Ashley Dean

Long-Term Structural and Functional Effects of Autologous Muscle Precursor Cell Therapy in a Nonhuman Primate Model of Urinary Sphincter Deficiency

PURPOSE We measured the long-term efficacy of autologous muscle precursor cell therapy in premenopausal female nonhuman primates with sustained urinary sphincter deficiency. MATERIALS AND METHODS Urinary sphincter deficiency was created in adult premenopausal female cynomolgus monkeys by selectively cauterizing and then transecting the pudendal innervation to the sphincter complex. The monkeys were then treated (18) or not treated (18) with intra-urinary sphincter injections of 5 million autologous green fluorescent protein labeled skeletal muscle precursor cells. Four untreated, uninjured monkeys served as controls. Maximal urethral pressure measurement and corresponding histological analysis of the structural and cellular components of the sphincter complex were performed up to 12 months after injection. RESULTS Cell treatment produced sustained (12 months) increases in resting, somatic nerve stimulated and adrenergic nerve stimulated maximal urethral pressure, and a greater percent of sphincter area occupied by muscle as well as a decrease in the sphincter area occupied by collagen compared to the untreated group (each p>0.05). These results were within control values (each p>0.05). By 3 months after injection green fluorescent protein positive cells were found in the skeletal muscle layer, expressing desmin and connexin-43, and in the smooth muscle layer, expressing α-smooth muscle actin and connexin-43, and they were incorporated into the subendothelial vasculature, expressing Von Willebrand factor. Cell injected sphincter tissue contained a mixture of green fluorescent protein positive cells and predominantly green fluorescent protein negative cells. CONCLUSIONS Injected skeletal muscle progenitor cells incorporated into the injured sphincter complex resulted in long-term structural and functional restoration of the injured sphincter complex in this nonhuman primate model.

The Dose-Effect Safety Profile of Skeletal Muscle Precursor Cell Therapy in a Dog Model of Intrinsic Urinary Sphincter Deficiency

Locally injected skeletal muscle precursor cells (skMPCs) integrate into and restore the muscle layers, innervation, vasculature, and function of the sphincter complex in animal models of intrinsic urinary sphincter deficiency (ISD). The goal of the present study was to test the dose‐effect safety profile of skMPC therapy in a dog model of ISD. Sphincter deficiency was created in 20 adult female dogs by surgically removing the skeletal muscle layer of the urinary sphincter complex. skMPCs isolated from the hind leg were expanded in culture and injected 4 weeks later into the sphincter complex at a dose of 25 million cells (n = 5), 50 million cells (n = 5), or 100 million cells (n = 5) per milliliter in a 2‐ml volume. Five dogs received no sphincter injection. The measures of maximal sphincter pressure, complete blood count, and blood chemistry were performed monthly until their sacrifice at 9 months. At that point, full necropsy was performed to assess the safety of the skMPC injections. Injection of different doses of cells had no effects on the body weight, blood cell count, or kidney or liver function test results (p > .05 among the skMPC doses). Some incidental pathologic features were found in the lower urinary tract in all groups and were most likely associated with repeat catheterization. The maximal urinary sphincter pressure was higher in the 50 million cells per milliliter treatment group than in the other experimental groups (p < .05). The findings of the present study have confirmed that urinary sphincter injection of skMPCs results in no significant local or systemic pathologic features within the dose range that improves sphincter pressures.

A nonhuman primate model of stable urinary sphincter deficiency.

PURPOSE The pathophysiology of urinary sphincter deficiency in women remains incompletely understood and current treatment options have limitations. Female nonhuman primates may represent a relevant animal model for studies of pathophysiology and treatment interventions because of their human-like reproductive and age associated stages of life (premenopause, perimenopause and postmenopause), lower urinary tract structure and bipedal posture. We developed and characterized a nonhuman primate model of defined injury to the urethral sphincter complex. MATERIALS AND METHODS We used 22 adult female cynomolgus monkeys in which injury to the sphincter complex was created by cauterizing and then transecting its pudendal innervation. Urodynamic studies were performed before and during pudendal and hypogastric nerve stimulation at baseline, and 3, 6 and 12 months after injury. We also analyzed sphincter structure in vivo by cystourethrography, and ex vivo by quantitative histology and immunohistochemistry at these time points. RESULTS Injury produced a 47% to 50% decrease in maximal urethral pressure (vs baseline p <0.05). It also abolished the increase in maximal urethral pressure in response to pudendal and hypogastric nerve stimulation (vs baseline p >0.05), which persisted more than 12 months after injury. Urodynamic changes were consistent with decreased skeletal and smooth muscle content, decreased nerve responses and an associated decrease in somatic and adrenergic innervation in the sphincter complex. CONCLUSIONS These structural and urodynamic changes are consistent with those in patients with stress urinary incontinence. They support the usefulness of nonhuman primates as translatable surrogates for pathophysiological studies of urinary sphincter deficiency and testing novel therapies for that condition.

Regenerative Medicine Therapies for Stress Urinary Incontinence

PURPOSE We summarize the current state of knowledge regarding cell therapy for stress urinary incontinence and introduce new approaches of using regenerative pharmacology as an adjunct or replacement for cell therapy. MATERIALS AND METHODS We reviewed the literature by searching PubMed®, Ovid and Biological Abstracts. The period searched was 1975 to December 2015. The inclusion terms separately or in combination were stress urinary incontinence, cell therapy, chemokine, vascularization, innervation, secretome and/or animal models. Epublished articles were not included. We did not exclude articles based on impact factor. RESULTS Cell therapy is currently proposed to restore functional muscle cells and aid in closure of the sphincter in women with sphincter associated incontinence. Clinical trials have included small numbers of patients and results have varied depending on the patient cohorts and the cells used. Results of preclinical studies have also varied but show a more favorable outcome. This difference was most likely explained by the fact that animal modeling is not directly translatable to the human condition. However, preclinical studies have identified an exciting new approach to regeneration of the urinary sphincter using the components of cells (secretomes) or chemokines that home reparative cells to sites of injury. CONCLUSIONS Cell therapy will continue to be explored. However, a regenerative pharmacological approach to the treatment of stress urinary incontinence holds the promise of bypassing the lengthy and expensive process of cell isolation and also increasing the availability of treatment in many clinical settings. This approach requires careful preclinical modeling and attention to its health benefit-to-risk ratio.

Cell versus Chemokine Therapy in a Nonhuman Primate Model of Chronic Intrinsic Urinary Sphincter Deficiency

PURPOSE Mixed efficacy results of autologous skeletal muscle precursor cell therapy in women with chronic intrinsic urinary sphincter deficiency have increased interest in the therapeutic value of alternative regenerative medicine approaches. The goal of this study was to compare the effects of the cell homing chemokine CXCL12 (C-X-C motif chemokine 12) and skeletal muscle precursor cells on chronic urinary sphincter regeneration in chronic intrinsic urinary sphincter deficiency. MATERIALS AND METHODS Five million autologous skeletal muscle precursor cells or 100 ng CXCL12 were injected in the urinary sphincter complex of adult female cynomolgus monkeys with chronic (6-month history) intrinsic urinary sphincter deficiency. These treatment groups of 3 monkeys per group were compared to a group of 3 with no intrinsic urinary sphincter deficiency and no injection, and a group of 3 with intrinsic urinary sphincter deficiency plus vehicle injection. Maximal urethral pressure was measured at rest, during stimulation of the urinary sphincter pudendal nerves at baseline and again 6 months after treatment. The monkeys were then necropsied. The urinary sphincters were collected for tissue analysis of muscle and collagen content, vascularization and motor endplates. RESULTS CXCL12 but not skeletal muscle precursor cells increased resting maximal urethral pressure in nonhuman primates with chronic intrinsic urinary sphincter deficiency compared to that in monkeys with intrinsic urinary sphincter plus vehicle injection (p >0.05). Skeletal muscle precursor cells and CXCL12 only partially restored pudendal nerve stimulated increases in maximal urethral pressure (p >0.05), sphincter vascularization and motor endplate expression in monkeys with chronic intrinsic urinary sphincter deficiency. Additionally, CXCL12 but not skeletal muscle precursor cell injections decreased collagen and increased the muscle content of urinary sphincter complex in monkeys with chronic intrinsic urinary sphincter deficiency compared to those with intrinsic urinary sphincter plus vehicle injection and no intrinsic urinary sphincter plus no injection (p <0.05 and >0.05, respectively). CONCLUSIONS These results raise questions about cell therapy for chronic intrinsic urinary sphincter deficiency and identify a chemokine treatment (CXCL12) as a potential alternative treatment of chronic intrinsic urinary sphincter deficiency.

Efficacy and Initial Safety Profile of CXCL12 Treatment in a Rodent Model of Urinary Sphincter Deficiency

Disappointing results of skeletal muscle precursor cell (skMPC) therapy for women with intrinsic urinary sphincter deficiency (ISD) associated urinary incontinence has increased interest in alternative sphincter regenerative approaches. This study was to measure the safety and efficacy of the cell homing chemokine CXCL12 versus skMPCs in a rat model of ISD. Thirty‐six adult female Sprague Dawley rats were divided into 6 treatment (Tx) conditions: (a) no ISD/noTx [Control]; (b) ISD/noTx; (c) ISD + skMPCs; (d) ISD + 3.5 mg CXCL12; (e) ISD + 7mg CXCL12; and (f) ISD + 14 mg CXCL12. Txs were injected directly into the sphincter complex 30 days post ISD and rats euthanized 30 days post Tx. Blood samples for measurements of kidney and liver function, white and red blood cell counts, were taken at baseline and at euthanasia. Leak point pressures (LPP) were measured prior to, and sphincter collagen/muscle content measured after, euthanasia. There were no effects of treatments on white or red/white blood cell counts, kidney/liver function tests or histopathology of the urinary sphincter complex or surrounding tissues. ISD lowered LPP 35% and sphincter muscle content by 17% versus control rats. CXCL12, but not skMPC injections, restored both LPP to control values in a dose‐dependent fashion. Both skMPCs and CXCL12 restored sphincter muscle content to control values. This chemokine approach may represent a novel therapeutic option for ISD and appears, at least short‐term, to produce little clinical or tissue pathology. Stem Cells Translational Medicine 2017;6:1740–1746

Nonhuman primate model of persistent erectile and urinary dysfunction following radical prostatectomy: Feasibility of minimally invasive therapy

Persistent urinary incontinence (UI) and/or erectile dysfunction (ED) occur in 30‐50% of post‐radical prostatectomy patients regardless of nerve sparing approaches. Identification of potential treatment options for these patients will require testing in an animal model that develops these chronic conditions. The objective was to characterize a nonhuman primate (NHP) model of persistent post‐prostatectomy ED and UI and then test the feasibility of periurethral injection of the chemokine CXCL‐12.

PD49-07 ENGINEERING OF CORPORAL TISSUE CONSTRUCTS USING NON-HUMAN PRIMATE CORPUS CAVERNOSAL SMOOTH MUSCLE AND ENDOTHELIAL CELLS FOR CLINICAL APPLICATIONS

METHODS: Invasive penile cancer cases from 2010-2012 were identified from the NCDB. Pathologic tumor stage was recorded including spongiosal versus carvernosal involvement. Differences in demographic (age, race, comorbid status) and pathologic features (size of tumor, grade, nodal status, LVI, histology, and extranodal extension) between T2 and T3 tumors were compared using c and t-tests. Univariate and multivariate logistic regression was performed to determine the odds of positive lymph nodes (pN+) at inguinal lymph node dissection (ILND) relative to T-stage. RESULTS: There were 367 T2 and 507 T3 patients with penile cancer. The proportion of cases with pN+ disease was 15%, 32%, 46% and 58% for T1, T2, T3 and T4 cases, respectively. Compared to T2 tumors, T3 tumors were larger (mean size 5.8 cm vs. 4.3 cm), more often treated with radical penectomy (36% vs 17%), had higher positive surgical margin rates (12% vs 9%), more aggressive pathology (32% vs 27% poorly differentiated), and were more likely to have lymphovascular invasion (42% vs 31%) (all p < 0.05). In univariate analysis, compared to T1 tumors, T2 (OR 2.8, 95% CI 1.9-4.2) and T3 (OR 4.7, 95% CI 3.3-6.8) were both associated with an increased risk of positive lymph nodes. Although in multivariate analysis, both T2 (OR 2.0, 95% CI 1.2-3.3) and T3 (OR 2.3, 95% CI 1.4-3.6) remained significantly associated with risk of positive lymph nodes compared to T1 disease, there was no increase in risk between T2 and T3 disease (OR 1.1, 95% CI 0.7-1.8, p 1⁄4 0.56). CONCLUSIONS: The proposed new AJCC staging system for the penile cancer distinguishes spongiosal (T2) from cavernosal (T3) involvement and identifies significant differences in pathologic features of the tumors (grade, LVI and size). There does not appear to be a difference in positive lymph node status between the two grades when other clinical and pathological variables are considered. Further study is required to confirm these findings and the prognostic implications of the proposed new staging system.

MP26-06 NON-HUMAN PRIMATE (NHP) MODEL OF URINARY INCONTINENCE AND ERECTILE DYSFUNCTION AFTER RADICAL PROSTATECTOMY

(PDE) inhibitors (PDE4i and PDE5i) in an experimental model of detrusor overactivity. METHODS: The experiments were performed on bladder strips of mice treated with L-NAME for 30 days. Chronic L-NAME administration leads to detrusor overactivity by NO deprivation. The following drugs were used: BRL 37344 (b3-AR agonist), tadalafil (PDE5i) and rolipram (PDE4i). After potassium-induced contraction, strips isolated from mice were exposed to increasing concentrations of each drug. In another series of experiments, prior to contraction, strips were incubated with either tadalafil or rolipram and then increasing concentrations of BRL 37344 were added. RESULTS: Cumulative concentration-response curves were constructed. Rolipram showed the best relaxation when compared to the other drugs (Figure 1). Rolipram increased the relaxing response of BRL 37344 in almost all concentrations, but no synergistic effect with tadalafil was observed (Figure 2). CONCLUSIONS: PDE inhibitors associated with the already proven effective b3-AR agonists may represent a new approach for patients with storage symptons. The relaxing effect of the b3-AR agonist was potentiated by PDE4i but not by PDE5i, suggesting cAMP plays an important role in DSM relaxation.

Regenerative Medicine Approaches to Repair Penile Structure and Function

Penile repair and reconstruction are needed for a variety of diseases and injuries, including Peyronie’s disease, hypospadias, epispadias, urethral stricture, penis fractures, burns, bites, infections, penetrating injuries, and blunt force trauma [1–4]. Damage to penile tissue often leads to loss of sexual function that can profoundly affect quality of life. When the penile defect or injury is sufficiently large, cannot be managed conservatively, and involves the urethra and rupture of the tunica albuginea and corpora cavernosum, various surgical procedures, such as penile prostheses and autograft implantation, are required. While cosmetic appearance may be improved with these procedures, restoration of spontaneous and natural erectile function is usually not achieved. This is often due to critical defect of the corpora cavernosa, which is responsible for erectile function.