This protocol provides a method that can be used to help assess, and in the future, help develop treatments for vascular calcification, which is the most significant predictor of cardiovascular disease. The ability to isolate EVs allows us to further study the mechanisms that lead to vascular calcification, and the phenotypical change of vascular smooth muscle cells. This technique provides insight into early molecular mechanisms of vascular smooth muscle cell-mediated calcification.
Understanding this allows therapeutic options to be studied for the understood mechanism. Any EVs of interest can be studied using this method. It is not exclusive to vascular smooth muscle cell, osteoblasts, or vascular calcification.
After euthanizing the mouse, lift the skin using tweezers, and make an incision down the midline. Remove any excess skin, or fat. Then remove the reproductive organs, bladder, and fat outside the midline.
Then move the gastrointestinal organs to the right-hand side and follow the intestines in the midline. Once found, lift the midline, and excise the gastrointestinal tract up to the stomach. Excise the kidneys by lifting them away from the midline.
Cut as close to the kidney as possible. Remove the liver by lifting the lobes and cutting away from the midline. Then perfuse the heart and aorta by injecting cold PBS into the right ventricle using a 10-milliliter syringe.
Wait for the lungs to inflate, and turn white. Remove the lungs and any excess diaphragm, or ribcage. To remove the bone, open a pair of scissors wide and place them at the lower region of the mouse above the tail.
Cut downward, lift the spine from the skin, and remove the fat from the sides of the midline. Once the skin is detached, excise the spine and intact organs by cutting right above the heart. Place the spine and intact organs in a beaker of PBS in the ice bucket if transporting to a stereoscope.
After moving the spine and organs into the dissecting dish, fill it with ice cold PBS, and pin down the spine and ribcage using needles. Under the dissection microscope, lift the heart carefully using tweezers, and begin cutting above the spine, and underneath the aorta. Continue this until the bottom of the spine is reached.
Remove the spine from the dissecting dish, and pin down the heart and any extraneous fat, or muscle. Starting from the area right below the heart, identify the esophagus, vena cava, and aorta. Remove the esophagus and vena cava to have a clear visual field of the aorta.
Using the micro-dissection forceps and scissors, begin lifting the adipose tissue, and cutting as close to the aorta as possible. Continue this down the medial line until all the aorta and the femoral arteries are exposed. At the heart, remove all the adipose tissue exposing the three branches at the aortic arch.
Remove the aortic root from the left ventricle by inserting the micro-dissection scissors into the left ventricle, and cutting the muscle surrounding the aorta. Once the aorta is isolated and cleaned, image the aorta using a near-infrared scanner to visualize vascular calcification. Using the custom MATLAB script, quantify the total signal of the calcium tracer normalized to the total scanned aorta area.
Direct the MATLAB to the location of the TIF files from the near-infrared scan of the aortas, open the individual files, and extract the pixel intensity values from the TIF files. Select the aorta with the greatest calcification as the scale maximum on the color mapped image. When the command, How many specimens are in the image, appears on the window, input the number of aortas in the current image, and select each aorta one by one.
Once an aorta is selected, MATLAB will create binary images with a mask of the total area and the calcified area of the aorta. The values from these masked images are then used to determine the total calcified area, the total area of the aorta, the percentage area positive for calcification, and the mean intensity of the calcified area. Immediately after scanning the aortas, pool two to three aortas to yield a sufficient protein concentration.
Incubate two to three pooled aortas in 1.5 milliliters of digestive solution for two hours at 37 degrees Celsius. Collect the solution and centrifuge at 1, 000 g for 15 minutes to remove cell debris. Next, to remove the microvesicles, centrifuge the supernatant again for 30 minutes at 33, 000 g.
Collect and assess the supernatant for calcification potential. After removing the cell debris from the conditioned collected medium by centrifugation, spin the collected supernatant at 33, 000 g for 30 minutes. Collect the supernatant and transfer it into a new tube.
Then add 1%of 300 millimolar sodium dihydrogen phosphate to the extracellular vesicle samples, and mix the solution by pipetting. Transfer 200 microliters of the mixture solution to a 96-well plate, and incubate the 96-well plate in the microplate reader at 37 degree Celsius. Set the plate reader to record the absorbance at a wavelength of 340 nanometers every one minute.
Pipette 300 microliters of the collagen solution into each well of an eight-well chamber glass, and incubate at 37 degrees Celsius and 5%carbon dioxide for 72 hours. After the incubation, add 300 microliters of DMEM as a control into the wells. Add 300 microliters of the extracellular vesicle medium samples collected previously to the remaining wells, and incubate for six days, as demonstrated earlier.
On the 10th day, pipette 2.5 microliters of the OsteoSense and DMEM mixture into each well, and incubate for 24 hours. At the end of the incubation, image the hydrogels with filters for OsteoSense fluorescence through the bottom of a coverglass chamber using an inverted microscope, or from the top with a long working distance objective. Assess the number of calcifications and the calcification size and the collected images using the particle analysis options available in ImageJ.
Then navigate to Image, select Adjust, and click Threshold to binarize the images, such that only the OsteoSense signal appears white. Then use the Analyze, and click Analyze Particles command to obtain information for each calcification in the image. Use the same threshold parameters for consistency for each image analyzed.
Near-infrared optical scanner imaging of the dissected aortas showed a higher OsteoSense signal throughout the aorta of a mouse with chronic kidney disease than the two aortas from control mice. The conditioned medium obtained from vascular smooth muscle cells cultured in a pro-calcific medium showed a higher absorbance at 340 nanometers than the control medium. The absorbance increase occurred 1.5 fold faster in the pro-calcific sample compared to a control sample.
The conditioned medium from cells cultured in pro-calcific conditions contained mineral deposits. When micro-dissecting the aorta, it's important to cut the fat surrounding the aorta carefully. You want as much of the aorta as possible to analyze, but this can be quite tedious.
Once EVs are isolated, additional tests, such as an alkaline phosphatase activity assay, or immuno body could be completed. These tests would further show the mechanisms and proteins present in EVs.
