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Targeted expression of plasminogen activator inhibitor(PAI)-1 to the stomach inhibits gut-brain signalling by the satiety hormone cholecystokinin (CCK)
No full textEnergy homeostasis is a tightly regulated system that is vital for survival involving anorectic and orexigenic signals. Obesity is a maladaptive response where the balance becomes disrupted. Obesity is one of the most concerning health problems of our time. It is no longer considered a consequence of a western lifestyle, with more developing countries now reporting an increased incidence of obesity and associated illnesses. While obesity itself can be debilitating and decrease quality of life, it is the associated comorbidities that are the main cause for concern; including type two diabetes, cancer and thrombo-occlusive diseases. One of the molecules thought to be responsible for occlusive events is plasminogen activator inhibitor (PAI)-1. This inhibitor of the plasminogen system is also reported to be up to 5 fold higher in obese subjects in plasma, and similar to leptin, is released from adipose tissue. PAI-1 is considered to play a protective role in circumstances of gastric mucosal attack, thus a transgenic mouse (PAI-1HKβ) was generated, with targeted expression of PAI-1 to the gastric parietal cells, to study this. However, an unexpected phenotype emerged, most notably hyperphagia and increased body weight, which formed the basis of these present studies.
The gut-brain axis is a major and well-studied regulator of energy homeostasis and this was the focus of this project. The PAI-1HKβ mice when compared to wild-type had decreased brain stem responses to the satiety hormone, Cholecystokinin (CCK). Brainstem responses were also attenuated in wild types pre-treated with exogenous PAI-1. Furthermore, it was shown that the urokinase plasminogen activator (uPA) receptor by which PAI-1 binds, was required to influence the observed decrease in brainstem responses.
CCK also has other physiological functions in the role of energy homeostasis, including gastric emptying. While delayed gastric emptying was observed following a protein rich liquid test meal in C57BL/6 mice, PAI-1HKβ mice had a blunted response. Blockade of the CCK1 receptor in C57BL/6 mice also attenuated the delay in gastric emptying. Moreover, exogenous PAI-1 attenuated CCK-mediated inhibition of gastric emptying. The PAI-1HKβ mice had an attenuated inhibition of gastric emptying of a non-nutrient containing liquid test meal in response to CCK. Treatment with gastrin was shown to increase plasma PAI-1 and attenuated delayed gastric emptying in C57BL/6 mice.
Food intake is stimulated by orexigens, most notably ghrelin, working via appetite-stimulating neurons in the arcuate nucleus. While ghrelin stimulated feeding in fed ad libitum C57BL/6 mice, PAI-1 increased feeding in previously fasted C57BL/6 mice only. This response to ghrelin and PAI-1 was also replicated in PAI-1 -/- mice, suggesting PAI-1 is not required for the orexigenic effect of ghrelin. Moreover, intrapertoneal (ip.) administered ghrelin increased fos expression in arcuate neurons of both C57BL/6 and PAI-1 -/- mice, whereas ip. PAI-1 did not.
Weight loss in the PAI-1HKβ mice appeared to reverse the insensitivity to CCK in terms of gastric emptying. PAI-1HKβ mice were also found to be insensitive to other gut-derived satiety hormones, suggesting gastric PAI-1 is an anti-satiety factor. However, mice null for wild-type gastric PAI-1 responded normally to CCK prior to feeding, indicating that wild type is necessary for CCK insensitivity in the PAI-1HKβ mice.
The current findings demonstrate that PAI-1 plays a role in the control of food intake. PAI-1 is an example of a novel anti-satiety factor that can modulate gut-brain signalling via the vagus nerve in order to preserve nutrient intake. This work provides a platform for future investigations into novel pathways implicated in the development and treatment of obesity
The neuroendocrine-like phenotype of gastric myofibroblasts and its significance in cancer.
No full textCell-cell communication, and specifically epithelial-mesenchymal signalling, is a key factor determining normal tissue development and organisation in hollow organs such as gastrointestinal tract. Mechanisms governing normal epithelial-mesenchymal communication have been studied for many years, but the changes occurring during tissue damage, infection and inflammation, and in cancer, remain unclear. Myofibroblasts are recognised as key mesenchymal cells involved in this communication in health and in disease. Myofibroblasts produce and secrete many proteins responsible for the assembly of extracellular matrix (ECM), tissue organisation and morphogenesis. Initial studies from this laboratory suggested that myofibroblasts might exhibit regulated secretion. The aim of this project was to determine the secretory mechanisms of myofibroblasts from the upper gut of normal and cancer tissues, and to address their significance in cancer.
Gastric myofibroblasts were shown to exhibit calcium-dependent secretion of the small ECM protein, Transforming growth factor-β inducible protein or TGFβig-h3, in response to acute stimulation (30 min) with insulin-like growth factor (IGF)s. Inhibitors of protein synthesis (actinomycin D and cycloheximide) and of protein transport from endoplasmic reticulum (ER) to Golgi (brefeldin A) inhibited basal, but not IGF-stimulated secretion, as seen from Western blot analyses of media. These observations support the idea that evoked secretion occurs from a pre-formed pool of vesicles.
Myofibroblasts from the upper gut showed differences in their secretory phenotype; specifically normal myofibroblasts from stomach exhibited regulated secretion, but their counterparts from oesophagus did not. Moreover, gastric cancer-associated myofibroblasts (CAMs) from patients with poor survival tend not to exhibit regulated secretion. These findings suggest a role for the tissue microenvironment in determining the secretory phenotype of myofibroblasts.
Secretome-wide analysis of myofibroblasts media collected after IGF stimulation revealed that about 85% of the secretome exhibits evoked release. The relevant proteins belonged to different classes including ECM proteins, ligands, binding proteins, carbohydrate-binding proteins, proteases and protease inhibitors. These data indicate that myofibroblasts may contribute to tissue organisation by rapid release of substances involved in re-modelling the tissue microenvironment.
The regulated secretory phenotype of myofibroblasts was associated with the expression of the chromogranin-like protein, secretogranin II. Knock-down of secretogranin II inhibited regulated secretion, whereas over-expression in myofibroblasts that lacked regulated released - induced it. Expression of secretogranin II by myofibroblasts coincided with the expression of dense core secretory vesicles that were similar to those found in neuroendocrine cells.
This work indicates that there is a neuroendocrine-like secretory phenotype in myofibroblasts, as illustrated by the expression of neuroendocrine cell protein secretogranin II and the presence of regulated secretion. However, not all normal myofibroblasts exhibit the regulated phenotype, and in gastric cancer the phenotype correlates with early stage of disease. These findings may be exploitable both in the development of novel therapies and in understanding cancer progression
Novel sites of CCK2R expression: Characterisation and functional significance of gastrin receptor expression in gastrointestinal myofibroblasts and human melanoma cancer cells
Full text linkMicroRNAs in gastric and oesophageal cancer-associated myofibroblasts
No full textMicroRNAs (miRNAs) are small RNAs of ~22 nucleotides in length that regulate gene expression post-transcriptionally. MicroRNAs regulate many biological processes, and may contribute to cancer initiation and progression. In the normal gastrointestinal mucosa, myofibroblasts are involved in wound healing, and in tumours, they play a role in cancer progression. The present study aimed to determine the miRNA expression profiles in different populations of myofibroblasts from gastric and oesophageal tissues, namely, cancer-associated myofibroblasts (CAMs), adjacent non-tumour tissue myofibroblasts (ATMs) and normal tissue myofibroblasts (NTMs), and to investigate the processes that are influenced by differential miRNA abundance in different myofibroblast populations. In addition, miRNA profiling of a putative myofibroblast precursor cell type, mesenchymal stromal cells (MSCs), was studied.
Global miRNA expression profiling of a panel of previously characterised gastric and oesophageal myofibroblasts was determined using locked nucleic acid (LNA) miRNA arrays. The microarray data for hsa-miR-29b, hsa-miR-181d, hsa-miR-214 and hsa-miR-424 were validated by quantitative RT-PCR. Using unsupervised miRNA datasets, principal component analysis (PCA) segregated gastric and oesophageal NTMs, and CAMs from gastric and oesophageal carcinomas. Analysis of global miRNA expression showed distinct profile of MSCs in comparison to gastric and oesophageal NTMs. Hierarchical clustering analysis of miRNAs exhibiting significantly different abundance revealed distinct clusters between CAMs and ATMs, CAMs and NTMs and between ATMs and NTMs.
In 12 paired samples of gastric CAMs and their corresponding ATMs, 12 miRNAs were significantly different with hsa-miR-181d being the most up-regulated miRNA in CAMs. Using already validated targets of 10 of these miRNAs, an analysis using MetaCore® indicated that the regulation of cell cycle progression and Wnt signalling were differentially affected. In a comparison of each CAM and its corresponding ATM, Wnt signalling was the only process that was significantly targeted by miRNAs that were different in abundance, in all 12 pairs.
Using previously determined gene expression data, pairwise analysis of 20 transcripts associated with Wnt signalling showed a significant increase in WNT5A and TGFB2 mRNAs in CAMs compared to their ATMs. Additionally, Western blot analysis showed increased expression of Wnt-5a in CAMs. The results of migration and EdU incorporation assays indicated that Wnt-5a induced the migration and proliferation of both CAMs and ATMs. The abundance of hsa-miR-181d was significantly increased in CAMs by Wnt-5a. Migration of AGS (gastric cancer) cells was stimulated by Wnt-5a and was inhibited in the presence of TIMP-3, a validated target of the miR-181 family. After hsa-miR-181d knockdown, the migration and proliferation of CAMs were significantly decreased, and stimulation with Wnt-5a reversed the migratory inhibition of hsa-miR-181d knockdown CAMs. Conditioned medium from hsa-miR-181d knockdown CAMs inhibited the migration of AGS cells.
The findings suggest that differential miRNA abundance is a feature of normal myofibroblasts from the stomach and oesophagus, and myofibroblasts from normal and cancer tissues. One consequence is increased Wnt-5a in gastric CAMs that is implicated in the regulation of cell migration, in health and disease, at least partly through modulation of hsa-miR-181d
The role of extracellular proteases in stromal-epithelial interactions in gastric cancer
No full textCancers of the upper gastrointestinal tract present at an advanced stage and carry a poor prognosis. Oesophageal and gastric tumours have a rich stroma composed of vascular cells, immune cells, and myofibroblasts, which promotes tumour growth, invasion and metastasis. In addition, mesenchymal stromal cells (MSCs) are recruited from the bone marrow to the tumour stroma; the mechanisms underpinning this have not yet been defined. Extracellular proteases play a role in cell migration, invasion, and cell signalling and are known to influence cancer growth in conflicting ways. Myofibroblasts present in normal tissue differ from those found in cancer. Cancer-associated myofibroblasts (CAMs) are known to modulate extracellular protease activity by secreting plasminogen activator inhibitor-1 (PAI-1), an inhibitor of the serine protease urokinase plasminogen activator, and matrix metalloproteinases (MMPs). This investigation studies the role of PAI-1 in gastric cancer and assesses the contribution of myofibroblast-derived MMPs to tumour growth. Finally, the role of chemerin in recruiting MSCs has been investigated.
The expression of PAI-1 in myofibroblasts was found to be higher than in gastric cancer cells. Overexpression of PAI-1 in gastric cancer cells resulted in decreased cell adhesion and decreased tumour growth in an in vivo subcutaneous xenograft model of gastric tumour growth.
The addition of gastric CAMs potentiated the growth of gastric cancer subcutaneous xenografts. This was not accounted for by differences in cell proliferation rate, apoptosis or final stromal content. Xenografts containing CAMs suppress the growth of a contralateral xenograft without CAMs, demonstrating that a long-range signal can be generated as a result of stromal-epithelial interactions.
MMP and cathepsin activity was compared between xenografts containing myofibroblasts to those without. MMP activity is increased in xenografts injected with CAMs, compared to those injected with myofibroblasts taken from normal stomach or those with gastric cancer cells alone. In an organotypic co-culture system, MMP inhibition resulted in a decrease in gastric cancer cell invasion.
The injection of fluorescently labelled MSCs injected resulted in homing of these cells to subcutaneous oesophageal tumours containing CAMs. Antagonism at the ChemR23 receptor inhibited this MSC homing to oesophageal xenografts containing CAMs.
This work emphasises the importance of assessing the contribution of specific proteases and their inhibitors in gastric cancer. The stroma is an important contributor to extracellular protease activity and myofibroblasts contribute both proteases and their inhibitors to the tumour microenvironment, resulting in the modulation of tumour growth and cell adhesion. MSCs are recruited to oesophageal tumours via a novel signalling pathway
Investigating the Impact of c-Cbl Deficiency in Adipose Tissue: Its Role in Insulin Sensitivity and Adipokine Production
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Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
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