Xiaopu Liu

Chronic Oral Administration of the Arginase Inhibitor 2(S)-amino-6-boronohexanoic Acid (ABH) Improves Erectile Function in Aged Rats

Arginase expression and activity have been noted to be heightened in conditions associated with erectile dysfunction, including aging. Previously, arginase inhibition by chronic administration of the arginase inhibitor 2-(S)-amino-6-boronohexanoic acid (ABH) has been shown to improve endothelial dysfunction in aged rats. The objective of this study was to assess whether chronic oral ABH administration affects cavernosal erectile function. Rats were divided into 4 groups: young control, young treated with arginase inhibitor, aged control, and aged treated with arginase inhibitor. Arginase activity was measured and presented as a proportion of young untreated rats. In vivo erectile responses to cavernous nerve stimulation were measured in all cohorts. The cavernous nerve was stimulated with a graded electrical stimulus, and the intracavernosal/mean arterial pressure ratios and total intracavernosal pressure were recorded. Arginase activity was elevated in the aged rats compared with young controls; however, arginase activity was significantly decreased in aged rats treated with ABH. With the addition of ABH, erectile responses improved in the aged rats (P < .05). Oral inhibition of arginase with ABH results in improved erectile function in aged rats, resulting in erectile hemodynamics similar to young rats. This represents the first documentation of systemic arginase inhibition positively affecting corporal cavernosal function.

Valproic acid prevents penile fibrosis and erectile dysfunction in cavernous nerve-injured rats

INTRODUCTION Bilateral cavernous nerve injury (BCNI) causes profound penile changes such as apoptosis and fibrosis leading to erectile dysfunction (ED). Histone deacetylase (HDAC) has been implicated in chronic fibrotic diseases. AIMS This study will characterize the molecular changes in penile HDAC after BCNI and determine if HDAC inhibition can prevent BCNI-induced ED and penile fibrosis. METHODS Five groups of rats (8-10 weeks, n = 10/group) were utilized: (i) sham; (ii and iii) BCNI 14 and 30 days following injury; and (iv and v) BCNI treated with HDAC inhibitor valproic acid (VPA 250 mg/kg; 14 and 30 days). All groups underwent cavernous nerve stimulation (CNS) to determine intracavernosal pressure (ICP). Penile HDAC3, HDAC4, fibronectin, and transforming growth factor-β1 (TGF-β1) protein expression (Western blot) were assessed. Trichrome staining and the fractional area of fibrosis were determined in penes from each group. Cavernous smooth muscle content was assessed by immunofluorescence to alpha smooth muscle actin (α-SMA) antibodies. MAIN OUTCOME MEASURES We measured ICP; HDAC3, HDAC4, fibronectin, and TGF-β1 protein expression; penile fibrosis; penile α-SMA content. RESULTS There was a voltage-dependent decline (P < 0.05) in ICP to CNS 14 and 30 days after BCNI. Penile HDAC3, HDAC4, and fibronectin were significantly increased (P < 0.05) 14 days after BCNI. There was a slight increase in TGF-β1 protein expression after BCNI. Histological analysis showed increased (P < 0.05) corporal fibrosis after BCNI at both time points. VPA treatment decreased (P < 0.05) penile HDAC3, HDAC4, and fibronectin protein expression as well as corporal fibrosis. There was no change in penile α-SMA between all groups. Furthermore, VPA-treated BCNI rats had improved erectile responses to CNS (P < 0.05). CONCLUSION HDAC-induced pathological signaling in response to BCNI contributes to penile vascular dysfunction. Pharmacological inhibition of HDAC prevents penile fibrosis, normalizes fibronectin expression, and preserves erectile function. The HDAC pathway may represent a suitable target in preventing the progression of ED occurring post-radical prostatectomy.

Caspase-3 dependent nitrergic neuronal apoptosis following cavernous nerve injury is mediated via RhoA and ROCK activation in major pelvic ganglion

Axonal injury due to prostatectomy leads to Wallerian degeneration of the cavernous nerve (CN) and erectile dysfunction (ED). Return of potency is dependent on axonal regeneration and reinnervation of the penis. Following CN injury (CNI), RhoA and Rho-associated protein kinase (ROCK) increase in penile endothelial and smooth muscle cells. Previous studies indicate that nerve regeneration is hampered by activation of RhoA/ROCK pathway. We evaluated the role of RhoA/ROCK pathway in CN regulation following CNI using a validated rat model. CNI upregulated gene and protein expression of RhoA/ROCK and caspase-3 mediated apoptosis in the major pelvic ganglion (MPG). ROCK inhibitor (ROCK-I) prevented upregulation of RhoA/ROCK pathway as well as activation of caspase-3 in the MPG. Following CNI, there was decrease in the dimer to monomer ratio of neuronal nitric oxide synthase (nNOS) protein and lowered NOS activity in the MPG, which were prevented by ROCK-I. CNI lowered intracavernous pressure and impaired non-adrenergic non-cholinergic-mediated relaxation in the penis, consistent with ED. ROCK-I maintained the intracavernous pressure and non-adrenergic non-cholinergic-mediated relaxation in the penis following CNI. These results suggest that activation of RhoA/ROCK pathway mediates caspase-3 dependent apoptosis of nitrergic neurons in the MPG following CNI and that ROCK-I can prevent post-prostatectomy ED.

Ex Vivo Model of Human Penile Transplantation and Rejection: Implications for Erectile Tissue Physiology

BACKGROUND Penile transplantation is a potential treatment option for severe penile tissue loss. Models of human penile rejection are lacking. OBJECTIVE Evaluate effects of rejection and immunosuppression on cavernous tissue using a novel ex vivo mixed lymphocyte reaction (MLR) model. DESIGN, SETTING, AND PARTICIPANTS Cavernous tissue and peripheral blood mononuclear cells (PBMCs) from 10 patients undergoing penile prosthesis operations and PBMCs from a healthy volunteer were obtained. Ex vivo MLRs were prepared by culturing cavernous tissue for 48h in media alone, in media with autologous PBMCs, or in media with allogenic PBMCs to simulate control, autotransplant, and allogenic transplant conditions with or without 1μM cyclosporine A (CsA) or 20nM tacrolimus (FK506) treatment. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS Rejection was characterized by PBMC flow cytometry and gene expression transplant array. Cavernous tissues were evaluated by histomorphology and myography to assess contraction and relaxation. Data were analyzed using two-way analysis of variance and unpaired Student t test. RESULTS AND LIMITATIONS Flow cytometry and tissue array demonstrated allogenic PBMC activation consistent with rejection. Rejection impaired cavernous tissue physiology and was associated with cellular infiltration and apoptosis. CsA prevented rejection but did not improve tissue relaxation. CsA treatment impaired relaxation in tissues cultured without PBMCs compared with media and FK506. Study limitations included the use of penile tissue with erectile dysfunction and lack of cross-matching data. CONCLUSIONS This model could be used to investigate the effects of penile rejection and immunosuppression. Additional studies are needed to optimize immunosuppression to prevent rejection and maximize corporal tissue physiology. PATIENT SUMMARY This report describes a novel ex vivo model of human penile transplantation rejection. Tissue rejection impaired erectile tissue physiology. This report suggests that cyclosporin A might hinder corporal physiology and that other immunosuppressant agents, such as FK506, might be better suited to penile transplantation.

Temporal changes in neurotrophic factors and neurite outgrowth in the major pelvic ganglion following cavernous nerve injury

Despite nerve‐sparing radical prostatectomy, nerve damage and erectile dysfunction (ED) prevail, and preventing neurodegeneration is of great importance. Neurotrophic factors and neurite outgrowth were characterized in major pelvic ganglia (MPG) following bilateral cavernous nerve injury (BCNI). Young male Sprague‐Dawley rats underwent sham or BCNI surgery, and the intracavernosal pressure to mean arterial pressure ratio was measured 2, 7, 14, 21, 30, and 60 days following injury (n = 8/group). MPG gene expression (qPCR) and Western blot were performed for glial cell line‐derived neurotrophic factor (GDNF), nerve growth factor (NGF), neurturin, neurotrophin (NT)−3, NT4, brain‐derived neurotrophic factor (BDNF), vascular endothelial growth factor, and activating transcription factor 3 (ATF3). Additional rats were injured, and MPGs were removed 24 hr, 48 hr, 3 days, and 7 days following BCNI (n = 3/group). MPGs were cultured in Matrigel, and neurite outgrowth was measured. Erections were impaired early and improved by 60 days in BCNI rats. GDNF, NGF, BDNF, and ATF3 gene expression was significantly increased and NT3 was decreased in MPGs following BCNI (48 hr to 21 days, P < 0.05). GDNF and NGF protein levels were elevated in 48‐hr BCNI rats. MPG neurite outgrowth from 24‐hr and 48‐hr BCNI was higher than sham (658 ± 19 μm, 607 ± 24 μm, 393 ± 23 μm, respectively, P < 0.05). Further studies examining the roles of neurotrophic factors in modulating signaling pathways may provide therapeutic avenues for neurogenically mediated ED.

Three-dimensional organoid culture reveals involvement of Wnt/β-catenin pathway in proliferation of bladder cancer cells

There has been increasing awareness of the importance of three-dimensional culture of cancer cells. Tumor cells growing as multicellular spheroids in three-dimensional culture, alternatively called organoids, are widely believed to more closely mimic solid tumors in situ. Previous studies concluded that the Wnt/β-catenin pathway is required for regeneration of the normal urothelium after injury and that β-catenin is upregulated in human bladder cancers, but no clear evidence has been advanced to support the idea that the Wnt/β-catenin pathway is directly involved in deregulated proliferation and the other malignant characteristics of bladder cancer cells. Here we report that the Wnt/β-catenin pathway activator, CHIR99021, promoted proliferation of established human bladder cancer cell lines when they were grown in organoid culture but not when they were grown in conventional adherent cultures. CHIR99021 activated Wnt/β-catenin pathway in bladder cancer cell lines in organoid culture. CHIR99021 also stimulated proliferation and the Wnt/b-catenin pathway in primary human bladder cancer organoids. RNAi-mediated knockdown of β-catenin blocked growth of organoids. The effects of CHIR99021 were associated with decreased expression of the urothelial terminal differentiation marker, cytokeratin 20. Our data suggest that the Wnt/β-catenin pathway is required for the proliferation of bladder cancer cells in three-dimensional organoid culture and provide a concrete example of why organoid culture is important for cancer research.

Early-stage Type 2 Diabetes Mellitus Impairs Erectile Function and Neurite Outgrowth From the Major Pelvic Ganglion and Downregulates the Gene Expression of Neurotrophic Factors

OBJECTIVE To assess neurite sprouting and gene expression of neurotrophic factors, nerve markers, and apoptosis in the major pelvic ganglia (MPGs) of rats with type 2 diabetes mellitus (T2DM) as it relates to erectile function. MATERIALS AND METHODS Male rats were fed high-fat diet for 2 weeks followed by 2 low-dose injections of streptozotocin (20 mg/kg). In 3 groups (controls, 3-week, or 5-week T2DM), erectile function was measured by ratios of intracavernosal pressure to mean arterial pressure after cavernous nerve stimulation. MPGs were harvested, and gene expressions of neurotrophic factor 3, nerve growth factor, glial cell line-derived neurotrophic factor, brain-derived neurotrophic factor, caspase-1, -3, -9, beta tubulin type III, and neuronal nitric oxide synthase were quantified by quantitative polymerase chain reaction. Additional MPGs were harvested and cultured in Matrigel. Neurite outgrowth from the MPG was evaluated at 48 hours after culture. RESULTS Erectile function was significantly decreased in all rats with T2DM. Gene expressions of neurotrophic factor 3, nerve growth factor, glial cell line-derived neurotrophic factor, and brain-derived neurotrophic factor were slightly lower at 3 weeks and significantly lower at 5 weeks after T2DM induction. Gene expression of apoptotic markers caspase-1, -3, -9, and neuronal markers beta tubulin type III and neuronal nitric oxide synthase remained unchanged. Rats with T2DM had shorter neurite length and less neurite sprouting than did the control MPG. CONCLUSION Early-stage T2DM downregulates neurotrophic factors, induces erectile dysfunction, and impairs MPG neurite outgrowth, suggesting that erectile dysfunction may be prevented by supplementing neurotrophic factors at early-stage T2DM.

Biomanufacturing Seamless Tubular and Hollow Collagen Scaffolds with Unique Design Features and Biomechanical Properties

A versatile process to develop designer collagen scaffolds for hollow and tubular tissue engineering applications is presented. This process creates seamless and biomechanically tunable scaffolds ranging from ureter-like microsized tubings to structures with highly customized lumens that resemble intestinal villi, fluid bladders, and alveolar sacs that together with stem cells can potentially be used in preclinical and clinical settings.

Ex vivo culture of tumor cells from N-methyl-N-nitrosourea-induced bladder cancer in rats: Development of organoids and an immortalized cell line

OBJECTIVE We ex vivo cultured primary tumor cells from N-methyl-N-nitrosourea (MNU)-induced bladder tumors in rats and established an immortalized cell line from them. MATERIALS AND METHODS Bladder tumors in rats were induced by instillation of MNU into the murine bladder. Primary tumor cells were prepared by the cancer-tissue originated spheroid method. An immortalized cell line was established by co-culture with fibroblasts. The cultured tumor cells were molecularly and functionally characterized by quantitative real-time polymerase chain reaction, Western blot, growth assay, and transwell migration assay. RESULTS Primary tumor cells were successfully prepared as multicellular spheroids from MNU-induced bladder tumors. The differentiation marker expression patterns observed in the original tumors were largely retained in the spheroids. We succeeded in establishing a cell line from the spheroids and named it T-MNU-1. Although basal markers (CK14 and CK5) were enriched in T-MNU-1 compared to the spheroids, T-MNU-1 expressed both luminal and basal markers. T-MNU-1 was able to migrate through a transwell. CONCLUSIONS Tumor cells in MNU-induced bladder tumors were successfully cultured ex vivo as organoids, and an immortalized cell line was also established from them. The ex vivo models offer a platform that enables analysis of intrinsic characteristics of tumor cells excluding influence of microenvironment in MNU-induced bladder tumors.

MP54-12 MOLECULAR CHARACTERIZATION OF N-METHYL-N-NITROSOUREA-INDUCED BLADDER UROTHELIAL TUMOR IN RATS

Silvia Selinski, Meinolf Blaszkewicz, Katja Ickstadt, Dortmund, Germany; Holger Gerullis, Oldenburg, Germany; Thomas Otto, Neuss, Germany; Emanuel Roth, Frank Volkert, Lutherstadt Wittenberg, Germany; Daniel Ovsiannikov, Koblenz, Germany; Thomas Reckwitz*, Oliver Moormann, Dortmund, Germany; Gergely Banfi, Peter Nyirady, Budapest, Hungary; Sita Vermeulen, Nijmegen, Netherlands; Montserrat Garcia-Closas, Bethesda, MD; Jonine Figueroa, Edinburgh, United Kingdom; Alison Johnson, Burlington, VT; Margaret Karagas, Hanover, NH; Manolis Kogevinas, Barcelona, Spain; Nuria Malats, Madrid, Spain; Molly Schwenn, Augusta, ME; Debra Silverman, Stella Koutros, Nathaniel Rothman, Bethesda, MD; Lambertus Kiemeney, Nijmegen, Netherlands; Jan Hengstler, Klaus Golka, Dortmund, Germany