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.
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 …
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 |