Carol A. Podlasek

Male accessory sex organ morphogenesis is altered by loss of function of Hoxd-13

The role of the Hox gene Hoxd‐13 in postnatal morphogenesis of the male accessory sex organs was examined by correlating the distribution and temporal regulation of expression in the accessory sex organs of postnatal mice with morphologic abnormalities of Hoxd‐13‐deficient transgenic mice. Previous studies of Hoxd‐13 expression in the perinatal period have shown a broad domain of expression in the lower genito‐urinary tract, with expression in both mesenchyme and epithelium; focal expression was also noted in the epithelium of the nascent ducts of the developing prostate. Quantitative RT‐PCR studies of Hoxd‐13 expression in the 5 day mouse confirm widespread expression in the accessory sex organs developing from both the Wolffian duct and the urogenital sinus. Expression is down‐regulated with age, and a detailed time course of expression in the developing prostate shows that the level of Hoxd‐13 expression correlates with morphogenetic activity in the development of the prostate ductal system. Transgenic Hoxd‐13‐deficient mice display multiple abnormalities in the male accessory sex organs. The most severe abnormalities were observed in organs exhibiting ductal branching during postnatal development and included diminished mesenchymal folding in the seminal vesicles, decreased size and diminished ductal branching in the ventral and dorsal prostate, and agenesis of the bulbourethral gland. We conclude that Hoxd‐13 expression in the postnatal period correlates with a period of intense morphogenetic activity in accessory sex organ development and that the function of Hoxd‐13 is evidenced by morphologic abnormalities in accessory sex organs of the Hoxd‐13‐deficient mutant. Dev. Dyn. 208:454–465, 1997.

Regeneration of the cavernous nerve by Sonic hedgehog using aligned peptide amphiphile nanofibers

SHH plays a significant role in peripheral nerve regeneration and has clinical potential to be used as a regenerative therapy for the CN in prostatectomy patients and in other patients with neuropathy of peripheral nerves. Efforts to regenerate the cavernous nerve (CN), which provides innervation to the penis, have been minimally successful, with little translation into improved clinical outcomes. We propose that, Sonic hedgehog (SHH), is critical to maintain CN integrity, and that SHH delivered to the CN by novel peptide amphiphile (PA) nanofibers, will promote CN regeneration, restore physiological function, and prevent penile morphology changes that result in erectile dysfunction (ED). We performed localization studies, inhibition of SHH signaling in the CN, and treatment of crushed CNs with SHH protein via linear PA gels, which are an innovative extended release method of delivery. Morphological, functional and molecular analysis revealed that SHH protein is essential to maintain CN architecture, and that SHH treatment promoted CN regeneration, suppressed penile apoptosis and caused a 58% improvement in erectile function in less than half the time reported in the literature. These studies show that SHH has substantial clinical application to regenerate the CN in prostatectomy and diabetic patients, that this methodology has broad application to regenerate any peripheral nerve that SHH is necessary for maintenance of its structure, and that this nanotechnology method of protein delivery may have wide spread application as an in vivo delivery tool in many organs.

HOXA-13 GENE MUTATION RESULTS IN ABNORMAL SEMINAL VESICLE AND PROSTATE DEVELOPMENT

The role of Hoxa-13 in postnatal morphogenesis of the male accessory sex organs was assessed by correlating the Hoxa-13 expression domain with phenotypic abnormalities in heterozygous Hypodactyly mutants. Hypodactyly is a naturally occurring semi-dominant mutation that results from a 50-base pair deletion in exon one of the Hoxa-13 allele. We demonstrate that Hoxa-13 is broadly expressed in the developing lower genitourinary tract and that the Hypodactyly mutation results in a specific phenotype characterized by decreased size and branching of the dorsolateral and ventral prostate and abnormal seminal vesicle morphology. This phenotype partially overlaps the genitourinary phenotype observed in Hoxd-13 deficient mice and comparison showed similar domains of Hoxa-13 and Hoxd-13 expression in the lower genitourinary tract. The similarity in expression and overlap in phenotype resulting from mutation is consistent with additive function and partial functional redundancy of Hoxa-13 and Hoxd-13 in male accessory sex organ development.

Characterization and localization of nitric oxide synthase isoforms in the BB/WOR diabetic rat.

PURPOSE Erectile dysfunction is a common pathological development in individuals with diabetes mellitus. Nitric oxide synthase (NOS) is essential for regulation of normal penile erection and NOS protein activity has been shown to be altered with diabetes. Several different isoforms and subtypes of NOS exist. However, little is known about how the distribution and abundance of these isoforms are altered with diabetes. We characterized the distribution and abundance of NOS isoforms and explored how they are altered with diabetes and result in erectile failure. MATERIALS AND METHODS In situ hybridization and quantitative reverse transcriptase-polymerase chain reaction were done to measure the abundance and distribution of NOS-Ia, NOS-Ib, NOS-Ic, NOS-II and NOS-III in control and diabetic (BB/WOR) rats. Protein was localized by immunohistochemical analysis and alterations in protein abundance with diabetes were examined by Western blot analysis. RESULTS NOS-I, NOS-II and NOS-III were observed in the endothelium lining the cavernous spaces and in the epithelium of the urethra. NOS-I protein was also present in the nerves of control and diabetic penes. We observed an increase in NOS-II expression around the dorsal nerves of diabetic penes, a decrease in NOS-III expression in diabetic pelvic ganglia and a decrease in NOS-Ib expression in the diabetic penis. NOS-I protein abundance was significantly decreased in diabetic pelvic ganglia. CONCLUSIONS To our knowledge this is the first report of regional differences in the distribution of NOS-III in the urethra and altered NOS-Ib gene expression with diabetes.

Hoxa‐10 deficient male mice exhibit abnormal development of the accessory sex organs

The role of mammalian Hox genes in regulating segmental patterning of axial structures and the limb is well established. A similar role in development of soft tissue organ systems has recently been suggested by observations linking several 5′ members of the HoxA and HoxD clusters to segmentation events and morphogenesis in the gastrointestinal and genitourinary systems. We have specifically examined the role of Hoxa‐10 in development of the male accessory sex organs by characterizing expression of Hoxa‐10 in the developing male reproductive tract and correlating expression to morphologic abnormalities in knockout mice deficient for Hoxa‐10 function. We report that Hoxa‐10 expression in the Wolffian duct and urogenital sinus is regionally restricted and temporally regulated. The domain of expression is defined anteriorly by the caudal epididymis and extends posteriorly to the prostatic anlagen of the urogenital sinus. Expression was maximal at E18 and down‐regulated postnatally, well before accessory sex organ morphogenesis is completed. Expression in the prostatic anlagen of the urogenital sinus cultured in vitro does not depend upon the presence of testosterone. Loss of Hoxa‐10 function is associated with diminished stromal clefting of the seminal vesicles and decreased size and branching of the coagulating gland. The ductal architecture of the coagulating gland was altered in approximately 30% of mutants examined and suggests a partial posterior morphologic transformation of the coagulating gland. We interpret these data to indicate that Hoxa‐10 is expressed in a region specific manner during late gestation and into the perinatal period and that Hoxa‐10 is required for normal accessory sex organ development. Dev Dyn 1999;214:1–12.

Analysis of NOS isoform changes in a post radical prostatectomy model of erectile dysfunction

Optimal treatment of erectile dysfunction (ED) following radical prostatectomy remains a subject of much controversy and is a significant concern for prostate cancer patients requiring surgical intervention. Neural stimulation involving nitric oxide synthase (NOS) is a crucial aspect of the normal erection process. In this study NOS isoform interaction was evaluated to improve our understanding of molecular changes pertaining to erection post radical prostatectomy. Bilateral cavernous nerve (CN) resected and control adult male Sprague-Dawley rats were killed 7, 14 and 21 days after injury. RT-PCR, in situ hybridization, Western blot and immunohistochemical analysis were used to evaluate changes in NOS isoform expression and distribution. NOS-I protein was dramatically decreased after CN injury while NOS-III and NOS-II remained unchanged. A profound decrease in smooth muscle and endothelium was observed in the corpora. To our knowledge this is the first report of differential altered NOS isoform protein abundance under conditions which mimic radical prostatectomy. These results show the importance of maintaining at least partial innervation of the penis after surgical intervention and that endothelial and smooth muscle changes resulting from loss of innervation may account for the ED observed in prostatectomy patients.

Regulation of Cavernous Nerve Injury Induced Apoptosis by Sonic Hedgehog

Abstract Thirty to eighty-seven percent of patients treated by radical prostatectomy experience erectile dysfunction (ED). The reduced efficacy of treatments in this population makes novel therapeutic approaches to treat ED essential. We propose that abundant apoptosis observed in penile smooth muscle when the cavernous nerve (CN) is cut (mimicking the neural injury which can result from prostatectomy) is a major contributing factor to ED development. We hypothesize that decreased Sonic hedgehog (SHH) signaling is a cause of ED in neurological models of impotence by increasing apoptosis in penile smooth muscle. We examined this hypothesis in a bilateral CN injury model of ED. We found that the active form of SHH protein was significantly decreased 1.2-fold following CN injury, that SHH inhibition causes a 12-fold increase in smooth muscle apoptosis in the penis, and that SHH treatment at the time of CN injury was able to decrease CN injury-induced apoptosis (1–3-fold) in a dose-dependent manner. These results show that SHH stabilizes the alterations of the corpora cavernosal smooth muscle following nerve injury.

Sonic hedgehog Cascade Is Required for Penile Postnatal Morphogenesis, Differentiation, and Adult Homeostasis

Abstract The penis is unique in that it undergoes morphogenesis and differentiation primarily in the postnatal period. For complex structures such as the penis to be made from undifferentiated precursor cells, proliferation, differentiation, and patterning are required. This process involves coordinated activity of multiple signals. Sonic hedgehog (Shh) forms part of a regulatory cascade that is essential for growth and morphogenesis of many tissues. It is hypothesized that the penis utilizes regulatory mechanisms similar to those of the limb and accessory sex organs to pattern penile postnatal morphogenesis and differentiation and that the Shh cascade is critical to this process. To test this hypothesis, Shh, BMP-4, Ptc, and Hoxa-10 localization and function were examined in Sprague-Dawley rat penes by means of quantitative reverse transcription polymerase chain reaction, in situ hybridization, immunohistochemistry, and Western blotting. These genes were expressed in the penis during postnatal morphogenesis in a spatially and temporally restricted manner in adjacent layers of the corpora cavernosal sinusoids. The function of Shh and BMP-4 is to establish and maintain corpora cavernosal sinusoids. The data suggest that Ptc and Hoxa-10 are also important in penile morphogenesis. The continuing function of Shh and targets of its signaling in maintaining penile homeostasis in the adult is significant because disruption of Shh signaling affects erectile function. This is the first report that demonstrates the significant role that Shh plays in establishing and maintaining penile homeostasis and how this relates to erectile function. These studies provide valuable insight that may be applied to improve treatment options for erectile dysfunction.

Altered Sonic Hedgehog Signaling Is Associated with Morphological Abnormalities in the Penis of the BB/WOR Diabetic Rat

Abstract Erectile dysfunction (ED) is a common and debilitating pathological development that affects up to 75% of diabetic males. Neural stimulation is a crucial aspect of the normal erection process. Nerve injury causes ED and disrupts signaling of the Sonic hedgehog (Shh) cascade in the smooth muscle of the corpora cavernosa. Shh and targets of its signaling establish normal corpora cavernosal morphology during postnatal differentiation of the penis and regulate homeostasis in the adult. Interruption of the Shh cascade in the smooth muscle of the corpora cavernosa results in extensive changes in corpora cavernosal morphology that lead to ED. Our hypothesis is that the neuropathy observed in diabetics causes morphological changes in the corpora cavernosa of the penis that result in ED. Disruption of the Shh cascade may be involved in this process. We tested this hypothesis by examining morphological changes in the penis, altered gene and protein expression, apoptosis, and bromodeoxyuridine incorporation in the BB/WOR rat model of diabetes. Extensive smooth muscle and endothelial degradation was observed in the corpora cavernosa of diabetic penes. This degradation accompanied profound ED, significantly decreased Shh protein in the smooth muscle of the corpora cavernosa, and increased penile Shh RNA expression in the intact penis (nerves, corpora, and urethra). Localization and expression of Shh targets were also disrupted in the corpora cavernosa. Increasing our understanding of the molecular mechanisms that regulate Shh signaling may provide valuable insight into improving treatment options for diabetic impotence.

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.