Development, establishment and validation of in vitro and ex vivo assays of vascular calcification

Jana Holmar; Heidi Noels; Michael Boehm; Shruti Bhargava; Joachim Jankowski; Setareh Orth-Alampour

doi:10.1016/j.bbrc.2020.05.085

Development, establishment and validation of in vitro and ex vivo assays of vascular calcification

Jana Holmar, Heidi Noels, Michael Boehm, Shruti Bhargava, Joachim Jankowski^*, Setareh Orth-Alampour

^*Corresponding author for this work

Research output: Contribution to journal › Article › Academic › peer-review

Abstract

Objective: Vascular calcification (VC) is one major complication in patients with chronic kidney disease, with a misbalance in calcium and phosphate metabolism playing crucial role. The mechanisms underlying VC have not been entirely revealed to date. As studies aiming at the identification and characterization of the involved mediators are highly relevant, we developed a standardized operating protocol for in vitro and ex vivo approaches in this study to aiming at the comparability of these studies.

Approach and results: We analyzed in vitro and ex vivo experimental conditions to study VC. Therefore, vascular smooth muscle cells were used for in vitro experiments and rat aorta for ex vivo experiments. The degree of calcification was estimated by quantification of calcium concentrations and by von Kossa staining. As a result, a step-by-step protocol for performing experiments on VC was established. We were able to demonstrate that the degree and the location of VC in vascular smooth muscle cells and aortic rings was highly dependent on the phosphate and CaCl2 concentration in the medium as well as the incubation time. Furthermore, the VC was reduced upon increasing fetal calf serum concentration in the medium.

Conclusion: In the current study, we developed and validated a standardized operating protocol for systematic in vitro and ex vivo analyses of medial calcification, which is essential for the comparability of the results of future studies. (C) 2020 The Authors. Published by Elsevier Inc.

Original language	English
Pages (from-to)	462-470
Number of pages	9
Journal	Biochemical and Biophysical Research Communications
Volume	530
Issue number	2
DOIs	https://doi.org/10.1016/j.bbrc.2020.05.085
Publication status	Published - 17 Sept 2020

Keywords

Vascular calcification
Vascular smooth muscle cells
Rat aorta
CaCl2
Phosphate
CALCIUM
SERUM
PHOSPHORUS
MECHANISMS

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@article{855078072f41474390f263ec7a5ff8fd,

title = "Development, establishment and validation of in vitro and ex vivo assays of vascular calcification",

abstract = "Objective: Vascular calcification (VC) is one major complication in patients with chronic kidney disease, with a misbalance in calcium and phosphate metabolism playing crucial role. The mechanisms underlying VC have not been entirely revealed to date. As studies aiming at the identification and characterization of the involved mediators are highly relevant, we developed a standardized operating protocol for in vitro and ex vivo approaches in this study to aiming at the comparability of these studies.Approach and results: We analyzed in vitro and ex vivo experimental conditions to study VC. Therefore, vascular smooth muscle cells were used for in vitro experiments and rat aorta for ex vivo experiments. The degree of calcification was estimated by quantification of calcium concentrations and by von Kossa staining. As a result, a step-by-step protocol for performing experiments on VC was established. We were able to demonstrate that the degree and the location of VC in vascular smooth muscle cells and aortic rings was highly dependent on the phosphate and CaCl2 concentration in the medium as well as the incubation time. Furthermore, the VC was reduced upon increasing fetal calf serum concentration in the medium.Conclusion: In the current study, we developed and validated a standardized operating protocol for systematic in vitro and ex vivo analyses of medial calcification, which is essential for the comparability of the results of future studies. (C) 2020 The Authors. Published by Elsevier Inc.",

keywords = "Vascular calcification, Vascular smooth muscle cells, Rat aorta, CaCl2, Phosphate, CALCIUM, SERUM, PHOSPHORUS, MECHANISMS",

author = "Jana Holmar and Heidi Noels and Michael Boehm and Shruti Bhargava and Joachim Jankowski and Setareh Orth-Alampour",

note = "Funding Information: The research was funded partly by the Estonian Research Council grant PUTJD66 and by the Estonian Ministry of Education and Research under institutional research financing IUT 19-2 (to J.H.). Furthermore, this study was supported by a grant from the German Research Foundation (DFG, SFB/TRR219 project C-04 to J.J., M − 05 to H.N. and S-01 to M.B.). S.B. was supported by the European Union's Horizon 2020 research and innovation program EU-ITN INTRICARE (722609) and S·O-A was supported by CaReSyAn (764474). The content of this article reflects only the views of its authors. The European Commission is not responsible for any use that may be made of the information it contains. Funding Information: The research was funded partly by the Estonian Research Council grant PUTJD66 and by the Estonian Ministry of Education and Research under institutional research financing IUT 19-2 (to J.H.). Furthermore, this study was supported by a grant from the German Research Foundation (DFG, SFB/TRR219 project C-04 to J.J., M − 05 to H.N. and S-01 to M.B.). S.B. was supported by the European Union{\textquoteright}s Horizon 2020 research and innovation program EU-ITN INTRICARE ( 722609 ) and S·O-A was supported by CaReSyAn ( 764474 ). The content of this article reflects only the views of its authors. The European Commission is not responsible for any use that may be made of the information it contains. Publisher Copyright: {\textcopyright} 2020 The Authors",

year = "2020",

month = sep,

day = "17",

doi = "10.1016/j.bbrc.2020.05.085",

language = "English",

volume = "530",

pages = "462--470",

journal = "Biochemical and Biophysical Research Communications",

issn = "0006-291X",

publisher = "Elsevier",

number = "2",

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TY - JOUR

T1 - Development, establishment and validation of in vitro and ex vivo assays of vascular calcification

AU - Holmar, Jana

AU - Noels, Heidi

AU - Boehm, Michael

AU - Bhargava, Shruti

AU - Jankowski, Joachim

AU - Orth-Alampour, Setareh

N1 - Funding Information: The research was funded partly by the Estonian Research Council grant PUTJD66 and by the Estonian Ministry of Education and Research under institutional research financing IUT 19-2 (to J.H.). Furthermore, this study was supported by a grant from the German Research Foundation (DFG, SFB/TRR219 project C-04 to J.J., M − 05 to H.N. and S-01 to M.B.). S.B. was supported by the European Union's Horizon 2020 research and innovation program EU-ITN INTRICARE (722609) and S·O-A was supported by CaReSyAn (764474). The content of this article reflects only the views of its authors. The European Commission is not responsible for any use that may be made of the information it contains. Funding Information: The research was funded partly by the Estonian Research Council grant PUTJD66 and by the Estonian Ministry of Education and Research under institutional research financing IUT 19-2 (to J.H.). Furthermore, this study was supported by a grant from the German Research Foundation (DFG, SFB/TRR219 project C-04 to J.J., M − 05 to H.N. and S-01 to M.B.). S.B. was supported by the European Union’s Horizon 2020 research and innovation program EU-ITN INTRICARE ( 722609 ) and S·O-A was supported by CaReSyAn ( 764474 ). The content of this article reflects only the views of its authors. The European Commission is not responsible for any use that may be made of the information it contains. Publisher Copyright: © 2020 The Authors

PY - 2020/9/17

Y1 - 2020/9/17

N2 - Objective: Vascular calcification (VC) is one major complication in patients with chronic kidney disease, with a misbalance in calcium and phosphate metabolism playing crucial role. The mechanisms underlying VC have not been entirely revealed to date. As studies aiming at the identification and characterization of the involved mediators are highly relevant, we developed a standardized operating protocol for in vitro and ex vivo approaches in this study to aiming at the comparability of these studies.Approach and results: We analyzed in vitro and ex vivo experimental conditions to study VC. Therefore, vascular smooth muscle cells were used for in vitro experiments and rat aorta for ex vivo experiments. The degree of calcification was estimated by quantification of calcium concentrations and by von Kossa staining. As a result, a step-by-step protocol for performing experiments on VC was established. We were able to demonstrate that the degree and the location of VC in vascular smooth muscle cells and aortic rings was highly dependent on the phosphate and CaCl2 concentration in the medium as well as the incubation time. Furthermore, the VC was reduced upon increasing fetal calf serum concentration in the medium.Conclusion: In the current study, we developed and validated a standardized operating protocol for systematic in vitro and ex vivo analyses of medial calcification, which is essential for the comparability of the results of future studies. (C) 2020 The Authors. Published by Elsevier Inc.

AB - Objective: Vascular calcification (VC) is one major complication in patients with chronic kidney disease, with a misbalance in calcium and phosphate metabolism playing crucial role. The mechanisms underlying VC have not been entirely revealed to date. As studies aiming at the identification and characterization of the involved mediators are highly relevant, we developed a standardized operating protocol for in vitro and ex vivo approaches in this study to aiming at the comparability of these studies.Approach and results: We analyzed in vitro and ex vivo experimental conditions to study VC. Therefore, vascular smooth muscle cells were used for in vitro experiments and rat aorta for ex vivo experiments. The degree of calcification was estimated by quantification of calcium concentrations and by von Kossa staining. As a result, a step-by-step protocol for performing experiments on VC was established. We were able to demonstrate that the degree and the location of VC in vascular smooth muscle cells and aortic rings was highly dependent on the phosphate and CaCl2 concentration in the medium as well as the incubation time. Furthermore, the VC was reduced upon increasing fetal calf serum concentration in the medium.Conclusion: In the current study, we developed and validated a standardized operating protocol for systematic in vitro and ex vivo analyses of medial calcification, which is essential for the comparability of the results of future studies. (C) 2020 The Authors. Published by Elsevier Inc.

KW - Vascular calcification

KW - Vascular smooth muscle cells

KW - Rat aorta

KW - CaCl2

KW - Phosphate

KW - CALCIUM

KW - SERUM

KW - PHOSPHORUS

KW - MECHANISMS

U2 - 10.1016/j.bbrc.2020.05.085

DO - 10.1016/j.bbrc.2020.05.085

M3 - Article

C2 - 32560961

SN - 0006-291X

VL - 530

SP - 462

EP - 470

JO - Biochemical and Biophysical Research Communications

JF - Biochemical and Biophysical Research Communications

IS - 2

ER -