This paper presents the methodology for obtaining and assessing vascular calcification by isolating murine aortas followed by extracting calcified extracellular vesicles to observe the mineralization potential.
Cardiovascular disease is the leading cause of death in the world, and vascular calcification is the most significant predictor of cardiovascular events; however, there are currently no treatment or therapeutic options for vascular calcification. Calcification begins within specialized extracellular vesicles (EVs), which serve as nucleating foci by aggregating calcium and phosphate ions. This protocol describes methods for obtaining and assessing calcification in murine aortas and analyzing the associated extracted EVs. First, gross dissection of the mouse is performed to collect any relevant organs, such as the kidneys, liver, and lungs. Then, the murine aorta is isolated and excised from the aortic root to the femoral artery. Two to three aortas are then pooled and incubated in a digestive solution before undergoing ultracentrifugation to isolate the EVs of interest. Next, the mineralization potential of the EVs is determined through incubation in a high-phosphate solution and measuring the light absorbance at a wavelength of 340 nm. Finally, collagen hydrogels are used to observe the calcified mineral formation and maturation produced by the EVs in vitro.
Calcification is the most significant predictor of cardiovascular disease mortality and morbidity1. Calcification alters the arterial wall mechanics due to the buildup of calcium and phosphate minerals2. In atherosclerosis, calcification can exacerbate local stress and result in plaque rupture, which is the leading cause of heart attacks. Medial calcification-often resulting from chronic kidney disease-is more widespread and leads to significant arterial stiffening, dysfunction, and cardiac overload2,3. Currently, there are no therapeutic options for the treatment or prevention of vascular calcification.
Vascular smooth muscle cells (VSMCs) adopt an osteoblast-like phenotype and release calcifying extracellular vesicles (EVs) that nucleate nascent minerals, thus driving calcification4,5,6. This process resembles the physiological mineralization of osteoblasts in bone7. Although the endpoint of mineralization is similar in the vascular wall and bone matrix, the mechanisms by which the calcifying EVs originate differ in the two tissues8. There are many types of models that are used to study vascular calcification. In vitro, cell culture models mimic the osteogenic transition of VSMCs and subsequent mineral formation with specialized media.
When studying in vivo calcification, the model used depends upon the type of calcification being studied. Hyperlipidemic mouse models are often used to study atherosclerotic calcification, which appears more focal within lipid-rich plaques9. In contrast, medial calcification is more widespread throughout the vasculature and is often studied using chronic kidney disease models that employ an adenine-rich diet regimen to induce renal failure or surgical techniques to remove significant portions of the kidneys10,11. Aggressive models of vascular calcification have used a combination of both hyperlipidemic and chronic kidney disease models12. This protocol provides a method for assessing vascular calcification in murine aortas for both medial and atherosclerotic calcification, extracting EVs from the aortic wall, and observing the mineralization potential in the EVs obtained from in vitro cell culture models. Future studies can use these procedures in mechanistic analyses of vascular calcification and to assess potential therapeutic interventions.
The in vivo work was approved and overseen by the Institutional Animal Care and Use Committee (IACUC) at Florida International University and conformed to current National Institutes of Health (NIH) guidelines. For this protocol, the procedure does not differ depending on the strain, weight, age, and sex of the mouse. The type of calcification being studied, diets, and treatments may alter the length of the study and the weight of the mouse used and may be dependent on a specific strain and sex of mouse used in the study. For this protocol, both male and female C57BL/6J mice were used, and they were fed a calcification diet. The mice were sacrificed between 20 weeks and 24 weeks of age.
1. Isolation and excision of the aorta
2. Isolating and extracting EVs from aortas
NOTE: Once the aortas have been isolated and removed, EVs can be extracted from the tissue. Using a digestive solution and multiple centrifuge cycles, EVs can be collected and used for many different techniques, including calcification assays, gel electrophoresis, and immunoblotting13. The protocol for isolating and extracting EVs is as follows:
3. Assessing the calcification potential of extracellular vesicles with light scattering absorbance
NOTE: To measure the real-time mineral formation of EVs, we used an assay originally developed to study mineral formation from growth plate cartilage EVs from cell culture14. Since calcium phosphate deposition is the hallmark of calcification, an increase in light scattering absorbance as the result of calcium phosphate compound formation is indicative of the calcification potential14. In vitro VSMC models are convenient for measuring the calcification potential of EVs. In this technique, a plate reader with a short wavelength filter can quantify the in vitro calcification of EVs. The absorbency reading is recorded at 340 nm, and a higher absorbance is indicative of more calcium phosphate mineral formation. The protocol for the light scattering absorbance assay is as follows:
4. Assessing the calcification mineral formation of extracellular vesicles with collagen hydrogels
NOTE: The aggregation of EVs and the formation of microcalcifications are observed through collagen hydrogels. These hydrogels act as a scaffold that mimics the collagen density observed in vivo15. This demonstrates the effect of collagen on calcification growth. The protocol for assessing the mineral formation of EVs in hydrogels is as follows:
Once the aortas are extracted, imaging using a near-infrared optical scanner shows a visual representation of the aorta as well as the vascular calcification (Figure 1). The pixel intensity values within the scanned fluorescent image represent the distribution of calcification and are shown here using a colored heatmap. Quantification methods include identifying a positive threshold and reporting the percentage area of the aorta with values greater than this threshold value and/or reporting the mean fluorescence intensity of the pixels within the aorta. As shown in Figure 2 and Figure 3, commercially available primary human coronary artery smooth muscle cells were used to demonstrate the techniques. Conditioned media from VSMCs can be used to measure the calcification potential of EVs. Mineral formation over time is measured by the light absorbance at 480 nm using a microplate reader (Figure 2A,B). A linear regression during the rapid phase of mineralization revealed that the absorbance increase occurred 1.5-fold faster in the pro-calcific sample compared to a control sample. Within collagen hydrogels, calcium deposits can be visualized by imaging Osteosense fluorescence (Figure 3A,B). Individual calcifications appear as Osteosense-positive regions within the hydrogel.
Figure 1: Imaged aortas following dissection. After scanning, calcification can be visualized within the aortas of C57BL/6J mice. A higher Osteosense signal is observed throughout the aorta of a mouse with chronic kidney disease (far left) compared to the two aortas from control mice (right). Please click here to view a larger version of this figure.
Figure 2: Mineral absorbance to measure the EV calcification potential. (A,B) Light scattering at 340 nm shows a higher absorbance in the conditioned medium obtained from VSMCs cultured in pro-calcific medium (dark line) compared to the normal control medium (light gray line). (C) A linear regression of the first 20 min of the assay shows faster mineral formation in the pro-calcific sample. Abbreviations: EV = extracellular vesicle; VSMCs = vascular smooth muscle cells. Please click here to view a larger version of this figure.
Figure 3: Mineral formation from conditioned media in collagen hydrogels. Conditioned media from (A) control cells and (B) cells cultured for 21 days in a pro-calcific medium were placed in collagen hydrogels. The imaging of the far-red Osteosense fluorescence shows (A) a minimal signal in the control sample and (B) the presence of minerals that formed from the conditioned medium of cells cultured in pro-calcific conditions. Scale = 5 µm. Please click here to view a larger version of this figure.
Supplemental File 1: A custom MATLAB script to quantify the total signal of the calcium tracer. Please click here to download this File.
When performing the protocol, it is important to note the critical steps for obtaining successful results. During the isolation of the murine aorta, it is vital that the perfusion is performed properly. When injecting the PBS, care must be taken not to puncture the right ventricle. This would cause the liquid to leak directly out of the ventricle and fail to circulate through the lungs, leaving blood within the aorta. Once the perfusion has been conducted properly and microdissection has begun, all the adipose and fatty tissue must be removed from the aorta before completion and scanning. Remaining fat will cause the aorta to appear larger when normalizing the data to the total tissue size.
Due to the limited size of the mouse aorta during dissection, it is common to pierce or cut the aorta in half. If this occurs, it is recommended to continue the dissection as normal. The aorta pieces can be placed close together for imaging. The aorta can also be placed into the digestive solution in pieces for EV isolation. When beginning the isolation of the EVs, multiple aortas are needed to yield sufficient protein. This limitation means more mice must be dissected to collect the appropriate amount of tissue to produce reliable results.
In the demonstrated method, we show how to measure the mineralization potential directly from EVs in conditioned media from VSMCs in culture. The light scattering protocol is a simple method used to measure the mineral formation of EVs in real time14. This provides insight into the period in which calcification occurs within EVs. The collagen hydrogels provide a platform to direct the aggregation and calcification of EVs for the three-dimensional analysis of mineralization15. Thus far, we have only performed these analyses with EVs from in vitro studies; however, in future studies, we will seek to adapt these methods to analyze the mineralization potential of EVs isolated from mouse aortas.
Cardiovascular disease is the leading cause of death in the world, with calcification being the most significant predictor of morbidity and mortality. By identifying the mechanisms through which EVs lead to calcification, future studies can be conducted focusing on controlling mineral growth as a therapeutic strategy by inhibiting the pathways that lead to the release of calcified EVs, as well as by directly interacting with the mineralization process.
The authors have nothing to disclose.
This work was supported by grants from the National Heart, Lung, and Blood Institute of the National Institutes of Health (NIH) (1R01HL160740 and 5 T32GM132054-04) and the Florida Heart Research Foundation. We would like to thank Kassandra Gomez for her help synthesizing and imaging the hydrogels.
8-well chambered coverglass | Thermo Scientific | 155409PK | |
10 mL Syringe | BD | 302995 | |
20 G 1 inch Needle | BD | 305175 | |
Collagen, High Concentraion, Rat Tail | Corning | 354249 | |
Collagenase | Worthington Biochemical | LS004174 | |
Curved Forceps | Roboz Surgical Instrument | RS-8254 | |
Dissection Dish | Living Systems Instrumentation | DD-90-S | |
Dissection Pan and Wax | United Scientific Supplies | DSPA01-W | |
DMEM | Cytiva | SH30022.FS | |
Isoflurane | Sigma-Aldrich | 26675-46-7 | |
LI-COR Odyssey | LI-COR | DLx | |
Micro Dissecting Curved Scissors (24 mm Blade) | Roboz Surgical Instrument | RS-5913 | |
Micro Dissecting Spring Scissors (13 mm Blade) | Roboz Surgical Instrument | RS-5677 | |
Micro Dissecting Spring Scissors (5 mm Blade) | Roboz Surgical Instrument | RS-5600 | |
Micro Dissecting Tweezers (0.10 x 0.06 mm Tip) | Roboz Surgical Instrument | RS-4976 | |
Optima MAX-TL Ultracentrifuge | Beckman Coulter | B11229 | |
OsteoSense 680EX | Perkin Elmer | NEV10020EX | |
Pierce Protease Inhibitor | Thermo Scientific | A32963 | |
Potassium Chloride | Fischer Chemical | P217 | |
RIPA Lysis and Extraction Buffer | G Biosciences | 786-489 | |
Sodium Chloride | Fischer Chemical | BP358 | |
Sodium Hydroxide | Thermo Scientific | A4782602 | |
Sodium phosphate monobasic | Sigma-Aldrich | S0751 | |
Sucrose | Sigma | S7903 | |
Synergy HTX Multimode Reader | Agilent | ||
Tissue culture plate, 96-well | Thermo Fisher | 167008 | |
T-Pins | United Scientific Supplies | TPIN02-PK/100 | |
Tris Hydrochloride | Fischer Chemical | BP153 |