(066) Targets of SHH Signaling, BMP4 and GREM1, are Downstream Regulators of Smooth Muscle in the Penis

Abstract

Abstract Introduction A key aspect of erectile dysfunction (ED) development is loss of innervation to the penis. The cavernous nerve (CN) is frequently damaged in prostatectomy and diabetic patients with ED, initiating changes in penile morphology including an acute and intense phase of apoptosis in penile smooth muscle followed by increasing collagen and fibrosis. This remodeling process alters penile architecture so that there is less smooth muscle in the corpora cavernosa, and what remains is less compliant/able to relax in response to normal neurotransmitter signaling, and ED results. Thus, novel therapies are needed which target the underlying remodeling process. We have shown previously that Sonic hedgehog (SHH) is a critical regulator of penile smooth muscle, and that SHH protein treatment suppresses penile remodeling after CN injury through an unknown mechanism. Objective We examine if part of the mechanism of how SHH preserves smooth muscle after CN injury involves bone morphogenetic protein 4 (BMP4) and GREMLIN (GREM1). Methods Primary cultures of smooth muscle cells were established from patients who had prostatectomy, diabetes, hypertension and Peyronie’s (control, n=18). Cultures were characterized by immunohistochemical analysis (IHC) for ACTA2, CD31, P4HB and nNOS. Smooth muscle cell growth was quantified in response to BMP4 and GREM1. Adult Sprague Dawley rats that were uninjured or underwent CN crush injury, were treated with BMP4, GREM1 or MSA (control) proteins via Affi-Gel beads (n=16) or peptide amphiphile (PA, n=26) for 3 and 14 days, and trichrome stain was performed. Sprague Dawley rats underwent CN injury (n=9) and SHH PA treatment for 1, 2 and 4 days (n=9) and western was performed assaying for BMP4 and GREM1 proteins in comparison to sham (n=3) controls. Adult Sprague Dawley rats were treated with 5E1 SHH inhibitor (n=6) or IgG (control, n=6) for 2 and 4 days, and BMP4 and GREM1 localization were examined by IHC. Statistics were performed by ANOVA with Scheffe’s posthoc test. Results Patient primary smooth muscle cultures were established. BMP-4 increased patient smooth muscle cell growth and GREM1 decreased growth. In rats BMP4 treatment via Aff-Gel beads and PA increased smooth muscle growth at 3 and 14 days of treatment. GREM1 treatment caused collagen and smooth muscle growth at 3 days, which switched to primarily collagen at 14 days. CN injury increased BMP4 and GREM1. SHH PA altered western band size suggesting alternative cleavage and range of signaling. SHH inhibition in rats changed BMP4 and GREM1 localization. Conclusions BMP4 treatment increases smooth muscle in patient cultures and in our rat model. BMP4 and GREM1 mediate a switch from smooth muscle to collagen induction. SHH treatment alters BMP4 and GREM1 localization and range of signaling, which can affect penile morphology. Whether GREM1 directly effects collagen induction or acts strictly through inhibition of BMP4 is unknown and requires further study. Part of the mechanism of how SHH regulates corpora cavernosal smooth muscle, involves BMP4 and GREM1. Understanding how SHH PA impacts penile morphology after CN injury will aid development of ED therapies. Disclosure No.