Vivian Cristofaro

Bladder Tissue Regeneration Using Acellular Bi-Layer Silk Scaffolds in a Large Animal Model of Augmentation Cystoplasty

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.

Inhibition of TNF-α Improves the Bladder Dysfunction That Is Associated With Type 2 Diabetes

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.

Loss of bladder smooth muscle caveolae in the aging bladder

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.

Increased Smooth Muscle Contractility in Mice Deficient for Neuropilin 2

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.

The impact of discrete modes of spinal cord injury on bladder muscle contractility

BackgroundPrior studies have compared the effect of spinal cord injury elicited using distinct approaches on motor and visceral function. However, the impact of such discrete modes of injury specifically on bladder muscle contractility has not been explored in detail. The goal of this study is to compare the impact of complete spinal cord transection versus clip compression at thoracic vertebra eight (T8) on bladder muscle contractility.MethodsRats underwent no treatment (Control), laminectomy (Sham, SH); complete extradural transection (TX); or cord compression with an aneurysm clip (CX). Bladders and spinal cords were harvested at 6 wk for contractility studies or histological analysis.ResultsDetrusor strips from TX and CX rats showed higher spontaneous activity than those from SH rats. Furthermore, the duration of the neurally-mediated contractile response was longer in TX and CX rats compared to controls and showed attenuated relaxation. No significant differences were observed between muscle strips from SH, TX or CX rats in response to KCl, ATP or phenylephrine. However, tissues from TX and CX rats showed a higher sensitivity to carbachol compared to that from SH animals.ConclusionsComplete SCI in rats either by cord transection or compression elicits qualitatively similar changes in bladder muscle contractility. Whereas cord transection is arguably easier to perform experimentally, cord compression better models the situation observed clinically, such that each approach has clear advantages and limitations.

Myosin Va Plays a Role in Nitrergic Smooth Muscle Relaxation in Gastric Fundus and Corpora Cavernosa of Penis

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.

Altered Caveolar Mediated Purinergic Signaling in Spontaneously Hypertensive Rats with Detrusor Overactivity

PURPOSE 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. MATERIALS AND METHODS 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. RESULTS 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. CONCLUSIONS 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.

Inosine attenuates spontaneous activity in the rat neurogenic bladder through an A2B pathway

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.

Regional distribution and molecular interaction of caveolins in bladder smooth muscle.

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Bi-layer Silk Fibroin Grafts Support Functional Tissue Regeneration in a Porcine Model of Onlay Esophagoplasty

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