS(5)

After 6 wk, smooth muscle lined sinusoidal tissue was abundant, the morphology of the corpora cavernosa was indistinguishable from PBS-treated controls (Fig. 6), and SHH protein was present in the sinusoidal tissue (data not shown).

CN Injury Induces Abundant Apoptosis

TUNEL staining was performed on control and CN-injured penes 2, 4, 7, and 21 days following CN injury in Sprague Dawley rat penes.