Ioana Alesutan1, Katharina Musculus1, Tatsiana Castor1, Kousi Alzoubi1, Jakob Voelkl1, Florian Läng1
1Department of Physiology, University of Tübingen, Gmelinstr. 5, 72076, Tübingen, Germany
Tóm tắt
<b><i>Background/Aims: </i></b>Excessive phosphate concentrations trigger vascular calcification, an active process promoted by osteoinduction of vascular smooth muscle cells (VSMCs) with increased expression and activity of transcription factor RUNX2 (Core-binding factor α1, CBFA1), alkaline phosphatase (ALPL), TGFß1, transcription factor NFAT5, and NFAT5-sensitive transcription factor SOX9. The osteoinductive signaling and vascular calcification of hyperphosphatemic klotho-hypomorphic mice could be reversed by treatment with NH<sub>4</sub>Cl, effects involving decrease of TGFß1 and inhibition of NFAT5-dependent osteoinductive signaling. Known effects of NH<sub>4</sub>Cl include alkalinization of acidic cellular compartments. The present study explored whether osteo-/chondrogenic signaling could be influenced by alkalinization of acidic cellular compartments following inhibition of the vacuolar H<sup>+</sup> ATPase with bafilomycin A1 or following dissipation of the pH gradient across the membranes of acidic cellular compartments with methylamine. <b><i>Methods: </i></b>Primary human aortic smooth muscle cells (HAoSMCs) were treated with high phosphate to trigger osteo-/chondrogenic signaling and calcification in the absence or presence of bafilomycin A1 or methylamine. Calcium content was determined using a QuantiChrom Calcium assay, ALP activity by a colorimetric assay and transcript levels by quantitative RT-PCR. <b><i>Results: </i></b>High phosphate increased significantly the calcium deposition, <i>CBFA1</i> and <i>ALPL</i> mRNA expression as well as alkaline phosphatase activity in HAoSMCs, all effects ameliorated by both, bafilomycin A1 and methylamine. High phosphate further significantly up-regulated the mRNA levels of <i>TGFB1, NFAT5</i> and <i>SOX9,</i> effects significantly blunted by additional treatment with bafilomycin A1 or methylamine. Treatment of HAoSMCs with human TGFß1 protein or high phosphate up-regulated <i>NFAT5, SOX9, CBFA1</i> and <i>ALPL</i> mRNA expression to similarly high levels which could not be further increased by combined treatment with high phosphate and TGFß1. Bafilomycin A1 failed to reverse the osteo-/chondrogenic signaling triggered by high phosphate together with TGFß1. <b><i>Conclusions: </i></b>Inhibition of the vacuolar H<sup>+</sup> ATPase or dissipation of the pH gradient across the membranes of acidic cellular compartments both disrupt osteo-/chondrogenic signaling and calcium deposition in VSMCs, observations supporting the hypothesis that vascular calcification requires acidic cellular compartments.
