Abstract
BackgroundCardiovascular disease including vascular calcification (VC) remains the leading cause of death in patients suffering from chronic kidney disease (CKD). The process of VC seems likely to be a tightly regulated process where vascular smooth muscle cells are playing a key role rather than just a mere passive precipitation of calcium phosphate. Characterisation of the chemical and crystalline structure of VC was mainly led in patients or animal models with CKD. Likewise, Mg2+ was found to be protective in living cells although a potential role for Mg2+ could not be excluded on crystal formation and precipitation. In this study, the crystal formation and the role of Mg2+ were investigated in an in vitro model of primary human aortic vascular smooth muscle cells (HAVSMC) with physical techniques.Methodology/Principal FindingsIn HAVSMC incubated with increased Ca x Pi medium, only calcium phosphate apatite crystals (CPA) were detected by Micro-Fourier Transform InfraRed spectroscopy (µFTIR) and Field Effect Scanning Electron Microscope (FE — SEM) and Energy Dispersive X-ray spectrometry (EDX) at the cell layer level. Supplementation with Mg2+ did not alter the crystal composition or structure. The crystal deposition was preferentially positioned near or directly on cells as pictured by FE — SEM observations and EDX measurements. Large µFTIR maps revealed spots of CPA crystals that were associated to the cellular layout. This qualitative analysis suggests a potential beneficial effect of Mg2+ at 5 mM in noticeably reducing the number and intensities of CPA µFTIR spots.Conclusions/SignificanceFor the first time in a model of HAVSMC, induced calcification led to the formation of the sole CPA crystals. Our data seems to exclude a physicochemical role of Mg2+ in altering the CPA crystal growth, composition or structure. Furthermore, Mg2+ beneficial role in attenuating VC should be linked to an active cellular role.
Highlights
Patients suffering from atherosclerosis, diabetes and chronic kidney disease (CKD) often present with vascular calcification (VC) [1,2,3]
Some results suggest that matrix calcification could be initiated through passive calcium phosphate deposition and that it could be the formation of calcium phosphate nanocrystals that triggers the osteogenic changes that are associated with the accumulation of extracellular, crystalline structures [7, 8], whereas other studies show that calcification could be initiated by the release of matrix vesicles and/or apoptotic bodies from vascular smooth muscle cells (VSMC) that act as nucleation points for forming crystals [9]
human aortic vascular smooth muscle cells (HAVSMC) were identified by their typical hill and valley morphology, and purity of the primary cell culture was further checked by immunocytochemistry using a monoclonal antibody against the α-smooth muscle actin protein 1A4 (Acta 2) (Santa Cruz Biotechnology). α-smooth muscle actin 1A4 is an isoform typical of smooth muscle cells (SMC) and is present in high amounts in vascular SMC [28]
Summary
Patients suffering from atherosclerosis, diabetes and chronic kidney disease (CKD) often present with vascular calcification (VC) [1,2,3]. The process of VC is not fully understood, but likely to be multifactorial [6] It appears to be a tightly regulated, active, cellular process rather than just a mere passive precipitation of calcium phosphate, that somewhat resembles normal bone formation. Cardiovascular disease including vascular calcification (VC) remains the leading cause of death in patients suffering from chronic kidney disease (CKD). The process of VC seems likely to be a tightly regulated process where vascular smooth muscle cells are playing a key role rather than just a mere passive precipitation of calcium phosphate. The crystal formation and the role of Mg2+ were investigated in an in vitro model of primary human aortic vascular smooth muscle cells (HAVSMC) with physical techniques
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