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* These authors contributed equally
Cardiovascular disease is the leading cause of death worldwide. Vascular calcification contributes substantially to the burden of cardiovascular morbidity and mortality. This protocol describes a simple method to quantify vascular smooth muscle cell-mediated calcium precipitation in vitro by fluorescent imaging.
Vascular calcification involves a series of degenerative pathologies, including inflammation, changes to cellular phenotype, cell death, and the absence of calcification inhibitors, that concomitantly lead to a loss of vessel elasticity and function. Vascular calcification is an important contributor to morbidity and mortality in many pathologies, including chronic kidney disease, diabetes mellitus, and atherosclerosis. Current research models to study vascular calcification are limited and are only viable at the late stages of calcification development in vivo. In vitro tools for studying vascular calcification use end-point measurements, increasing the demands on biological material and risking the introduction of variability to research studies. We demonstrate the application of a novel fluorescently labeled probe that binds to in vitro calcification development on human vascular smooth muscle cells and determines the real-time development of in vitro calcification. In this protocol, we describe the application of our newly developed calcification assay, a novel tool in disease modeling that has potential translational applications. We envisage this assay to be relevant in a broader spectrum of mineral deposition research, including applications in bone, cartilage, or dental research.
Vascular calcification (VC) is an independent risk factor for cardiovascular morbidity and mortality1,2,3. Long considered a passive chemical process of ectopic mineral deposition, it now appears a modifiable tissue healing response involving the active contribution of various cells including activated vascular smooth muscle cells (hVSMC) as a driver of the disease4,5. In vivo VC can be measured by multislice CT scans as an assessment of atherosclerotic burden6,7,8. Currently, a paradigm shift is underway, wherein VC severity is becoming recognized as a risk factor in cardiovascular disease, type II diabetes, chronic kidney disease, and ageing9,10,11,12,13,14,15.
hVSMCs are the most abundant cell type in the cardiovascular system and a principal actor in the development of VC. In vitro hVSMC-induced calcification is a widely used disease model to study cardiovascular disease16,17. However, most protocols for the detection of in vitro calcification use end-point measurements that can limit data acquisition, require greater use of cellular material, and can slow research. Common methods for the detection of in vitro hVSMC calcification include the o-cresolphthalein assay, which measures solubilized calcium deposition against total protein and requires cell lysis18. Also, Alizarin Red staining is used, which binds directly to calcium deposits on fixed cells or tissue19. To study hVSMC calcification over time with either o-cresolphthalein or Alizarin Red requires batches of replicates per time point, increasing the demand on biological material, and in turn, increasing the chance of variability.
In this paper, we detail the method for the application of a novel assay that utilizes hVSMCs with a fluorescent imaging probe to determine in vitro VC progression as well as function as a singular end-stage calcification assay. We previously demonstrated that this assay is directly comparable to the o-cresolphthalein and Alizarin Red methods and can be used to distinguish between varying culture conditions20. In addition to real-time measurements, this assay may be used to determine the propensity of serum or plasma samples as a surrogate marker for clinical VC development20. This will aid in the application of biological strategies of cardiovascular sciences and disease modeling. A further application of the assay may be as a translational BioHybrid system to assess VC severity or progression from blood constituents such as serum or plasma.
1. Cell seeding, maintenance, and calcification induction
2. Calcification detection via imaging
NOTE: The following protocol provides the general steps to be taken in preparation, imaging, and data analysis. Screenshots supporting the instructions for each step using an automated imaging platform and corresponding image analysis software (see Table of Materials for details) are provided in Supplemental File 2 and Supplemental File 3. Other imaging instruments and image processing tools may be used to apply this protocol. However, repeated imaging at the same location in each well is crucial for meaningful data acquisition. Creating a protocol to image calcification and re-use at every imaging step is necessary for obtaining reproducible results. The first time applying the method, follow the steps below to prepare before the imaging.
3. Data analysis
NOTE: For detailed screenshots on how to perform the data analysis using an automated imaging platform and corresponding image analysis software (see Table of Materials for details), please see Supplemental File 4. If using alternative imaging instruments or analysis software, the images should be exported and batch processed ensuring that the exposure, fluorescence threshold, or intensity are adjusted equally for all images in a comparative data set.
The outcome includes original images of HOECHST-stained nuclei, RFP-labeled calcification, and brightfield images. Different stages of calcification ranging from low (Figure 2) to high (Figure 3) may be detected and analyzed. Calcification can usually be spotted as black speckles using light microscopy (Figure 2D and Figure 3B, arrows indicate calcification), which are useful for primary assessment...
In this manuscript, we describe a semi-automated method for in vitro calcification determination. For this method, three critical steps of hVSMC calcification should be optimized. First, cellular density is critical for hVSMC calcification development. Low densities of hVSMCs will result in slow or no calcification and cell death due to the lack of cell-to-cell contact and the stress that is induced under calcifying conditions21. High cellular densities result in over-confluency, after wh...
Leon Schurgers has received institutional grants from Bayer, Boehringer Ingelheim, NattoPharma, and IDS. Leon Schurgers owns shares in Coagulation Profile. Willi Jahnen-Dechent is a co-founder and shareholder of CALCISCON AG.
This research was funded by the European Union's Horizon 2020 research and innovation programs under the Marie Sklodowska-Curie grant agreement No 722609 and 764474, NWO ZonMw (MKMD 40-42600-98-13007). This research was supported by BioSPX. WJ-D received funding from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) TRR219-project ID 322900939 and project ID 403041552
Name | Company | Catalog Number | Comments |
Calcium chloride, 93%, anhydrous | Thermo Fisher Scientific | 349615000 | |
Costar 6-well Clear TC-treated well plates | Corning | 3516 | |
Cytation 3 System | BioTek, Abcoude, The Netherlands | ||
Fetal Bovine Serum | Merck | F7524-100ML | |
Fetuin-A-Alexa Fluor-546 | Prepared in-house | ||
Gen5 Software v3.10 | BioTek | ||
Gibco Medium 199 | Thermo Fisher Scientific | 11150059 | |
Hoechst 33342, Trihydrochloride | Thermo Fisher Scientific | H3570 | |
PBS (10X), pH 7.4 | Thermo Fisher Scientific | 70011044 | |
Penicillin-Streptomycin | Thermo Fisher Scientific | 15140122 | |
Trypsin-EDTA (0.05%), phenol red | Thermo Fisher Scientific | 25300062 |
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