Alan P. Kypson

Readily Available Tissue-Engineered Vascular Grafts

Nonimmunogenic, tissue-engineered vascular grafts stored long-term maintain their patency, strength, and function after transplant in large-animal models. Grow Your Own Blood Vessels Growing your own vegetables may be a well-established approach for a healthier life, but growing blood vessels for surgical transplantation is a more unusual pastime. But the idea of growing a readily available supply of blood vessels for surgical transplant into patients requiring, for example, a cardiac bypass or dialysis is not as far-fetched as it sounds. Although a patient’s own blood vessels can sometimes be used for the graft, often this is not possible. Engineered autologous blood vessels can be grown from endothelial cells taken from the patient and cultured on scaffolds, but this process takes 9 months or more, and often the patients cannot wait that long for surgery. Enter Dahl and her team with a new approach that provides readily available, off-the-shelf vascular grafts that retain their strength and patency during long-term storage and function successfully after vascular surgery in baboon and dog animal models. The authors grew their human vascular grafts by culturing smooth muscle cells from human cadavers (that is, allogeneic cells) on tubular scaffolds made from a biodegradable polymer called polyglycolic acid (PGA). The smooth muscle cells produced collagen and other molecules that formed an extracellular matrix. When the scaffold degraded, fully formed vascular grafts were left behind. The investigators then stripped the cells from the grafts, using detergent to make sure the grafts would not elicit an immune response when transplanted. These human vascular grafts were 6 mm or greater in diameter and retained their strength, elasticity, and patency even after storage in phosphate-buffered saline solution for a year. The human vascular grafts were tested in a baboon model of arteriovenous bypass in which the graft formed a direct conduit between an artery and a vein (an approach that enables human patients with kidney disease to undergo dialysis). The authors showed that the grafts in baboons restored blood flow and retained their patency and strength for up to 6 months. When the grafts were removed and examined histologically, they did not show evidence of fibrosis, calcification, or thickening of the vessel wall intima. But the authors wanted to test engineered vascular grafts with smaller diameters, which are often plagued by thrombi (blood clots) after transplant. To do this, they turned to a dog model of peripheral and coronary artery bypass, surgeries that require smaller-diameter vascular grafts. Using dog smooth muscle cells cultured on PGA scaffolds, they created canine vascular grafts with small diameters (3 to 4 mm). They then seeded these grafts with endothelial cells (from the dogs due to be recipients) because an endothelial cell lining helps to prevent blood clot formation. Using the engineered grafts, the investigators then conducted either peripheral or coronary artery bypass in the dog recipients and showed that they functioned effectively for at least 1 month. Together, these results demonstrate that durable vascular grafts derived from allogeneic donors and rendered nonimmunogenic by removal of donor cells are suitable for surgical transplant. The added advantage of being able to store these off-the-shelf vascular grafts long-term in a simple saline solution means that these can be made ahead of time and then are ready to go whenever they are needed. Growing blood vessels for a healthier life is as real as the home-grown asparagus in your garden. Autologous or synthetic vascular grafts are used routinely for providing access in hemodialysis or for arterial bypass in patients with cardiovascular disease. However, some patients either lack suitable autologous tissue or cannot receive synthetic grafts. Such patients could benefit from a vascular graft produced by tissue engineering. Here, we engineer vascular grafts using human allogeneic or canine smooth muscle cells grown on a tubular polyglycolic acid scaffold. Cellular material was removed with detergents to render the grafts nonimmunogenic. Mechanical properties of the human vascular grafts were similar to native human blood vessels, and the grafts could withstand long-term storage at 4°C. Human engineered grafts were tested in a baboon model of arteriovenous access for hemodialysis. Canine grafts were tested in a dog model of peripheral and coronary artery bypass. Grafts demonstrated excellent patency and resisted dilatation, calcification, and intimal hyperplasia. Such tissue-engineered vascular grafts may provide a readily available option for patients without suitable autologous tissue or for those who are not candidates for synthetic grafts.

Substrate-Specific Derangements in Mitochondrial Metabolism and Redox Balance in the Atrium of the Type 2 Diabetic Human Heart

OBJECTIVES The aim of this study was to determine the impact of diabetes on oxidant balance and mitochondrial metabolism of carbohydrate- and lipid-based substrates in myocardium of type 2 diabetic patients. BACKGROUND Heart failure represents a major cause of death among diabetic patients. It has been proposed that derangements in cardiac metabolism and oxidative stress may underlie the progression of this comorbidity, but scarce evidence exists in support of this mechanism in humans. METHODS Mitochondrial oxygen (O(2)) consumption and hydrogen peroxide (H(2)O(2)) emission were measured in permeabilized myofibers prepared from samples of the right atrial appendage obtained from nondiabetic (n = 13) and diabetic (n = 11) patients undergoing nonemergent coronary artery bypass graft surgery. RESULTS Mitochondria in atrial tissue of type 2 diabetic individuals show a sharply decreased capacity for glutamate and fatty acid-supported respiration, in addition to an increased content of myocardial triglycerides, as compared to nondiabetic patients. Furthermore, diabetic patients show an increased mitochondrial H(2)O(2) emission during oxidation of carbohydrate- and lipid-based substrates, depleted glutathione, and evidence of persistent oxidative stress in their atrial tissue. CONCLUSIONS These findings are the first to directly investigate the effects of type 2 diabetes on a panoply of mitochondrial functions in the human myocardium using cellular and molecular approaches, and they show that mitochondria in diabetic human hearts have specific impairments in maximal capacity to oxidize fatty acids and glutamate, yet increased mitochondrial H(2)O(2) emission, providing insight into the role of mitochondrial dysfunction and oxidative stress in the pathogenesis of heart failure in diabetic patients.

Reduction of Ischemia/Reperfusion Injury With Bendavia, a Mitochondria-Targeting Cytoprotective Peptide

Background Manifestations of reperfusion injury include myocyte death leading to infarction, contractile dysfunction, and vascular injury characterized by the “no-reflow” phenomenon. Mitochondria-produced reactive oxygen species are believed to be centrally involved in each of these aspects of reperfusion injury, although currently no therapies reduce reperfusion injury by targeting mitochondria specifically. Methods and Results We investigated the cardioprotective effects of a mitochondria-targeted peptide, Bendavia (Stealth Peptides), across a spectrum of experimental cardiac ischemia/reperfusion models. Postischemic administration of Bendavia reduced infarct size in an in vivo sheep model by 15% (P=0.02) and in an ex vivo guinea pig model by 38% to 42% (P<0.05). In an in vivo rabbit model, the extent of coronary no-reflow was assessed with Thioflavin S staining and was significantly smaller in the Bendavia group for any given ischemic risk area than in the control group (P=0.0085). Myocardial uptake of Bendavia was ≈25% per minute, and uptake remained consistent throughout reperfusion. Postischemic recovery of cardiac hemodynamics was not influenced by Bendavia in any of the models studied. Isolated myocytes exposed to hypoxia/reoxygenation showed improved survival when treated with Bendavia. This protection appeared to be mediated by lowered reactive oxygen species–mediated cell death during reoxygenation, associated with sustainment of mitochondrial membrane potential in Bendavia-treated myocytes. Conclusions Postischemic administration of Bendavia protected against reperfusion injury in several distinct models of injury. These data suggest that Bendavia is a mitochondria-targeted therapy that reduces reperfusion injury by maintaining mitochondrial energetics and suppressing cellular reactive oxygen species levels. (J Am Heart Assoc. 2012;1:e001644 doi: 10.1161/JAHA.112.001644.)

Quantum dot labeling of mesenchymal stem cells

BackgroundMesenchymal stem cells (MSCs) are multipotent cells with the potential to differentiate into bone, cartilage, fat and muscle cells and are being investigated for their utility in cell-based transplantation therapy. Yet, adequate methods to track transplanted MSCs in vivo are limited, precluding functional studies. Quantum Dots (QDs) offer an alternative to organic dyes and fluorescent proteins to label and track cells in vitro and in vivo. These nanoparticles are resistant to chemical and metabolic degradation, demonstrating long term photostability. Here, we investigate the cytotoxic effects of in vitro QD labeling on MSC proliferation and differentiation and use as a cell label in a cardiomyocyte co-culture.ResultsA dose-response to QDs in rat bone marrow MSCs was assessed in Control (no-QDs), Low concentration (LC, 5 nmol/L) and High concentration (HC, 20 nmol/L) groups. QD yield and retention, MSC survival, proinflammatory cytokines, proliferation and DNA damage were evaluated in MSCs, 24 -120 hrs post QD labeling. In addition, functional integration of QD labeled MSCs in an in vitro cardiomyocyte co-culture was assessed. A dose-dependent effect was measured with increased yield in HC vs. LC labeled MSCs (93 ± 3% vs. 50% ± 15%, p < 0.05), with a larger number of QD aggregates per cell in HC vs. LC MSCs at each time point (p < 0.05). At 24 hrs >90% of QD labeled cells were viable in all groups, however, at 120 hrs increased apoptosis was measured in HC vs. Control MSCs (7.2% ± 2.7% vs. 0.5% ± 0.4%, p < 0.05). MCP-1 and IL-6 levels doubled in HC MSCs when measured 24 hrs after QD labeling. No change in MSC proliferation or DNA damage was observed in QD labeled MSCs at 24, 72 and 120 hrs post labeling. Finally, in a cardiomyocyte co-culture QD labeled MSCs were easy to locate and formed functional cell-to-cell couplings, assessed by dye diffusion.ConclusionFluorescent QDs label MSC effectively in an in vitro co-culture model. QDs are easy to use, show a high yield and survival rate with minimal cytotoxic effects. Dose-dependent effects suggest limiting MSC QD exposure.

Increased propensity for cell death in diabetic human heart is mediated by mitochondrial-dependent pathways

Progressive energy deficiency and loss of cardiomyocyte numbers are two prominent factors that lead to heart failure in experimental models. Signals that mediate cardiomyocyte cell death have been suggested to come from both extrinsic (e.g., cytokines) and intrinsic (e.g., mitochondria) sources, but the evidence supporting these mechanisms remains unclear, and virtually nonexistent in humans. In this study, we investigated the sensitivity of the mitochondrial permeability transition pore (mPTP) to calcium (Ca(2+)) using permeabilized myofibers of right atrium obtained from diabetic (n = 9) and nondiabetic (n = 12) patients with coronary artery disease undergoing nonemergent coronary revascularization surgery. Under conditions that mimic the energetic state of the heart in vivo (pyruvate, glutamate, malate, and 100 μM ADP), cardiac mitochondria from diabetic patients show an increased sensitivity to Ca(2+)-induced mPTP opening compared with nondiabetic patients. This increased mPTP Ca(2+) sensitivity in diabetic heart mitochondria is accompanied by a substantially greater rate of mitochondrial H(2)O(2) emission under identical conditions, despite no differences in respiratory capacity under these conditions or mitochondrial enzyme content. Activity of the intrinsic apoptosis pathway mediator caspase-9 was greater in diabetic atrial tissue, whereas activity of the extrinsic pathway mediator caspase-8 was unchanged between groups. Furthermore, caspase-3 activity was not significantly increased in diabetic atrial tissue. These data collectively suggest that the myocardium in diabetic patients has a greater overall propensity for mitochondrial-dependent cell death, possibly as a result of metabolic stress-imposed changes that have occurred within the mitochondria, rendering them more susceptible to insults such as Ca(2+) overload. In addition, they lend further support to the notion that mitochondria represent a viable target for future therapies directed at ameliorating heart failure and other comorbidities that come with diabetes.

Monoamine Oxidase is a Major Determinant of Redox Balance in Human Atrial Myocardium and is Associated With Postoperative Atrial Fibrillation

Background Onset of postoperative atrial fibrillation (POAF) is a common and costly complication of heart surgery despite major improvements in surgical technique and quality of patient care. The etiology of POAF, and the ability of clinicians to identify and therapeutically target high‐risk patients, remains elusive. Methods and Results Myocardial tissue dissected from right atrial appendage (RAA) was obtained from 244 patients undergoing cardiac surgery. Reactive oxygen species (ROS) generation from multiple sources was assessed in this tissue, along with total glutathione (GSHt) and its related enzymes GSH‐peroxidase (GPx) and GSH‐reductase (GR). Monoamine oxidase (MAO) and NADPH oxidase were observed to generate ROS at rates 10‐fold greater than intact, coupled mitochondria. POAF risk was significantly associated with MAO activity (Quartile 1 [Q1]: adjusted relative risk [ARR]=1.0; Q2: ARR=1.8, 95% confidence interval [CI]=0.84 to 4.0; Q3: ARR=2.1, 95% CI=0.99 to 4.3; Q4: ARR=3.8, 95% CI=1.9 to 7.5; adjusted Ptrend=0.009). In contrast, myocardial GSHt was inversely associated with POAF (Quartile 1 [Q1]: adjusted relative risk [ARR]=1.0; Q2: ARR=0.93, 95% confidence interval [CI]=0.60 to 1.4; Q3: ARR=0.62, 95% CI=0.36 to 1.1; Q4: ARR=0.56, 95% CI=0.34 to 0.93; adjusted Ptrend=0.014). GPx also was significantly associated with POAF; however, a linear trend for risk was not observed across increasing levels of the enzyme. GR was not associated with POAF risk. Conclusions Our results show that MAO is an important determinant of redox balance in human atrial myocardium, and that this enzyme, in addition to GSHt and GPx, is associated with an increased risk for POAF. Further investigation is needed to validate MAO as a predictive biomarker for POAF, and to explore this enzymes potential role in arrhythmogenesis.

Obesity in a model of gpx4 haploinsufficiency uncovers a causal role for lipid-derived aldehydes in human metabolic disease and cardiomyopathy

Objective Lipid peroxides and their reactive aldehyde derivatives (LPPs) have been linked to obesity-related pathologies, but whether they have a causal role has remained unclear. Glutathione peroxidase 4 (GPx4) is a selenoenzyme that selectively neutralizes lipid hydroperoxides, and human gpx4 gene variants have been associated with obesity and cardiovascular disease in epidemiological studies. This study tested the hypothesis that LPPs underlie cardio-metabolic derangements in obesity using a high fat, high sucrose (HFHS) diet in gpx4 haploinsufficient mice (GPx4+/−) and in samples of human myocardium. Methods Wild-type (WT) and GPx4+/− mice were fed either a standard chow (CNTL) or HFHS diet for 24 weeks, with metabolic and cardiovascular parameters measured throughout. Biochemical and immuno-histological analysis was performed in heart and liver at termination of study, and mitochondrial function was analyzed in heart. Biochemical analysis was also performed on samples of human atrial myocardium from a cohort of 103 patients undergoing elective heart surgery. Results Following HFHS diet, WT mice displayed moderate increases in 4-hydroxynonenal (HNE)-adducts and carbonyl stress, and a 1.5-fold increase in GPx4 enzyme in both liver and heart, while gpx4 haploinsufficient (GPx4+/−) mice had marked carbonyl stress in these organs accompanied by exacerbated glucose intolerance, dyslipidemia, and liver steatosis. Although normotensive, cardiac hypertrophy was evident with obesity, and cardiac fibrosis more pronounced in obese GPx4+/− mice. Mitochondrial dysfunction manifesting as decreased fat oxidation capacity and increased reactive oxygen species was also present in obese GPx4+/− but not WT hearts, along with up-regulation of pro-inflammatory and pro-fibrotic genes. Patients with diabetes and hyperglycemia exhibited significantly less GPx4 enzyme and greater HNE-adducts in their hearts, compared with age-matched non-diabetic patients. Conclusion These findings suggest LPPs are key factors underlying cardio-metabolic derangements that occur with obesity and that GPx4 serves a critical role as an adaptive countermeasure.

Clinical Outcomes in Patients With Prolonged Intensive Care Unit Length of Stay After Cardiac Surgical Procedures

BACKGROUND Advances in critical care medicine have allowed for improved care of patients requiring prolonged intensive care unit length of stay (prICULOS) after cardiac operations, yet little is known regarding their eventual outcomes. The purpose of this study was to examine short- and long-term outcomes in patients undergoing cardiac operations with prICULOS. METHODS All cases of coronary artery bypass grafting (CABG), aortic valve, mitral valve, and combined CABG/valve surgical procedures performed at a single institution from July 2002 to July 2007 were identified. All-cause mortality in patients discharged alive from the hospital was determined until December 2007 through linkage with the Social Security Death Index. Patients who experienced intraoperative death or those with missing or invalid social security numbers were excluded. The definition of prICULOS was total ICULOS greater than 7 days. RESULTS A total of 3,478 patients met inclusion criteria. One hundred thirty-seven of three thousand four hundred seventy-eight patients (3.9%) experienced prICULOS. These patients were more likely to be older than 70 years (55.5% versus 30.5%; p<0.0001) and to have had recent myocardial infarction (28.5% versus 20.1%; p=0.02), previous cardiac operation (18.3% versus 6.9%; p<0.0001), and emergent status (9.5% versus 1.6%; p<0.0001). They experienced greater in-hospital mortality (37.2% versus 1.7%; p<0.0001) and those who were discharged alive had worse long-term survival (log-rank, p<0.0001). After risk adjustment, prICULOS emerged as a significant predictor of in-hospital death (odds ratio [OR] 20.9; 95% confidence interval [CI], 12.9-33.7) and decreased long-term survival (hazard ratio [HR] 2.9; 95% CI, 2.0-4.3). CONCLUSIONS Patients with prICULOS after cardiac operations have worse overall outcomes. These data may be used to inform these patients and their families of realistic expectations regarding their clinical course.

Robotic mitral valve surgery

Traditionally mitral valve surgery has been performed via median sternotomy. However, a renaissance in cardiac surgery is occurring. Cardiac operations are being performed through smaller and alternative incisions with enhanced technological assistance. Specifically, minimally invasive mitral valve surgery has become standard for many surgeons. At our institution, we have developed a robotic mitral surgery program with the da Vinci(TM) telemanipulation system. This system allows the surgeon to perform complex mitral valve operations through small port sites rather than a traditional median sternotomy. Our techniques and initial results are reported as is a brief overview of the evolution of robotic cardiac surgery.

Effect of Peripheral Arterial Disease and Race on Survival After Coronary Artery Bypass Grafting

BACKGROUND Although peripheral arterial disease (PAD) is more prevalent among blacks, the effect of race on long-term survival after coronary artery bypass grafting (CABG) has not been examined in this population. METHODS A retrospective cohort study was conducted of CABG patients between 1992 and 2011. Long-term survival was compared in patients with and without PAD and stratified by race. Hazard ratios (HR) and 95% confidence intervals were computed using a Cox regression model. RESULTS Of 13,053 patients who underwent CABG, 1,501 (11%) had PAD, comprising 311 blacks and 1,190 whites. Median follow-up was 8.3 years. Long-term survival differed by race (no PAD: HR, 1.0; white PAD: adjusted HR, 1.5, 95% confidence interval, 1.4 to 1.6; black PAD: adjusted HR, 2.1, 95% confidence interval, 1.8 to 2.5; p < 0.0001 for trend). CONCLUSIONS Risk of death after CABG was comparatively higher among black PAD patients. This finding provides useful outcome information for surgeons and their patients.