Mineral Composition of Phosphate-Induced Calcification in a Rat Aortic Tissue Culture Model

Abstract

Aim: High phosphorus conditions promote vascular calcification (VC) in both chronic kidney disease (CKD) patients and experimental models. However, the composition of medial calcification has not been accurately determined, so the objective of this study was to evaluate the mineral composition of calcification in a tissue culture model, not a cell culture system. Methods: Aortic rings obtained from male Sprague-Dawley rats were incubated in serum-supplemented medium for 10 days. The inorganic phosphate (Pi) concentration of the medium was increased to induce VC, which was assessed by histology, imaging, and spectroscopy. The mineral composition of the calcification was analyzed using Fourier transform infrared (FTIR) spectroscopic imaging, scanning electron microscopy (SEM), and energy dispersive X-ray spectroscopy (EDX) mapping. Results: The calcium content significantly increased only in aortic rings cultured for 10 days in the high-Pi medium (HiP: 3.8 mmol/L). The concentration of the phosphate transporter Pit-1 in the aortic tissue exposed to HiP was higher than that in the control incubated sections. The FTIR images and spectra indicated that PO4 3 was mostly distributed as hydroxyapatite in the medial calcification of aortic rings cultured in HiP. A small quantity of carbonate was identified. The SEM -EDX overlay map demonstrated that phosphorus and calcium simultaneously accumulated and localized in the area of medial calcification induced by exposure to HiP. Conclusion: This is the first report of accurate determination of the chemical composition of aortic medial calcification. Exposure to high Pi concentration augments aortic calcification via an increase in Pit-1, which mainly contains calcium phosphate.

Critical Parameters of the In Vitro Method of Vascular Smooth Muscle Cell Calcification

“…Thus, the relevant value for the onset of spontaneous precipitation should be that of ACP supersaturation (which depends on ACP solubility and the ionic product of the ions forming the ACP precipitate), rather than that of HAP, as it has been argued for the in vivo condition [ 22 ]. While HPO 4 2- is the only phosphate species in DCPD, several characterization techniques, such as 31 P NMR [ 23 ] and X-ray diffraction, have shown that ACP [ 24 ] is mostly, if not only, composed of PO 4 3- , which concurs with a recent report on calcification in vitro that used massive calcification [ 25 ]. This is crucial for predicting precipitation at a certain pH, because the concentration of HPO 4 2- barely changes in the pH range of interest (7.4–8.7), so DCPD supersaturation is not modified.…”

Phenotypic Modulation of Cultured Primary Human Aortic Vascular Smooth Muscle Cells by Uremic Serum

“…Many aspects regarding the complex process of vascular calcification and phenotypic remodeling secondary to CKD and uremia remain unanswered. The process has been studied using different in vitro models, including intact vessels (Shroff et al, 2008 ), aortic rings (Lomashvili et al, 2004 ; Sonou et al, 2015 ), or dispersed VSMC, mainly obtained from bovine or rat thoracic aorta (Chen et al, 2006a ; Villa-Bellosta et al, 2007 ; Hortells et al, 2015 ). In spite of the limitations associated with cultured VSMC, such as the lack of extracellular elastin fibers (Lin et al, 2011 ) and the diversity of phenotypes that can co-exist in culture (Patel et al, 2016 ), this model has been extensively used to study vascular calcification.…”

Calcium phosphate bions: towards a pathogenetic concept

“…Кроме того, сниженный уровень ингибиторов эктопической кальцификации фетуина-А и альбумина в крови также ассоциирован с повышенным риском развития ишемической болезни сердца [4,5]. В то же время свободные ионы кальция и фосфора могут приводить к прямой кальцификации средней оболочки сосудов (медии), однако не вызывают собственно атеросклероза [6,7] -хронического воспалительного процесса, ха-ОБЗОРНЫЕ СТАТЬИ VOL. 5, № 1, 2020 ® рактеризующегося формированием гетерогенных бляшек из клеток, внеклеточного матрикса и липидов во внутренней оболочке артерий [8,9].…”