Untersuchungen zur Pathobiochemie der extrazellulären Matrix bei Pseudoxanthoma elasticum (PXE)

Pseudoxanthoma elasticum (PXE) is a rare inheritable disorder that is characterized by extensive remodelling of the extracellular matrix (ECM). PXE is caused by mutations in the ABCC6 gene. By now, no conclusive pathophysiological connection between ABCC6 and the clinical findings of the disease could be established. Particularly, the molecular mechanisms leading to the ECM alterations are unknown. The clinical picture of PXE is highly variable, with age at disease onset and the number and magnitude of its symptoms differing considerably among patients. The individual disease phenotype is believed to be determined by environmental and genetic factors. In order to identify genetic cofactors of PXE, 29 polymorphisms in four candidate genes, namely MMP2, MMP9, MMP14 and VEGFA, have been examined in this study. Comparison of allelic frequencies showed association of the polymorphisms c.-1575G>A, c.-1306C>T and c.-790T>G in the promoter of the MMP2 gene with PXE. Additionally, polymorphisms in the VEGFA gene could be shown to be associated with the retinopathy frequently accompanying PXE. Moreover, logistic regression analysis identified the c.-460T and the c.674C alleles of VEGFA as independent risk factors for the development of severe retinopathy. An extensive analysis of the expression of ECM genes by dermal fibroblasts from PXE patients was performed and the data were compared with gene expression data of fibroblasts originating from healthy donors. The results showed significant alterations of the expression of several ECM genes as well as of genes connected to glycosaminoglycan (GAG) synthesis in PXE patients. Furthermore, a variation of the gene expression profile during prolonged in vitro cultivation could be demonstrated. Using siRNA mediated gene knockdown, ABCC6 deficiency was induced in normal dermal fibroblasts. In this model, gene expression variations that had been found in cells from PXE patients could be reproduced, thereby establishing a direct connection with lack of ABCC6 activity. In another part of this work, [Delta]-disaccharides derived from GAGs produced by PXE fibroblasts were analyzed using a HPLC method and compared with the [Delta]-disaccharide composition of GAGs produced by normal dermal fibroblasts. The results showed an increased GAG synthesis by PXE fibroblasts which was accompanied by increased activity of xylosyltransferase, the initial enzyme in GAG biosynthesis. Both findings also occurred in normal dermal fibroblasts treated with siRNA against ABCC6. These findings point towards a direct interaction between ABCC6 deficiency and modified GAG synthesis. Analysis of serum concentrations of matrix metalloproteinases in PXE patients and healthy control subjects showed significantly increased levels of MMP-2 and MMP-9 in the serum of PXE patients. For the first time, these results show an increased proteolytic potential in the serum of PXE patients, thereby corroborating the involvement of matrix metalloproteinases in PXE pathogenesis. Finally, by transfection with a plasmid coding for the catalytic subunit of human telomerase, fibroblasts from PXE patients were immortalized and a PXE fibroblast cell line was established, which is ready to be used as a cell model for studying consequences of ABCC6 deficiency. In summary, the results of the present work provide new insights into the mechanisms and genes involved in ECM remodelling, which is the hallmark of PXE.

Level thesis:degree_level

thesis.doctoral

Grantor dc:publisher

Universität Bielefeld

Year

2010

Author dc:creator

• Zarbock, Ralf

Repository record source_url

https://pub.uni-bielefeld.de/record/2304823

OAI identifier oai:identifier

oai:pub.uni-bielefeld.de:2304823