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

Abstract

BackgroundVascular calcification (VC) is primarily studied using cultures of vascular smooth muscle cells. However, the use of very different protocols and extreme conditions can provide findings unrelated to VC. In this work we aimed to determine the critical experimental parameters that affect calcification in vitro and to determine the relevance to calcification in vivo.Experimental Procedures and ResultsRat VSMC calcification in vitro was studied using different concentrations of fetal calf serum, calcium, and phosphate, in different types of culture media, and using various volumes and rates of change. The bicarbonate content of the media critically affected pH and resulted in supersaturation, depending on the concentration of Ca2+ and Pi. Such supersaturation is a consequence of the high dependence of bicarbonate buffers on CO2 vapor pressure and bicarbonate concentration at pHs above 7.40. Such buffer systems cause considerable pH variations as a result of minor experimental changes. The variations are more critical for DMEM and are negligible when the bicarbonate concentration is reduced to ¼. Particle nucleation and growth were observed by dynamic light scattering and electron microscopy. Using 2mM Pi, particles of ~200nm were observed at 24 hours in MEM and at 1 hour in DMEM. These nuclei grew over time, were deposited in the cells, and caused osteogene expression or cell death, depending on the precipitation rate. TEM observations showed that the initial precipitate was amorphous calcium phosphate (ACP), which converts into hydroxyapatite over time. In blood, the scenario is different, because supersaturation is avoided by a tightly controlled pH of 7.4, which prevents the formation of PO4 3--containing ACP.ConclusionsThe precipitation of ACP in vitro is unrelated to VC in vivo. The model needs to be refined through controlled pH and the use of additional procalcifying agents other than Pi in order to reproduce calcium phosphate deposition in vivo.

Identifying early pathogenic events during vascular calcification in uremic rats

“…15 Third, with the logical objective of simplifying this complexity, the scientific community has been using in vitro methods of calcification that, unfortunately, were unrelated to cell-mediated ectopic calcification. 14 In this work, our purpose has been to disentangle the complexity of VC by using an experimental approach that has sufficient clarity: we have focused on the very early pathogenic steps, while assuming that the complexity of advanced calcification is a consequence of an increasing and self-complicating morbid process. We chose the evolution of artery calcium content as from nephrectomy as the baseline for a time-course study of pathogenic events until a frank calcification was observed.…”

“…In the search for a less complex model, the in vitro methods of Pi-induced calcification were discarded. 14 Therefore, for the sake of simplicity we have studied the very early stages of aortic calcification during CKD using the 5/6-nephrectomized (NX) rat model, in which rats were fed a Pi-rich diet. The aorta calcium content, the specific gene expression changes, serum and urine parameters, and protein expression and activities were analyzed at different times as from nephrectomy.…”

Associations between calcium-phosphate metabolism and coronary artery calcification; a cross sectional study of a middle-aged general population

“…In line with our findings, Reynold et al [25] found that calcium was a stronger inducer of calcification than phosphate in an in vitro study of aortic rings from humans exposed to various phosphate and calcium concentrations with CPP held constant. However, in vitro studies of the pathophysiology of calcium-phosphate disturbances and vascular calcification, conducted to imitate the conditions of chronic kidney disease with elevated extracellular calcium and phosphate and have been criticised for their un-physiological conditions [26], making extrapolation of these data difficult.…”

Matrix vesicles from chondrocytes and osteoblasts: Their biogenesis, properties, functions and biomimetic models

“…Several inhibitors of mineralization including PP i [102], phosphorylated osteopontin (OPN) [103], matrix Gla protein [104, 105], and fetuin A [106] can prevent apatite formation. The formation of apatite is preceded by a nucleation step [1, 98–101, 107] and/or by an intermediate formation of ACP [100, 101, 108, 109]. Apatite in a crystalline phase is induced by a nucleation step, even if it is generated from ACP.…”