Einfluss einer ABCC6-Defizienz auf die Remodellierung der extrazellulären Matrix in in vitro und ex vivo Gewebemodellen
Plümers R (2023)
Bielefeld: Universität Bielefeld.
Bielefelder E-Dissertation | Deutsch
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Plümers, Ricarda
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Abstract / Bemerkung
The pathobiochemical processes of the heterogeneous disease pseudoxanthoma elasticum (PXE) include changes in lipid homeostasis, remodeling of the extracellular matrix (ECM), ectopic calcification, and inflammation. Symptomatically, these are reflected peripherally in the dermis, retina, and vascular walls. Mutations in the ATP binding cassette subfamily C member 6 (ABCC6) gene are the cause of PXE. ABCC6 is primarily expressed by the liver. It has therefore been postulated that PXE is a metabolic disease. While previous studies focused on the effects of individual pathobiochemical processes in different tissue or cell types, the goal of this work was to establish new cell models and further develop already established models to investigate a more comprehensive impact of ABCC6 on an organism.
CRISPR Cas9 systems were established to create ABCC6-deficient cell lines via genome editing in human immortalized hepatocytes and human adipocytes. Human fibroblasts isolated from PXE patients and tissues from an Abcc6-/- mouse model were used for comparison purposes.
The results of the analysis of these ABCC6-deficient models identified reduced uptake of triglycerides and cholesterol due to reduced expression of the surface proteins cluster of differentiation 36 and scavenger receptor class B member 1 involved in these processes. Deficiencies in reverse cholesterol transport due to ABCC6 deficiency were manifested by reduced low density lipoprotein receptor gene expression in the hepatocytic models and by lower expression of the cholesterol transporter ABCA1 in the adipocytes. Increased levels of cholesterol in gallbladders and of cholic acid in serum indicated a shifted metabolite profile in bile acid metabolism and disruption of enterohepatic circulation in the Abcc6-/- mouse model.
Remodeling of the ECM in PXE fibroblasts in two- and three-dimensional culture proved to be particularly dependent on the expression of a gene cluster of matrix metalloproteinases (MMPs). In addition, dysregulated expression of ECM components such as collagens was detectable in the hABCC6-deficient hepatocytes. Coculture confirmed the direct influence of hepatocytes on the expression of hMMP1, hMMP3, and hMMP12 and thus on ECM of fibroblasts. Increased expression of MMP under calcifying culture conditions highlighted the relationship between these pathobiochemical mechanisms. The increase in calcification of fibroblast cultures after inhibition of MMP, shown by enhanced staining of mineral deposits by alizarin red, raised the hypothesis that MMP make elastic fibers accessible to controlled, possibly osteopontin-dependent calcification.
The hABCC6-deficient hepatocytes were characterized by induced expression of the calcification inhibitor matrix γ-carboxyglutamate protein, reduced expression of the cell cycle inhibitor p21, and lower expression of complement components C3 and C1r, among others. Their phenotype can therefore be described as anti-calcifying and anti-inflammatory and contrasts with the phenotype of PXE fibroblasts.
In summary, changes in lipid and bile acid metabolism and remodeling of the ECM appear as systemic influencing factors of PXE. In contrast, peripheral tissue calcification and inflammation appear to be primarily shaped locally by the cellular milieu and are presumably influenced secondarily by the systemic aberrations in the progressive course of PXE.
The results of the analysis of these ABCC6-deficient models identified reduced uptake of triglycerides and cholesterol due to reduced expression of the surface proteins cluster of differentiation 36 and scavenger receptor class B member 1 involved in these processes. Deficiencies in reverse cholesterol transport due to ABCC6 deficiency were manifested by reduced low density lipoprotein receptor gene expression in the hepatocytic models and by lower expression of the cholesterol transporter ABCA1 in the adipocytes. Increased levels of cholesterol in gallbladders and of cholic acid in serum indicated a shifted metabolite profile in bile acid metabolism and disruption of enterohepatic circulation in the Abcc6-/- mouse model.
Remodeling of the ECM in PXE fibroblasts in two- and three-dimensional culture proved to be particularly dependent on the expression of a gene cluster of matrix metalloproteinases (MMPs). In addition, dysregulated expression of ECM components such as collagens was detectable in the hABCC6-deficient hepatocytes. Coculture confirmed the direct influence of hepatocytes on the expression of hMMP1, hMMP3, and hMMP12 and thus on ECM of fibroblasts. Increased expression of MMP under calcifying culture conditions highlighted the relationship between these pathobiochemical mechanisms. The increase in calcification of fibroblast cultures after inhibition of MMP, shown by enhanced staining of mineral deposits by alizarin red, raised the hypothesis that MMP make elastic fibers accessible to controlled, possibly osteopontin-dependent calcification.
The hABCC6-deficient hepatocytes were characterized by induced expression of the calcification inhibitor matrix γ-carboxyglutamate protein, reduced expression of the cell cycle inhibitor p21, and lower expression of complement components C3 and C1r, among others. Their phenotype can therefore be described as anti-calcifying and anti-inflammatory and contrasts with the phenotype of PXE fibroblasts.
In summary, changes in lipid and bile acid metabolism and remodeling of the ECM appear as systemic influencing factors of PXE. In contrast, peripheral tissue calcification and inflammation appear to be primarily shaped locally by the cellular milieu and are presumably influenced secondarily by the systemic aberrations in the progressive course of PXE.
Jahr
2023
Seite(n)
190
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https://pub.uni-bielefeld.de/record/2979561
Zitieren
Plümers R. Einfluss einer ABCC6-Defizienz auf die Remodellierung der extrazellulären Matrix in in vitro und ex vivo Gewebemodellen. Bielefeld: Universität Bielefeld; 2023.
Plümers, R. (2023). Einfluss einer ABCC6-Defizienz auf die Remodellierung der extrazellulären Matrix in in vitro und ex vivo Gewebemodellen. Bielefeld: Universität Bielefeld.
Plümers, Ricarda. 2023. Einfluss einer ABCC6-Defizienz auf die Remodellierung der extrazellulären Matrix in in vitro und ex vivo Gewebemodellen. Bielefeld: Universität Bielefeld.
Plümers, R. (2023). Einfluss einer ABCC6-Defizienz auf die Remodellierung der extrazellulären Matrix in in vitro und ex vivo Gewebemodellen. Bielefeld: Universität Bielefeld.
Plümers, R., 2023. Einfluss einer ABCC6-Defizienz auf die Remodellierung der extrazellulären Matrix in in vitro und ex vivo Gewebemodellen, Bielefeld: Universität Bielefeld.
R. Plümers, Einfluss einer ABCC6-Defizienz auf die Remodellierung der extrazellulären Matrix in in vitro und ex vivo Gewebemodellen, Bielefeld: Universität Bielefeld, 2023.
Plümers, R.: Einfluss einer ABCC6-Defizienz auf die Remodellierung der extrazellulären Matrix in in vitro und ex vivo Gewebemodellen. Universität Bielefeld, Bielefeld (2023).
Plümers, Ricarda. Einfluss einer ABCC6-Defizienz auf die Remodellierung der extrazellulären Matrix in in vitro und ex vivo Gewebemodellen. Bielefeld: Universität Bielefeld, 2023.
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