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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 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.
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....
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
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 5µg/kg/min. Systolic function is analyzed by measuring wall thickness percentage (wall .......
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.......
This work was supported by the VA Merit Review #I01 BX000760 (RFK) from the United States (U.S.) Department of Veterans Affairs BLR&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.
....Name | Company | Catalog Number | Comments |
5 Ethibond | Ethicon | MG46G | Suture |
# 40 clipper blade | Oster | 078919-016-701 | Remove hair from surgery sites |
0 Vicryl | Ethicon | J208H | Suture |
1 mL Syringe | Medtronic/Covidien | 1188100777 | Administer injectable agents |
1" medical tape | Medline | MMM15271Z | Secure wound dressing and IV catheters |
1000mL 0.9% Sodium chloride | Baxter | 2B1324X | IV replacement fluid |
12 mL Syringe | Medtronic/Covidien | 8881512878 | Administer injectable agents |
18 ga needles | BD | 305185 | Administration of injectable agents |
20 ga needles | BD | 305175 | Administration of injectable agents |
20 mL Syringe | Medtronic/Covidien | 8881520657 | Administer injectable agents |
2-0 Vicryl | Ethicon | J317H | Suture |
250 mL 0.9% saline | Baxter | UE1322D | Replacement IV Fluid |
3 mL Syinge | Medtronic/Covidien | 1180300555 | Administer injectable agents |
3-0 Vicryl | Ethicon | VCP824G | Suture |
36” Pressure monitoring tubing | Smith’s Medical | MX563 | Connect art. Line to transducer |
4.0 mm ID endotracheal tube | Medline | DYND43040 | Establish airway for Hibernation |
4-0 Tevdek II Strands | Deknatel | 7-922 | Suture to secure constrictor around LAD |
48” Pressure monitoring tubing | Smith’s Medical | MX564 | Connect art. Line to transducer |
500mL 0.9% Sodium chloride | Baxter | 2B1323Q | Drug delivery, Provide mist for Blower Mister |
6 mL Syringe | Medtronic/Covidien | 1180600777 | Administer injectable agents |
6.0 mm ID endotracheal tube | Mallinckrodt | 86049 | Establish airway for Revasc,MRI and Termination |
6.5 mm ID endotracheal tube | Medline | DYND43065 | Establish airway for Revasc,MRI and Termination |
6” pressure tubing line | Smith’s Medical | MX560 | Collect bone marrow |
60 mL Syringe | Medtronic/Covidien | 8881560125 | Administer injectable agents |
7.0 mm ID endotracheal tube | Medline | DYND43070 | Establish airway for Revasc,MRI and Termination |
7-0 Prolene | Ethicon | M8702 | Suture |
Advanced DMEM (1X) | ThermoFisher Scientific | 12491023 | |
Alcohol Prep pads | MedSource | MS-17402 | Skin disinfectant |
Amicon Ultra-15 Centrifugal Filter Unit | Millipore Sigma | UFC910024 | |
Anesthesia Machine | Drager | Fabious Trio | maintains general anesthesia |
Anesthesia Machine + ventilator | DRE Drager- Fabius Tiro | DRE0603FT | Deliver Oxygen and inhalant to patient |
Anesthesia Monitor | Phillips Intellivue | MP70 | Multiparameter for patient safety |
Arterial Line Kit | Arrow | ASK-04510-HF | Femoral catheter for blood pressure monitoring |
Artificial Tears | Rugby | 0536-1086-91 | Lubricate eyes to prevent corneal drying |
Bair Hugger | 3M | Model 505 | Patient Warming system |
Basic pack | Medline | DYNJP1000 | Sterile drapes and table cover |
Blood Collection Tubes- green top | Fisher Scientific | 02-689-7 | Collect microsphere blood samples |
Blower Mister Kit | Medtronic/Covidien | 22120 | Clears surgical field for vessel anastomosis |
BODIPY TR Ceramide | ThermoFisher Scientific | D7540 | |
Bone marrow needle- 25mm 15 ga IO needle | Vidacare | 9001-VC-005 | Collect bone marrow |
Bone Wax | Medline | ETHW31G | Hemostasis of cut bone |
Bovie Cautery hand piece | Covidien | E2516 | Hemostasis |
Bupivicaine | Pfizer | 00409-1161-01 | Local Anesthetic |
Buprenorphine 0.3 mg/mL | Sigma Aldrich | B9275 | Pre operative Analgesic for survivial procedures |
Cell Scrapers | Corning | 353085 | |
Cephazolin 1 gr | Pfizer | 00409-0805-01 | Antibiotic |
Chest Tube | Covidien | 8888561043 | Evacuates air from chest cavity |
Cloroprep | Becton Dickenson | 260815 | Surgical skin prep |
Corning bottle-top vacuum Filter System (500mL) | Millipore Sigma | 430758 | |
CPT tube | BD | 362753 | MSC isolation from bone marrow |
Delrin Constrictor | U of MN | Custom made | Creates stenosis of LAD |
Dermabond | Ethicon | DNX12 | Skin adhesive |
DMEM (1X) Dulbecco's Modified Eagle Medium, HEPES | ThermoFisher Scientific | 12430062 | |
Dobutamine 12.5 mg/mL | Pfizer | 00409-2344-01 | Increases blood pressure and heart rate during the second microsphere blood collection |
ECG Pads | DRE | 1496 | Monitor heart rhythm |
Exosome-Depleted FBS | ThermoFisher Scientific | A2720801 | |
Falcon Disposable Polystyrene Serological Pipets, Sterile, 10mL | Fisher Scientific | 13-675-20 | |
Femoral and carotid introducer | Cordis- J&J | 504606P | femoral and carotis cannulas |
Fetal Bovine Serum, Heat Inactivated, Gibco FBS | ThermoFisher Scientific | 16140089 | |
Flo-thru 1.0 | Baxter | FT-12100 | used to anastomos LIMA to L |
Flo-thru 1.25 | Baxter | FT-12125 | FT-12125 |
Flo-thru 1.5 | Baxter | FT-12150 | FT-12150 |
Flo-thru 2.0 | Baxter | FT-12200 | FT-12200 |
GlutaMAX Supplement | ThermoFisher Scientific | 35050061 | |
Hair Clipper | Oster | 078566-011-002 | Remove hair from surgery sites |
Helistat collagen sponge | McKesson | 570973 1690ZZ | Sponge for embedding exosomes |
Heparin | Pfizer | 0409-2720-03 | anticoaggulant |
Histology Jars | Fisher Scientific | 316-154 | Formalin for tissue samples |
HyClone Characterized Fetal Bovine Serum (FBS) | Cytiva | SH30071.03 | |
Hypafix | BSN Medical | 4210 | Secure wound dressing and IV catheters |
Isoflurane | Sigma Aldrich | CDS019936 | General Anesthestic- Inhalant |
IV Tubing for Blower Mister | Carefusion | 42493E | Adapts to IV Fluids for Blower/Mister |
Jelco 18 ga IV catheter | Smiths medical | 4054 | IV access in Revasc, MRI and Term |
Lidocaine 2% | Pfizer | 00409-4277-01 | Local Anesthetic/ antiarrthymic |
Ligaclips | Ethicon | MSC20 | Surgical Staples for LIMA takedown |
Long blade for laryngoscope | DRE | 12521 | Allows for visualization of trachea for intubation |
Meloxicam 5 mg/mL | Boehringer Ingelheim | 141-219 | Post operative Analgesic |
Microsphere pump | Collect blood samples from femoral introducer | ||
Monopolar Cautery | Covidien | Valleylab™ FT10 | Hemostasis |
Nanosight NS 300 | Malvern Panalytical | MAN0541-03-EN | |
NTA 3.1.54 software | Malvern Panalytical | MAN0520-01-EN-00 | |
OPVAC Synergy II | Terumo Cardiovascular System | 401-230 | Heart positioner and Stabilizer |
Oxygen Tank E cylinder | various | various | Used for Blower Mister if anesthesia machine doesn't have auxiliary flow meter |
PBS, pH 7.2 | ThermoFisher Scientific | 20012050 | |
Penicillin-Streptomycin-Neomycin (PSN) Antibiotic Mixture | ThermoFisher Scientific | 15640055 | |
Pigtail 145 catheter 6 French | Boston Scientific | 08641-41 | Measure LV pressures |
Pressure Transducer | various | Must adapt to anesthesia monitor | Monitor direct arterial pressures |
Propofol | Diprivan | 269-29 | Induction agent |
Roncuronium | Mylan | 67457-228-05 | Neuromuscular blocking agent |
SR Buprenorphine 10 mg/mL | Abbott Labs | NADA 141-434 | Post operative Analgesic |
Sterile Saline 20 mL | Fisher Scientific | 20T700220 | Flush for IV catheters |
Sternal Saw/ Necropsy Saw | Thermo Fisher | 812822 | Used to open chest cavity |
Stop Cocks | Smith Medical | MX5311L | 2 to connect to pig tail |
Succinylcholine 20 mg/mL | Pfizer | 00409-6629-02 | Neuromuscular blocking agent |
Suction tubing | Medline | DYND50223 | |
Suction Container | Medline | DYNDCL03000 | |
Surgery pack with chest retractor | various | See pack list | Femoral cut down and median sternotomy |
Surgical Instruments | various | See pack list | Femoral and carotid cutdowns and sternotomy |
Surgical Spring Clip | Applied Medical | A1801 | Clamp end of LIMA after takedown |
Syringe pump | Harvard | Delivers IV Dobutamine infusion | |
SYTO RNASelect Green Fluorescent cell Stain - 5 mM Solution in DMSO | Millipore Sigma | S32703 | |
Telazol 100 mg/mL | Fort Dodge | 01L60030 | Pre operative Sedative |
Telpha pad | Covidien | 2132 | Sterile wound dressing |
Timer | Time collection of blood samples | ||
Total Exosome Isolation Reagent (from cell culture media) | ThermoFisher Scientific | 4478359 | |
TPP Tissue Culture Flask, T75, Filter Cap w/ 0.22uM PTFE | ThermoFisher Scientific | TP90076 | |
Triple Antibiotic Ointment | Johnson & Johnson | 23734 | Topical over wound |
Vicryl mesh | Ethicon | VKML | Patch for epicardial cell application |
Vortex | Mix microspheres | ||
Xylazine 100 mg/mL | Vedco | 468RX | Pre operative Sedative/ analgesic |
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