Can Urinary Bladder Innervation Be Restored After Outlet Obstruction and Denervation?

Transurethral resection of the prostate, or other methods to decrease outlet resistance usually leads to relief of symptoms in patients with bladder outlet obstruction (BOO). If symptoms of underactivity persist after normalization of outflow conditions, treatment options are limited. In this review, we hypothesize, based on results from basic research, what might become treatment options for such patients in the future. The primary local treatment will still aim at reducing outlet obstruction. We speculate that local secondary treatment in the future might include transplantation of stem cells or mature bladder ganglion cells into the bladder wall. There has been some success in transplanting ganglion cells into the rat bladder. The ganglion cells will sprout into the surrounding tissue but functional connections between the axons of the transplanted neurons, and the detrusor smooth muscle have so far not been demonstrated. Neurotrophins or neurotrimin might be injected into the bladder wall to increase the sprouting of existing or transplanted neurons. Stem cell transplantation has been performed and improves detrusor function, but it has so far, been difficult to demonstrate transplanted stem cells. BOO, persisting detrusor underactivity, and decreased nerve density are often combined with inflammatory activity of the lower urinary tract. NLR family pyrin domain containing 3 (NLRP3) and its messenger RNA (mRNA) as well as cyclooxygenase-2 (Cox-2) mRNA are increased in obstructed bladders. Systemic treatment with the NLRP3 inhibitor glyburide normalized nerve density in rat bladder, and, to some extent, bladder function. It is unclear whether Cox-2 is involved in the decreased nerve density following obstruction, but Cox-2 mRNA increases 5-fold in obstructed bladder. Future therapy against bladder underactivity remaining following relief of obstruction includes either systemic treatment, perhaps by anti-inflammatory drugs, or local treatment by injection of stem cells, mature ganglion cells, and/or neurotrophins or neurotrimin into the bladder wall.

Molecular and Morphological Characteristics of the De-Obstructed Rat Urinary Bladder-An Update.

Many patients with outlet obstruction secondary to prostatic enlargement have lower urinary tract symptoms (LUTSs) and an increased frequency of micturition. The standard treatment is transurethral resection of the prostate (TURP), which alleviates obstruction and symptoms. However, after TURP, 20-40 percent of patients continue to experience LUTSs. The aim of the present study in rats was to identify the mechanisms that do not normalize after the removal of the obstruction and that could explain the persisting symptoms. We had microarray data from control, obstructed, and de-obstructed female rat bladders, which made it possible to study 14,553 mRNA expressions. We also had a bank of electron micrographs from similar detrusors. Microarrays: There were significant differences between the control and obstructed bladders for 1111 mRNAs. The obstructed and de-obstructed bladders differed significantly for 1059 mRNAs. The controls and the de-obstructed bladders differed significantly for 798 mRNAs. We observed many mRNAs that were increased in the obstructed bladder and then decreased to control levels after de-obstruction, and many mRNAs that were decreased in the obstructed bladder and then increased following de-obstruction. mRNAs that were significantly higher or lower in the de-obstructed bladder than in the control bladder were also found. Ultrastructure: The detrusor cells in the obstructed bladders had cross-sectional areas that were much larger than those in the controls. The control cells had smooth outlines and similar cross-sectional areas. The de-obstructed detrusor cells had larger cross-sectional areas than the controls, as well as corrugated surfaces. The cell areas varied, suggesting that the shrinkage of the de-obstructed cells was not even. We did not find any points of contact of the gap junction plaque type between the detrusor cells. There were abundant finger-like processes between the detrusor cells in the obstructed and in de-obstructed bladders, which were only occasionally found in the control detrusors. They are the only possible localization for gap junction channels. The de-obstructed rat bladder is not an organ with properties intermediate between those of the control and obstructed bladders. Instead, de-obstructed bladders have gene expressions, morphologies, and functional properties of the individual cells and their organization, which make them distinctly different from both control and obstructed bladders.

Early history of skin preservation and transplantation; the role of Carl August Ljunggren.

During the late 19th and the early 20th century there was an unprecedented development in medical research. Tissue and cell culture rapidly developed into areas with many contributing scientists. The same is true for tissue transplantation. When these achievements are described afterwards in a historical context and a mainline development is constructed, there are researchers whose pioneering work is forgotten. The present paper attempts to correct this and to present a correct description of the start of tissue preservation and transplantation. We have traced relevant original publications in international journals between 1870 and 1920. The traditional view is that Alexis Carrel was the first He received a Nobel Prize 1912 for his work on vascular suture and the transplantation of blood vessels and organs. The same year he published an article on human skin storage and transplantation. This was more than a decade later than Carl August Ljunggren (1860-1934) who 1898 published his pioneering but long forgotten work on human skin preservation and transplantation, and with a vision of tissue banks. Our article contains a brief biography of Ljunggren, and further reconstructs the processes that resulted in the lack of awareness today of his achievements. Conclusion: Carl August Ljunggren was the first to preserve human skin in vitro for prolonged periods, followed by transplantation of the specimens to other patients. He was also the first to propose the use of tissue banks.

[Treatment of erectile dysfunction - from pill to implant]. / Behandling av erektil dysfunktion ­ från piller till implantat - Region Skåne har kapat kön till implantatkirurgi utan att urologisk cancerkirurgi påverkats.

The article gives an overview of erectile mechanisms and erectile dysfunction (ED). Current treatment of ED is presented. Most of the patients with ED should be treated by their primary care physician. Urologists should be involved only when treatment has failed, and when erectile implants might be an option. In Skåne the waiting list for these patients has been eliminated by using the operating capacity of a smaller hospital.

Antagonistic relationship between the unfolded protein response and myocardin-driven transcription in smooth muscle.

Smooth muscle cells (SMCs) are characterized by a high degree of phenotypic plasticity. Contractile differentiation is governed by myocardin-related transcription factors (MRTFs), in particular myocardin (MYOCD), and when their drive is lost, the cells become proliferative and synthetic with an expanded endoplasmic reticulum (ER). ER is responsible for assembly and folding of secreted proteins. When the load on the ER surpasses its capacity, three stress sensors (activating transcription factor 6 [ATF6], inositol-requiring enzyme 1α [IRE1α]/X-box binding protein 1 [XBP1], and PERK/ATF4) are activated to expand the ER and increase its folding capacity. This is referred to as the unfolded protein response (UPR). Here, we hypothesized that there is a reciprocal relationship between SMC differentiation and the UPR. Tight negative correlations between SMC markers (MYH11, MYOCD, KCNMB1, SYNPO2) and UPR markers (SDF2L1, CALR, MANF, PDIA4) were seen in microarray data sets from carotid arterial injury, partial bladder outlet obstruction, and bladder denervation, respectively. The UPR activators dithiothreitol (DTT) and tunicamycin (TN) activated the UPR and reduced MYOCD along with SMC markers in vitro. The IRE1α inhibitor 4µ8C counteracted the effect of DTT and TN on SMC markers and MYOCD expression. Transfection of active XBP1s was sufficient to reduce both MYOCD and the SMC markers. MRTFs also antagonized the UPR as indicated by reduced TN and DTT-mediated induction of CRELD2, MANF, PDIA4, and SDF2L1 following overexpression of MRTFs. The latter effect did not involve the newly identified MYOCD/SRF target MSRB3, or reduced production of either XBP1s or cleaved ATF6. The UPR thus counteracts SMC differentiation via the IRE1α/XBP1 arm of the UPR and MYOCD repression.

Identification of the intermediate filament protein synemin/SYNM as a target of myocardin family coactivators.

Myocardin (MYOCD) is a critical regulator of smooth muscle cell (SMC) differentiation, but its transcriptional targets remain to be exhaustively characterized, especially at the protein level. Here we leveraged human RNA and protein expression data to identify novel potential MYOCD targets. Using correlation analyses we found several targets that we could confirm at the protein level, including SORBS1, SLMAP, SYNM, and MCAM. We focused on SYNM, which encodes the intermediate filament protein synemin. SYNM rivalled smooth muscle myosin (MYH11) for SMC specificity and was controlled at the mRNA and protein levels by all myocardin-related transcription factors (MRTFs: MYOCD, MRTF-A/MKL1, and MRTF-B/MKL2). MRTF activity is regulated by the ratio of filamentous to globular actin, and SYNM was accordingly reduced by interventions that depolymerize actin, such as latrunculin treatment and overexpression of constitutively active cofilin. Many MRTF target genes depend on serum response factor (SRF), but SYNM lacked SRF-binding motifs in its proximal promoter, which was not directly regulated by MYOCD. Furthermore, SYNM resisted SRF silencing, yet the time course of induction closely paralleled that of the SRF-dependent target gene ACTA2. SYNM was repressed by the ternary complex factor (TCF) FLI1 and was increased in mouse embryonic fibroblasts lacking three classical TCFs (ELK1, ELK3, and ELK4). Imaging showed colocalization of SYNM with the intermediate filament proteins desmin and vimentin, and MRTF-A/MKL1 increased SYNM-containing intermediate filaments in SMCs. These studies identify SYNM as a novel SRF-independent target of myocardin that is abundantly expressed in all SMCs.

Scientific language trends among Swedish urologists and surgeons 1900-1955.

PURPOSE: Before English took the lead as the prime scientific language among northern European urologists and surgeons, German was widely regarded as the "lingua franca". This shift has to date not been systematically reconstructed. This article provides insights into the question how political and social factors influence how physicians communicate with each other, what they read, and how the constellations of international scientific communities in medicine change over time. METHODS: Through a language analysis of more than 2000 articles, including their references, in major Swedish medical journals as well as surgical doctoral dissertations defended at Swedish universities, this paper explores scientific language trends during the first half of the twentieth century among Swedish physicians for the first time on a large scale. RESULTS AND CONCLUSIONS: The study shows that Swedish urologists and surgeons generally did not switch to English during the years immediately after the First World War, as has been documented in other countries. After a decrease during the first 10 years after the First World War, the German language dominated among Swedish urologists and surgeons from the 1930s until the early 1940s, when English first dominated at large. The rapidity of this process shows that almost all surgical researchers had changed from German to English within just a few years.

Nexilin/NEXN controls actin polymerization in smooth muscle and is regulated by myocardin family coactivators and YAP.

Nexilin, encoded by the NEXN gene, is expressed in striated muscle and localizes to Z-discs, influencing mechanical stability. We examined Nexilin/NEXN in smooth muscle cells (SMCs), and addressed if Nexilin localizes to dense bodies and dense bands and whether it is regulated by actin-controlled coactivators from the MRTF (MYOCD, MKL1, MKL2) and YAP/TAZ (YAP1 and WWTR1) families. NEXN expression in SMCs was comparable to that in striated muscles. Immunofluorescence and immunoelectron microscopy suggested that Nexilin localizes to dense bodies and dense bands. Correlations at the mRNA level suggested that NEXN expression might be controlled by actin polymerization. Depolymerization of actin using Latrunculin B repressed the NEXN mRNA and protein in bladder and coronary artery SMCs. Overexpression and knockdown supported involvement of both YAP/TAZ and MRTFs in the transcriptional control of NEXN. YAP/TAZ and MRTFs appeared equally important in bladder SMCs, whereas MRTFs dominated in vascular SMCs. Expression of NEXN was moreover reduced in situations of SMC phenotypic modulation in vivo. The proximal promoter of NEXN conferred control by MRTF-A/MKL1 and MYOCD. NEXN silencing reduced actin polymerization and cell migration, as well as SMC marker expression. NEXN targeting by actin-controlled coactivators thus amplifies SMC differentiation through the actin cytoskeleton, probably via dense bodies and dense bands.

Array profiling reveals contribution of Cthrc1 to growth of the denervated rat urinary bladder.

Bladder denervation and bladder outlet obstruction are urological conditions that cause bladder growth. Transcriptomic surveys in outlet obstruction have identified differentially expressed genes, but similar studies following denervation have not been done. This was addressed using a rat model in which the pelvic ganglia were cryo-ablated followed by bladder microarray analyses. At 10 days following denervation, bladder weight had increased 5.6-fold, and 2,890 mRNAs and 135 micro-RNAs (miRNAs) were differentially expressed. Comparison with array data from obstructed bladders demonstrated overlap between the conditions, and 10% of mRNAs changed significantly and in the same direction. Many mRNAs, including collagen triple helix repeat containing 1 ( Cthrc1), Prc1, Plod2, and Dkk3, and miRNAs, such as miR-212 and miR-29, resided in the shared signature. Discordantly regulated transcripts in the two models were rare, making up for <0.07% of all changes, and the gene products in this category localized to the urothelium of normal bladders. These transcripts may potentially be used to diagnose sensory denervation. Western blotting demonstrated directionally consistent changes at the protein level, with increases of, e.g., Cthrc1, Prc1, Plod2, and Dkk3. We chose Cthrc1 for further studies and found that Cthrc1 was induced in the smooth muscle cell (SMC) layer following denervation. TGF-ß1 stimulation and miR-30d-5p inhibition increased Cthrc1 in bladder SMCs, and knockdown and overexpression of Cthrc1 reduced and increased SMC proliferation. This work defines common and distinguishing features of bladder denervation and obstruction and suggests a role for Cthrc1 in bladder growth following denervation.

Similar regulatory mechanisms of caveolins and cavins by myocardin family coactivators in arterial and bladder smooth muscle.

Caveolae are membrane invaginations present at high densities in muscle and fat. Recent work has demonstrated that myocardin family coactivators (MYOCD, MKL1), which are important for contractile differentiation and cell motility, increase caveolin (CAV1, CAV2, CAV3) and cavin (CAVIN1, CAVIN2, CAVIN3) transcription, but several aspects of this control mechanism remain to be investigated. Here, using promoter reporter assays we found that both MKL1/MRTF-A and MKL2/MRTF-B control caveolins and cavins via their proximal promoter sequences. Silencing of MKL1 and MKL2 in smooth muscle cells moreover reduced CAV1 and CAVIN1 mRNA levels by well over 50%, as did treatment with second generation inhibitors of MKL activity. GATA6, which modulates expression of smooth muscle-specific genes, reduced CAV1 and CAV2, whereas the cavins were unaffected or increased. Viral overexpression of MKL1 and myocardin induced caveolin and cavin expression in bladder smooth muscle cells from rats and humans and MYOCD correlated tightly with CAV1 and CAVIN1 in human bladder specimens. A recently described activator of MKL-driven transcription (ISX) failed to induce CAV1/CAVIN1 which may be due to an unusual transactivation mechanism. In all, these findings further support the view that myocardin family coactivators are important transcriptional drivers of caveolins and cavins in smooth muscle.

Neurite outgrowth in cultured mouse pelvic ganglia - Effects of neurotrophins and bladder tissue.

Neurotrophic factors regulate survival and growth of neurons. The urinary bladder is innervated via both sympathetic and parasympathetic neurons located in the major pelvic ganglion. The aim of the present study was to characterize the effects of the neurotrophins nerve growth factor (NGF), brain derived neurotrophic factor (BDNF) and neurotrophin 3 (NT-3) on the sprouting rate of sympathetic and parasympathetic neurites from the female mouse ganglion. The pelvic ganglion was dissected out and attached to a petri dish and cultured in vitro. All three factors (BDNF, NT-3 and NGF) stimulated neurite outgrowth of both sympathetic and parasympathetic neurites although BDNF and NT-3 had a higher stimulatory effect on parasympathetic ganglion cells. The neurotrophin receptors TrkA, TrkB and TrkC were all expressed in neurons of the ganglia. Co-culture of ganglia with urinary bladder tissue, but not diaphragm tissue, increased the sprouting rate of neurites. Active forms of BDNF and NT-3 were detected in urinary bladder tissue using western blotting whereas tissue from the diaphragm expressed NGF. Neurite outgrowth from the pelvic ganglion was inhibited by a TrkB receptor antagonist. We therefore suggest that the urinary bladder releases trophic factors, including BDNF and NT-3, which regulate neurite outgrowth via activation of neuronal Trk-receptors. These findings could influence future strategies for developing pharmaceuticals to improve re-innervation due to bladder pathologies.

Cavin-3 (PRKCDBP) deficiency reduces the density of caveolae in smooth muscle.

Cavins belong to a family of proteins that contribute to the formation of caveolae, which are membrane organelles with functional roles in muscle and fat. Here, we investigate the effect of cavin-3 ablation on vascular and urinary bladder structure and function. Arteries and urinary bladders from mice lacking cavin-3 (knockout: KO) and from controls (wild type: WT) were examined. Our studies revealed that the loss of cavin-3 resulted in ∼40% reduction of the caveolae protein cavin-1 in vascular and bladder smooth muscle. Electron microscopy demonstrated that the loss of cavin-3 was accompanied by a reduction of caveolae abundance by 40-45% in smooth muscle, whereas the density of caveolae in endothelial cells was unchanged. Vascular contraction in response to an α1-adrenergic agonist was normal but nitric-oxide-dependent relaxation was enhanced, in parallel with an increased relaxation on direct activation of soluble guanylyl cyclase (sGC). This was associated with an elevated expression of sGC, although blood pressure was similar in WT and KO mice. Contraction of the urinary bladder was not affected by the loss of cavin-3. The proteomic response to outlet obstruction, including STAT3 phosphorylation, the induction of synthetic markers and the repression of contractile markers were identical in WT and KO mice, the only exception being a curtailed induction of the Golgi protein GM130. Loss of cavin-3 thus reduces the number of caveolae in smooth muscle and partly destabilizes cavin-1 but the functional consequences are modest and include an elevated vascular sensitivity to nitric oxide and slightly disturbed Golgi homeostasis in situations of severe cellular stress.

Assessing the contribution of thrombospondin-4 induction and ATF6α activation to endoplasmic reticulum expansion and phenotypic modulation in bladder outlet obstruction.

Phenotypic modulation of smooth muscle cells is a hallmark of disease. The associated expansion of endoplasmic reticulum (ER) volume remains unexplained. Thrombospondin-4 was recently found to promote ATF6α activation leading to ER expansion. Using bladder outlet obstruction as a paradigm for phenotypic modulation, we tested if thrombospondin-4 is induced in association with ATF6α activation and ER expansion. Thrombospondin-4 was induced and ATF6α was activated after outlet obstruction in rodents. Increased abundance of spliced of Xbp1, another ER-stress sensor, and induction of Atf4 and Creb3l2 was also seen. Downstream of ATF6α, Calr, Manf, Sdf2l1 and Pdi increased as did ER size, whereas contractile markers were reduced. Overexpression of ATF6α, but not of thrombospondin-4, increased Calr, Manf, Sdf2l1 and Pdi and caused ER expansion, but the contractile markers were inert. Knockout of thrombospondin-4 neither affected bladder growth nor expression of ATF6α target genes, and repression of contractile markers was the same, even if ATF6α activation was curtailed. Increases of Xbp1s, Atf4 and Creb3l2 were similar. Our findings demonstrate reciprocal regulation of the unfolded protein response, including ATF6α activation and ER expansion, and reduced contractile differentiation in bladder outlet obstruction occurring independently of thrombospondin-4, which however is a sensitive indicator of obstruction.

MicroRNAs in Bladder Outlet Obstruction: Relationship to Growth and Matrix Remodelling.

The discovery of microRNAs (miRNAs), which are ~22 nucleotide RNAs that inhibit protein synthesis in a sequence-specific manner and are present in a range of species, has born hope of new therapeutic strategies. miRNAs play important roles in development and disease, but they remain poorly studied in uropathologies beyond cancer. Here, we discuss biological functions of miRNAs in the lower urogenital tract. A special focus is on miRNAs that change in bladder outlet obstruction (BOO). This is a condition that affects nearly one third of all men over 60 years and that involves growth and fibrosis of the urinary bladder. Animal models of BOO, such as that in rat, have been developed, and they feature a massive 6-fold bladder growth over 6 weeks. Using microarrays, we have charted the miRNAs that change during the time course of this process and identified several with important modulatory roles. We discuss known and predicted functions of miR-1, miR-29, miR-30, miR-132/212, miR-204 and miR-221, all of which change in BOO. The majority of the miRNA-mediated influences in BOO are expected to favour growth. We also outline evidence that miR-29 represents a key effector molecule in a generic response to mechanical distension that is designed to counteract exaggerated organ deformation via effects on matrix deposition and stiffness. We conclude that miRNAs play important roles in bladder remodelling and growth and that they may be targeted pharmacologically to combat diseases of the lower urinary tract.

Non-uniform changes in membrane receptors in the rat urinary bladder following outlet obstruction.

The aim of the present study was to investigate the expression and distribution of membrane receptors after bladder outlet obstruction (BOO). Partial bladder outlet obstruction (BOO) was induced in female rats and bladders were harvested after either 10 days or 6 weeks of BOO. The expression of different receptors was surveyed by microarrays and corroborated by immunohistochemistry and western blotting. A microarray experiment identified 10 membrane receptors that were differentially expressed compared to sham-operated rats including both upregulated and downregulated receptors. Four of these were selected for functional experiments on the basis of magnitude of change and relevance to bladder physiology. At 6 weeks of BOO, maximal contraction was reduced for neuromedin B and vasopressin (AVP), consistent with reductions of receptor mRNA levels. Glycine receptor-induced contraction on the other hand was increased and receptor mRNA expression was accordingly upregulated. Maximal relaxation by the ß3-adrenergic receptor agonist CL316243 was reduced as was the receptor mRNA level. Immunohistochemistry supported reduced expression of neuromedin B receptors, V1a receptors and ß3-adrenergic receptors, but glycine receptor expression appeared unchanged. Western blotting confirmed repression of V1a receptors and induction of glycine receptors in BOO. mRNA for vasopressin was detectable in the bladder, suggesting local AVP production. We conclude that changes in receptor expression following bladder outlet obstruction are non-uniform. Some receptors are upregulated, conferring increased responsiveness to agonist, whereas others are downregulated, leading to decreased agonist-induced responses. This study might help to select pharmacological agents that are effective in modulating lower urinary tract symptoms in BOO.

Vasopressin-induced mouse urethral contraction is modulated by caveolin-1.

Caveolae are 50-100nm large invaginations in the cell membrane that are considered to play roles in receptor signaling. Here we aimed to investigate the expression and distribution of the arginine-vasopressin (AVP) V1a receptor and its functional dependence on caveolin-1 (Cav1) in the mouse urethra. Female Cav1 knockout (KO) and wild type (WT) mice were used, and urethral preparations were micro-dissected for mechanical experiments. Methyl-ß-cyclodextrin (mßcd) was used to deplete cholesterol and to disrupt caveolae. Protein expression and localization was determined using immunofluorescence and western blotting and transcript expression was determined by qRT-PCR. We found that Cav1 and AVP V1a receptors were expressed in urethral smooth muscle cells with apparent co-localization at the cell membrane. AVP caused urethral contraction that was inhibited by the V1a receptor antagonist SR49059. Concentration-response curves for AVP were right-shifted and maximal contraction was reduced in Cav1 KO mice and after mßcd treatment. In addition to caveolin-1 we also detected caveolin-2, cavin-1 and cavin-3 in the mouse urethra by western blotting. Caveolin-2, cavin-1 and cavin-3 as well as V1a receptor expression was reduced in KO urethra. We conclude that AVP regulates urethral contractility via the V1a receptor through a Cav1-dependent mechanism involving, in part, altered V1a receptor expression.

Detrusor induction of miR-132/212 following bladder outlet obstruction: association with MeCP2 repression and cell viability.

The microRNAs (miRNAs) miR-132 and miR-212 have been found to regulate synaptic plasticity and cholinergic signaling and recent work has demonstrated roles outside of the CNS, including in smooth muscle. Here, we examined if miR-132 and miR-212 are induced in the urinary bladder following outlet obstruction and whether this correlates with effects on gene expression and cell growth. Three to seven-fold induction of miR-132/212 was found at 10 days of obstruction and this was selective for the detrusor layer. We cross-referenced putative binding sites in the miR-132/212 promoter with transcription factors that were predicted to be active in the obstruction model. This suggested involvement of Creb and Ahr in miR-132/212 induction. Creb phosphorylation (S-133) was not increased, but the number of Ahr positive nuclei increased. Moreover, we found that serum stimulation and protein kinase C activation induced miR-132/212 in human detrusor cells. To identify miR-132/212 targets, we correlated the mRNA levels of validated targets with the miRNA levels. Significant correlations between miR-132/212 and MeCP2, Ep300, Pnkd and Jarid1a were observed, and the protein levels of MeCP2, Pnkd and Ache were reduced after obstruction. Reduction of Ache however closely matched a 90% reduction of synapse density arguing that its repression was unrelated to miR-132/212 induction. Importantly, transfection of antimirs and mimics in cultured detrusor cells increased and decreased, respectively, the number of cells and led to changes in MeCP2 expression. In all, these findings show that obstruction of the urethra increases miR-132 and miR-212 in the detrusor and suggests that this influences gene expression and limits cell growth.