The authors thank the support of Institutional Program E015; and Fondo Sectorial FOSSIS-CONACYT, SALUD-2014-1-233947.

This protocol meets the institutional guidelines from the human research Ethics Committee.
1. Coronary Angiography, Ultrasound, and Blood Sampling
<ol>
	<li>Request baseline clinical and demographic information before coronary intervention. Collect the individual&#39;s data: age, sex, current smoking status, body mass index (BMI), high blood pressure, dyslipidemia, diabetes mellitus, medications, and the indication for current coronary angiography.</li>
	<li>Perform coronary angiography through heart catheterization using a radial approach. This procedure should be performed under a fluoroscopy guide in the hemodynamics room by expert cardiologists. 
	NOTE: Identify evaluable vessels. For the present study, evaluable vessels were defined as arteries with sections larger than 1.5 mm and lumen stenosis of more than 50%.</li>
	<li>Advance the intravascular ultrasound catheter to the region of interest and record images. Use the appropriate software to locate and measure the smallest luminal area.</li>
	<li>Use a coronary catheter to collect 10 mL of blood from the closest location to the plaque.</li>
	<li>After patient discharge, schedule periodical medical evaluations to follow up study endpoints. If telephone contact is not possible or a physician visit is delayed for longer than 2 months, request an authorized person (previously designed) to verify the study endpoints. 
	NOTE: Consider any of the following a MACE: 1) cardiovascular death, 2) new myocardial infarction, 3) unstable angina prompting an unscheduled medical visit within 24 h, 4) stent restenosis as demonstrated by coronary angiography, 5) episodes of decompensated heart failure requiring clinical attention.</li>
</ol>
2. Determination of Circulating MPCs (Figure 2)
<ol>
	<li>Process the blood within 1 h from collection. Transfer 6 mL of the collected blood to a 15 mL conical tube and dilute 1:1 (v/v) with 1x phosphate buffered saline (PBS), pH = 7.4.</li>
	<li>Add 2 mL of density gradient medium to three test tubes. Carefully transfer three equal volume aliquots of diluted blood into each test tube containing the density gradient medium. 
	NOTE: The total volume of density gradient medium and diluted blood should not exceed three-fourths of the test tube maximal capacity.</li>
	<li>Centrifuge at 1,800 x g, 4 &#176;C for 30 min. Transfer the band at the interface between the layers into a new tube. Add 2 mL of PBS and centrifuge at 1,800 x g, 4 &#176;C for 6 min. The pellet will contain the MPCs.</li>
	<li>Wash the pellet several times. Aspirate off the previous solution and gently resuspend the cell pellet in fresh PBS. For subsequent washes, centrifuge at 1,800 x g, 4 &#176;C for 2 min. Repeat the process 6x.</li>
	<li>Resuspend the cell pellet in 1 mL of PBS. Mix 20 &#181;L of the cell suspension with 0.4% trypan blue, diluted 1:1 (v/v). Apply a drop to a hemocytometer and count the unstained cells under a light microscope.</li>
	<li>Proceed to MPCs determination. Label 5 mL flow cytometry tubes and aliquot out 1 x 106 cells per tube. Prepare the corresponding isotype-matched control antibodies. Centrifuge at 1,800 x g, 4 &#176;C for 6 min and discard the supernatant.</li>
	<li>Add the primary antibody diluted in 100 &#181;L of an antibody incubation solution consisting of 1x PBS (pH = 7.4), 2 mM ethylenediaminetetraacetic acid (EDTA), and 0.05% bovine serum albumin (BSA). Resuspend for 10 s and incubate for 20 min at 4 &#176;C, light-protected. The protocol may be paused at this step by fixing the lymphocytes in 4% paraformaldehyde in PBS and storing samples up to 24 h at 4 &#176;C. 
	NOTE: The final concentrations of the primary antibodies used in the present protocol were CD45 1:50, CD34 1:20, KDR 1:50, CD184 1:20, CD133 1:50.</li>
	<li>Centrifuge at 1,800 x g, 4 &#176;C for 2 min and discard the supernatant. Resuspend in 500 &#181;L of 1x PBS (pH = 7.4), 2 mM EDTA.</li>
	<li>Perform flow cytometry analysis. Use isotype-matched control antibodies to set up the background staining. Then, select lymphocytes spread at the FSC/SSC plot, trying to exclude residual granulocytes, cellular debris, and other particles, which are usually located in the lower, left-distributed in the plot. Such distribution is considered as 100%.</li>
	<li>Use a gate containing a high number of cells with common immunophenotype CD45+ and CD34+. For double positive immunophenotypes, use a gate previously identifying CD45+, CD34+, with the addition of either KDR (VEGFR-2)+, CD133+, or CD184+. Identify the MPC subpopulations by their specific cell surface markers. Report as the percentage of gated events.</li>
	<li>Identify the main subpopulations of MPCs. In the present study the main immunophenotypes were CD45+CD34+CD133+, CD45+CD34+CD184+, CD45+CD34+CD133+CD184+, CD45+CD34+KDR+, CD45+CD34+KDR+CD133+, and CD45+CD34+KDR+CD184. 
	NOTE: The cell surface markers used were CD45 (lymphocytes), CD34 (endothelial and/or vascular cells), KDR (VEGFR-2, membrane marker of endothelial cells), CD133 (endothelial progenitor cells), and CD184 (hematopoietic stem cells and endothelial cells).</li>
</ol>
3. Determination of Plasma Soluble Biomarkers
<ol>
	<li>Use an enzyme-linked immunosorbent assay (ELISA) to determine the concentration of SICAM-1 and MMP-9 (Figure 3, upper row).
	<ol>
		<li>Centrifuge the blood samples at 3,000 x g, room temperature for 5 min and collect the plasma.</li>
		<li>Label the standards, sample tubes, and control tubes. Equilibrate the pre-coated wells in the assay plate by washing 2x with the washing buffer provided in the ELISA kit.</li>
		<li>Transfer the standards, samples, and controls to the wells. Seal and incubate at 37 &#176;C for 90 min. 
		NOTE: Do not let the wells dry completely.</li>
		<li>Discard the contents and add the biotin-detection antibody. Seal and incubate at 37 &#176;C for 60 min.</li>
		<li>Discard the contents and wash 3x. Seal the plaque and incubate sequentially with streptavidine working solution followed by tetramethylbenzidine substrate at 37 &#176;C for 30 min, light-protected. Wash 3x between incubations. When the color develops, add the stop solution, and read the optical density absorbance in a microplate ELISA reader.</li>
	</ol>
	</li>
	<li>Use an immuno-magnetic multiplexing assay to determine the concentration of tumor necrosis factor alpha (TNF&#945;) and interleukin 1 beta (IL-1&#946;) (Figure 3, lower row).
	<ol>
		<li>Label the standards, sample tubes, and control tubes.</li>
		<li>Vortex the magnetic beads vials for 30 s. Transfer the bead suspension to appropriately sized tubes, and then to the wells in the multiplexing assay plate. Periodic vortexing avoids precipitation of the beads.</li>
		<li>Securely insert the hand-held magnetic plate washer. Wait 2 min for the beads to accumulate on the bottom of each well and quickly invert both the hand-held magnetic plate washer and plate assembly, over a sink or waste container. Remember to use the hand-held magnetic plate washer to maintain the beads inside the wells.</li>
		<li>Add 150 &#181;L of wash buffer into each well and wait 30 s to allow the beads to accumulate on the bottom. Discard the contents as in step 3.2.3. Then, add 25 &#181;L of universal assay buffer (provided in the kit) followed by 25 &#181;L of prepared standards, samples, and controls.</li>
		<li>Seal the plate and incubate for least 60 min at room temperature, light-protected, with constant shaking at 500 rpm. Alternatively, incubate overnight at 4 &#176;C, light-protected, with constant shaking at 500 rpm if possible.</li>
		<li>Wash 2x by adding 150 &#181;L of wash buffer and wait 30 s. Discard the contents by inserting the hand-held magnetic plate washer. Wait 2 min and invert over a sink or waste container.</li>
		<li>Incubate sequentially with 25 &#181;L of detection antibody mixture followed by 25 &#181;L of streptavidin-PE solution at room temperature for 30 min, sealed and light-protected, with constant shaking at 500 rpm. Wash 2x between incubations, as described in step 3.2.6.</li>
		<li>Obtain the readings. Add 120 &#181;L of reading buffer. Seal the plate and incubate 5 min at room temperature, light-protected, with constant shaking at 500 rpm. Run the reading on a multiplexing assay reader. Adjust the reading parameters according to each analyte.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Cassar, A., Holmes, D. R., Rihal, C. S., Gersh, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+coronary+artery+disease:+diagnosis+and+management.">Chronic coronary artery disease: diagnosis and management.</a> Mayo Clinic Proceedings. 84 (12), 1130-1146 (2009).</li><li>Regueiro, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mobilization+of+endothelial+progenitor+cells+in+acute+cardiovascular+events+in+the+PROCELL+study:+time-course+after+acute+myocardial+infarction+and+stroke.">Mobilization of endothelial progenitor cells in acute cardiovascular events in the PROCELL study: time-course after acute myocardial infarction and stroke.</a> Journal of Molecular and Cellular Cardiology. 80, 146-155 (2015).</li><li>Sen, S., McDonald, S. P., Coates, P. T., Bonder, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+progenitor+cells:+novel+biomarker+and+promising+cell+therapy+for+cardiovascular+disease.">Endothelial progenitor cells: novel biomarker and promising cell therapy for cardiovascular disease.</a> Clinical Science (Lond). 120 (7), 263-283 (2011).</li><li>Samman Tahhan, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progenitor+Cells+and+Clinical+Outcomes+in+Patients+With+Acute+Coronary+Syndromes.">Progenitor Cells and Clinical Outcomes in Patients With Acute Coronary Syndromes.</a> Circulation Research. 122 (11), 1565-1575 (2018).</li><li>Tomuli&#263;, V., Gobi&#263;, D., Luli&#263;, D., &#381;idan, D., Zaputovi&#263;, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soluble+adhesion+molecules+in+patients+with+acute+coronary+syndrome+after+percutaneous+coronary+intervention+with+drug-coated+balloon,+drug-eluting+stent+or+bare+metal+stent.">Soluble adhesion molecules in patients with acute coronary syndrome after percutaneous coronary intervention with drug-coated balloon, drug-eluting stent or bare metal stent.</a> Medical Hypotheses. 95, 20-23 (2016).</li><li>Jaumdally, R., Varma, C., Macfadyen, R. J., Lip, G. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronary+sinus+blood+sampling:+an+insight+into+local+cardiac+pathophysiology+and+treatment?.">Coronary sinus blood sampling: an insight into local cardiac pathophysiology and treatment?.</a> European Heart Journal. 28 (8), 929-940 (2007).</li><li>Kremastinos, D. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracoronary+cyclic-GMP+and+cyclic-AMP+during+percutaneous+transluminal+coronary+angioplasty.">Intracoronary cyclic-GMP and cyclic-AMP during percutaneous transluminal coronary angioplasty.</a> International Journal of Cardiology. 53 (3), 227-232 (1996).</li><li>Karube, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+cytokine+levels+by+coronary+sinus+blood+sampling+during+cardiac+surgery+with+cardiopulmonary+bypass.">Measurement of cytokine levels by coronary sinus blood sampling during cardiac surgery with cardiopulmonary bypass.</a> American Society for Artificial Internal Organs Journal. 42 (5), M787-M791 (1996).</li><li>Truong, Q. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronary+sinus+biomarker+sampling+compared+to+peripheral+venous+blood+for+predicting+outcomes+in+patients+with+severe+heart+failure+undergoing+cardiac+resynchronization+therapy:+the+BIOCRT+study.">Coronary sinus biomarker sampling compared to peripheral venous blood for predicting outcomes in patients with severe heart failure undergoing cardiac resynchronization therapy: the BIOCRT study.</a> Heart Rhythm. 11 (12), 2167-2175 (2014).</li><li>Su&#225;rez-Cuenca, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronary+circulating+mononuclear+progenitor+cells+and+soluble+biomarkers+in+the+cardiovascular+prognosis+after+coronary+angioplasty.">Coronary circulating mononuclear progenitor cells and soluble biomarkers in the cardiovascular prognosis after coronary angioplasty.</a> Journal of Cellular and Molecular Medicine. 23 (7), 4844-4849 (2019).</li><li>Su&#225;rez-Cuenca, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relation+of+Coronary+Artery+Lumen+with+Baseline,+Post-angioplasty+Coronary+Circulating+Pro-Inflammatory+Cytokines+in+Patients+with+Coronary+Artery+Disease.">Relation of Coronary Artery Lumen with Baseline, Post-angioplasty Coronary Circulating Pro-Inflammatory Cytokines in Patients with Coronary Artery Disease.</a> Angiology Open Access. 7, 01 (2019).</li><li>Schmidt-Lucke, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+circulating+endothelial+progenitor+cells+using+the+modified+ISHAGE+protocol.">Quantification of circulating endothelial progenitor cells using the modified ISHAGE protocol.</a> PLoS One. 5 (1), e13790 (2010).</li><li>Moyer, C. F., Sajuthi, D., Tulli, H., Williams, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+IL-1+alpha+and+IL-1+beta+by+arterial+cells+in+atherosclerosis.">Synthesis of IL-1 alpha and IL-1 beta by arterial cells in atherosclerosis.</a> American Journal of Pathology. 138 (4), 951-960 (1991).</li><li>Morales-Portano, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Echocardiographic+measurements+of+epicardial+adipose+tissue+and+comparative+ability+to+predict+adverse+cardiovascular+outcomes+in+patients+with+coronary+artery+disease.">Echocardiographic measurements of epicardial adipose tissue and comparative ability to predict adverse cardiovascular outcomes in patients with coronary artery disease.</a> International Journal of Cardiovascular Imaging. 34 (9), 1429-1437 (2018).</li><li>Huang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+progenitor+cells+correlated+with+oxidative+stress+after+mild+traumatic+brain+injury.">Endothelial progenitor cells correlated with oxidative stress after mild traumatic brain injury.</a> Yonsei Medical Journal. 58 (5), 1012-1017 (2017).</li></ol>

The authors have nothing to disclose.

Blood collection from the affected coronary artery may be difficult. Sometimes, the coronary artery is barely accessible. In this case, sampling from the venous sinus may be an alternative. We performed validation tests comparing circulating biomarkers in coronary artery vs. venous sinus, with no significant differences. However, the performance of circulating biomarkers was validated only for coronary sampling. Therefore, the performance of biomarkers obtained from the venous sinus remains to be explored.
<p class="...

Coronary angioplasty and stenting represent a salvage procedure for patients with coronary artery disease (CAD). However, major adverse cardiovascular events (MACEs), including cardiovascular death, myocardial infarction, coronary restenosis, and episodes of angina or decompensate heart failure, may occur months after coronary intervention, prompting unscheduled visits to the hospital. MACEs are common worldwide and their morbi-mortality is high1.
Coronary ischemic injury induces early vascular response and reparative mechanisms involving mobilization of MPCs due to their differentiation ability and/or angio-reparative potential, as well as the production of soluble molecules like intercellular adhesion molecules (ICAMs), matrix metalloproteinases (MMPs), and reactive oxygen species, reflecting cell adhesion, tissue remodeling, and oxidative stress. Although intrinsic vascular features like plaque characteristics and coronary artery complexity have been used to predict MACEs, some studies have suggested that biomarkers related to the mechanisms of injury and repair occurring in the coronary endothelium could be very useful for the early identification and prognosis of cardiovascular events in patients with CAD submitted to coronary angioplasty2,3,4,5.
Continuous interest in understanding the mechanisms underlying CAD injury and repair has motivated investigators to study intracoronary circulating biomarkers, because coronary sampling more closely reflects vascular damage and repair6. However, characterization of coronary biomarkers in human studies has been scarce7,8,9. Therefore, the purpose of the present study was to describe a method to determine the amount of coronary circulating MPCs and soluble molecules, reflecting both vascular injury and repair, and to show whether these biomarkers are associated with MACEs and the clinical prognosis of CAD patients that underwent coronary angioplasty. This method is based on the use of vascular-related, circulating MPCs and soluble molecules obtained by sampling locations closest to the vessel damage. It may also be useful for clinical studies for lower limb ischemia, stroke, vasculitis, venous thrombosis, and other injuries involving vascular injury and repair.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>BSA</td>
			<td>Roche</td>
			<td>10735086001</td>
			<td>Bovine Serum Albumin (BSA) as a buffering agent, stabilizer, standard and for blending.</td>
		</tr>
		<tr>
			<td>Calibration Beads</td>
			<td>Miltenyi Biotec / MACS</td>
			<td>#130-093-607</td>
			<td>MACQuant calibration beads are supplied in aqueous solution containing 0.05% sodium azide. 3.5 ml for up to 100 tests</td>
		</tr>
		<tr>
			<td>CD133/1 (AC133)-PE</td>
			<td>Milteny Biotec / MACS</td>
			<td>#130-080-801</td>
			<td>Antibody conjugated to R-Phycoerythrin in PBS/EDTA buffer</td>
		</tr>
		<tr>
			<td>CD184 (CXCR4)-PE-VIO770</td>
			<td>Miltenyi Biotec / MACS</td>
			<td>#130-103-798</td>
			<td>Monoclonal, Isotype recombinant human IgG1, conjugated</td>
		</tr>
		<tr>
			<td>CD309 (VEGFR-2/KDR)-APC</td>
			<td>Miltenyi Biotec / MACS</td>
			<td>#130-093-601</td>
			<td>Antibody conjugated to R-Phycoerythrin in PBS/EDTA buffer</td>
		</tr>
		<tr>
			<td>CD34-FITC</td>
			<td>Miltenyi Biotec / MACS</td>
			<td>#130-081-001</td>
			<td>The monoclonal antibody clone AC136 detecs a class III epitope of the CD34</td>
		</tr>
		<tr>
			<td>CD45- VioBlue</td>
			<td>Miltenyi Biotec / MACS</td>
			<td>#130-092-880</td>
			<td>Monoclonal CD45 Antibody, human conjugated</td>
		</tr>
		<tr>
			<td>Conical Tubes</td>
			<td>Thermo SCIENTIFIC</td>
			<td>#339651</td>
			<td>15ml conical centrifuge tubes</td>
		</tr>
		<tr>
			<td>Cytometry Tubes</td>
			<td>FALCON Corning Brand</td>
			<td>#352052</td>
			<td>5 mL Polystyrene Round-Bottom Tube. 12x75 style. Sterile.</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>BIO-RAD</td>
			<td>#161-0729</td>
			<td>Heavy metals, (as Pb) &#60;10ppm, Fe &#60;0.01%, As &#60;1ppm, Insolubles &#60;0.005%</td>
		</tr>
		<tr>
			<td>Improved Neubauer</td>
			<td>Without brand</td>
			<td>Without catalog number</td>
			<td>Hemocytometer for cell counting. (range 0.1000mm, 0.0025mm2)</td>
		</tr>
		<tr>
			<td>K2 EDTA Blood Collection Tubes</td>
			<td>BD Vacutainer</td>
			<td>#367863</td>
			<td>Lilac plastic vacutainer tube (K2E) 10.8mg, 6 mL.</td>
		</tr>
		<tr>
			<td>Lymphoprep</td>
			<td>Stemcell Technologies</td>
			<td>01-63-12-002-A</td>
			<td>Sterile and checked on the presence of endotoxins. Density: 1.077&#177;0.001g/mL</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>SIGMA-ALDRICH</td>
			<td>#SZBF0920V</td>
			<td>Fixation of biological samples, (powder, 95%)</td>
		</tr>
		<tr>
			<td>Pipette Transfer 1,3mL</td>
			<td>CRM Globe</td>
			<td>PF1016, PF1015</td>
			<td>The transfer pipette is a tool that facilitates liquid transfer with greater accuracy.</td>
		</tr>
		<tr>
			<td>Test Tubes</td>
			<td>KIMBLE CHASE</td>
			<td>45060 13100</td>
			<td>Heat-resistant test tubes. SIZE/CAP 13 x 100 mm</td>
		</tr>
	</tbody>
</table>

coronary progenitor cells and soluble biomarkers in cardiovascular prognosis after coronary angioplasty

Major adverse cardiovascular events (MACEs) negatively impact the cardiovascular prognosis of patients undergoing coronary angioplasty due to coronary ischemic injury. The extent of coronary damage and the mechanisms of vascular repair are factors influencing the future development of MACEs. Intrinsic vascular features like the plaque characteristics and coronary artery complexity have demonstrated prognostic information for MACEs. However, the use of intracoronary circulating biomarkers has been postulated as a convenient method for the early identification and prognosis of MACEs, as they more closely reflect dynamic mechanisms involving coronary damage and repair. Determination of coronary circulating biomarkers during angioplasty, such as the number of subpopulations of mononuclear progenitor cells (MPCs) as well as the concentration of soluble molecules reflecting inflammation, cell adhesion, and repair, allows for assessment of future developments and the prognosis of MACEs 6 months post coronary angioplasty. This method is highlighted by its translational nature and better performance than peripheral blood circulating biomarkers regarding prediction of MACEs and its effect on the cardiovascular prognosis, which may be applied for risk stratification of patients with coronary artery disease undergoing angioplasty.

Coronary, venous sinus, and peripheral blood were collected from 52 patients that underwent coronary angiography (Figure 1) and showed a high prevalence of hypertension and dyslipidemia. At the clinical follow-up, 11 (21.1%) MACEs occurred 6 months after coronary angiography: death (n = 1), angina requiring hospital attendance (n = 6), myocardial infarction (n = 2), and/or evidence of heart failure (n = 4).
<p class="jove_content" fo:keep-together.within-...

Watch this Scientific Journal Video about Coronary Progenitor Cells and Soluble Biomarkers in Cardiovascular Prognosis after Coronary Angioplasty at JoVE.com

Coronary Progenitor Cells and Soluble Biomarkers in Cardiovascular Prognosis after Coronary Angioplasty

Development of major adverse cardiovascular events, which impact cardiovascular prognosis after coronary angioplasty, are influenced by the extent of coronary damage and vascular repair. The use of novel coronary cellular and soluble biomarkers, reactive to vascular damage and repair, are useful to predict the development of MACEs and prognosis.

Major adverse cardiovascular events (MACEs) negatively impact the cardiovascular prognosis of patients undergoing coronary angioplasty due to coronary ...

coronary-progenitor-cells-soluble-biomarkers-cardiovascular-prognosis

Research

JoVE Journal

Medicine

5.1K Views.  Experimental Metabolism and Clinical Research Laboratory & Regenerative Medicine and Tissue Engineering Laboratory. Development of major adverse cardiovascular events, which impact cardiovascular prognosis after coronary angioplasty, are influenced by the extent of coronary damage and vascular repair. The use of novel coronary cellular and soluble biomarkers, reactive to vascular damage and repair, are useful to predict the development of MACEs and prognosis.

Video: Coronary Progenitor Cells and Soluble Biomarkers in Cardiovascular Prognosis after Coronary Angioplasty

Modified Technique for Coronary Artery Ligation in Mice

Myocardial infarction (MI) is one of the most important causes of mortality in humans1-3. In order to improve morbidity and mortality in patients with MI we need better knowledge about pathophysiology of myocardial ischemia. This knowledge may be valuable to define new therapeutic targets for innovative cardiovascular therapies4. Experimental MI model in mice is an increasingly popular small-animal model in preclinical research in which MI is induced by means of permanent or temporary ligation of left coronary artery (LCA)5. In this video, we describe the step-by-step method of how to induce experimental MI in mice.
The animal is first anesthetized with 2% isoflurane. The unconscious mouse is then intubated and connected to a ventilator for artificial ventilation. The left chest is shaved and 1.5 cm incision along mid-axillary line is made in the skin. The left pectoralis major muscle is bluntly dissociated until the ribs are exposed. The muscle layers are pulled aside and fixed with an eyelid-retractor. After these preparations, left thoracotomy is performed between the third and fourth ribs in order to visualize the anterior surface of the heart and left lung. The proximal segment of LCA artery is then ligated with a 7-0 ethilon suture which typically induces an infarct size ~40% of left ventricle. At the end, the chest is closed and the animals receive postoperative analgesia (Temgesic, 0.3 mg/50 ml, ip). The animals are kept in a warm cage until spontaneous recovery.

The surgical procedure used to induce experimental myocardial infarction in mice begins with left thoracotomy between the third and the fourth ribs in order to visualize the anterior surface of the heart and left lung. The left coronary artery is ligated, the chest is closed and the mouse is allowed to recover spontaneously.

Myocardial infarction (MI) is one of the most important causes of mortality in humans1-3. In order to improve morbidity and mortality in patients with MI ...

Retrograde Perfusion and Filling of Mouse Coronary Vasculature as Preparation for Micro Computed Tomography Imaging

Visualization of the vasculature is becoming increasingly important for understanding many different disease states. While several techniques exist for imaging vasculature, few are able to visualize the vascular network as a whole while extending to a resolution that includes the smaller vessels1,2. Additionally, many vascular casting techniques destroy the surrounding tissue, preventing further analysis of the sample3-5. One method which circumvents these issues is micro-Computed Tomography (&mu;CT). &mu;CT imaging can scan at resolutions &lt;10 microns, is capable of producing 3D reconstructions of the vascular network, and leaves the tissue intact for subsequent analysis (e.g., histology and morphometry)6-11. However, imaging vessels by ex vivo &mu;CT methods requires that the vessels be filled with a radiopaque compound. As such, the accurate representation of vasculature produced by &mu;CT imaging is contingent upon reliable and complete filling of the vessels. In this protocol, we describe a technique for filling mouse coronary vessels in preparation for &mu;CT imaging.
Two predominate techniques exist for filling the coronary vasculature: in vivo via cannulation and retrograde perfusion of the aorta (or a branch off the aortic arch) 12-14, or ex vivo via a Langendorff perfusion system 15-17. Here we describe an in vivo aortic cannulation method which has been specifically designed to ensure filling of all vessels. We use a low viscosity radiopaque compound called Microfil which can perfuse through the smallest vessels to fill all the capillaries, as well as both the arterial and venous sides of the vascular network. Vessels are perfused with buffer using a pressurized perfusion system, and then filled with Microfil. To ensure that Microfil fills the small higher resistance vessels, we ligate the large branches emanating from the aorta, which diverts the Microfil into the coronaries. Once filling is complete, to prevent the elastic nature of cardiac tissue from squeezing Microfil out of some vessels, we ligate accessible major vascular exit points immediately after filling. Therefore, our technique is optimized for complete filling and maximum retention of the filling agent, enabling visualization of the complete coronary vascular network &#x2013; arteries, capillaries, and veins alike.

Visualization of the coronary vessels is critical to advancing our understanding of cardiovascular diseases. Here we describe a method for perfusing murine coronary vasculature with a radiopaque silicone rubber (Microfil), in preparation for micro-Computed Tomography (&mu;CT) imaging.

Visualization of the vasculature is becoming increasingly important for understanding many different disease states. While several techniques exist for ...

Permanent Ligation of the Left Anterior Descending Coronary Artery in Mice: A Model of Post-myocardial Infarction Remodelling and Heart Failure

Heart failure is a syndrome in which the heart fails to pump blood at a rate commensurate with cellular oxygen requirements at rest or during stress. It is characterized by fluid retention, shortness of breath, and fatigue, in particular on exertion. Heart failure is a growing public health problem, the leading cause of hospitalization, and a major cause of mortality. Ischemic heart disease is the main cause of heart failure.
Ventricular remodelling refers to changes in structure, size, and shape of the left ventricle. This architectural remodelling of the left ventricle is induced by injury (e.g., myocardial infarction), by pressure overload (e.g., systemic arterial hypertension or aortic stenosis), or by volume overload. Since ventricular remodelling affects wall stress, it has a profound impact on cardiac function and on the development of heart failure. A model of permanent ligation of the left anterior descending coronary artery in mice is used to investigate ventricular remodelling and cardiac function post-myocardial infarction. This model is fundamentally different in terms of objectives and pathophysiological relevance compared to the model of transient ligation of the left anterior descending coronary artery. In this latter model of ischemia/reperfusion injury, the initial extent of the infarct may be modulated by factors that affect myocardial salvage following reperfusion. In contrast, the infarct area at 24 hr after permanent ligation of the left anterior descending coronary artery is fixed. Cardiac function in this model will be affected by 1) the process of infarct expansion, infarct healing, and scar formation; and 2) the concomitant development of left ventricular dilatation, cardiac hypertrophy, and ventricular remodelling.
Besides the model of permanent ligation of the left anterior descending coronary artery, the technique of invasive hemodynamic measurements in mice is presented in detail.

Heart failure is the leading cause of hospitalization and a major cause of mortality. A model of permanent ligation of the left anterior descending coronary artery in mice is applied to investigate ventricular remodelling and cardiac dysfunction post-myocardial infarction. The technique of invasive hemodynamic measurements in mice is presented.

Heart failure is a syndrome in which the heart fails to pump blood at a rate commensurate with cellular oxygen requirements at rest or during stress. It ...

Ultrasound Based Assessment of Coronary Artery Flow and Coronary Flow Reserve Using the Pressure Overload Model in Mice

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow reserve (CFR) in humans. Reduced CFR is accompanied by marked intramyocardial and pericoronary fibrosis and is used as an indication of the severity of dysfunction. This study explores, step-by-step, the real-time changes measured in the coronary flow velocity, CFR and systolic to diastolic peak velocity (S/D) ratio in the setting of an aortic banding model in mice. By using a Doppler transthoracic imaging technique that yields reproducible and reliable data, the method assesses changes in flow in the septal coronary artery (SCA), for a period of over two weeks in mice, that previously either underwent aortic banding or thoracotomy. 
During imaging, hyperemia in all mice was induced by isoflurane, an anesthetic that increased coronary flow velocity when compared with resting flow. All images were acquired by a single imager. Two ratios, (1) CFR, the ratio between hyperemic and baseline flow velocities, and (2) systolic (S) to diastolic (D) flow were determined, using a proprietary software and by two independent observers. Importantly, the observed changes in coronary flow preceded LV dysfunction as evidenced by normal LV mass and fractional shortening (FS). 
The method was benchmarked against the current gold standard of coronary assessment, histopathology. The latter technique showed clear pathologic changes in the coronary artery in the form of peri-coronary fibrosis that correlated to the flow changes as assessed by echocardiography. 
The study underscores the value of using a non-invasive technique to monitor coronary circulation in mouse hearts. The method minimizes redundant use of research animals and demonstrates that advanced ultrasound-based indices, such as CFR and S/D ratios, can serve as viable diagnostic tools in a variety of investigational protocols including drug studies and the study of genetically modified strains.

Coronary flow reserve (CFR) is useful for assessment of myocardial oxygen demand and evaluation of cardiovascular risk. This study establishes a step-by-step transthoracic Doppler echocardiographic (TTDE) method for longitudinal monitoring of the changes in CFR, as measured from coronary artery in mice, under the experimental pressure overload of aortic banding.

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow ...

Identifying Coronary Artery Calcification on Non-gated Computed Tomography Scans

Coronary artery calcification (CAC) provides an objective measure of coronary artery disease and can readily be identified on non-gated computed tomography (CT) scans with a high correlation with gated cardiac CT scans. This standardized protocol takes a step-wise approach to not only optimizing an image for the identification of calcification but also to distinguishing CAC from other common causes of calcification in the cardiac silhouette. Recognition of CAC on non-gated CT scans helps to identify a very powerful prognostic factor that can influence therapeutic interventions or downstream diagnostic testing without requiring a gated cardiac scan. These non-gated CT scans are often acquired as part of the routine care of the patient, and this data is readily available without another dose of&#160;ionizing radiation. This protocol allows for the precise and accurate extraction of this data for the purposes of retrospective data analysis in clinical research studies, but also in the clinical evaluation and management of patients.

Here, we present a protocol to reliably and systematically identify coronary artery calcification (CAC) on non-gated computed tomography (CT) scans of the chest or abdomen. CAC provides an objective measure of coronary artery disease for both research and clinical purposes.

Coronary artery calcification (CAC) provides an objective measure of coronary artery disease and can readily be identified on non-gated computed ...

Evaluation of Coronary Flow Reserve After Myocardial Ischemia Reperfusion in Rats

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via recanalization of the coronary artery is the most effective strategy for reducing the size of ischemic myocardium. The coronary microvasculature cannot be visualized and imaged&#160;in vivo, but there are several invasive and noninvasive techniques that can be used to assess parameters which depend directly on coronary microvascular function. The endothelial function after ischemia reperfusion can be assessed also at the level of the coronary circulation via the coronary flow reserve (CFR).&#160;In this study, peak velocity of left anterior descending (LAD) coronary arteries was measured in rats in vivo via Transthoracic Doppler Echocardiography during resting and stress challenge (induced by Dobutamine). A normal heart can increase its coronary blood flow up to four times above the resting values during stress induction. Following ischemia reperfusion, we found a significantly diminished CFR, which can be used as a marker of coronary microvascular dysfunction. CFR has opened a window on the importance of microvascular dysfunction and has been shown to predict cardiovascular risk independent of whether the severe obstructive disease is present.

Coronary flow reserve (CFR), is defined as the ratio of maximal coronary blood flow to the resting coronary blood flow. We present a protocol for evaluating CFR in rats via ultrasound, which offers the opportunity to predict cardiovascular risk factors in the absence of obstructive coronary disease.

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via ...

In Vitro Model of Coronary Angiogenesis

Here, we describe an in vitro culture assay to study coronary angiogenesis. Coronary vessels feed the heart muscle and are of clinical importance. Defects in these vessels represent severe health risks such as in atherosclerosis, which can lead to myocardial infarctions and heart failures in patients. Consequently, coronary artery disease is one of the leading causes of death worldwide. Despite its clinical importance, relatively little progress has been made on how to regenerate damaged coronary arteries. Nevertheless, recent progress has been made in understanding the cellular origin and differentiation pathways of coronary vessel development. The advent of tools and technologies that allow researchers to fluorescently label progenitor cells, follow their fate, and visualize progenies in vivo have been instrumental in understanding coronary vessel development. In vivo studies are valuable, but have limitations in terms of speed, accessibility, and flexibility in experimental design. Alternatively, accurate in vitro models of coronary angiogenesis can circumvent these limitations and allow researchers to interrogate important biological questions with speed and flexibility. The lack of appropriate in vitro model systems may have hindered the progress in understanding the cellular and molecular mechanisms of coronary vessel growth. Here, we describe an in vitro culture system to grow coronary vessels from the sinus venosus (SV) and endocardium (Endo), the two progenitor tissues from which many of the coronary vessels arise. We also confirmed that the cultures accurately recapitulate some of the known in vivo mechanisms. For instance, we show that the angiogenic sprouts in culture from SV downregulate COUP-TFII expression similar to what is observed in vivo. In addition, we show that VEGF-A, a well-known angiogenic factor in vivo, robustly stimulates angiogenesis from both the SV and Endo cultures. Collectively, we have devised an accurate in vitro culture model to study coronary angiogenesis.

In vitro models of coronary angiogenesis can be utilized for the discovery of the cellular and molecular mechanisms of coronary angiogenesis. In vitro explant cultures of sinus venosus and endocardium tissues show robust growth in response to VEGF-A and display a similar pattern of COUP-TFII expression as in vivo.

Here, we describe an in vitro culture assay to study coronary angiogenesis. Coronary vessels feed the heart muscle and are of clinical importance. Defects ...

Novel Percutaneous Approach for Deployment of 3D Printed Coronary Stenosis Implants in Swine Models of Ischemic Heart Disease

Minimally invasive methods for creating models of focal coronary narrowing in large animals are challenging. Rapid prototyping using three-dimensionally (3D) printed coronary implants can be employed to percutaneously create a focal coronary stenosis. However, reliable delivery of the implants can be difficult without the use of ancillary equipment. We describe the use of a mother-and-child coronary guide catheter for stabilization of the implant and for effective delivery of the 3D printed implant to any desired location along the length of the coronary vessel. The focal coronary narrowing was confirmed under coronary cineangiography and the functional significance of the coronary stenosis was assessed using gadolinium-enhanced first-pass cardiac perfusion MRI. We showed that reliable delivery of 3D printed coronary implants in swine models (n = 11) of ischemic heart disease can be achieved through repurposing mother-and-child coronary guide catheters. Our technique simplifies the percutaneous delivery of coronary implants to create closed-chest swine models of focal coronary artery stenosis and can be performed expeditiously, with a low procedural failure rate.

We describe a novel, cost-effective, and efficient technique for percutaneous delivery of three-dimensionally printed coronary implants to create closed-chest swine models of ischemic heart disease. The implants were fixed in place using a mother-and-child extension catheter with high success rate.

Minimally invasive methods for creating models of focal coronary narrowing in large animals are challenging. Rapid prototyping using three-dimensionally ...

Predicting Amputation using Local Circulating Mononuclear Progenitor Cells in Angioplasty-treated Patients with Critical Limb Ischemia

Critical limb ischemia (CLI) represents an advanced stage of the peripheral arterial disease. Angioplasty improves the blood flow to the lower limb; however, some patients irreversibly progress to limb amputation. The extent of vascular damage and the mechanisms of vascular repair are factors affecting post-angioplasty outcome. Mononuclear Progenitor Cells (MPCs) are reactive to vascular damage and repair, with the ability to reflect vascular diseases. The present protocol describes quantification of MPCs obtained from blood circulation from vessel close to the angioplasty site, as well as its relationship with endothelial dysfunction and its predictive ability for limb amputation in the next 30 days after angioplasty in patients with CLI.

Lower limb amputation may occur even after angioplasty of obstructed vessels in Critical Limb Ischemia (CLI). Mononuclear Progenitor Cells (MPCs) reflect vascular repair. The present protocol describes the quantification of MPCs from circulation close to angioplasty, and its relationship with endothelial dysfunction and prediction of lower limb amputation.

Critical limb ischemia (CLI) represents an advanced stage of the peripheral arterial disease. Angioplasty improves the blood flow to the lower limb; ...

Interventional Diagnostic Procedure: A Practical Guide for the Assessment of Coronary Vascular Function

Approximately 40% of patients undergoing invasive coronary angiography for investigation of angina are found to have no obstructive coronary artery disease (ANOCA). Abnormal coronary function underlies coronary vasomotion syndromes including coronary endothelial dysfunction, microvascular angina, vasospastic angina, post-PCI angina and myocardial infarction with no obstructive coronary arteries (MINOCA). Each of these endotypes are distinct subgroups, characterized by specific disease mechanisms. Diagnostic criteria and linked therapy for these conditions are now established by expert consensus and clinical guidelines.
Coronary function tests are performed as an adjunctive interventional diagnostic procedure (IDP) in appropriately selected patients during coronary angiography. This aids differentiation of patients according to endotype. The IDP includes two distinct components: a diagnostic guidewire test and a pharmacological coronary reactivity test. The tests last approximately 5 minutes for the former and 10-15 minutes for the latter. Patient safety and staff education are key.
The diagnostic guidewire test measures parameters of coronary flow limitation (fractional flow reserve [FFR], coronary flow reserve [CFR], microvascular resistance [index of microvascular resistance (IMR)], basal resistance index, and vasodilator function [CFR, resistive reserve ratio (RRR)]).
The pharmacological coronary reactivity test measures the vasodilator potential and propensity to vasospasm of both the main coronary arteries and the micro-vessels. It involves intra-coronary infusion of acetylcholine and glyceryl trinitrate (GTN). Acetylcholine is not licensed for parenteral use and is therefore prescribed on a named-patient basis. Vasodilatation is the normal, expected response to infusion of physiological concentrations of acetylcholine. Vascular spasm represents an abnormal response, which supports the diagnosis of vasospastic angina.
The purpose of this practical guide is to provide information on the preparation and administration of the IDP in clinical practice. It discusses some key preparation and safety considerations, as well as tips for procedural success. The IDP supports stratified medicine for a personalized approach to health and wellbeing.

The purpose of this practical guide is to provide information on the preparation and administration of an interventional diagnostic procedure in clinical practice. It discusses some key preparation and safety considerations, as well as tips for procedural success.

Approximately 40% of patients undergoing invasive coronary angiography for investigation of angina are found to have no obstructive coronary artery ...