Optimisation of cell and ex vivo culture conditions to study vascular calcification

Abstract

Medial vascular calcification (MVC) is a highly prevalent disease associated with a high risk of severe, potentially lethal, complications. While animal studies may not systematically be circumvented, in vitro systems have been proven useful to study disease physiopathology. In the context of MVC, the absence of a clinically relevant standardized in vitro method prevents the appropriate comparison and overall interpretation of results originating from different experiments. The aim of our study is to establish in vitro models mimicking in vivo vascular calcification and to select the best methods to unravel the mechanisms involved in MVC. Human aortic smooth muscle cells and rat aortic rings were cultured in different conditions. The influence of fetal calf serum (FCS), alkaline phosphatase, phosphate and calcium concentrations in the medium were evaluated. We identified culture conditions, including the herein reported Aorta Calcifying Medium (ACM), which allowed a reproducible and specific medial calcification of aortic explants. Studying cells and aortic explants cultured, the involvement of bone morphogenetic protein 2 (BMP2) pathway, fibrosis and apoptosis processes in in vitro MVC were demonstrated. Expression of osteoblastic markers was also observed suggesting the occurrence of transdifferentiation of smooth muscle cells to osteoblasts in our models. The use of these models will help researchers in the field of vascular calcification to achieve reproducible results and allow result comparison in a more consistent way.

Research Models for Studying Vascular Calcification

“…Furthermore, sodium pyruvate, insulin or calciferol are frequently used supplements in calcification media. A recent work compared the influence of different factors such as phosphate, calcium and FBS on in vitro calcification yield and reproducibility [59].…”

“…Currently, aortic rings from rats and mice are commonly used for studying vessel calcification under various conditions. The stimulation time varies from 3-14 days [6,24,25,28,59,[62][63][64][65]. Although the utilization of aortic rings comes closer to the physiologic setting, a multitude of influencing factors is still lost.…”

Vascular Calcification in Different Arterial Beds in Ex Vivo Ring Culture and in Vivo Rat Model

“…After one week of acclimatization, rats were anesthetized with iso urane (2% in O 2 ) and killed by decapitation before harvesting the vessels for ex vivo experimentations. Methods are more precisely described in a previous work 19 .…”

“…To study vascular calci cation, in-vitro smooth muscle cell culture is often used 19 . This approach, however, cannot be used to investigate the susceptibility of different arteries to calci cation.…”

Uremic Toxins and Vascular Calcification–Missing the Forest for All the Trees

“…Also, for Ca 2+ or Pi, concentration, donor (e.g., β-Glycerophosphate vs. Na 2 HPO 4 ), exposure time and the use of foetal bovine serum (FBS) are subject to differences. A proposal for standardization has recently been put forward, however consensus has not been reached yet [ 285 ]. Additionally, the concentration of the URMs to which the VSMC are exposed matters and should ideally reflect/mimic the clinical situation.…”