Normal Adult Pancreas Research Articles

3D genomic mapping reveals multifocality of human pancreatic precancers.

Pancreatic intraepithelial neoplasias (PanINs) are the most common precursors of pancreatic cancer, but their small size and inaccessibility in humans make them challenging to study1. Critically, the number, dimensions and connectivity of human PanINs remain largely unknown, precluding important insights into early cancer development. Here, we provide a microanatomical survey of human PanINs by analysing 46 large samples of grossly normal human pancreas with a machine-learning pipeline for quantitative 3D histological reconstruction at single-cell resolution. To elucidate genetic relationships between and within PanINs, we developed a workflow in which 3D modelling guides multi-region microdissection and targeted and whole-exome sequencing. From these samples, we calculated a mean burden of 13 PanINs per cm3 and extrapolated that the normal intact adult pancreas harbours hundreds of PanINs, almost all with oncogenic KRAS hotspot mutations. We found that most PanINs originate as independent clones with distinct somatic mutation profiles. Some spatially continuous PanINs were found to contain multiple KRAS mutations; computational and in situ analyses demonstrated that different KRAS mutations localize to distinct cell subpopulations within these neoplasms, indicating their polyclonal origins. The extensive multifocality and genetic heterogeneity of PanINs raises important questions about mechanisms that drive precancer initiation and confer differential progression risk in the human pancreas. This detailed 3D genomic mapping of molecular alterations in human PanINs provides an empirical foundation for early detection and rational interception of pancreatic cancer.

Abstract A045: Persistence of the splanchnic gene signature along the mesenchymal cell trajectory during pancreatic cancer progression

Abstract In pancreatic ductal adenocarcinoma (PDAC), cancer associated fibroblasts (CAFs) play critical and complex roles in the tumor microenvironment. While fibroblasts are sparse in the normal adult pancreas, CAFs are a major component in the desmoplastic stroma of PDAC and may account for half of the entire tumor tissue. Here we aimed to investigate the origin, diversification, and function of CAFs. We constructed a dual-DNA-recombinase mouse genetic model, which allowed for genetic alternations in pancreatic epithelial cells and fibroblasts independently and simultaneously. The splanchnic mesenchyme is a layer of tissue adjacent to the pancreatic endoderm during fetal development. Our lineage tracing studies demonstrated that the splanchnic mesenchyme is the fetal origin of adult pancreatic fibroblasts during homeostasis and tumorigenesis. Notably, the other two postulated CAF sources, bone marrow and epithelial cells, have minimal contributions to fibroblasts. We further compared mice carrying no genetic mutations, only Kras mutation, and Kras/p53 dual mutations in pancreatic epithelial cells. We found that fibroblasts in all three conditions are derived from the splanchnic mesenchyme, suggesting a continuous mesenchymal trajectory in coordination with the pancreatic epithelium. Additionally, single cell transcriptomic analysis indicated persistent and dynamic gene expressions along the pancreatic mesenchymal trajectory during development, homeostasis, precancer lesion and cancer. Intriguingly, certain splanchnic factors are expressed in adult pancreatic fibroblasts in temporally and spatially distinct patterns. To determine the function of splanchnic factors in fibroblasts, we constructed mouse genetic models to delete GATA6 specifically in CAFs, which resulted in increased tumor burden in the pancreas. This suggests a non-cell autonomous function of GATA6 in CAFs to restrain pancreatic cancer progression. In summary, this study delineated a continuous cell trajectory of the mesenchymal lineage in the pancreas across different life stages. Furthermore, persistent gene expressions along the mesenchymal trajectory contributes to pancreatic CAF heterogeneity. Importantly, such persistence may constitute an inherent mechanism to restrain pancreatic cancer. Citation Format: Lu Han, Tom Walter, Joseph Beaudet, Caroline Everett, Julia Piermattei, Alex Adams, Kun Fang, Michael Zimmermann, Angela Mathison, Raul Urrutia, Victor Jin, Gustavo Leone, Michael Ostrowski. Persistence of the splanchnic gene signature along the mesenchymal cell trajectory during pancreatic cancer progression [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A045.

In Situ Analysis Reveals That CFTR Is Expressed in Only a Small Minority of β-Cells in Normal Adult Human Pancreas.

Although diabetes affects 40% to 50% of adults with cystic fibrosis, remarkably little is known regarding the underlying mechanisms leading to impaired pancreatic β-cell insulin secretion. Efforts toward improving the functional β-cell deficit in cystic fibrosis-related diabetes (CFRD) have been hampered by an incomplete understanding of whether β-cell function is intrinsically regulated by cystic fibrosis transmembrane conductance regulator (CFTR). Definitively excluding meaningful CFTR expression in human β-cells in situ would contribute significantly to the understanding of CFRD pathogenesis. To determine CFTR messenger ribonucleic acid (mRNA) and protein expression within β-cells in situ in the unmanipulated human pancreas of donors without any known pancreatic pathology. In situ hybridization for CFTR mRNA expression in parallel with insulin immunohistochemical staining and immunofluorescence co-localization of CFTR with insulin and the ductal marker, Keratin-7 (KRT7), were undertaken in pancreatic tissue blocks from 10 normal adult, nonobese deceased organ donors over a wide age range (23-71 years) with quantitative image analysis. CFTR mRNA was detectable in a mean 0.45% (range 0.17%-0.83%) of insulin-positive cells. CFTR protein expression was co-localized with KRT7. One hundred percent of insulin-positive cells were immunonegative for CFTR. For the first time, in situ CFTR mRNA expression in the unmanipulated pancreas has been shown to be present in only a very small minority (<1%) of normal adult β-cells. These data signal a need to move away from studying endocrine-intrinsic mechanisms and focus on elucidation of exocrine-endocrine interactions in human cystic fibrosis.

Antibodies to a CA 19-9 Related Antigen Complex Identify SOX9 Expressing Progenitor Cells In Human Foetal Pancreas and Pancreatic Adenocarcinoma

The Sialyl Lewis A antigen, or CA 19-9, is the prototype serum biomarker for adenocarcinoma of the pancreas. Despite extensive clinical study of CA 19-9 in gastrointestinal malignancies, surprisingly little is known concerning the specific cell types that express this marker during development, tissue regeneration and neoplasia. SOX9 is a transcription factor that plays a key role in these processes in foregut tissues. We report the biochemistry and tissue expression of the GCTM-5 antigen, a pancreatic cancer marker related to, but distinct from, CA19-9. This antigen, defined by two monoclonal antibodies recognising separate epitopes on a large glycoconjugate protein complex, is co-expressed with SOX9 by foregut ductal progenitors in the developing human liver and pancreas, and in pancreatic adenocarcinoma. These progenitors are distinct from cell populations identified by DCLK1, LGR5, or canonical markers of liver and pancreatic progenitor cells. Co-expression of this antigen complex and SOX9 also characterises the ductal metaplasia of submucosal glands that occurs during the development of Barrett’s oesophagus. The GCTM-5 antigen complex can be detected in the sera of patients with pancreatic adenocarcinoma. The GCTM-5 epitope shows a much more restricted pattern of expression in the normal adult pancreas relative to CA19-9. Our findings will aid in the identification, characterisation, and monitoring of ductal progenitor cells during development and progression of pancreatic adenocarcinoma in man.

The MET Receptor Tyrosine Kinase Confers Repair of Murine Pancreatic Acinar Cells following Acute and Chronic Injury.

Acinar cells represent the primary target in necroinflammatory diseases of the pancreas, including pancreatitis. The signaling pathways guiding acinar cell repair and regeneration following injury remain poorly understood. The purpose of this study was to determine the importance of Hepatocyte Growth Factor Receptor/MET signaling as an intrinsic repair mechanism for acinar cells following acute damage and chronic alcohol-associated injury. Here, we generated mice with targeted deletion of MET in adult acinar cells (MET-/-). Acute and repetitive pancreatic injury was induced in MET-/- and control mice with cerulein, and chronic injury by feeding mice Lieber-DeCarli diets containing alcohol with or without enhancement of repetitive pancreatic injury. We examined the exocrine pancreas of these mice histologically for acinar death, edema, inflammation and collagen deposition and changes in the transcriptional program. We show that MET expression is relatively low in normal adult pancreas. However, MET levels were elevated in ductal and acinar cells in human pancreatitis specimens, consistent with a role for MET in an adaptive repair mechanism. We report that genetic deletion of MET in adult murine acinar cells was linked to increased acinar cell death, chronic inflammation and delayed recovery (regeneration) of pancreatic exocrine tissue. Notably, increased pancreatic collagen deposition was detected in MET knockout mice following repetitive injury as well alcohol-associated injury. Finally, we identified specific alterations of the pancreatic transcriptome associated with MET signaling during injury, involved in tissue repair, inflammation and endoplasmic reticulum stress. Together, these data demonstrate the importance of MET signaling for acinar repair and regeneration, a novel finding that could attenuate the symptomology of pancreatic injury.

Islet Cells Serve as Cells of Origin of Pancreatic Gastrin-Positive Endocrine Tumors.

The cells of origin of pancreatic gastrinomas remain an enigma, since no gastrin-expressing cells are found in the normal adult pancreas. It was proposed that the cellular origin of pancreatic gastrinomas may come from either the pancreatic cells themselves or gastrin-expressing cells which have migrated from the duodenum. In the current study, we further characterized previously described transient pancreatic gastrin-expressing cells using cell lineage tracing in a pan-pancreatic progenitor and a pancreatic endocrine progenitor model. We provide evidence showing that pancreatic gastrin-expressing cells, found from embryonic day 12.5 until postnatal day 7, are derived from pancreatic Ptf1a(+) and neurogenin 3-expressing (Ngn3(+)) progenitors. Importantly, the majority of them coexpress glucagon, with 4% coexpressing insulin, indicating that they are a temporary subpopulation of both alpha and beta cells. Interestingly, Men1 disruption in both Ngn3 progenitors and beta and alpha cells resulted in the development of pancreatic gastrin-expressing tumors, suggesting that the latter developed from islet cells. Finally, we detected gastrin expression using three human cohorts with pancreatic endocrine tumors (pNETs) that have not been diagnosed as gastrinomas (in 9/34 pNETs from 6/14 patients with multiple endocrine neoplasia type 1, in 5/35 sporadic nonfunctioning pNETs, and in 2/20 sporadic insulinomas), consistent with observations made in mouse models. Our work provides insight into the histogenesis of pancreatic gastrin-expressing tumors.

The correlation of apparent diffusion coefficient values with gender and age in normal adult pancreas

Objective To investigate the effect of gender and age on the apparent diffusion coefficient (ADC) of normal adult pancreas. Methods A total of 383 patients with normal pancreas (290 male,93 female,range from 21 to 78 years of age) were enrolled in this study.The subjects were divided into four groups based on different age (≤40 years,41-50 years,51-60 years and>60 years) with patient number of 56,108,139 and 80,respectively.Breath-hold single-shot echo-planar DWI (b value=0,500 s/mm2) was performed to determine ADCs on all patients.The average ADCs was calculated by four ADCs measured from the head to tail part of the pancreas in each patient.Patients with different age or gender were analyzed by independent-samples t test.Effect of gender on ADCs was analyzed using the Mann-Whitney U test.Relationship between ADCs and age was analyzed using Spearman rank-order correlation test,and Kruskal-Wallis H test was used to compare the ADCs among 4 age groups. Results The median pancreatic ADC values in female group (n=93) [1.60×10-3mm2/s (1.47×10-3-1.77×10-3) mm2/s] was higher than that in male group (n=290) [1.57×10-3mm2/s (1.41×10-3-1.74×10-3) mm2/s].Mann-Whitney U test results showed the mean ADCs was similar between the two groups (Z=1.335,P=0.182).The age distribution was similar between the male[ (52±10) years of age]and female[ (51±11) years of age] groups (t=0.267,P=0.790).The age spectrum showed that there was no correlation between the average ADC values and age (r=0.016,P=0.752).The median ADC values of the four age groups were 1.58×10-3,1.54×10-3,1.59×10-3and 1.57×10-3mm2/s,respectively.Kruskal-Wallis H test showed no significant difference of mean ADCs among the age groups (χ2=2.15,P=0.542). Conclusion There is no correlation of ADCs between age and gender in normal adult pancreas. Key words: Pancreas; Diffusion magnetic resonance imaging; Age factors; Sex factors

Diffusion-weighted MRI of the normal adult pancreas: The effect of age on apparent diffusion coefficient values

To investigate and characterize the effect of age on apparent diffusion coefficient (ADC) values in the normal adult pancreas using diffusion-weighted magnetic resonance imaging (DWI). Five hundred and fifty-nine adult patients without pancreatic disease, ranging from 20-81 years of age (mean 50.9 years; 436 men, 123 women), were included in this study. Breath-hold single-shot echo-planar DWI (b-values = 0, 500 s/mm(2)) was employed to determine the ADCs across all patients. Dependency of ADCs on age was characterized using a Spearman rank-order correlation test. Across the age spectrum, there was no significant correlation between ADC and age (p = 0.409). The findings of the present study suggest that the effect of age on ADCs can be excluded from the diagnosis of pancreatic diseases and design of future studies using breath-hold single-shot DWI and ADCs (as calculated with b-values of 0 and 500 s/mm(2)).

Age-related changes in normal adult pancreas: MR imaging evaluation

ObjectiveTo investigate age-related changes in normal adult pancreas as identified by magnetic resonance imaging (MRI). Materials and methodsWe examined 115 patients without pancreatic diseases (21–90 years) who underwent upper abdominal MRI to evaluate the normal pancreatic MRI findings related to aging. The parameters examined were the pancreatic anteroposterior (AP) diameter, pancreatic lobulation, pancreatic signal intensity (SI), depiction of the main pancreatic duct (MPD), grade of the visual SI decrease on the opposed-phase T1-weighted images compared with in-phase images, and enhancement effect of the pancreas in the arterial phase of dynamic imaging. ResultsThe pancreatic AP diameter significantly reduced (head, p=0.0172; body, p=0.0007; tail, p<0.0001), and lobulation (p<0.0001) and parenchymal fatty change (p<0.0001) became more evident with aging. No significant correlation was observed between aging and pancreatic SI, however the SI on the in-phase T1-weighted images tended to decrease with aging. No significant correlation was observed between aging and the depiction of the MPD as well as aging and contrast enhancement. ConclusionMRI findings of pancreatic atrophy, lobulation, and fatty degeneration are characteristic changes related to aging, and it is necessary to recognize these changes in the interpretation of abdominal MRI in patients with and without pancreatic disease.

Abstract 352: ERG protein is a marker for vascular tumors and prostatic adenocarcinoma

Abstract ERG belongs to the ETS-family of transcription factors and is expressed in subsets of Ewing sarcomas, myeloid leukemias and prostate carcinomas due to chromosomal translocations. Normal expression of ERG has been detected in vascular endothelial cells. In this study we examined ERG-expression in selected adult and embryonic normal tissues, vascular tumors, and non-vascular tumors as controls. A mouse monoclonal antibody (CPDR ERG-MAb) that specifically recognizes ERG oncoprotein was developed at the Center for Prostate Disease Research. We have utilized CPDR ERG-MAb with a Leica automatic immunostainer in combination with the Leica's avidin-biotin free polymer-based detection system. Diaminobenzidine was used the chromogen. Tissues that demonstrated nuclear ERG-immunoreactivity were scored, and only nuclear immunoreactivity was counted as positive. Approximately 10% of examined tissues lacked any ERG-immunoreactivity suggesting that this epitope may be sensitive to sub-optimal preservation. In normal adult skin, tonsil, thymus, liver, kidney, pancreas and, stomach, and colon nuclear ERG-immunoreactivity was restricted to vascular endothelial cells. In human and mouse embryonic tissues wide-spread immonolocalization of ERG was observed in endothelial cells and restricted expression in precartillage, and hematopoietic tissues. Nuclear ERG-positivity was consistently observed in benign vascular tumors (n = 6, at minimum in each category): capillary and cavernous hemangiomas of different types, papillary endothelial hyperplasia, and lymphangioma. In spindle cell hemangioma, the endothelial component was positive and spindle cells negative. In malignant vascular tumors, majority of spindle cells and endothelial cells in Kaposi sarcomas (n = 11) and tumor cells in epithelioid hemangioendothelioma (n = 11) and angiosarcoma (n = 17), were positive. All non-vascular tumors (minimum 5 cases of each): glomus tumor, epithelioid sarcoma, synovial sarcoma, undifferentiated sarcoma, metastatic malignant melanoma, undifferentiated carcinomas of lung and small intestine, pulmonary adenocarcinomas, and malignant mesotheliomas were negative. Consistent with our previous report, In prostatic adenocarcinomas ERG oncoprotein was present in 45% of the examined cases, with no reactivity in non-neoplastic prostate. ERG oncoprotein is a promising marker for prostatic adenocarcinoma in sepration of it from no-neoplastic prostate and other adenocarcinomas. Our recent study revealed that detecting ERG may also be utilized for the diagnosis of angiosarcoma, epithelioid hemangioendothelioma, and endothelial components of hemangiomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 352. doi:10.1158/1538-7445.AM2011-352

Sox9+ ductal cells are multipotent progenitors throughout development but do not produce new endocrine cells in the normal or injured adult pancreas

One major unresolved question in the field of pancreas biology is whether ductal cells have the ability to generate insulin-producing β-cells. Conclusive examination of this question has been limited by the lack of appropriate tools to efficiently and specifically label ductal cells in vivo. We generated Sox9CreER(T2) mice, which, during adulthood, allow for labeling of an average of 70% of pancreatic ductal cells, including terminal duct/centroacinar cells. Fate-mapping studies of the Sox9(+) domain revealed endocrine and acinar cell neogenesis from Sox9(+) cells throughout embryogenesis. Very small numbers of non-β endocrine cells continue to arise from Sox9(+) cells in early postnatal life, but no endocrine or acinar cell neogenesis from Sox9(+) cells occurs during adulthood. In the adult pancreas, pancreatic injury by partial duct ligation (PDL) has been suggested to induce β-cell regeneration from a transient Ngn3(+) endocrine progenitor cell population. Here, we identify ductal cells as a cell of origin for PDL-induced Ngn3(+) cells, but fail to observe β-cell neogenesis from duct-derived cells. Therefore, although PDL leads to activation of Ngn3 expression in ducts, PDL does not induce appropriate cues to allow for completion of the entire β-cell neogenesis program. In conclusion, although endocrine cells arise from the Sox9(+) ductal domain throughout embryogenesis and the early postnatal period, Sox9(+) ductal cells of the adult pancreas no longer give rise to endocrine cells under both normal conditions and in response to PDL.

Id3 modulates cellular localization of bHLH Ptf1‐p48 protein

Ptf1-p48 is a pancreas-specific bHLH transcriptional protein, which, in the normal adult pancreas, shows a restricted expression in acinar cells where it is predominantly localized in the nucleus and activates the transcription of exocrine-specific genes. Ptf1-p48 partners with two proteins to form the PTF1 active complex: a bHLH E-protein and suppressor of hairless RBP-J. Cytoplasmic mislocalization of Ptf1-p48 has been reported in pancreatic pathologies, suggesting its contribution in the early steps of pancreatic carcinogenesis. The aim of the our work was to elucidate the mechanisms regulating Ptf1-p48 subcellular localization. We hypothesized a role of Id proteins acting in a dominant-negative fashion by heterodimerizing with bHLH proteins. We reproduced Ptf1-p48 cytoplasmic mislocalization in acinar AR4-2J cells and demonstrated that a proliferative signal elicited by gastrin leads to increases in Id3 protein expression and levels of Id3/E47 and Id3/Ptf1-p48 interactions, and a decrease in the level of E47/Ptf1-p48 interaction. By contrast, Id3 silencing reversed the cytoplasmic mislocalization of Ptf1-p48 induced by gastrin. As E47 is responsible for the nuclear import of the PTF1 complex, disruption of this complex via Id3 interactions with both E47 and Ptf1-p48 appears to induce cytoplasmic mislocalization of Ptf1-p48. We then found that Ptf1-p48 is either absent or mislocalized in the cytoplasm and Id3 is overexpressed in human and murine pancreatic preneoplastic lesions. Our data provide novel insight into the regulation of Ptf1-p48 function and provide evidence that Ptf1-p48 cytoplasmic mislocalization and Id3 overexpression are early events in pancreatic cancer progression.

Evidence for Epithelial-Mesenchymal Transition in Adult Human Pancreatic Exocrine Cells

It has been shown that adult pancreatic ductal cells can dedifferentiate and act as pancreatic progenitors. Dedifferentiation of epithelial cells is often associated with the epithelial-mesenchymal transition (EMT). In this study, we investigated the occurrence of EMT in adult human exocrine pancreatic cells both in vitro and in vivo. Cells of exocrine fraction isolated from the pancreas of brain-dead donors were first cultured in suspension for eight days. This led to the formation of spheroids, composed of a principal population of cells with duct-like phenotype. When cultivated in tissue culture-treated flasks, spheroid cells exhibited a proliferative capacity and coexpressed epithelial (cytokeratin7 and cytokeratin19) and mesenchymal (vimentin and alpha-smooth muscle actin) markers as well as marker of progenitor pancreatic cells (pancreatic duodenal homeobox factor-1) and surface markers of mesenchymal stem cells. The switch from E-cadherin to N-cadherin associated with Snail1 expression suggested that these cells underwent EMT. In addition, we showed coexpression of epithelial and mesenchymal markers in ductal cells of one normal adult pancreas and three type 2 diabetic pancreases. Some of the vimentin-positive cells were found to coexpress glucagon or amylase. These results point to the occurrence of EMT, which may take place on dedifferentiation of ductal cells during the regeneration or renewal of human pancreatic tissues.

Expression of prostate stem cell antigen and Claudin-4 in human pancreatic carcinoma

To investigate the expressions of prostate stem cell antigen (PSCA) and Claudin-4 in human pancreatic carcinoma and to discuss its role in the ontogenesis of pancreatic cancer. Pancreatic carcinoma tissue microarray was constructed, containing 100 cores of 10 normal adult pancreas tissues, 12 chronic pancreatitis tissues, and 78 pancreatic carcinomas. The expressions of PSCA and Claudin-4 were detected using immunohistochemical method and the relationship between PSCA and Claudin-4 and the pancreatic carcinoma was analyzed. The positive expression rates of PSCA and Claudin-4 protein in pancreatic carcinoma were 79. % and 88. % respectively. PSCA and Claudin-4 staining were more intense in malignant cells than in chronic pancreatic tissues and normal adult pancreas tissues. No evidence was found for an association between expressions of PSCA and Claudin-4 and other variables, including gender, age at surgery, and tumor grade. The expressions of PSCA and Claudin-4 are related to the pancreatic carcinomas. PSCA and Claudin-4 play a role in the development of pancreatic cancer, but PSCA and Claudin-4 are not correlated with the clinical pathology of tumor.

Expression of Id1 in adult, regenerating and developing pancreas

Several key transcription factors are necessary for alpha cell development in the pancreas. In this study, we describe the expression of Inhibition of DNA-binding protein 1 (Id1) in the developing as well as the normal adult pancreas. We found co-expression of Id1 with bone morphogenetic protein (BMP) receptor in alpha cells. Inhibition of BMP4 signaling with a specific neutralizing antibody slightly decreases the proportion of glucagon cells in the adult pancreas but had a significant effect in a model of pancreas regeneration. In late embryonic pancreas, Id1 co-localized with GATA4, a transcription factor known for its critical function in glucagon cell development. However, in early postnatal period, the expression of Id1 and GATA4 diverged with Id1 identified in glucagon cells and GATA4 restricted to the acinar pancreas.

Peptide Products of the Neurotrophin-Inducible Gene vgf Are Produced in Human Neuroendocrine Cells from Early Development and Increase in Hyperplasia and Neoplasia

Although the neurotrophin-inducible gene vgf is expressed in mammalian neurons and endocrine cells, limited data is available in man. The objective of the study was to map proVGF peptides in human endocrine cells during development, adulthood, hyperplasia, and tumors. Antisera were generated against peptides related to internal cleavage or cleavage-amidation sites (rat proVGF(422-430) and human proVGF(298-306)-NH2) and the proVGF C-terminal ending (human proVGF(607-615)). Developing and normal adult endocrine cells, hyperplastic endocrine lesions (thyroid, parathyroid, lung, and stomach), and 120 tumors (102 endocrine) were studied. Immunogold electron microscopy was performed on normal adult pancreas and gut, and Western blotting was performed on extracts of control tissues and endocrine tumors. proVGF fragments were revealed in developing pituitary, gut, pancreas, and adrenal medulla from 10 gestational weeks, in normal adult pituitary and adrenal medulla, pancreatic glucagon, and insulin cells and gut serotonin cells, in hyperplastic thyroid calcitonin cells, lung P cells, gastric enterochromaffin-like cells, and gastrin cells, and in 88 of 102 endocrine tumors. At electron microscopy proVGF immunoreactivity was restricted to electron-dense granules. Western blotting revealed large molecular weight forms and cleavage fragments in both control tissues and tumor extracts. proVGF-related peptides are present in endocrine cells early during development and adulthood and increase in hyperplasia and tumors, and proVGF fragments could be novel diagnostic tools for endocrine cells and related lesions, including tumors.

Bone marrow mesenchymal stem cells express a restricted set of functionally active chemokine receptors capable of promoting migration to pancreatic islets

AbstractBone marrow–derived mesenchymal stem cells (BM-MSCs) are stromal cells with the ability to proliferate and differentiate into many tissues. Although they represent powerful tools for several therapeutic settings, mechanisms regulating their migration to peripheral tissues are still unknown. Here, we report chemokine receptor expression on human BM-MSCs and their role in mediating migration to tissues. A minority of BM-MSCs (2% to 25%) expressed a restricted set of chemokine receptors (CXC receptor 4 [CXCR4], CX3C receptor 1 [CX3CR1], CXCR6, CC chemokine receptor 1 [CCR1], CCR7) and, accordingly, showed appreciable chemotactic migration in response to the chemokines CXC ligand 12 (CXCL12), CX3CL1, CXCL16, CC chemokine ligand 3 (CCL3), and CCL19. Using human pancreatic islets as an in vitro model of peripheral tissue, we showed that islet supernatants released factors able to attract BM-MSCs in vitro, and this attraction was principally mediated by CX3CL1 and CXCL12. Moreover, cells with features of BM-MSCs were detected within the pancreatic islets of mice injected with green fluorescent protein (GFP)–positive BM. A population of bona fide MSCs that also expressed CXCR4, CXCR6, CCR1, and CCR7 could be isolated from normal adult human pancreas. This study defines the chemokine receptor repertoire of human BM-MSCs that determines their migratory activity. Modulation of homing capacity may be instrumental for harnessing the therapeutic potential of BM-MSCs.

Immune cells, pancreas development, regeneration and type 1 diabetes

Macrophages (MΦs), dendritic cells (DCs) and lymphocytes are involved in the pathogenesis of type 1 diabetes (T1D). However, the presence of these cells is not specific for a diabetes-prone background. MΦs are also constituents of the normal fetal, neonatal and adult pancreas. We hypothesize that MΦs, DCs and lymphocytes have a role in pancreas and islet development because immune cells (particularly MΦs) are known to participate in the morphogenesis of various organs. In addition, we hypothesize that a defective function of immune cells generates an aberrant islet morphogenesis in T1D-prone individuals or animals. In the post-weaning period (a period of islet remodelling), the same defective function of immune cells might precipitate the pro-inflammatory peri-islet microenvironment that favours islet autoimmune reactivity.

Expression of serum response factor (SRF) in normal and regenerating pancreas

Background: SRF is a transcription factor that controls mesodermal differentiation. To date, there has been no report demonstrating the role of SRF in endodermally derived tissue. Genetic ablation of SRF results in embryonic lethality precluding analysis of SRF in pancreatic development. We sought to determine the expression of SRF in the normal adult rodent pancreas and during pancreatic regeneration (PR). Methods: Pancreata from adult rodents were harvested and fixed in 4% paraformaldehyde. Sections were stained for SRF expression using a polyclonal antibody. 90% pancreatectomies were performed and regenerating pancreatic remnants harvested at 0, 24, 72 hrs and 7 days. Tissues were processed and stained as above. Sham laparotomies served as controls. Results: Nuclear staining for SRF was observed in normal pancreatic ductal, acinar, and islet cells. Staining was stronger in islet cells compared to ductal and acinar cells. Double staining for insulin demonstrated islet SRF staining was predominately within the beta cells. During PR, SRF expression was increased with maximal expression at 36 hours (Fig 1B). SRF expression continued to be increased compared to baseline and sham at 7 days. In contrast to normal pancreata, SRF expression in the regenerating pancreas was found predominately in the acinar cytoplasm (AC). Conclusion: For the first time, SRF expression is observed in all three cellular components of the pancreas. During PR, SRF expression is increased and redistributed to the acinar cytoplasm. The data suggests SRF may be an important regulator of pancreatic growth, replication, and differentiation.

Netrin-1 expression in fetal and regenerating rat pancreas and its effect on the migration of human pancreatic duct and porcine islet precursor cells.

We investigated the expression and function of netrin-1, a diffusible laminin-like protein known to regulate neuronal-cell migration in the pancreas. We questioned whether this factor regulates migration of pancreatic epithelial cells and whether this could be involved in islet neogenesis. We studied fetal and adult rat pancreas wherein duct ligation induced islet neogenesis. Netrin-1 expression was analysed by RT-PCR, western blot and immunohistochemistry. In vitro cell migration was measured with a human pancreatic duct cell line (CAPAN-2) and with fetal porcine islet cells. We also studied the expression of two netrin-receptors, neogenin and deleted in colorectal cancer. We found a transient expression of netrin-1 mRNA and protein in fetal pancreas from E15 to E18, and in adult pancreas after duct ligation. In normal adult pancreas there was very little netrin-1 expression. Netrin-1 expression was observed both in endocrine and exocrine cells. At the immunohistochemical level, it was expressed by islet cells during tissue regeneration. We could show that netrin-1 increases the migration of fetal islet cells and of a ductal cell line, mainly via a chemokinetic effect. From the two well-established netrin receptors, DCC and neogenin, we only found neogenin to be expressed in the pancreas. Neogenin expression coincided with the period of netrin-1 up-regulation. Netrin-1 is involved in pancreatic morphogenesis and tissue remodelling and plays a role in the regulation of duct-cell and fetal-islet cell migration. This can be of importance in islet regeneration, where migration of islet precursors takes place.