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No description available.

Thesis (M.D.)—Boston University

Myocardial infarction is a prominent medical problem in the world today. Current treatments are limited and do not strive to regenerate the myocardial tissue that is lost post-infarction. Human mesenchymal stem cells (hMSCs) have been shown to improve cardiac function when implanted post-infarction. The effectiveness of stem cell therapy largely depends on the delivery method. Current delivery methods are insufficient due to their low cell engraftment rate and inability to target the endocardium, where most myocardial infarctions occur. Biological microthreads are a promising new local cell delivery method that may improve upon these current limitations. We hypothesize that biological microthreads will increase efficiency of hMSC delivery to the beating rat heart compared to intramyocardial injection. To test our hypothesis we seeded biological microthreads in vitro with 100 ìL of cell suspension (100,000 hMSCs). After one day, an average of 11,806 ± 3,932 hMSCs were counted on the biological microthreads. The biological microthreads were attached to suture needles to allow targeted delivery to the rat heart (in the left ventricular wall). Human mesenchymal stem cells were loaded with quantum dots prior to seeding the biological microthread bundles or delivery to the rat heart via injection. For intramyocardial injection, a cell suspension containing 10,000 hMSCs (35 ìL) was injected into the myocardial wall using a 100 ìL syringe. The delivery efficiency of each method was determined by sectioning the heart into 8 µm thick sections and analyzing three sections every sixty sections (24 µm every 480 µm) for quantum dot loaded hMSCs. These sections were stained with Hoechst dye and quantum dot loaded cells in the heart sections were manually counted. The delivery efficiency of each biological microthread implantation was calculated by dividing the number of counted quantum dot loaded hMSCs in the heart wall by the average number of hMSCs on the biological microthread bundles (normalized to the length that was implanted in the heart wall) after 24 hours. The delivery efficiency of intramyocardial injection was calculated by dividing the number of counted quantum dot loaded hMSCs in the heart wall by 10,000 (the number of cells injected). Biological microthread mediated hMSC delivery had a significantly higher delivery efficiency (66.6 ± 11.1%) compared to intramyocardial injection (11.8 ± 6.25%) after 1 hour (p < 0.05). Biological microthread implantation tracking illustrated that we were able to deliver hMSCs to the myocardium and endocardium of the left ventricular wall for hMSC delivery. This study illustrates that biological microthreads can serve as an efficient means of delivering hMSCs to the infarcted heart. Unlike the currently utilized delivery methods, biological microthreads can target the infarcted layer of the left ventricular wall and maximize hMSC engraftment to that layer.

Rapid restoration of blood flow to ischemic myocardium is essential, however it causes further injury called reperfusion injury. Apoptosis contributes significantly to cardiomyocyte cell death during ischemia reperfusion injury, in which caspase family proteases play an essential role as they are the executioners of apoptosis. Caspase inhibitors showed promising cardioprotective results when administered before ischemia or at the start of reperfusion. However, before applying them in pre clinical studies of myocardial ischemia, several investigations needed to be taken to determine their therapeutic window post reperfusion, their effect on functional recovery of myocardium post ischemia, their mechanism of action. Methods Isolated perfused rat hearts were subjected to 35 min ischemia followed by 2 hr reperfusion where caspase inhibitors [broad spectrum caspase inhibitor (ZVAD, 0.1µM), specific caspase 3 inhibitor (DEVD, 0.07µM)] were added at the start of reperfusion, 15, 30 and 60 min after starting reperfusion at the presence or absence of Wortmannin (WORT, 100nM, PI3-kinase inhibitor). Hearts underwent triphenyl tetrazolium staining for infarct size assessment, or were frozen for Western blot analysis. Freshly isolated adult rat ventricular myocytes were subjected to 6 hr hypoxia followed by either 18 hr, where caspase inhibitors (ZVAD, 25µM and DEVD, 25µM) were added at the start of reoxygenation, 15, 30 and 60 min after starting reoxygenation at the presence or absence of Wortmannin (WORT, 100nM). Cardiomyocytes were analysed for viability, apoptosis, necrosis and intracellular caspase-3 activity using flow cytometry analysis. Isolated adult rat ventricular papillary muscles were subjected to 35 min hypoxia followed by 100 min reperfusion where caspase inhibitors [ZVAD (0.1 µM, 2.5µM) and DEVD (2.5µM)] were added at the start of reperfusion throughout. Power output was measured using work loop technique.

The main purpose of this study was to determine the impact of depression and anxiety on mortality, CHD incidence, and quality-of-life in patients hospitalised for an acute myocardial infarction (MI). Questionnaires, including the Beck Depression Inventory (BDI) and the State-Trait Anxiety Inventory were completed during hospitalisation by 288 MI patients, and four months and 12 months after discharge among survivors. Quality-of-life was assessed at both follow-up points using the Dartmouth COOP charts. Twenty-five (8.7%) patients died, 22 of cardiac causes, during the four month follow-up. Six further fatalities occurred between four and 12 months following MI. Symptoms of depression and anxiety did not predict either cardiac or all-cause mortality, or CHD incidence at either follow-up point. Indices of disease severity predicted both four month and 12 month mortality and CHD incidence. In a subset of seven patients who died prior to discharge, depressive symptoms did predict mortality, but the association did not withstand correction for severity of infarction. Multiple regression analyses revealed that baseline depression and state anxiety, as well as severity of infarction, predicted both four and 12 month quality-of-life. In addition, partner status and living alone also predicted four and 12 month quality-of-life, respectively. Attendance at rehabilitation was positively associated with quality-of-life at both four and 12 months, and negatively associated with 12 month CHD morbidity. In conclusion, depression and anxiety were not significant predictors of mortality, or CHD incidence, during the first year following MI but they were predictive of four and 12 month quality-of-life among survivors.

Background: Myocardial infarction (MI), also known as heart attack, is the leading cause of morbidity and mortality among the heart diseases spectrum. It results from an insufficient supply of blood to the heart muscles. According to the United States (U.S.) Centers for Disease Control and Prevention (CDC), about 610 000 people die of heart disease in the U.S. every year. Myocardial infarction contributes 370 000 of these deaths annually. Every 40 seconds, someone in the U.S. experience heart attack. This burden is disproportionately distributed within the U.S. population. The rate of heart disease in Central Appalachia is 249 per 100 000, 42% higher than the national rate. Exploring further within the region, rural areas experience higher heart disease mortality rates; 27% higher than the region’s metro counties. According to 2018 America Health Ranking, the prevalence of heart attack in Tennessee is 5.9%, compared to the 4.9% nationwide, with the majority of the burden seen among adults aged ≥65 years and with a 1:1.8 female to male ratio. Patients with heart disease often have other comorbid conditions such as peripheral arterial disease (PAD), hypertension, diabetes, dyslipidemias, which contribute immensely to this chronic condition. Therefore, the aim of this study is to explore the association between cardiovascular comorbidities such as diabetes mellitus, hypertension and dyslipidemia, and the prevalence of heart attack among patients with PAD in rural Central Appalachia. Methods: We used a cross-sectional data of patients diagnosed with PAD in the Central Appalachian region. A total of 13455 patients with PAD were recruited using ICD 9 and 10 search terms for PAD from the electronic medical records (EMR) system between January 1, 2008, and April 30, 2018. Descriptive statistics of the variables were extracted. The association between the comorbidities, including hypertension, diabetes, dyslipidemia, body mass index(BMI) and the prevalence of MI was determined using a binomial logistic regression model. All analysis was done using IBM SPSS statistics 25. Results: Of the total 13455 patients with PAD, 3045 had MI (37.7% female and 62.3% male) with a mean age of 69±10.5years. While 93% had hypertension, 56% had diabetes. For the lipids, the mean of HDL, Cholesterol, and LDL among participants with a history of MI is 40.99mg/dl±13, 156.32mg/dl±45, 82.08mg/dl±36.35 respectively. The results of binomial logistic regression with stratification based on gender shows that female patients with diabetes had 86% increased odds of MI [OR: 1.858, C.I: 1.308-2.638, p-value=0.001), and for female hypertensives, 4.51 times increased odds of MI was found (C.I: 1.576-12.895, p-value=0.005). The male diabetics and hypertensives showed a similarly increased odds of MI with (OR 1.138, C.I: 0.870-1.489 p-value=0.345) and (OR 3.697C.I: 1.559-8.736, p-value=0.003) respectively. No significant association was found among the various lipid profiles examined. Conclusion: The results showed that female PAD patients with hypertension and diabetes have a significantly increased likelihood of having MI. In contrast, male with PAD also showed increased likelihood (although to a lesser degree) of MI in those with hypertension, but not those with diabetes. These findings underscore the importance of a proactive approach to preventive care and adequate control among PAD patients with diabetes and hypertension in a bid to curbing the morbidity and mortality associated with myocardial infarction among residents in Central Appalachia.

An analysis of snow removal injury data from the National Electronic Injury Surveillance System (NEISS) revealed a persistent gender gap in injuries and deaths during snow clearing activities. In general, men, those who identified as White and those aged 60-79 represented the vast majority of injuries and deaths sustained during automated snow removal. Injuries and deaths from manual snow clearing had greater representation across gender lines, as well as across various age groups and race categories. This indicates that a greater cross-section of society relies on the standard shovel in comparison to the snow blower for snow removal. The most likely injuries sustained during shoveling were to the neck and back, while hand and finger injuries were far more common during the use of a snow blower. Similar percentages of cardiac (30%) and non-cardiac chest injuries (70%) were found for both manual and automated modes of snow removal. While the majority of cardiac chest injuries were in those aged 40-59 for shoveling and 60-79 for snow blowing, the majority of cardiac fatalities were in those aged 60-79 for both methods of snow removal. Daily all-cause mortality and daily deaths from acute heart attacks showed a weak but inverse relationship to daily maximum, minimum and average temperatures. Mortality related to temperatures had significant lag effects for two days. Daily all-cause and heart attack mortality were also significantly related to the depth of the existing snowpack. Snow to liquid ratios indicating differences between heavy, wet snow and dry, powdery snow were not significant. However, the water equivalent of the existing snowpack was significantly related to daily mortality. Comparisons between all age and elderly mortality showed weaker and opposite relationships for the elderly group suggesting the use of protective behaviors such as cold and snow avoidance.

Activation of A3 adenosine receptors has been shown to protect the myocardium from ischaemia reperfusion injury in a number of animal models. The PI3K - AKT and MEK1/2 - ERK1/2 cell survival pathways have been shown to play a critical role in regulating myocardial ischaemia reperfusion injury. In this study we investigated whether the A3 adenosine receptor agonist 2-CL-IB-MECA protects the myocardium from ischaemia reperfusion injury, when administered at reperfusion or post reperfusion and whether the protection involved the PI3K – AKT or MEK 1/2 – ERK1/2 cell survival pathways. In the Langendorff model of ischaemia reperfusion injury isolated perfused rat hearts underwent 35 minutes of ischaemia and 120 minutes of reperfusion. Administration of 2-CL-IB-MECA (1nM) at reperfusion significantly decreased infarct size to risk ratio compared to non-treated ischeamic reperfused control hearts. This protection was abolished in the presence of the PI3K inhibitor Wortmannin or MEK1/2 inhibitor UO126. Western blot analysis determined that administration of 2-CL-IB-MECA (1 nM) upregulated ERK1/2 phosphorylation. In the adult rat cardiac myocyte model of hypoxia/reoxygenation cells underwent 6 hours of hypoxia and 18 hours of reoxygenation. Administration of 2-CL-IB-MECA (1 nM) at the onset of reoxygenation significantly decreased cellular apoptosis and necrosis. Administration of 2-CL-IB-MECA (1nM) in the presence of the Wortmannin or UO126 significantly reversed this anti-apoptotic effect and anti-necrotic effect. Our data further showed that 2-CL-IB-MECA protects myocytes subjected to hypoxia/reoxygenation injury via decreasing cleaved-caspase 3 activity that was abolished in presence of the PI3K inhibitor but not in the presence of the MEK1/2 inhibitor UO126. Administration of 2-CL-IB-MECA (100nM) at the onset of reperfusion also significantly decreased infarct size to risk ratio in the ischaemic reperfused rat heart compared to controls that was reversed in the presence of Wortmannin or Rapamycin. This protection was associated with an increase in PI3K-AKT / p70S6K / BAD phosphorylation. 2-CL-IB-MECA (100nM) administered at reoxygenation also significantly protected adult rat cardiac myocytes from hypoxia/reoxygenation injury 28 in an anti-apoptotic and anti-necrotic manner. This anti-apoptotic/necrotic effect of 2-CL-IB-MECA was abolished in the presence Wortmannin. Furthermore, that this protection afforded by 2-CL-IB-MECA (100nM) when administered at reoxygenation was associated with a decrease in cleaved caspase 3 activity that was abolished in the presence of the Wortmannin Interestingly, postponing the administration of 2-CL-IB-MECA to 15 or 30 minutes after the onset of reperfusion significantly protected the isolated perfused rat heart from ischaemia reperfusion injury in a Wortmannin and UO126 sensitive manner. This protection was associated with an increase in AKT and ERK1/2 phosphorylation. Administration of the A3 agonist 2-CL-IB-MECA 15 or 30 minutes after the onset of reoxygenation significantly protected isolated adult rat cardiac myocytes subjected to 6 hours of hypoxia and 18 hours of reoxygenation from injury in an anti-apoptotic/necrotic manner. This anti-apoptotic was abolished upon PI3K inhibition with Wortmannin or MEK1/2 inhibition with UO126. The anti-necrotic effect of 2-CL-IB-MECA when administered 15 or 30 minutes post-reperfusion was not abolished in the presence of the inhibitors. Delaying the administration of 2-CL-IB-MECA to 15 or 30 minutes after reoxygenation was associated with a decrease in cleaved-caspase 3 activity that was abolished in the presence of Wortmannin but not in the presence of the MEK 1/2inhibitor UO126. Collectively, we have demonstrated for the first time that administration of 2-CL-IB-MECA at the onset of reperfusion protects the ischaemic reperfused rat myocardium from lethal ischaemia reperfusion injury in a PI3K and MEK1/2 sensitive manner. Delaying the administration of 2-CL-IB-MECA to 15 or 30 minutes after the onset of reperfusion of reoxygenation also significantly protects the isolated perfused rat heart from ischaemia reperfusion injury and the adult rat cardiac myocyte from hypoxia/reoxygenation injury in an anti apoptotic / necrotic manner. Furthermore, that this protection is associated with recruitment of the PI3K-AKT and MEK1/2 – ERK1/2 cell survival pathways.

This literature review posits that the transcription factor Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is an attractive candidate therapeutic target in the setting of acute myocardial infarction (AMI). This transcription factor binds to antioxidant response elements (ARE) in the promoter region of a battery of genes that collectively encode an array of antioxidant, phase II drug metabolism, metabolically stabilizing, and overall cytoprotective enzymes, facilitating their transcription at basal levels and increasing transcription in response to various cellular stressors. Following a brief background tutorial on normal cardiac myocyte cellular physiology, key events that occur early in ischemia and reperfusion are outlined and integrated. These include ionic and metabolic dysregulation, electron transport chain uncoupling, mitochondrial depolarization, and the generation of reactive oxygen species (ROS). Abrupt changes in response to ischemia prime opening of the mitochondrial permeability transition pore (MPTP) and cardiac myocytes to generate a burst of ROS upon reperfusion–two key events that contribute to the umbrella term ischemia-reperfusion injury (IRI). How ROS damage cells is then outlined, and through a ROS-centric viewpoint, a case will be made as to how exogenous upregulation of Nrf2 could protect and/or salvage at-risk tissue immediately subjected to infarction and neighboring tissue in the peri-infarct zone (PIZ). The history of how Nrf2 came to be known as the "master regulator of oxidative stress" is reviewed, as well as the discovery of the canonical mechanism of Nrf2 regulation via Kelch-like ECH-associated protein 1 (Keap1) and other alternative mechanisms of endogenous Nrf2 regulation. Finally, compiling interdisciplinary evidence from research publications around the world, the benefits of therapeutically targeting Nrf2 are considered given the timescale and context of acute MI. Drug delivery methods, potential challenges, and limitations are then considered. Cardiac tissue is a dynamic substrate that exhibits changes for up to 90 days after AMI and patient outcomes are directly related to the extent of tissue lost following infarction/reperfusion. Targeting Nrf2 addresses an unmet need, as current clinical therapies focus on precluding occlusions and prompt reperfusion of infarcted tissue, but do not explicitly target at-risk tissue following infarcts and/or present-day reperfusion methodologies.