SHH regulates penile morphology and smooth muscle through a mechanism involving BMP4 and GREM1.

BACKGROUND: The cavernous nerve (CN) is frequently damaged in prostatectomy and diabetic patients with erectile dysfunction (ED), initiating changes in penile morphology including an acute and intense phase of apoptosis in penile smooth muscle and increased collagen, which alter penile architecture and make corpora cavernosa smooth muscle less able to relax in response to neurotransmitters, resulting in ED. AIM: Sonic hedgehog (SHH) is a critical regulator of penile smooth muscle, and SHH treatment suppresses penile remodeling after CN injury through an unknown mechanism; we examine if part of the mechanism of how SHH preserves smooth muscle after CN injury involves bone morphogenetic protein 4 (BMP4) and gremlin1 (GREM1). METHODS: Primary cultures of smooth muscle cells were established from prostatectomy, diabetic, hypertension and Peyronie's (control) (N = 18) patients. Cultures were characterized by ACTA2, CD31, P4HB, and nNOS immunohistochemical analysis. Patient smooth muscle cell growth was quantified in response to BMP4 and GREM1 treatment. Adult Sprague Dawley rats underwent 1 of 3 surgeries: (1) uninjured or CN-injured rats were treated with BMP4, GREM1, or mouse serum albumin (control) proteins via Affi-Gel beads (N = 16) or peptide amphiphile (PA) (N = 26) for 3 and 14 days, and trichrome stain was performed; (2) rats underwent sham (N = 3), CN injury (N = 9), or CN injury and SHH PA treatment for 1, 2, and 4 days (N = 9). OUTCOMES: Western analysis for BMP4 and GREM1 was performed; (3) rats were treated with 5E1 SHH inhibitor (N = 6) or IgG (control; N = 6) for 2 and 4 days, and BMP4 and GREM1 localization was examined. Statistics were performed by analysis of variance with Scheffé's post hoc test. RESULTS: BMP4 increased patient smooth muscle cell growth, and GREM1 decreased growth. In rats, BMP4 treatment via Affi-Gel beads and PA increased smooth muscle at 3 and 14 days of treatment. GREM1 treatment caused increased collagen and smooth muscle at 3 days, which switched to primarily collagen at 14 days. CN injury increased BMP4 and GREM1, while SHH PA altered Western band size, suggesting alternative cleavage and range of BMP4 and GREM1 signaling. SHH inhibition in rats increased BMP4 and GREM1 in fibroblasts. CLINICAL IMPLICATIONS: Understanding how SHH PA preserves and regenerates penile morphology after CN injury will aid development of ED therapies. STRENGTHS AND LIMITATIONS: SHH treatment alters BMP4 and GREM1 localization and range of signaling, which can affect penile morphology. CONCLUSION: Part of the mechanism of how SHH regulates corpora cavernosa smooth muscle involves BMP4 and GREM1.

BMP4 and GREM1 are targets of SHH signaling and downstream regulators of collagen in the penis.

BACKGROUND: Cavernous nerve (CN) injury, caused by prostatectomy and diabetes, initiates a remodeling process (smooth muscle apoptosis and increased collagen) in the corpora cavernosa of the penis of patients and animal models that is an underlying cause of erectile dysfunction (ED), and the Sonic hedgehog (SHH) pathway plays an essential role in the response of the penis to denervation, as collagen increases with SHH inhibition and decreases with SHH treatment. AIM: We examined if part of the mechanism of how SHH prevents penile remodeling and increased collagen with CN injury involves bone morphogenetic protein 4 (BMP4) and gremlin1 (GREM1) and examined the relationship between SHH, BMP4, GREM1, and collagen in penis of ED patients and rat models of CN injury, SHH inhibition, and SHH, BMP4, and GREM1 treatment. METHODS: Corpora cavernosa of Peyronie's disease (control), prostatectomy, and diabetic ED patients were obtained (N = 30). Adult Sprague Dawley rats (n = 90) underwent (1) CN crush (1-7 days) or sham surgery; (2) CN injury and BMP4, GREM1, or mouse serum albumin (control) treatment via Affi-Gel beads or peptide amphiphile (PA) for 14 days; (3) 5E1 SHH inhibitor, IgG, or phosphate-buffered saline (control) treatment for 2 to 4 days; or (4) CN crush with mouse serum albumin or SHH for 9 days. OUTCOMES: Immunohistochemical and Western analysis for BMP4 and GREM1, and collagen analysis by hydroxyproline and trichrome stain were performed. RESULTS: BMP4 and GREM1 proteins were identified in corpora cavernosa smooth muscle of prostatectomy, diabetic, and Peyronie's patients, and in rat smooth muscle, sympathetic nerve fibers, perineurium, blood vessels, and urethra. Collagen decreased 25.4% in rats with CN injury and BMP4 treatment (P = .02) and increased 61.3% with CN injury and GREM1 treatment (P = .005). Trichrome stain showed increased collagen in rats treated with GREM1. Western analysis identified increased BMP4 and GREM1 in corpora cavernosa of prostatectomy and diabetic patients, and after CN injury (1-2 days) in our rat model. Localization of BMP4 and GREM1 changed with SHH inhibition. SHH treatment increased the monomer form of BMP4 and GREM1, altering their range of signaling. CLINICAL IMPLICATIONS: A better understanding of penile remodeling and how fibrosis occurs with loss of innervation is essential for development of novel ED therapies. STRENGTHS AND LIMITATIONS: The relationship between SHH, BMP4, GREM1, and collagen is complex in the penis. CONCLUSION: BMP4 and GREM1 are downstream targets of SHH that impact collagen and may be useful in collaboration with SHH to prevent penile remodeling and ED.

Pathway analysis of microarray data from corpora cavernosal tissue of patients with a prostatectomy or Peyronie disease in comparison with a cavernous nerve-injured rat model of erectile dysfunction.

INTRODUCTION: Patients with a prostatectomy are at high risk of developing erectile dysfunction (ED) that is refractory to phosphodiesterase type 5 inhibitors. The cavernous nerve (CN) is frequently damaged during prostatectomy, causing loss of innervation to the penis. This initiates corpora cavernosal remodeling (apoptosis and fibrosis) and results in ED. AIM: To aid in the development of novel ED therapies, the current aim was to obtain a global understanding of how signaling mechanisms alter in the corpora cavernosa with loss of CN innervation that results in ED. METHODS: Microarray and pathway analysis were performed on the corpora cavernosal tissue of patients with a prostatectomy (n = 3) or Peyronie disease (control, n = 3). Results were compared with an analysis of a Sprague-Dawley rat CN injury model (n = 10). RNA was extracted by TRIzol, DNase treated, and purified by a Qiagen Mini Kit. Microarray was performed with the Human Gene 2.0 ST Array and the RU34 rat array. Differentially expressed genes were identified through several analytic tools (ShinyGO, Ingenuity, WebGestalt) and databases (GO, Reactome). A 2-fold change was used as the threshold for differential expression. OUTCOMES: Pathways that were altered (up- or downregulated) in response to CN injury in the prostatectomy patients and a rat CN injury model were determined. RESULTS: Microarray identified 197 differentially expressed protein-coding genes in the corpora cavernosa from the prostatectomy cohort, with 100 genes upregulated and 97 genes downregulated. Altered signaling pathways that were identified that affect tissue morphology included the following: neurologic disease, cell death and survival, tissue and cellular development, skeletal and muscle development and disorders, connective tissue development and function, tissue morphology, embryonic development, growth and proliferation, cell-to-cell signaling, and cell function and maintenance. These human pathways have high similarity to those observed in the CN-injured rat ED model. CLINICAL IMPLICATIONS: Significant penile remodeling continues in patients long after the acute surgical injury to the CN takes place, offering the opportunity for clinical intervention to reverse penile remodeling and improve erectile function. STRENGTHS AND LIMITATIONS: Understanding how signaling pathways change in response to CN injury and how these changes translate to altered morphology of the corpora cavernosa and ensuing ED is critical to identify strategic targets for therapy development. CONCLUSION: Altered signaling in pathways that regulate tissue homeostasis, morphogenesis, and development was identified in penes of patients with a prostatectomy, and competitive forces of apoptosis and proliferation/regeneration were found to compete to establish dominance after CN injury. How these pathways interact to regulate penis tissue homeostasis is a complex process that requires further investigation.

Pathway Enrichment Analysis of Microarray Data Fom Human Penis of Diabetic and Peyronie's Patients, in Comparison With Diabetic Rat Erectile Dysfunction Models.

BACKGROUND: Erectile dysfunction (ED) is a debilitating medical condition in which current treatments are minimally effective in diabetic patients due to neuropathy of the cavernous nerve, a peripheral nerve that innervates the penis. Loss of innervation causes apoptosis of penile smooth muscle, remodeling of corpora cavernosa (penile erectile tissue) morphology, and ED. AIM: In this study, microarray and pathway analysis were used to obtain a global understanding of how signaling mechanisms are altered in diabetic patients and animal models as ED develops, in order to identify novel targets for disease management, and points of intervention for clinical therapy development. METHODS AND OUTCOMES: Human corpora cavernosal tissue was obtained from diabetic (n = 4) and Peyronie's (control, n = 3) patients that were undergoing prosthesis implant to treat ED, and BB/WOR diabetic (n = 5) and resistant (n = 5) rats. RNA was extracted using TRIzol, DNase treated, and purified by Qiagen mini kit. Microarray was performed using the Human Gene 2.0 ST Array. (i) Alterations in patient and diabetic rat pathway signaling were examined using several analytical tools (ShinyGO, Metascape, WebGestalt, STRING) and databases, (ii) Strengths/weaknesses of the different pathway analysis tools were compared, and (iii) Comparison of human and rat (BB/WOR and Streptozotocin) pathway analysis was performed. Two technical replicates were performed. P value (FDR) < .15 was used as threshold for differential expression. FDR < 0.05 was considered significant. RESULTS: Microarray identified 182 differentially expressed protein-coding genes. Pathway analysis revealed similar enrichments with different analytical tools. Down regulated pathways include development, tubular structure, sprouting, cell death, ischemia, angiogenesis, transcription, second messengers, and stem cell differentiation. ED patients, who have diabetes, incur significant loss of normal regulatory processes required for repair and replacement of injured corpora cavernosal tissue. Combined with loss of apoptotic regulatory mechanisms, this results in significant architectural remodeling of the corpora cavernosa, and loss of regenerative capacity in the penis. CLINICAL TRANSLATION: This first report of microarray and pathway analysis in human corpora cavernosa, is critical for identification of novel pathways pertinent to ED and for validating animal models. STRENGTHS AND LIMITATIONS: The analysis of tissue specific gene expression profiles provides a means of understanding drivers of disease and identifying novel pathways for clinical intervention. CONCLUSION: Penis from diabetic ED patients lacks capacity for maintenance of corpora cavernosal architecture and regeneration, which are critical points for intervention for therapy development. Searl T, Ohlander S, McVary KT, et al., Pathway Enrichment Analysis of Microarray Data Fom Human Penis of Diabetic and Peyronie's Patients, in Comparison With Diabetic Rat Erectile Dysfunction Models. J Sex Med 2022;19:37-53.

Sonic Hedgehog Signaling in Primary Culture of Human Corpora Cavernosal Tissue From Prostatectomy, Diabetic, and Peyronie's Patients.

BACKGROUND: Cavernous nerve (CN) injury causes penile remodeling, including smooth muscle apoptosis and increased collagen, which results in erectile dysfunction (ED), and prevention of this remodeling is critical for novel ED therapy development. AIM: We developed 2 peptide amphiphile (PA) hydrogel delivery vehicles for Sonic hedgehog (SHH) protein to the penis and CN, which effectively suppress penile distrophic remodeling (apoptosis and fibrosis), in vivo in a rat CN injury model, and the aim of this study is to determine if SHH PA can be used to regenerate human corpora cavernosal smooth muscle deriving from multiple ED origins. METHODS: Corpora cavernosal tissue was obtained from prostatectomy, diabetic, hypertension, cardiovascular disease and Peyronie's (control) patients (n = 21). Primary cultures (n = 21) were established, and corpora cavernosal cells were treated with SHH protein, MSA (control), 5E1 SHH inhibitor, and PBS (control). Growth was quantified by counting the number of cells at 3-4 days. Statistics were performed by ANOVA with Scheffe's post hoc test. Concentration of SHH protein for maximal growth was optimized, and a more active SHH protein examined. OUTCOMES: Cultures were characterized by immunohistochemical analysis with ACTA2, CD31, nNOS and P4HB, and smooth muscle was quantified in comparison to DAPI. RESULTS: Cultures established were >97% smooth muscle. SHH protein increased growth of smooth muscle cells from prostatectomy, diabetic, and Peyronie's patients in a similar manner (49%-51%), and SHH inhibition decreased growth (20%-33%). There was no difference in growth using 25 ug and 10 ug SHH protein, suggesting a threshold concentration of SHH protein above which smooth muscle growth is enhanced. A more active lipid modified SHH peptide further enhanced growth (15%), indicating a more robust growth response. SHH increased growth in smooth muscle cells from hypertension (37%) and cardiovascular disease (32%) patients. SHH protein increased growth under normal and high glucose conditions, suggesting that high glucose conditions that may be present in under controlled diabetic patients would not detract from SHH regenerative capacity. CLINICAL IMPLICATIONS: SHH PA would be beneficial to enhance smooth muscle regeneration in patients with ED of multiple etiologies. STRENGTHS AND LIMITATIONS: Understanding how human corpora cavernosal tissue responds to SHH treatment is critical for clinical translation of SHH PA to ED patients. CONCLUSION: Corpora cavernosal smooth muscle from all ED patients responded to SHH treatment with increased growth. Stupp, SI. Sonic Hedgehog Signaling in Primary Culture of Human Corpora Cavernosal Tissue From Prostatectomy, Diabetic, and Peyronie's Patients. J Sex Med 2022;19:1228-1242.

Caspase Signaling in ED Patients and Animal Models.

BACKGROUND: Current treatments for erectile dysfunction (ED) are ineffective in prostatectomy and diabetic patients due to cavernous nerve (CN) injury, which causes smooth muscle apoptosis, penile remodeling, and ED. Apoptosis can occur via the intrinsic (caspase 9) or extrinsic (caspase 8) pathway. AIM: We examined the mechanism of how apoptosis occurs in ED patients and CN injury rat models to determine points of intervention for therapy development. METHODS AND OUTCOMES: Immunohistochemical and western analyses for caspase 3-cleaved, caspase-8 and caspase-9 (pro and active forms) were performed in corpora cavernosal tissue from Peyronie's, prostatectomy and diabetic ED patients (n = 33), penis from adult Sprague Dawley rats that underwent CN crush (n = 24), BB/WOR diabetic and control rats (n = 8), and aged rats (n = 9). RESULTS: Caspase 3-cleaved was observed in corpora cavernosa from Peyronie's patients and at higher abundance in prostatectomy and diabetic tissues. Apoptosis takes place primarily through the extrinsic (caspase 8) pathway in penis tissue of ED patients. In the CN crushed rat, caspase 3-cleaved was abundant from 1-9 days after injury, and apoptosis takes place primarily via the intrinsic (caspase 9) pathway. Caspase 9 was first observed and most abundant in a layer under the tunica, and after several days was observed in the lining of and between the sinuses of the corpora cavernosa. Caspase 8 was initially observed at low abundance in the rat corpora cavernosa and was not observed at later time points after CN injury. Aged and diabetic rat penis primarily exhibited intrinsic mechanisms, with diabetic rats also exhibiting mild extrinsic activation. CLINICAL TRANSLATION: Knowing how and when to intervene to prevent the apoptotic response most effectively is critical for the development of drugs to prevent ED, morphological remodeling of the corpora cavernosa, and thus, disease management. STRENGTHS AND LIMITATIONS: Animal models may diverge from the signaling mechanisms observed in ED patients. While the rat utilizes primarily caspase 9, there is a significant flux through caspase 8 early on, making it a reasonable model, as long as the timing of apoptosis is considered after CN injury. CONCLUSIONS: Apoptosis takes place primarily through the extrinsic caspase 8 dependent pathway in ED patients and via the intrinsic caspase 9 dependent pathway in commonly used CN crush ED models. This is an important consideration for study design and interpretation that must be taken into account for therapy development and testing of drugs, and our therapeutic targets should ideally inhibit both apoptotic mechanisms. Martin S, Harrington DA, Ohlander S, et al. Caspase Signaling in ED Patients and Animal Models. J Sex Med 2021;18:711-722.

Peptide amphiphile nanofiber hydrogel delivery of Sonic hedgehog protein to the penis and cavernous nerve suppresses intrinsic and extrinsic apoptotic signaling mechanisms, which are an underlying cause of erectile dysfunction.

Erectile dysfunction (ED) is a common and debilitating condition with high impact on quality of life. An underlying cause of ED is apoptosis of penile smooth muscle, which occurs with cavernous nerve injury, in prostatectomy, diabetic and aging patients. We are developing peptide amphiphile (PA) nanofiber hydrogels as an in vivo delivery vehicle for Sonic hedgehog protein to the penis and cavernous nerve to prevent the apoptotic response. We examine two important aspects required for clinical application of the biomaterials: if SHH PA suppresses intrinsic (caspase 9) and extrinsic (caspase 8) apoptotic mechanisms, and if suppressing one apoptotic mechanism forces apoptosis to occur via a different mechanism. We show that SHH PA suppresses both caspase 9 and 8 apoptotic mechanisms, and suppressing caspase 9 did not shift signaling to caspase 8. SHH PA has significant clinical potential as a preventative ED therapy, by management of intrinsic and extrinsic apoptotic mechanisms.

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 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.

Role of Nanotechnology in Erectile Dysfunction Treatment.

INTRODUCTION: The biological importance of nanotechnology-based delivery vehicles for in vivo tissue regeneration is gaining acceptance by the medical community; however, its relevance and incorporation into the treatment of sexual dysfunction are evolving and have not been well evaluated. AIM: To provide scientific evidence examining the use of state-of-the-art nanotechnology-based delivery methodology in the treatment of erectile dysfunction (ED) in animal models and in patients. METHODS: This review assessed the current basic science literature examining the role of nanotechnology-based delivery vehicles in the development of potential ED therapies. RESULTS: There are four primary areas where nanotechnology has been applied for ED treatment: (i) topical delivery of drugs for on-demand erectile function, (ii) injectable gels into the penis to prevent morphologic changes after prostatectomy, (iii) hydrogels to promote cavernous nerve regeneration or neuroprotection, and (iv) encapsulation of drugs to increase erectile function (primarily of phosphodiesterase type 5 inhibitors). CONCLUSION: Basic science studies provide evidence for a significant and evolving role for nanotechnology in the development of therapies for ED and suggest that properly administered nano-based therapies might be advantageous for treating male sexual dysfunction.

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.

Translational Perspective on the Role of Testosterone in Sexual Function and Dysfunction.

INTRODUCTION: The biological importance of testosterone is generally accepted by the medical community; however, controversy focuses on its relevance to sexual function and the sexual response, and our understanding of the extent of its role in this area is evolving. AIM: To provide scientific evidence examining the role of testosterone at the cellular and molecular levels as it pertains to normal erectile physiology and the development of erectile dysfunction and to assist in guiding successful therapeutic interventions for androgen-dependent sexual dysfunction. METHODS: In this White Paper, the Basic Science Committee of the Sexual Medicine Society of North America assessed the current basic science literature examining the role of testosterone in sexual function and dysfunction. RESULTS: Testosterone plays an important role in sexual function through multiple processes: physiologic (stimulates activity of nitric oxide synthase), developmental (establishes and maintains the structural and functional integrity of the penis), neural (development, maintenance, function, and plasticity of the cavernous nerve and pelvic ganglia), therapeutically for dysfunctional regulation (beneficial effect on aging, diabetes, and prostatectomy), and phosphodiesterase type 5 inhibition (testosterone supplement to counteract phosphodiesterase type 5 inhibitor resistance). CONCLUSION: Despite controversies concerning testosterone with regard to sexual function, basic science studies provide incontrovertible evidence for a significant role of testosterone in sexual function and suggest that properly administered testosterone therapy is potentially advantageous for treating male sexual dysfunction.

Basic Science Evidence for the Link Between Erectile Dysfunction and Cardiometabolic Dysfunction.

INTRODUCTION: Although clinical evidence supports an association between cardiovascular/metabolic diseases (CVMD) and erectile dysfunction (ED), scientific evidence for this link is incompletely elucidated. AIM: This study aims to provide scientific evidence for the link between CVMD and ED. METHODS: In this White Paper, the Basic Science Committee of the Sexual Medicine Society of North America assessed the current literature on basic scientific support for a mechanistic link between ED and CVMD, and deficiencies in this regard with a critical assessment of current preclinical models of disease. RESULTS: A link exists between ED and CVMD on several grounds: the endothelium (endothelium-derived nitric oxide and oxidative stress imbalance); smooth muscle (SM) (SM abundance and altered molecular regulation of SM contractility); autonomic innervation (autonomic neuropathy and decreased neuronal-derived nitric oxide); hormones (impaired testosterone release and actions); and metabolics (hyperlipidemia, advanced glycation end product formation). CONCLUSION: Basic science evidence supports the link between ED and CVMD. The Committee also highlighted gaps in knowledge and provided recommendations for guiding further scientific study defining this risk relationship. This endeavor serves to develop novel strategic directions for therapeutic interventions.

Sonic hedgehog is neuroprotective in the cavernous nerve with crush injury.

INTRODUCTION: The cavernous nerve (CN) is commonly injured during prostatectomy, resulting in erectile dysfunction (ED). Although peripheral nerves have a limited ability to regenerate, a return of function typically does not occur due to irreversible down stream morphological changes in the penis that result from CN injury. We have shown in previous studies that sonic hedgehog (SHH) is critical for CN regeneration and improves erectile function after crush injury. AIMS: Examine a new direction, to determine if SHH is neuroprotective to the pelvic ganglia (PG)/CN after crush injury. A secondary focus is to examine if SHH signaling decreases with age in the PG/CN. METHODS: Sprague-Dawley rats underwent bilateral CN crush and SHH and glial fibrillary acidic protein were quantified by western analysis of the PG/CN (N = 6 rats at each time point) at 1, 2, 4, 7, and 14 days, and the apoptotic index was measured in the penis. SHH was quantified by western in the PG/CN with blockade of anterograde transport (N = 4 rats) in comparison to mouse IgG (N = 4 rats). If SHH is neuroprotective was examined at 4 (N = 14 rats) and 7 days (N = 16 rats) of treatment after CN crush. SHH protein was quantified in aging (P200-300, N = 5 rats) PG/CN in comparison to normal adult (P115-120, N = 3 rats) PG/CN. Main Outcome Measures. SHH pathway was examined in PG via immunohistochemistry, in situ, western, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). RESULTS: SHH is neuroprotective in the PG/CN with injury. SHH localization in the PG/CN suggests SHH interaction in neuronal/glial signaling. SHH protein is significantly decreased in the PG/CN after crush injury and in the aged PG/CN. Signals from the PG are required to maintain SHH in the CN. CONCLUSIONS: There is a window of opportunity immediately after nerve insult in which manipulation of SHH signaling in the nerve microenvironment can affect long-term regeneration outcome.

Sonic Hedgehog regulates brain-derived neurotrophic factor in normal and regenerating cavernous nerves.

INTRODUCTION: The cavernous nerve (CN) is commonly injured during prostatectomy. Manipulation of the nerve microenvironment is critical to improve regeneration and develop novel erectile dysfunction therapies. Sonic hedgehog (SHH) treatment promotes CN regeneration. The mechanism of how this occurs is unknown. Brain-derived neurotrophic factor (BDNF) facilitates return of erectile function after CN injury and it has been suggested in cortical neurons and the sciatic nerve that BDNF may be a target of SHH. AIM: To determine if SHH promotes CN regeneration through a BDNF-dependent mechanism. METHODS: Sprague Dawley rats underwent (i) bilateral CN crush (N = 15); (ii) SHH treatment of pelvic ganglia (PG)/CN (N = 10); (iii) SHH inhibition in PG/CN (N = 14 rats); (iv) CN crush with SHH treatment of PG/CN (N = 10 rats); (v) CN crush with SHH treatment and BDNF inhibition (N = 14 rats); and (vi) CN injury and SHH treatment of the penis (N = 23). MAIN OUTCOME MEASURES: BDNF and glial fibrillary acidic protein were quantified in PG/CN by Western, and a t-test was used to determine differences. RESULTS: In normal rats SHH inhibition in the PG/CN decreased BDNF 34% and SHH treatment increased BDNF 36%. BDNF was increased 44% in response to SHH treatment of crushed CNs, and inhibition of BDNF in crushed CNs treated with SHH protein hampers regeneration. CONCLUSIONS: SHH regulates BDNF in the normal and regenerating PG/CN. BDNF is part of the mechanism of how SHH promotes regeneration, thus providing an opportunity to further manipulate the nerve microenvironment with combination therapy to enhance regeneration.

Peptide amphiphile nanofiber delivery of sonic hedgehog protein to reduce smooth muscle apoptosis in the penis after cavernous nerve resection.

INTRODUCTION: Erectile dysfunction (ED) is a serious medical condition that affects 16-82% of prostate cancer patients treated by radical prostatectomy and current treatments are ineffective in 50-60% of prostatectomy patients. The reduced efficacy of treatments makes novel therapeutic approaches to treat ED essential. The secreted protein Sonic hedgehog (SHH) is a critical regulator of penile smooth muscle and apoptosis that is decreased in cavernous nerve (CN) injury and diabetic ED models. Past studies using Affi-Gel beads have shown SHH protein to be effective in suppressing apoptosis caused by CN injury. AIM: We hypothesize that SHH protein delivered via novel peptide amphiphile (PA) nanofibers will be effective in suppressing CN injury-induced apoptosis. METHODS: Adult Sprague Dawley rats (n=50) were used to optimize PA injection in vivo. PA with SHH protein (n=16) or bovine serum albumin (BSA) (control, n=14) was injected into adult rats that underwent bilateral CN cut. Rats were sacrificed at 2, 4, and 7 days. Alexa Fluor-labeled SHH protein was used to determine the target of SHH signaling (n=3). MAIN OUTCOME MEASURES: Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and semiquantitative immunohistochemical analysis for SHH protein and cluster differentiation protein three (CD3) were performed. RESULTS: SHH-PA caused a 25% and 16% reduction in apoptosis at 4 and 7 days after CN injury and a 9.3% and 19% increase in SHH protein at 4 and 7 days after CN injury. CD3 protein was not observed in SHH-PA-treated penis. In vitro, 73% of SHH protein diffused from PA within 6 days. Labeled SHH was observed in smooth muscle. CONCLUSIONS: PA technology is effective in delivering SHH protein to the penis and SHH is effective in suppressing CN injury-induced apoptosis. These results suggest substantial translational potential of this methodology and show that only a short duration of SHH treatment is required to impact the apoptotic index.

Analysis of testosterone effects on sonic hedgehog signaling in juvenile, adolescent and adult sprague dawley rat penis.

INTRODUCTION: Smooth muscle apoptosis is a major contributing factor to erectile dysfunction (ED) development in prostatectomy and diabetic patients and animal models. A critical regulator of penile smooth muscle and apoptosis is Sonic hedgehog (SHH). The SHH protein is decreased in ED models and SHH treatment of cavernous nerve (CN) injured rats prevents smooth muscle apoptosis. A close association between androgen deficiency and ED has been suggested in the literature, but few studies have examined the molecular effects on penile smooth muscle and on known signaling mechanisms that regulate morphology. Aim. Examine testosterone and SHH interaction in eugonadal adult, adolescent and juvenile rats by performing castration studies and treatment with supraphysiological testosterone. METHODS: The eugonadal adult Sprague Dawley rats were either treated with testosterone for 7 or 14 days (N = 14) or were castrated for 4 or 7 days (N = 12). The juvenile rats were treated with testosterone for 8 days (N = 7). The adolescent rats were castrated and sacrificed at P88 (N = 8). The control rats had empty vehicle (N = 22) or sham surgery (N = 20). MAIN OUTCOME MEASURES: The active form of SHH protein and mRNA were quantified by semi-quantitative immunohistochemical analysis and real-time reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: Testosterone treatment did not alter SHH signaling in juvenile rats. Shh mRNA increased 3.2-fold and SHH protein increased 1.2-fold in rats castrated during puberty. In adult rats, castration decreased Shh mRNA 3.2-fold but did not alter SHH protein. Testosterone supplement in adult rats increased Shh mRNA 2.3-fold and decreased SHH protein 1.3-fold. CONCLUSIONS: SHH signaling is independent of testosterone in normal juvenile rats and is sensitive to testosterone during adolescence, while testosterone supplement in the adult adversely impacts SHH signaling in a very similar manner to that observed with CN injury.

Sonic hedgehog, apoptosis, and the penis.

INTRODUCTION: Smooth muscle apoptosis in the penis is common in prostatectomy patients and animal models of erectile dysfunction (ED). A critical regulator of smooth muscle apoptosis in the penis is the secreted protein Sonic hedgehog (SHH). Since SHH protein treatment of the penis prevents cavernous nerve (CN) injury-induced apoptosis, SHH has the potential to treat post-prostatectomy apoptosis. However, little is known about how SHH signaling is regulated in the adult penis. AIM: The goal of this review is to examine what is known about SHH signaling in the penis, to offer insight as to how SHH inhibition induces apoptosis in penile smooth muscle, and to define the role of the SHH pathway in maintaining CN integrity. METHODS: Information presented in this review was derived from a literature search using the National Library of Medicine PubMed Services. Search terms included SHH, apoptosis, smooth muscle, penis, ED, pelvic ganglia, corpora cavernosa, CN, regeneration, Schwann cell, neural activity, and transport. RESULTS: In this review, we have discussed the role of the CN in regulation of SHH abundance and apoptosis induction in the penis, and have examined the function and localization of SHH signaling in the CN. CONCLUSION: There is substantial potential to develop SHH for delivery to the penis of prostatectomy patients at the time of surgery in order to prevent apoptosis induction and long-term ED development. Studies are in progress that will identify if SHH may be used as a regenerative therapy to speed CN regeneration.