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The expression of aquaporins (AQPs) in the fetal porcine urinary tract and its relation to gestational age has not been established. Tissue samples from the renal pelvis, ureter, bladder and urethra were obtained from porcine fetuses. Samples were examined by RT-PCR (AQPs 1-11), QPCR (AQPs positive on RT-PCR), and immunohistochemistry. Bladder samples were additionally examined by Western blotting. RNA was extracted from 76 tissue samples obtained from 19 fetuses. Gestational age was 60 (n=11) or 100 days (n=8). PCR showed that AQP1, 3, 9 and 11 mRNA was expressed in all locations. The expression of AQP3 increased significantly at all four locations with gestational age, whereas AQP11 significantly decreased. AQP1 expression increased in the ureter, bladder and urethra. AQP9 mRNA expression increased in the urethra and bladder, but decreased in the ureter. AQP5 was expressed only in the urethra. Immunohistochemistry showed AQP1 staining in sub-urothelial vessels at all locations. Western blotting analysis confirmed increased AQP1 protein levels in bladder samples during gestation. Expression levels of AQP1, 3, 5, 9 and 11 in the urinary tract change during gestation, and further studies are needed to provide insights into normal and pathophysiological water handling mechanisms in the fetus.

Erectile dysfunction (ED) is a prevalent medical condition affecting 18 million men and their sexual partners in the United States alone. In the majority of patients, ED is related to alterations in the flow of blood to or from the penis. Undeniably, significant progress has been made in understanding the multifactorial mechanisms that modulate erectile capacity and predispose one to ED, and this, in turn, has led to the availability of more effective treatment options. Nonetheless, all current therapies have untoward side effects, and moreover, there are still no satisfactory treatments for many patients with ED. Further enhancements in the treatment of ED would logically result from both early intervention and more detailed mechanistic insight into the characteristics of the disease process per se . This fact underscores the importance of improved understanding of the initiation, development and progression of ED. However, to do so requires longitudinal studies on animal models that more closely approximate the corresponding clinical features and time course of human disease. The goal of this report is twofold. First, to provide a brief general overview of the applicability of commonly used animal models for the study of ED. The second and primary goal is to highlight the scientific rationale for using non-human primates to evaluate the impact of atherosclerosis-induced vascular disease on the penile and systemic circulatory systems. This latter goal seems especially relevant in light of the recent literature documenting a link between ED and systemic vascular disease, a finding that has major implications in an aging US male population consuming a high fat diet.

Despite considerable advances, both the central regulation of erection with processing of various stimuli, and the different steps involved in neurotransmission, impulse propagation and intracellular transduction of neural signals in penile smooth muscles, are still incompletely known. Centrally as well as peripherally, many transmitters and transmitter systems are involved. Dopamine, nitric oxide, oxytocin and ACTH/alpha-MSH, seem to have a facilitatory role, whereas serotonin may be either facilitatory or inhibitory, and enkephalins are inhibitory. Peripherally, the balance between contractant (eg noradrenaline, endothelins, angiotensins) and relaxant (eg NO, VIP and related peptides, prostanoids) factors controls the degree of contraction of the smooth muscle of the corpora cavernosa, and determines the functional state of the penis. Neurogenic NO is considered the most important factor for relaxation of penile vessels and corpus cavernosum. The roles of other putative transmitters/mediators and of various intracellular mechanisms, producing relaxation of vascular and corpus cavernosum smooth muscle, have not been established. For example, recent findings have suggested a role of Rho/Rho-kinase in the regulation of cavernosal tone, and that Rho-kinase antagonism could be a new potential principle for the treatment of erectile dysfunction. Further research in this area may be rewarding.

The former perception of the urothelium as an impermeable barrier has been revised during the last decade, as increasing evidence of changes in urine composition during its passage of the urinary tract has been presented. Since differences in urothelial permeability between upper and lower urinary tract have been found, our aim is to demonstrate whether changes in urine composition occur during passage through the ureter. We studied consecutive urine samples from both renal pelvises in six pigs and compared them to samples from the bladder and distal ureter. We further sampled urine during storage in the bladder at a fixed volume. All samples were analysed by measuring osmolality and pH, along with the concentration of the following parameters: Na+, K+, Cl-, creatinine, urea. Urine alkalinity increased significantly during passage of the ureter. Creatinine concentration, pH and K+ increased significantly during the passage from pelvis to the bladder. All other parameters increased non-significantly during the passage to the bladder. The increase in concentration was more pronounced at low concentrations in the pelvis. During storage in the bladder, there was a significant increase in urea concentration. Changes in the composition of urine occur during its passage from the renal pelvis to the bladder and during storage in the bladder. Despite the brief transit time, significant changes in alkalinity were found already during passage through the ureter.

Research in the field of erectile function and dysfunction has continued to expand rapidly. Based on the information available, some directions for future erectile dysfunction therapies can be identified. The first direction is improvement of current therapeutic principles. A second generation of orally active phosphodiesterase (PDE) inhibitors is being introduced, and further developments within this field can be expected. The recent introduction of apomorphine has opened the way for new dopamine receptor agonists. The second direction is combinations of existing therapeutic principles. Combinations of apomorphine and sildenafil and apomorphine and alpha(1)-adrenoceptor (AR) antagonists, for example, seem attractive and may have a therapeutic potential in patients not responding satisfactorily to single-drug treatment. Nitrosylated alpha(1)-AR antagonists, combining nitric oxide donation and alpha(1)- or alpha(2)-AR antagonism, are currently being evaluated. The third direction is new targets within the central nervous system. Melanocortin receptor agonists have shown promise not only in animal models, but also in preliminary studies in humans. Other possible targets, such as growth hormone-releasing peptide receptors, are being explored. The fourth direction is new peripheral targets. Rho-kinase antagonism and non-nitric oxide-mediated stimulation of soluble guanylyl cyclase have been suggested as possible new principles for drug development. The fourth direction is gene therapy. Progress has been made in intracavernosal somatic gene therapy and will probably continue. Still, problems remain, and advantages over conventional pharmacological therapies have to be demonstrated. The final direction is prevention strategies. Strategies to prevent cavernosal degeneration and/or to restore cavernosal function will be one of the most exciting challenges for future research.

The aim of this work was to characterize the effect of experimental diabetes on neurotransmission in rat vagina. Female Sprague-Dawley rats were divided into two groups: non-diabetic controls (NDM, n=38) and diabetics (DM, n=38). DM was produced by intraperitoneal injection of streptozotocin. Eight weeks later the animals were killed, the distal part of the vagina was removed, and smooth muscle strips were prepared for functional organ bath experiments and for measurement of nitric oxide synthase (NOS) activity. In DM preparations, the EC(50) value for noradrenaline (NA) was significantly increased (P<0.05) and the maximal contractile response decreased (P=0.001). In preparations precontracted with NA, the NO donor SNAP and calcitonin gene-related peptide (CGRP) caused concentration-dependent relaxations, which were significantly decreased (P<0.001) in the DM group. Electrical stimulation of nerves (EFS) caused frequency-dependent contractions, which were significantly lower in DM than in NDM strips (P<0.001). SNAP and CGRP concentration-dependently inhibited EFS evoked contractions in both NDM and DM preparations. The inhibition was significantly lower (P<0.05) in the DM group. In NDM preparations precontracted with NA, EFS evoked frequency-dependent relaxations; such relaxations were inhibited or reduced in DM. Treatment with the NOS inhibitor, L-NOARG 0.1 mM, abolished relaxations in all preparations or produced contraction in DM preparations. Calcium-dependent NOS activity was not significantly different in the DM and NDM groups. However, the DM animals showed a small but significant increase in calcium-independent NOS-activity (P<0.05). Diabetes interferes with adrenergic-, cholinergic- and NANC-neurotransmitter mechanisms in the smooth muscle of the rat vagina. The changes in the nitrergic neurotransmission are not due to reduction in NOS-activity, but seem to be due to interference with later steps in the L-arginine/NO/guanylate cyclase/cGMP system.

The sympathetic nervous system is important for penile function: it mediates detumescence and may contribute to the maintenance of the penis in a non-erect state. The sympathetic preganglionic neurons are found in the intermediolateral gray matter of the spinal cord. Postganglionic neurons are located to the sympathetic chain ganglia, the inferior mesenteric, hypogastric and pelvic ganglia, and possibly to ganglia near the target organ. Sympathetic fibres can be found in the pelvic, cavernous, and pudendal nerves. Stimulation of the sympathetic pathways to the penis may, however, also produce erection. It has been suggested that the suprasacral vasodilator pathway is a sympathetic cholinergic pathway, operating through cholinergic neurons in the pelvic plexus. In the penis, there is a rich sympathetic, adrenergic innervation of the corpus cavernosum (CC) and the vasculature, and in particular of the helicine arteries. Sympathetic, adrenergic nerves also contain neuropeptide Y. Parasympathetic cholinergic nerves, which mediate CC relaxation and erection, contain not only acetylcholine, but also vasoactive intestinal polypeptide, nitric oxide synthase, and probably other mediators and/or mediator-synthesizing enzymes. Activation of sympathetic adrenergic nerves causes release of noradrenaline, acting on alpha-adrenoceptors in the trabecular smooth muscle of the CC and in penile vessels. The role of interactions between different transmitters and mediators, released from nerves or generated locally, in the regulation of contraction and relaxation of CC and penile vessels, needs further study.

Reflexive erection initiated by recruitment of penile afferents, involves both autonomic and somatic efferents. The reflex is mediated at the spinal cord level, modulated by supraspinal influences, and may use several transmitters. Dopamine, acetylcholine, nitric oxide, and peptides, such as oxytocin and ACTH/alpha-MSH, seem to have a facilitatory role, whereas serotonin may be either facilitatory or inhibitory, and enkephalins are inhibitory. Peripherally, the balance between contractant and relaxant factors controls the degree of contraction of the smooth muscle of the corpora cavernosa, and determines the functional state of the penis. Noradrenaline contracts both corpus cavernosum and penile vessels via stimulation of alpha1-adrenoceptors. The role of endothelins in the control of penile smooth muscle tone is presently unclear. Neurogenic nitric oxide (NO) is considered the most important factor for relaxation of penile vessels and corpus cavernosum. The role of other mediators, released from nerves or endothelium has not been definitely established. International Journal of Impotence Research (2000) 12, Suppl 4, S26-S33.

Background:Immunomodulating treatment in rheumatoid arthritis (RA) prevent aberrant proliferation and activation of CD4+T cells by various mechanisms. Yet, drug choice remains empirical. In RA, unrepaired DNA damage promote the arthritogenic potential of effector T cells [1]. DNA damage response (DDR) combines cell cycle check points, DNA repair and DNA damage tolerance. It is controlled epigenetically by the Switch/Sucrose NonFermentable complex (SWI/SNF or BRG1) at chromatin marked by histone H3 tail modifications. DDR has a strong IFN adherence [2].Objectives:This study investigates if the molecular signature of DNA damage is connected to high BRG1 expression in CD4+ cells of RA patients and how it is affected by immunomodulating drugs.Methods:Transcriptome of BRG1hi CD4+ cells of RA patients (n=24) were identified by RNAseq followed by DESeq2 analysis. RA treatment effect was identified in paired CD4+ T cells of patients pre- and post-treatment with abatacept (n=14, GSE121827, 24 weeks), tocilizumab (n=6, GSE113156, 24 weeks), and methotrexate (MTX, n=28, GSE176440, 12 weeks), by RNAseq. Treatment responsive differentially expressed genes (trDEG) were identified by DESeq2.DNA sequences binding H3K4me3, H3K27me3 and H3K27ac were obtained through immunoprecipitation and sequencing (ChIPseq). ChIPseq overlap indicating bivalent chromatin regions (BvCR) was identified by R package ChIPPeakAnno. Tag normalization enabled comparison between different ChIPseq to identify the dominant H3 modification. Genes connected to overlapped ChIPseq peaks (BvCR-dependent) within cis-regulatory elements were retrieved from GeneHancer database. Annotation to DDR pathway was done using Enrichr. Weighted gene correlation network analysis was performed using R package WGCNA. To identify IFN-sensitive genes, CD4+ cells sorted from blood (n=4) were cultured with IFNγ (50ng/mL) for 72h. Transcriptome was analysed by RNAseq followed by DESeq2.Results:Analysis of BRG1hiCD4+ cells revealed 8358 DEG compared to BRG1loCD4+ cells, which had accumulation of immune checkpoint proteins PD1, PD-L1 and CTLA4, and chemokine receptors CCR1, CCR5 and CD69, indicating a PD1+CD4+ cell phenotype promoting RA pathogenesis. The DDR pathway was significantly enriched in DEG of BRG1hiCD4+ cells (FDR=1.5e-23) and included the canonical SWI complex (ARID1A/B, SMARCA2), PBAF complex (PBRM1, ARID2) and the common subunits (BRG1, SMARCC1/C2, SMARCD1/D3). DNA damage marker γH2AX and classic DNA repair proteins FANCI, MSH2, MSH6, and MRE11 were significantly upregulated in BRG1hiCD4+ cells, while ATM was repressed.Immunomodulating treatment affected 40% of BRG1hi DEG. Among those, MTX reduced expression of PD1 (log2FC=-0.35, p=4.2e-4) and increased CD86 (log2FC=0.63, p=0.054), while abatacept and tocilizumab had no effect on PD1 expression.One-fourth of trDEG were BvCR-dependent and 40% (682 genes) were IFNγ−sensitive, with a majority regulated by H3K4me3-dominated BvCR. MTX affected 71% of these IFNγ-sensitive genes (484 genes) making a significant impact in epigenetic processes in RA CD4 cells. Among the DDR pathway trDEG were numerous SWI complex subunits, while BRG1 expression was unaffected. WGCNA of IFNγ-sensitive genes in BRG1hi cells (682 genes) showed downregulation of chromatin remodeling (GO:0006338, ARID1B, CHD7, SATB1) and upregulation of cell cycle phase transition (GO:0044772, CDKN1A, WEE1, MYC).In the trDEG under BvCR control in BRG1hi cells, H3K4me3-BvCR regulated 15 genes annotated to apoptotic DNA damage signaling (GO:0042771, DYRK2, PML). Genes controlled by H3K4me3-BvCR were mostly downregulated by immunomodulation, with MTX having the largest effect.Conclusion:BRG1hiCD4+ cells embody the activated DNA damage response and promote RA pathogenesis. MTX treatment affected the DNA damage response controlled by IFN signaling in CD4+T cells through the SWI/BRG1 complex. This makes a significant impact in epigenetic processes in RA CD4 cells and implies that high expression of BRG1 in RA recognize MTX-responsive RA patients.REFERENCES:[1] Li, Y. et al. Immunity 45, 903–916 (2016).[2] Begg, K. A. G., et al. DNA Repair 133, 103609 (2024).Acknowledgements:NIL.Disclosure of Interests:None declared.

Background:Epigenetic processes promote development of pathogenic cell types in rheumatoid arthritis (RA), but precise molecular mechanisms behind these effects remain largely unexplored. Survivin has recently been identified as important regulator of histone deposition on chromatin preventing accumulation of the repressive H3K27me3 mark (1,2).Objectives:In this study, we investigate if survivin affects acetylation of histone H3K27 in CD4+ T cells, and how this is associated with effect of immunosuppressive RA treatment.Methods:Chromatin of CD4+ cells (n=12) treated with or without the survivin-inhibitor YM155 was immunoprecipitated with antibodies to histone H3K27ac and sequenced (ChIP-seq, Illumina). Peaks with change >30% in deposition of H3K27ac upon YM155-treatment were annotated to the genomic regulatory elements (RE) via GeneHancer database, which also gave information of genes connected to these RE. Transcriptomics of CD4+ cells isolated from RA patients treated with MTX (n=18), TNFi (n=10), JAKi (n=24) and having no DMARDs (n=7), by RNA sequencing, deposited GSE201669. The genes differentially expressed (DEG, nominal p<0.05) upon JAKi-treatment compared to other treatments were identified by DESeq2 (R-studio, Bioconductor). External transcriptome datasets of CD4+ cells isolated from patients before and after treatment with MTX (n = 28, GSE176440), abatacept (n = 14, GSE121827), and tocilizumab (n = 12, GSE113156). Treatment affected DEG were identified by DESeq2 (R-studio, Bioconductor). The enrichment analysis of DEG for biological processes within Gene ontology library were analyzed through String database. Over-representation analysis was done at http://cpdb.molgen.mpg.de.Results:Inhibiting survivin with YM155 in CD4+ cells induced a significant (>30%) change in 17% (1967 of 11529 peaks) of H3K27ac-peaks. These survivin-sensitive H3K27ac peaks were located within 339 cis-RE, connected to 1022 protein-coding genes expressed in CD4+ cells. Biological pathway analysis revealed that the top biological processes enriched among the these 1022 genes were mRNA metabolic process (GO:0016071 FDR = 0.0043), Cellular response to DNA damage stimulus (GO:0006974 FDR = 0.0029), DNA metabolic process (GO:0006259 FDR 0.033), RNA processing (GO:0006396 FDR 0.00058), RNA metabolic process (GO:0016070 FDR = 2.70e-05), and Cellular response to stress (GO:0033554 FDR = 0.00099). Together, these processes has a significant overlap and comprised 28% (286 of 1022) of the genes connected to the changed H3K27ac- genes.Immunosuppressive treatment had a significant imprint on H3K27ac connected genes, which were found over-represented among the DEG after treatment with MTX (p = 6e-49, OR = 1.77), abatacept (p = 1.6e-25, OR = 2.31), tocilizumab (p = 1.9e-7, OR = 1.51) and JAKi (p = 0, OR = 3.52) (all by Enrichr: Epigenomics roadmap). Furthermore, we found that JAKi and Mtx affected the same processes of transcription, RNA processing and translation; TGFb and SMAD signaling; NOTCH signaling; and DNA damage response as controlled by the genes connected to changed H3K27ac (JAKi p = 0.010 OR = ∞, Mtx p = 0.0001 OR = 3.2).In the top enriched GO:BP of the genes connected to changed H3K27ac, the DEG of the treatments were enriched to different extent (Table 1). In total, JAKi affected 219, abatacept 17, tocilizubam 17 and MTX 48 of the genes within the top GO:BP.Conclusion:Immunosuppressive treatment affects genes under the epigenetic control in transcription, RNA processing and DNA damage response exerted through the survivin-sensitive H3K27ac deposition. Molecular signature of CD4+T cells reflecting activation of these processes could assist individual treatment choice in RA patients.Table 1.REFERENCES:[1] Jensen, M.. et al. (2023) Survivin prevents the polycomb repressor complex 2 from methylating histone 3 lysine 27. iScience, 26, 106976.[2] Erlandsson, M.C.. et al. (2022) Survivin promotes a glycolytic switch in CD4(+) T cells by suppressing the transcription of PFKFB3 in rheumatoid arthritis. iScience, 25, 105526.Acknowledgements:NIL.Disclosure of Interests:None declared.