transforming-growth-factor-beta and Cystitis

To investigate the mechanism of bladder nerve hyperplasia and fibrosis in the patients with ketamine-associated cystitis (KC).. Sixteen patients with severe KC, six patients with mild KC, and five patients with localized invasive bladder cancer served as control patients. Bladder mucosa specimens were taken during the operations, and the specimens were stained for nerve growth factor (NGF) and S-100 to evaluated nerve hyperplasia. The quantitative Western blot analysis was performed for NGF, brain-derived neurotrophic factor (BDNF), growth-associated protein 43 (GAP-43), tropomyosin receptor kinase A and B (TrkA and TrkB), transforming growth factor-β (TGF-β), phosphorylated extracellular signal-regulated kinases (p-ERK), protein kinase B (p-Akt), and glycogen synthase kinase 3β (p-GSK-3β).. The results demonstrated diffuse NGF expression in KC bladder epithelium, lamina propria, and muscle. The GAP-43, NGF, BDNF, TrkA, TGF-β, p-ERK, P-AKT, and p-GSK-3β expression in the bladder mucosa specimens of patients with severe KC was significantly higher than in patients with mild KC and control patients. Expression of neurotrophins was significantly correlated with bladder capacity and pain. NGF and BDNF expression were significantly higher in the KC bladder specimens with strongly positive S-100 staining. TGF-β expression in the bladder specimens was significantly correlated with neurotrophins, p-ERK, P-AKT, and p-GSK-3β levels.. Our findings indicate upregulation of neurotrophins, TGF-β, and activation of the cell proliferation kinases plays an important role in nerve hyperplasia and fibrosis mechanisms in severe KC bladders. The neurotrophins and TGF-β interact as cause and effect, leading to bladder hypersensitivity and fibrosis in severe KC.

Topics: Adult; Aged; Brain-Derived Neurotrophic Factor; Cystitis; Excitatory Amino Acid Antagonists; Female; Fibrosis; Humans; Hyperplasia; Ketamine; Male; Middle Aged; Mucous Membrane; Nerve Growth Factors; Transforming Growth Factor beta; Up-Regulation; Urinary Bladder; Urinary Bladder Neoplasms; Young Adult

Partial bladder outlet obstruction is a multifactorial urological condition in which hypoxia plays a significant role. We recently investigated hypoxia's role as a single stressor and found that hypoxia induced an intense inflammatory and profibrotic switch in bladder smooth muscle cells (bSMCs). With the immunomodulatory capacity of mesenchymal stem cells (MSCs), we aimed to investigate if the hypoxia-signaling pathways can be mitigated using MSCs.. Bladder smooth muscle cells were cultured in 3% oxygen tension for 72 h with either the direct or indirect co-culture with bone marrow derived MSCs. High pore density transwells were used for indirect co-cultures. Total RNA was extracted for gene expression analysis and the Mesoscale multiplex assay was used for secreted cytokines and growth factor measurements. Total collagen contents were determined using the Sirius Red collagen assay.. Hypoxia induced increase of HIF3α, VEGF, TGFβ1, TNFα, IL-1β, IL-6, αSMA, and total collagen expression and decreased IL-10 levels in bSMCs. Both direct and indirect MSCs co-cultures inhibited > 50% of hypoxia-induced TGFβ1 and IL-6 expression (p < 0.005) in a HIF-independent manner. Also, both MSCs co-culture techniques induced > 200% increase in IL-10 protein (p < 0.005) and inhibited hypoxia-induced αSMA, collagen I and III transcripts as well as total collagen proteins (p < 0.0001). Contrastingly, the hypoxia-induced IL-1β and TNFα were inhibited by only the direct co-cultures (p < 0.05).. MSCs co-culture with bSMCs potently mitigates hypoxia-induced inflammatory and profibrotic pathways. This work has elucidated the role of cell-cell contact and paracrine immunomodulatory mechanisms of MSCs action and opened avenues for therapeutic intervention.

Topics: Actins; Apoptosis Regulatory Proteins; Basic Helix-Loop-Helix Transcription Factors; Cell Communication; Cell Hypoxia; Cells, Cultured; Collagen; Cystitis; Extracellular Matrix Proteins; Fibrosis; Humans; Interleukin-1beta; Interleukin-6; Mesenchymal Stem Cells; Myocytes, Smooth Muscle; Repressor Proteins; RNA; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Urinary Bladder; Urinary Bladder Neck Obstruction; Vascular Endothelial Growth Factor A

The sensory components of the urinary bladder are responsible for the transduction of bladder filling and are often impaired with neurological injury or disease. Elevated extracellular ATP contributes, in part, to bladder afferent nerve hyperexcitability during urinary bladder inflammation or irritation. Transforming growth factor-β1 (TGF-β1) may stimulate ATP release from the urothelium through vesicular exocytosis mechanisms with minimal contribution from pannexin-1 channels to increase bladder afferent nerve discharge. Bladder afferent nerve hyperexcitability and urothelial ATP release with CYP-induced cystitis is decreased with TGF-β inhibition. These results establish a causal link between an inflammatory mediator, TGF-β, and intrinsic signalling mechanisms of the urothelium that may contribute to the altered sensory processing of bladder filling.. The afferent limb of the micturition reflex is often compromised following bladder injury, disease and inflammatory conditions. We have previously demonstrated that transforming growth factor-β (TGF-β) signalling contributes to increased voiding frequency and decreased bladder capacity with cystitis. Despite the functional presence of TGF-β in bladder inflammation, the precise mechanisms of TGF-β mediating bladder dysfunction are not yet known. Thus, the present studies investigated the sensory components of the urinary bladder that may underlie the pathophysiology of aberrant TGF-β activation. We utilized bladder-pelvic nerve preparations to characterize bladder afferent nerve discharge and the mechanisms of urothelial ATP release with distention. Our findings indicate that bladder afferent nerve discharge is sensitive to elevated extracellular ATP during pathological conditions of urinary bladder inflammation or irritation. We determined that TGF-β1 may increase bladder afferent nerve excitability by stimulating ATP release from the urothelium via vesicular exocytosis mechanisms with minimal contribution from pannexin-1 channels. Furthermore, blocking aberrant TGF-β signalling in cyclophosphamide-induced cystitis with TβR-1 inhibition decreased afferent nerve hyperexcitability with a concomitant decrease in urothelial ATP release. Taken together, these results establish a role for purinergic signalling mechanisms in TGF-β-mediated bladder afferent nerve activation that may ultimately facilitate increased voiding frequency. The synergy between intrinsic urinary bladder signalling mechanisms and an inflammatory mediator provides novel insight into bladder dysfunction and supports new avenues for therapeutic intervention.

Topics: Adenosine Triphosphate; Animals; Connexins; Cyclophosphamide; Cystitis; Male; Mice, Inbred C57BL; Nerve Tissue Proteins; Protein Serine-Threonine Kinases; Purinergic Antagonists; Pyridoxal Phosphate; Receptor, Transforming Growth Factor-beta Type I; Receptors, Purinergic; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta; Urinary Bladder; Urothelium

To investigate the progression of urodynamic changes, as well as histological and biochemical outcomes over a prolonged period of partial bladder outlet obstruction (pBOO) in an animal model with physiologically relevant pBOO.. Healthy, adult, female Fischer rats underwent surgical creation of a pBOO for either 2, 4, 8, or 13 weeks and were compared with sham-operated rats. Urodynamic measurements were used to compare bladder volumes and pressure. Tissue was grossly analysed with light microscopy and bladder weights and thicknesses were compared. Reverse transcription-polymerase chain reaction for collagen, transforming growth factor β (TGF-β), connective tissue growth factor (CTGF), hypoxia inducible factor 1α (HIF-1α), and platelet-derived growth factor (PDGF-A) was performed on all samples, as well as immunohistochemistry (IHC) for α-smooth muscle actin (α-SMA). Finally, mass spectrometry was used to quantify the collagen content of the bladders as a measure of fibrosis.. After induction of pBOO, all rats remained healthy. Initial urodynamics showed an increase in capacity while maintaining normal pressures, but then deteriorated into small capacity, high-pressure bladders. Haematoxylin and eosin (H&E) staining showed an initial inflammatory response, and this was confirmed with significantly increased mRNA levels of TGF-β, CTGF, HIF-1α, and PDGF. The progression to smooth muscle hypertrophy was evident on H&E and confirmed with increased bladder mass and thickness. IHC for α-SMA showed a progressive increase associated with the elevated bladder pressures. Masson's trichrome and mass spectrometry showed a progressive increase in collagen to 13 weeks.. With this model, we have effectively replicated the clinical scenario, with significant pathophysiological changes occurring insidiously in otherwise healthy rats. We believe that our observed changes represent distinct phases of bladder decompensation; with an initial inflammatory response to the stress of the pBOO, smooth muscle hypertrophy to overcome the increased urethral resistance, and eventual decompensation to fibrosis. The time course of the inflammatory markers implies the need for early intervention to prevent this cascade. Novel strategies targeting these observed physiological responses could lead to improved preventative strategies, with respect to biochemical pathways and the time course of their initiation.

Topics: Animals; Collagen; Connective Tissue Growth Factor; Cystitis; Disease Models, Animal; Disease Progression; Female; Fibrosis; Hypoxia-Inducible Factor 1, alpha Subunit; Immunohistochemistry; Muscle, Smooth; Platelet-Derived Growth Factor; Rats; Rats, Inbred F344; Reverse Transcriptase Polymerase Chain Reaction; Transforming Growth Factor beta; Urinary Bladder; Urinary Bladder Neck Obstruction; Urodynamics

Stimulation of sensory nerves can lead to release of peptides such as substance P (SP) and consequently to neurogenic inflammation. We studied the role of bacterial lipopolysaccharide (LPS) in regulating SP-induced inflammation. Experimental cystitis was induced in female mice by intravesical instillation of SP, LPS, or fluorescein-labeled LPS. Uptake of fluorescein-labeled LPS was determined by confocal analysis, and bladder inflammation was determined by morphological analysis. SP was infused into the bladders of some mice 24 h after exposure to LPS. In vitro studies determined the capacity of LPS and SP to induce histamine and cytokine release by the bladder. LPS was taken up by urothelial cells and distributed systemically. Twenty-four hours after instillation of LPS or SP, bladder inflammation was characterized by edema and leukocytic infiltration of the bladder wall. LPS pretreatment enhanced neutrophil infiltration induced by SP, increased in vitro release of histamine, tumor necrosis factor-alpha, and interferon-gamma, and significantly reduced transforming growth factor-beta1 release. These findings suggest that LPS amplifies neurogenic inflammation, thereby playing a role in the pathogenesis of neurogenic cystitis.

Topics: Administration, Intravesical; Animals; Contrast Media; Cystitis; Disease Models, Animal; Drug Synergism; Female; Fluorescein; Histamine; Interferon-gamma; Lipopolysaccharides; Mice; Mice, Inbred BALB C; Neurons, Afferent; Neutrophils; Substance P; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Necrosis Factor-alpha; Urinary Bladder; Urothelium