Mareike Dabisch-Ruthe1, Patricia Kuzaj1, Christian Götting2, Cornelius Knabbe1, Doris Hendig3. 1. Institut für Laboratoriums- und Transfusionsmedizin, Herz- und Diabeteszentrum Nordrhein-Westfalen, Universitätsklinik der Ruhr-Universität Bochum, Bad Oeynhausen, Germany. 2. MVZ Labor Limbach Nürnberg, Nürnberg, Germany. 3. Institut für Laboratoriums- und Transfusionsmedizin, Herz- und Diabeteszentrum Nordrhein-Westfalen, Universitätsklinik der Ruhr-Universität Bochum, Bad Oeynhausen, Germany. Electronic address: dhendig@hdz-nrw.de.
Abstract
BACKGROUND: Pseudoxanthoma elasticum (PXE) is a rare hereditary disorder characterized by late onset and progressive calcification of elastic fibers in skin, eyes and the cardiovascular system, exemplifying a model for conditions characterized by soft tissue calcification. OBJECTIVE: The aim of our study was to characterize cellular inorganic pyrophosphate (PPi) homeostasis in PXE. METHODS: Gene expression of PPi metabolizing enzymes was determined by quantitative real-time PCR after incubation up to 21 days with or without addition of Na2HPO4. Extracellular and cytosolic PPi concentrations were measured by enzyme-linked bioluminescence assay. ALP and ENPP1 activity was determined spectrophotometrically. We further established a human cell culture model suitable for investigating PXE and related disorders without addition of artificial calcification triggers. RESULTS: Independently of the experimental conditions, PXE fibroblasts revealed a higher degree of matrix calcification. We observed that matrix calcification was associated with altered gene expression of PPi metabolizing enzymes in PXE fibroblasts. In this context, PXE fibroblasts exhibited significantly higher expression of ALP and OPN and reduced mRNA expression and activity of ENPP1. Here, for the first time cytosolic and extracellular PPi levels were shown to be strongly reduced in PXE fibroblasts. We further showed that PPi concentration in bovine and human sera additives had a strong impact on matrix calcification. In a last experimental line, we demonstrated that addition of PPi analogs reduced matrix calcification of PXE fibroblasts most likely by reducing ALP and OPN mRNA expression, restoring ENPP1 activity and subsequently elevating PPi concentrations. CONCLUSION: The results of our study along with recent findings point to the essential role of PPi as the central regulatory metabolites preventing matrix calcification in PXE. But what remains to be determined is the underlying molecular mechanism leading to depletion of PPi in PXE. We further suggest that supplementation of PPi analogs might counteract pathological calcification in PXE and related disorders.
BACKGROUND: Pseudoxanthoma elasticum (PXE) is a rare hereditary disorder characterized by late onset and progressive calcification of elastic fibers in skin, eyes and the cardiovascular system, exemplifying a model for conditions characterized by soft tissue calcification. OBJECTIVE: The aim of our study was to characterize cellular inorganic pyrophosphate (PPi) homeostasis in PXE. METHODS: Gene expression of PPi metabolizing enzymes was determined by quantitative real-time PCR after incubation up to 21 days with or without addition of Na2HPO4. Extracellular and cytosolic PPi concentrations were measured by enzyme-linked bioluminescence assay. ALP and ENPP1 activity was determined spectrophotometrically. We further established a human cell culture model suitable for investigating PXE and related disorders without addition of artificial calcification triggers. RESULTS: Independently of the experimental conditions, PXE fibroblasts revealed a higher degree of matrix calcification. We observed that matrix calcification was associated with altered gene expression of PPi metabolizing enzymes in PXE fibroblasts. In this context, PXE fibroblasts exhibited significantly higher expression of ALP and OPN and reduced mRNA expression and activity of ENPP1. Here, for the first time cytosolic and extracellular PPi levels were shown to be strongly reduced in PXE fibroblasts. We further showed that PPi concentration in bovine and human sera additives had a strong impact on matrix calcification. In a last experimental line, we demonstrated that addition of PPi analogs reduced matrix calcification of PXE fibroblasts most likely by reducing ALP and OPN mRNA expression, restoring ENPP1 activity and subsequently elevating PPi concentrations. CONCLUSION: The results of our study along with recent findings point to the essential role of PPi as the central regulatory metabolites preventing matrix calcification in PXE. But what remains to be determined is the underlying molecular mechanism leading to depletion of PPi in PXE. We further suggest that supplementation of PPi analogs might counteract pathological calcification in PXE and related disorders.
Authors: Shira G Ziegler; Carlos R Ferreira; Elena Gallo MacFarlane; Ryan C Riddle; Ryan E Tomlinson; Emily Y Chew; Ludovic Martin; Chen-Ting Ma; Eduard Sergienko; Anthony B Pinkerton; José Luis Millán; William A Gahl; Harry C Dietz Journal: Sci Transl Med Date: 2017-06-07 Impact factor: 17.956
Authors: Ana M Blazquez-Medela; Pierre J Guihard; Jiayi Yao; Medet Jumabay; Aldons J Lusis; Kristina I Boström; Yucheng Yao Journal: FEBS Open Bio Date: 2015-03-23 Impact factor: 2.693
Authors: Patricia Kuzaj; Joachim Kuhn; Ryan D Michalek; Edward D Karoly; Isabel Faust; Mareike Dabisch-Ruthe; Cornelius Knabbe; Doris Hendig Journal: PLoS One Date: 2014-09-29 Impact factor: 3.240