Biomedical subjects

Luc Bouwens

Publications and source records attributed to Luc Bouwens.

12 recordsLinked to original sources

PubMed2006

Expression of the Notch signaling pathway and effect on exocrine cell proliferation in adult rat pancreas.

When pancreatic tissue is injured after duct obstruction, acinoductal metaplasia is observed. Similar metaplastic changes occur when exocrine pancreatic cells are isolated and cultured. We demonstrate that under these experimental conditions the exocrine acinar cells lose their differentiated characteristics: expression of the acinar transcription factors p48/Ptf1alpha and Mist1 is decreased or lost, whereas expression of the embryonic transcription factor Pdx1 is increased. The receptors Notch1 and Notch2, members of the DSL family of Notch ligands, and the target genes in the Notch-signaling pathway Hes1, Hey1, and Hey2 become strongly up-regulated. We noted also reduced expression of Sel1L, a Notch repressor that is normally highly expressed in exocrine pancreas. Stimulation of Notch by its ligand Jagged1 diminished the proliferation of cultured metaplastic exocrine cells. Chemical inhibition of Notch signaling resulted in increased proliferation and induction of the cell-cycle regulator p21Cip1. This effect seems to be Hes1-independent and mainly coincides with decreased Hey1 and Hey2 mRNA expression. In conclusion, we demonstrate that during acinoductal metaplasia the Notch-signaling pathway is activated concomitantly with changes in transcription factor expression of pancreatic acinar cells. In addition, we show that Notch signaling is implicated in the suppression of proliferation of these metaplastic exocrine cells. The latter may be important in protection from neoplastic transformation.

Animals↗

PubMed2005

Metaplasia in the pancreas.

There is currently much interest in the possibility to treat chronic diseases by cell replacement or regenerative therapies. Most of these studies focus on the manipulation of undifferentiated stem cells. However, tissue repair and regeneration can also be achieved by differentiated cells, which, in certain conditions, can even transdifferentiate to other cell types. Such transdifferentiations can lead to tissue metaplasia. The pancreas is an organ wherein metaplasia has been well investigated and for which experimental models have been recently developed allowing to unravel the molecular basis of transdifferentiation. Pancreatic metaplasias studied so far include the conversion of exocrine acinar cells to duct cells, exocrine cells to endocrine islet cells, endocrine cells to duct cells, and acinar cells to hepatocytes. Epitheliomesenchymal transitions have also been described. The available evidence indicates that mature cells can be reprogrammed by specific environmental cues inducing the expression of cell type-specific transcription factors. For example, the glucocorticoid hormone dexamethasone induces pancreatic transdifferentiation to hepatocytes, whereas the combination of epidermal growth factor and leukemia-inhibitory factor induces exocrine-endocrine transdifferentiation in vitro. Further unravelling of the involved signal transduction pathways, transcription factor networks, and chromatin modifications is required to manipulate metaplasia at will and to apply it in tissue repair or regeneration.

Animals↗

PubMed2005

Regulation of pancreatic beta-cell mass.

Beta-cell mass regulation represents a critical issue for understanding diabetes, a disease characterized by a near-absolute (type 1) or relative (type 2) deficiency in the number of pancreatic beta cells. The number of islet beta cells present at birth is mainly generated by the proliferation and differentiation of pancreatic progenitor cells, a process called neogenesis. Shortly after birth, beta-cell neogenesis stops and a small proportion of cycling beta cells can still expand the cell number to compensate for increased insulin demands, albeit at a slow rate. The low capacity for self-replication in the adult is too limited to result in a significant regeneration following extensive tissue injury. Likewise, chronically increased metabolic demands can lead to beta-cell failure to compensate. Neogenesis from progenitor cells inside or outside islets represents a more potent mechanism leading to robust expansion of the beta-cell mass, but it may require external stimuli. For therapeutic purposes, advantage could be taken from the surprising differentiation plasticity of adult pancreatic cells and possibly also from stem cells. Recent studies have demonstrated that it is feasible to regenerate and expand the beta-cell mass by the application of hormones and growth factors like glucagon-like peptide-1, gastrin, epidermal growth factor, and others. Treatment with these external stimuli can restore a functional beta-cell mass in diabetic animals, but further studies are required before it can be applied to humans.

Animals↗

PubMed2005

Expression of regulatory genes for pancreas development during murine embryonic stem cell differentiation.

Insulin-producing cells derived from embryonic stem cells could be surrogates for beta cells in diabetes therapy. However, their derivation remains hard to achieve with current protocols which rely on initial embryoid body formation. We assume that factors known to inhibit pancreas development contribute to this limitation in vitro. To evaluate this hypothesis, embryoid bodies were examined after different culture periods by real time RT-PCR to profile the expression of genes known to regulate embryonic pancreas development. Our data indicate that transcripts for pancreas markers (insulin, glucagon and amylase ) were expressed during differentiation, but the highest levels achieved were at least 10(5) times lower than in the adult mouse pancreas. Notch signalling was activated as suggested by Delta, Jagged, Ngn3 and NeuroD1 profiles. However, Sonic hedgehog, a known inhibitor of pancreas induction in vivo drastically increased in day 6 embryoid bodies, while Inhibin betaA and betaB were down-regulated and follistatin up-regulated. Members of the Fibroblast- and Transforming Growth Factor families which pattern the endoderm were expressed at low levels, while those that inhibit pancreas development were highly transcribed. The profile of pancreas regulators expressed in embryoid bodies is therefore not compatible with differentiation of pancreatic and insulin-producing cells. These findings provide an explanation for the limited derivation of such cells to date, in addition to basic information for establishing novel differentiation protocols.

Animals↗

PubMed2004

Interactions between rat colon carcinoma cells and Kupffer cells during the onset of hepatic metastasis.

Liver sinusoids harbor populations of 2 important types of immunocompetent cells, Kupffer cells (KCs) and natural killer (NK) cells, which are thought to play an important role in controlling hepatic metastasis in the first 24 hr upon arrival of the tumor cells in the liver. We studied the early interaction of KCs, NK and CC531s colon carcinoma cells in a syngeneic rat model by confocal laser scanning microscopy. Results showed a minority of KCs (19% periportal and 7% pericentral) involved in the interaction with 94% of tumor cells and effecting the phagocytosis of 92% of them. NK cell depletion decreased the phagocytosis of tumor cells by KCs by 33% over a period of 24 hr, leaving 35% of the cancer cells free, as compared to 6% in NK-positive rats. Surviving cancer cells were primarily located close to the Glisson capsule, suggesting that metastasis would initiate from this region.

Animals↗

PubMed2004

Plasticity in the adult rat pancreas: transdifferentiation of exocrine to hepatocyte-like cells in primary culture.

Under certain experimental conditions, hepatocytes can arise in the pancreas. It has been suggested that the pancreas retains a source of hepatocyte progenitor cells. However, such cells have not been yet identified in the adult pancreas. We describe here the transdifferentiation of primary rat pancreatic exocrine cells into hepatocyte-like cells during 5 days of tissue culture in the presence of dexamethasone (DX). Using reverse-transcription polymerase chain reaction and immunocytochemistry, it was observed that DX treatment induced albumin RNA and protein expression in the cells. Coexpression of albumin and amylase, and the absence of cell proliferation, demonstrated a direct transdifferentiation of acinar cells to hepatocytic cells. CCAAT enhancer-binding protein-ss protein, a liver-enriched transcription factor that is considered to be the master switch in pancreatohepatic transdifferentiation, and alpha-fetoprotein were markedly upregulated in the cells after treatment with DX. We compared transcriptional profiles of freshly isolated exocrine cells and DX-treated cells using oligonucleotide microarrays and found that multiple liver-specific genes are induced along with albumin, and that certain pancreatic genes are downregulated in the DX-treated cells. In conclusion, these observations support the notion of plasticity in the adult pancreas and that exocrine cells can be reprogrammed to transdifferentiate into other cell types such as hepatocytes.

Age Factors↗

PubMed2004

Mechanisms of beta cell death during restricted and unrestricted enterovirus infection.

Coxsackie B virus (CVB-5) infections potentially trigger and accelerate pancreatic beta cell damage leading to type 1 diabetes. In vivo, all viruses face natural resistance mediated by various host factors which restrict the progression of infection. Thus, the aims of this study were to generate a tissue culture model of restricted coxsackie B virus infection in primary islet cells by preventing the production of viral progeny with a selective inhibitor of viral RNA replication and to investigate the mechanisms of virus-induced islet cell death during productive and restricted infective conditions. Cultured foetal porcine islet cells were infected effectively with the prototype strain of coxsackievirus B5 (CVB-5). Nuclear viability stainings and electron microscopy showed productive infection to result in dominantly necrotic cell death with additional slight induction of apoptosis during the 7 days of follow-up. The restricted conditions were created by addition of guanidine-hydrochloride (G-HCl) into culture medium. At 1 mM concentration, it significantly protected the infected cells from necrosis and thus maintained high viability. This was associated with increased significantly apoptosis. In perifusion analysis, the cellular ability to release insulin was reduced, although the metabolic integrity was preserved as shown by MTT-analysis and cellular ATP levels. These data show that restriction of CVB-5 replication with G-HCl protects islet cells against virus-induced necrosis. However, restriction of viral replication shifts the mechanism of cell death from necrosis toward apoptosis. A slowly progressing subclinical infection of islets could thus lead to increased beta-cell apoptosis.

Adenosine Triphosphate↗

PubMed2004

Islet morphogenesis and stem cell markers.

The mechanism of islet neogenesis remains poorly understood, despite its potential applications in regenerative or replacement therapies for the treatment of insulin-dependent diabetes. During fetal development of the mouse or rat, the majority of islet cells are formed in late gestation (E18-21) by the process of neogenesis from precursor cells. The precursor cells are organized as ducts that actively proliferate and express high levels of specific cytokeratin (CK) proteins. Transitional cells coexpressing islet hormones and CK are frequent and disappear shortly after birth, to reappear only in conditions in which pancreas or islet regeneration has been induced. Islet morphogenesis is thought to operate mainly through the budding of islet cells from ducts, followed by their migration away from the duct to form clusters. Single islet cells are indeed frequent in the fetal and regenerating pancreas, but they also occur in normal tissue, especially in the human pancreas. A different neogenic mechanism, observed in the fetal rat, consists in the proliferation of ductal cells resulting in large aggregates. Starting from the middle of the aggregate, cells differentiate into islet cells and gradually lose their proliferative activity and other ductal characteristics. In adult pancreas, islets are in close contact with at least one duct or ductule. Such a direct duct-islet axis becomes even more evident in regeneration models, such as duct ligation. In these models, a metaplastic transformation of the exocrine pancreas to so-called pseudoductal complexes is seen. Surviving exocrine cells acquire a metaplastic phenotype, which resembles the fetal protodifferentiated state. They start to express CK, the beta-cell transcription factor Pdx1, the neuroendocrine/islet cell markers PGP9.5 and the CCKB receptor for gastrin, and they show pronounced proliferative activity and islet neogenesis. We hypothesize that these de-differentiated or metaplastic exocrine cells (acinar and ductal), acquire a multipotential state and can serve as islet precursors.

Animals↗

PubMed2003

Exocrine cell transdifferentiation in dexamethasone-treated rat pancreas.

Injured pancreatic tissue, for example, after duct ligation, undergoes remodeling, which involves the replacement of exocrine acini by duct-like structures. This acinoductal metaplasia is probably at least partly due to transdifferentiation of amylase-positive, cytokeratin-20 (CK20)-negative acinar cells into amylase-negative, CK20-positive duct-like cells. Due to the kinetics of these phenotypic changes, however, it has not been possible to demonstrate transitional stages of differentiation, which would express both markers at the same time. We took advantage of the fact that dexamethasone treatment inhibits the loss of amylase from acinar cells to demonstrate transitional cells co-expressing amylase and CK20. This was found both in vivo, where duct-ligation induced metaplasia, and in vitro, after isolation of acini. In addition, we found evidence for an acinar-to-islet conversion under the form of transitional cells co-expressing amylase and insulin. These observations strengthen the notion that fully differentiated cells, such as exocrine pancreatic cells, retain the capacity to undergo important phenotypic switches. This finding could have applications in tissue engineering or cell replacement strategies.

Amylases↗

PubMed2003

Specific and combined effects of insulin and glucose on functional pancreatic beta-cell mass in vivo in adult rats.

We investigated the specific and associated effects of insulin and glucose on beta-cell growth and function in adult rats. By combining simultaneous infusion either of glucose and/or insulin or glucose and diazoxide, three groups of rats were constituted: hyperglycemic-hyperinsulinemic rats (high glucose-high insulin), hyperglycemic-euinsulinemic rats (high glucose), and euglycemic-hyperinsulinemic rats (high insulin). All the infusions lasted 48 h. Control rats were infused with 0.9% NaCl (saline controls). In all groups, beta-cell mass was significantly increased, compared with controls (by 70% in high glucose-high insulin rats, 65% in high glucose rats, and 50% in high insulin rats). The stimulation of neogenesis was suggested by the high number of islets budding from pancreatic ducts in high glucose-high insulin and high glucose rats and by the presence of numerous clusters of few beta-cells within the exocrine pancreas in high insulin rats. beta-Cell hypertrophy was observed only in high glucose-high insulin rats. The rate of beta-cell proliferation was similar to that of controls in high glucose-high insulin rats after a 48-h glucose infusion, dropped dramatically in high insulin rats, and dropped to a lesser extent in high glucose rats. In high glucose-high insulin and high glucose rats, beta-cell mass increase was related to a higher beta-cell responsiveness to glucose in vitro as measured by islet perifusion studies, whereas in high insulin rats, no significant enhancement of glucose induced insulin secretion could be noticed. The data show that glucose and insulin may have specific stimulating effects on beta-cell growth and function in vivo in adult rats independently of the influence they exert each other on their respective plasma concentration.

Age Factors↗

PubMed2002

Nestin expression in pancreatic stellate cells and angiogenic endothelial cells.

Nestin is an intermediate filament protein expressed by neuroepithelial stem cells and which has been proposed to represent also a marker for putative islet stem cells. The aim of this study was to characterize the cell type(s) expressing nestin in the rat pancreas. By immunohistochemistry, nestin positivity was localized exclusively in mesenchymal cells of normal and regenerating adult pancreas. In the latter condition, the number of nestin-positive cells and the intensity of nestin immunoreactivity were greatly increased. Most nestin-positive cells had the morphology of stellate cells, a type of pericyte associated with blood vessels which has been previously reported to occur in liver and pancreas. In addition, nestin positivity was present in endothelial cells from neocapillaries during pancreas regeneration, and in all blood vessels during morphogenesis in fetal pancreas. Nestin expression was not found in the ductal epithelial cells from which islet cells originate in fetal and regenerating pancreas. In primary pancreatic tissue explants, nestin-positive mesenchymal cells rapidly attached to plastic and proliferated. These cells also expressed desmin, vimentin, and glial fibrillary acidic protein which are known to represent stellate cell markers. In summary, nestin in the pancreas is primarily a marker for reactive stellate cells, or pericytes, and endothelial cells during active angiogenesis.

Animals↗

PubMed2002

Gastrin stimulates beta-cell neogenesis and increases islet mass from transdifferentiated but not from normal exocrine pancreas tissue.

It is still unclear which factors regulate pancreatic regeneration and beta-cell neogenesis and which precursor cells are involved. We evaluated the role of intravenously infused gastrin in regenerating pancreas of duct-ligated rats. The ligation of exocrine ducts draining the splenic half of the pancreas resulted in acinoductal transdifferentiation within the ligated part but not in the unligated part. We found that infusion of gastrin from day 7 to 10 postligation resulted in a doubling of the beta-cell mass in the ligated part as measured by morphometry. This increase in insulin-expressing cells was not associated with increased proliferation, hypertrophy, or reduced cell death of the beta-cells. Furthermore, we found an increased percentage of single, extra-insular beta-cells and small beta-cell clusters induced by gastrin infusion. These changes occurred only in the ligated part of the pancreas, where transdifferentiation of the exocrine acinar cells to ductlike cells (metaplasia) had occurred, and was not found in the normal unaffected pancreatic tissue. In conclusion, we demonstrate that administration of gastrin stimulates beta-cell neogenesis and expansion of the beta-cell mass from transdifferentiated exocrine pancreas.

Animals↗