Shawn Choe

Sonic hedgehog delivery from self-assembled nanofiber hydrogels reduces the fibrotic response in models of erectile dysfunction ☆

UNLABELLED Erectile dysfunction (ED) is a serious medical condition in which current treatments are ineffective in prostatectomy and diabetic patients, due to injury to the cavernous nerve (CN), which causes irreversible remodeling of the penis (decreased smooth muscle and increased collagen), through a largely undefined mechanism. We propose that sonic hedgehog (SHH) and neural innervation, are indispensable regulators of collagen in the penis, with decreased SHH protein being an integral component of the fibrotic response to loss of innervation. We examined collagen abundance and morphology in control (Peyronies), prostatectomy and diabetic patients, and in rat models of penile development, CN injury, SHH inhibition and under regenerative conditions, utilizing self-assembling peptide amphiphile (PA) nanofiber hydrogels for SHH delivery. Collagen abundance increased in penis of ED patients. In rats, collagen increased with CN injury in a defined time frame independent of injury severity. An inverse relationship between SHH and collagen abundance was identified; SHH inhibition increased and SHH treatment decreased penile collagen. SHH signaling in the pelvic ganglia (PG)/CN is important to maintain CN integrity and when inhibited, downstream collagen induction occurs. Collagen increased throughout penile development and with age, which is important when considering how to treat fibrosis clinically. These studies show that SHH PA treatment reduces collagen under regenerative post-prostatectomy conditions, indicating broad application for ED prevention in prostatectomy, diabetic and aging patients and in other peripheral nerve injuries. The PA nanofiber protein vehicle may be widely applicable as an in vivo delivery tool. STATEMENT OF SIGNIFICANCE We use self-assembling peptide amphiphiles (PA) as biological delivery vehicles to prevent cavernous nerve (CN) injury induced erectile dysfunction (ED). These versatile hydrogels were molecularly pre-programmed for sonic hedgehog (SHH) protein delivery, either from an injectable solution with fast, in situ assembly into a soft hydrogel, or by highly aligned monodomain nanofiber bundles. We used PAs to examine a novel neuronal component to collagen regulation and the role of SHH in the fibrotic response to CN injury. SHH perturbation in the penis or the CN, selectively impacts collagen, with SHH inhibition increasing and SHH treatment suppressing collagen. These results suggest that SHH treatment by PA has translational potential to suppress collagen induction and remodelling, an irreversible component of ED development.

Peptide amphiphile nanofiber hydrogel delivery of sonic hedgehog protein to the cavernous nerve to promote regeneration and prevent erectile dysfunction.

Erectile dysfunction (ED) has high impact on quality of life in prostatectomy, diabetic and aging patients. An underlying mechanism is cavernous nerve (CN) injury, which causes ED in up to 80% of prostatectomy patients. We examine how sonic hedgehog (SHH) treatment with innovative peptide amphiphile nanofiber hydrogels (PA), promotes CN regeneration after injury. SHH and its receptors patched (PTCH1) and smoothened (SMO) are localized in PG neurons and glia. SMO undergoes anterograde transport to signal to downstream targets. With crush injury, PG neurons degenerate and undergo apoptosis. SHH protein decreases, SMO localization changes to the neuronal cell surface, and anterograde transport stops. With SHH treatment SHH is taken up at the injury site and undergoes retrograde transport to PG neurons, allowing SMO transport to occur, and neurons remain intact. SHH treatment prevents neuronal degeneration, maintains neuronal, glial and downstream target signaling, and is significant as a regenerative therapy.

MP43-14 SONIC HEDGEHOG REGULATION OF SPROUTING IN PENILE PROJECTING NEURONS

response was measured by cavernosal nerve stimulation. The relaxant and contractile responses of CC were obtained from in vitro studies. Western blotting and immunohistochemistry were used to evaluate protein expression and localization of neuronal nitric oxide synthase (NOS), endothelial NOS, CSE, CBS, oxidative stress (inducible NOS and nuclear factor kappa B), hypoxia (hypoxia-inducible factor-1a) and fibrosis (transforming growth factor beta 1) markers. The relative area of smooth muscle to collagen using Masson trichrome staining was determined. RESULTS: Our data were reported in Table 1. Combined treatment completely restored enhanced bladder weight, decreased in vivo erectile responses, in vitro electrical field stimulation (EFS)-mediated and endothelium-dependent acetylcholine (ACh)-induced relaxation of CC in the PBOO group, while partial improvement with monotherapy. Contractile responses of CC in obstructed rats were lower than in control rats, which were increased in all treatments groups (Table 1). The combined treatment modulated alteration of protein expressions and decreased ratio of smooth muscle to collagen in obstructed rats. CONCLUSIONS: We firstly indicated that successful combination with H2S donor and PDE5i therapy provided the recovery of erectile function, the progression of ischemia-related functional and morphological penile changes in obstruction. H2S and NO most probably may have a synergistic effect on the structure of penile tissue and molecular regulation of erectile function, and be beneficial for improving clinical outcomes in men with ED and BPH/LUTS.

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

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.

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.

MP42-07 SONIC HEDGEHOG REGULATION OF RHABDOSPHINCTER MUSCLE

persists following the resolution of colitis, and that linaclotide, an FDA approved guanylate cyclase-C (GC-C) agonist, is able to attenuate these changes [3]. We hypothesise that these CCH-induced changes are the result of altered sensitivity of afferents both within the colon and bladder wall and within the dorsal root ganglion (DRG), and that oral linaclotide administration may act to reduce this hypersensitivity. METHODS: We investigated healthy C57BL/6J mice and mice with CCH, 28 days after intra-colonic TNBS administration. CCH mice were randomly assigned to either chronic linaclotide (3mg/kg/day) or placebo (water) administration, consisting of a once daily oral gavage for 2 weeks prior to experimentation. Ex-vivo electrophysiological recordings determined bladder afferent and contractile sensitivity to abMe-ATP (30mM), carbachol (1mM), and capsaicin (10mM) as well as whole cell patch clamp of retrogradely traced bladder DRG neurons in all four groups. RESULTS: Bladder DRG from mice with CCH display hyperexcitability, with a significant reduction in rheobase compared to controls (p 0.01). CCH mice also display significantly enhanced afferent responses to abMe-ATP (p 0.001), carbachol (p 0.001), and capsaicin (p 0.001). CCH mice treated with linaclotide display attenuated DRG hyperexcitability and normalised afferent responses to agonists (p 0.01) compared to placebo (p 0.01). CONCLUSIONS: Mice with CCH also display increased bladder afferent excitability within both the DRG and bladder wall, indicating cross-organ sensitisation. Chronic oral administration of linaclotide, a locally acting GC-C agonist that inhibits colonic nociceptors, reverses these colitis-induced changes in bladder afferent sensitivity. Common sensory pathways may allow agents that reduce abdominal pain to improve urological symptoms. 1. Lamb, K., et al. AJPGI, 2006. 2. Ustinova et al, Neurourology and Urodynamics, 2010. 3. Grundy, L., et al., MP28-06. The Journal of Urology, 2016.

MP45-08 CAVERNOUS NERVE CRUSH INJURY INDUCES APOPTOSIS IN THE PELVIC PLEXUS INCLUDING PELVIC AND HYPOGASTRIC NERVES

on Matrigel in growth factor reduced medium for three to five days. Pelvic ganglia/CN were exposed to Affi-Gel beads containing: 1.) SHH protein, 2.) 5e1 and cyclopamine SHH inhibitors, and 3.) SHH protein delivered by PA. Additional pelvic ganglia/CN tissue underwent CN crush and were exposed to SHH protein or mouse serum albumin protein (control) by PA in vivo for 4 days with an additional 4 days in culture. Sprouting was evaluated for number of sprouts and their length, and by immunohistochemical analysis for sprouting markers (GAP43 and nNOS). RESULTS: Sprouting of neurons in the pelvic ganglia and CN were increased with SHH treatment. Sprouts were more abundant, longer in length, and had increased branching, in comparison to controls. Sprouting was even further enhanced in CN injured nerves with SHH treatment. Sprouting did not occur in the presence of either SHH inhibitor. The CN had similar sprouting potential at 4 and 9 days after crush injury. SHH induced sprouting even when not delivered to the CN until 4 days after injury. Sprouts continued to grow in organ culture once initiated with SHH PA in vivo. Localization of SHH delivery makes a difference in sprouting potential. Sprouts formed in response to SHH treatment stained strongly for nNOS protein. CONCLUSIONS: SHH PA treatment promotes CN regeneration by enhancing sprouting of pelvic ganglia and CN neurons. Understanding the mechanism of SHH PA action on neuronal tissue is critical for translation to prostatectomy patients and to further enhance regeneration.

AB300. SPR-27 Sonic hedgehog promotes sprouting of neurons in the pelvic ganglia and cavernous nerve during regeneration

Objective We’ve shown in previous studies that sonic hedgehog (SHH) protein delivered by nanoparticle based peptide amphiphile (PA) hydrogels to the cavernous nerve (CN) at the time of crush injury (mimicking prostatectomy), are neuroprotective and promote CN regeneration in a rat model. The mechanism of how SHH promotes CN regeneration is unknown. We hypothesize that SHH promotes sprouting of CN axons, in order to enhance nerve regeneration. We examine this hypothesis in an in vitro organ culture model. Methods The caudal portion of the pelvic ganglia (innervates penis) and CN were dissected from adult Sprague Dawley rats (n=47) and placed in Matrigel in growth factor reduced medium and were grown for three to five days. Pelvic ganglia were exposed to Affi-Gel beads containing: (I) SHH protein; (II) 5e1 and cyclopamine SHH inhibitors; and (III) SHH protein delivered by PA. Additional pelvic ganglia/CN tissue underwent CN crush and were exposed to SHH protein or PBS/mouse serum albumin (MSA) protein. Sprouting was evaluated for number of sprouts and their length, and by immunohistochemical analysis for sprouting markers (GAP43 and nNOS). Results Sprouting of pelvic ganglia and CN axons was increased with SHH treatment. Sprouts were more abundant, longer in length, with larger arborization of sprouts, in comparison to controls. More sprouting was promoted with SHH treatment of CN injured nerves. The CN had similar sprouting potential at 4 and 9 days after crush injury. Localization of SHH delivery makes a difference in sprouting potential. Conclusions The mechanism of how SHH PA treatment promotes CN regeneration, involves enhanced sprouting of pelvic ganglia and CN neurons. Understanding the mechanism of SHH PA action on neuronal tissue is critical for translation to prostatectomy patients and to further enhance regeneration. Funding Source(s) NIH/NIDDK DK079184

AB307. SPR-34 Optimization of sonic hedgehog delivery from self-assembled nanofiber hydrogels

Objective Sonic hedgehog (SHH) protein delivered by nanoparticle based peptide amphiphile (PA) hydrogels to the penis suppress apoptosis in a rat cavernous nerve (CN) resection model. We examine the hypothesis that SHH PA will suppress morphology changes in the penis in a CN crush model that more readily reflects injury observed in prostatectomy patients. Optimization of delivery conditions is essential for clinical translation. Methods Bilateral CN crush was performed on Sprague Dawley rats (n=67) and SHH or mouse serum albumin (MSA) (control) protein was delivered by PA injected into the corpora cavernosa. Rats were sacrificed after 4 and 9 days. 2X SHH protein was also assayed at 4 days. A second SHH PA injection at 5 days occurred prior to sacrifice at 9 days. Additional rats had SHH or MSA delivered to both the penis and CN by PA. TUNEL and hydroxyproline assay were performed. Results Apoptosis increased 54% 4 days after injury (P=0.0001). SHH PA suppressed apoptosis 27% at 4 days after CN injury (P=0.005). 2X SHH protein suppressed apoptosis 29% (P=0.003). Apoptosis increased 21% at 9 days after injury (P=0.014). Two SHH PA injections decreased apoptosis 22% at 9 days (P=0.021), while one SHH PA injection was indistinguishable from controls (P=0.830). SHH delivery to penis and CN decreased apoptosis 27% (P=0.0001). Conclusions Apoptosis suppression was similar in CN resection and crush models in response to SHH treatment. One SHH PA injection suppressed apoptosis until protein was depleted. Increasing the duration of SHH treatment by a second SHH PA injection, suppress apoptosis longer. Optimization of SHH PA delivery is essential for translation to prostatectomy patients to prevent ED. Funding Source(s) NIH/NIDDK DK079184