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Appropriate coordination of smooth muscle contraction and relaxation is essential for normal colonic motility. The impact of perturbed motility ranges from moderate, in conditions such as colitis, to potentially fatal in the case of pseudo-obstruction. The mechanisms underlying aberrant motility and the extent to which they can be targeted pharmacologically are incompletely understood. This study identified colonic smooth muscle as a major site of expression of neuropilin 2 (Nrp2) in mice and humans. Mice with inducible smooth muscle-specific knockout of Nrp2 had an increase in evoked contraction of colonic rings in response to carbachol at 1 and 4 weeks following initiation of deletion. KCl-induced contractions were also increased at 4 weeks. Colonic motility was similarly enhanced, as evidenced by faster bead expulsion in Nrp2-deleted mice versus Nrp2-intact controls. In length-tension analysis of the distal colon, passive tension was similar in Nrp2-deficient and Nrp2-intact mice, but at low strains, active stiffness was greater in Nrp2-deficient animals. Consistent with the findings in conditional Nrp2 mice, Nrp2-null mice showed increased contractility in response to carbachol and KCl. Evaluation of selected proteins implicated in smooth muscle contraction revealed no significant differences in the level of α-smooth muscle actin, myosin light chain, calponin, or RhoA. Together, these findings identify Nrp2 as a novel regulator of colonic contractility that may be targetable in conditions characterized by dysmotility. more...

INTRODUCTION AND OBJECTIVE:Neurogenic and idiopathic detrusor overactivity (DO) is currently treated with onabotulinumtoxin A (BoNT/A) as third line therapy. In cases of treatment failure, alternat... more...

Introduction: Neuropilin-1 (NRP1) is a transmembrane receptor present in vascular smooth muscle cells (VSMC) that mediates the inhibition of Rho signaling by binding the Class 3 Semaphorin (SEMA) ligand SEMA3A. Hypothesis: We hypothesize that loss of NRP1 in VSMC mitigates SEMA3A-induced Rho inhibition, thereby increasing vascular tone and blood pressure in vivo . Methods: Male and female adult mice (8-12 weeks) with inducible, smooth muscle cell-specific deletion of NRP1 (SM22a-Cre ERT2 X Nrp1 flox/flox ) were examined. Following recombination using 4-hydroxy tamoxifen (SM- NRP1 KO), systolic blood pressure (SBP) was measured using a tail cuff and compared to age- and sex-matched mice that did not receive tamoxifen (control). Aortic vascular reactivity and expression of key proteins in the Rho signaling cascade were measured using ex vivo tension myography and western blotting, respectively. Results: SBP was significantly increased in SM- NRP1 KO mice following recombination compared to control mice (SBP: 136.5 ± 10.9 vs 112.9 ± 5.6 mmHg; p=0.0006). Contractile responses in aortas of SM- NRP1 KO mice to phenylephrine (p=0.025), KCl (p=0.012), and the thromboxane agonist U44619 (p=0.019) were significantly enhanced compared to controls. Expression of total myosin light chain and LIMK-2 proteins were increased in SM- NRP1 KO compared to control aortas. In vitro , treatment of murine primary VSMC expressing NRP1 with SEMA3A decreased angiotensin II-induced Rho-GTP activation. Additionally, control and SM- NRP1 KO mice (starting at 2 weeks post-recombination) were administered angiotensin II (490 ng/kg/day) for 4 weeks. While there was no significant difference in SBP at weeks 1 and 2, SM- NRP1 KO mice had significantly lower SBP at weeks 3 and 4 following angiotensin II infusion compared to controls (Week 4 SBP: 150 ± 1.4 vs 130.5 ± 2.5 mmHg; p=0.02), suggesting a low ejection fraction and cardiac dysfunction in these mice. In support of this observation, mRNA expression of atrial natriuretic peptide was increased in hearts of angiotensin II-infused SM- NRP1 KO mice. Conclusion: Our data suggest that VSMC NRP1 regulates basal tone and blood pressure, and that loss of NRP1 causes hypertension and exacerbates cardiac dysfunction. more...

Surgical reconstruction of tubular esophageal defects with autologous gastrointestinal segments is the gold standard treatment to replace damaged or diseased esophageal tissues. Unfortunately, this approach is associated with adverse complications, including dysphagia, donor-site morbidity, and in some cases patient death. Bilayer silk fibroin (BLSF) scaffolds were investigated as alternative, acellular grafts for tubular esophagoplasty in a porcine defect model for 3 months of implantation. Adult Yucatan mini-swine (n = 5) were subjected to esophageal reconstruction with tubular BLSF grafts (2 cm in length) in combination with transient esophageal stenting for 2 months followed by a 1-month period, where the graft site was unstented. All animals receiving BLSF grafts survived and were capable of solid food consumption, however strictures were noted at graft regions in 60% of the experimental cohort between 2 and 3 months postop and required balloon dilation. In addition, fluoroscopic analysis showed peristaltic function in only 1/5 neotissues. Following swine harvest at 3 months, ex vivo tissue bath evaluations revealed that neoconduits exhibited contractile responses to carbachol, electric field stimulation, and KCl, whereas sodium nitroprusside and isoproterenol induced relaxation effects. Histological (Masson's Trichrome) and immunohistochemical analyses of regenerated tissue conduits showed a stratified, squamous epithelium expressing pan-cytokeratins buttressed by a vascularized lamina propria containing a smooth muscle-rich muscularis mucosa surrounded by a muscularis externa. Neuronal density, characterized by the presence of synaptophysin-positive boutons, was significantly lower in neotissues in comparison to nonsurgical controls. BLSF scaffolds represent a promising platform for the repair of tubular esophageal defects, however improvements in scaffold design are needed to reduce the rate of complications and improve the extent of constructive tissue remodeling. IMPACT STATEMENT: The search for a superior “off-the-shelf” scaffold capable of repairing tubularesophageal defects as well as overcoming limitations associated with conventional autologous gastrointestinal segments remains elusive. The purpose of this study was to investigate the performance of an acellular, bilayer silk fibroin graft (BLSF) for tubular esophagoplasty in a porcine model. Our results demonstrated that BLSF scaffolds supported the formation of tubular neotissues with innervated, vascularized epithelial and muscular components capable of contractile and relaxation responses. BLSF scaffolds represent a promising platform for esophageal tissue engineering. more...

INTRODUCTION AND OBJECTIVE:Suprasacral spinal cord injury (SCI) is associated with profound remodeling of the bladder wall and with the development of neurogenic detrusor overactivity (NDO). Howeve... more...

The ability of bi-layer silk fibroin (BLSF) matrices to mediate functional tissue formation in porcine bladders subjected to acute partial bladder outlet obstruction (pBOO) was investigated. Sixteen female swine were fitted with a transient urinary catheter containing controlled release valves capable of producing either mild (m-pBOO, 35 ± 10 cmH(2)O, N = 5) or severe (s-pBOO, 70 ± 15 cmH(2)O, N = 11) urinary outlet resistance for 2 or 4 weeks, respectively. After obstructive insults, augmentation cystoplasty was performed with BLSF grafts and animals were harvested up to 3 months postrepair. Urodynamic evaluations of swine after m-pBOO and s-pBOO injury displayed significant reductions in bladder capacity reflecting 63% ± 19% and 39% ± 13% of noninjured levels, while the s-pBOO cohort demonstrated a 61% decline in bladder compliance from baseline. By 3 months postreconstruction, bladder capacity and compliance in the augmented s-pBOO group were significantly increased over post-pBOO values reflecting 79% ± 19% and 171% ± 75% of baseline quantities, respectively. Neotissues with contractile properties were present at graft sites in both injury groups and displayed SM22α(+) smooth muscle, pan-cytokeratin(+) epithelia, vessels lined with CD31(+) endothelial cells, and neurofilament 200(+) nerve trunks. We demonstrate that BLSF scaffolds can support improvements in bladder capacity and compliance and promote the formation of neotissues in diseased bladders. IMPACT STATEMENT: The search for an ideal “off-the-shelf” biomaterial for augmentation cystoplasty remains elusive and current scaffold configurations are hampered by mechanical and biocompatibility restrictions. In addition, preclinical evaluations of potential scaffold designs for bladder repair are limited by the lack of tractable large animal models of obstructive bladder disease that can mimic clinical pathology. The results of this study describe a novel, minimally invasive, porcine model of partial bladder outlet obstruction that simulates clinically relevant phenotypes. Utilizing this model, we demonstrate that acellular, bi-layer silk fibroin grafts can support the formation of vascularized, innervated bladder tissues with functional properties. more...

In the above-cited article, the “CTR 12w” image in Fig. 2 F was inadvertently duplicated in Fig. 7 F …

Spinal cord injury (SCI) caused by trauma or disease leads to motor and sensory abnormalities that depend on the level, severity and duration of the lesion. The most obvious consequence of SCI is paralysis affecting lower and upper limbs. SCI also leads to loss of bladder and bowel control, both of which have a deleterious, life-long impact on the social, psychological, functional, medical and economic well being of affected individuals. Currently, there is neither a cure for SCI nor is there adequate management of its consequences. Although medications provide symptomatic relief for the complications of SCI including muscle spasms, lower urinary tract dysfunction and hyperreflexic bowel, strategies for repair of spinal injuries and recovery of normal limb and organ function are still to be realized. In this review, we discuss experimental evidence supporting the use of the naturally occurring purine nucleoside inosine to improve the devastating sequelae of SCI. Evidence suggests inosine is a safe, novel agent with multifunctional properties that is effective in treating complications of SCI and other neuropathies. more...

Surgical management of long-gap esophageal defects with autologous gastrointestinal tissues is frequently associated with adverse complications including organ dysmotility, dysphagia, and donor site morbidity. In order to develop alternative graft options, bi-layer silk fibroin (SF) scaffolds were investigated for their potential to support functional tissue regeneration in a rodent model of esophageal repair. Onlay esophagoplasty was performed with SF matrices (N = 40) in adult rats for up to 2 m of implantation. Parallel groups consisted of animals implanted with small intestinal submucosa (SIS) scaffolds (N = 22) or sham controls receiving esophagotomy alone (N = 20). Sham controls exhibited a 100% survival rate while rats implanted with SF and SIS scaffolds displayed respective survival rates of 93% and 91% prior to scheduled euthanasia. Animals in each experimental group were capable of solid food consumption following a 3 d post-op liquid diet and demonstrated similar degrees of weight gain throughout the study period. End-point μ-computed tomography at 2 m post-op revealed no evidence of contrast extravasation, fistulas, strictures, or diverticula in any of the implant groups. Ex vivo tissue bath studies demonstrated that reconstructed esophageal conduits supported by both SF and SIS scaffolds displayed contractile responses to carbachol, KCl and electrical field stimulation while isoproterenol produced tissue relaxation. Histological (Masson's trichrome and hematoxylin and eosin) and immunohistochemical (IHC) evaluations demonstrated both implant groups produced de novo formation of skeletal and smooth muscle bundles positive for contractile protein expression [fast myosin heavy chain (MY32) and α-smooth muscle actin (α-SMA)] within the graft site. However, SF matrices promoted a significant 4-fold increase in MY32+ skeletal muscle and a 2-fold gain in α-SMA+ smooth muscle in comparison to the SIS cohort as determined by histomorphometric analyses. A stratified squamous, keratinized epithelium expressing cytokeratin 5 and involucrin proteins was also present at 2 m post-op in all experimental groups. De novo innervation and vascularization were evident in all regenerated tissues indicated by the presence of synaptophysin (SYP38)+ boutons and vessels lined with CD31 expressing endothelial cells. In respect to SIS, the SF group supported a significant 4-fold increase in the density of SYP38+ boutons within the implant region. Evaluation of host tissue responses revealed that SIS matrices elicited chronic inflammatory reactions and severe fibrosis throughout the neotissues, in contrast to SF scaffolds. The results of this study demonstrate that bi-layer SF scaffolds represent promising biomaterials for onlay esophagoplasty, capable of producing superior regenerative outcomes in comparison to conventional SIS scaffolds. more...

Aims Mounting evidence indicates that a variety of factors released from the urothelium or suburothelium can modulate smooth muscle activity. Although the relationship between the mucosa and smooth muscle has been investigated, little is known about the pathophysiologic changes in detrusor-mucosa interactions in neurogenic bladders. The goal of the study was to determine the impact of the mucosa on evoked responses in spinal cord injured (SCI) bladders. Methods Urinary bladders were obtained from 6wk SCI rats or age-matched uninjured controls. Ex vivo isometric tension studies were performed and muscarinic receptor expression was measured in bladder tissue with and without mucosa. Results The magnitude and area of nerve evoked responses in SCI tissue with mucosa was higher than without mucosa. The duration and decay time of nerve-evoked responses were longer in SCI than control tissue irrespective of the mucosa. The level of the muscarinic M2 receptor was decreased in the mucosa of SCI bladders. Conclusions Detrusor-mucosa interactions are substantially altered in the neurogenic bladder. After spinal cord injury, an excitatory modulation of smooth muscle contraction by the mucosa emerges, and could be targeted via intravesical treatment in the context of neurogenic bladder dysfunction. more...

Surgical repair of caustic oesophageal injuries with autologous gastrointestinal segments is often associated with dysmotility, dysphagia and donor site morbidity, and therefore alternative graft options are needed. Bilayer silk fibroin (BLSF) scaffolds were assessed for their ability to support functional restoration of damaged oesophageal tissues in a rat model of onlay oesophagoplasty. Transient exposure of isolated oesophageal segments with 40% NaOH led to corrosive oesophagitis and a 91% reduction in the luminal cross-sectional area of damaged sites. Oesophageal repair with BLSF matrices was performed in injured rats (n = 27) as well as a nondiseased cohort (n = 12) for up to 2 months after implantation. Both implant groups exhibited >80% survival rates, displayed similar degrees of weight gain, and were capable of solid food consumption following a 3-day liquid diet. End-point μ-computed tomography of repaired sites demonstrated a 4.5-fold increase in luminal cross-sectional area over baseline injury levels. Reconstructed oesophageal conduits from damaged and nondiseased animals produced comparable contractile responses to KCl and electric field stimulation while isoproterenol generated similar tissue relaxation responses. Histological and immunohistochemical evaluations of neotissues from both implant groups showed formation of a stratified, squamous epithelium with robust cytokeratin expression as well as skeletal and smooth muscle layers positive for contractile protein expression. In addition, synaptophysin positive neuronal junctions and vessels lined with CD31 positive endothelial cells were also observed at graft sites in each setting. These results provide preclinical validation for the use of BLSF scaffolds in reconstructive strategies for oesophageal repair following caustic injury. more...

Partial circumferential, full thickness defects of the esophagus can occur as a result of organ perforation or tumour resection, or during surgical reconstruction of strictured segments. Complications associated with autologous tissue flaps conventionally utilized for defect repair necessitate the development of new graft options. In this study, bi-layer silk fibroin (BLSF) scaffolds were investigated for their potential to support functional restoration of partial circumferential defects in a porcine model of esophageal repair. Onlay thoracic esophagoplasty with BLSF matrices (~3 x 1.5 cm) was performed in adult swine (N = 6) for 3 months of implantation. All animals receiving BLSF grafts survived with no complications and were capable of solid food consumption. Radiographic esophagrams revealed preservation of organ continuity with no evidence of contrast extravasation or strictures. Fluoroscopic analysis demonstrated peristaltic contractions. Ex vivo tissue bath studies displayed contractile responses to carbachol, electric field stimulation, and KCl while isoproterenol produced tissue relaxation. Histological and immunohistochemical evaluations of neotissues showed a stratified, squamous epithelium, a muscularis mucosa composed of smooth muscle bundles, and a muscularis externa organized into circular and longitudinal layers, with a mix of striated skeletal muscle fascicles interspersed with smooth muscle. De novo innervation and vascularization were observed throughout the graft sites and consisted of synaptophysin-positive neuronal boutons and vessels lined with CD31-positive endothelial cells. The results of this study demonstrate that BLSF scaffolds can facilitate constructive remodeling of partial circumferential, full thickness esophageal defects in a large animal model. Copyright © 2017 John WileySons, Ltd. more...

Although slow gastric emptying (gastroparesis) is a well-known complication of chronic hyperglycemia in diabetes mellitus (DM), it recently has become clear that rapid gastric emptying also is a frequent and important diabetic complication. In contrast, acute hyperglycemia causes slow gastric emptying, and acute hypoglycemia causes rapid gastric emptying. Rapid gastric emptying is frequent in T2DM; however, it may also occur in T1DM, particularly in the early stages of the disease, but may persist even into late stages. Recent studies suggest that usually, the stomach restricts the emptying of nutrients to 1–4 kcals/min. This restriction is due to the action of the gastric ‘braking’ hormones such as GLP-1, leptin, and amylin acting via the gastric inhibitory vagal motor circuit (GIVMC). Disruption of this braking system leads to rapid gastric emptying. Acute hyperglycemia also slows gastric emptying by stimulating the GIVMC, while acute hypoglycemia causes rapid gastric emptying by stimulating the gastric excitatory vagal motor circuit (GEVMC). In contrast, chronic hyperglycemia causes rapid gastric emptying by inducing oxidative stress in the stomach wall that disrupts inhibitory neuromuscular transmission and increases the contractility of the smooth muscle, while chronic hyperglycemia may also cause slow gastric emptying via severe inflammatory stress caused by proinflammatory macrophages and reduce contractility of the smooth muscle. There is a bidirectional relationship between blood glucose and gastric emptying. Thus, rapid gastric emptying may lead to a sizeable postprandial spike, and slow gastric emptying may blunt it. Postprandial hyperglycemia is involved in the development, progression, and complications of DM. Correction of fast gastric emptying involves agents that activate GIVMC and the use of gastric ‘braking’ hormones or their analogs. Recognition and treatment of rapid gastric emptying may contribute to better management of postprandial hyperglycemia and prevention of some diabetic complications. more...

Acellular scaffolds derived from Bombyx mori silk fibroin were investigated for their ability to support functional tissue regeneration in a porcine model of augmentation cystoplasty. Two bi-layer matrix configurations were fabricated by solvent-casting/salt leaching either alone (Group 1) or in combination with silk film casting (Group 2) to yield porous foams buttressed by heterogeneous surface pore occlusions or homogenous silk films, respectively. Bladder augmentation was performed with each scaffold group (6 × 6 cm(2)) in juvenile Yorkshire swine for 3 m of implantation. Augmented animals exhibited high rates of survival (Group 1: 5/6, 83%; Group 2: 4/4, 100%) and voluntary voiding over the course of the study period. Urodynamic evaluations demonstrated mean increases in bladder capacity over pre-operative levels (Group 1: 277%; Group 2: 153%) which exceeded nonsurgical control gains (144%) encountered due to animal growth.In addition, animals augmented with both matrix configurations displayed increases in bladder compliance over pre-operative levels(Group 1: 357%; Group 2: 338%) similar to growth-related elevations observed in non-surgical controls (354%) [corrected]. Gross tissue evaluations revealed that both matrix configurations supported extensive de novo tissue formation throughout the entire original implantation site which exhibited ultimate tensile strength similar to nonsurgical counterparts. Histological and immunohistochemical analyses showed that both implant groups promoted comparable extents of smooth muscle regeneration and contractile protein (α-smooth muscle actin and SM22α) expression within defect sites similar to controls. Parallel evaluations demonstrated the formation of a transitional, multi-layered urothelium with prominent cytokeratin, uroplakin, and p63 protein expression in both matrix groups. De novo innervation and vascularization processes were evident in all regenerated tissues indicated by synaptophysin-positive neuronal cells and vessels lined with CD31 expressing endothelial cells. Ex vivo organ bath studies demonstrated that regenerated tissues supported by both silk matrices displayed contractile responses to carbachol, α,β-methylene-ATP, KCl, and electrical field stimulation similar to controls. Our data detail the ability of acellular silk scaffolds to support regeneration of innervated, vascularized smooth muscle and urothelial tissues within 3 m with structural, mechanical, and functional properties comparable to native tissue in a porcine model of bladder repair. more...

Neurogenic detrusor overactivity (NDO) is among the most challenging complications of spinal cord injury (SCI). A recent report by us demonstrated an improvement in NDO in SCI rats following chronic systemic treatment with the purine nucleoside inosine. The objective of this study was to investigate the mechanism of action of inosine underlying improvement of NDO. Male Sprague-Dawley rats underwent complete spinal cord transection at T8. Inosine (1 mM) delivered intravesically to SCI rats during conscious cystometry significantly decreased the frequency of spontaneous non-voiding contractions. In isolated tissue assays, inosine (1 mM) significantly decreased the amplitude of spontaneous activity (SA) in SCI bladder muscle strips. This effect was prevented by a pan-adenosine receptor antagonist CGS15943, but not by A1 or A3 receptor antagonists. The A2A antagonist ZM241385 and A2B antagonist PSB603 prevented the effect of inosine. The effect of inosine was mimicked by the adenosine receptor agonist NECA and the A2B receptor agonist BAY60-6583. The inhibition of SA by inosine was not observed in the presence of the BK antagonist, iberiotoxin, but persisted in the presence of KATP and SK antagonists. These findings demonstrate that inosine acts via an A2B receptor-mediated pathway that impinges on specific potassium channel effectors. more...

Chronic urethral obstruction and the ensuing bladder wall remodeling can lead to diminished bladder smooth muscle (BSM) contractility and debilitating lower urinary tract symptoms. No effective pharmacotherapy exists to restore BSM contractile function. Neuropilin 2 (Nrp2) is a transmembrane protein that is highly expressed in BSM. Nrp2 deletion in mice leads to increased BSM contraction. We determined whether genetic ablation of Nrp2 could restore BSM contractility following obstruction. Partial bladder outlet obstruction (pBOO) was created by urethral occlusion in mice with either constitutive and ubiquitous, or inducible smooth muscle-specific deletion of Nrp2, and Nrp2-intact littermates. Mice without obstruction served as additional controls. Contractility was measured by isometric tension testing. Nrp2 deletion prior to pBOO increased force generation in BSM 4 weeks following surgery. Deletion of Nrp2 in mice already subjected to pBOO for 4 weeks showed increased contractility of tissues tested 6 weeks after surgery compared with nondeleted controls. Assessment of tissues from patients with urodynamically defined bladder outlet obstruction revealed reduced NRP2 levels in obstructed bladders with compensated compared with decompensated function, relative to asymptomatic controls. We conclude that downregulation of Nrp2 promotes BSM force generation. Neuropilin 2 may represent a novel target to restore contractility following obstruction. more...

What's known on the subject? and What does the study add? Caveolae are specialised regions of bladder smooth muscle (BSM) cell membranes where specific signalling pathways are regulated. Caveolin proteins are involved in caveolar biogenesis and function as signal transduction regulators. Expression of caveolin-1, -2, and -3 has been previously identified in the bladder; however, the distribution and relative expression of these proteins have not been defined. The present data show significant differences in the spatial distribution of caveolin proteins throughout the bladder wall. Region dependent variations in the co-localisation of caveolin subtypes in detrusor SM were also detected. These findings support the premise that the unique spatial pattern of caveolin proteins associated with BSM cells may enable regionally distinct functional responses to common stimuli. OBJECTIVE • To determine the regional expression profile of caveolin isoforms (integral membrane proteins abundant in caveolae), the spatial relationships among caveolin proteins within specific smooth muscle (SM) regions and the extent of their molecular interactions in bladder SM (BSM). MATERIALS AND METHODS • Regional differences in the expression of caveolin family members were determined by quantitative reverse transcriptase-polymerase chain reaction and Western blot of RNA and protein extracted from the base, body and dome of rat bladders. • To evaluate the distribution of caveolin-1 (Cav-1), Cav-2 and Cav-3 within the bladder, longitudinal tissue sections from the base to dome were processed for confocal microscopy and quantified for intensity of immunoreactivity (IR) and extent of co-localisation. • Interactions among Cav-1, Cav-2 and Cav-3 were determined by co-immunoprecipitation. RESULTS • Differential expression of Cav-1 and Cav-3 was detected among bladder regions, with lowest expression in the bladder base relative to the dome. • Cav-1 was highly expressed in all regions, although an increase in IR from submucosa to serosa was detected in each region. • The distribution of Cav-2 IR generally paralleled Cav-1, but progressively decreased from submucosa to serosa in each region. • Cav-3 expression predominated in the medial region of BSM increasing progressively from base to dome, but was poorly expressed in the outer SM layer particularly in the dome. • Cav-1 co-precipitated extensively with both Cav-2 and Cav-3. Co-precipitation between Cav-3 and Cav-2 was also detected. CONCLUSIONS • The isoform-specific spatial distribution and distinct molecular interactions among caveolins in BSM may contribute to the contractile heterogeneity of BSM cells and facilitate differential modulation of responses to local stimuli. • As BSM caveolae regulate key signalling processes involved in contraction, altered expression of caveolin proteins may generate a regional imbalance in contraction/relaxation responses, thus leading to bladder dysfunction. more...

Diabetic bladder dysfunction (DBD) is common and affects 80% of diabetic patients. However, the molecular mechanisms underlying DBD remain elusive because of a lack of appropriate animal models. We demonstrate DBD in a mouse model that harbors hepatic-specific insulin receptor substrate 1 and 2 deletions (double knockout [DKO]), which develops type 2 diabetes. Bladders of DKO animals exhibited detrusor overactivity at an early stage: increased frequency of nonvoiding contractions during bladder filling, decreased voided volume, and dispersed urine spot patterns. In contrast, older animals with diabetes exhibited detrusor hypoactivity, findings consistent with clinical features of diabetes in humans. The tumor necrosis factor (TNF) superfamily genes were upregulated in DKO bladders. In particular, TNF-α was upregulated in serum and in bladder smooth muscle tissue. TNF-α augmented the contraction of primary cultured bladder smooth muscle cells through upregulating Rho kinase activity and phosphorylating myosin light chain. Systemic treatment of DKO animals with soluble TNF receptor 1 (TNFRI) prevented upregulation of Rho A signaling and reversed the bladder dysfunction, without affecting hyperglycemia. TNFRI combined with the antidiabetic agent, metformin, improved DBD beyond that achieved with metformin alone, suggesting that therapies targeting TNF-α may have utility in reversing the secondary urologic complications of type 2 diabetes. more...

Aims Caveolae are specialized regions of the cell membrane that modulate signal transduction and alterations in these structures affect bladder smooth muscle (BSM) contraction. Since bladder dysfunctions are common in the elderly, we evaluated the effect of aging on the morphology of caveolae and caveolin protein expression in BSM. Methods Caveolar morphology (number, size, and depth) in BSM was determined from electron microscopy images of young (10 weeks), adult (6-month old), and old (12-month old) rat urinary bladders. Changes in expression levels of caveolin proteins with age were investigated by Western blot and immunofluorescence microscopy. Caveolin-3 gene expression was determined by real-time RT-PCR in young and 19-month-old rat bladders. Results Twelve-month-old animals exhibited 50% fewer BSM caveolae compared to young (P more...

Megaesophagus is defined as the abnormal enlargement or dilatation of the esophagus, characterized by a lack of normal contraction of the esophageal walls. This is called achalasia when associated with reduced or no relaxation of the lower esophageal sphincter (LES). To date, there are few naturally occurring models for this disease. A colony of transgenic (Pvrl3-Cre) rats presented with megaesophagus at 3 to 4 months of age; further breeding studies revealed a prevalence of 90% of transgene-positive animals having megaesophagus. Affected rats could be maintained on a total liquid diet long term and were shown to display the classic features of dilated esophagus, closed lower esophageal sphincter, and abnormal contractions on contrast radiography and fluoroscopy. Histologically, the findings of muscle degeneration, inflammation, and a reduced number of myenteric ganglia in the esophagus combined with ultrastructural lesions of muscle fiber disarray and mitochondrial changes in the striated muscle of these animals closely mimic that seen in the human condition. Muscle contractile studies looking at the response of the lower esophageal sphincter and fundus to electrical field stimulation, sodium nitroprusside, and L-nitro-L-arginine methyl ester also demonstrate the similarity between megaesophagus in the transgenic rats and patients with achalasia. No primary cause for megaesophagus was found, but the close parallel to the human form of the disease, as well as ease of care and manipulation of these rats, makes this a suitable model to better understand the etiology of achalasia as well as study new management and treatment options for this incurable condition., National Institutes of Health (U.S.) (Grant T32OD011141), National Institutes of Health (U.S.) (Grant P30ES002109) more...

3.3%). The potency of solabegron was also significantly increased in the presence of both oxybutynin and tolterodine with IC50 values of 0.05 M and 0.03 M, respectively, versus vehicle (0.14 M). The efficacy of CL-316,243 was also significantly increased in the presence of oxybutynin and tolterodine, (Emax values of 84.7 2.7% and 91.3 1.5%, respectively) compared to vehicle (Emax 68.6 3.0%). However, the potency of CL-316,243 was only significantly affected in the presence of oxybutynin (IC50 value of 1.91 nM compared with 7.36 nM for vehicle). In the presence of oxybutynin, tolterodine or solifenacin, the potency of mirabegron was significantly increased with IC50 values of 0.87 M, 0.33 M and 0.77 M, respectively versus vehicle (1.71 M), while no significant effects on efficacy were observed. CONCLUSIONS: The current results demonstrate that the combination of antimuscarinics and 3-adrenoceptor agonists can result in increased efficacy and potency and supports the hypothesis that combining these compound classes in the clinic could have beneficial effects in treating urinary bladder dysfunction. more...

INTRODUCTION AND OBJECTIVES: Bladder smooth muscle (BSM) cells release Angiotensin II (AngII) in response to mechanical stress and the angiotensin system is upregulated with bladder obstruction. AngII-mediated bladder contractions require intact caveolae, membrane microdomains involved in signal transduction regulation. These microdomains were found to be altered in hypertrophic detrusor muscle from obstructed bladders. This study investigated the effect of AngII on caveolin expression and whether changes in caveolins induced by AngII are associated with functional alterations. METHODS: Rat bladder tissue without mucosa was challenged for 8 hours with exogenous AngII, or with continuous electrical field stimulation (EFS) in the presence or absence of the AngII receptor-1 (AT1R) antagonist losartan (los). Changes in amplitude of contractions in response to treatments were recorded during the 8 hours. The effect of EFS on the release of endogenous AngII was determined by enzyme immunoassay. After stimulation, caveolin gene and protein expression was determined by real time rt-PCR and western blot. The interaction between AT1R and caveolin proteins was investigated by immunoprecipitation. RESULTS: Exposure of BSM tissue to AngII upregulated Cav-1, Cav-2 and Cav-3 gene and protein expression compared to non-stimulated tissue. Caveolin gene expression was similarly upregulated after 8 hours of EFS, but remained unchanged if EFS was delivered in the presence of los. Release of endogenous AngII was increased in EFS stimulated tissue compared to control. Continuous AngII exposure increased the amplitude of spontaneous activity compared to non-stimulated tissue. In addition, the amplitude of EFSinduced contractions was augmented after 8 hours. However, the increase in EFS-induced contractions was prevented by los. AT1R co-precipitated with Cav-1, Cav-2 and Cav-3. CONCLUSIONS: The caveolin protein-AT1R interaction is consistent with the localization of AT1R in caveolae and its regulation by caveolin proteins. The effect of AT1R inhibition on caveolin expression during mechanical perturbations indicates that autocrine/paracrine AngII positively regulates caveolin expression. These findings suggest that endogenous AngII released by BSM initiates a positive feedback response in which AT1R-activated detrusor contractions that are facilitated by caveolae become augmented by an enhanced caveolin-AT1R interaction. These findings may have important implications for pathophysiologic processes associated with altered AngII secretion such as the hypertrophic response to bladder obstruction. more...

Objective Under physiologic conditions in animals and pathologic conditions in humans, purinergic mechanisms contribute significantly to detrusor contractions. Although the activation of P2X1 receptors (P2X1R) accounts for the largest portion of bladder smooth muscle (BSM) responses to ATP, previous reports have shown that P2X1R antagonists do not completely abolish the purinergic component of neurogenic contractions, suggesting the presence of other P2XR subtypes on BSM. P2X4R has been identified in bladder tissue, however whether this receptor is functionally relevant warrants investigation. The aim of this study was to examine the extent of P2X4R expression in BSM tissue and to investigate its pharmacological contribution to ATP-mediated detrusor contractions. Methods P2X4R mRNA and protein expression was investigated in mouse bladder tissue (without mucosa) and in cultured BSM cells by real-time RT-PCR and western blotting respectively. In vitro isometric tension studies were performed in mouse BSM strips without mucosa. Purinergic detrusor contractions were elicited by administration of α-β-methylene-ATP (αβmATP), and the purinergic component of neurogenic contractions induced by electrical field stimulations (EFS) was isolated by pre-treatment with the muscarinic receptor antagonist atropine. The inhibitory effect of two P2X4R selective antagonists, 5-BDBD and BX430, on the αβmATP- and EFS-induced contractions was investigated in the presence of P2X1R antagonist NF449. In addition, the effect of the P2X4R positive modulator ivermectin (IVC) on αβmATP responses was investigated. Data were analyzed by a repeated measures analysis of variance. Results P2X4R mRNA was detected in smooth muscle from both bladder tissue and cultured BSM cells, although its expression was significantly lower than P2X1R expression. Immunoreactivity for P2X4R was detected in lysates from both mouse BSM tissue and smooth muscle cells. Functional studies indicated that although P2X1R activation is predominantly responsible for purinergic contractions in mouse detrusor, a significant portion of the contractile response to both αβmATP (22.3±7%) and EFS (27.5±4% of purinergic component of EFS) was resistant to P2X1R inhibition. This NF449-resistant component was abolished by administration of P2X4R antagonists 5-BDBD or BX430. In addition, responses to αβmATP increased significantly upon administration of IVC. Conclusions The expression of P2X4R in detrusor smooth muscle together with the identification of a P2X4R-sensitive component of bladder contractions suggest that the activation of this P2X receptor subtype could significantly contribute to ATP-mediated BSM responses. P2X4R may thus potentially represent a novel target for the management of detrusor dysfunctions associated with alterations in purinergic signaling. Funding Source(s) Department of Veterans Affairs, Research Service BX001790; BX002806 more...

The intracellular motor protein myosin Va is involved in nitrergic neurotransmission possibly by trafficking of neuronal nitric oxide synthase (nNOS) within the nerve terminals. In this study, we examined the role of myosin Va in the stomach and penis, proto-typical smooth muscle organs in which nitric oxide (NO) mediated relaxation is critical for function. We used confocal microscopy and co-immunoprecipitation of tissue from the gastric fundus (GF) and penile corpus cavernosum (CCP) to localize myosin Va with nNOS and demonstrate their molecular interaction. We utilized in vitro mechanical studies to test whether smooth muscle relaxations during nitrergic neuromuscular neurotransmission is altered in DBA (dilute, brown, non-agouti) mice which lack functional myosin Va. Myosin Va was localized in nNOS-positive nerve terminals and was co-immunoprecipitated with nNOS in both GF and CCP. In comparison to C57BL/6J wild type (WT) mice, electrical field stimulation (EFS) of precontracted smooth muscles of GF and CCP from DBA animals showed significant impairment of nitrergic relaxation. An NO donor, Sodium nitroprusside (SNP), caused comparable levels of relaxation in smooth muscles of WT and DBA mice. These normal postjunctional responses to SNP in DBA tissues suggest that impairment of smooth muscle relaxation resulted from inhibition of NO synthesis in prejunctional nerve terminals. Our results suggest that normal physiological processes of relaxation of gastric and cavernosal smooth muscles that facilitate food accommodation and penile erection, respectively, may be disrupted under conditions of myosin Va deficiency, resulting in complications like gastroparesis and erectile dysfunction. more...

What's known on the subject? and What does the study add? Caveolae are specialised regions of bladder smooth muscle (BSM) cell membranes where specific signalling pathways are regulated. Caveolin proteins are involved in caveolar biogenesis and function as signal transduction regulators. Expression of caveolin-1, -2, and -3 has been previously identified in the bladder; however, the distribution and relative expression of these proteins have not been defined. The present data show significant differences in the spatial distribution of caveolin proteins throughout the bladder wall. Region dependent variations in the co-localisation of caveolin subtypes in detrusor SM were also detected. These findings support the premise that the unique spatial pattern of caveolin proteins associated with BSM cells may enable regionally distinct functional responses to common stimuli.• To determine the regional expression profile of caveolin isoforms (integral membrane proteins abundant in caveolae), the spatial relationships among caveolin proteins within specific smooth muscle (SM) regions and the extent of their molecular interactions in bladder SM (BSM).• Regional differences in the expression of caveolin family members were determined by quantitative reverse transcriptase-polymerase chain reaction and Western blot of RNA and protein extracted from the base, body and dome of rat bladders. • To evaluate the distribution of caveolin-1 (Cav-1), Cav-2 and Cav-3 within the bladder, longitudinal tissue sections from the base to dome were processed for confocal microscopy and quantified for intensity of immunoreactivity (IR) and extent of co-localisation. • Interactions among Cav-1, Cav-2 and Cav-3 were determined by co-immunoprecipitation.• Differential expression of Cav-1 and Cav-3 was detected among bladder regions, with lowest expression in the bladder base relative to the dome. • Cav-1 was highly expressed in all regions, although an increase in IR from submucosa to serosa was detected in each region. • The distribution of Cav-2 IR generally paralleled Cav-1, but progressively decreased from submucosa to serosa in each region. • Cav-3 expression predominated in the medial region of BSM increasing progressively from base to dome, but was poorly expressed in the outer SM layer particularly in the dome. • Cav-1 co-precipitated extensively with both Cav-2 and Cav-3. Co-precipitation between Cav-3 and Cav-2 was also detected.• The isoform-specific spatial distribution and distinct molecular interactions among caveolins in BSM may contribute to the contractile heterogeneity of BSM cells and facilitate differential modulation of responses to local stimuli. • As BSM caveolae regulate key signalling processes involved in contraction, altered expression of caveolin proteins may generate a regional imbalance in contraction/relaxation responses, thus leading to bladder dysfunction. more...

Neuropilins (NRPs) are transmembrane receptors that bind class 3 semaphorins and VEGF family members to regulate axon guidance and angiogenesis. Although expression of NRP1 by vascular smooth muscle cells (SMCs) has been reported, NRP function in smooth muscle (SM) in vivo is unexplored. Using Nrp2(+/LacZ) and Nrp2(+/gfp) transgenic mice, we observed robust and sustained expression of Nrp2 in the SM compartments of the bladder and gut, but no expression in vascular SM, skeletal muscle, or cardiac muscle. This expression pattern was recapitulated in vitro using primary human SM cell lines. Alterations in cell morphology after treatment of primary visceral SMCs with the NRP2 ligand semaphorin-3F (SEMA3F) were accompanied by inhibition of RhoA activity and myosin light chain phosphorylation, as well as decreased cytoskeletal stiffness. Ex vivo contractility testing of bladder muscle strips exposed to electrical stimulation or soluble agonists revealed enhanced tension generation of tissues from mice with constitutive or SM-specific knockout of Nrp2, compared with controls. Mice lacking Nrp2 also displayed increased bladder filling pressures, as assessed by cystometry in conscious mice. Together, these findings identify Nrp2 as a mediator of prorelaxant stimuli in SMCs and suggest a novel function for Nrp2 as a regulator of visceral SM contractility. more...

intensities analysed on Image J. Two or one-factor anovas were utilised as applicable, with post-hoc t-tests. RESULTS: DNC increased localization of DNMT3A to the nucleus of BSMC, in contrast to NC cells, which expressed only cytoplasmic DNMT3A. The JAK2/STAT inhibitor AG490 significantly reduced DNMT3A nuclear localization (P 0.001), without changing SMA expression and proliferation compared to control. Hypoxia enhanced DNMT3A and 3B localization to the nucleus on DNC, but had no effect on NC. Aza prevented DNC-induced proliferation (p 0.05). On DNC, Sonic hedgehog (SHH) upregulated expression of SMA, but SHH increased absolute levels of both nuclear and cytoplasmic DNMT3A (p 0.05). CONCLUSIONS: Matrix exquisitely regulates DNMT3A localization and expression, and influences differentiation in BSMCs exposed to denatured matrix /growth factors or SHH. That nuclear expression of DNMT does not always correspond to increased SMA expression suggests that DNA methylation may not directly act to induce BSMC de-differentiation, though it may influence both proliferation on DNC and differentiation by SHH. Future work will examine how expression of other SMC markers is affected by shRNA against specific DNMTs in a context specific manner. more...

Loss of bladder smooth muscle caveolae, which are membrane invaginations involved in signaling regulation, is associated with detrusor dysfunction. We investigated whether caveolar loss in bladder smooth muscle from SHR rats contributes to detrusor overactivity by dysregulating caveolar mediated signaling.Caveolar density and caveolin-1 protein expression were compared between SHR and WKY rats by ultrastructural and molecular analysis. The functional effects of caveolar depletion achieved by methyl-β-cyclodextrin on neurogenic and agonist induced contractions, and spontaneous activity in isolated bladder tissue were also compared between the strains. P2X1 receptor and caveolin-1 interaction was investigated by confocal microscopy, co-immunoprecipitation and proximity ligation assay.Bladder smooth muscle caveolar density and caveolin-1 expression were decreased in SHR vs WKY rats. Responses to α-β-methylene adenosine triphosphate at baseline were lower in SHR than in WKY rats. Methyl-β-cyclodextrin significantly decreased α-β-methylene adenosine triphosphate responses in WKY rats but had less effect in SHR rats. Methyl-β-cyclodextrin decreased the amplitude of the purinergic component of neurally mediated contractions in each strain but had no effect on the cholinergic component. Bladder spontaneous activity was significantly higher in SHR than in WKY rats. Exposure to methyl-β-cyclodextrin or P2X1 receptor antagonist significantly increased spontaneous activity in WKY rats but had no effect in SHR rats. P2X1 receptor and caveolin-1 were co-localized and co-precipitated in bladder smooth muscle tissue.Caveolar depletion in WKY bladders results in a functional phenotype analogous to that of overactive SHR bladder. The intrinsically decreased caveolae in SHR rats causes loss of the caveolar mediated regulation of purinergic signaling and augmented spontaneous activity, conditions that may lead to detrusor overactivity. more...

Caveolae are specialized regions of the cell membrane that modulate signal transduction and alterations in these structures affect bladder smooth muscle (BSM) contraction. Since bladder dysfunctions are common in the elderly, we evaluated the effect of aging on the morphology of caveolae and caveolin protein expression in BSM.Caveolar morphology (number, size, and depth) in BSM was determined from electron microscopy images of young (10 weeks), adult (6-month old), and old (12-month old) rat urinary bladders. Changes in expression levels of caveolin proteins with age were investigated by Western blot and immunofluorescence microscopy. Caveolin-3 gene expression was determined by real-time RT-PCR in young and 19-month-old rat bladders.Twelve-month-old animals exhibited 50% fewer BSM caveolae compared to young (P 0.01). The area of caveolae was significantly decreased at 6 and 12 months. Despite a decrease in the number of BSM caveolae at 12 months, the expression of caveolin-1 and cavin-1 were unaltered with age. In contrast, caveolin-2 and caveolin-3 protein expression and immunoreactivity were reduced in BSM at 6 and 12 months of age. Caveolin-3 gene expression was also downregulated at 19 months compared to young animals.Biological aging significantly decreases BSM caveolae number and morphology with associated selective alteration in caveolin protein expression. Since caveolae are protected membrane regions that regulate signal transduction, age-related alterations in caveolae and caveolin protein expression could alter BSM contractility resulting in bladder dysfunctions of the elderly. more...

lowed by reoxygenation with 21% oxygen for one hour; cycling in this manner for 48 hours. After 48 hours, samples were collected and processed for fluorometric and enzyme immunoassay of cell injury and transmission electron microscopy. RESULTS: Lipid peroxidation was found in cells exposed to hypoxia and oxidative stress while protein oxidation was evident in cellular oxidative stress only. The levels of oxidatively modified products increased after cell exposure to oxidative stress while increased nitrosative products were evident in both hypoxic and oxidative stress conditions. Cellular antioxidant capacity decreased in oxidative stress but remained unchanged in hypoxia. Forty-eight hours of hypoxia and oxidative stress had no significant effect on cell senescence. Electron microscopy of cells showed thickened deformed cell membrane, swollen mitochondria and enlarged endoplasmic reticulum (ER) in cells exposed to hypoxia. Distorted partially lost cell membrane with increased caveolae, enlarged mitochondria with degraded or lost cristae, splintered ER, and increased cytoplasmic lysosomes and multivesicular bodies were evident in oxidative stress. CONCLUSIONS: Human bladder smooth muscle cells are highly sensitive to hypoxic and oxidative stress conditions exhibiting specific biological and ultrastructural reactions. Bladder smooth muscle cell reactions to hypoxia are consistent with cell survival signaling to cope with lack of oxygen. Changes in oxidative stress correspond to extensive damage, cellular ultrastructural disintegration and deterioration of the subcellular elements. more...