SCREENING AND RECRUITMENT OF SUBJECTS WITH ALPHA GLOBIN DELETIONS To being studying the function of endothelial alpha globin in humans, we need to identify individuals who have genetic deletions of alpha globin. Deletional mutation in the alpha globin gene are common in many parts of the world. We have chosen to focus on the alpha -3.7 deletion because it affects Africans and African-Americans and has an established protective effect against many complications of sickle cell disease. About 27.5% of African Americans have a deletion of one of four alpha globin genes; 1.9% have two of four genes deleted. We wish to identify men and women who have two of hour alpha globin genes deleted, but are otherwise healthy. To facilitate the screening of healthy volunteers for the presence of two alpha globin -3.7 kb deletions, we are developing quantitative PCR based molecular diagnostics that we can perform in our laboratory. We plan to screen 1,000 - 2,000 young healthy African Americans at mobile fields sites (health fairs, college campuses, etc) as well as by mail. We have written the clinical protocol for this project and it has passed the first levels of review. VASCULAR FUNCTION IN HEALTHY SUBJECTS WITH ALPHA GLOBIN DELETIONS Alpha globin appears to restrict the diffusion of the vasodilator nitric oxide from endothelium to smooth muscle. We wish to test whether alpha globin regulates blood pressure and blood flow in healthy humans. We have designed a study in which we will compare healthy young African American individuals with no alpha globin deletions against those with two alpha globin deletions. We will examine several endpoints that we hypothesize will be affected by loss of endothelial alpha globin: 1) blood pressure, 2) orthostatic blood pressure, 3) shear stress-induced vasodilation, and 4) response to vasoactive infusions. We have written the protocol for these studies which is through the first level of review; in addition we have applied for a U01 grant with the University of Virginia to examine this mechanism in detail via both clinical investigations and experiments with inducible tissue specific knock out in mice. EFFECT OF ALPHA GLOBIN DELETION ON RENAL HEMODYNAMICS IN THE MOUSE We have developed techniques to quantify the expression of alpha globin in tissue and to control for the potential confounding effects of alpha globin transcripts present in circulating reticulocytes (immature red blood cells). We have found that kidney tissue has a high level of alpha globin in tissue that is not derived from blood cells, and that is silenced in mice with genetic deletion of Hba-a1. We hypothesize that deletion of alpha globin from endothelium will affect nitric oxide signaling in renal endothelial cells and alter renal hemodynamics, sodium excretion, and local NO production. To evaluate this hypothesis we are measuring glomerular filtration, sodium excretion and NO production in kidneys of mice, comparing alpha globin intact with alpha globin deleted animals. This will determine whether alpha globin regulates renal function in mice. If so, next we will examine how alpha globin deletion can mitigate the renal injury associated with sickle cell disease. HIGH THROUGHPUT SCREENING OF COMPOUNDS THAT INTERRUPT ENOS/ALPHA GLOBIN INTERACTIONS The ability of alpha globin to limit the diffusion of nitric oxide appears to be dependent on formation of a macro molecular complex with endothelial nitric oxide synthase. Disruption of this complex with a mimetic peptide greatly increases the amount of NO that leaves the endothelium. This presents a druggable target: displacing alpha globin from eNOS with a small molecule should increase NO signaling in the vasculature. To identify these molecules, we are developing a FRET (Forster resonance energy transfer) assay to rapidly screen compounds that displace alpha globin from eNOS. Our goal is to identify existing or novel drugs that disrupt this interaction, test them in model systems, and then bring promising leads to safety testing in humans. Our goal is to develop a new class of drug that increases endothelial nitric oxide signaling. This could potentially benefit patients with blood and vascular diseases such as sickle cell disease or malaria. RED CELL BIOMECHANICS The conventional hypothesis is that alpha thalassemia protects against the complications of sickle cell disease by changing the red blood cell. Specifically, in sickle cell disease, alpha thalassemia is associated with smaller red blood cells, fewer irreversibly sickled cells, and fewer reticulocytes. We have developed a microfluidic device to evaluate red blood cell biomechanic properties quantitatively. We can apply this device and analytical approach to answer several questions: 1) does alpha thalassemia improve the ability of red blood cells to flow through narrow channels; 2) do red cell biomechanical properties changes during the course of sickle cell disease pain crisis; 3) what impact do white blood cells and reticulocytes have on capillary transit. ENDOTHELIAL BARRIER INTEGRITY IN A MOUSE MODEL OF SICKLE CELL DISEASE One of the functions of endothelium is to provide a selectively permeable barrier in the blood vessel wall, regulating the transit of cells and signaling molecules between blood and tissues. The repetitive ischemia-reperfusion injury that occurs in sickle cell disease can causes endothelial injury, but the status of endothelial barrier integrity in sickle cell disease is unknown. We have begun to evaluate endothelial barrier integrity in a mouse model of sickle cell disease by infusing fluorescently labeled albumin and then examining extravasation into tissues. Initially, we are examining the extent and distribution of endothelial permeability in tissues post mortem using fluorometry of tissue homogenates. In the future we will use in vivo microscopy to examine the kinetics of tracers as they move from the vascular compartment into tissue. We hope to use this model to study factors that are responsible for loss of endothelial barrier integrity, and also to study therapeutic interventions that restore or protect the endothelial barrier. BUILDING CLINICAL RESEARCH CAPACITY AT THE CENTRE RECHERCHE ET DE LUTTE CONTRE LA DREPANOCYTOSE (CRLD) IN BAMAKO, MALI The NHLBI Sickle Cell Branch is a leading center for clinical studies of sickle cell disease in the United States. However, sickle cell disease is a global disease, and there is an urgent need to improve the diagnosis and care of children with sickle cell disease who are living in sub-Saharan Africa. To this end, we have identified a clinical research partner organization in Bamako, Mali, the CRLD. The CRLD sees approximately 5,000 patients with sickle cell disease on a regular basis. They provide comprehensive specialty care for patients with sickle cell disease, including molecular diagnosis, education, preventive care, and treatment. We are working with the CRLD to establish IRB-approved natural history protocols to enable the collection of data and specimens from patients, implement electronic capture of clinical data, and provide research training for their staff and fellows. Together we hope to build the clinical research capacity of the CRLD so that we can address questions of public health concern for Malian children with sickle cell disease.

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