Targeted deletion of von-Hippel-Lindau in the proximal tubule conditions the kidney against early diabetic kidney disease.

Diabetic kidney disease (DKD) is the leading cause of end-stage renal disease. Glomerular hyperfiltration and albuminuria subject the proximal tubule (PT) to a subsequent elevation of workload, growth, and hypoxia. Hypoxia plays an ambiguous role in the development and progression of DKD and shall be clarified in our study. PT-von-Hippel-Lindau (Vhl)-deleted mouse model in combination with streptozotocin (STZ)-induced type I diabetes mellitus (DM) was phenotyped. In contrary to PT-Vhl-deleted STZ-induced type 1 DM mice, proteinuria and glomerular hyperfiltration occurred in diabetic control mice the latter due to higher nitric oxide synthase 1 and sodium and glucose transporter expression. PT Vhl deletion and DKD share common alterations in gene expression profiles, including glomerular and tubular morphology, and tubular transport and metabolism. Compared to diabetic control mice, the most significantly altered in PT Vhl-deleted STZ-induced type 1 DM mice were Ldc-1, regulating cellular oxygen consumption rate, and Zbtb16, inhibiting autophagy. Alignment of altered genes in heat maps uncovered that Vhl deletion prior to STZ-induced DM preconditioned the kidney against DKD. HIF-1α stabilization leading to histone modification and chromatin remodeling resets most genes altered upon DKD towards the control level. These data demonstrate that PT HIF-1α stabilization is a hallmark of early DKD and that targeting hypoxia prior to the onset of type 1 DM normalizes renal cell homeostasis and prevents DKD development.

Localization Pattern of Dispatched Homolog 2 (DISP2) in the Central and Enteric Nervous System.

Dispatched homolog (DISP) proteins have been implicated in the regulation of hedgehog signaling during embryologic development. Although DISP2 has recently been associated with neuronal development and control of cognitive functions, its localization pattern in the mammalian central and peripheral nervous system has not yet been investigated. In this study, the Disp2 expression profile was assessed in human tissues from publicly available transcriptomic datasets. The DISP2 localization pattern was further characterized in the human and rat central nervous system (CNS), as well as within the colonic enteric nervous system (ENS) using dual-label immunohistochemistry. Colocalization of DISP2 with neuronal and glial markers was additionally analyzed in murine primary ENS culture. At transcriptomic level, DISP2 expression was predominant in neuronal cell types of the CNS and ENS. DISP2 immunoreactivity was mainly located within PGP9.5-positive neurons rather than in S100-positive glial cells throughout the nervous system. Investigation of human and rat brain tissues, colonic specimens, and murine ENS primary cultures revealed that DISP2 was located in neuronal cell somata, as well as along neuronal processes both in the human and murine CNS and ENS. Our results indicate that DISP2 is prominently localized within neuronal cells of the CNS and ENS and support putative functions of DISP2 in these tissues.

The genetic deletion of the Dual Specificity Phosphatase 3 (DUSP3) attenuates kidney damage and inflammation following ischaemia/reperfusion injury in mouse.

AIM: Dual Specificity Phosphatase 3 (DUSP3) regulates the innate immune response, with a putative role in angiogenesis. Modulating inflammation and perfusion contributes to renal conditioning against ischaemia/reperfusion (I/R). We postulate that the functional loss of DUSP3 is associated with kidney resistance to I/R. METHODS: Ten C57BL/6 male WT and Dusp3-/- mice underwent right nephrectomy and left renal I/R (30 min/48 hours). Renal injury was assessed based on serum levels of urea (BUN) and Jablonski score. The expression of CD31 and VEGF vascular markers was quantified by RT-qPCR and immuno-staining. Renal resistivity index (RRI) was measured in vivo by Doppler ultrasound. Comparative phosphoproteomics was conducted using IMAC enrichment of phosphopeptides. Inflammatory markers were quantified at both mRNA and protein levels in ischaemic vs non-ischaemic kidneys in WT vs Dusp3-/- . RESULTS: At baseline, we located DUSP3 in renal glomeruli and endothelial cells. CD31-positive vascular network was significantly larger in Dusp3-/- kidneys compared to WT, with a lower RRI in Dusp3-/- mice. Following I/R, BUN and Jablonski score were significantly lower in Dusp3-/- vs WT mice. Phosphoproteomics highlighted a down-regulation of inflammatory pathways and up-regulation of phospho-sites involved in cell metabolism and VEGF-related angiogenesis in Dusp3-/- vs WT ischaemic kidneys. Dusp3-/- ischaemic kidneys showed decreased mRNA levels of CD11b, TNF-α, KIM-1, IL-6, IL-1ß and caspase-3 compared to controls. The numbers of PCNA-, F4-80- and CD11b-positive cells were reduced in Dusp3-/- vs WT kidneys post-I/R. CONCLUSION: Genetic inactivation of Dusp3 is associated with kidney conditioning against I/R, possibly due to attenuated inflammation and improved perfusion.

Behind every smile there's teeth: Cathepsin B's function in health and disease with a kidney view.

Cathepsin B (CatB) is a very abundant lysosomal protease with endo- and carboxydipeptidase activities and even ligase features. In this review, we will provide a general characterization of CatB and describe structure, structure-derived properties and location-dependent proteolytic actions. We depict CatB action within lysosome and its important roles in lysosomal biogenesis, lysosomal homeostasis and autophagy rendering this protease a key player in orchestrating lysosomal functions. Lysosomal leakage and subsequent escape of CatB into the cytosol lead to harmful actions, e.g. the role in activating the NLPR3 inflammasome, affecting immune responses and cell death. The second focus of this review addresses CatB functions in the kidney, i.e. the glomerulus, the proximal tubule and collecting duct with strong emphasis of its role in pathology of the respective segment. Finally, observations regarding CatB functions that need to be considered in cell culture will be discussed. In conclusion, CatB a physiologically important molecule may, upon aberrant expression in different cellular context, become a harmful player effectively showing its teeth behind its smile.

Expression Profiling of Rectal Biopsies Suggests Altered Enteric Neuropathological Traits in Parkinson's Disease Patients.

Still little is known about the nature of the gastrointestinal pathological alterations occurring in Parkinson's disease (PD). Here, we used multiplexed mRNA profiling to measure the expression of a panel of 770 genes related to neuropathological processes in deep submucosal rectal biopsies of PD patients and healthy controls. Altered enteric neuropathological traits based on the expression of 22 genes related to neuroglial and mitochondrial functions, vesicle trafficking and inflammation was observed in 9 out of 12 PD patients in comparison to healthy controls. These results provide new evidences that intestinal neuropathological alterations may occur in a large proportion of PD patients.

Alpha Synuclein Connects the Gut-Brain Axis in Parkinson's Disease Patients - A View on Clinical Aspects, Cellular Pathology and Analytical Methodology.

Parkinson's disease (PD) is marked by different kinds of pathological features, one hallmark is the aggregation of α-synuclein (aSyn). The development of aSyn pathology in the substantia nigra is associated to the manifestation of motor deficits at the time of diagnosis. However, most of the patients suffer additionally from non-motor symptoms, which may occur already in the prodromal phase of the disease years before PD is diagnosed. Many of these symptoms manifest in the gastrointestinal system (GIT) and some data suggest a potential link to the occurrence of pathological aSyn forms within the GIT. These clinical and pathological findings lead to the idea of a gut-brain route of aSyn pathology in PD. The identification of pathological aSyn in the intestinal system, e.g., by GIT biopsies, is therefore of highest interest for early diagnosis and early intervention in the phase of formation and propagation of aSyn. However, reliable methods to discriminate between physiological and pathological forms of enteral aSyn on the cellular and biochemical level are still missing. Moreover, a better understanding of the physiological function of aSyn within the GIT as well as its structure and pathological aggregation pathways are crucial to understand its role within the enteric nervous system and its spreading from the gut to the brain. In this review, we summarize clinical manifestations of PD in the GIT, and discuss biochemical findings from enteral biopsies. The relevance of pathological aSyn forms, their connection to the gut-brain axis and new developments to identify pathologic forms of aSyn by structural features are critically reviewed.

Many Patients With Irritable Bowel Syndrome Have Atypical Food Allergies Not Associated With Immunoglobulin E.

BACKGROUND & AIMS: Confocal laser endomicroscopy (CLE) is a technique that permits real-time detection and quantification of changes in intestinal tissues and cells, including increases in intraepithelial lymphocytes and fluid extravasation through epithelial leaks. Using CLE analysis of patients with irritable bowel syndrome (IBS), we found that more than half have responses to specific food components. Exclusion of the defined food led to long-term symptom relief. We used the results of CLE to detect reactions to food in a larger patient population and analyzed duodenal biopsy samples and fluid from patients to investigate mechanisms of these reactions. METHODS: In a prospective study, 155 patients with IBS received 4 challenges with each of 4 common food components via the endoscope, followed by CLE, at a tertiary medical center. Classical food allergies were excluded by negative results from immunoglobulin E serology analysis and skin tests for common food antigens. Duodenal biopsy samples and fluid were collected 2 weeks before and immediately after CLE and were analyzed by histology, immunohistochemistry, reverse transcription polymerase chain reaction, and immunoblots. Results from patients who had a response to food during CLE (CLE+) were compared with results from patients who did not have a reaction during CLE (CLE-) or healthy individuals (controls). RESULTS: Of the 108 patients who completed the study, 76 were CLE+ (70%), and 46 of these (61%) reacted to wheat. CLE+ patients had a 4-fold increase in prevalence of atopic disorders compared with controls (P = .001). Numbers of intraepithelial lymphocytes were significantly higher in duodenal biopsy samples from CLE+ vs CLE- patients or controls (P = .001). Expression of claudin-2 increased from crypt to villus tip (P < .001) and was up-regulated in CLE+ patients compared with CLE- patients or controls (P = .023). Levels of occludin were lower in duodenal biopsy samples from CLE+ patients vs controls (P = .022) and were lowest in villus tips (P < .001). Levels of messenger RNAs encoding inflammatory cytokines were unchanged in duodenal tissues after CLE challenge, but eosinophil degranulation increased, and levels of eosinophilic cationic protein were higher in duodenal fluid from CLE+ patients than controls (P = .03). CONCLUSIONS: In a CLE analysis of patients with IBS, we found that more than 50% of patients could have nonclassical food allergy, with immediate disruption of the intestinal barrier upon exposure to food antigens. Duodenal tissues from patients with responses to food components during CLE had immediate increases in expression of claudin-2 and decreases in occludin. CLE+ patients also had increased eosinophil degranulation, indicating an atypical food allergy characterized by eosinophil activation.

Altered enteric expression of the homeobox transcription factor Phox2b in patients with diverticular disease.

Background: Diverticular disease, a major gastrointestinal disorder, is associated with modifications of the enteric nervous system, encompassing alterations of neurochemical coding and of the tyrosine receptor kinase Ret/GDNF pathway. However, molecular factors underlying these changes remain to be determined. Objectives: We aimed to characterise the expression of Phox2b, an essential regulator of Ret and of neuronal subtype development, in the adult human enteric nervous system, and to evaluate its potential involvement in acute diverticulitis. Methods: Site-specific gene expression of Phox2b in the adult colon was analysed by quantitative polymerase chain reaction. Colonic specimens of adult controls and patients with diverticulitis were subjected to quantitative polymerase chain reaction for Phox2b and dual-label immunochemistry for Phox2b and the neuronal markers RET and tyrosine hydroxylase or the glial marker S100ß. Results: The results indicate that Phox2b is physiologically expressed in myenteric neuronal and glial subpopulations in the adult enteric nervous system. Messenger RNA expression of Phox2b was increased in patients with diverticulitis and both neuronal, and glial protein expression of Phox2b were altered in these patients. Conclusions: Alterations of Phox2b expression may contribute to the enteric neuropathy observed in diverticular disease. Future studies are required to characterise the functions of Phox2b in the adult enteric nervous system and to determine its potential as a therapeutic target in gastrointestinal disorders.

Genome-wide association analysis of diverticular disease points towards neuromuscular, connective tissue and epithelial pathomechanisms.

OBJECTIVE: Diverticular disease is a common complex disorder characterised by mucosal outpouchings of the colonic wall that manifests through complications such as diverticulitis, perforation and bleeding. We report the to date largest genome-wide association study (GWAS) to identify genetic risk factors for diverticular disease. DESIGN: Discovery GWAS analysis was performed on UK Biobank imputed genotypes using 31 964 cases and 419 135 controls of European descent. Associations were replicated in a European sample of 3893 cases and 2829 diverticula-free controls and evaluated for risk contribution to diverticulitis and uncomplicated diverticulosis. Transcripts at top 20 replicating loci were analysed by real-time quatitative PCR in preparations of the mucosal, submucosal and muscular layer of colon. The localisation of expressed protein at selected loci was investigated by immunohistochemistry. RESULTS: We discovered 48 risk loci, of which 12 are novel, with genome-wide significance and consistent OR in the replication sample. Nominal replication (p<0.05) was observed for 27 loci, and additional 8 in meta-analysis with a population-based cohort. The most significant novel risk variant rs9960286 is located near CTAGE1 with a p value of 2.3×10-10 and 0.002 (ORallelic=1.14 (95% CI 1.05 to 1.24)) in the replication analysis. Four loci showed stronger effects for diverticulitis, PHGR1 (OR 1.32, 95% CI 1.12 to 1.56), FAM155A-2 (OR 1.21, 95% CI 1.04 to 1.42), CALCB (OR 1.17, 95% CI 1.03 to 1.33) and S100A10 (OR 1.17, 95% CI 1.03 to 1.33). CONCLUSION: In silico analyses point to diverticulosis primarily as a disorder of intestinal neuromuscular function and of impaired connective fibre support, while an additional diverticulitis risk might be conferred by epithelial dysfunction.

Persistent Increased Enteric Glial Expression of S100ß is Associated With Low-grade Inflammation in Patients With Diverticular Disease.

BACKGROUND: Diverticular disease (DD) is a common gastrointestinal inflammatory disorder associated with an enteric neuropathy. Although enteric glial cells (EGCs) are essential regulators of intestinal inflammation and motility functions, their contribution to the pathophysiology of DD remains unclear. Therefore, we analyzed the expression of specific EGC markers in patients with DD. MATERIALS AND METHODS: Expression of the glial markers S100ß, GFAP, Sox10, and Connexin 43 was analyzed by real-time quantitative PCR in colonic specimens of patients with DD and in that of controls. Protein expression levels of S100ß, GFAP, and Connexin 43 were further analyzed using immunohistochemistry in the submucosal and myenteric plexus of patients with DD and in that of controls. Expression of the inflammatory cytokines tumor necrosis factor-α and interleukin-6 was quantified using qPCR, and infiltration of CD3+ lymphocytes was determined using immunohistochemistry. RESULTS: Expression of S100ß was increased in the submucosal and myenteric plexus of patients with DD compared with that in controls, whereas expression of other glial factors remained unchanged. This increased expression of S100ß was correlated to CD3+ lymphocytic infiltrates in patients with DD, whereas no correlation was observed in controls. CONCLUSIONS: DD is associated with limited but significant alterations of the enteric glial network. The increased expression of S100ß is associated with a persistent low-grade inflammation reported in patients with DD, further emphasizing the role of EGCs in intestinal inflammation.

Impaired Expression of Neuregulin 1 and Nicotinic Acetylcholine Receptor ß4 Subunit in Diverticular Disease.

Neuregulin 1 (NRG1) regulates the expression of the nicotinic acetylcholine receptor (nAChR) and is suggested to promote the survival and maintenance of the enteric nervous system (ENS), since deficiency of its corresponding receptor complex ErbB2/ErbB3 leads to postnatal colonic aganglionosis. As diverticular disease (DD) is associated with intestinal hypoganglionosis, the NRG1-ErbB2/ErbB3 system and the nAChR were studied in patients with DD and controls. Samples of tunica muscularis of the sigmoid colon from patients with DD (n = 8) and controls (n = 11) were assessed for mRNA expression of NRG1, ErbB2, and ErbB3 and the nAChR subunits α3, α5, α7, ß2, and ß4. Site-specific gene expression levels of the NRG1-ErbB2/3 system were determined in myenteric ganglia harvested by laser microdissection (LMD). Localization studies were performed by immunohistochemistry for the NRG1-ErbB2/3 system and nAChR subunit ß4. Rat enteric nerve cell cultures were stimulated with NRG1 or glial-cell line derived neurotrophic factor (GDNF) for 6 days and mRNA expression of the aforementioned nAchR was measured. NRG1, ErbB3, and nAChR subunit ß4 expression was significantly down-regulated in both the tunica muscularis and myenteric ganglia of patients with DD compared to controls, whereas mRNA expression of ErbB3 and nAChR subunits ß2, α3, α5, and α7 remained unaltered. NRG1, ErbB3, and nAChR subunit ß4 immunoreactive signals were reduced in neuronal somata and the neuropil of myenteric ganglia from patients with DD compared to control. nAChR subunit ß4 exhibited also weaker immunoreactive signals in the tunica muscularis of patients with DD. NRG1 treatment but not GDNF treatment of enteric nerve cell cultures significantly enhanced mRNA expression of nAchR ß4. The down-regulation of NRG1 and ErbB3 in myenteric ganglia of patients with DD supports the hypothesis that intestinal hypoganglionosis observed in DD may be attributed to a lack of neurotrophic factors. Regulation of nAChR subunit ß4 by NRG1 and decreased nAChR ß4 in patients with DD provide evidence that a lack of NRG1 may affect the composition of enteric neurotransmitter receptor subunits thus contributing to the intestinal motility disorders previously reported in DD.

Laser-Capture Microdissection for Layer-Specific Analysis of Enteric Ganglia.

The enteric nervous system (ENS) is the division of the autonomic nervous system that innervates the gastrointestinal (GI) tract and controls central intestinal functions such as peristalsis and fluid movement. Enteric nerve cell bodies (neurons and glia) are predominantly organized in ganglionated networks that are present along the entire length of the GI tract in multiple tissue layers. Most cell bodies are organized in the myenteric plexus allocated between the longitudinal and the circular muscle layers or in the submucosal plexus between muscle tissue and mucosa. The site-specific characteristics of these enteric nerve cells have traditionally been analyzed via imaging techniques. Laser-capture microdissection (LCM) offers the prospect of site-specifically analyzing the gene expression profiles of these different subpopulations. This protocol addresses critical aspects of handling intestinal tissue for ENS dissection, such as the optimal quick-staining procedure, suitable laser settings, and limits of tissue material required to successfully dissect and analyze tissue layers for gene expression.

No neuronal loss, but alterations of the GDNF system in asymptomatic diverticulosis.

BACKGROUND: Glial cell line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor known to promote the survival and maintenance of neurons not only in the developing but also in the adult enteric nervous system. As diverticular disease (DD) is associated with reduced myenteric neurons, alterations of the GDNF system were studied in asymptomatic diverticulosis (diverticulosis) and DD. METHODS: Morphometric analysis for quantifying myenteric ganglia and neurons were assessed in colonic full-thickness sections of patients with diverticulosis and controls. Samples of tunica muscularis (TM) and laser-microdissected myenteric ganglia from patients with diverticulosis, DD and controls were analyzed for mRNA expression levels of GDNF, GFRA1, and RET by RT-qPCR. Myenteric protein expression of both receptors was quantified by fluorescence-immunohistochemistry of patients with diverticulosis, DD, and controls. RESULTS: Although no myenteric morphometric alterations were found in patients with diverticulosis, GDNF, GFRA1 and RET mRNA expression was down-regulated in the TM of patients with diverticulosis as well as DD. Furthermore GFRA1 and RET myenteric plexus mRNA expression of patients with diverticulosis and DD was down-regulated, whereas GDNF remained unaltered. Myenteric immunoreactivity of the receptors GFRα1 and RET was decreased in both asymptomatic diverticulosis and DD patients. CONCLUSION: Our data provide evidence for an impaired GDNF system at gene and protein level not only in DD but also during early stages of diverticula formation. Thus, the results strengthen the idea of a disturbed GDNF-responsiveness as contributive factor for a primary enteric neuropathy involved in the pathogenesis and disturbed intestinal motility observed in DD.

Establishment and Characterization of an SV40 Large T Antigen-Transduced Porcine Colonic Epithelial Cell Line.

Continuous cell lines have become indispensable tools that have enabled investigations into cellular mechanisms by increasing experimental reproducibility and sample availability, and decreasing the use of experimental animals. To facilitate studies of epithelial barrier function of the porcine colon, we aimed to establish an epithelial cell line with an extended replicative capacity. Cells were isolated from the proximal colon of a 3-week-old piglet and transduced using a recombinant retroviral vector construct containing the simian virus 40 large T antigen (SV40 TAg). We established a clonal epithelial cell line, referred to as PoCo83-3, that stably expressed the SV40 TAg, verified at mRNA and protein levels. PoCo83-3 showed epithelial cell-specific features, such as cobblestone-like morphology, dome structure formation, the presence of apical microvilli, and the expression of keratin 18, E-cadherin and the tight junction-associated proteins zonula occludens-1, occludin, and claudin-1. To validate PoCo83-3 as an in vitro model in epithelial barrier research, proinflammatory cytokine-inducible alterations in barrier integrity were demonstrated by incubating the cells with TNF-α and IFN-γ for 48 h. These cytokine treatments promoted a decreased transepithelial electrical resistance. In summary, PoCo83-3 exhibited an extended life span and a differentiated phenotype while maintaining epithelial characteristics. Based on these results, we present this cell line as a valuable in vitro model for investigations of epithelial barrier function in the porcine colon.

Distinct pattern of enteric phospho-alpha-synuclein aggregates and gene expression profiles in patients with Parkinson's disease.

Phosphorylated alpha-synuclein (p-α-syn) containing Lewy bodies (LBs) and Lewy neurites (LNs) are neuropathological hallmarks of Parkinson's disease (PD) in the central nervous system (CNS). Since they have been also demonstrated in the enteric nervous system (ENS) of PD patients, the aim of the study was to analyze enteric p-α-syn positive aggregates and intestinal gene expression. Submucosal rectal biopsies were obtained from patients with PD and controls and processed for dual-label-immunohistochemistry for p-α-syn and PGP 9.5. p-α-syn positive aggregates in nerve fibers and neuronal somata were subjected to a morphometric analysis. mRNA expression of α-syn and dopaminergic, serotonergic, VIP (vaso intestinal peptide) ergic, cholinergic, muscarinergic neurotransmitter systems were investigated using qPCR. Frequency of p-α-syn positive nerve fibers was comparable between PD and controls. Although neuronal p-α-syn positive aggregates were detectable in both groups, total number and area of p-α-syn positive aggregates were increased in PD patients as was the number of small and large sized aggregates. Increased expression of dopamine receptor D1, VIP and serotonin receptor 3A was observed in PD patients, while serotonin receptor 4 and muscarinic receptor 3 (M3R) were downregulated. M3R expression correlated negative with the number of small sized p-α-syn positive aggregates. The findings strengthen the hypothesis that the CNS pathology of increased p-α-syn in PD also applies to the ENS, if elaborated morphometry is applied and give further insights in altered intestinal gene expression in PD. Although the mere presence of p-α-syn positive aggregates in the ENS should not be regarded as a criterion for PD diagnosis, elaborated morphometric analysis of p-α-syn positive aggregates in gastrointestinal biopsies could serve as a suitable tool for in-vivo diagnosis of PD.

A Tunable Scaffold of Microtubular Graphite for 3D Cell Growth.

Aerographite (AG) is a novel carbon-based material that exists as a self-supportive 3D network of interconnected hollow microtubules. It can be synthesized in a variety of architectures tailored by the growth conditions. This flexibility in creating structures presents interesting bioengineering possibilities such as the generation of an artificial extracellular matrix. Here we have explored the feasibility and potential of AG as a scaffold for 3D cell growth employing cyclic RGD (cRGD) peptides coupled to poly(ethylene glycol) (PEG) conjugated phospholipids for surface functionalization to promote specific adhesion of fibroblast cells. Successful growth and invasion of the bulk material was followed over a period of 4 days.

Cardiomyocyte behavior on biodegradable polyurethane/gold nanocomposite scaffolds under electrical stimulation.

Following a myocardial infarction (MI), cardiomyocytes are replaced by scar tissue, which decreases ventricular contractile function. Tissue engineering is a promising approach to regenerate such damaged cardiomyocyte tissue. Engineered cardiac patches can be fabricated by seeding a high density of cardiac cells onto a synthetic or natural porous polymer. In this study, nanocomposite scaffolds made of gold nanotubes/nanowires incorporated into biodegradable castor oil-based polyurethane were employed to make micro-porous scaffolds. H9C2 cardiomyocyte cells were cultured on the scaffolds for one day, and electrical stimulation was applied to improve cell communication and interaction in neighboring pores. Cells on scaffolds were examined by fluorescence microscopy and scanning electron microscopy, revealing that the combination of scaffold design and electrical stimulation significantly increased cell confluency of H9C2 cells on the scaffolds. Furthermore, we showed that the gene expression levels of Nkx2.5, atrial natriuretic peptide (ANF) and natriuretic peptide precursor B (NPPB), which are functional genes of the myocardium, were up-regulated by the incorporation of gold nanotubes/nanowires into the polyurethane scaffolds, in particular after electrical stimulation.

Expression and function of Neuregulin 1 and its signaling system ERBB2/3 in the enteric nervous system.

Neuregulin 1 (NRG1) is suggested to promote the survival and maintenance of the enteric nervous system (ENS). As deficiency in its corresponding receptor signaling complex ERBB2/ERBB3 leads to postnatal colonic hypo/aganglionosis we assessed the distributional and expressional pattern of the NRG1-ERBB2/ERBB3 system in the human colon and explored the neurotrophic capacity of NRG1 on cultured enteric neurons. Site-specific mRNA expression of the NRG1-ERBB2/3 system was determined in microdissected samples harvested from enteric musculature and ganglia. Localization of NRG1, ERBB2 and ERBB3 was determined by dual-label-immunohistochemistry using pan-neuronal and pan-glial markers. Morphometric analysis was performed on NRG1-stimulated rat enteric nerve cultures to evaluate neurotrophic effects. mRNA expression of the NRG1-ERBB2/3 system was determined by qPCR. Co-localization of NRG1 with neuronal or synaptic markers was analyzed in enteric nerve cultures stimulated with glial cell line-derived neurotrophic factor (GDNF). The NRG1 system was expressed in both neurons and glial cells of enteric ganglia and in nerve fibers. NRG1 significantly enhanced growth parameters in enteric nerve cell cultures and ErB3 mRNA expression was down-regulated upon NRG1 stimulation. GDNF negatively regulates ErbB2 and ErbB3 mRNA expression. The NRG1-ERBB2/3 system is physiologically present in the human ENS and NRG1 acts as a neurotrophic factor for the ENS. The down-regulation of ErbB3/ErbB2 in GDNF stimulated nerve cell cultures points to an interaction of both neurotrophic factors. Thus, the data may provide a basis to assess disturbed signaling components of the NRG1 system in enteric neuropathies.

[Diverticular disease - new insights into pathogenesis]. / Divertikelkrankheit - Neues zur Pathogenese.

Diverticular disease is associated with a high incidence, morbidity and burden of the healthcare system. However, the pathogenesis is not yet satisfactorily clarified and thought to be multifactorial. Non-influenceable risk factors include increasing age, genetic predisposition and rare congenital connective tissue diseases. Influenceable risk factors are low-fiber diet, increased meat consumption and obesity. Alterations of connective tissue lead to a weakening of preformed emergence sites of diverticula ("loci minoris resistentiae") and may explain the increased incidence of diverticular disease in diseases caused by a systematic connective tissue disorder. The impact of neuromuscular alterations and disturbed colonic motility on triggering diverticula formation has been previously underestimated. Moreover, intestinal innervation disorders are considered to be responsable for persisting recurrent pain symptoms in chronic diverticular disease.

Alpha-synuclein is associated with the synaptic vesicle apparatus in the human and rat enteric nervous system.

BACKGROUND AND AIMS: Aggregation of alpha-synuclein (a-syn) has been implicated in the development of neurodegenerative diseases including its spread from the enteric nervous system (ENS) to the brain. Physiologically, a-syn is located at the presynapse and might be involved in regulating of neurotransmission. Therefore, the aim of the study was to characterize the physiological ontogenetic and locoregional expression pattern of a-syn in the ENS and its association with the synaptic vesicle apparatus. MATERIAL AND METHODS: Ontogenetic mRNA expression of a-syn and synaptophysin was determined in the rat intestine. Myenteric plexus cultures treated with glial cell line-derived neurotrophic factor (GDNF) were assessed for mRNA expression of a-syn, co-localization of a-syn with the pan-neuronal marker PGP 9.5 and the synaptic vesicle marker synaptophysin and studied by scanning electron microscopy (SEM). Human colonic specimens were subjected to co-localization studies of a-syn with synaptophysin. RESULTS: a-syn and synaptophysin intestinal gene expression levels were highest during early postnatal life and also detectable at adult age. a-syn was co-localized with PGP 9.5 and synaptophysin in myenteric plexus cultures and up-regulated after GDNF treatment. SEM confirmed the presence of neuronal varicosities to which a-syn was associated. Consistently, a-syn and synaptophysin showed partial co-localization in the human ENS. CONCLUSIONS: The ontogenetic and cellular expression pattern as well as the regulation by GNDF give evidence that a-syn is physiologically associated to the synaptic vesicle apparatus. The data suggest that a-syn is involved in the regulation of synaptic plasticity in the ENS during early postnatal life and adult age.