Serotonin regulates mouse cranial neural crest migration.

Serotonergic agents (uptake inhibitors, receptor ligands) cause significant craniofacial malformations in cultured mouse embryos suggesting that 5-hydroxytryptamine (serotonin) (5-HT) may be an important regulator of craniofacial development. To determine whether serotonergic regulation of cell migration might underly some of these effects, cranial neural crest (NC) explants from embryonic day 9 (E9) (plug day = E1) mouse embryos or dissociated mandibular mesenchyme cells (derived from NC) from E12 embryos were placed in a modified Boyden chamber to measure effects of serotonergic agents on cell migration. A dose-dependent effect of 5-HT on the migration of highly motile cranial NC cells was demonstrated, such that low concentrations of 5-HT stimulated migration, whereas this effect was progressively lost as the dose of 5-HT was increased. In contrast, most concentrations of 5-HT inhibited migration of less motile, mandibular mesenchyme cells. To investigate the possible involvement of specific 5-HT receptors in the stimulation of NC migration, several 5-HT subtype-selective antagonists were used to block the effects of the most stimulatory dose of 5-HT (0.01 microM). Only NAN-190 (a 5-HT1A antagonist) inhibited the effect of 5-HT, suggesting involvement of this receptor. Further evidence was obtained by using immunohistochemistry with 5-HT receptor antibodies, which revealed expression of the 5-HT1A receptor but not other subtypes by migrating NC cells in both embryos and cranial NC explants. These results suggest that by activating appropriate receptors 5-HT may regulate migration of cranial NC cells and their mesenchymal derivatives in the mouse embryo.

Transendothelial migration of neutrophils involves integrin-associated protein (CD47).

Inflammation is a primary pathological process. The development of an inflammatory reaction involves the movement of white blood cells through the endothelial lining of blood vessels into tissues. This process of transendothelial cell migration of neutrophils has been shown to involve neutrophil beta 2 integrins (CD18) and endothelial cell platelet-endothelium cell adhesion molecules (PECAM-1; CD31). We now show that F(ab')2 fragments of the monoclonal antibody B6H12 against integrin-associated protein (IAP) blocks the transendothelial migration of neutrophils stimulated by an exogenous gradient of the chemokine interleukin 8 (IL-8; 60% inhibition), by the chemotactic peptide N-formyl-methionylleucylphenylalanine (FMLP; 76% inhibition), or by the activation of the endothelium by the cytokine tumor necrosis factor alpha (98% inhibition). The antibody has two mechanisms of action: on neutrophils it prevents the chemotactic response to IL-8 and FMLP, and on endothelium it prevents an unknown but IL-8-independent process. Blocking antibodies to IAP do not alter the expression of adhesion proteins or production of IL-8 by endothelial cells, and thus the inhibition of neutrophil transendothelial migration is selective. These data implicate IAP as the third molecule essential for neutrophil migration through endothelium into sites of inflammation.

Étude de la migration des populations de lymphocytes B du sang de patients infectés par le virus d’immunodéficience humaine (VIH)

La dérégulation du compartiment de cellules B est une conséquence importante de l’infection par le virus de l’immunodéficience humaine (VIH-1). On observe notamment une diminution des nombres de lymphocytes B sanguins ainsi qu’une variation des fréquences relatives des différentes populations de lymphocytes B chez les individus infectés par rapport aux contrôles sains. Notre laboratoire a précédemment démontré l’implication des cellules dendritiques dans la dérégulation des lymphocytes B via la roduction excessive de BLyS/BAFF, un stimulateur des cellules B. De plus, lors l’études menées chez la souris transgénique présentant une maladie semblable au SIDA, et chez la souris BLyS/BAFF transgénique, l’infection au VIH-1 fut associée à une expansion de la zone marginale (MZ) de la rate. De façon intéressante, nous observons chez les contrôleurs élites une diminution de la population B ‘mature’ de la MZ. Il s’agit du seul changement important chez les contrôleurs élites et reflète possiblement un recrutement de ces cellules vers la périphérie ainsi qu’une implication dans des mécanismes de contrôle de l’infection. Pour tenter d’expliquer et de mieux comprendre ces variations dans les fréquences des populations B, nous avons analysé les axes chimiotactiques CXCL13-CXCR5, CXCL12-CXCR4/CXCR7, CCL20-CCR6 et CCL25-CCR9. L’étude longitudinale de cohortes de patients avec différents types de progression clinique ou de contrôle de l’infection démontre une modulation des niveaux plasmatiques de la majorité des chimiokines analysées chez les progresseurs rapides et classiques. Au contraire, les contrôleurs élites conservent des niveaux normaux de chimiokines, démontrant leur capacité à maintenir l’homéostasie. La migration des populations de cellules B semble être modulée selon la progression ou le contrôle de l’infection. Les contrôleurs élites présentent une diminution de la population B ‘mature’ de la MZ et une augmentation de la fréquence d’expression du récepteur CXCR7 associé à la MZ chez la souris, suggérant un rôle important des cellules de la MZ dans le contrôle de l’infection au VIH-1. De façon générale, les résultats dans cette étude viennent enrichir nos connaissances du compartiment de cellules B dans le contexte de l’infection au VIH-1 et pourront contribuer à élaborer des stratégies préventives et thérapeutiques contre ce virus.

Investigation du rôle de Myo9b dans la migration des interneurones GABAergiques corticaux

Les encéphalopathies épileptogènes sont des maladies graves de l’enfance associant une épilepsie, souvent réfractaire, et un retard de développement. Les mécanismes sous-tendant ces maladies sont peu connus. Cependant, nous postulons que ces épilepsies puissent être causées par une dysfonction du réseau inhibiteur. En effet, des défauts de migration ou de maturation des interneurones GABAergiques (INs) corticaux induisent l’épilepsie, tant chez l’humain que chez la souris. Dans le but d’étudier les causes génétiques des encéphalopathies épileptogènes sporadiques inexpliquées, le laboratoire de la Dre Rossignol a procédé au séquençage d’exome entier d’une cohorte d’enfants atteints. Cela a permis d’identifier, chez un patient, une nouvelle mutation de novo, possiblement pathogène, dans le gène MYO9b. MYO9b est impliqué dans la migration de cellules immunitaires et cancéreuses et est exprimée durant le développement cérébral. Nous émettons l’hypothèse voulant que MYO9b puisse être importante pour la migration des INs corticaux. Les résultats présentés dans ce mémoire démontrent que Myo9b est exprimé dès le stade embryonnaire par les progéniteurs des INs corticaux et que son expression se restreint aux INs dans le cortex mature. De plus, nous démontrons que la répression ex vivo de Myo9b sélectivement dans les INs au sein de tranches corticales organotypiques embryonnaires mène à des défauts morphologiques majeurs de ces cellules en migration. En effet, ces cellules présentent une morphologie multipolaire et des neurites rostraux plus longs et plus complexes. Ces changements morphologiques pourraient avoir un impact majeur sur la migration des INs et ainsi perturber le développement des réseaux inhibiteurs.

Modelling oxygen diffusion and cell growth in a porous, vascularising scaffold for soft tissue engineering applications

Soft tissue engineering presents significant challenges compared to other tissue engineering disciplines such as bone, cartilage or skin engineering. The very high cell density in most soft tissues, often combined with large implant dimensions, means that the supply of oxygen is a critical factor in the success or failure of a soft tissue scaffold. A model is presented for oxygen diffusion in a 15-60 mm diameter dome-shaped scaffold fed by a blood vessel loop at its base. This model incorporates simple models for vascular growth, cell migration and the effect of cell density on the effective oxygen diffusivity. The model shows that the dynamic, homogeneous cell seeding method often employed in small-scale applications is not applicable in the case of larger scale scaffolds such as these. Instead, we propose the implantation of a small biopsy of tissue close to a blood supply within the scaffold as a technique more likely to be successful. Crown Copyright (c) 2005 Published by Elsevier Ltd. All rights reserved.

Robo1 regulates the development of major axon tracts and interneuron migration in the forebrain

The Slit genes encode secreted ligands that regulate axon branching, commissural axon pathfinding and neuronal migration. The principal identified receptor for Slit is Robo ( Roundabout in Drosophila). To investigate Slit signalling in forebrain development, we generated Robo1 knockout mice by targeted deletion of exon 5 of the Robo1 gene. Homozygote knockout mice died at birth, but prenatally displayed major defects in axon pathfinding and cortical interneuron migration. Axon pathfinding defects included dysgenesis of the corpus callosum and hippocampal commissure, and abnormalities in corticothalamic and thalamocortical targeting. Slit2 and Slit1/2 double mutants display malformations in callosal development, and in corticothalamic and thalamocortical targeting, as well as optic tract defects. In these animals, corticothalamic axons form large fasciculated bundles that aberrantly cross the midline at the level of the hippocampal and anterior commissures, and more caudally at the medial preoptic area. Such phenotypes of corticothalamic targeting were not observed in Robo1 knockout mice but, instead, both corticothalamic and thalamocortical axons aberrantly arrived at their respective targets at least 1 day earlier than controls. By contrast, in Slit mutants, fewer thalamic axons actually arrive in the cortex during development. Finally, significantly more interneurons ( up to twice as many at E12.5 and E15.5) migrated into the cortex of Robo1 knockout mice, particularly in both rostral and parietal regions, but not caudal cortex. These results indicate that Robo1 mutants have distinct phenotypes, some of which are different from those described in Slit mutants, suggesting that additional ligands, receptors or receptor partners are likely to be involved in Slit/Robo signalling.

Mechanical stimulation alters pleiotrophin and aggrecan expression by human intervertebral disc cells and influences their capacity to stimulate endothelial migration

Study Design. The influence of mechanical load on pleiotrophin (PTM) and aggrecan expression by intervertebral disc (IVD) cells, and the effects of disc cell conditioned medium on endothelial cell migration was investigated. Objective. To examine possible interactions of mechanical loads and known pro- and antiangiogenic factors, which may regulate disc angiogenesis during degeneration. Summary of Background Data. Pleiotrophin expression can be influenced by mechanical stimulation and has been associated with disc vascularization. Disc aggrecan inhibits endothelial cell migration, suggesting an antiangiogenic role. A possible interplay between these factors is unknown. Methods. The influence of the respective predominant load (cyclic strain for anulus fibrosus and hydrostatic pressure for nucleus pulposus cells) on PTN and aggrecan expression by IVD cells was determined by real-time RT-PCR and Western blotting (PTN only). The effects of IVD cell conditioned medium on endothelial cell migration were analyzed in a bioassay using human microvascular endothelial (HMEC-1) cells. Results. Application of both mechanical loads resulted in significant alterations of gene expression of PTN (+67%, P = 0.004 in anulus cells; +29%, P = 0.03 in nucleus cells) and aggrecan (+42%, P = 0.03 in anulus cells, -25%, P = 0.03 in nucleus cells). These effects depended on the cell type, the applied load, and timescale. Conditioned media of nucleus pulposus cells enhanced HMEC-1 migration, but this effect was diminished after 2.5 MPa hydrostatic pressure, when aggrecan expression was diminished, but not 0.25 MPa, when expression levels were unchanged. Conclusion. Mechanical loading influences PTN expression by human IVD cells. Conditioned media from nucleus pulposus cell cultures stimulated HMEC-1 endothelial cell migration. This study demonstrates that the influence of mechanical loads on vascularization of the human IVD is likely to be complex and does not correlate simply with altered expression of known pro- and antiangiogenic factors.

Mesenchymal stem cell-conditioned medium accelerates skin wound healing:an in vitro study of fibroblast and keratinocyte scratch assays

We have used in vitro scratch assays to examine the relative contribution of dermal fibroblasts and keratinocytes in the wound repair process and to test the influence of mesenchymal stem cell (MSC) secreted factors on both skin cell types. Scratch assays were established using single cell and co-cultures of L929 fibroblasts and HaCaT keratinocytes, with wound closure monitored via time-lapse microscopy. Both in serum supplemented and serum free conditions, wound closure was faster in L929 fibroblast than HaCaT keratinocyte scratch assays, and in co-culture the L929 fibroblasts lead the way in closing the scratches. MSC-CM generated under serum free conditions significantly enhanced the wound closure rate of both skin cell types separately and in co-culture, whereas conditioned medium from L929 or HaCaT cultures had no significant effect. This enhancement of wound closure in the presence of MSC-CM was due to accelerated cell migration rather than increased cell proliferation. A number of wound healing mediators were identified in MSC-CM, including TGF-beta1, the chemokines IL-6, IL-8, MCP-1 and RANTES, and collagen type I, fibronectin, SPARC and IGFBP-7. This study suggests that the trophic activity of MSC may play a role in skin wound closure by affecting both dermal fibroblast and keratinocyte migration, along with a contribution to the formation of extracellular matrix.

The role of heterodimerization between VEGFR-1 and VEGFR-2 in the regulation of endothelial cell homeostasis

VEGF-A activity is tightly regulated by ligand and receptor availability. Here we investigate the physiological function of heterodimers between VEGF receptor-1 (VEGFR-1; Flt-1) and VEGFR-2 (KDR; Flk-1) (VEGFR(1-2)) in endothelial cells with a synthetic ligand that binds specifically to VEGFR(1-2). The dimeric ligand comprises one VEGFR-2-specific monomer (VEGF-E) and a VEGFR-1-specific monomer (PlGF-1). Here we show that VEGFR(1-2) activation mediates VEGFR phosphorylation, endothelial cell migration, sustained in vitro tube formation and vasorelaxation via the nitric oxide pathway. VEGFR(1-2) activation does not mediate proliferation or elicit endothelial tissue factor production, confirming that these functions are controlled by VEGFR-2 homodimers. We further demonstrate that activation of VEGFR(1-2) inhibits VEGF-A-induced prostacyclin release, phosphorylation of ERK1/2 MAP kinase and mobilization of intracellular calcium from primary endothelial cells. These findings indicate that VEGFR-1 subunits modulate VEGF activity predominantly by forming heterodimer receptors with VEGFR-2 subunits and such heterodimers regulate endothelial cell homeostasis.

Autocrine activity of soluble Flt-1 controls endothelial cell function and angiogenesis

Background - The negative feedback system is an important physiological regulatory mechanism controlling angiogenesis. Soluble vascular endothelial growth factor (VEGF) receptor-1 (sFlt-1), acts as a potent endogenous soluble inhibitor of VEGF- and placenta growth factor (PlGF)-mediated biological function and can also form dominant-negative complexes with competent full-length VEGF receptors. Methods and results - Systemic overexpression of VEGF-A in mice resulted in significantly elevated circulating sFlt-1. In addition, stimulation of human umbilical vein endothelial cells (HUVEC) with VEGF-A, induced a five-fold increase in sFlt-1 mRNA, a time-dependent significant increase in the release of sFlt-1 into the culture medium and activation of the flt-1 gene promoter. This response was dependent on VEGF receptor-2 (VEGFR-2) and phosphoinositide-3'-kinase signalling. siRNA-mediated knockdown of sFlt-1 in HUVEC stimulated the activation of endothelial nitric oxide synthase, increased basal and VEGF-induced cell migration and enhanced endothelial tube formation on growth factor reduced Matrigel. In contrast, adenoviral overexpression of sFlt-1 suppressed phosphorylation of VEGFR-2 at tyrosine 951 and ERK-1/-2 MAPK and reduced HUVEC proliferation. Preeclampsia is associated with elevated placental and systemic sFlt-1. Phosphorylation of VEGFR-2 tyrosine 951 was greatly reduced in placenta from preeclamptic patients compared to gestationally-matched normal placenta. Conclusion - These results show that endothelial sFlt-1 expression is regulated by VEGF and acts as an autocrine regulator of endothelial cell function.

Importance of tissue transglutaminasenitrosylation in fibroblasts migration and MMPregulation

As an extracellular second messenger, nitric oxide (NO) mediates the modification of proteins through nitrosylation of cysteine andtyrosine residues. Tissue Transglutaminase (TG2) is a Ca2+ activated, sulfhydryl rich protein with 18 free cysteine residues, which catalyzes ε-(γ glutamyl)lysine crosslink between extracellular and intracellular proteins. NO can nitrosylate up to 15 of the cysteine residues in TG2, leading to the irreversible inactivation of the enzyme activity. The interplay between these two agents was revealed for the first time by our study showing that NO inhibited the TG2-induced transcriptional activation of TGFb1and extracellular matrix (ECM) protein synthesis by nitrosylating TG2 in an inactive confirmation with inert catalytic activity. However, nitrosylated TG2 was still able to serve as a novel cell adhesion protein. In the light of our previous findings, in this study we aim to elucidate the NO modified function of TG2 in cell migration using an in vitro model mimicking the tissue matrix remodeling phases of wound healing. Using transfected fibroblasts expressing TG2 under the control of the tetracycline-off promoter, we demonstrate that upregulation of TG2 expression and activity inhibited the cell migration through the activation of TGFβ1. Increased TG2 activity led to arise in the biosynthesis and activity of the gelatinases, MMP-2 andMMP-9, while decreasing the biosynthesis and activity of the col-lagenases MMP-1a and MMP-13. NO donor S-Nitroso-N-acetyl-penicillamine (SNAP) treatment relieved the TG2 obstructed-cellmigration by blocking the TG2 enzyme activity. In addition,decrease in TG2 activity due to nitrosylation led to an inhibition of TGFβ1, which in turn affected the pattern of MMP activation. Recent evidence suggests that, once in complex with fibronectin in the ECM, TG2 can interact with syndecan-4 or integrinβ-1and regulate the cell adhesion. In the other part of this study, the possible role of nitrosylated TG2 on the regulation of cell migration during wound healing was investigated with respect to its interactions with integrin β1 (ITGβ1) and syndecan-4 (SDC4). Treatment with TG2 inhibitor Z-DON resulted in a 50% decrease in the TG2 interaction with ITGB1 and SDC4, while increasing concentrations of SNAP firstly led to a substantial decrease and then completely abolished the TG2/ITGβ1 and TG2/SDC4 complex formation on the cell surface. Taken together, data obtained from this study suggests that nitrosylation of TG2 leads to a change not only in the binding partners of TG2 on cell surface but also in TGFβ1-dependent MMP activation, which give rise to an increase in the migration potential of fibroblasts.

Intracellular Signaling and Trafficking in Cancer: Role of Rab5-GTPase in Migration and Invasion of Breast Cells

Metastasis is characterized pathologically by uncontrolled cell invasion, proliferation, migration and angiogenesis. Steroid hormones, such as estrogen, and growth factors, which include insulin growth factor I/II (IGF-1/IGF-2) therapy has been associated with most if not all of the features of metastasis. It has been determined that IGF-1 increases cell survival of cancer cells and potentiate the effect of E2 and other ligand growth factors on breast cancer cells. However not much information is available that comprehensively expounds on the roles of insulin growth factor receptor (IGFR) and Rab GTPases may play in breast cancer. The latter, Rab GTPases, are small signaling molecules and critical in the regulation of many cellular processes including cell migration, growth via the endocytic pathway. This research involves the role of Rab GTPases, specifically Rab5 and its guanine exchange factors (GEFs), in the promotion of cancer cell migration and invasion. Two important questions abound: Are IGFR stimulation and downstream effect involved the endocytic pathway in carcinogenesis? What role does Rab5 play in cell migration and invasion of cancer cells? The hypothesis is that growth factor signaling is dependent on Rab5 activity in mediating the aggressiveness of cancer cells. The goal is to demonstrate that IGF-1 signaling is dependent on Rab5 function in breast cancer progression. Here, the results thus far, have shown that while activation of Rab5 may mediate increased cell proliferation, migration and invasion in breast cancer cells, the Rab5 GEF, RIN1 interacts with the IGFR thereby facilitating migration and invasion activities in breast cells. Furthermore, endocytosis of the IGFR in breast cancer cells seems to be caveolin dependent as the data has shown. This taken together, the data shows that IGF-1 signaling in breast cancer cells relies on IGF-1R phosphorylation, caveolae internalization and sequestration to the early endosome RIN1 function and Rab5 activation.

Role for cell-to-cell communication in stem cell specification toward pancreatic progenitors: relevance to the design of novel therapies for diabetes.

Thesis (Ph.D.)--University of Washington, 2016-08

Analysis and modelling of immune cell behaviour in lymph nodes based on multi-photon imaging

This thesis presents quantitative studies of T cell and dendritic cell (DC) behaviour in mouse lymph nodes (LNs) in the naive state and following immunisation. These processes are of importance and interest in basic immunology, and better understanding could improve both diagnostic capacity and therapeutic manipulations, potentially helping in producing more effective vaccines or developing treatments for autoimmune diseases. The problem is also interesting conceptually as it is relevant to other fields where 3D movement of objects is tracked with a discrete scanning interval. A general immunology introduction is presented in chapter 1. In chapter 2, I apply quantitative methods to multi-photon imaging data to measure how T cells and DCs are spatially arranged in LNs. This has been previously studied to describe differences between the naive and immunised state and as an indicator of the magnitude of the immune response in LNs, but previous analyses have been generally descriptive. The quantitative analysis shows that some of the previous conclusions may have been premature. In chapter 3, I use Bayesian state-space models to test some hypotheses about the mode of T cell search for DCs. A two-state mode of movement where T cells can be classified as either interacting to a DC or freely migrating is supported over a model where T cells would home in on DCs at distance through for example the action of chemokines. In chapter 4, I study whether T cell migration is linked to the geometric structure of the fibroblast reticular network (FRC). I find support for the hypothesis that the movement is constrained to the fibroblast reticular cell (FRC) network over an alternative 'random walk with persistence time' model where cells would move randomly, with a short-term persistence driven by a hypothetical T cell intrinsic 'clock'. I also present unexpected results on the FRC network geometry. Finally, a quantitative method is presented for addressing some measurement biases inherent to multi-photon imaging. In all three chapters, novel findings are made, and the methods developed have the potential for further use to address important problems in the field. In chapter 5, I present a summary and synthesis of results from chapters 3-4 and a more speculative discussion of these results and potential future directions.

Dissecting signaling pathways that control glia migration in the developing Drosophila retina

The function of a complex nervous system relies on an intricate interaction between neurons and glial cells. However, as glial cells are generally born distant from the place where they settle, molecular cues are important to direct their migration. Glial cell migration is important in both normal development and disease, thus current research in the laboratory has been focused on dissecting regulatory events underlying that crucial process. With this purpose, the Drosophila eye imaginal disc has been used as a model. In response to neuronal photoreceptor differentiation, glial cells migrate from the CNS into the eye disc where they act to correctly wrap axons. To ensure proper development, attractive and repulsive signals must coordinate glial cell migration. Importantly, one of these signals is Bnl, a Fibroblast Growth Factor (FGF) ligand expressed by retinal progenitor cells that was suggested to act as a non-autonomous negative regulator of excessive glial cell migration (overmigration) by binding and activating the Btl receptor expressed by glial cells. Through the experimental results described in chapter 3 we gained a detailed insight into the function of bnl in eye disc growth, photoreceptor development, and glia migration. Interestingly, we did not find a direct correlation between the defects on the ongoing photoreceptors and the glia overmigration phenotype; however, bnl knockdown caused apoptosis of eye progenitor cells what was strongly correlated with glia migration defects. Glia overmigration due to Bnl down-regulation in eye progenitor cells was rescued by inhibiting the pro-apoptotic genes or caspases activity, as well as, by depleting JNK or Dp53 function in retinal progenitor cells. Thus, we suggest a cross-talk between those developmental signals in the control of glia migration at a distance. Importantly, these results suggest that Bnl does not control glial migration in the eye disc exclusively through its ability to bind and activate its receptor Btl in glial cells. We also discuss possible biological roles for the glia overmigration in the bnl knockdown background. Previous results in the lab showed an interaction between dMyc, a master regulator of tissue growth, and Dpp, a Transforming Growth Factor-β important for retinal patterning and for accurate glia migration into the eye disc. Thus, we became interested in understanding putative relationships between Bnl and dMyc. In chapter 4, we show that they positively cooperate in order to ensure proper development of the eye disc. This work highlights the importance of the FGF signaling in eye disc development and reveals a signaling network where a range of extra- and intra-cellular signals cooperate to non-autonomously control glial cell migration. Therefore, such inter-relations could be important in other Drosophila cellular contexts, as well as in vertebrate tissue development.