Nitric Oxide and Endothelial Function in Patients with Malaria

National Institute Of Allergy And Infectious Diseases

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Abstract

ELUCIDATING THE FUNCTION OF ENDOTHELIAL GLOBINS IN THE HUMAN CIRCULATORY SYSTEM Nitric oxide is a signaling molecule that is released by endothelial cells and acts on vascular smooth muscle cells to regulate vessel diameter and therefore blood flow and blood pressure. Recent work has identified alpha globin as a regulator of endothelial nitric oxide (NO) signaling. We need to learn how this novel mechanism of endothelial NO regulation works in the human circulatory system in order to (1) understand normal human vascular physiology, (2) define the vascular manifestations of common inherited deletions of the alpha globin genes, and (3) design or discover novel molecules that target endothelial alpha globin to modulate nitric oxide signaling in small vessels. Our long-term goal is to translate the knowledge we gain into new treatments for diseases characterized by vascular dysfunction, such as malaria, sickle cell anemia, and chronic vascular disease. To understand how alpha globin regulates nitric oxide signaling between endothelial cells and vascular smooth muscle cells, we are screening healthy African-American volunteers for a -3.7 kb deletion that inactivates one of the alpha globin genes. We are studying vasoreactivity and nitric oxide signaling directly in small arteries obtained by biopsy. In addition, we are performing genetic association studies at the population level in a range of disease where endothelial nitric oxide signaling may be important. These human studies are supported by experiments in mouse models in which we have inactivated one or more alpha globin genes and by biochemical studies of alpha globin with its potential binding partners. HUMAN ISOLATED VESSEL STUDIES In order to study the role of alpha globin in regulating small vessel reactivity and function, we have initiated a clinical protocol to screen healthy African Americans for alpha thalassemia, a common condition caused by inheriting one or two deletions of the alpha globin gene locus. Based on our analysis of a population sample of African Americans in the REGARDS Study, 27% of the African American population are carriers of the alpha globin deletion (i.e., heterozygotes), and 4% are homozygous for the alpha globin deletion. Our goal is to screen 1,000-2,000 healthy African Americans to develop a cohort of approximately 30 individuals who are homozygous for the deletion and 30 age and gender-matched controls. We have established a CLIA-compliant laboratory to conduct the genetic testing and return the results to the participants with appropriate genetic counseling. We have thus far screened over 350 individuals and returned the genetic testing results to them. We are engaging these individuals in more detailed studies to elucidate the function of alpha globin. By comparing vasoreactivity of individuals with different genetically-determined levels of alpha globin expression, we can test the hypothesis that alpha globin regulates NO signaling in the human vasculature. We are also elucidating the expression and function of alpha globin in isolated human arteries. Through a collaboration with Jeremy Davis, the NIH Surgeon-in-Chief, we obtain omentum tissue from individuals undergoing abdominal surgery here at the NIH Clinical Center. We microdissect small vessels (less than 100 micrometers in diameter) from these tissue specimens and examine alpha globin gene expression, subcellular protein localization/co-localization, and vasoreactivity. In addition, we perform subcutaneous fat biopsies on individuals with genomic deletions in the alpha globin genes to determine whether this common alpha globin gene deletion affects vascular expression of alpha globin, protein concentration, and vasoreactivity. These studies will reveal whether alpha globin regulates endothelial nitric oxide signaling in human arteries. HUMAN POPULATION STUDIES In addition to the studies of freshly isolated human arteries, we are studying the impact of alpha globin gene deletions on common diseases at the human population level. An association between alpha globin gene copy number and vascular disease outcomes would imply that alpha globin has an important role in vascular health. To address this question, we have partnered with the investigators of the REasons for Geographic and Racial Differences in Stroke (REGARDS) Study. This study includes approximately 11,000 African Americans who have been followed for more than ten years to evaluate their vascular disease status. We have determined the alpha globin genotype of each participant using a novel digital droplet PCR approach that we developed in our laboratory. We are now performing candidate gene genetic association studies to determine whether alpha globin gene deletions are associated with changes in vascular disease severity and outcomes. To understand how alpha globin genotype affects vascular function during an episode of malaria, we are collaborating with a Nicholas Anstey at the Menzies School of Health Research. His research team has measured vascular function using near-infrared spectroscopy in patients who were acutely ill with malaria. We are analyzing genomic DNA from each patient to determine the number of functional alpha globin genes present. We have developed a panel of assays to identify the six most common deletions or mutations present in people from Malaysia and Indonesia. We will analyze the data for an association between functional alpha globin copy number and vasodilatory response. This collaborative effort will reveal whether alpha globin deletions alter vascular function during a malaria infection. MURINE MODEL STUDIES In order to study the function of alpha globin in the circulatory system, we are developing mouse models. The mouse models are important because they provide an opportunity to isolate the vascular effects of alpha globin gene expression from the red blood cell effects. We do this by exploiting differences in cell-specific expression of the Hba1 and Hba2 genes or by engineering tissue-specific knockouts. To understand which alpha globin genes are expressed in the blood vessel wall, and which genes are expressed in red blood cells, we developed locus-specific molecular probes that measure the expression of the Hba1 and Hba2 genes separately. We have identified which gene is predominantly expressed in the vasculature and which gene is predominantly expressed in the developing red blood cell. Next, we have used CRISPR-Cas9 to inactivate Hba1, Hba2, or both. We will study the vascular changes in these models to understand both the immediate function of alpha globin in the vasculature as well as how changes in vascular alpha globin expression alter blood pressure and susceptibility to/severity of malaria and sickle cell disease. BIOCHEMICAL STUDIES OF ALPHA GLOBIN AND ENDOTHELIAL NITRIC OXIDE SYNTHASE Our working model is that alpha globin binds to eNOS to limit NO diffusion. We have measured the binding affinity of purified alpha globin with a recombinant eNOS oxygenase domain. Our next step is to use this knowledge of the biophysical interactions of alpha globin and eNOS to screen for or design a small molecule that destabilizes the alpha globin/eNOS complex. We are developing assays to screen compounds that destabilize the alpha globin / eNOS complex. Compounds that destabilize this complex would be predicted to acutely increase the amount of NO that diffuses from endothelium to smooth muscle, causing vasodilation. IN SUMMARY, we are taking a multidisciplinary approach to elucidate the role of endothelial alpha globin as a regulator of small artery vasoreactivity, understand the impact of alpha globin gene deletions on common diseases, and develop a new class of therapeutics that modulate endothelial nitric oxide signaling.