Optimization of Sonic Hedgehog Delivery to the Penis from Self-Assembling Nanofiber Hydrogels to Preserve Penile Morphology after Cavernous Nerve Injury.

Erectile dysfunction (ED) is a significant medical condition, with high impact on patient quality of life. Current treatments are minimally effective in prostatectomy, diabetic and aging patients due to injury to the cavernous nerve (CN); loss of innervation causes extensive smooth muscle (SM) apoptosis, increased collagen and ED. Sonic hedgehog (SHH) is a critical regulator of penile SM. We developed a self-assembling peptide amphiphile (PA) nanofiber hydrogel for extended release of SHH protein to the penis after CN injury, to suppress SM apoptosis. In this study we optimize the animal model, SHH concentration, duration of suppression, and location of delivery, to maximize SM preservation. SHH treatment suppressed apoptosis and preserved SM 48%. Increased SHH duration preserved SM 100%. Simultaneous penis/CN delivery increased SM 127%. Optimization of SHH PA delivery is essential for clinical translation to ED patients, and the PA vehicle has wide applicability as an in vivo delivery tool.

Sonic hedgehog regulation of cavernous nerve regeneration and neurite formation in aged pelvic plexus.

INTRODUCTION: Erectile dysfunction (ED) is a significant health concern that greatly impacts quality of life, and is common in men as they age, impacting 52% of men between the ages of 40 and 70. A significant underlying cause of ED development is injury to the cavernous nerve (CN), a peripheral nerve that innervates the penis. CN injury also occurs in up to 82% of prostatectomy patients. We recently showed that Sonic hedgehog (SHH) protein delivered by peptide amphiphile (PA) nanofiber hydrogel to the CN and penis of a prostatectomy model of CN injury, is neuroprotective, accelerates CN regeneration, improves erectile function ~60%, preserves penile smooth muscle 56% and suppresses collagen deposition 30%. This regenerative potential is substantial in an adult prostatectomy model (P120). However prostatectomy patients are typically older (61.5 ±â€¯9.6 years) and our models should mimic patient conditions more effectively when considering translation. In this study we examine regenerative potential in an aged prostatectomy model (P200-329). METHODS: The caudal portion of the pelvic ganglia (MPG) and CN were dissected from adult (n = 11), and aged (n = 13) Sprague Dawley rats, and were grown in organ culture 3 days. Uninjured and 2 day CN crushed MPG/CN were exposed to Affi-Gel beads containing SHH protein, PBS (control), or 5e1 SHH inhibitor. Neurites were quantified by counting the number of growth cones normalized by tissue perimeter (mm) and immunohistochemistry for SHH, patched1 (PTCH1), smoothened (SMO), GLI1-3, and GAP43 were performed. RESULTS: SHH treatment increased neurites 3.5-fold, in uninjured adult, and 5.7-fold in aged rats. Two days after CN crush, SHH treatment increased neurites 1.8-fold in adult rats and 2.5-fold in aged rats. SHH inhibition inhibited neurite formation in uninjured MPG/CN but not in 2 day CN crushed MPG/CN. PTCH1 and SMO (SHH receptors), and SHH transcriptional activators/repressors, GLI1-3, were abundant in aged MPG/CN with unaltered localization. ROCK1 was induced with SHH treatment. CONCLUSIONS: Reintroduction of SHH protein in an aged prostatectomy model is even more effective in promoting neurite formation/CN regeneration than in the adult. The first 48 h after CN injury are a critical window when growth factors are released, that impact later neurite formation. These studies are significant because most prostatectomy patients are not young and healthy, as with adult rats, so the aged prostatectomy model will more accurately simulate ED patient response. Understanding how neurite formation changes with age is critical for clinical translation of SHH PA to prostatectomy patients.

Pelvic and hypogastric nerves are injured in a rat prostatectomy model, contributing to development of stress urinary incontinence.

Urinary incontinence affects 40% of elderly men, is common in diabetic patients and in men treated for prostate cancer, with a prevalence of up to 44%. Seventy-two percent of prostatectomy patients develop stress urinary incontinence (SUI) in the first week after surgery and individuals who do not recover within 6 months generally do no regain function without intervention. Incontinence has a profound impact on patient quality of life and a critical unmet need exists to develop novel and less invasive SUI treatments. During prostatectomy, the cavernous nerve (CN), which provides innervation to the penis, undergoes crush, tension, and resection injury, resulting in downstream penile remodeling and erectile dysfunction in up to 85% of patients. There are other nerves that form part of the major pelvic ganglion (MPG), including the hypogastric (HYG, sympathetic) and pelvic (PN, parasympathetic) nerves, which provide innervation to the bladder and urethra. We examine if HYG and PNs are injured during prostatectomy contributing to SUI, and if Sonic hedgehog (SHH) regulatory mechanisms are active in the PN and HYG nerves. CN, PN, HYG and ancillary (ANC) of uninjured, sham and CN crush/MPG tension injured (prostatectomy model) adult Sprague Dawley rats (n = 37) were examined for apoptosis, sonic hedgehog (SHH) pathway, and intrinsic and extrinsic apoptotic mechanisms. Fluorogold tracing from the urethra/bladder was performed. PN and HYG response to SHH protein was examined in organ culture. TUNEL, immunohistochemical analysis for caspase-3 cleaved, -8, -9, SHH, Patched and Smoothened (SHH receptors), and neurite formation, were examined. Florogold positive neurons in the MPG were reduced with CN crush. Apoptosis increased in glial cells of the PN and HYG after CN crush. Caspase 9 was abundant in glial cells (intrinsic), while caspase-8 was not observed. SHH and its receptors were abundant in neurons and glia of the PN and HYG. SHH treatment increased neurite formation. PN and HYG injury occur concomitant with CN injury during prostatectomy, likely contributing to SUI. PN and HYG response to SHH treatment indicates an avenue for intervention to promote regeneration and prevent SUI.

Sonic hedgehog regulation of human rhabdosphincter muscle:Potential implications for treatment of stress urinary incontinence.

AIMS: Rhabdosphincter (RS) muscle injury occurs during prostatectomy, and is a leading cause of stress urinary incontinence (SUI). Current SUI treatments engender significant side effects, which negatively impact patient quality of life. Thus an unmet need exists to develop novel RS regeneration methods. We have shown that Sonic hedgehog (SHH) is a critical regulator of penile smooth muscle, and we have developed novel peptide amphiphile nanofiber hydrogel delivery of SHH protein to the penis to regenerate smooth muscle after prostatectomy induced injury. If similar SHH signaling mechanisms regulate RS muscle homeostasis, this innovative technology may be adapted for RS regeneration post-prostatectomy. We examine the SHH pathway in human RS muscle. METHODS: Human RS obtained during radical cystoprostatectomy (n = 13), underwent SHH pathway analysis. Primary cultures were established (n = 5), and RS cells were treated with SHH protein, SHH inhibitor, or PBS (control). Immunohistochemical analysis for SHH pathway, skeletal muscle actin, and trichrome stain were performed. RS growth was quantified at 3 and 6 days. RESULTS: SHH, it is receptors patched and smoothened, and transcriptional activators, GLI proteins, were identified in human RS muscle. At 3 and 6 days, RS cells increased 62% and 78% (P = 0.0001) with SHH treatment and decreased 40% (P = 0.0001) and 18% (P = 0.039) with SHH inhibition. CONCLUSIONS: The SHH pathway was identified in human RS. RS growth increased with SHH treatment, indicating intervention may be possible to enhance RS regeneration, and impact SUI. Peptide amphiphile delivery of SHH may be applicable for RS regeneration and SUI prevention.

Peptide amphiphile delivery of sonic hedgehog protein promotes neurite formation in penile projecting neurons.

Erectile dysfunction (ED) critically impacts quality of life in prostatectomy, diabetic and aging patients. The underlying mechanism involves cavernous nerve (CN) damage, resulting in ED in 80% of prostatectomy patients. Peptide amphiphile (PA) nanofiber hydrogel delivery of sonic hedgehog (SHH) protein to the injured CN, improves erectile function by 60% at 6 weeks after injury, by an unknown mechanism. We hypothesize that SHH is a regulator of neurite formation. SHH treatment promoted extensive neurite formation in uninjured and crushed CNs, and SHH inhibition decreased neurites >80%. Most abundant neurites were observed with continuous SHH PA treatment of crushed CNs. Once induced with SHH, neurites continued to grow. SHH rescued neurite formation when not given immediately. SHH is a critical regulator of neurite formation in peripheral neurons under uninjured and regenerative conditions, and SHH PA treatment at the time of injury/prostatectomy provides an exploitable avenue for intervention to prevent ED.