Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCA362

Poster Communications

Fibulin-3 ameliorates phosphate-induced vascular smooth muscle cell calcification through inhibition of oxidative stress

T. Luong1, N. Schelski1, B. Boehme1, M. Makridakis2, A. Vlahou2, F. Lang3, B. Pieske1,4,5, I. Alesutan1,4, J. Voelkl1

1. Center for Cardiovascular Research Cardiology CVK, Charité - Universitätsmedizin Berlin, Berlin, Germany. 2. Biomedical Research Foundation, Academy of Athens, Athens, Greece. 3. Department of Physiology I, Eberhard-Karls University, Tübingen, Germany. 4. Berlin Institute of Health, Berlin, Germany. 5. Department of Internal Medicine and Cardiology, DHZB, Berlin, Germany.

Background: Medial vascular calcification induced by hyperphosphatemia is associated with cardiovascular events and mortality in chronic kidney disease (CKD) patients. This process is actively promoted by osteo-/chondrogenic transdifferentiation of vascular smooth muscle cells (VSMCs) and involves cellular oxidative stress. Fibulin-3, an extracellular matrix protein, inhibits vascular oxidative stress in hypertension. Thus, this study investigated the effects of Fibulin-3 on phosphate-induced VSMC calcification and the possible underlying mechanisms. Methods: Primary human aortic smooth muscle cells (HAoSMCs) were treated with control or β-glycerophosphate without or with recombinant human Fibulin-3 protein and hydrogen peroxide as an exogenous source of oxidative stress. Calcification was analyzed by quantification of calcium content and Alizarin Red staining, the mRNA and protein expression by quantitative RT-PCR and Western blotting, respectively and the antioxidant capacity and ALPL activity by colorimetric methods. Results: Phosphate treatment increased mineral deposition, tissue-nonspecific alkaline phosphatase (ALPL) activity and mRNA expression of osteogenic and chondrogenic markers MSX2, CBFA1, SOX9 and ALPL in HAoSMCs. All these effects were blunted by additional treatment with Fibulin-3, suggesting that Fibulin-3 is able to interfere with osteo-/chondrogenic transdifferentiation and calcification of HAoSMCs. Furthermore, Fibulin-3 reversed the decrease in total antioxidant capacity and up-regulation of oxidative stress markers NOX4 and CYBA mRNA expression in HAoSMCs during high phosphate conditions. Accordingly, Fibulin-3 suppressed the expression of oxidative stress downstream effectors PAI-1 and matrix metalloproteinases MMP2 and MMP9 as well as apoptosis marker BAX/BLC2 mRNA expression ratio in phosphate treated HAoSMCs. The inhibitory effects of Fibulin-3 on phosphate-induced osteo-/chondrogenic transdifferentiation of HAoSMCs were suppressed by additional treatment with hydrogen peroxide. Conclusions: Fibulin-3 inhibits endogenous oxidative stress in VSMCs during hyperphosphatemia and, thus, interferes with phosphate-induced osteo-/chondrogenic transdifferentiation and calcification of VSMCs. Up-regulation or supplementation of Fibulin-3 may be beneficial in reducing the progression of vascular calcification during hyperphosphatemic conditions such as CKD.

Where applicable, experiments conform with Society ethical requirements