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Calcification of Vascular Smooth Muscle Cells and Imaging of Aortic Calcification and Inflammation
Summary May 31st, 2016
Vascular calcification is an important predictor of and contributor to human cardiovascular disease. This protocol describes methods for inducing calcification of cultured primary vascular smooth muscle cells and for quantifying calcification and macrophage burden in animal aortas using near-infrared fluorescence imaging.
Transcript
The overall goal of this procedure is to visualize aortic vascular calcification ex vivo and to model in vivo vascular calcification using cultured primary murine aortic smooth muscle cells. This method can help answer key questions in the field of vascular disease biology by providing an in vitro model to study the molecular mechanisms of vascular calcification and allowing the assessment of vascular calcification and macrophage inflammation in vivo. The main advantage of this technique is that it allows for the sensitive quantification and co-localization of macrophage activity with calcium deposits in atherosclerotic lesions even at early states of the disease.
Demonstrating this procedure in addition to Caitlin and myself will be Robert Tainsh a technician in our laboratory. To begin this procedure, disinfect the tail with an alcohol swab and locate the tail veins laterally. Next, apply slight forward pressure on the syringe as a 30-gauge needle is advanced into the tail without resistance.
Then inject 100 microliters of calcium NIR and/or 100 microliters of cathepsin NIR at a steady rate. Withdraw the needle after five seconds, and harvest the aortas three to 24 hours after injection. Under a dissecting microscope, remove the peritoneum to reveal the abdominal organs.
Subsequently, remove the gastrointestinal organs taking care not to transect the aorta. After that, make a lateral incision in the anterior diaphragm and continue the incision across the abdomen. Using dissection scissors, release the rib cage by cutting through the sides of the ribs and removing the soft tissue adherent to the superior portion of the sternum.
Then remove the rib cage and reveal the lungs. Carefully, remove the lungs. Next remove the trachea and esophagus with care ensuring the the aorta remains intact.
Subsequently remove the soft tissue surrounding the aorta from the iliac bifurcation to the aortic arch. Be careful when removing the peri-aortic fat. After that, remove the remaining fat and soft tissue surrounding the thoracic aorta and large branches of the aortic arch.
Next, remove the thymus. Now remove the heart from the thoracic cavity by carefully detaching it from the proximal aorta. Then transect the distal aorta at the iliac bifurcation.
Using an insulin needle, inject normal saline into the aorta from the aortic arch to wash out the remaining blood cells. Detach the aorta along with the aortic arch vessels and completely remove it from the body. Place the aorta in normal saline solution on ice until ready for imaging.
In this step, place the aortas in cold HBSS until the dissections are complete. In a sterile tissue culture hood, transfer the aortas to the aortic digestion solution in the 35 X 10 millimeter tissue culture dishes. Then incubate them at 37 degree Celsius for 30 minutes with gentle intermittent rocking.
After digestion, the aortas should exhibit a stretched or frayed appearance. Under a dissection microscope, remove the outer adventitious layer of an aorta while keeping the medial layer intact. To do so, peel away the outer layer of the aorta at one end and remove it from the underlying medial layer like a sock.
Once the adventitious layer has been removed, incubate the remaining aorta in a new culture dish with cell culture media at 37 degree Celsius for two to four hours. Afterward, cut the aorta into one to two millimeter wide rings. Transfer the rings in a new tissue culture dish with aortic digestion solution and incubate at 37 degree Celsius with gentle intermittent rocking for 120 minutes.
During the incubation, pipette the solution up and down several times to resuspend the cells. Afterward, add five milliliters of warm cell culture media to the digestion solution and transfer it to a 15 milliliter conical tube. Centrifuge the tube for five minutes at 200 G.Then aspirate the media and resuspend the cells into the desired volume of cell culture media.
To induce calcification in vascular smooth muscle cells, transfer the cells to the six well culture plate and allow them to adhere. Next refeed the cells in calcification media. Incubate the cells at 37 degrees Celsius for at least seven days.
Afterward, aspirate the media from the cell culture plate. Wash the cells once in the ionized water. Fix the cells by placing them in one milliliter of 10%formalin at room temperature for 20 minutes.
Then remove the formalin and wash the fixed cells with distilled water for five minutes. Next incubate the cells in one milliliter of 5%silver nitrate solution under a 60 to 100 watt bulb for one to two hours. Afterward, aspirate the silver nitrate solution and wash with distilled water for five minutes.
After five minutes, remove the unreacted silver by placing the cells in one milliliter of 5%sodium thiosulphate solution in distilled water for five minutes. Subsequently, rinse the cells with distilled water for five minutes. The cells stained with von Kossa are now ready for imaging with standard inverted light microscopy.
In this figure, wild type MGP knockout mice and LDLR knockout mice were injected with calcium NIR and Cathepsin NIR via tail vein injection and aortas were harvested 24 hours later and assessed with NIR florescence imaging. Wild type mice exhibit virtually no aortic calcification or macrophage presence. The aortas from LDLR knockout mice have extensive calcification that co-localizes with macrophage accumulation.
In contrast, MGP knockout mice have aortic calcification that occurs in the absence of macrophage infiltration. In this experiment, cultured aortic VSMCs were isolated from MGP knockout mice and infected with adenovirus expressing either GFP or MGP. Aortic VSMCs isolated from wild type mice, were transfected with either control scrambled siRNA or siRNA targeting MGP.
Cells were grown in calcification media A for seven days and subsequently fixed and stained with von Kossa. Calcium staining was quantified using ImageJ software after background subtraction. After its development, these techniques have enabled scientists to image atherosclerosis and vascular calcification ex vivo.
And to model vascular calcification in vitro. That's opening up the possibility of testing the effects of novel drug treatments on vascular disease. After watching this video, you should have a good understanding of how to isolate aortas in primary murine aortic vascular smooth muscle cells, visualize atherosclerotic plaques using near infrared fluorescent imaging probes and calcified vascular smooth muscles in vitro.
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