Qualitativer Nachweis von CCM1, CCM2 und CCM3 sowie die Analyse morphologischer und proliferativer Veränderungen nach Verringerung der Expression von CCM1 in HUV Endothelzellen

Kavernome sind vaskuläre Neubildungen im zentralen Nervensystem, welche aus dilatierten, dünnwandigen, sinusoidalen Blutgefäßen bestehen. Histologisch zeigen sich nur anormal vorhandene oder gänzlich fehlende Tight Junctions der Endothelzellen und teilweise fehlende astrozytische Fortsätze, was zu e...

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Bibliographic Details
Main Author: Hansen, Bodil
Contributors: Sure, Ulrich (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2009
Online Access:PDF Full Text
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Cerebral cavernous malformations are vascular neoplasms in the central nervous system. They consist of dilated, thinwalled, sinusoidal bloodvessels. Histologically the tight junctions of these endothelial cells forming the cerebral cavernous malformation are abnormal or totaly missing, also the appendages of the astrozytes can be partly missing, which results wider gaps between the cells. Main Symptoms are cerebral bleeding, convulsion or focal neurological deficencies. The prevalence of cerebral cavernous malformation averages at about 0,5%, of these only 20 to 30% present with clinical symptoms. Cerebral cavernous malformations can occur sporadic or can be autosomal dominant inherited. The familial cases are associated with loss of function mutations within three genes. Mutations of CCM1 are responsible for 40 to 50%, of CCM2 for 10 to 20% and mutations of CCM3 for about 40% of the familial cases. The model of genesis of the cerebral cavernous malformation builds on the two-hit mechanisms established by Knudson, and is supported by Ccm1+/- Trp53-/- mice. CCM1 (KRIT1) as well as CCM2 (Malcavernin) are integrated in β1 integrine signaling, which among other factors regulates adhesion, migration and proliferation of cells. Also both genes are involved in activation of p38MAPK. P38Mapk-/- mice have significant defects in plazental angiogenesis and development of bloodvessels, especially in brain vessels. Moreover CCM3 (PDCD10) interacts with ERK signaling, which also affects proliferation and transformation of cells. Aim of the present work was to detect the expression of all three CCM genes and to test the hypothesis that endothelial cells play a major role in the pathogenesis of cerebral cavernous malformations. Moreover we wanted to created a tissue culture system, to detect functional changes in endothelial cells after downregulation of CCM1. Using real-time RT-PCR we found the expression of all three CCM genes in HUV endothelial cells. A tissue culture system was established also in HUV endothelial cells, where we were able to downregulate CCM1 by transient transfection with short interfering RNA. Morphologically knock down cells were bigger and more fusiform compared to the control cells. The cells also formed more restiform structures. Similar observations were made in cells isolated from cerebral cavernous malformations in angiogenesis assays. The results of this thesis confirm, that the loss of function of CCM1 in endothelial cells plays a major role in changes leading to the pathogenesis of cerebral cavernous malformations. The accomplished proliferationassay showed a reduction of proliferation, which demonstrates that the loss of function of CCM1 has a direct effect on cellproliferation.