(084) Analysis of BMP4 and GREMLIN as targets of SHH signaling and regulators of the collagen axis in the penis.

Abstract Introduction Increased collagen deposition occurs in erectile dysfunction (ED) patients and animal models, and the underlying causes of these fibrotic changes have not been well defined. The Sonic hedgehog (SHH) pathway is critical for the response of the penis to denervation. Decreased SHH in the penis due to cavernous nerve (CN) injury, causes smooth muscle apoptosis and ED. As smooth muscle is lost, increased collagen occurs by a largely unknown mechanism. We have shown that collagen abundance is responsive to SHH signaling in the penis. Microarray analysis identified increased GREMLIN (BMP4 antagonist) in corpora cavernosa of ED patients, and SHH is a regulator of GREMLIN in the limb bud. Objective We hypothesize that SHH regulation of collagen occurs through a BMP4/GREMLIN dependent mechanism and will examine BMP4 and GREMLIN signalling in corpora cavernosa of ED patients and a rat prostatectomy model. Study of how collagen induction occurs after prostatectomy would be useful to determine the underlying mechanism of penile remodeling. Methods Corpora cavernosal tissue (n=36) from prostatectomy, diabetic and Peyronie’s (control) patients were examined by immunohistochemical and western analysis for BMP-4 and GREMLIN proteins. Localization was determined by dual staining with ACTA2 and CD31. Collagen was quantified by hydroxyproline assay and trichrome stain with Image J analysis. Primary cultures of patient corpora cavernosa were established and treated with BMP4 and GREMLIN proteins, and smooth muscle cell growth was quantified. Sprague dawley rat penis was treated in vivo with SHH protein by peptide amphiphile, 5E1 SHH inhibitor, BMP4 and GREMLIN via Affi-Gel beads. Collagen was quantified. Results BMP-4 and GREMLIN were identified in smooth muscle of Peyronie’s, prostatectomy and diabetic ED patients. Western analysis identified increased BMP4 and GREMLIN proteins in prostatectomy and diabetic patients where collagen is increased. Primary culture of corpora cavernosal smooth muscle cells showed an increase in growth with BMP4 and decreased growth with GREMLIN treatment. This trend was more pronounced in cells from hypertension and cardiovascular disease patients. BMP4 and GREMLIN proteins increased 1-2 days after CN injury in a rat prostatectomy model that showed decreasing SHH in the penis. SHH inhibition in the penis increases BMP4, GREMLIN and collagen. SHH treatment of the penis after CN injury in the rat decreases collagen, BMP4 and GREMLIN. Conclusions SHH is decreased in corpora cavernosa of prostatectomy and diabetic ED patients while BMP4 and GREMLIN are increased. Decreased SHH increases BMP4 protein and collagen, suggesting a possible mechanism for fibrotic penile remodelling after CN injury. This study is the first step in understanding the mechanism of how SHH regulates collagen abundance in the penis, which is important for clinical translation. Disclosure No