Differential Post-Translational Modifications of Proteins in Bladder Ischemia.

Clinical and basic research suggests that bladder ischemia may be an independent variable in the development of lower urinary tract symptoms (LUTS). We have reported that ischemic changes in the bladder involve differential expression and post-translational modifications (PTMs) of the protein's functional domains. In the present study, we performed in-depth analysis of a previously reported proteomic dataset to further characterize proteins PTMs in bladder ischemia. Our proteomic analysis of proteins in bladder ischemia detected differential formation of non-coded amino acids (ncAAs) that might have resulted from PTMs. In-depth analysis revealed that three groups of proteins in the bladder proteome, including contractile proteins and their associated proteins, stress response proteins, and cell signaling-related proteins, are conspicuously impacted by ischemia. Differential PTMs of proteins by ischemia seemed to affect important signaling pathways in the bladder and provoke critical changes in the post-translational structural integrity of the stress response, contractile, and cell signaling-related proteins. Our data suggest that differential PTMs of proteins may play a role in the development of cellular stress, sensitization of smooth muscle cells to contractile stimuli, and deferential cell signaling in bladder ischemia. These observations may provide the foundation for future research to validate and define clinical translation of the modified biomarkers for precise diagnosis of bladder dysfunction and the development of new therapeutic targets against LUTS.

Molecular Regulation of Concomitant Lower Urinary Tract Symptoms and Erectile Dysfunction in Pelvic Ischemia.

Aging correlates with greater incidence of lower urinary tract symptoms (LUTS) and erectile dysfunction (ED) in the male population where the pathophysiological link remains elusive. The incidence of LUTS and ED correlates with the prevalence of vascular risk factors, implying potential role of arterial disorders in concomitant development of the two conditions. Human studies have revealed lower bladder and prostate blood flow in patients with LUTS suggesting that the severity of LUTS and ED correlates with the severity of vascular disorders. A close link between increased prostatic vascular resistance and greater incidence of LUTS and ED has been documented. Experimental models of atherosclerosis-induced chronic pelvic ischemia (CPI) showed increased contractile reactivity of prostatic and bladder tissues, impairment of penile erectile tissue relaxation, and simultaneous development of detrusor overactivity and ED. In the bladder, short-term ischemia caused overactive contractions while prolonged ischemia provoked degenerative responses and led to underactivity. CPI compromised structural integrity of the bladder, prostatic, and penile erectile tissues. Downstream molecular mechanisms appear to involve cellular stress and survival signaling, receptor modifications, upregulation of cytokines, and impairment of the nitric oxide pathway in cavernosal tissue. These observations may suggest pelvic ischemia as an important contributing factor in LUTS-associated ED. The aim of this narrative review is to discuss the current evidence on CPI as a possible etiologic mechanism underlying LUTS-associated ED.

Formation of Double Stranded RNA Provokes Smooth Muscle Contractions and Structural Modifications in Bladder Ischemia.

Purpose: Growing evidence suggests that ischemia provokes detrusor overactivity and degenerative responses in the bladder. Underlying mechanisms appear to involve modification of smooth muscle contractile rudiments by hypoxia, redox, cellular stress and cell survival signaling. Downstream pathways of cellular stress and stress response molecules eliciting bladder dysfunction in ischemia remain largely elusive. Our goal was to define the role of double stranded RNA (dsRNA), a stress response molecule provoked by redox, in ischemia mediated bladder dysfunction. Methods: A rat model of pelvic ischemia along with a cell culture hypoxia model were used to investigate the expression levels, functional consequences, structural aspects, and regulatory mechanisms of dsRNA in the bladder. Gene and protein expression were examined by reverse transcription polymerase chain reaction (RT-PCR), dot blot, and Western blotting, respectively. Tissue structure and function were assessed using histological staining and organ bath. Regulatory mechanisms were analyzed in cultured bladder smooth muscle cells. Results: The data presented here provide the first evidence of the formation of dsRNA in the overactive bladder. dsRNA is a cellular stress response molecule that sensitizes smooth muscle and regulates inflammatory and degenerative rejoinders. Our data suggest that the production of dsRNA in the bladder is provoked by ischemia. Formation of dsRNA appears to augment bladder smooth muscle contractions and provoke fibrotic and apoptotic responses. Downstream actions of dsRNA in the bladder may involve upregulation of dsRNA-activated protein kinase R (PKR) and caspase-3, the executioner of apoptosis. Conclusion: Activation of dsRNA/PKR pathway may play a role in sensitization of bladder smooth muscle cells to contractile stimuli, whereas dsRNA and caspase-3 crosstalk appear to modulate cellular stress and instigate degenerative responses in bladder ischemia. These observations suggest the role of dsRNA in bladder dysfunction and may open new perspectives to overcome overactive smooth muscle contractions and structural damage in the bladder.

Cellular Stress and Molecular Responses in Bladder Ischemia.

The concept of bladder ischemia as a contributing factor to detrusor overactivity and lower urinary tract symptoms (LUTS) is evolving. Bladder ischemia as a consequence of pelvic arterial atherosclerosis was first documented in experimental models and later in elderly patients with LUTS. It was shown that early-stage moderate ischemia produces detrusor overactivity, while prolonged severe ischemia provokes changes consistent with detrusor underactivity. Recent studies imply a central role of cellular energy sensors, cellular stress sensors, and stress response molecules in bladder responses to ischemia. The cellular energy sensor adenosine monophosphate-activated protein kinase was shown to play a role in detrusor overactivity and neurodegeneration in bladder ischemia. The cellular stress sensors apoptosis signal-regulating kinase 1 and caspase-3 along with heat shock proteins were characterized as important contributing factors to smooth muscle structural modifications and apoptotic responses in bladder ischemia. Downstream pathways seem to involve hypoxia-inducible factor, transforming growth factor beta, vascular endothelial growth factor, and nerve growth factor. Molecular responses to bladder ischemia were associated with differential protein expression, the accumulation of non-coded amino acids, and post-translational modifications of contractile proteins and stress response molecules. Further insight into cellular stress responses in bladder ischemia may provide novel diagnostic and therapeutic targets against LUTS.

Impairment of AMPK-α2 augments detrusor contractions in bladder ischemia.

PURPOSE: Ischemia disrupts cellular energy homeostasis. Adenosine monophosphate-activated protein kinase alpha-2 (AMPK-α2) is a subunit of AMPK that senses cellular energy deprivation and signals metabolic stress. Our goal was to examine the expression levels and functional role of AMPK-α2 in bladder ischemia. MATERIALS AND METHODS: Iliac artery atherosclerosis and bladder ischemia were engendered in apolipoprotein E knockout rats by partial arterial endothelial denudation using a balloon catheter. After eight weeks, total and phosphorylated AMPK-α2 expression was analyzed by western blotting. Structural integrity of AMPK-α2 protein was assessed by Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS). Functional role of AMPK-α2 was examined by treating animals with the AMPK activator 5-aminoimidazole-4-carboxamide-1-beta-D ribofuranoside (AICAR). Tissue contractility was measured in the organ bath and bladder nerve density was examined by immunostaining. RESULTS: Total AMPK-α2 expression increased in bladder ischemia, while phosphorylated AMPK-α2 was significantly downregulated. LC-MS/MS suggested post-translational modification of AMPK-α2 functional domains including phosphorylation sites, suggesting accumulation of catalytically inactive AMPK-α2 in bladder ischemia. Treatment of rats with AICAR diminished the force of overactive detrusor contractions and increased bladder capacity but did not have a significant effect on the frequency of bladder contractions. AICAR diminished contractile reactivity of ischemic tissues in the organ bath and prevented loss of nerve fibers in bladder ischemia. CONCLUSIONS: Ischemia induces post-translational modification of AMPK-α2 protein. Impairment of AMPK-α2 may contribute to overactive detrusor contractions and loss of nerve fibers in bladder ischemia. AMPK activators may have therapeutic potential against detrusor overactivity and neurodegeneration in bladder conditions involving ischemia.

Post-Translational Modification Networks of Contractile and Cellular Stress Response Proteins in Bladder Ischemia.

Molecular mechanisms underlying bladder dysfunction in ischemia, particularly at the protein and protein modification levels and downstream pathways, remain largely unknown. Here we describe a comparison of protein sequence variations in the ischemic and normal bladder tissues by measuring the mass differences of the coding amino acids and actual residues crossing the proteome. A large number of nonzero delta masses (11,056) were detected, spanning over 1295 protein residues. Clustering analysis identified 12 delta mass clusters that were significantly dysregulated, involving 30 upregulated (R2 > 0.5, ratio > 2, p < 0.05) and 33 downregulated (R2 > 0.5, ratio < -2, p < 0.05) proteins in bladder ischemia. These protein residues had different mass weights from those of the standard coding amino acids, suggesting the formation of non-coded amino acid (ncAA) residues in bladder ischemia. Pathway, gene ontology, and protein-protein interaction network analyses of these ischemia-associated delta-mass containing proteins indicated that ischemia provoked several amino acid variations, potentially post-translational modifications, in the contractile proteins and stress response molecules in the bladder. Accumulation of ncAAs may be a novel biomarker of smooth muscle dysfunction, with diagnostic potential for bladder dysfunction. Our data suggest that systematic assessment of global protein modifications may be crucial to the characterization of ischemic conditions in general and the pathomechanism of bladder dysfunction in ischemia.

Post-Translational Modification of Human Histone by Wide Tolerance of Acetylation.

Histone acetylation adds an acetyl group on the lysine residue commonly found within the N-terminal tail protruding from the histone core of the nucleosome, and is important for chromosome structure and function in gene transcription and chromatin remodeling. Acetylation may also occur on other residues additional to lysine, but have not been thoroughly investigated at the proteomics level. Here we report a wide tolerance acetylation study mimicking the addition of 42 ± 0.5 Da delta mass modification on undefined amino acid residues of histones by shotgun proteomics using liquid chromatography-tandem mass spectrometry. A multi-blind spectral alignment algorithm with a wide peptide tolerance revealed frequent occurrence of 42 ± 0.5 Da modifications at lysine (K), serine (S) and threonine (T) residues in human histones from kidney tissues. Precision delta mass analysis identified acetylation (42.011 ± 0.004 Da) and trimethylation (42.047 ± 0.002 Da) modifications within the delta mass range. A specific antibody was produced to validate the acetylated T22 of human histone H3 (H3T22ac) by immune assays. Thus, we demonstrated that the wide tolerance acetylation approach identified histone acetylation as well as modification variants commonly associated with acetylation at undefined residues additional to lysine.

Mitochondrial stress and activation of PI3K and Akt survival pathway in bladder ischemia.

PURPOSE: Detrusor overactivity contributes to bothersome constellation of lower urinary tract symptoms (LUTS) in men and women as they age. However, the underlying mechanisms of non-obstructive detrusor overactivity and LUTS remain largely unknown. Growing evidence suggests that ischemia may be an independent factor in the development of non-obstructive bladder dysfunction. Our goal was to determine the effects of ischemia on detrusor function and voiding behavior and define redox-mediated cellular stress and cell survival signaling in the ischemic bladder. MATERIALS AND METHODS: Male Sprague Dawley rats were randomly divided into treatment (n=8) and control (n=8) groups. In the treatment group, iliac artery atherosclerosis and chronic bladder ischemia were induced. At 8 weeks after bladder ischemia, voiding patterns were examined in metabolic cages, cystometrograms were recorded in conscious animals, and then bladder blood flow was measured under general anesthesia. Bladder tissues were processed for assessment of transcription factors, markers of cellular and mitochondrial stress, mitochondrial respiration, and cell survival signaling pathway. RESULTS: Atherosclerotic occlusive disease spread from the common iliac arteries to the internal iliac and vesical arteries and produced sustained bladder ischemia. Studies in metabolic cages showed increased micturition frequency and decreased voided volume in bladder ischemia. Conscious cystometrograms produced consistent data showing significant increase in micturition frequency and decreased voided volume and bladder capacity. Voiding behavior and cystometric changes in bladder ischemia were associated with significant decrease in DNA binding activity of Nrf2, significant increase in cellular levels of stress protein Hsp70 and mitochondrial stress protein GRP75, and significant decrease in mitochondrial oxygen consumption and upregulation of PI3K and Akt expression. CONCLUSION: Chronic bladder ischemia may be a mediating variable in the development of detrusor overactivity in the non-obstructive bladder. The mechanism may involve ischemia-induced cellular stress, Nrf2 functional deficit, depression of mitochondrial respiration, and upregulation of PI3K/Akt cell survival signaling pathway.

Label-free quantitative proteomics unravels the importance of RNA processing in glioma malignancy.

Glioma, one of the most common cancers in human, is classified to different grades according to the degrees of malignancy. Glioblastoma (GBM) is known to be the most malignant (Grade IV) whereas low-grade astrocytoma (LGA, Grade II) is relatively benign. The mechanism underlying the pathogenesis and progression of glioma malignancy remains unclear. Here we report a quantitative proteomic study to elucidate the differences between GBM and LGA using liquid chromatography and tandem mass spectrometry followed by label-free quantification. A total of 136 proteins were differentially expressed in GBM for at least five folds in comparison with LGA. Ontological analysis revealed a close correlation between GBM-associated proteins and RNA processing. Interaction network analysis indicated that the GBM-associated proteins in the RNA processing were linked to crucial signaling transduction modulators including epidermal growth factor receptor (EGFR), signal transducer and activator of transcription 1 (STAT1), and mitogen-activated protein kinase 1 (MAPK1), which were further connected to the proteins important for neuronal structural integrity, development and functions. Upregulation of 40S ribosomal protein S5 (RPS5), Ferritin Heavy chain (FTH1) and STAT1, and downregulation of tenascin R (TNR) were validated as representatives by immune assays. In summary, we revealed a panel of GBM-associated proteins and the important modulators centered at the RNA-processing network in glioma malignancy that may become novel biomarkers and help elucidate the underlying mechanism.

LC-MS/MS Analysis Unravels Deep Oxidation of Manganese Superoxide Dismutase in Kidney Cancer.

Manganese superoxide dismutase (MNSOD) is one of the major scavengers of reactive oxygen species (ROS) in mitochondria with pivotal regulatory role in ischemic disorders, inflammation and cancer. Here we report oxidative modification of MNSOD in human renal cell carcinoma (RCC) by the shotgun method using data-dependent liquid chromatography tandem mass spectrometry (LC-MS/MS). While 5816 and 5571 proteins were identified in cancer and adjacent tissues, respectively, 208 proteins were found to be up- or down-regulated (p < 0.05). Ontological category, interaction network and Western blotting suggested a close correlation between RCC-mediated proteins and oxidoreductases such as MNSOD. Markedly, oxidative modifications of MNSOD were identified at histidine (H54 and H55), tyrosine (Y58), tryptophan (W147, W149, W205 and W210) and asparagine (N206 and N209) residues additional to methionine. These oxidative insults were located at three hotspots near the hydrophobic pocket of the manganese binding site, of which the oxidation of Y58, W147 and W149 was up-regulated around three folds and the oxidation of H54 and H55 was detected in the cancer tissues only (p < 0.05). When normalized to MNSOD expression levels, relative MNSOD enzymatic activity was decreased in cancer tissues, suggesting impairment of MNSOD enzymatic activity in kidney cancer due to modifications. Thus, LC-MS/MS analysis revealed multiple oxidative modifications of MNSOD at different amino acid residues that might mediate the regulation of the superoxide radicals, mitochondrial ROS scavenging and MNSOD activity in kidney cancer.

Yersinia pestis acetyltransferase-mediated dual acetylation at the serine and lysine residues enhances the auto-ubiquitination of ubiquitin ligase MARCH8 in human cells.

Lysine acetylation is known as a post translational modification (PTM) by histone acetyltransferases (HAT) that modifies histones and non-histone proteins to regulate gene expression. Serine acetylation, however, is reported in mammalian hosts by serine acetyltransferase of Yersinia pestis (YopJ) during infection. The protein target and cellular function of bacterial YopJ in mammalian systems are not fully addressed. Here we report dual acetylation at the serine and lysine residues by transiently expressed serine acetyltransferase YopJ mimicking Y. pestis infection in HeLa cells. Using shotgun proteomics followed by label-free quantification, we demonstrate an increase of dual acetylation in YopJ transfected human cells, including 10 Ser- (YopJ/non-YopJ 1.3-fold, p = 0.02) and 8 Lys- (YopJ/non-YopJ 3.5-fold, p = 0.00003) acetylation sites. Specifically, YopJ expression augments acetylation of membrane-associated E3 ubiquitin ligase MARCH8 at the serine residue Sac44, Sac71 and Sac253, and the lysine residue Kac247 and Kac252. YopJ-mediated Ser- and Lys-acetylation of MARCH8 is further confirmed by Western blotting using the specific antibodies against MARCH8 Sac71 and pan-acetyl lysine. Functional study demonstrates that YopJ-mediated Ser- and Lys-acetylation affects the auto-ubiquitination of MARCH8. The mutant C172A of YopJ previously shown to abolish the acetyltransferase activity also reduces Ser- and Lys-acetylation and diminishes the auto-ubiquitination of MARCH8. In support, MARCH8 is indeed acetylated at serine and lysine in vitro by purified YopJ but the activity is reduced by the C172A mutant in YopJ. Our study provides evidence that bacterial serine acetyltransferase YopJ mediates Ser- and Lys-acetylation and affects auto-ubiquitination of ubiquitin ligase MARCH8 in human cells.

Corrigendum: Correction of title spelling. Progressive changes in detrusor function and micturition patterns with chronic bladder ischemia.

[This corrects the article on p. 249 in vol. 57.].

Progressive changes in detrusor function and micturition patterns with chronic bladder ischemia.

PURPOSE: Lower urinary tract symptoms (LUTS) are bothersome constellation of voiding symptoms in men and women as they age. Multiple factors and comorbidities are attributed to this problem but underlying mechanisms of nonobstructive nonneurogenic detrusor overactivity, detrusor underactivity and LUTS remain largely unknown. Our goal was to characterize detrusor function and voiding patterns in relation to muscarinic receptors expression, nerve fiber density, and neural ultrastructure in chronic bladder ischemia. MATERIALS AND METHODS: Iliac artery atherosclerosis and bladder ischemia were produced in male Sprague-Dawley rats. At 8 and 16 weeks after ischemia, micturition patterns and cystometrograms were recorded in conscious rats then bladder blood flow and nonvoiding spontaneous contractions were measured under general anesthesia. Bladder tissues were processed for Western blotting, immunostaining, and transmission electron microscopy. RESULTS: Bladder responses to ischemic insult depended on the duration of ischemia. Micturition patterns and cystometric changes at 8-week ischemia suggested detrusor overactivity, while voiding behavior and cystometrograms at 16-week ischemia implied abnormal detrusor function resembling underactivity. Upregulation of muscarinic M2 receptor was found after 8- and 16 weeks of ischemia. Downregulation of M3 and upregulation of M1 were detected at 16-week ischemia. Neural structural damage and marked neurodegeneration were found after 8 and 16 weeks of ischemia, respectively. CONCLUSIONS: Prolonged ischemia may be a mediating variable in progression of overactive bladder to dysfunctional patterns similar to detrusor underactivity. The mechanism appears to involve differential expression of M1, M2, and M3 receptors, neural structural injury, and progressive loss of nerve fibers.

Unraveling molecular effects of ADAR1 overexpression in HEK293T cells by label-free quantitative proteomics.

ADAR1 is a double-stranded RNA (dsRNA) editing enzyme that specifically converts adenosine to inosine. ADAR1 is ubiquitously expressed in eukaryotes and participate in various cellular processes such as differentiation, proliferation and immune responses. We report here a new proteomics study of HEK293T cells with and without ADAR1 overexpression. The up- and down-regulated proteins by ADAR1 overexpression are identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) followed by label-free protein quantification. Totally 1,495 proteins (FDR < 0.01) are identified, among which 211 are up- and 159 are down-regulated for at least 1.5-fold (n = 3, p < 0.05). Gene ontology analysis reveals that these ADAR1-regulated proteins are involved in protein translation and cell cycle regulation. Bioinformatics analysis identifies a closely related network consistent for the protein translation machinery and a tightly connected network through proliferating cell nuclear antigen (PCNA)-interactions. Up-regulation of the proteins in the PCNA-mediated cell proliferation network is confirmed by Western blotting. In addition, ADAR1 overexpression is confirmed to increase cell proliferation in HEK293T cells and A549 cells. We conclude that ADAR1 overexpression modulates the protein translation and cell cycle networks through PCNA-mediated protein-protein interaction to promote cell proliferation in HEK293 cells.

Activation of innate antiviral immune response via double-stranded RNA-dependent RLR receptor-mediated necroptosis.

Viruses induce double-stranded RNA (dsRNA) in the host cells. The mammalian system has developed dsRNA-dependent recognition receptors such as RLRs that recognize the long stretches of dsRNA as PAMPs to activate interferon-mediated antiviral pathways and apoptosis in severe infection. Here we report an efficient antiviral immune response through dsRNA-dependent RLR receptor-mediated necroptosis against infections from different classes of viruses. We demonstrated that virus-infected A549 cells were efficiently killed in the presence of a chimeric RLR receptor, dsCARE. It measurably suppressed the interferon antiviral pathway but promoted IL-1ß production. Canonical cell death analysis by morphologic assessment, phosphatidylserine exposure, caspase cleavage and chemical inhibition excluded the involvement of apoptosis and consistently suggested RLR receptor-mediated necroptosis as the underlying mechanism of infected cell death. The necroptotic pathway was augmented by the formation of RIP1-RIP3 necrosome, recruitment of MLKL protein and the activation of cathepsin D. Contributing roles of RIP1 and RIP3 were confirmed by gene knockdown. Furthermore, the necroptosis inhibitor necrostatin-1 but not the pan-caspase inhibitor zVAD impeded dsCARE-dependent infected cell death. Our data provides compelling evidence that the chimeric RLR receptor shifts the common interferon antiviral responses of infected cells to necroptosis and leads to rapid death of the virus-infected cells. This mechanism could be targeted as an efficient antiviral strategy.

Alu RNA accumulation in hyperglycemia augments oxidative stress and impairs eNOS and SOD2 expression in endothelial cells.

Endothelial dysfunction resulting from oxidative stress and inflammation plays a dominant role in hyperglycemia-induced vasculopathy. While double-stranded RNA (dsRNA) accumulates in redox and inflammatory conditions, its precise role in hyperglycemia-associated endothelial dysfunction remains unclear. This study aimed to investigate whether and how endogenous dsRNA contributes to endothelial dysfunction via oxidative stress. We used a dsRNA-specific antibody J2 to detect and immunoprecipitate cellular dsRNA. Acquired dsRNA was recognized by cDNA library construction and DNA sequencing. Quantitative PCR, ELISA and immunoassays were performed to identify changes induced by acquired dsRNA in primary human umbilical vein endothelial cells (HUVEC). Our data showed that endogenous dsRNA homologous to Alu Sc subfamily accumulated in hyperglycemic HUVEC. Comparing Alu-transfected HUVEC with high-glucose treated HUVEC, we found that Alu RNA elicited the production of reactive oxygen species (ROS) and up-regulated interleukin-1ß (IL-1ß) expression and secretion in a similar manner as high-glucose treatment. Moreover, Alu RNA impeded the expression of endothelial nitric oxide synthase (eNOS) and superoxide dismutase 2 (SOD2), increased ROS production and activated nuclear factor NFκB by chemically scavenging ROS and inactivation of NFκB. The repressed expression of eNOS and SOD2 resulted from Alu RNA-mediated negative regulatory mechanisms. Our study uncovered endogenous Alu RNA accumulation in hyperglycemic endothelial cells that provoked endothelial oxidative stress and dysfunction by suppressing SOD2 and eNOS expression at both transcription and translation levels via NFκB signaling pathway. These findings suggest a novel regulatory mechanism that involves endogenous dsRNA in endothelial oxidative stress and dysfunction.

LUTS in pelvic ischemia: a new concept in voiding dysfunction.

Lower urinary tract symptoms (LUTS) are a group of voiding symptoms affecting both genders as they age. Traditionally, LUTS in men were commonly attributed to bladder outlet obstruction (BOO) due to benign prostatic enlargement (BPE). It was later shown that, in approximately one-third to more than one-half of cases, LUTS in men are not associated with BOO. Urodynamic changes in the male bladder and symptom scores in aging men were found to be identical to their age-matched female counterparts. These observations suggested that LUTS in the elderly do not necessarily relate to BOO and may result from local changes in bladder muscle, nerves, and blood vessels. However, aging factors predisposing to bladder dysfunction and LUTS remain unknown. Growing evidence suggests that aging-associated pelvic ischemia may be a primary factor in the development of nonobstructed nonneurogenic overactive bladder and LUTS. First identified in experimental models and later in clinical studies, pelvic ischemia has been shown to compromise the lower urinary tract structure and lead to dysfunction. Structural and functional consequences of bladder and prostate ischemia have been documented in animal models. Clinical studies have shown that bladder and prostate blood flow decreases with aging. The severity of LUTS in elderly patients correlates with the degrees of bladder ischemia. LUTS improvement with α blockers has been associated with increased bladder blood flow. Pelvic ischemia may be an independent factor in nonobstructed nonneurogenic bladder instability and LUTS. Further research into the pathophysiology of LUTS in pelvic ischemia may lead to better management of this problem in the elderly population.

Unraveling Molecular Differences of Gastric Cancer by Label-Free Quantitative Proteomics Analysis.

Gastric cancer (GC) has significant morbidity and mortality worldwide and especially in China. Its molecular pathogenesis has not been thoroughly elaborated. The acknowledged biomarkers for diagnosis, prognosis, recurrence monitoring and treatment are lacking. Proteins from matched pairs of human GC and adjacent tissues were analyzed by a coupled label-free Mass Spectrometry (MS) approach, followed by functional annotation with software analysis. Nano-LC-MS/MS, quantitative real-time polymerase chain reaction (qRT-PCR), western blot and immunohistochemistry were used to validate dysregulated proteins. One hundred forty-six dysregulated proteins with more than twofold expressions were quantified, 22 of which were first reported to be relevant with GC. Most of them were involved in cancers and gastrointestinal disease. The expression of a panel of four upregulated nucleic acid binding proteins, heterogeneous nuclear ribonucleoprotein hnRNPA2B1, hnRNPD, hnRNPL and Y-box binding protein 1 (YBX-1) were validated by Nano-LC-MS/MS, qRT-PCR, western blot and immunohistochemistry assays in ten GC patients' tissues. They were located in the keynotes of a predicted interaction network and might play important roles in abnormal cell growth. The label-free quantitative proteomic approach provides a deeper understanding and novel insight into GC-related molecular changes and possible mechanisms. It also provides some potential biomarkers for clinical diagnosis.

Quantitative Proteomic Analysis of Differentially Expressed Proteins and Downstream Signaling Pathways in Chronic Bladder Ischemia.

PURPOSE: Growing evidence suggests that ischemia may contribute to aging associated bladder dysfunction and lower urinary tract symptoms. Our goal was to determine the effects of chronic ischemia on bladder proteomic profiles and characterize downstream signaling pathways. MATERIALS AND METHODS: Bilateral iliac artery atherosclerosis and chronic bladder ischemia were created in male Sprague Dawley® rats. At 8 weeks cystometrograms were obtained. Ischemic and control bladder tissues were then processed for label-free quantitative proteomic analysis. GO (Gene Ontology) and IPA (Ingenuity® Pathway Analysis) software were used to classify altered proteins in bladder ischemia. Western blot was done to confirm differentially expressed proteins. Tissue structure was examined by transmission electron microscopy. RESULTS: Chronic ischemia resulted in detrusor instability and noncompliance. Proteomic analysis revealed a total of 4,277 proteins in ischemic and 4,602 in control bladder tissues. In ischemic bladders 359 and 66 proteins were differentially expressed with a greater than twofold and fivefold change, respectively. On GO analysis differentially expressed proteins were associated with molecular signaling mechanisms underlying proteolysis and degenerative processes. Pathway and network analysis of ischemic tissues suggested that altered proteins are involved in ubiquitination, Nrf2 mediated oxidative stress response, cell death, glucose metabolism and cytoskeleton remodeling. Western blot verified changes in 4 representative proteins, including Nedd4l, Mpo, Ca3 and Fkbp5. Altered proteomic profile of the bladder was associated with widespread ultrastructural damage. CONCLUSIONS: Alterations of bladder proteomic profiles in ischemia may provide new insight into molecular pathways underlying bladder dysfunction and lower urinary tract symptoms in pelvic atherosclerosis.

Structural modifications of the prostate in hypoxia, oxidative stress, and chronic ischemia.

PURPOSE: Clinical studies have reported a correlation between pelvic ischemia and voiding dysfunction in elderly men. The aim of this study was to identify and compare prostate structural modifications in cultured cells and in a rabbit model after exposure to hypoxia, oxidative stress, and chronic ischemia. MATERIALS AND METHODS: Cultured human prostate smooth muscle cells (SMCs), epithelial cells (ECs), and stromal cells (SCs) were incubated under normoxia, hypoxia, and oxidative stress conditions by use of a computerized oxycycler system. We developed a rabbit model of chronic prostate ischemia by creating aorto-iliac arterial atherosclerosis. Markers of oxidative stress were examined by using fluorometric analysis and enzyme immunoassay. Prostate structure was examined by using Masson's trichrome staining and transmission electron microscopy (TEM). RESULTS: Lipid peroxidation was found in SMCs exposed to hypoxia and in all cell types exposed to oxidative stress. We identified protein oxidation in ECs exposed to hypoxia and in all cell types exposed to oxidative stress. Markers indicating oxidative damage were present in chronically ischemic rabbit prostate tissue. These reactions were associated with DNA damage. Prostate ischemia resulted in epithelial atrophy, loss of smooth muscle, and diffuse fibrosis. TEM showed swollen mitochondria with degraded cristae, loss of membrane, loss of Golgi bodies, degenerated nerves, and disrupted cell-to-cell junctions. CONCLUSIONS: Human prostate cells exhibited differential reactions to hypoxia and oxidative stress with widespread DNA damage. Structural modifications in ischemic prostate tissue were similar to those in cells exposed to oxidative stress. Structural changes due to ischemia and oxidative stress may contribute to prostatic noncompliance in aging men.