Using 99mTc-(V)-DMSA to follow the vascular calcification process in vascular smooth muscle cells based on pit-1 expression.

Abstract

Background: Vascular calcification is an established feature of atherosclerosis process. The sodium/phosphate transporter PiT-1 acts as a biosensor in vascular calcification of VSMCs. [^99mTc]-Pentavalent dimercaptosuccinic acid (^99mTc-(V)-DMSA) was mediated by PiT-1 transporter in tumoral cells and we propose its evaluation in a vascular calcification in vitro model. The aim of this study was to determine if ^99mTc-(V)-DMSA can follow the vascular calcification process in vascular smooth muscle cells (VSMCs) based on PiT-1 expression.

Methods: From a rat aortic VSMC cell line (A7r5), we set up a model of calcification within 7 days using a calcifying medium containing a high inorganic phosphate concentration. Phosphocalcic deposits were monitored with Alizarin red and Von Kossa staining and with phase contrast microscopy. PiT-1 expression was evaluated with an immunofluorescence assay and osteopontin expression, with whole cell ELISA assay. ^99mTc-(V)-DMSA uptake was measured in control and calcifying conditions and compared with optical microscopy evaluation.

Results: Under hyperphosphatemia conditions, the VSMC cells progressively overexpressed osteopontin protein, PiT-1 transporter, and synthetized mineralized matrix with phosphocalcic deposition. ^99mTc-(V)-DMSA uptake was to 2.8±2.08%DA/mg-protein in control cells and 42±24%DA/mg-protein in calcified cells (P<0.001). PiT-1 inhibition with phosphonoformic acid completely reverse the calcium deposition as well as the ^99mTc-(V)-DMSA uptake. These results demonstrated that ^99mTc-(V)-DMSA in-vitro uptake is mediated by PiT-1 transporter and follow the VSMC calcification process.

Conclusions: These preliminary in-vitro results showed ^99mTc-(V)-DMSA uptake follow the phospho-calcic deposition mediated by PiT-1 transporter. This radiotracer may have some potential to detect changes of VSMC metabolism occurring in the atherosclerosis process.