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Cellular proliferation, differentiation and survival in response to extracellular signals are controlled by the signal transduction pathway of Ras, Raf and MAP kinase. The Raf proteins are serine/threonine kinases with essential function in growth/differentiation/survival - related signal transduction events. In mammals, three functional (A-, B-, and C-Raf) genes were described. Biochemical studies suggest overlapping and differential utilization of Raf isozymes. However, the frequent co-expression of Raf isozymes and their multiple activators and effectors impedes the full understanding of their specific roles. The elucidation of these roles is important due to the involvement of the Ras/Raf/MEK/MAP kinase cascade in human disorders especially in tumor development and progression. B-Raf was shown to posses the strongest kinase activity among Raf kinases and display antiapoptotic properties. Mice deficient in B-Raf show overall growth retardation and die between E10.5 and E12.5 of vascular defects caused by excessive death of differentiated endothelial cells. To elucidate the redundancy of Raf isozymes during embryonic development and to rescue B-Raf-/- (KO) phenotype, B-Raf alleles were disrupted by introducing A-Raf cDNA under the control of endogenous B-Raf promoter. The resulting BRaf A-Raf/A-Raf (KIN) phenotype depends on genetic background. The living embryos displaying normal development but size reduction were found with low incidence at E12.5d-16.5d. All of them displayed the rescue of vascular system. One adult p20 mouse without any visible defects in development and behavior was obtained. On the other hand, the processes of neurogenesis and neural precursors migration in survived embryos were disturbed which led in some cases to underdevelopment of different brain compartments. TUNEL and cell proliferation (PCNA staining) assays revealed more apoptotic (E13.5d) and less proliferating(E12.5d cells within ventricular and sub-ventricular zones of brain ventricles and in striatum of KIN embryos. In addition, more apoptotic cells were detected in many other tissues of E13.5d and in lung of E16.5d KIN embryos but not in adult KIN mouse. p20 KIN mouse demonstrated reduced fraction of neural precursor cells in sub-granular zone of hippocampus and mature neurons in olfactory bulb. The other processes of neurogenesis were not disturbed in adult KIN animal. Fibroblasts obtained from KIN embryos demonstrated less proliferative ability and were more susceptible to apoptotic stimuli compared to WT. This was accompanied by the reduction of active ERK and Akt required for survival, and with decrease of inactive phosphorylated BAD. The kinetic of both ERK and Akt phosphorylation upon serum stimulation was delayed. All these data indicate that moderate A-Raf kinase activity can prevent the endothelial apoptosis but is not enough to completely rescue the other developmental consequences.
The mammalian Vasodilator Stimulated Phosphoprotein (VASP) is a founding member of the Ena/VASP family of proteins that includes Drosophila Enabled (ena), the mammalian Ena homologue (Mena) and the Ena-VASP-like protein (Evl). VASP was initially discovered and characterized as a substrate for cGMP- and cAMP-dependent protein kinases (cGKs and cAKs). Ena/VASP proteins are involved in Actin-filament formation, plasma membrane protrusion, acceleration of Actin-based motility of Listeria and the establishment of cell-cell adhesion. Moreover, Ena/VASP proteins have been implicated as inhibitory factors in repulsive axon guidance and inhibition of plasma membrane activity and random motility in fibroblast. In order to study the physiological function of VASP, VASP-deficient mice had been generated in the laboratory by homologous recombination. VASP-/- mice showed hyperplasia of megakaryocytes in the bone marrow and spleen and a two-fold increase in thrombin- and collagen-induced platelet activation. To further investigate the cellular function of VASP, I established cardiac fibroblast cell lines derived from both wild type and VASP-/- mice. Both cell lines presented similar growth rates and normal contact dependent-growth inhibition but showed differences in morphology, migration and adhesion. Adherent VASP-/- cells, despite normal Mena and Evl expression levels, were highly spread. VASP-/- cells covered about twice the substrate surface area as wild type cells, while the cell volumes were unchanged. This shape difference suggests that VASP is involved in the regulation of spreading. Since the small GTPases Rac and Cdc 42 and their effector p21-activated kinase (Pak) are key regulators of lamellipodia formation and cell spreading, I analyzed this signalling pathway in VASP-/- cells stimulated with Platelet Derived Growth Factor-BB (PDGF-BB) or fetal calf serum. In wild type cells Rac and Pak were rapidly and transiently activated by PDGF or serum; however, in the absence of VASP both Rac and Pak activation was dramatically prolonged. The Rac/Pak pathway is known to play an essential role in cell motility. VASP deficient cells showed compromised migration and reorientation in a wound healing assay, probably due to enhanced Rac activity. The spreading phenotype, compromised migration and the effect observed on the Rac and Pak activities were reverted in VASP-/- cells stably transfected with full lenght human VASP, indicating a VASP dependent modulation of the Rac/Pak pathway and Rac/Pak regulated processes. Moreover, adhesion and detachment of VASP-deficient cells were significantly slower when compared to wild type cells. Preincubation of VASP+/+ cells with a cGMP analog accelerated adhesion. This acceleration did not take place in the VASP-/- cells, suggesting a VASP dependent effect. The second part of this work focused on VASP function in platelets. On the one hand I investigated the possibility of VASP-dependent Rac regulation in mouse platelets. Murine platelets are a good model for studying Rac regulation since they express high levels of VASP but not Mena/Evl and since VASP-deficient platelets show an increased platelet activation. Rac was activated by platelet agonists which was inhibited by preincubation with cGMP and cAMP analogs. Initial results which need to be extended showed that the cGMPcaused inhibition of Rac activation was VASP-dependent. Finally, in vivo platelet adhesion (platelet-vessel wall interactions) was studied using VASP-deficient mice. These studies demonstrated in-vivo that VASP down regulates platelet adhesion to the vascular wall under both physiological and pathophysiological conditions.
In vitro and in vivo studies on the activating platelet collagen receptor glycoprotein VI in mice
(2003)
The work summarized here focused on the characterization of the murine platelet collagen receptor glycoprotein (GP) VI and was performed to evaluate its potential as an antithrombotic target. The first mAb against (mouse) GPVI, JAQ1, was generated and used to demonstrate that GPVI requires the FcRgamma-chain for its expression and function and that this receptor is the central molecule in collagen-induced platelet activation. Blocking the major collagen binding site on GPVI with JAQ1 revealed the presence of a second activatory epitope within collagen. Additionally, the collagen receptor integrin alpha2beta1 was found to be required for activation via this second pathway but not to be essential for collagen-induced activation of normal platelets. In studies with mice expressing reduced levels of the GPVI-FcRgamma-complex, differential responses to GPVI ligands were observed. Most importantly, the striking difference between platelet responses to collagen and the GPVI specific synthetic collagen related peptide (CRP) confirmed the supportive role of other collagen receptor(s) on platelets. Irrespective of yet undefined additional receptors, studies with mice deficient in GPVI (FcRgamma-chain) or alpha2beta1 showed that GPVI, but not alpha2beta1 is essential for platelet-collagen interaction. Based on these results, the model of platelet attachment to collagen was revised establishing GPVI as the initial activating receptor which upregulates the activity of integrins, thus enabling firm attachment of platelets to the ECM. While the mAb JAQ1 had only limited inhibitory effects on collagen-induced activation in vitro, its in vivo application to mice resulted in completely abolished platelet responses to collagen and the GPVI specific agonists CRP and convulxin. This effect was found to be due to antibody-induced irreversible down-regulation of GPVI on circulating platelets for at least two weeks. Further studies revealed that GPVI depletion occurs independently of the targeted epitope on the receptor and does not require the divalent form of IgG as it was also induced by mAbs (JAQ2, JAQ3) or the respective Fab fragments directed against epitopes distinct from the major collagen binding site. The internalization of GPVI in vivo resulted in a long-term protection of the mice from lethal collagen-dependent thromboembolism whereas it had only moderate effects on the bleeding time, probably because the treatment did not affect other activation pathways. These results establish GPVI as a potential pharmacological target for the prevention of ischemic cardiovascular diseases and may open the way for a completely new generation of antithrombotics.