GDNF family receptors in peripheral target innervation and hormone production

Glial cell line-derived neurotrophic factor (GDNF) family ligands: GDNF, neurturin, persephin and artemin, signal through a receptor tyrosine kinase Ret by binding first to a co-receptor (GFRα1-4) that is attached to the plasma membrane. The GDNF family factors can support the survival of various peripheral and central neuronal populations and have important functions also outside the nervous system, especially in kidney development. Activating mutations in the RET gene cause tumours in neuroendocrine cells, whereas inactivating mutations in RET are found in patients with Hirschsprung s disease (HSCR) characterized by loss of ganglionic cells along the intestine. The aim of this study was to examine the in vivo functions of neurturin receptor GFRα2 and persephin receptor GFRα4 using knockout (KO) mice. Mice lacking GFRα2 grow poorly after weaning and have deficits in parasympathetic and enteric innervation. This study shows that impaired secretion of the salivary glands and exocrine pancreas contribute to growth retardation in GFRα2-KO mice. These mice have a reduced number of intrapancreatic neurons and decreased cholinergic innervation of the exocrine pancreas as well as reduced excitatory fibres in the myenteric plexus of the small intestine. This study also demonstrates that GFRα2-mediated Ret signalling is required for target innervation and maintenance of soma size of sympathetic cholinergic neurons and sensory nociceptive IB4-binding neurons. Furthermore, lack of GFRα2 in mice results in deficient perception of temperatures above and below thermoneutrality and in attenuated inflammatory pain response. GFRα4 is co-expressed with Ret predominantly in calcitonin-producing thyroid C-cells in the mouse. In this study GFRα4-deficient mice were generated. The mice show no gross developmental deficits and have a normal number of C-cells. However, young but not adult mice lacking GFRα4 have a lower production of calcitonin in thyroid tissue and consequently, an increased bone formation rate. Thus, GFRα4/Ret signalling may regulate calcitonin production. In conclusion, this study reveals that GFRα2/Ret signalling is crucial for the development and function of specific components of the peripheral nervous system and that GFRα4-mediated Ret signalling is required for controlling transmitter synthesis in thyroid C-cells.

VEGFR-2 and VEGFR-3 Specific Signaling in Lymphangiogenesis and Angiogenesis

The circulatory system consists of the blood and lymphatic vessels. While blood vessels transport oxygen, cells, and nutrients to tissues, the lymphatic vessels collect fluid, cells, and plasma proteins from tissues to return back to the blood circulation. Angiogenesis, the growth of new blood vessels from pre-existing ones, is an important process involved in several physiological conditions such as inflammation, wound healing, and embryonic development. Furthermore, angiogenesis is found in many pathological conditions such as atherosclerosis and the growth and differentiation of solid tumors. Many tumor types spread via lymphatic vessels to form lymph node metastasis. The elucidation of the molecular players coordinating development of the vascular system has provided an array of tools for further insight of the circulatory system. The discovery of the Vascular Endothelial Growth Factor (VEGF) family members and their tyrosine kinase receptors (VEGFRs) has facilitated the understanding of the vasculature in different physiological and pathological situations. The VEGFRs are expressed on endothelial cells and mediate the growth and maintenance of both the blood and lymphatic vasculatures. This study was undertaken to address the role of VEGFR-2 specific signaling in maturation of blood vessels during neoangiogenesis and in lymphangiogenesis. We also wanted to differentiate between VEGFR-2 and VEGFR-3 specific signaling in lymphangiogenesis. We found that specific VEGFR-2 stimulation alone by gene therapeutic methods is not sufficient for production of mature blood vessels. However, VEGFR-2 stimulation in combination with expression of platelet-derived growth factor D (PDGF-D), a recently identified member of the PDGF growth factor family, was capable of stabilizing these newly formed vessels. Signaling through VEGFR-3 is crucial during developmental lymphangiogenesis, but we showed that the lymphatic vasculature becomes independent of VEGFR-3 signaling after the postnatal period. We also found that VEGFR-2 specific stimulation cannot rescue the loss of lymphatic vessels when VEGFR-3 signaling is blocked and that VEGFR-2 specific signals promote lymphatic vessel enlargement, but are not involved in vessel sprouting to generate new lymphatic vessels in vivo, in contrast to the VEGFR-2 dependent sprouting observed in blood vessels. In addition, we compared the inhibitory effects of a small molecular tyrosine kinase inhibitor of VEGFR-2 vs. VEGFR-3 specific signaling in vitro and in vivo. Our results showed that the tyrosine kinase inhibitor could equally affect physiological and pathological processes dependent on VEGFR-2 and VEGFR-3 driven angiogenesis or lymphangiogenesis. These results provide new insights into the VEGFR specific pathways required for pre- and postnatal angiogenesis as well as lymphangiogenesis, which could provide important targets and therapies for treatment of diseases characterized by abnormal angiogenesis or lymphangiogenesis.

Lymphatic vascular maturation : Roles of FOXC2, NFATc1 and liprin ß1

The blood vascular system is a closed circulatory system, responsible for delivering oxygen and nutrients to the tissues. In contrast, the lymphatic vascular system is a blind-ended transport system that collects the extravasated tissue fluid from the capillary beds, and transports it back to the blood circulation. Failure in collecting or transporting the lymph, due to defects in the lymphatic vasculature, leads to accumulation of extra fluid in the tissues, and consequently to tissue swelling lymphedema. The two vascular systems function in concert. They are structurally related, but their development is regulated by separate, however overlapping, molecular mechanisms. During embryonic development, blood vessels are formed by vasculogenesis and angiogenesis, processes largely mediated by members of the vascular endothelial growth factor (VEGF) family and their tyrosine kinase receptors. The lymphatic vessels are formed after the cardiovascular system is already functional. This process, called lymphangiogenesis, is controlled by distinct members of the VEGF family, together with the transcription factors Prox1 and Sox18. After the primary formation of the vessels, the vasculature needs to mature and remodel into a functional network of hierarchically organized vessels: the blood vasculature into arteries, capillaries and veins; and the lymphatic vasculature into lymphatic capillaries, responsible for the uptake of the extravasated fluid from the tissues, and collecting vessels, responsible for the transport of the lymph back to the blood circulation. A major event in the maturation of the lymphatic vasculature is the formation of collecting lymphatic vessels. These vessels are characterized by the presence of intraluminal valves, preventing backflow of the lymph, and a sparse coverage of smooth muscle cells, which help in pumping the lymph forward. In our study, we have characterized the molecular and morphological events leading to formation of collecting lymphatic vessels. We found that this process is regulated cooperatively by the transcription factors Foxc2 and NFATc1. Mice lacking either Foxc2 or active NFATc1 fail to remodel the primary lymphatic plexus into functional lymphatic capillaries and collecting vessels. The resulting vessels lack valves, display abnormal expression of lymphatic molecules, and are hyperplastic. Moreover, the lymphatic capillaries show aberrant sprouting, and are abnormally covered with smooth muscle cells. In humans, mutations in FOXC2 lead to Lymphedema-Distichiasis (LD), a disabling disease characterized by swelling of the limbs due to insufficient lymphatic function. Our results from Foxc2 mutant mice and LD patients indicate that the underlying cause for lymphatic failure in LD is agenesis of collecting lymphatic valves and aberrant recruitment of periendothelial cells and basal lamina components to lymphatic capillaries. Furthermore, we show that liprin β1, a poorly characterized member of the liprin family of cytoplasmic proteins, is highly expressed in lymphatic endothelial cells in vivo, and is required for lymphatic vessel integrity. These data highlight the important role of FOXC2, NFATc1 and liprin β1 in the regulation of lymphatic development, specifically in the maturation and formation of the collecting lymphatic vessels. As damage to collecting vessels is a major cause of lymphatic dysfunction in humans, our results also suggest that FOXC2 and NFATc1 are potential targets for therapeutic intervention.

Biological mechanisms behind clozapine-induced agranulocytosis

Clozapine is the most effective drug in treating therapy-resistant schizophrenia and may even be superior to all other antipsychotics. However, its use is limited by a high incidence (approximately 0.8%) of a severe hematological side effect, agranulocytosis. The exact molecular mechanism(s) of clozapine-induced agranulocytosis is still unknown. We investigated the mechanisms behind responsiveness to clozapine therapy and the risk of developing agranulocytosis by performing an HLA (human leukocyte antigens) association study in patients with schizophrenia. The first group comprised patients defined by responsiveness to first-generation antipsychotics (FGAs) (n= 19). The second group was defined by a lack of response to FGAs but responsiveness to clozapine (n=19). The third group of patients had a history of clozapine-induced granulocytopenia or agranulocytosis (n=26). Finnish healthy blood donors served as controls (n= 120). We found a significantly increased frequency of HLA-A1 among patients who were refractory to FGAs but responsive to clozapine. We also found that the frequency of HLA-A1 was low in patients with clozapine-induced neutropenia or agranulocytosis. These results suggest that HLA-A1 may predict a good therapeutic outcome and a low risk of agranulocytosis and therefore HLA typing may aid in the selection of patients for clozapine therapy. Furthermore, in a subgroup of schizophrenia, HLA-A1 may be in linkage disequilibrium with some vulnerability genes in the MHC (major histocompatibility complex) region on chromosome 6. These genes could be involved in antipsychotic drug response and clozapine-induced agranulocytosis. In addition, we investigated the effect of clozapine on gene expression in granulocytes by performing a microarray analysis on blood leukocytes of 8 schizophrenic patients who had started clozapine therapy for the first time. We identified an altered expression in 4 genes implicated in the maturation or apoptosis of granulocytes: MPO (myeloperoxidase precursor), MNDA (myeloid cell nuclear differentiation antigen), FLT3LG (Fms-related tyrosine kinase 3 ligand) and ITGAL (antigen CD11A, lymphocyte function-associated antigen 1). The altered expression of these genes following clozapine administration may suggest their involvement in clozapine-induced agranulocytosis. Finally, we investigated whether or not normal human bone marrow mesenchymal stromal cells (MSC) are sensitive to clozapine. We treated cultures of human MSCs and human skin fibroblasts with 10 µM of unmodified clozapine and with clozapine bioactivated by oxidation. We found that, independent of bioactivation, clozapine was cytotoxic to MSCs in primary culture, whereas clozapine at the same concentration stimulated the growth of human fibroblasts. This suggests that direct cytotoxicity to MSCs is one possible mechanism by which clozapine induces agranulocytosis.

NK-and T-cell function in chronic myeloid leukemia patients treated with interferon-? monotherapy

Chronic myeloid leukemia (CML) is one of the most studied human malignancies. It is caused by an autonomously active tyrosine kinase BCR-ABL, which is a result from a translocation between chromosomes 9 and 22 in the hematopoietic stem cell. As an outcome, a Philadelphia (Ph) chromosome is formed. BCR-ABL causes disturbed cell proliferation among other things. Although targeted tyrosine kinase inhibitor therapy has been developed in the beginning of the millenium and the survival rate has increased significantly, it is still not known why some patients benefit more from the treatment than others. Furthermore, the therapy is not considered to be curative. Before the era of tyrosine kinase inhibitors, the first-line treatment for CML was interferon-? (IFN-?). However, only a small proportion of patients benefitted from the treatment. Of these patients, a few were able to discontinue the treatment without renewal of the disease. The mechanism of IFN-? is not completely understood, but it is believed that differences in the immune system can be one of the reasons why some patients have better therapy response. Kreutzman, Rohon et al. have recently discovered that patients who have been able to stop IFN-? treatment have an increased number of NK- and T-cells. They also have a unique clonal T-cell population and more cytotoxic CD8+ T-cells and less CD4+ T-cells. The aim of this master’s thesis was to study the function of T- and NK-cells in IFN-? treated patients. Although it was shown earlier that IFN-? treated patients have increased NK-cell count, the function of these cells was unknown. Therefore, we have now investigated the killing potential of patients’ NK-cells, their activation status and cell surface antigen expression. In addition, we have also studied the activation status of patients’ T-cells and their cytotoxic properties. We observed that NK-cells from patients treated with IFN-? are unable to kill leukemic cells (K562) than NK-cells from healthy controls. In addition, patients on IFN-? treatment have more active T-cells and their NK-cells have an undifferentiated immunoregulatory phenotype. Patients that have been able to stop the treatment have anergic T-and NK-cells. As a conclusion our results suggest that IFN-? therapy induces increased NK-cell count, NK-cell immunoregulatory functions and more active T-cells. After stopping IFN-? therapy, NK- and T-cells from CML patients restore anergy typical for CML.

Hypertension, kardiotoxicitet och njursvikt i samband med tyrosinkinashämmaren sunitinib

Nybildning av blodkärl från tidigare existerande kärl, angiogenes, är ett väsentligt skede vid tumörtillväxt. Denna process regleras av bland annat tillväxtfaktorer, var av den vaskulära endoteliala tillväxtfaktorn har en central roll. Hämning av angiogenes kan ske antingen extracellulärt med hjälp av humaniserade monoklonala antikroppar eller intracellulärt med hjälp av småmolekylära hämmaren. Sunitinib är en småmolekylär multikinashämmare och inhiberar flera tyrosinkinasreceptorer som påverkar tumörtillväxten och metastasutvecklingen vid cancer. Sunitinibs främsta indikationer är gastrointestinala stromacellstumörer, metastaserad njurcellscancer och neuroendokrina tumörer i bukspottskörteln. Behandling med tyrosinkinashämmare orsakar biverkningar som hypertension, kardiotoxicitet och njursvikt, vilka antas bero på de hämmande effekterna på mål som inte är väsentliga för anti-cancer-aktiviteten (”off-target” biverkningar). Bland annat AMP-aktiverat proteinkinas (AMPK), ett kinas som upprätthåller metabolisk homeostas i hjärtat, inhiberas av sunitinib och antas framkalla kardiovaskulära biverkningar. För att reducera ”off-target” biverkningar strävar man till att hitta alternativ som minskar de skadliga effekterna utan att den terapeutiska aktiviteten försvagas. Bland annat ett begränsat kaloriintag har uppvisat skyddande effekt på hjärtat via mekanismer sammankopplade till ökad resistens mot oxidativ stress, inflammation och mitokondriell dysfunktion, samt avtagande apoptos och autofagi. Detta sker delvis genom aktivering av enzymet Sirt1. Syftet med den här studien var att undersöka ifall kaloribegränsning skyddar mot kardiovaskulära och renala biverkningar inducerade av sunitinib hos råttor. Dessutom studerades vilka signalkedjor i cellen som medverkar. I studien användes 40 spontant hypertensiva råttor samt 10 normotensiva Wistar-Kyoto råttor. Försöksdjuren delades in i fem grupper beroende på behandling; I WKY kontroll, II SHR kontroll, III SHR + kaloribegränsning 70 %, IV SHR + sunitinib 3 mg/kg och V SHR + sunitinib 3 mg/kg + kaloribegränsning 70 %. Behandlingsperioden var åtta veckor. Blodtrycket mättes varje vecka med svansmanchett, urinutsöndringen undersöktes vecka 4 och vecka 8 med metabolismburar, ultraljudsundersökning av hjärtat utfördes sista veckan och blodkärlens respons till acetylkolin och natriumnitroprussid studerades i samband med avlivning. Proteinerna Sirt1 och AMPK analyserades i hjärtat med Western blotting samt förekomsten av makrofagmarkören ED1 i njurarna med immunhistokemi. Studien visade att sunitinibdosen 3 mg/kg är mycket väl tolererbar hos råttor eftersom sunitinib inte orsakade högre blodtryck, kraftigare hypertrofi eller mer omfattande njurskada jämfört med obehandlade SHR- grupper. Utgående från resultaten kan man också konstatera att kaloribegränsningen har positiva kardiovaskulära effekter.