scholarly journals Inhibition of DNA methylation during chronic obstructive bladder disease (COBD) improves function, pathology and expression

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Martin Sidler ◽  
K. J. Aitken ◽  
Jia-Xin Jiang ◽  
Priyank Yadav ◽  
Erin Lloyd ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Dna Methyltransferase ◽  
Contractile Function ◽  
Ex Vivo ◽  
Outlet Obstruction ◽  
Bladder Function ◽  
Chronic Obstructive ◽  
Sprague Dawley ◽  
Prostate Hyperplasia ◽  
Bladder Disease

AbstractPartial bladder outlet obstruction due to prostate hyperplasia or posterior urethral valves, is a widespread cause of urinary dysfunction, patient discomfort and also responsible for immense health care costs. Even after removal or relief of obstruction, the functional and pathologic aspects of obstruction remain as a chronic obstructive bladder disease (COBD). Epigenetic changes, such as DNA methylation, contribute to the persistent character of many chronic diseases, and may be altered in COBD. We tested whether candidate genes and pathways and the pathophysiology of COBD were affected by a hypomethylating agent, decitabine (DAC). COBD was created in female Sprague-Dawley rats by surgical ligation of the urethra for 6 weeks, followed by removal of the suture. Sham ligations were performed by passing the suture behind the urethra. After removal of the obstruction or sham removal, animals were randomized to DAC treatment (1 mg/kg/3-times/week intraperitoneally) or vehicle (normal saline). Bladder function was non-invasively tested using metabolic cages, both one day prior to de-obstruction at 6 weeks and prior to sacrifice at 10 weeks. Residual volume and bladder mass were measured for each bladder. Bladders were examined by immunostaining as well as qPCR. The effects of DNA methyltransferase (DNMT)-3A knockout or overexpression on smooth muscle cell (SMC) function and phenotype were also examined in bladder SMC and ex vivo culture. Residual volumes of the DAC treated group were not significantly different from the NS group. Compared to COBD NS, COBD DAC treatment helped preserve micturition volume with a significant recovery of the voiding efficiency (ratio of the maximum voided volume/maximum bladder capacity) by one third (Fig. 1, p > 0.05). Brain-derived neurotrophic factor (BDNF) variants 1 and 5 were upregulated by COBD and significantly reduced by DAC treatment. Deposition of collagen in the COBD bladder was reduced by DAC, but gross hypertrophy remained. In bladder SMC, DNMT3A overexpression led to a loss of contractile function and phenotype. In bladders, persistently altered by COBD, inhibition of DNA-methylation enhances functional recovery, unlike treatment during partial obstruction, which exacerbates obstructive pathology. The underlying mechanisms may relate to the gene expression changes in BDNF and their effects on signaling in the bladder.

scholarly journals Nerve transfer for restoration of lower motor neuron-lesioned bladder function. Part 3: Correlation between histological changes and nerve evoked contractions

2021 ◽  
Author(s):  
Mary F Barbe ◽  
Courtney L Testa ◽  
Geneva E. Cruz ◽  
Nagat Frara ◽  
Ekta Tiwari ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Contractile Function ◽  
Ex Vivo ◽  
Bladder Function ◽  
Detrusor Muscle ◽  
Axon Density ◽  
Spinal Roots ◽  
One Year ◽  
Intramural Ganglia ◽  
Stimulation Of

We determined the effect of pelvic organ decentralization and reinnervation one year later on urinary bladder histology and function. Nineteen canines underwent decentralization by bilateral transection of all coccygeal and sacral (S) spinal roots, dorsal roots of lumbar (L)7 and hypogastric nerves. After exclusions, 8 were reinnervated 12 months post-decentralization with obturator-to-pelvic and sciatic-to-pudendal nerve transfers, then euthanized 8-12 months later; four served as long-term decentralized only animals. Before euthanasia, pelvic or transferred nerves and L1-S3 spinal roots were stimulated and maximum detrusor pressure (MDP) recorded. Bladder specimens were collected for histological and ex vivo smooth muscle contractility studies. Both reinnervated and decentralized animals showed less or denuded urothelium, fewer intramural ganglia, and more inflammation and collagen, than controls, although percent muscle was maintained. In reinnervated animals, pgp9.5+ axon density was higher, compared to decentralized animals. Ex vivo smooth muscle contractions in response to KCl correlated positively with submucosal inflammation, detrusor muscle thickness, pgp9.5+ axon density. In vivo, reinnervated animals showed higher MDP after stimulation of L1-L6 roots, compared to their transected L7-S3 roots, and reinnervated and decentralized animals showed lower MDP than controls after stimulation of nerves (due likely to fibrotic nerve encapsulation). MDP correlated negatively with detrusor collagen and inflammation, and positively with pgp9.5+ axon density and intramural ganglia numbers. These results demonstrate that bladder function can be improved by transfer of obturator nerves to pelvic nerves at one year after decentralization, although the fibrosis and inflammation that developed were associated with decreased contractile function.

Abstract 508: TNFα Downregulates DNA Methylation and Increases Pulmonary Arterial Smooth Muscle Cell (PASMC) Proliferation

Hypertension ◽  
2012 ◽  
Vol 60 (suppl_1) ◽  
Author(s):  
Patrick Crosswhite ◽  
Zhongjie Sun
Keyword(s):  
Dna Methylation ◽  
Histone Acetylation ◽  
Dna Methyltransferase ◽  
Pulmonary Arteries ◽  
Cell Number ◽  
Superoxide Production ◽  
Tnf Alpha ◽  
Sprague Dawley ◽  
Sprague Dawley Rats ◽  
Pulmonary Arterial

Background: Chronic cold exposure triggers robust TNFα production in the lungs and pulmonary arteries (PAs) and causes PA remodeling in rats. A hallmark of the PA remodeling is the over-proliferation of PASMCs. The purpose of this experiment was to investigate potential mechanisms of TNF-alpha induced PASMC proliferation. Methods and Results: PASMCs were isolated from 8-week-old male Sprague Dawley rats and treated with 0, 20 or 200 ng/ml TNFα for 24 or 48 hours. After treatment, cell number, superoxide production, histone acetylation and DNA methylation were assessed. TNFα treatment significantly increased the number of PASMCs, indicating excessive proliferation. TNFα also increased NADPH oxidase activity and superoxide production compared with untreated controls. TNFα treatment however did not affect histone acetylation at either dose. Interestingly, DNA methylation was significantly decreased by TNFα treatment compared to controls. Unexpectedly, the activity of DNA methyltransferase-1, the major enzyme for DNA methylation, was not altered by TNFα treatment. Further investigation using QRT-RT-PCR revealed that TNFα upregulated several factors including Gadd45α that regulate DNA de-methylation. Conclusions: Our results show that TNFα treatment induced cell proliferation and increased superoxide production in PASMCs. TNFα decreased DNA methylation and upregulated mediators of de-methylation in PASMCs. These findings suggest that TNF-alpha induced PASMCs proliferation may involve the DNA methylation mechanism. (Supported by NIH HL077490 and AHA 11PRE7830040).

Strain Induced Bladder Smooth Muscle Remodeling

2007 ◽  
Author(s):  
Rebecca A. Long ◽  
Aron Parekh ◽  
Michael S. Sacks
Keyword(s):  
Smooth Muscle ◽  
Ex Vivo ◽  
Bladder Wall ◽  
Outlet Obstruction ◽  
Tissue Remodeling ◽  
Bladder Function ◽  
Compensatory Mechanism ◽  
Bladder Smooth Muscle ◽  
Cord Injury

Multiple urinary bladder wall (UBW) pathologies, such as overactive bladder, bladder outlet obstruction, spinal cord injury (SCI) and related neurogenic disorders, and diabetes result in tissue remodeling marked by hypertrophic bladder smooth muscle cells (BSMC) and altered extra-cellular matrix components. This remodeling results in changes in UBW biomechanical properties leading to altered bladder function. Our previous studies have revealed that during the initial areflexic phase of SCI the UBW undergoes profound remodeling that appears to be a compensatory mechanism for the increased wall stretch resulting from over-distension [1, 2]. Remodeling in the bladder wall results in changes in biomechanics and ultimately the ability of the organ to normally fill and void [3]. The stimuli and precise mechanisms that are responsible for bladder remodeling in SCI and the aforementioned pathologies remain unknown. The objective of the present study is to determine the effects of varied in vitro strain on ECM production in the ex vivo rat bladder as a first step toward understanding tissue remodeling in response to strain.

Effects of Mixtures of Polychlorinated Biphenyls, Methylmercury, and Organochlorine Pesticides on Hepatic DNA Methylation in Prepubertal Female Sprague-Dawley Rats

2009 ◽  
Vol 28 (4) ◽  
pp. 294-307 ◽  
Author(s):  
Daniel Desaulniers ◽  
Gong-hua Xiao ◽  
Hong Lian ◽  
Yong-Lai Feng ◽  
Jiping Zhu ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Polychlorinated Biphenyls ◽  
Organochlorine Pesticides ◽  
Dna Methyltransferase ◽  
Female Offspring ◽  
High Dose ◽  
Mrna Levels ◽  
Postnatal Exposure ◽  
Sprague Dawley ◽  
Pregnant Rats

DNA methylation is one of the epigenetic mechanisms that regulates gene expression, chromosome structure, and stability. Our objective was to determine whether the DNA methylation system could be a target following in utero and postnatal exposure to human blood contaminants. Pregnant rats were dosed daily from gestation day 1 until postnatal day 21 with 2 dose levels of either organochlorine pesticides (OCP; 0.019 or 1.9 mg/kg/day), methylmercury chloride (MeHg; 0.02 or 2 mg/kg/day), polychlorinated biphenyls (PCBs; 0.011 or 1.1 mg/kg/day), or a mixture (Mix; 0.05, or 5 mg/kg/day) including all 3 groups of chemicals. Livers from 1 female offspring per litter were collected at postnatal day 29. Hepatic analysis revealed that the mRNA abundance for DNA methyltransferase (DNMT)-1, -3a, and -3b were significantly reduced by the high dose of PCB, that the high dose of MeHg also reduced mRNA levels for DNMT-1, and -3b, but that OCP had no significant effects compared with control. The high dose of PCB and Mix reduced the abundance of the universal methyl donor S-adenosylmethionine, and Mix also reduced global genome DNA methylation (5-methyl-deoxycytidine/5-methyl-deoxycytidine + deoxycytidine). The latter is consistent with pyrosequencing methylation analysis, revealing that the high-dose groups (except OCP) generally decreased the methylation of CpG sites (position -63 to -29) in the promoter of the tumor suppressor gene p16INK4a. Overall, these hepatic results suggest that the DNA methylation system can be affected by exposure to high doses of blood contaminants, and that OCP is the least potent chemical group from the investigated mixtures.

5-Azacytidine Reduces Leukemic Burden in a Xenograft Model of Juvenile Myelomonocytic Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1655-1655
Author(s):  
Christopher Felix Krombholz ◽  
Angelina Meier ◽  
Konrad Aumann ◽  
Silvia Fluhr ◽  
Matthias Kollek ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Bone Marrow ◽  
Dna Methyltransferase ◽  
Ex Vivo ◽  
Xenograft Model ◽  
Patient Specific ◽  
Juvenile Myelomonocytic Leukemia ◽  
Methylation Patterns ◽  
Myelomonocytic Leukemia

Abstract Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative disorder of early childhood with often fatal outcome. Despite many attempts to develop alternative treatment options allogeneic hematopoietic stem cell transplantation (HSCT) remains the only curative modality. In the past our group has linked the prognosis of JMML to differential DNA methylation patterns (Olk-Batz, Blood 2011;117:4871-80 and Poetsch, Epigenetics 2014;9:1252-60), suggesting a key role of epigenetic modifications in JMML pathophysiology. To overcome the lack of suitable preclinical JMML research models we have developed an ex vivo JMML xenotransplantation system using neonatal Rag2-/- gamma-c-/- mice. Transplantation of 1x106 primary JMML cells resulted in stable xenologous engraftment and reproduced a characteristic JMML phenotype including myelomonocytic expansion; infiltration of spleen, liver and, notably, lung; splenomegaly; and reduced survival (median 26 weeks). Persistent human engraftment and leukemic organ infiltration was confirmed by both flow cytometry and immunohistology. Ras pathway mutations present in xenotransplanted patient samples were invariably confirmed in engrafted tissues. In addition, the model sustained serial transplantations and can therefore be used to amplify scarce patient material. We first tested if DNA methylation patterns in JMML cells were stable even after xenologous engraftment because such stability would be a prerequisite if the model were to be used for preclinical investigation of DNA methyltransferase inhibitors. JMML cells before xenotransplantation and those retrieved from the bone marrow of engrafted mice were profiled for global CpG methylation using Illumina 450K arrays. DNA methylation patterns in JMML were patient-specific and surprisingly robust in functional regions over several months of engraftment time (on average, 0.29% of 30877 promoters and 0.25 % of 30725 intragenic regions were called as "differentially methylated" between source and xenograft; 0.2 β-value change cutoff). These findings confirm the suitability of the xenograft model to investigate JMML epigenetics and, more importantly, indicate that patient-specific epigenetic profiles originate in leukemia-initiating stem cells, reinforcing a fundamental role of these alterations in JMML biology. Our group recently published a retrospective case series demonstrating unprecedented clinical efficacy of the DNA methyltransferase inhibitor 5-azacytidine (5AC) to induce partial or complete remissions in JMML before allogeneic HSCT (Cseh, Blood 2015;125:2311-3). To further investigate the drug on the preclinical level we administered 5AC to Rag2-/- gamma-c-/- mice xenografted with primary JMML cells. After a leukemia establishment phase the mice were divided into treatment or mock groups and treated with 5AC (3mg/kg body weight i.p., N=6) or saline (N=6) for 2 cycles (1 dose daily for 5 days; 9 days of recovery). This regimen was tolerated well by the animals. We found that 5AC reduced JMML infiltration in all organs analyzed, with most pronounced effects in spleen (human CD45+ fraction of all CD45+ cells, 0.24% +/- 0.04% vs 39.78% +/- 10.72%; p<0.01) and lung (0.41% +/-0.18% vs 42.88% +/-8.42%; p<0.01). The proportion of early progenitor cells (CD34+) within the human leukemia population in murine bone marrow was dramatically reduced after 5AC treatment (7.89% +/-0.74% vs 32.65% +/-3.76%; p<0.01) while the amount of granulocytes increased simultaneously (44.90% +/-1.74% vs 9.35% +/-1.95%; p<0.01). These findings suggest a loss of JMML cells induced by forced differentiation of more immature cells into mature myelomonocytic cells with reduced proliferation potential. Bisulfite pyrosequencing of the human BMP4 promoter CpG island, a locus frequently hypermethylated in JMML, showed significantly reduced DNA methylation in JMML cells retrieved from 5AC-treated mice (31.32% +/-2.66% vs 52.46% +/-1.39%; p<0.001). In summary we created an ex vivo JMML xenograft model in immunodeficient mice that reflects many important aspects of this disorder and proved its usefulness for preclinical research of DNA methyltransferase inhibition because of extraordinary stability of leukemic DNA methylation patterns. 5AC showed clear preclinical efficacy in this model, supporting its further development in clinical treatment strategies for JMML. Disclosures No relevant conflicts of interest to declare.

scholarly journals Role of GABRD Gene Methylation in the Nucleus Accumbens in Heroin-Seeking Behavior in Rats

2021 ◽  
Vol 11 ◽  
Author(s):  
Qingxiao Hong ◽  
Wenjin Xu ◽  
Zi Lin ◽  
Jing Liu ◽  
Weisheng Chen ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Nucleus Accumbens ◽  
Dna Methyltransferase ◽  
Opposite Effect ◽  
Heroin Addiction ◽  
Gamma Aminobutyric Acid ◽  
Sprague Dawley ◽  
Self Administration ◽  
Drug Induced ◽  
Seeking Behavior

Epigenetic modifications such as DNA methylation play important roles in regulating gene expression and may mediate neuroplasticity and lead to drug-induced aberrant behaviors. Although several brain regions and neurobiological mechanisms have been suggested to be involved in these processes, there is remarkably little known about the effects of DNA methylation on heroin-seeking behavior. Using a Sprague-Dawley rat model, we show that heroin self-administration resulted in gamma-aminobutyric acid type A receptor subunit delta (GABRD) gene hypomethylation, which was associated with transcriptional upregulation of GABRD in the nucleus accumbens (NAc). Systemic l-methionine (MET) administration significantly strengthened the reinstatement of heroin-seeking behavior induced by heroin priming, whereas intra-NAc injections of the DNA methyltransferase (DNMT) inhibitor 5-aza-2′-deoxycytidine (5-Aza-dC) had the opposite effect on heroin-seeking. Meanwhile, 5-Aza-dC treatment decreased DNA methylation and upregulated the expression of GABRD in the NAc, whereas MET had the opposite effect. Our results also reveal that 5-Aza-dC might alter the methylation landscape of the GABRD gene by directly repressing DNMT1 and DNMT3A expression. Furthermore, reinstatement of heroin-seeking behavior was significantly inhibited by directly overexpressing GABRD and remarkably reinforced by GABRD gene silencing in the NAc. Collectively, these results suggest that targeting the GABRD gene and its methylation might represent a novel pharmacological strategy for treating heroin addiction and relapse.

Exposure to glyphosate and tetrachlorvinphos induces cytotoxicity and global DNA methylation in human cells

2021 ◽  
pp. 074823372110331
Author(s):  
Hacer Ergun ◽  
Akin Cayir
Keyword(s):  
Dna Methylation ◽  
Environmental Protection Agency ◽  
Dna Methyltransferase ◽  
Molecular Mechanisms ◽  
A549 Cells ◽  
Lung Epithelial Cells ◽  
Control Group ◽  
Sprague Dawley ◽  
Sprague Dawley Rats ◽  
Liver And Kidney

Two organophosphate pesticides—glyphosate and tetrachlorvinphos—have been announced as carcinogens to humans by various authorities, including the European Chemical Agency and the Environmental Protection Agency. We aimed to investigate molecular mechanisms associated with carcinogenicity and to examine changes in global m5C DNA methylation and cytotoxic potential in A549 lung epithelial cells in response to glyphosate and tetrachlorvinphos, and differential gene expression of m5C DNA methyltransferase genes in Sprague Dawley rats to Roundup (commercial formulation of glyphosate). Global m5C level significantly increased after 1500 μM glyphosate exposure for 24 h. We determined that exposure to tetrachlorvinphos did not significantly increase the m5C level in A549 cells for 24 h. Additionally, we did not observe significant DNA methylation alteration for both pesticides after 12 h exposure. In the animal study, we observed that DNA methyltransferase genes (DNMT3b and DNMT3a) showed significantly higher expression in Roundup-exposed rats than the control group in the liver and kidney. We also observed that a significant cytotoxic effect was determined after the treatment of the cells with higher concentrations of glyphosate and tetrachlorvinphos. Our results revealed that DNA methylation could be modified by exposure to glyphosate and that exposure to Roundup was associated with the differential expression level of m5C DNA methylation methyltransferase. Finally, exposure to both pesticides increased cytotoxicity.

scholarly journals Upregulation of TRPC6 Mediated by PAX6 Hypomethylation Is Involved in the Mechanical Allodynia Induced by Chemotherapeutics in Dorsal Root Ganglion

2020 ◽  
Vol 23 (4) ◽  
pp. 257-267 ◽  
Author(s):  
Xiang-Zhong Zhang ◽  
De-Xing Luo ◽  
Xiao-Hui Bai ◽  
Huan-Huan Ding ◽  
Meng Liu ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Mechanical Allodynia ◽  
Dna Methyltransferase ◽  
Molecular Mechanisms ◽  
Transient Receptor Potential ◽  
Quantitative Polymerase Chain Reaction ◽  
Receptor Potential ◽  
Pax6 Gene ◽  
Sprague Dawley ◽  
Painful Neuropathy

Abstract Background Although the action mechanism of antineoplastic agents is different, oxaliplatin, paclitaxel, or bortezomib as first-line antineoplastic drugs can induce painful neuropathy. In rodents, mechanical allodynia is a common phenotype of painful neuropathy for 3 chemotherapeutics. However, whether there is a common molecular involved in the different chemotherapeutics-induced painful peripheral neuropathy remains unclear. Methods Mechanical allodynia was tested by von Frey hairs following i.p. injection of vehicle, oxaliplatin, paclitaxel, or bortezomib in Sprague-Dawley rats. Reduced representation bisulfite sequencing and methylated DNA immunoprecipitation were used to detect the change of DNA methylation. Western blot, quantitative polymerase chain reaction, chromatin immunoprecipitation, and immunohistochemistry were employed to explore the molecular mechanisms. Results In 3 chemotherapeutic models, oxaliplatin, paclitaxel, or bortezomib accordantly upregulated the expression of transient receptor potential cation channel, subfamily C6 (TRPC6) mRNA and protein without affecting the DNA methylation level of TRPC6 gene in DRG. Inhibition of TRPC6 by using TRPC6 siRNA (i.t., 10 consecutive days) relieved mechanical allodynia significantly following application of chemotherapeutics. Furthermore, the downregulated recruitment of DNA methyltransferase 3 beta (DNMT3b) at paired box protein 6 (PAX6) gene led to the hypomethylation of PAX6 gene and increased PAX6 expression. Finally, the increased PAX6 via binding to the TPRC6 promoter contributes to the TRPC6 increase and mechanical allodynia following chemotherapeutics treatment. Conclusions The TRPC6 upregulation through DNMT3b-mediated PAX6 gene hypomethylation participated in mechanical allodynia following application of different chemotherapeutic drugs.

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