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  • 1
    Keywords: CORTICOTROPIN-RELEASING HORMONE, EPITHELIAL STEM-CELLS, EXPRESSION, gene regulation, GENE-EXPRESSION
    Abstract: The controls of human keratin expression in situ remain to be fully elucidated. Here, we have investigated the effects of the neurohormone prolactin (PRL) on keratin expression in a physiologically and clinically relevant test system: organ-cultured normal human hair follicles (HFs). Not only do HFs express a wide range of keratins, but they are also a source and target of PRL. Microarray analysis revealed that PRL differentially regulated a defined subset of keratins and keratin-associated proteins. Quantitative immunohistomorphometry and quantitative PCR confirmed that PRL up-regulated expression of keratins K5 and K14 and the epithelial stem cell-associated keratins K15 and K19 in organ-cultured HFs and/or isolated HF keratinocytes. PRL also up-regulated K15 promoter activity and K15 protein expression in situ, whereas it inhibited K6 and K31 expression. These regulatory effects were reversed by a pure competitive PRL receptor antagonist. Antagonist alone also modulated keratin expression, suggesting that "tonic stimulation" by endogenous PRL is required for normal expression levels of selected keratins. Therefore, our study identifies PRL as a major, clinically relevant, novel neuroendocrine regulator of both human keratin expression and human epithelial stem cell biology in situ.
    Type of Publication: Journal article published
    PubMed ID: 20103718
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  • 2
    Keywords: ANGIOGENESIS ; CANCER ; CELLS ; GROWTH ; tumor ; BLOOD ; CELL ; IN-VIVO ; MODEL ; MODELS ; POPULATION ; DIFFERENTIATION ; TUMORS ; MICE ; DENDRITIC CELLS ; BIOLOGY ; MATURATION ; MOUSE ; HUMAN-TUMORS ; SURVEILLANCE ; immune response ; IMMUNE-RESPONSE ; IMMUNITY ; IMMUNOTHERAPY ; MOUSE MODEL ; CHILDREN ; PRECURSORS ; microenvironment ; PROGRAM ; fibroblast ; TUMOR-GROWTH ; ABLATION ; SUPPLEMENTATION ; DEPLETION ; vasculogenesis ; PROGENITORS ; immune cells ; CELL BIOLOGY ; BOSTON ; IMMUNE
    Abstract: Dendritic cells (DCs)-immunomodulatory cells that initiate adaptive immune responses-have recently been shown to exert proangiogenic effects when infiltrating the tumor microenvironment. As tumors that escape immune surveillance inhibit DC maturation, we explored whether maturation status determines their ability to promote angiogenesis and whether angiogenesis depends on the presence of DCs. Using mouse xenograft models of human tumors, we show that fast-growing "angiogenic" tumors are infiltrated by a more immature DC population than respective dormant avascular tumors. Accordingly, supplementation of immature DCs, but not mature DCs, enhanced tumor growth. When DCs were mixed with Matrigel and injected subcutaneously into mice, only immature DCs promoted the ingrowth of patent blood vessels. Notably, depletion of DCs in a transgenic mouse model that allows for their conditional ablation completely abrogated basic fibroblast growth factor-induced angiogenesis in Matrigel plugs, and significantly inhibited tumor growth in these mice. Because immature DCs actively promote angiogenesis and tumor growth, whereas DC maturation or ablation suppresses this response, we conclude that angiogenesis is dependent on the presence of immature DCs. Thus, cancer immunotherapies that promote DC maturation may act by both augmenting the host immune response to the tumor and by suppressing tumor angiogenesis.-Fainaru, O., Almog, N., Yung, C. W., Nakai, K., Montoya-Zavala, M., Abollahi, A., D'Amato, R., Ingber, D. E. Tumor growth and angiogenesis are dependent on the presence of immature dendritic cells. FASEB J. 24, 1411-1418 (2010). www.fasebj.org
    Type of Publication: Journal article published
    PubMed ID: 20008545
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  • 3
    Abstract: Lentiviral expression vectors are powerful tools for gene therapy and long-term gene expression/repression in the mammalian brain. However, no specificity of transduction has been reported so far in the central nervous system. Here we have developed a novel system to achieve a neuronal subtype specific expression in either dopaminergic (DA) or GABAergic neurons. We employed a delivery strategy by which the transgene is not expressed until its activation by Cre recombinase. We successfully tested the system in vitro and then used this novel lentivector, containing loxP sites, in 2 different transgenic mouse lines expressing Cre either in DA or in GABAergic neurons. In both lines the reporter gene was detected exclusively in Cre-positive cells, demonstrating that with this experimental approach we were able to achieve completely specific expression of transgenes delivered by lentiviral vectors. This universal system can be applied to all neural subtypes making use of the growing number of specific Cre driver lines.- Tolu, S., Avale, M. E., Nakatani, H., Pons, S., Parnaudeau, S., Tronche, F., Vogt, A., Monyer, H., Vogel, R., de Chaumont, F., Olivo-Marin, J.-C., Changeux, J.-P., Maskos, U. A versatile system for the neuronal subtype specific expression of lentiviral vectors.
    Type of Publication: Journal article published
    PubMed ID: 19858094
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  • 4
    Keywords: CELLS ; PROTEIN ; COMPONENTS ; MICE ; KERATINOCYTES ; COLLAGEN TYPE-IV ; EPITHELIAL MORPHOGENESIS ; MOUSE NIDOGEN-2 ; BINDING-SITE ; LAMININ GAMMA-1 CHAIN
    Abstract: Nidogen-1 and nidogen-2 are homologous proteins found in all basement membranes (BMs). They show comparable binding activities in vitro and partially redundant functions in vivo. Previously, we showed that in skin organotypic cocultures, BM formation was prevented in the absence of nidogens and that either nidogen was able to rescue this failure. We now dissected the two nidogens to identify the domains required for BM deposition. For that purpose, HaCaT cells were grown on collagen matrices containing nidogen-deficient, murine fibroblasts. After addition of nidogen-1 or nidogen-2 protein fragments comprising different binding domains, BM deposition was analyzed by immunofluorescence and electron microscopy. We could demonstrate that the rod-G3 domain of nidogen-2 was sufficient to achieve deposition of BM components at the epidermal-collagen interface. In contrast, for nidogen-1, both the G2 and G3 domains were required. Immunoblot analysis confirmed that all BM components were present in comparable amounts under all culture conditions. This finding demonstrates that nidogens, although homologous proteins, exert their effect on BM assembly through different binding domains, which may in turn result in alterations of BM structure and functions, thus providing an explanation for the phenotypical differences observed between nidogen-1 and -2 deficient mice.
    Type of Publication: Journal article published
    PubMed ID: 22623588
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  • 5
    Keywords: RECEPTOR ; EXPRESSION ; MODELS ; PROTEIN ; DIFFERENTIATION ; DOMAIN ; MIGRATION ; CD34 ; INHIBITS SPROUTING ANGIOGENESIS ; EXHIBIT
    Abstract: Given the need for robust and cost-efficient in vitro models to study angiogenesis and reproducibly analyze potential pro- and antiangiogenic compounds in preclinical studies, we developed a 3-dimensional in vitro angiogenesis assay that is based on collagen gel-embedded, size-defined spheroids generated from cultured human umbilical vein endothelial cells (HUVECs). Despite its wide distribution, limitations, sensitivity, robustness, and improvements, the capacity of this assay for functional screening purposes has not been elucidated thus far. By using time-lapse video microscopy, we show that tip cells lead the formation of capillary-like and partially lumenized sprouts originating from the spheroids. Angiogenic sprouting from spheroids generated from 5 different primary cultured human endothelial cell types was induced by physiologic concentrations of vascular endothelial cell growth factor 165. Based on this assay system, we determined the capacity of 880 approved drugs to interfere with or boost angiogenic sprouting, thereby assessing their putative angiogenesis-related side effects or novel applications. However, although this assay allowed for a rapid and reproducible determination of functional IC50 values of individual compounds, the sprouting results were partially affected by the HUVEC passage number and donor variability. To overcome this limitation, immortalized HUVECs (iHUVECs) showing a more homogenous response in terms of proliferation and sprouting over multiple population doublings were used in the course of this study. Collectively, the spheroid-based angiogenesis assay provides a sensitive and versatile tool to study the impact of pro- and antiangiogenic determinants on multiple steps of the angiogenic cascade. It is compatible with different endothelial cell types and allows use of iHUVECs to improve its overall robustness.-Heiss, M., Hellstrom, M., Kalen, M., May, T., Weber, H., Hecker, M., Augustin, H. G., Korff, T. Endothelial cell spheroids as a versatile tool to study angiogenesis in vitro.
    Type of Publication: Journal article published
    PubMed ID: 25857554
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  • 6
    Abstract: T-cell lymphopenia is a major risk factor for autoimmunity. Here we describe congenic Lewis (LEW) rats with a loss-of-function mutation in the Gimap5 gene, leading to a 92% reduction in peripheral T-cell numbers. Gimap5-deficient LEW rats developed eosinophilic autoimmune gastroenteritis accompanied by a 40-fold increase in IgE serum levels. This phenotype was ameliorated by antibiotic treatment, indicating a critical role of the microbial flora in the development of inflammatory bowel disease. Interestingly, Gimap5-deficient LEW rats showed strongly aggravated experimental autoimmune encephalomyelitis (EAE) after immunization with guinea pig myelin basic protein. This phenotype, however, persisted after antibiosis, confirming that the enhanced CNS autoimmune response in T-cell lymphopenic Gimap5-deficient LEW rats was unrelated to the composition of the microbial flora. Rather, it seems that it was caused by the 7-fold increase in the percentage of activated T cells producing IL-17 and IFN-gamma, and the skewed T-cell receptor (TCR) repertoire, both of which were the result of T-cell lymphopenia and not affected by antibiosis. This notion was supported by the observation that adoptive T-cell transfer corrected the TCR repertoire and improved EAE. Collectively, our findings confirm a critical albeit differential role of T-cell lymphopenia in the susceptibility to organ-specific autoimmune responses.-Fischer, H. J., Witte, A.-K., Walter, L., Grone, H.-J., van den Brandt, J., Reichardt, H. M. Distinct roles of T-cell lymphopenia and the microbial flora for gastrointestinal and CNS autoimmunity.
    Type of Publication: Journal article published
    PubMed ID: 26740263
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  • 7
  • 8
    Keywords: RECEPTOR ; ANGIOGENESIS ; CELLS ; ENDOTHELIAL-CELLS ; GROWTH ; proliferation ; CELL ; ENDOTHELIAL GROWTH-FACTOR ; TYROSINE KINASE ; MICE ; TRANSDUCTION ; COMPLEX ; COMPLEXES ; MECHANISM ; BINDING ; BIOLOGY ; MOLECULAR-BIOLOGY ; PHOSPHORYLATION ; signal transduction ; SIGNAL ; FORM ; ISOFORM ; MUTANT ; SIGNAL-TRANSDUCTION ; EFFICIENT ; DISPLAY ; TUMOR ANGIOGENESIS ; AFFINITY ; VEGF ; LACKING ; BINDS ; signaling ; molecular biology ; molecular ; ENHANCER ; RE ; INCREASE ; endothelial cells ; AMINO-ACID ; ENHANCEMENT ; USA ; function ; vascular endothelial growth factor ; ENDOTHELIAL-CELL ; INHIBIT ; vasculogenesis ; neuropilin-1 ; ENDOTHELIAL GROWTH ; KDR ; binding affinity ; semaphorin
    Abstract: The neuropilin-1 (np1) receptor binds the 165 amino-acid form of vascular endothelial growth factor(165) (VEGF(165)) and functions as an enhancer that potentiates VEGF(165) signaling via the VEGFR-2 tyrosine-kinase receptor. To study the mechanism by which neuropilins potentiate VEGF activity we produced a VEGF(165) mutant (VEGF(165KF)) that binds to neuropilins but displays a much lower affinity toward VEGFR-1 and VEGFR-2. VEGF(165KF) failed to induce VEGFR-2 phosphorylation in cells lacking neuropilins. However, in the presence of np1, VEGF(165KF) bound weakly to VEGFR-2, induced VEGFR-2 phosphorylation, and activated ERK1/2. Interestingly, VEGF(165KF) did not promote formation of VEGFR-2/np1 complexes nor did high concentrations of VEGF(165KF) inhibit VEGF165 induced formation of such complexes, suggesting that VEGF(165) does not stabilize VEGFR-2/np1 complexes by forming bridges spanning VEGFR-2 and np1. VEGF(121) is a VEGF form that does not bind to neuropilins. Surprisingly, both np1 and neuropilin-2 (np2) enhanced VEGF(121)-induced phosphorylation of VEGFR-2 and VEGF121-induced proliferation of endothelial cells. The enhancement of VEGF(121) activity by np1 was accompanied by a 10-fold increase in binding affinity. to, VEGFR-2 and was not associated with the formation of new VEGFR-2/np1 complexes. These observations suggest that neuropilins enhance the activity of VEGF forms that do not bind to neuropilins, indicate that np2 is a functional VEGF receptor, and imply that spontaneously formed VEGFR-2/np1 complexes suffice for efficient neuropilin mediated enhancement of VEGF activity
    Type of Publication: Journal article published
    PubMed ID: 17185751
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  • 9
    Abstract: Vascular endothelial cells are characterized by a high degree of functional and phenotypic plasticity, which is controlled both by their pericellular microenvironment and their intracellular gene expression programs. To gain further insight into the mechanisms regulating the endothelial cell phenotype, we have compared the responses of lymphatic endothelial cells (LECs) and blood vascular endothelial cells (BECs) to vascular endothelial growth factors (VEGFs). VEGFR-3-specific signals are sufficient for LEC but not BEC proliferation, as shown by the ability of the specific ligand VEGF-C156S to stimulate cell cycle entry only in LECs. On the other hand, we found that VEGFR-3 stimulation did not induce LEC cell shape changes typical of VEGFR-2-stimulated LECs, indicating receptor-specific differences in the cytoskeletal responses. Genes induced via VEGFR-2 also differed between BECs and LECs: angiopoietin-2 (Ang-2) was induced via VEGFR-2 in BECs and LECs, but the smooth muscle cell (SMC) chemoattractant BMP-2 was induced only in BECs. Both BECs and LECs were able to promote SMC chemotaxis, but contact with SMCs led to down-regulation of VEGFR-3 expression in BECs in a 3-dimensional coculture system. This was consistent with the finding that VEGFR-3 is down-regulated in vivo at sites of endothelial cell-pericyte/smooth muscle cell contacts. Collectively, these data show intrinsic cell-specific differences of BEC and LEC responses to VEGFs and identify a pericellular regulatory mechanism for VEGFR-3 down-regulation in endothelial cells.
    Type of Publication: Journal article published
    PubMed ID: 14597670
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  • 10
    Keywords: brain ; CELLS ; EXPRESSION ; PATHWAY ; MICE ; MOUSE ; CELL-DEATH ; TUMOR-SUPPRESSOR GENE ; CRE RECOMBINASE ; ANIMAL-MODELS ; MAMMALIAN TARGET ; neurodegeneration ; mTOR ; NIGROSTRIATAL SYSTEM ; MOTOR DEFICITS ; NUCLEOLAR DISRUPTION
    Abstract: Parkinson's disease (PD) is a progressive age-related movement disorder that results primarily from the selective loss of midbrain dopaminergic (DA) neurons. Symptoms of PD can be induced by genetic mutations or by DA neuron-specific toxins. A specific ablation of an essential factor controlling ribosomal RNA transcription, TifIa, in adult mouse DA neurons represses mTOR signaling and leads to progressive neurodegeneration and PD-like phenotype. Using an inducible Cre system in adult mice, we show here that the specific ablation of Pten in adult mouse DA neurons leads to activation of mTOR pathway and is neuroprotective in genetic (TifIa deletion) and neurotoxin-induced (MPTP or 6OHDA) mouse models of PD. Adult mice with DA neuron-specific Pten deletion exhibit elevated expression of tyrosine hydroxylase, a rate-limiting enzyme in the dopamine biosynthesis pathway, associated with increased striatal dopamine content, and increased mRNA levels of Foxa2, Pitx3, En1, Nurr1, and Lmx1b-the essential factors for maintaining physiological functions of adult DA neurons. Pten deletion attenuates the loss of tyrosine hydroxylase-positive cells after 6OHDA treatment, restores striatal dopamine in TifIa-knockout and MPTP-treated mice, and rescues locomotor impairments caused by TifIa loss. Inhibition of Pten-dependent functions in adult DA neurons may represent a promising PD therapy.-Domanskyi, A., Geissler, C., Vinnikov, I. A., Alter, H., Schober, A., Vogt, M. A., Gass, P., Parlato, R., Schutz, G. Pten ablation in adult dopaminergic neurons is neuroprotective in Parkinson's disease models.
    Type of Publication: Journal article published
    PubMed ID: 21593433
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