Lymphangiogenesis and angiogenesis during human fetal pancreas development

Background: The complex endocrine and exocrine functionality of the human pancreas depends on an efficient fluid transport through the blood and the lymphatic vascular systems. The lymphatic vasculature has key roles in the physiology of the pancreas and in regulating the immune response, both important for developing successful transplantation and cell-replacement therapies to treat diabetes. However, little is known about how the lymphatic and blood systems develop in humans. Here, we investigated the establishment of these two vascular systems in human pancreas organogenesis in order to understand neovascularization in the context of emerging regenerative therapies. Methods: We examined angiogenesis and lymphangiogenesis during human pancreas development between 9 and 22 weeks of gestation (W9-W22) by immunohistochemistry. Results: As early as W9, the peri-pancreatic mesenchyme was populated by CD31-expressing blood vessels as well as LYVE1- and PDPN-expressing lymphatic vessels. The appearance of smooth muscle cell-coated blood vessels in the intra-pancreatic mesenchyme occurred only several weeks later and from W14.5 onwards the islets of Langerhans also became heavily irrigated by blood vessels. In contrast to blood vessels, LYVE1- and PDPN-expressing lymphatic vessels were restricted to the peri-pancreatic mesenchyme until later in development (W14.5-W17), and some of these invading lymphatic vessels contained smooth muscle cells at W17. Interestingly, between W11-W22, most large caliber lymphatic vessels were lined with a characteristic, discontinuous, collagen type IV-rich basement membrane. Whilst lymphatic vessels did not directly intrude the islets of Langerhans, three-dimensional reconstruction revealed that they were present in the vicinity of islets of Langerhans between W17-W22. Conclusion: Our data suggest that the blood and lymphatic machinery in the human pancreas is in place to support endocrine function from W17-W22 onwards. Our study provides the first systematic assessment of the progression of lymphangiogenesis during human pancreatic development

β-Cell Generation: Can Rodent Studies Be Translated to Humans?

β-cell replacement by allogeneic islet transplantation is a promising approach for patients with type 1 diabetes, but the shortage of organ donors requires new sources of β cells. Islet regeneration in vivo and generation of β-cells ex vivo followed by transplantation represent attractive therapeutic alternatives to restore the β-cell mass. In this paper, we discuss different postnatal cell types that have been envisaged as potential sources for future β-cell replacement therapy. The ultimate goal being translation to the clinic, a particular attention is given to the discrepancies between findings from studies performed in rodents (both ex vivo on primary cells and in vivo on animal models), when compared with clinical data and studies performed on human cells

Oxidative Stress Leads to β-Cell Dysfunction Through Loss of β-Cell Identity

Pancreatic β-cell failure is a critical event in the onset of both main types of diabetes mellitus but underlying mechanisms are not fully understood. β-cells have low anti-oxidant capacity, making them more susceptible to oxidative stress. In type 1 diabetes (T1D), reactive oxygen species (ROS) are associated with pro-inflammatory conditions at the onset of the disease. Here, we investigated the effects of hydrogen peroxide-induced oxidative stress on human β-cells. We show that primary human β-cell function is decreased. This reduced function is associated with an ER stress response and the shuttling of FOXO1 to the nucleus. Furthermore, oxidative stress leads to loss of β-cell maturity genes MAFA and PDX1, and to a concomitant increase in progenitor marker expression of SOX9 and HES1. Overall, we propose that oxidative stress-induced β-cell failure may result from partial dedifferentiation. Targeting antioxidant mechanisms may preserve functional β-cell mass in early stages of development of T1D

Conversion of Mature Human β-Cells Into Glucagon-Producing α-Cells

Conversion of one terminally differentiated cell type into another (or transdifferentiation) usually requires the forced expression of key transcription factors. We examined the plasticity of human insulin-producing β-cells in a model of islet cell aggregate formation. Here, we show that primary human β-cells can undergo a conversion into glucagon-producing α-cells without introduction of any genetic modification. The process occurs within days as revealed by lentivirus-mediated β-cell lineage tracing. Converted cells are indistinguishable from native α-cells based on ultrastructural morphology and maintain their α-cell phenotype after transplantation in vivo. Transition of β-cells into α-cells occurs after β-cell degranulation and is characterized by the presence of β-cell–specific transcription factors Pdx1 and Nkx6.1 in glucagon+ cells. Finally, we show that lentivirus-mediated knockdown of Arx, a determinant of the α-cell lineage, inhibits the conversion. Our findings reveal an unknown plasticity of human adult endocrine cells that can be modulated. This endocrine cell plasticity could have implications for islet development, (patho)physiology, and regeneration

Expansion of Adult Human Pancreatic Tissue Yields Organoids Harboring Progenitor Cells with Endocrine Differentiation Potential.

Generating an unlimited source of human insulin-producing cells is a prerequisite to advance β cell replacement therapy for diabetes. Here, we describe a 3D culture system that supports the expansion of adult human pancreatic tissue and the generation of a cell subpopulation with progenitor characteristics. These cells display high aldehyde dehydrogenase activity (ALDHhi), express pancreatic progenitors markers (PDX1, PTF1A, CPA1, and MYC), and can form new organoids in contrast to ALDHlo cells. Interestingly, gene expression profiling revealed that ALDHhi cells are closer to human fetal pancreatic tissue compared with adult pancreatic tissue. Endocrine lineage markers were detected upon in vitro differentiation. Engrafted organoids differentiated toward insulin-positive (INS+) cells, and circulating human C-peptide was detected upon glucose challenge 1 month after transplantation. Engrafted ALDHhi cells formed INS+ cells. We conclude that adult human pancreatic tissue has potential for expansion into 3D structures harboring progenitor cells with endocrine differentiation potential

Single-cell transcriptomics links loss of human pancreatic β-cell identity to er stress

The maintenance of pancreatic islet architecture is crucial for proper β-cell function. We previously reported that disruption of human islet integrity could result in altered β-cell identity. Here we combine β-cell lineage tracing and single-cell transcriptomics to investigate the mechanisms underlying this process in primary human islet cells. Using drug-induced ER stress and cytoskeleton modification models, we demonstrate that altering the islet structure triggers an unfolding protein response that causes the downregulation of β-cell maturity genes. Collectively, our findings illus-trate the close relationship between endoplasmic reticulum homeostasis and β-cell phenotype, and strengthen the concept of altered β-cell identity as a mechanism underlying the loss of functional β-cell mass

Distinct Merkel Cell Polyomavirus Molecular Features in Tumour and Non Tumour Specimens from Patients with Merkel Cell Carcinoma

Merkel Cell Polyomavirus (MCPyV) is associated with Merkel Cell carcinoma (MCC), a rare, aggressive skin cancer with neuroendocrine features. The causal role of MCPyV is highly suggested by monoclonal integration of its genome and expression of the viral large T (LT) antigen in MCC cells. We investigated and characterized MCPyV molecular features in MCC, respiratory, urine and blood samples from 33 patients by quantitative PCR, sequencing and detection of integrated viral DNA. We examined associations between either MCPyV viral load in primary MCC or MCPyV DNAemia and survival. Results were interpreted with respect to the viral molecular signature in each compartment. Patients with MCC containing more than 1 viral genome copy per cell had a longer period in complete remission than patients with less than 1 copy per cell (34 vs 10 months, P = 0.037). Peripheral blood mononuclear cells (PBMC) contained MCPyV more frequently in patients sampled with disease than in patients in complete remission (60% vs 11%, P = 0.00083). Moreover, the detection of MCPyV in at least one PBMC sample during follow-up was associated with a shorter overall survival (P = 0.003). Sequencing of viral DNA from MCC and non MCC samples characterized common single nucleotide polymorphisms defining 8 patient specific strains. However, specific molecular signatures truncating MCPyV LT were observed in 8/12 MCC cases but not in respiratory and urinary samples from 15 patients. New integration sites were identified in 4 MCC cases. Finally, mutated-integrated forms of MCPyV were detected in PBMC of two patients with disseminated MCC disease, indicating circulation of metastatic cells. We conclude that MCPyV molecular features in primary MCC tumour and PBMC may help to predict the course of the disease

Thérapie génique (stratégie suicide humanisée et évaluation du transfert de genes par vecteurs lentiviraux)

Gene therapy is a new approach of Medicine creating the notion of "gene-medicine". During my thesis, I mainly participated to development and evaluation of four essential components of gene therapy strategies : transfer, expression, and control of the therapeutic gene, as well as the future of the therapeutic targets, the transduced cells. In a first part of this work, we try to develop a system of induced and targeted apoptosis. This system relies on the expression of an inducible caspase 8 controlled by a "specific" promoter, and transfered in target cells by a viral vector. The main point of this strategy is the evaluation of an inducible caspase 8 as a "therapeutic" gene. We compared two transcriptional regulation systems, and a post-translational one. We eventually proposed a modified form of human caspase 8 inducible by an homodimerization regulated system as an alternative system to suicide gene HSV-tk. We also considered controlling expression of this proapoptotic gene with a promoter "specifically" active in cells transformed by a mutated form of Ras, our first cellular model. In a second part, we were interested in gene transfer systems, and more particularly in entiviral vectors. Our first study model was the 3T3-L1 adipocyte system, one of the main cellular model used in obesity and diabetes fields of research, but hardly transfectable by usual transfection methods. We show that HIV-1 derived lentiviral vectors efficiently transduce quiescent and fully differentiated 3T3-L1 adipocytes, as well as 3T3-L1 dividing pre-adipocytes. Furthermore, contrary to adenoviruses, there's no overt signs of cytotoxicity, as confirmed by the glucose uptake physiological assay. Moreover, we checked that capacity to differentiate is not affected by the transduction of 3T3-L1 pre-adipocytes. We thus show that 3rd generation of lentiviral vectors are the system of choice for genetic modification of 3T3-L1 pre-adipocytes and mature adipocytes. Finally, we established several collaborations offering attracting applications for such a system, not only as gene vectorisation system, but also as a stable ectopic expression system that can be combined with other technologies like siRNA. To conclude, it's important to note that if clinical applications in this kind of approaches are long-term prospects, even very long-term, for security and efficiency reasons, such technologies can find multiple applications in various fields of research as a commun tool of molecular biology.La thérapie génique est une nouvelle approche de la médecine créant la notion de gènes-médicaments. Au cours de ma thèse, j'ai participé principalement au développement et à l'évaluation de quatre composants essentiels d'une approche de thérapie génique : le transfert, l'expression et le contrôle du gène thérapeutique, ainsi que le devenir des cibles thérapeutiques, les cellules transduites. Dans une première partie de notre travail, nous avons tenté de développer un système d'apoptose inductible et ciblée. Ce système est basé sur une caspase inductible dont l'expression est contrôlée par un promoteur " spécifique ", et transférée dans la cellule cible par un vecteur viral. Le point central de cette stratégie a été l'évaluation d'une caspase 8 inductible comme gène " thérapeutique ". Nous avons comparé pour cela des systèmes de régulation transcriptionnelle ainsi qu'un système de régulation post-traductionnelle, et nous proposons finalement une caspase 8 humaine inductible par un système d'homodimérisation régulée comme une alternative au gène suicide HSV-tk. Nous avons également considéré le contrôle de l'expression de ce gène grâce à un promoteur " spécifique " de cellules transformées par une forme activée de Ras, notre premier modèle d'étude. Dans une deuxième partie, nous nous sommes intéressés au système de transfert de matériel génétique et plus particulièrement aux vecteurs lentiviraux. Notre premier modèle d'étude a été le système adipocytaire 3T3-L1, un des principaux modèles cellulaires pour les études concernant l'obésité, le diabète, mais très difficilement transfectable par les méthodes usuelles. Nous montrons que des adipocytes 3T3-L1 quiescents et pleinement différenciés, aussi bien que des pré-adipocytes 3T3-L1 en division, sont efficacement transduits par les vecteurs lentiviraux dérivés du HIV-1. De plus, contrairement aux adénovirus, les lentivirus n'engendrent aucun signe de cytotoxicité apparent, comme le confirme le test physiologique d'absorption du glucose en réponse à l'insuline. Nous avons également vérifié que la transduction des pré-adipocytes 3T3-L1 n'affecte en rien leur capacité à se différencier. Nous montrons donc que les vecteurs lentiviraux de 3ème génération constituent le système de transfert de choix pour la modification génétique des pré-adipocytes et adipocytes matures 3T3-L1. Enfin, nous avons établi plusieurs collaborations offrant des applications attrayantes pour un tel système, non seulement en tant que stratégie de vectorisation de gènes, mais aussi comme outil d'expression ectopique stable pouvant notamment être combiné avec d'autres technologies comme les siRNA. En conclusion, il est important de noter que si les applications cliniques de ce type d'approche restent des perspectives à long terme, voire très long terme pour cause d'aspects sécuritaires et d'efficacité encore insuffisants, une telle technologie peut trouver de multiples applications dans des domaines très divers comme outil commun de biologie moléculaire.NICE-BU Sciences (060882101) / SudocSudocFranceF

Mitogen-activated protein kinase (MAPK) phosphatase-1 and -4 attenuate p38 MAPK during dexamethasone-induced insulin resistance in 3T3-L1 adipocytes

Prolonged use of glucocorticoids induces pronounced insulin resistance in vivo. In vitro, treatment of 3T3-L1 adipocytes with dexamethasone for 48 h reduces the maximal level of insulin- and stress (arsenite)-induced glucose uptake by approximately 50%. Although phosphatidylinositol 3-kinase signaling was slightly attenuated, phosphorylation of its downstream effectors such as protein kinase B and protein kinase C-lambda remained intact. Nor was any effect of dexamethasone treatment observed on insulin- or arsenite-induced translocation of glucose transporter 4 (GLUT4) toward the plasma membrane. However, for a maximal response to either arsenite- or insulin-induced glucose uptake in these cells, functional p38 MAPK signaling is required. Dexamethasone treatment markedly attenuated p38 MAPK phosphorylation coincident with an up-regulation of the MAPK phosphatases MKP-1 and MKP-4. Employing lentivirus-mediated ectopic expression in fully differentiated 3T3-L1 adipocytes demonstrated a differential effect of these phosphatases: whereas MKP-1 was a more potent inhibitor of insulin-induced glucose uptake, MKP-4 more efficiently inhibited arsenite-induced glucose uptake. This coincided with the effects of these phosphatases on p38 MAPK phosphorylation, i.e. MKP-1 and MKP-4 attenuated p38 MAPK phosphorylation by insulin and arsenite, respectively. Taken together, these data provide evidence that in 3T3-L1 adipocytes dexamethasone inhibits the activation of the GLUT4 in the plasma membrane by a p38 MAPK-dependent process, rather than in a defect in GLUT4 translocation per s

Lentiviral vectors efficiently transduce quiescent mature 3T3-L1 adipocytes

Obesity is associated with many serious afflictions such as cardiovascular disease, cancer, and diabetes. One of the main cellular systems used to study the underlying physiological and biological processes is the 3T3-L1 preadipocyte differentiation model. However, studies on 3T3-L1 adipocytes are hampered by the fact that genetic modification of mature adipocytes is notoriously difficult. In this report, we evaluated the use of lentivirus-mediated gene transfer into 3T3-L1 mature adipocytes. We demonstrate that quiescent, fully differentiated 3T3-L1 adipocytes as well as 3T3-L1 preadipocytes can be efficiently transduced with HIV-1-derived lentiviral vectors. Upon transduction using LV-PGK-GFP lentiviral vector at 100 ng p24 per 10(5) cells, more than 95% of the 3T3-L1 adipocytes in the culture expressed the GFP reporter gene. There were no overt signs of toxicity or cytopathogenicity in the cultures. Furthermore, modification of undifferentiated preadipocytes did not affect their capacity to differentiate. In addition, insulin-induced glucose uptake was not affected by the procedure. In contrast, adenoviral-mediated gene transfer into 3T3-L1 adipocytes is associated with marked cytopathogenicity. From these data, we conclude that lentiviral vectors are the gene-transfer system of choice for genetic modification of mature adipocytes. The availability of an efficient vector system may stimulate the use of adipose tissue as a target for gene therapy in obesity and other disorder