ELUCIDATING THE FUNCTION OF ENDOTHELIAL ALPHA GLOBIN IN THE HUMAN CIRCULATORY SYSTEM Nitric oxide is a cell-to-cell 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) recognize the vascular manifestations of inherited deletions of the alpha globin genes, a spectrum of disease called alpha thalassemia; and (3) design or discover molecules that target endothelial alpha globin to modulate nitric oxide signaling in small vessels. Our ultimate goal is to translate the knowledge we gain into new treatments for diseases caused 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 taking a variety of approaches involving human population studies as well studies of isolated blood vessels obtained from humans. These human studies are supported by experiments in mouse models and biochemical studies of alpha globin and its potential binding partners. HUMAN POPULATION STUDIES At the human population level, we are interested in whether genomic deletions of alpha globin may alter nitric oxide signaling and therefore affect susceptibility to vascular conditions such as high blood pressure, kidney disease, stroke, and myocardial infarction. An association between alpha globin gene copy number and these vascular disease manifestations 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 find that alpha globin genotype does not alter the risk of developing hypertension, but may be associated with diastolic blood pressure. We have also found an association between alpha globin genotype and kidney disease.We will continue on to examine the relationship between alpha globin genotype and stroke or myocardial infarction. Together, these studies will tell us whether alpha globin deletions are associated with a change in vascular disease risk. In order to support future studies of 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 200 individuals and returned the results to them. We hope to engage 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. 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. HUMAN ISOLATED VESSEL STUDIES In addition to the population epidemiologic approach, we are also studying the expression and function of alpha globin in isolated human vessels. Through a collaboration with Jeremy Davis, the NIH Surgeon-in-Chief, we have obtained tissue from individuals undergoing abdominal surgery here at the NIH Clinical Center. We have microdissected small vessels (less than 100 micrometers in diameter) from these tissue specimens and examined alpha globin gene expression. We found high levels of alpha globin transcripts in these vessels that appear to be coming from vascular cells rather than blood cells. Next, we will examine gene expression from vessels obtained from subcutaneous fat biopsies taken from individuals with deletions in the alpha globin genes to determine whether this common alpha globin gene deletion affects vascular expression of alpha globin. If so, then we intend to compare the vasoreactivity of vessels taken from individual with low alpha globin expression against the vasoreactivity of vessels obtained from healthy controls with normal levels of alpha globin expression. These studies will reveal whether alpha globin regulates endothelial nitric oxide signaling. MURINE MODEL STUDIES In order to study the function of alpha globin the circulatory system, we are developing mouse models. First, 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. Based on this information, we are currently developing new knockout mice individually targeting each alpha globin gene. 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 ITS PUTATIVE PARTNER, 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. In collaboration with scientists at the NCATS, we are developing assays to screen compounds that destabilize binding of alpha globin / eNOS. Compounds that destabilize this complex would be predicted to acutely increase the amount of NO that diffuses from endothelium to smooth muscle, causing vasodilation.
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