Name: Author Spotlight: Enhancing Coronary Artery Revascularization
Uploaded: 2023-09-15T13:40:00
Description: Watch this Scientific Journal Video about Surgical Porcine Model of Chronic Myocardial Ischemia Treated by Exosome-laden Collagen Patch and Off-pump Coronary Artery Bypass Graft at JoVE.com

This work was supported by the VA Merit Review #I01 BX000760 (RFK) from the United States (U.S.) Department of Veterans Affairs BLR&#38;D and U.S. Department of Veterans Affairs grant #I01 BX004146 (TAB). We also gratefully acknowledge the support of the University of Minnesota Lillehei Heart Institute. The contents of this work do not represent the views of the U.S. Department of Veterans Affairs of the United States Government.

The Institutional Animal Care and Use Committees (IACUC) of the Minneapolis VA Medical Center and the University of Minnesota have approved all of the animal studies. The current National Institutes of Health (NIH) guidelines for the use and care of laboratory animals were followed.
1. Isolation of mesenchymal stem cells and preparation and characterization of exosomes
<ol>
	<li>Isolation of bone marrow derived mesenchymal stem cells (MSCs)
	<ol>
		<li>Obtain 30-50 mL of ste...

Adjunctive Exosome Therapy: Enhancing Myocardial Recovery Post-CABG Surgery

<ol>
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J., 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=Prognostic+Value+and+Interplay+Between+Myocardial+Tissue+Velocities+in+Patients+Undergoing+Coronary+Artery+Bypass+Grafting.">Prognostic Value and Interplay Between Myocardial Tissue Velocities in Patients Undergoing Coronary Artery Bypass Grafting.</a> The American Journal of Cardiology. 144, 37-45 (2021).</li><li>Virani, S. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+and+their+potential+as+cardiac+therapeutics.">Mesenchymal stem cells and their potential as cardiac therapeutics.</a> Circulation Research. 95 (1), 9-20 (2004).</li><li>Campos-Silva, 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=High+sensitivity+detection+of+extracellular+vesicles+immune-captured+from+urine+by+conventional+flow+cytometry.">High sensitivity detection of extracellular vesicles immune-captured from urine by conventional flow cytometry.</a> Scientific Reports. 9 (1), 2042 (2019).</li><li>Hocum Stone, L. 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This study presents the first porcine model of chronically ischemic myocardium, in which it was shown that treatment with an MSC-derived exosome laden collagen patch during surgical revascularization recovers diastolic and systolic function upon inotropic stimulation potentially by targeting mitochondrial recovery. Previously, it was demonstrated that in a large animal model of HIB the diastolic and systolic function, as measured by cardiac MRI, remains impaired and only slightly improves with revascularization without c...

Globally, severe CAD affects over a hundred million patients, and although the mortality rate has decreased, it remains one of the leading causes of death1,2. CAD has a wide clinical spectrum from myocardial infarction (MI) to ischemia with preserved viability. Most pre-clinical research focuses on MI, characterized by the presence of infarcted tissue as it is possible to study in small and large animal models. However, that model does not address patients with preserved viability and amenable to revascularization. Most patients undergoing CABG have decreased blood supply and limited function while maintaining variability in contractile reserve and viability3. Without treatment, these patients can progress to advanced heart failure and sudden death, especially during increased workload4. Among these patients, coronary artery bypass graft (CABG) is an effective therapy but may not result in complete functional recovery5. Importantly, diastolic dysfunction, which is a marker for worse clinical outcomes, fails to recover after revascularization suggesting the need for novel adjuvant therapies during CABG6,7. Currently, there are no clinically available adjuvant interventions used with CABG to restore cardiomyocytes to full functional capacity. This is a major therapeutic gap given that many patients progress to advanced heart failure despite appropriate revascularization8.
An innovative porcine model of chronic myocardial ischemia that is amenable to CABG, to mimic clinical CAD experience was created9. Swine provide a good model of heart disease over other large animals as they do not have epicardial bridging collaterals so stenosis of the LAD alone results in regional ischemia10. In this study, 16-week-old female Yorkshire-Landrace pigs were used. In this model, the LAD was revascularized with off-pump CABG using the left internal mammary artery (LIMA) graft (Supplementary Table 1). Percutaneous coronary intervention (PCI) is not possible to open the stenosis as the constrictor is a rigid device. Cardiac magnetic resonance imaging (MRI) is used to assess global and regional function, coronary anatomy, and tissue viability. Cardiac MRI analysis showed diastolic function, characterized by peak filling rate (PFR) remains impaired despite CABG6. The mechanism of diastolic dysfunction likely relates to impaired mitochondrial bioenergetics and collagen formation in HIB that persist following CABG11.
Mesenchymal stem cells (MSC) provide therapeutic signaling through exosomes to improve myocardial recovery when applied during CABG. In this swine model and parallel in vitro studies, it was shown that placement of an epicardial MSC vicryl patch during CABG recovers contractile function with increase in key mitochondrial proteins namely PGC-1&#945;12, an important regulator of mitochondrial energy metabolism13. The in vitro model allowed us to investigate the signaling mechanism of MSCs on impaired mitochondrial function. Exosomes are secreted stable microvesicles (50-150 nm) that contain protein or nucleic acids including microRNA (miRNA)14. Recent in vitro data suggest that MSC-derived exosomes are an important signaling mechanism necessary for recovery of mitochondrial respiration.
Stem cell derived exosomes are promising adjunctive therapeutics as they are readily accessible, can be commercially produced, and lack ethical conflicts. In consideration of clinical translation, a collagen patch embedded with MSC-derived exosomes was created that can be surgically sutured to the hibernating region of myocardium. It was demonstrated that there is sustained delivery of exosomes using this patch and it provides a cell-free regenerative therapy with paracrine signaling mechanism that targets mitochondrial recovery and enhance mitochondrial biogenesis15. This procedure provides the pre-clinical model to study the impact of MSC-derived therapies to improve cardiac function by means of enhancing mitochondrial function and reducing inflammation at the time of revascularization and reverse the myocyte adaptations to chronic ischemia.
In this study, a surgical method of off-pump CABG using LIMA to LAD anastomosis to bypass the area of proximal LAD stenosis mimicking the standard treatment for patients with CAD is shown. As an adjunctive therapy with CABG, the surgical application of MSC-derived exosome embedded collagen patch on the ischemic region of the myocardium was demonstrated. This surgical model can be used to study the physiologic responses to the paracrine effect seen with use of an exosome patch as well as the molecular mechanisms of recovery.

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			<td>5 Ethibond</td>
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			<td>0 Vicryl</td>
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			<td>J208H</td>
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			<td>1 mL Syringe</td>
			<td>Medtronic/Covidien</td>
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			<td>VCP824G</td>
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			<td>Smith&#8217;s Medical</td>
			<td>MX563</td>
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			<td>ThermoFisher Scientific</td>
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			<td>Bupivicaine</td>
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			<td>Buprenorphine 0.3 mg/mL</td>
			<td>Sigma Aldrich</td>
			<td>B9275</td>
			<td>Pre operative Analgesic for survivial procedures</td>
		</tr>
		<tr>
			<td>Cell Scrapers</td>
			<td>Corning</td>
			<td>353085</td>
			<td></td>
		</tr>
		<tr>
			<td>Cephazolin 1 gr</td>
			<td>Pfizer</td>
			<td>00409-0805-01</td>
			<td>Antibiotic</td>
		</tr>
		<tr>
			<td>Chest Tube</td>
			<td>Covidien</td>
			<td>8888561043</td>
			<td>Evacuates air from chest cavity</td>
		</tr>
		<tr>
			<td>Cloroprep</td>
			<td>Becton Dickenson</td>
			<td>260815</td>
			<td>Surgical skin prep</td>
		</tr>
		<tr>
			<td>Corning bottle-top vacuum Filter System (500mL)</td>
			<td>Millipore Sigma</td>
			<td>430758</td>
			<td></td>
		</tr>
		<tr>
			<td>CPT tube</td>
			<td>BD</td>
			<td>362753</td>
			<td>MSC isolation from bone marrow</td>
		</tr>
		<tr>
			<td>Delrin Constrictor</td>
			<td>U of MN</td>
			<td>Custom made</td>
			<td>Creates stenosis of LAD</td>
		</tr>
		<tr>
			<td>Dermabond</td>
			<td>Ethicon</td>
			<td>DNX12</td>
			<td>Skin adhesive</td>
		</tr>
		<tr>
			<td>DMEM (1X) Dulbecco&#39;s Modified Eagle Medium, HEPES</td>
			<td>ThermoFisher Scientific</td>
			<td>12430062</td>
			<td></td>
		</tr>
		<tr>
			<td>Dobutamine 12.5 mg/mL</td>
			<td>Pfizer</td>
			<td>00409-2344-01</td>
			<td>Increases blood pressure and heart rate during the second microsphere blood collection</td>
		</tr>
		<tr>
			<td>ECG Pads</td>
			<td>DRE</td>
			<td>1496</td>
			<td>Monitor heart rhythm</td>
		</tr>
		<tr>
			<td>Exosome-Depleted FBS</td>
			<td>ThermoFisher Scientific</td>
			<td>A2720801</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon Disposable Polystyrene Serological Pipets, Sterile, 10mL</td>
			<td>Fisher Scientific</td>
			<td>13-675-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Femoral and carotid introducer</td>
			<td>Cordis- J&#38;J</td>
			<td>504606P</td>
			<td>femoral and carotis cannulas</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, Heat Inactivated, Gibco FBS</td>
			<td>ThermoFisher Scientific</td>
			<td>16140089</td>
			<td></td>
		</tr>
		<tr>
			<td>Flo-thru 1.0</td>
			<td>Baxter</td>
			<td>FT-12100</td>
			<td>used to anastomos LIMA to L</td>
		</tr>
		<tr>
			<td>Flo-thru 1.25</td>
			<td>Baxter</td>
			<td>FT-12125</td>
			<td>FT-12125</td>
		</tr>
		<tr>
			<td>Flo-thru 1.5</td>
			<td>Baxter</td>
			<td>FT-12150</td>
			<td>FT-12150</td>
		</tr>
		<tr>
			<td>Flo-thru 2.0</td>
			<td>Baxter</td>
			<td>FT-12200</td>
			<td>FT-12200</td>
		</tr>
		<tr>
			<td>GlutaMAX Supplement</td>
			<td>ThermoFisher Scientific</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>Hair Clipper</td>
			<td>Oster</td>
			<td>078566-011-002</td>
			<td>Remove hair from surgery sites</td>
		</tr>
		<tr>
			<td>Helistat collagen sponge</td>
			<td>McKesson</td>
			<td>570973 1690ZZ</td>
			<td>Sponge for embedding exosomes</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Pfizer</td>
			<td>&#160;0409-2720-03</td>
			<td>anticoaggulant</td>
		</tr>
		<tr>
			<td>Histology Jars</td>
			<td>Fisher Scientific</td>
			<td>316-154</td>
			<td>Formalin for tissue samples</td>
		</tr>
		<tr>
			<td>HyClone Characterized Fetal Bovine Serum (FBS)</td>
			<td>Cytiva</td>
			<td>SH30071.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Hypafix</td>
			<td>BSN Medical</td>
			<td>4210</td>
			<td>Secure wound dressing and IV catheters</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Sigma Aldrich</td>
			<td>CDS019936</td>
			<td>General Anesthestic- Inhalant</td>
		</tr>
		<tr>
			<td>IV Tubing for Blower Mister</td>
			<td>Carefusion</td>
			<td>42493E</td>
			<td>Adapts to IV Fluids for Blower/Mister</td>
		</tr>
		<tr>
			<td>Jelco 18 ga IV catheter</td>
			<td>Smiths medical</td>
			<td>4054</td>
			<td>IV access in Revasc, MRI and Term</td>
		</tr>
		<tr>
			<td>Lidocaine 2%</td>
			<td>Pfizer</td>
			<td>00409-4277-01</td>
			<td>Local Anesthetic/ antiarrthymic</td>
		</tr>
		<tr>
			<td>Ligaclips</td>
			<td>Ethicon</td>
			<td>MSC20</td>
			<td>Surgical Staples for LIMA takedown</td>
		</tr>
		<tr>
			<td>Long blade for laryngoscope</td>
			<td>DRE</td>
			<td>12521</td>
			<td>Allows for visualization of trachea for intubation</td>
		</tr>
		<tr>
			<td>Meloxicam 5 mg/mL</td>
			<td>Boehringer Ingelheim</td>
			<td>141-219</td>
			<td>Post operative Analgesic</td>
		</tr>
		<tr>
			<td>Microsphere pump</td>
			<td></td>
			<td></td>
			<td>Collect blood samples from femoral introducer</td>
		</tr>
		<tr>
			<td>Monopolar Cautery</td>
			<td>Covidien</td>
			<td>Valleylab&#8482; FT10</td>
			<td>Hemostasis</td>
		</tr>
		<tr>
			<td>Nanosight NS 300</td>
			<td>Malvern Panalytical</td>
			<td>MAN0541-03-EN</td>
			<td></td>
		</tr>
		<tr>
			<td>NTA 3.1.54 software</td>
			<td>Malvern Panalytical</td>
			<td>MAN0520-01-EN-00</td>
			<td></td>
		</tr>
		<tr>
			<td>OPVAC Synergy II</td>
			<td>Terumo Cardiovascular System</td>
			<td>401-230</td>
			<td>Heart positioner and Stabilizer</td>
		</tr>
		<tr>
			<td>Oxygen Tank E cylinder</td>
			<td>various</td>
			<td>various</td>
			<td>Used for Blower Mister if anesthesia machine doesn&#39;t have auxiliary flow meter</td>
		</tr>
		<tr>
			<td>PBS, pH 7.2</td>
			<td>ThermoFisher Scientific</td>
			<td>20012050</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin-Neomycin (PSN) Antibiotic Mixture</td>
			<td>ThermoFisher Scientific</td>
			<td>15640055</td>
			<td></td>
		</tr>
		<tr>
			<td>Pigtail 145 catheter 6 French</td>
			<td>Boston Scientific</td>
			<td>08641-41</td>
			<td>Measure LV pressures</td>
		</tr>
		<tr>
			<td>Pressure Transducer</td>
			<td>various</td>
			<td>Must adapt to anesthesia monitor</td>
			<td>Monitor direct arterial pressures</td>
		</tr>
		<tr>
			<td>Propofol</td>
			<td>Diprivan</td>
			<td>269-29</td>
			<td>Induction agent</td>
		</tr>
		<tr>
			<td>Roncuronium</td>
			<td>Mylan</td>
			<td>67457-228-05</td>
			<td>Neuromuscular blocking agent</td>
		</tr>
		<tr>
			<td>SR Buprenorphine 10 mg/mL</td>
			<td>Abbott Labs</td>
			<td>NADA 141-434</td>
			<td>Post operative Analgesic</td>
		</tr>
		<tr>
			<td>Sterile Saline 20 mL</td>
			<td>Fisher Scientific</td>
			<td>20T700220</td>
			<td>Flush for IV catheters</td>
		</tr>
		<tr>
			<td>Sternal Saw/ Necropsy Saw</td>
			<td>Thermo Fisher</td>
			<td>812822</td>
			<td>Used to open chest cavity</td>
		</tr>
		<tr>
			<td>Stop Cocks</td>
			<td>Smith Medical</td>
			<td>MX5311L</td>
			<td>2 to connect to pig tail</td>
		</tr>
		<tr>
			<td>Succinylcholine 20 mg/mL</td>
			<td>Pfizer</td>
			<td>00409-6629-02</td>
			<td>Neuromuscular blocking agent</td>
		</tr>
		<tr>
			<td>Suction&#160; tubing</td>
			<td>Medline</td>
			<td>DYND50223</td>
			<td></td>
		</tr>
		<tr>
			<td>Suction Container</td>
			<td>Medline</td>
			<td>DYNDCL03000</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgery pack with chest retractor</td>
			<td>various</td>
			<td>See pack list</td>
			<td>Femoral cut down and median sternotomy</td>
		</tr>
		<tr>
			<td>Surgical Instruments</td>
			<td>various</td>
			<td>See pack list</td>
			<td>Femoral and carotid cutdowns and sternotomy</td>
		</tr>
		<tr>
			<td>Surgical Spring Clip</td>
			<td>Applied Medical</td>
			<td>A1801</td>
			<td>Clamp end of LIMA after takedown</td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>Harvard</td>
			<td></td>
			<td>Delivers IV Dobutamine infusion</td>
		</tr>
		<tr>
			<td>SYTO RNASelect Green Fluorescent cell Stain - 5 mM Solution in DMSO</td>
			<td>Millipore Sigma</td>
			<td>S32703</td>
			<td></td>
		</tr>
		<tr>
			<td>Telazol 100 mg/mL</td>
			<td>Fort Dodge</td>
			<td>01L60030</td>
			<td>Pre operative Sedative</td>
		</tr>
		<tr>
			<td>Telpha pad</td>
			<td>Covidien</td>
			<td>2132</td>
			<td>Sterile wound dressing</td>
		</tr>
		<tr>
			<td>Timer</td>
			<td></td>
			<td></td>
			<td>Time collection of blood samples</td>
		</tr>
		<tr>
			<td>Total Exosome Isolation Reagent (from cell culture media)</td>
			<td>ThermoFisher Scientific</td>
			<td>4478359</td>
			<td></td>
		</tr>
		<tr>
			<td>TPP Tissue Culture Flask, T75, Filter Cap w/ 0.22uM PTFE</td>
			<td>ThermoFisher Scientific</td>
			<td>TP90076</td>
			<td></td>
		</tr>
		<tr>
			<td>Triple Antibiotic Ointment</td>
			<td>Johnson &#38; Johnson</td>
			<td>23734</td>
			<td>Topical over wound</td>
		</tr>
		<tr>
			<td>Vicryl mesh</td>
			<td>Ethicon</td>
			<td>VKML</td>
			<td>Patch for epicardial cell application</td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td></td>
			<td></td>
			<td>Mix microspheres</td>
		</tr>
		<tr>
			<td>Xylazine 100 mg/mL</td>
			<td>Vedco</td>
			<td>468RX</td>
			<td>Pre operative Sedative/ analgesic</td>
		</tr>
	</tbody>
</table>

surgical porcine model of chronic myocardial ischemia treated by exosome-laden collagen patch and off-pump coronary artery bypass graft

Chronic myocardial ischemia resulting from progressive coronary artery stenosis leads to hibernating myocardium (HIB), defined as myocardium that adapts to reduced oxygen availability by reducing metabolic activity, thereby preventing irreversible cardiomyocyte injury and infarction. This is distinct from myocardial infarction, as HIB has the potential for recovery with revascularization. Patients with significant coronary artery disease (CAD) experience chronic ischemia, which puts them at risk for heart failure and sudden death. The standard surgical intervention for severe CAD is coronary artery bypass graft surgery (CABG), but it has been shown to be an imperfect therapy, yet no adjunctive therapies exist to recover myocytes adapted to chronic ischemia. To address this gap, a surgical model of HIB using porcine that is amenable to CABG and mimics the clinical scenario was used. The model involves two surgeries. The first operation involves implanting a 1.5 mm rigid constrictor on the left anterior descending (LAD) artery. As the animal grows, the constrictor gradually causes significant stenosis resulting in reduced regional systolic function. Once the stenosis reaches 80%, the myocardial flow and function are impaired, creating HIB. An off-pump CABG is then performed with the left internal mammary artery (LIMA) to revascularize the ischemic region. The animal recovers for one month to allow for optimal myocardial improvement prior to sacrifice. This allows for physiologic and tissue studies of different treatment groups. This animal model demonstrates that cardiac function remains impaired despite CABG, suggesting the need for novel adjunctive interventions. In this study, a collagen patch embedded with mesenchymal stem cell (MSC)-derived exosomes was developed, which can be surgically applied to the epicardial surface distal to LIMA anastomosis. The material conforms to the epicardium, is absorbable, and provides the scaffold for the sustained release of signaling factors. This regenerative therapy can stimulate myocardial recovery that does not respond to revascularization alone. This model translates to the clinical arena by providing means of physiological and mechanistic explorations regarding recovery in HIB.

Author Spotlight: Enhancing Coronary Artery Revascularization

Surgical Porcine Model of Chronic Myocardial Ischemia Treated by Exosome-laden Collagen Patch and Off-pump Coronary Artery Bypass Graft

Following revascularization, coronary angiography is performed to assess for LAD stenosis (greater than 80%) and patency of the LIMA-LAD graft (Figure 1). Four weeks following the revascularization surgery and placement of the exosome-laden collagen patch, cardiac MRI is performed to assess for systolic and diastolic function of the heart at rest and under stress using low-dose dobutamine infusion at 5&#181;g/kg/min. Systolic function is analyzed by measuring wall thickness percentage (wall ...

Watch this Scientific Journal Video about Surgical Porcine Model of Chronic Myocardial Ischemia Treated by Exosome-laden Collagen Patch and Off-pump Coronary Artery Bypass Graft at JoVE.com

This study presents a surgical porcine model of chronic myocardial ischemia due to progressive coronary artery stenosis, resulting in impaired cardiac function without infarction. Following ischemia, animals undergo off-pump coronary artery bypass graft with epicardial placement of stem cells-derived exosomes-laden collagen patch. This adjunctive therapy improves myocardial function and recovery.

Chronic myocardial ischemia resulting from progressive coronary artery stenosis leads to hibernating myocardium (HIB), defined as myocardium that adapts ...

The overall goal of this surgical procedure is to create a surgical model of coronary artery revascularization using LIMA to LAD bypass, and the use of exosome-laden collagen patch as an adjunct to demonstrate physiological improvement in cardiac function. Our in-vivo model is a single-vessel model that does not include any comorbidities, which is not representative of the typical clinical situation for patients undergoing treatment for multi-vessel disease. However, despite this limitation, our model has consistently produced reliable results and has maintained low mortality rates.We developed a clinically relevant adjuvant therapy to be used during CABG and aim to understand the cellular mechanisms driving the myocardial functional improvement in chronic myocardial ischemia. Complete recovery of ischemic tissue appears to hinge on restoration of mitochondrial capabilities and reduced inflammation;representing a novel area of study.