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 thro...

<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>

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 ...

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.

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

Research

JoVE Journal

Medicine

Coronary Artery Ligation and Intramyocardial Injection in a Murine Model of Infarction

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo. With the advent of genetic modifications to the whole organism or the myocardium and an array of biological and/or synthetic materials, there is great potential for any combination of these to assuage the extent of acute ischemic injury and impede the onset of heart failure pursuant to myocardial remodeling. 
Here we present the methods and materials used to reliably perform this microsurgery and the modifications involved for temporary (with reperfusion) or permanent coronary artery occlusion studies as well as intramyocardial injections. The effects on the heart that can be seen during the procedure and at the termination of the experiment in addition to histological evaluation will verify efficacy. 
Briefly, surgical preparation involves anesthetizing the mice, removing the fur on the chest, and then disinfecting the surgical area. Intratracheal intubation is achieved by transesophageal illumination using a fiber optic light. The tubing is then connected to a ventilator. An incision made on the chest exposes the pectoral muscles which will be cut to view the ribs. For ischemia/reperfusion studies, a 1 cm piece of PE tubing placed over the heart is used to tie the ligature to so that occlusion/reperfusion can be customized. For intramyocardial injections, a Hamilton syringe with sterile 30gauge beveled needle is used. When the myocardial manipulations are complete, the rib cage, the pectoral muscles, and the skin are closed sequentially. Line block analgesia is effected by 0.25% marcaine in sterile saline which is applied to muscle layer prior to closure of the skin. The mice are given a subcutaneous injection of saline and placed in a warming chamber until they are sternally recumbent. They are then returned to the vivarium and housed under standard conditions until the time of tissue collection. At the time of sacrifice, the mice are anesthetized, the heart is arrested in diastole with KCl or BDM, rinsed with saline, and immersed in fixative. Subsequently, routine procedures for processing, embedding, sectioning, and histological staining are performed. 
Nonsurgical intubation of a mouse and the microsurgical manipulations described make this a technically challenging model to learn and achieve reproducibility. These procedures, combined with the difficulty in performing consistent manipulations of the ligature for timed occlusion(s) and reperfusion or intramyocardial injections, can also affect the survival rate so optimization and consistency are critical.

Numerous genetic manipulations and/or intramyocardial injections of genes, proteins, cells, and/or biomaterials are superimposed upon the dimension of time in studies of acute ischemia/ reperfusion injury and chronic remodeling in mice. This video illustrates the microsurgical procedures for ischemia/reperfusion, permanent coronary artery ligation, and intramyocardial injection studies.

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo.  With the advent of genetic modifications to ...

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 ...

Direct Re-implantation of Left Coronary Artery into the Aorta in Adults with Anomalous Origin of Left Coronary Artery from the Pulmonary Artery (ALCAPA)

Anomalous origin of the left coronary artery from the pulmonary artery (ALCAPA) is a rare congenital anomaly which is one of leading causes of myocardial ischemia and infarction in children. If left untreated, it results in a 90% mortality rate in the first year of life. In patients who survive to the adulthood, the coronary steal phenomenon and retrograde left-sided coronary flow provide a substrate for chronic subendocardial ischemia, which may lead to left ventricular dysfunction, ischemic mitral regurgitation, malignant ventricular arrhythmias, and sudden cardiac death. The average age of life-threatening presentation is 33 years and of sudden cardiac death 31 years. Therefore, surgical correction is highly recommended as soon as the diagnosis is made, regardless of age. In adult-type ALCAPA originating from the right-facing sinus of the pulmonary artery, direct re-implantation of the ALCAPA into the aorta is the more physiologically sound repair technique to re-establish the dual-coronary perfusion system and is recommended. This protocol describes the technique of direct re-implantation of adult-type ALCAPA into the aorta.

Direct Re-implantation of Left Coronary Artery into the Aorta in Adults with Anomalous Origin of Left Coronary Artery from the Pulmonary Artery ALCAPA

Surgical correction of ALCAPA is highly recommended, regardless of age or the degree of intercoronary collateralization. This protocol presents a technique for the direct re-implantation of adult-type ALCAPA into the aorta to re-establish the dual-coronary perfusion. Whenever feasible, direct re-implantation is preferred to other surgical correction techniques.

Anomalous origin of the left coronary artery from the pulmonary artery (ALCAPA) is a rare congenital anomaly which is one of leading causes of myocardial ...

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 ...