Analysis of Extracellular Vesicle-Mediated Vascular Calcification Using In Vitro and In Vivo Models

Summary

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.

Abstract

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.

Introduction

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 overload^2,3. 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 calcification^4,5,6. 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 kidneys^10,11. 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.

Protocol

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 …

Representative Results

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 …

Discussion

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 …

Materials

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