Transgenic models to study TGF-[beta] function in hematopoiesis

Studies of hematopoietic pathologies involving the growth factor TGF-β have provided important evidence of its keyrole in the regulation of human hematopoietic stem/progenitor cell quiescence, proliferation, and differentiation. The inactivation of one of the various genes involved in the TGF-β signal transduction pathway may represent a possible mechanism by which some early hematopoietic progenitors, which are normally quiescent, escape from cellcycle inhibition. Abnormalities in the expression of TGF-β receptors have been described in proliferative syndromes including both early myeloid and lymphocytic leukemia 1,2. In these cases the loss of the growth inhibitory TGF-β signal might provide a selective advantage to the malignant cell. Additional autocrine TGF-β production and thereby inhibition of neighboring cells leads to an overgrowth of the malignant clone. In patients with myeloproliferative disorders, reduced mRNA levels of the TGF-β signaling components Smad4 and type II TGF-β receptor were reported 3-5 further establishing a role of abolished TGF-β signaling in the pathogenesis of hematopoietic malignancies. The role of TGF-β in the regulation of hematopoiesis has also been analyzed in vivo using different mouse models. For example, the administration of TGF-β in mice revealed an inhibition of thrombopoiesis and erythropoiesis 6. A variety of knockout mice have been generated to study the effect of TGF- β in vivo. The most of these approaches were hampered by the early lethality of the knockout like in the case of the Smad proteins and the TGF-β receptors I and II 7,8. Homozygous TGF- β1 knockout mice have a 50% intrauterine death rate because of severe developmental retardation. The other 50% die within several weeks after birth due to a severe inflammatory autoimmune disease 9. Nevertheless, TGF-β knockout mice display defective hematopoiesis with elevated platelet counts and reduced numbers of erythroid cells 9. However, as most of the knockout approaches for TGF-β signaling components resulted in early embryonic lethality, the exact functions of the different elements of the TGF-β signaling cascade in hematopoiesis are still controversial. In this thesis work I describe different transgenic approaches to gain insight into the function of TGF-β signaling components in hematopoiesis, with a focus on megakaryopoiesis. In the first part I describe the generation of a transgenic mouse strain for the tissue-specific deletion of target genes in megakaryocytes and platelets. Many of the genes potentially involved in megakaryopoiesis are difficult to study by conventional knockout approaches, as they are ubiquitiously expressed and therefore their germline deletion is embryonically lethal. One way to circumvent the obstacles of early embryonic lethality is the use of the Cre/loxP system for tissue-restricted target gene deletion 10. Hence, we generated a transgenic mouse for the megakaryocyte-specific expression of the Cre recombinase. As short plasmid based transgenes are often hampered by position variegation effects, like mosaic expression or transgene silencing, we decided to modify a large genomic DNA fragment using ETrecombination in E.coli 11. The coding sequence of the Cre recombinase was placed under the control of the Pf4 gene embedded in a 100kB bacterial artificial chromosome (BAC). The modified BAC-insert was used to generate PF4Cre transgenic lines. Analysis of the resulting transgenic lines revealed differences in tissue-specific expression of the Cre recombinase, dependent on copy numbers. Accordingly, strains with low copy numbers revealed very specific Cre expression in megakaryocytes and platelets, while strains with higher copy numbers displayed ectopic Cre expression. The evaluation of excision efficiency in megakaryocytes of the different PF4Cre strains revealed that the strain with 5 integrations excised with 90%, whereas the strains with 1 or 2 copies excised with 60-70% efficiency. However, I used these strains to delete the TGF-β signaling components type II TGF- β receptor (TBRII) and Smad4 in megakaryocytes by mating the PF4Cre strains with either TBRIIlox/lox or Smad4lox/lox mice. Homozygous offspring was analyzed for peripheral blood counts. Surprisingly, no change in the numbers of circulating platelets was detected in any of these mice in comparison to control mice. I confirmed these results using the transgenic Mx1Cre mouse for inducible deletion of target genes in hematopoietic stem cells. Again, no changes in the numbers of circulating platelets were detected neither in TBRIIlox/lox-Mx1Cre mice, nor in Smad4lox/lox-Mx1Cre mice. Together these results argue against an involvement of TGF-β signaling components in the onset of myeloproliferative disorders and additionally reveal that TGF-β signaling is dispensable for functional megakaryopoiesis. In a second mouse model we intended to disrupt Smad-mediated TGF-β signaling in hematopoiesis by the induced deletion of the TGF-β signal transducer Smad4. We used the Mx1Cre transgenic strain to induce Smad4 deletion in the bone marrow of Smad4lox/lox- Mx1Cre mice. Smad4 deleted mice developed a severe haemolytic anemia 4-5 weeks after the induction of Cre recombinase expression, accompanied by extramedullary hematopoiesis and splenomegaly. Anemia in Smad4lox/lox-Mx1Cre mice was not autoimmune-mediated as revealed by a negative direct antiglobulin test (DAT). The hyperplasia of the spleens in Smad4lox/lox-Mx1Cre mice was due to a massive increase of immature myeloid cells. FACS analysis revealed the myeloid cells in the spleen are TER119high/CD71high erythroblasts, which argues for a maturation block in erythropoiesis as the cause for anemia in Smad4lox/lox- Mx1Cre mice. Transplantation of Smad4lox/lox-Mx1Cre bone marrow into lethally irradiated C57BL/6 recipients revealed that the anemia is not transplantable and thus can be compensated by host-derived factors. Furthermore, Smad4lox/lox-Mx1Cre bone marrow transplanted recipients did not develop a wasting syndrome. This is in complete contrast to the previously described induced deletion of TBRII and TBRI in TBRIIlox/lox- and TBRIlox/lox- Mx1Cre mice. In both of these mouse models deletion of the TGF-β signaling caused a severe inflammatory phenotype, which is transplantable. Together, these results implicate that the autoimmune phenotype in TGF-β receptor deleted mice is not Smad-mediated, as Smad4 is the quintessential for signaling through activated Smads. In the last part of my thesis I describe the generation of a new tool to study gene function in human hematopoietic stem/progenitor cells. For this purpose I took advantage of the rapid advances in the RNA-interference field and the demonstrated capability of lentiviruses to infect non-cycling human hematopoietic stem cells. I modified a lentiviral vector by the insertion of a expression cassette for short-interfering RNAs (siRNA), which drives siRNA expression under the control of the H1 promotor. Originally thought to target TBRII in human hematopoietic stem cells, the system was first established to target the human p53 mRNA, as a functional siRNA sequence for this target was available at that time. Human cord blood derived CD34+ cells were infected with the lentiviral construct pWPXLp53si and p53 mRNA from infected cells was analyzed by quantitative real-time PCR. Infection efficiencies were typically around 50% as revealed by the enhanced green fluorescent protein reporter gene (EGFP). Infected CD34+ cells not only revealed p53 mRNA reduction to 3-10% of the control levels, but also functional p53 silencing was demonstrated by the increased resistance to apoptotic stimuli of pWPXLp53si-infected CD34+ cells. We also demonstrated that the lentiviral system was able to silence p53 in early hematopoietic progenitors by growing infected CD34+ cells under long-term culture initiating cell (LTC-IC) conditions. In summary, we revealed that lentiviral delivery of siRNA can be used for efficient and stable gene silencing in human hematopoietic progenitors. This system will be very valuable to study the function of key regulatory genes in human hematopoiesis

Anwendung und Optimierung verschiedener Verfahren zur Herstellung von 3D-Resonatoren aus III-V- und II-VI-Halbleitern

Es wurden zwei verschiedene Typen von Resonatoren hergestellt. Pillars und pyramidenförmige Strukturen. Werden nun zwei solcher Resonatoren über einen schmalen Kanal miteinander gekoppelt,so weist das Spektrum der optischen Moden deutliche Analogien zu dem elektronischer Zustände in zweiatomigen Molekülen. Der Schwerpunkt dieser Dissertation liegt deshalb auf dem Finden und Entwickeln von Methoden, um Resonatoren mit 3D-Einschluss zu erhalten

Suppressive Effects of Vascular Endothelial Growth Factor-B on Tumor Growth in a Mouse Model of Pancreatic Neuroendocrine Tumorigenesis

Peer reviewe

Toll-Like Receptor 2 Induced Angiogenesis and Invasion Is Mediated through the Tie2 Signalling Pathway in Rheumatoid Arthritis

BACKGROUND: Angiogenesis is a critical early event in inflammatory arthritis, facilitating leukocyte migration into the synovium resulting in invasion and destruction of articular cartilage and bone. This study investigates the effect of TLR2 on angiogenesis, EC adhesion and invasion using microvascular endothelial cells and RA whole tissue synovial explants ex-vivo. METHODS: Microvascular endothelial cells (HMVEC) and RA synovial explants ex vivo were cultured with the TLR2 ligand, Pam3CSK4 (1 µg/ml). Angiopoietin 2 (Ang2), Tie2 and TLR2 expression in RA synovial tissue was assessed by immunohistology. HMVEC tube formation was assessed using Matrigel matrix assays. Ang2 was measured by ELISA. ICAM-1 cell surface expression was assessed by flow cytometry. Cell migration was assessed by wound repair scratch assays. ECM invasion, MMP-2 and -9 expression were assessed using transwell invasion chambers and zymography. To examine if the angiopoietin/Tie2 signalling pathway mediates TLR2 induced EC tube formation, invasion and migration assays were performed in the presence of a specific neutralising anti-Tie2mAb (10 ug/ml) and matched IgG isotype control Ab (10 ug/ml). RESULTS: Ang2 and Tie2 were localised to RA synovial blood vessels, and TLR2 was localised to RA synovial blood vessels, sub-lining infiltrates and the lining layer. Pam3CSK4 significantly increased angiogenic tube formation (p<0.05), and upregulated Ang2 production in HMVEC (p<0.05) and RA synovial explants (p<0.05). Pam3CSK4 induced cell surface expression of ICAM-1, from basal level of 149±54 (MFI) to 617±103 (p<0.01). TLR-2 activation induced an 8.8±2.8 fold increase in cell invasion compared to control (p<0.05). Pam3CSK4 also induced HMVEC cell migration and induced MMP-2 and -9 from RA synovial explants. Neutralisation of the Ang2 receptor, Tie2 significantly inhibited Pam3CSK4-induced EC tube formation and invasion (p<0.05). CONCLUSION: TLR2 activation promotes angiogenesis, cell adhesion and invasion, effects that are in part mediated through the Tie2 signalling pathway, key mechanisms involved in the pathogenesis of RA