Mechanosensitivity Is a Characteristic Feature of Cultured Suburothelial Interstitial Cells of the Human Bladder.
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ABSTRACT:
SUBMITTER: Neuhaus J
PROVIDER: S-EPMC7432121 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundSuburothelial myofibroblasts (sMF) are located underneath the urothelium in close proximity to afferent nerves. They express purinergic receptors and show calcium transients in response to ATP. Therefore they are supposed to be involved in afferent signaling of the bladder fullness. Since ATP concentration is likely to be very low during the initial filling phase, we hypothesized that sMF Ca(2+) activity is affected even at very low ATP concentrations. We investigated ATP induced modulation of spontaneous activity, intracellular calcium response and purinergic signaling in cultured sMF.Methodology/principal findingsMyofibroblast cultures, established from cystectomies, were challenged by exogenous ATP in presence or absence of purinergic antagonist. Fura-2 calcium imaging was used to monitor ATP (10(-16) to 10(-4) mol/l) induced alterations of calcium activity. Purinergic receptors (P2X1, P2X2, P2X3) were analysed by confocal immunofluorescence. We found spontaneous calcium activity in 55.18% ± 1.65 of the sMF (N = 48 experiments). ATP significantly increased calcium activity even at 10(-16) mol/l. The calcium transients were partially attenuated by subtype selective antagonist (TNP-ATP, 1 µM; A-317491, 1 µM), and were mimicked by the P2X1, P2X3 selective agonist α,β-methylene ATP. The expression of purinergic receptor subtypes in sMF was confirmed by immunofluorescence.Conclusions/significanceOur experiments demonstrate for the first time that ATP can modulate spontaneous activity and induce intracellular Ca(2+) response in cultured sMF at very low concentrations, most likely involving P2X receptors. These findings support the notion that sMF are able to register bladder fullness very sensitively, which predestines them for the modulation of the afferent bladder signaling in normal and pathological conditions.
| S-EPMC3187810 | biostudies-literature
Project description:Enhancing cellular mechanosensitivity is recognized as a novel tool for successful musculoskeletal tissue engineering. We examined the hypothesis that mechanosensitivity of human mesenchymal stem cells (hMSCs) is enhanced on nanotopographic substrates relative to flat surfaces. hMSCs were cultured on polymer-demixed, randomly distributed nanoisland surfaces with varying island heights and changes in intracellular calcium concentration, [Ca(2+)](i), in response to fluid flow induced shear stress were quantifide. Stem cells cultured on specific scale nanotopographies displayed greater intracellular calcium responses to fluid flow. hMSCs cultured on 10-20nm high nanoislands displayed a greater percentage of cells responding in calcium relative to cells cultured on flat control, and showed greater average [Ca(2+)](i) increase relative to cells cultured on other nanoislands (45-80nm high nanoislands). As [Ca(2+)](i) is an important regulator of downstream signaling, as well as proliferation and differentiation of hMSCs, this observation suggests that specific scale nanotopographies provide an optimal milieu for promoting stem cell mechanotransduction activity. That mechanical signals and substrate nanotopography may synergistically regulate cell behavior is of significant interest in the development of regenerative medicine protocols.
| S-EPMC3614341 | biostudies-literature
Project description:AimsManaging bladder pressure in patients with neurogenic bladders is needed to improve rehabilitation options, avoid upper tract damage, incontinence, and their associated co-morbidities and mortality. Current methods of determining bladder contractions are not amenable to chronic or ambulatory settings. In this study we evaluated detection of bladder contractions using a novel piezoelectric catheter-free pressure sensor placed in a suburothelial bladder location in animals.MethodsWired prototypes of the pressure monitor were implanted into 2 nonsurvival (feline and canine) and one 13-day survival (canine) animal. Vesical pressures were obtained from the device in both suburothelial and intraluminal locations and simultaneously from a pressure sensing catheter in the bladder. Intravesical pressure was monitored in the survival animal over 10 days from the suburothelial location and necropsy was performed to assess migration and erosion.ResultsIn the nonsurvival animals, the average correlation between device and reference catheter data was high during both electrically stimulated bladder contractions and manual compressions (r = 0.93±0.03, r = 0.89±0.03). Measured pressures correlated strongly (r = 0.98±0.02) when the device was placed in the bladder lumen. The survival animal initially recorded physiologic data, but later this deteriorated. However, endstage intraluminal device recordings correlated (r = 0.85±0.13) with the pressure catheter. Significant erosion of the implant through the detrusor was found.ConclusionsThis study confirms correlation between suburothelial pressure readings and intravesical bladder pressures. Due to device erosion during ambulatory studies, a wireless implant is recommended for clinical rehabilitation applications.
| S-EPMC5218553 | biostudies-literature
Project description:BackgroundThe last decade identified cytokines as one group of major local cell signaling molecules related to bladder dysfunction like interstitial cystitis (IC) and overactive bladder syndrome (OAB). Gap junctional intercellular communication (GJIC) is essential for the coordination of normal bladder function and has been found to be altered in bladder dysfunction. Connexin (Cx) 43 and Cx45 are the most important gap junction proteins in bladder smooth muscle cells (hBSMC) and suburothelial myofibroblasts (hsMF). Modulation of connexin expression by cytokines has been demonstrated in various tissues. Therefore, we investigate the effect of interleukin (IL) 4, IL6, IL10, tumor necrosis factor-alpha (TNF?) and transforming growth factor-beta1 (TGF?1) on GJIC, and Cx43 and Cx45 expression in cultured human bladder smooth muscle cells (hBSMC) and human suburothelial myofibroblasts (hsMF).Methodology/principal findingsHBSMC and hsMF cultures were set up from bladder tissue of patients undergoing cystectomy. In cytokine stimulated cultured hBSMC and hsMF GJIC was analyzed via Fluorescence Recovery after Photo-bleaching (FRAP). Cx43 and Cx45 expression was assessed by quantitative PCR and confocal immunofluorescence. Membrane protein fraction of Cx43 and Cx45 was quantified by Dot Blot. Upregulation of cell-cell-communication was found after IL6 stimulation in both cell types. In hBSMC IL4 and TGF?1 decreased both, GJIC and Cx43 protein expression, while TNF? did not alter communication in FRAP-experiments but increased Cx43 expression. GJ plaques size correlated with coupling efficacy measured, while Cx45 expression did not correlate with modulation of GJIC.Conclusions/significanceOur finding of specific cytokine effects on GJIC support the notion that cytokines play a pivotal role for pathophysiology of OAB and IC. Interestingly, the effects were independent from the classical definition of pro- and antiinflammatory cytokines. We conclude, that connexin regulation involves genomic and/or post-translational events, and that GJIC in hBSMC and hsMF depend of Cx43 rather than on Cx45.
| S-EPMC3107230 | biostudies-literature
Project description:We evaluated the effect of serum-free and xeno-cultivation (SFXFM) on the characterization, proliferation, and differentiation properties of human nasal stem cells (airway tissue; hTMSCs). hTMSCs were isolated from 10 patients, after which patient samples were separated into two groups, an SFXFM group and a control group. The control group was treated with bovine serum-containing medium. FACS analysis revealed that SFXFM-cultured hTMSCs maintained a characteristic mesenchymal stem cell phenotype. hTMSC proliferation was not influenced by SFXFM. In addition, upregulation of IL-8 and GM-CSF and downregulation of RANTES expression were shown in response to SFXFM. Moreover, two-lineage differentiation properties (osteocyte and adipocyte) of hTMSCs were enhanced under SFXFM. Finally, the genetic stability of SFXFM-cultured hTMSCs was demonstrated by normal karyotype results. SFXFM enables good expansion, multipotentiality, and normal genotype maintenance of MSCs. Moreover, this approach serves as a substitute to conventional media for the cultivation of capable MSCs for upcoming medical applications.
| S-EPMC5648157 | biostudies-literature
Project description:Background and purposeIn suburothelial venules of rat bladder, pericytes (perivascular cells) develop spontaneous Ca(2+) transients, which may drive the smooth muscle wall to generate spontaneous venular constrictions. We aimed to further explore the morphological and functional characteristics of pericytes in the mouse bladder.Experimental approachThe morphological features of pericytes were investigated by electron microscopy and fluorescence immunohistochemistry. Changes in diameters of suburothelial venules were measured using video microscopy, while intracellular Ca(2+) dynamics were visualized using Fluo-4 fluorescence Ca(2+) imaging.Key resultsA network of α-smooth muscle actin immunoreactive pericytes surrounded venules in the mouse bladder suburothelium. Scanning electron microscopy revealed that this network of stellate-shaped pericytes covered the venules, while transmission electron microscopy demonstrated that the venular wall consisted of endothelium and adjacent pericytes, lacking an intermediate smooth muscle layer. Pericytes exhibited spontaneous Ca(2+) transients, which were accompanied by phasic venular constrictions. Nicardipine (1 μM) disrupted the synchrony of spontaneous Ca(2+) transients in pericytes and reduced their associated constrictions. Residual asynchronous Ca(2+) transients were suppressed by cyclopiazonic acid (10 μM), 2-aminoethoxydiphenyl borate (10 μM), U-73122 (1 μM), oligomycin (1 μM) and SKF96365 (10 μM), but unaffected by ryanodine (100 μM) or YM-244769 (1 μM), suggesting that pericyte Ca(2+) transients rely on Ca(2+) release from the endoplasmic reticulum via the InsP(3) receptor and also require Ca(2+) influx through store-operated Ca(2+) channels.Conclusions and implicationsThe pericytes in mouse bladder can generate spontaneous Ca(2+) transients and contractions, and thus have a fundamental role in promoting spontaneous constrictions of suburothelial venules.
| S-EPMC3525874 | biostudies-literature
Project description:The upper (suburothelial) lamina propria (ULP) is a distinct region in the human bladder with dense populations of interstitial cells (IC), fine vascular networks and variable development of muscularis mucosae (MM). It is more and more obvious that the ULP plays an important role in bladder physiology and bladder disease, and in the present study we have quantified changes in the cellular key players of the ULP in bladders from patients with carcinoma in situ (CIS), multiple sclerosis (MS) and bladder pain syndrome (BPS). Tissue samples for the different patient groups were obtained from radical cystectomy-specimens. Standardized immunohistochemistry with a panel of specific cell markers was used to characterise the ULP cellular structures, followed by digitalised morphometry and quantitative staining analysis. Alterations in the ULP area were most pronounced in MS bladders, but also present in BPS and CIS bladders. We observed an increased thickness and increased variability in thickness of the ULP IC area in MS and BPS bladders; a significantly increased development of MM in MS bladders; a changed organization of vascular plexuses in the lamina propria in most pathologic bladders and a changed phenotype of ULP IC: a significantly decreased expression of progesterone receptor in MS bladders and a trend towards decreased expression of alpha-smooth muscle actin in BPS bladders. We show here for the first time the presence of disease-specific changes in organisation and/or phenotype of the different key players of the ULP area in human bladder. The present findings further support the hypothesis that the ULP area is involved and altered in different bladder diseases.
| S-EPMC4431865 | biostudies-literature
Project description:Oligodendrocytes produce and repair myelin, which is critical for the integrity and function of the central nervous system (CNS). Oligodendrocyte and oligodendrocyte progenitor cell (OPC) biology is modulated in vitro by mechanical cues within the magnitudes observed in vivo. In some cases, these cues are sufficient to accelerate or inhibit terminal differentiation of murine oligodendrocyte progenitors. However, our understanding of oligodendrocyte lineage mechanobiology has been restricted primarily to animal models to date, due to the inaccessibility and challenges of human oligodendrocyte cell culture. Here, we probe the mechanosensitivity of human oligodendrocyte lineage cells derived from human induced pluripotent stem cells. We target phenotypically distinct stages of the human oligodendrocyte lineage and quantify the effect of substratum stiffness on cell migration and differentiation, within the range documented in vivo. We find that human oligodendrocyte lineage cells exhibit mechanosensitive migration and differentiation. Further, we identify two patterns of human donor line-dependent mechanosensitive differentiation. Our findings illustrate the variation among human oligodendrocyte responses, otherwise not captured by animal models, that are important for translational research. Moreover, these findings highlight the importance of studying glia under conditions that better approximate in vivo mechanical cues. Despite significant progress in human oligodendrocyte derivation methodology, the extended duration, low yield, and low selectivity of human-induced pluripotent stem cell-derived oligodendrocyte protocols significantly limit the scale-up and implementation of these cells and protocols for in vivo and in vitro applications. We propose that mechanical modulation, in combination with traditional soluble and insoluble factors, provides a key avenue to address these challenges in cell production and in vitro analysis.
| S-EPMC7420028 | biostudies-literature
Project description:BackgroundIncidence of urinary tract infections is elevated in patients with diabetes mellitus. Those patients show increased levels of the saturated free fatty acid palmitate. As recently shown metabolic alterations induced by palmitate include production and secretion of the pro-inflammatory cytokine interleukine-6 (IL-6) in cultured human bladder smooth muscle cells (hBSMC). Here we studied the influence of palmitate on vital cell properties, for example, regulation of cell proliferation, mitochondrial enzyme activity and antioxidant capacity in hBSMC, and analyzed the involvement of major cytokine signaling pathways.Methodology/principal findingsHBSMC cultures were set up from bladder tissue of patients undergoing cystectomy and stimulated with palmitate. We analyzed cell proliferation, mitochondrial enzyme activity, and antioxidant capacity by ELISA and confocal immunofluorescence. In signal transduction inhibition experiments we evaluated the involvement of NF-?B, JAK/STAT, MEK1, PI3K, and JNK in major cytokine signaling pathway regulation. We found: (i) palmitate decreased cell proliferation, increased mitochondrial enzyme activity and antioxidant capacity; (ii) direct inhibition of cytokine receptor by AG490 even more strongly suppressed cell proliferation in palmitate-stimulated cells, while counteracting palmitate-induced increase of antioxidant capacity; (iii) in contrast knockdown of the STAT3 inhibitor SOCS3 increased cell proliferation and antioxidant capacity; (iv) further downstream JAK/STAT3 signaling cascade the inhibition of PI3K or JNK enhanced palmitate induced suppression of cell proliferation; (v) increase of mitochondrial enzyme activity by palmitate was enhanced by inhibition of PI3K but counteracted by inhibition of MEK1.Conclusions/significanceSaturated free fatty acids (e.g., palmitate) cause massive alterations in vital cell functions of cultured hBSMC involving distinct major cytokine signaling pathways. Thereby, certain cytokines might counteract the palmitate-induced downregulation of cell proliferation and vitality. This could be an important link to clinical findings of increased risk of metabolic related bladder diseases such as overactive bladder (OAB) and bladder pain syndrome/interstitial cystitis (BPS/IC).
| S-EPMC3396599 | biostudies-literature
Project description:Stress is associated with exacerbated symptoms in patients with interstitial cystitis/bladder pain syndrome (IC/BPS). To investigate the mechanism of stress implicated on IC/BPS, we investigated expression of stress-response receptor corticotropin-releasing hormone receptor (CRHR) in bladder from IC/BPS patients. Twenty-three IC/BPS patients with Hunner's lesion (HIC), 51 IC/BPS patients without Hunner's lesion (NHIC), and 24 patients with stress urinary incontinence as controls were enrolled. Cystoscopic biopsies of bladder wall including mucosa and submucosa were obtained from all patients. Western blotting was used to investigate the bladder expression of the CRHR1 and CRHR2. Immunochemical staining revealed CRHR1 expression was mainly located in the submucosa while CRHR2 expression was mainly in uroepithelial cells. Compared to control subjects, the CRHR1 expression was significantly higher, while CRHR2 expression was significantly lower in IC/BPS patients. Further analysis of patients with HIC, NHIC, and control subjects showed that bladder in patients with HIC had significantly higher expressions of CRHR1 and significantly lower CRHR2. CRHR2 expression was significantly negatively correlated with O'Leary-Sant score and bladder pain. Our results indicate dysregulation of bladder CRHR1 and CRHR2 in patients with IC/BPS, and suggest CRH signaling may be associated with IC/BPS symptoms.
| S-EPMC6915757 | biostudies-literature