Although angioplasty helps patients with coronary artery disease, cardiovascular events can still occur after the procedure. As MPCs and soluble molecules are obtained from coronary circulation, they can be correlated to the degree of vascular damage and repair. Coronary MPC and soluble mediator levels therefore can be used to estimate cardiovasculary events and prognosis in patients with coronary artery disease undergoing coronary angioplasties.
MPCs and soluble molecules may also be used to estimate complications and prognoses that occur under different ischemic settings, such as recurrent limb ischemiary stroke. Demonstrating the procedure will be Eduardo Vera-Gomez and Alejandro Hernandez-Patricio, lab technicians, and Karen De La Vega-Moreno and Carlos Zamora-Aleman, undergrad students. Also demonstrating will be Gabriela Alexandra Dominguez-Perez, a Master's of Science student, Alberto Melchor-Lopez, a PhD student, and Mario Antonio Tellez-Gonzalez, a Master's of Science collaborator.
Within one hour of collection, transfer six milliliters of the harvested blood sample to a 15 milliliter conical tube and dilute the blood at a one to one ratio in PBS. Add two milliliters of density gradient medium to three test tubes and carefully transfer three equal aliquots of diluted blood into each tube of density gradient medium. Separate the cells by centrifugation and transfer the cells at the interface to a new tube.
Wash the collected cells one time in two milliliters of PBS for six minutes, followed by six washes in two milliliters of fresh PBS per wash for two minutes. After the last wash, re-suspend the MPC-containing cell pellet in one milliliter of PBS for counting and add one times ten to the six cells to labeled five milliliter flow cytometry tubes. Pellet the cells by centrifugation and add 100 microliters of the appropriate antibody of interest, diluted in antibody incubation solution to each tube.
Gently mix the cells with each added antibody cocktail for 10 seconds, and place the samples at four degrees Celsius, protected from light, for 20 minutes. At the end of the incubation, centrifuge the samples and re-suspend the pellets in 500 microliters of PBS supplemented with two millimolar EDTA. Using isotype-matched controls to set up the background staining, analyze the samples by flow cytometry according to standard protocols, gating on the lymphocytes in a forward by side scatter plot, to exclude residual granulocytes, cellular debris, and other particles.
Set a gate to contain a high number of cells with a CD45-positive CD34-positive phenotype. Next, use the CD45-positive CD34-positive gate to select for kinase insert domain receptor CD133 or CD184-positive cells. The MPC sub-populations can then be identified by their specific cell surface markers and reported as the percentage of gated events.
To determine the concentration of soluble biomarkers in the collected blood samples, first, wash the appropriate pre-coated ELISA plates two times with the kit-provided wash buffer and label the standard, sample, and control tubes. Centrifuge the blood sample to separate the blood components and aliquot the plasma into the sample tubes. Transfer the standards, samples, and controls to the appropriate wells and seal and incubate the plate at 37 degrees Celsius for 90 minutes.
At the end of the incubation, discard the well contents and add the biotin detection antibody to each well. After a one hour incubation at 37 degrees Celsius, discard the plate contents and wash the wells three times with fresh wash buffer per wash. After the last wash, incubate the samples with Streptavidin working solution for 30 minutes at 37 degrees Celsius followed by three washes, as demonstrated.
After the last wash, treat the samples with tetramethylbenzidine substrate for 30 minutes at 37 degrees Celsius. When the color develops, add stop solution from the kit and read the optical density absorbance in a microplate ELISA reader. To determine the concentration of tumor necrosis factor alpha, and interleukin-1 beta, using immunomagnetic multiplexing, label the standard, sample, and control tubes and vortex the magnetic bead vials for 30 seconds.
Add beads to the appropriate wells of the multiplexing assay plate. When all of the beads have been added, securely insert the handheld magnetic plate washer, and wait two minutes for the beads to accumulate on the bottom of each well, before quickly inverting both the handheld magnetic plate washer, and the plate assembly over a waste container. Next, add 150 microliters of wash buffer to each well and wait 30 seconds to allow the beads to accumulate on the bottom.
Discard the contents again as just demonstrated and add 25 microliters of universal assay buffer and 25 microliters of the prepared standards, samples, and controls. Seal the plate for an at least 60 minute incubation, protected from light at room temperature, and 500 rotations per minute. At the end of the incubation, add 150 microliters of wash buffer to each well and allow the beads to settle for 30 seconds.
Then, use the plate washer to discard the well contents and label each well with 25 microliters of detection antibody mixture followed by two washes, as demonstrated. After the second wash, incubate the samples with 25 microliters of Streptavidin PE for 30 minutes at room temperature. At the end of the incubation, add 120 microliters of reading buffer to each well and seal the plate for a five minute incubation, protected from light at room temperature, for 500 rotations per minute.
Then, load the plate onto a multiplexing assay reader and adjust the reading parameters according to each analyte. Coronary, venus-sinus, and peripheral blood were collected from 52 patients that underwent coronary angiography and demonstrated a high prevalence of hypertension and dyslipidemia. The baseline coronary concentration of most MPCs was significantly lower in patients who developed major adverse cardiovascular events, with a larger decrease in MPC subpopulations CD34-positive CD133-positive, and CD45-positive, CD34-positive, CD133-positive, CD184-positive.
Likewise, patients who developed major adverse cardiovascular events had an increased baseline in coronary amounts of soluble ICAM-1 and lower amounts of metallomatrix protein 9. Coronary MPCs and soluble ICAM-1 demonstrated a prognostic ability for major adverse cardiovascular event-free survival. Interestingly, the expression of tumor necrosis factor alpha demonstrated variations according to the time of measurement and the location of the coronary sampling based on a comparison of different lumen areas at the same coronary artery, using intravascular ultrasound.
During MPC isolation, be sure to transfer the blood onto the density gradient, and to collect the cells from the interface after centrifugation carefully. Periodic vortexing prevents bead precipitation and using a magnetic plate washer helps to keep the beads inside the wells during washes. Coronary MPCs and soluble molecules are useful for stratifying the risk of cardiovascular events during follow up after angioplasty, assisting in the provision of secondary preventions to high-risk individuals as this technique reflects the pathophysiological process that occurs during the cease of solution, it might be useful for conditions in which vascular biology plays a role.
