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Red Blood Ageing and the Ability of their to Protect Human Endothelial Cells from Oxidative Stress Llewellyn R. Delsarte, Charlotte Lawson, Nripendra R. Bose, Department of Chemistry and Biochemistry, Spelman College, Atlanta, GA

Abstract Discussion Red blood cells (RBCs) have various roles that are important in making sure that the body Figure 1: Comparison graph of Day 2 and Day 6 The findings suggest that the calcium ionophore treated RBC sample showed an functions properly. They are responsible for delivering oxygen and removing carbon dioxide and microparticle formation for Sample 1 increase in microparticle formation and that the other samples did not reflect a other waste gases to and from the body. Studies have shown that storing blood for extended significant increase in microparticle formation. The in the samples that amounts of time leads to more oxygen entering the blood, causing oxidative stress and also did not include calcium ionophore showed relatively the same amount of formation of microparticles. Small in size (<1 micron), microparticles are membrane vesicles 2500 microparticles from the first day (Day 1) to the last day (Day 27). Since it was known that are released by RBCs and other types of cells such as , leukocytes, and that there would be an increase in microparticle formation with the inhibition of Erk endothelial cells. It has been shown that microparticles are increased in the plasma of patients 2000 with cardiovascular disease, however conflicting evidence has lead to the idea that the activity and the treatment of calcium ionophore, it was quite simple to see if the correlations do hold true over time. It was a similar case with the addition of the increased release of microparticles may be harmful to patient care. Other research findings 1500 suggest that microparticles derived from RBCs are actually protective against pro-inflammatory calpain inhibitor except it is expected that there will be a decrease in microparticle formation. From the observed data, it suggested that there was only an observed signals in endothelial cells. The aim of this project was to test the hypothesis that microparticles 1000 from younger red blood cells protect against oxidative stress signals in human endothelial cells, increase in the microparticle events among the RBC samples that contained only and as they age, if they enhance the oxidative stress response. 500 calcium ionophore. The primary research methods used for this study were the staining of RBC membranes with antibodies and analysis with a flow cytometer, measuring reactive oxygen species production 0 The other data for the RBC samples do not seem to align with the initial hypothesis through reactive oxygen species (ROS) assays, and observing the structural changes of the red Day 2 Day 2 Day 2 Day 2 Ca, Day 2 Ca, Day 6 Day 6 Ca Day 6 Day 6 Ca, Day 6 Ca, Untreated Calcium CaIono Calp,Iono Calp,Iono, Untreated CaIono Calp,Iono Calp,Iono, made by other research. There was also evidence in the cellular structure of the RBC U0 U0 blood cells using bright field microscopy. Results suggested that there was only an observed samples that much change did not occur in regards to microparticle formation. The increase in the microparticle events among the RBC samples that contained only calcium major difference between the samples from day 2 and day 27 is that the day 27 ionophore. The other data for the RBC samples do not seem to support the initial hypothesis that was made. Further research needs to be completed to prove whether microparticles RBCs samples seemed to have lysed and there is no particular shape, where as the contain protective properties or if they contribute to cardiovascular diseases. Figure 2: Comparison graph of Day 2 and Day day 2 RBCs samples seem to have a distinct shape. 6 microparticle formation for Sample 2 Keywords: red blood cells, microparticles, oxidative stress Conclusion

7000 Introduction In this study, multiple research methods were utilized and data was generated. 6000 However, there were multiple factors that contributed to some of the Coronary Heart Disease (CHD) is the most common form of cardiovascular disease. CHD occurs 5000 inaccuracies of the study. For example, the ROS assays did not prove to be a when the arteries supplying blood to the heart become narrow and stiffen due to the build up of plaque

[1]. The formation of plaque is also known as atherosclerosis, a chronic inflammatory condition, 4000 successful procedure because of some procedural errors that occurred. characterized by monocyte recruitment to the wall of the large arteries, endothelial dysfunction, and Series1 For future projects pertaining to this research study, a ROS needs to be oxidative stress, and secretion of pro-inflammatory mediators. 3000 Red blood cells (RBCs) have various roles that are important in making sure that the body functions completed to test the initial hypothesis regarding if microparticle exists as properly. They are responsible for delivering oxygen and removing carbon dioxide and other waste 2000 reactive oxygen species. This would then address the issues on if gases to and from the body. Red blood cells obtain their bright red color from the hemoglobin, 1000 microparticles are a result of cardiovascular disease and if they do have pro- which contains iron and helps transport oxygen and carbon dioxide with ease. As blood passes through the lungs, oxygen molecules attach to the hemoglobin. Then, as blood moves through the inflammatory properties that prevent oxidative stress. 0 body’s tissues, the hemoglobin frees the oxygen to the cells. Carbon dioxide then attaches to the Day 2 Day 2 Day 2 Day 2 Ca, Day 2 Ca, Day 6 Day 6 Ca Day 6 Day 6 Ca, Day 6 Ca, Untreated Calcium CaIono Calp,Iono Calp,Iono, Untreated CaIono Calp,Iono Calp,Iono, hemoglobin, which transports the CO2 away [2]. U0 U0 Research has shown that storing blood for extended amounts of time leads to more oxygen entering the blood, causing oxidative stress, and also formation of microparticles. Microparticles are membrane vesicles that are released by RBCs and other types of cells such as platelets, leukocytes, and Figure 3: Comparison of Day 2 and Day 27 endothelial cells. They are 1mm in diameter and possess pro-inflammatory and pro-coagulant Acknowledgements properties [3]. Their formation is due to a calcium influx that is frequently caused by apoptosis and cell RBC samples from microscope. activation [4]. Although this is a normal process that happens in the body frequently, the formation of

microparticles is believed to increase over time in stored blood. Sample 2 While some research studies have suggested that there is an increase in microparticle formation in the This research was based upon work supported by the National Science Foundation under plasma of patients with cardiovascular diseases, others have suggested that microparticles formed Grant #HRD-0963629 (G-STEM) and the U.S. Department of Education; Student Aid and from RBCs actually protect against pro-inflammatory sign in human endothelial cells. Endothelial cells Fiscal Responsibility Act; Title III Grant (SAFRA, Part F). Any opinions, findings and form the inner lining of a blood vessel and provide an anticoagulant barrier between the vessel wall conclusions or recommendations expressed in this material are those of the authors and and the blood. However, conflicting evidence shows that RBC-derived microparticles have a key role in the processes of inflammation, coagulation, and vascular function, which are all involved in the do not necessarily reflect the views of the National Science Foundation or the U.S. pathogenesis of cardiovascular diseases [4], [5]. The primary aim of this project was to test the Department of Education. Any opinions, findings and conclusions or recommendations

hypothesis that microparticles from younger red blood cells protect against oxidative stress signals in Day 2 Unt. Day 27 Unt expressed in this material are those of the authors and do not necessarily reflect the human endothelial cells, and as they age, whether they enhance the oxidative stress response. This views of the National Science Foundation. This research was performed at the Royal study consisted of using a cocktail of different inhibitors in order to observe the effect it had on the Veterinary College located in Camden-London, England during June 16, 2013 through presence of microparticles over a period of time. July 27, 2013 under the mentorship of Dr. Charlotte Lawson. I would like to thank Dr. Lawson, Pierre, Royal Veterinary College, Arcadia University, Dr. Terezinha Galvao, Dr. Materials and Methods Nripendra Bose, Ms. Karen Clay, G-STEM Program, and Spelman College.

Materials: Day 2 Calcium Day 27 Calcium

Two blood packs with different blood types were collected and rationed into four 50 mL test tubes for References the purpose of this study. 1x phosphate buffered saline solution was used to dilute the blood samples and other used as a solvent for other solutions. [1] New York State Department of Health. “Type of Cardiovascular Disease” Coronary Heart Disease. 2012 The following inhibitors were used to test the activity of microparticles: 1µM calcium ionophore, 2.5µM http://www.health.ny.gov/diseases/cardiovascular/heart_disease/types_of_cv.htm calcium chloride solution, 1µM mitogen-activated protein kinase (MEK) inhibitor (U0126), and 1µM 24/8/2013 calpain inhibitor. Day 2 Calcium Ionophore Day 27 Calcium Ionophore [2] The Franklin Institute. “Red Blood Cells: Riding on the Red Road”. 2013 http://www.fi.edu/learn/heart//blood/red.html. 1x Annexin buffer, sizing beads, enumeration beads were used to prepare the control tubes, as well as 16/7/2013. the samples. The controls and samples were placed in FACS tubes to be analyzed by the flow [3] Rubin, Olivier (2012). Microparticles: Clinical Relevance. Transfusion Medicine and Hemotherapy. 343. cytometer. The antibodies anti-CD235 (gylcophorin A), anti-CD61 (intergrin b3/glycoprotein llla) and annexin V-Cy7 were added to the RBC supernatants for preparation for microparticle analysis with the [4] VanWijk, Marja J., E. VanBavel, A. Sturk, R. Nieuwland (2003). Microparticles in Cardiovascular Diseases. Cardiovascular flow cytometer. Research 59, 277-287

For preparation of the ROS assays, white opaque 96 well plates were plated with human umbilical vein Day 2 Ca, CaIono, Calp, U0126 Day 27 Ca, CaIono, Calp, [5] Kriebardis, Anastasios (2012). Cell-derived microparticles in stored blood products: innocent-bystanders or effective endothelial cells (HUVEC), then later was incubated with 100 µM DHR, M199/10% FCS, and 20 µM mediators of post-transfusion reactions? 27, 25-39 hydrogen peroxide solution. [6] Riley, Roger S. Principles and Applications of , 3, 1-15 Methods: [7] Held, Paul (2010). An introduction to Reactive Oxygen Species- Measurement of ROS in Cells. 1-14 1. Isolation of Red Blood Cells from Whole Blood Research Blood Pack 2. Staining of Scrambled RBC Membranes with Antibodies, Annexin V-Cy7, and Merocyanine (MC540) [8] Uy, Benedict (2011). Measurement of Reactive Oxygen Species in the Culture Media Using Acridan 3. Measurement of Reactive Oxygen Species Production using Dihydrorhodamine- 1,2,3 (DHR) Day 2 Ca, Calp, CaIono, U0126 Day Ca, Calp, CaIono, U0126