In humans, pruritus (itch) is a common but poorly understood symptom in numerous skin and systemic diseases. Endothelin 1 (ET-1) evokes histamine-independent pruritus in mammals through activation of ...its cognate G protein-coupled receptor endothelin A receptor (ETAR). Here, we have identified neural endothelin-converting enzyme 1 (ECE-1) as a key regulator of ET-1-induced pruritus and neural signaling of itch. We show here that ETAR, ET-1, and ECE-1 are expressed and colocalize in murine dorsal root ganglia (DRG) neurons and human skin nerves. In murine DRG neurons, ET-1 induced internalization of ETAR within ECE-1-containing endosomes. ECE-1 inhibition slowed ETAR recycling yet prolonged ET-1-induced activation of ERK1/2, but not p38. In a murine itch model, ET-1-induced scratching behavior was substantially augmented by pharmacological ECE-1 inhibition and abrogated by treatment with an ERK1/2 inhibitor. Using iontophoresis, we demonstrated that ET-1 is a potent, partially histamine-independent pruritogen in humans. Immunohistochemical evaluation of skin from prurigo nodularis patients confirmed an upregulation of the ET-1/ETAR/ECE-1/ERK1/2 axis in patients with chronic itch. Together, our data identify the neural peptidase ECE-1 as a negative regulator of itch on sensory nerves by directly regulating ET-1-induced pruritus in humans and mice. Furthermore, these results implicate the ET-1/ECE-1/ERK1/2 pathway as a therapeutic target to treat pruritus in humans.
Introduction
Neuroendocrine neoplasms (NEN) are rare and heterogeneous epithelial tumors, occurring throughout the body. For gastroenteropancreatic (GEP)‐NEN, rising incidence rates were reported for ...the last decades, with underlying causes remaining largely unexplained. We evaluated NEN trends stratifying by their histologic subtypes.
Methods
Incident cases of GEP‐NEN diagnosed between 2005 and 2019 were retrieved from the prospective, population‐based Bavarian Cancer Registry. GEP‐NEN were divided in their histologic subtypes, that is, neuroendocrine tumors (NET) G1, NET G2/G3, other NET versus small‐cell neuroendocrine carcinoma (NEC), large‐cell NEC, and other NEC. We calculated annual age‐standardized incidence rates (ASIRs) per 100,000 persons for the total of GEP‐NEN, NEN histologic subtypes, and tumor sites. We used an annual percentage change (APC) approach including a joinpoint analysis to investigate NEN incidence trends.
Results
ASIR of GEP‐NEN rose from 2.2 in 2005 to 4.8 in 2019, characterized by a significant increase until 2012 (APC 2005–2012: 10.1%), followed by modest rise (APC 2012–2019: 1.5%). In the last decade, this increase was mainly driven by the rise of NET G1 and G2/G3, while incidence for NEC declined. Over the study period, ASIR increased significantly for all GEP‐sites except the colon. APCs were largest for the stomach, the appendix, the pancreas, and the rectum.
Conclusions
This study found a significant increase in the incidence of GEP‐NET. Though this development may partially be attributable to the increased use of advanced detection techniques and changes in NEN classification, further research should also focus on the identification of NEN risk factors.
Incidence trends of gastroenteropancreatic neuroendocrine neoplasms (GEP‐NEN) between 2005 and 2019 were analyzed, stratified by cancer site and by histologic subtype. Findings indicated a significant increase in age‐standardized incidence rates for all GEP‐sites with exception of the colon. The increase was mainly driven by the rise of neuroendocrine tumors, while neuroendocrine carcinoma declined.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
A trial fibrillation (AF), the most common human cardiac arrhythmia, is associated with abnormal intracellular Ca2+ handling. Diastolic Ca2+ release from the sarcoplasmic reticulum via "leaky" ...ryanodine receptors (RyR2s) is hypothesized to contribute to arrhythmogenesis in AF, but the molecular mechanisms are incompletely understood. Here, we have shown that mice with a genetic gain-of-function defect in Ryr2 (which we termed Ryr2R176Q/+ mice) did not exhibit spontaneous AF but that rapid atrial pacing unmasked an increased vulnerability to AF in these mice compared with wild-type mice. Rapid atrial pacing resulted in increased Ca2+/calmodulin-dependent protein kinase II (CaMKII) phosphorylation of RyR2, while both pharmacologic and genetic inhibition of CaMKII prevented AF inducibility in Ryr2R176Q/+ mice. This result suggests that AF requires both an arrhythmogenic substrate (e.g., RyR2 mutation) and enhanced CaMKII activity. Increased CaMKII phosphorylation of RyR2 was observed in atrial biopsies from mice with atrial enlargement and spontaneous AF, goats with lone AF, and patients with chronic AF. Genetic inhibition of CaMKII phosphorylation of RyR2 in Ryr2S2814A knockin mice reduced AF inducibility in a vagotonic AF model. Together, these findings suggest that increased RyR2-dependent Ca2+ leakage due to enhanced CaMKII activity is an important downstream effect of CaMKII in individuals susceptible to AF induction.
Directed cardiac differentiation of human pluripotent stem cells (hPSCs) enables disease modeling, investigation of human cardiogenesis, as well as large‐scale production of cardiomyocytes (CMs) for ...translational purposes. Multiple CM differentiation protocols have been developed to individually address specific requirements of these diverse applications, such as enhanced purity at a small scale or mass production at a larger scale. However, there is no universal high‐efficiency procedure for generating CMs both in two‐dimensional (2D) and three‐dimensional (3D) culture formats, and undefined or complex media additives compromise functional analysis or cost‐efficient upscaling. Using systematic combinatorial optimization, we have narrowed down the key requirements for efficient cardiac induction of hPSCs. This implied differentiation in simple serum and serum albumin‐free basal media, mediated by a minimal set of signaling pathway manipulations at moderate factor concentrations. The method was applicable both to 2D and 3D culture formats as well as to independent hPSC lines. Global time‐course gene expression analyses over extended time periods and in comparison with human heart tissue were used to monitor culture‐induced maturation of the resulting CMs. This suggested that hPSC‐CMs obtained with our procedure reach a rather stable transcriptomic state after approximately 4 weeks of culture. The underlying gene expression changes correlated well with a decline of immature characteristics as well as with a gain of structural and physiological maturation features within this time frame. These data link gene expression patterns of hPSC‐CMs to functional readouts and thus define the cornerstones of culture‐induced maturation. Stem Cells 2015;33:1456–1469
Fluoropyrimidine c (5-fluorouracil 5FU) increasingly represents the chemotherapeutic backbone for neoadjuvant, adjuvant, and palliative treatment of pancreatic ductal adenocarcinoma (PDAC). Even in ...combination with other agents, 5FU efficacy remains transient and limited. One explanation for the inadequate response is insufficient and nonspecific delivery of 5FU to the tumor.
We designed, generated, and characterized 5FU-incorporated systematic evolution of ligands by exponential enrichment (SELEX)–selected epidermal growth factor receptor (EGFR)-targeted aptamers for tumor-specific delivery of 5FU to PDAC cells and tested their therapeutic efficacy in vitro and in vivo.
5FU-EGFR aptamers reduced proliferation in a concentration-dependent manner in mouse and human pancreatic cancer cell lines. Time-lapsed live imaging showed EGFR-specific uptake of aptamers via clathrin-dependent endocytosis. The 5FU-aptamer treatment was equally effective in 5FU-sensitive and 5FU-refractory PDAC cell lines. Biweekly treatment with 5FU-EGFR aptamers reduced tumor burden in a syngeneic orthotopic transplantation model of PDAC, in an autochthonously growing genetically engineered PDAC model (LSL-KrasG12D/+;LSL-Trp53flox/+;Ptf1a-Cre KPC), in an orthotopic cell line–derived xenograft model using human PDAC cells in athymic mice (CDX; Crl:NU-Foxn1nu), and in patient-derived organoids. Tumor growth was significantly attenuated during 5FU-EGFR aptamer treatment in the course of follow-up.
Tumor-specific targeted delivery of 5FU using EGFR aptamers as the carrier achieved high target specificity; overcame 5FU resistance; and proved to be effective in a syngeneic orthotopic transplantation model, in KPC mice, in a CDX model, and in patient-derived organoids and, therefore, represents a promising backbone for pancreatic cancer chemotherapy in patients. Furthermore, our approach has the potential to target virtually any cancer entity sensitive to 5FU treatment by incorporating 5FU into cancer cell–targeting aptamers as the delivery platform.
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Neuroendocrine tumours (NET) of the gastroenteropancreatic system comprise a malignant entity with a low incidence. Only limited information is available on long-term clinical outcome and clinically ...applicable prognostic factors. We performed a retrospective analysis of a large, well-characterized centre-based patient cohort of 399 patients with histologically proven NET. Data were analysed according to epidemiological, clinical and histopathological characteristics. Detailed survival analyses using the Kaplan–Meier method were performed. Prognostic factors were tested by log-rank testing and independent risk factors were analysed using a Cox regression model. In the studied cohort, primary tumours originated in the fore-, mid- and hindgut in 46.1, 37.1 and 4.5% respectively. Extra-intestinal or unknown primary tumours were present in 8.4 and 10.5% respectively. Distant metastasis was present at initial diagnosis in 69.4%. Most frequent metastatic sites were liver (85%), peritoneal cavity (18%), bones (8%), other intra-abdominal sites (6%) and lungs (4%). Overall, 5- and 10-year survival rates were 78 and 63% respectively. Time to progression after initial diagnosis was significantly shorter in pancreatic as compared with ileal NET. Survival analysis revealed significantly better clinical outcome for primary tumours smaller than 25 mm, absence of metastasis, absence of any clinical symptoms, positive immunohistochemical staining for chromogranin A and a lower Ki67 index. These results were confirmed as independent by multivariate analysis. Therefore, this large retrospective analysis of a well-documented cohort of patients with NET demonstrates several prognostic factors of clinical relevance and wide availability, which should be considered for risk stratification in the management of NET.
BACKGROUND:A structural, electrical and metabolic atrial remodeling is central in the development of atrial fibrillation (AF) contributing to its initiation and perpetuation. In the heart, HDACs ...(histone deacetylases) control remodeling associated processes like hypertrophy, fibrosis, and energy metabolism. Here, we analyzed, whether the HDAC class I/IIa inhibitor valproic acid (VPA) is able to attenuate atrial remodeling in CREM-IbΔC-X (cAMP responsive element modulator isoform IbΔC-X) transgenic mice, a mouse model of extensive atrial remodeling with age-dependent progression from spontaneous atrial ectopy to paroxysmal and finally long-lasting AF.
METHODS:VPA was administered for 7 or 25 weeks to transgenic and control mice. Atria were analyzed macroscopically and using widefield and electron microscopy. Action potentials were recorded from atrial cardiomyocytes using patch-clamp technique. ECG recordings documented the onset of AF. A proteome analysis with consecutive pathway mapping identified VPA-mediated proteomic changes and related pathways.
RESULTS:VPA attenuated many components of atrial remodeling that are present in transgenic mice, animal AF models, and human AF. VPA significantly (P<0.05) reduced atrial dilatation, cardiomyocyte enlargement, atrial fibrosis, and the disorganization of myocyte’s ultrastructure. It significantly reduced the occurrence of atrial thrombi, reversed action potential alterations, and finally delayed the onset of AF by 4 to 8 weeks. Increased histone H4-acetylation in atria from VPA-treated transgenic mice verified effective in vivo HDAC inhibition. Cardiomyocyte-specific genetic inactivation of HDAC2 in transgenic mice attenuated the ultrastructural disorganization of myocytes comparable to VPA. Finally, VPA restrained dysregulation of proteins in transgenic mice that are involved in a multitude of AF relevant pathways like oxidative phosphorylation or RhoA (Ras homolog gene family, member A) signaling and disease functions like cardiac fibrosis and apoptosis of muscle cells.
CONCLUSIONS:Our results suggest that VPA, clinically available, well-tolerated, and prescribed to many patients for years, has the therapeutic potential to delay the development of atrial remodeling and the onset of AF in patients at risk.
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