Expression and Localization of Alpha Globin in the Vascular Endothelium

Abstract

RationaleAlpha globin was recently discovered in the endothelium of murine resistance arteries, where it regulates diffusion of nitric oxide. In humans, alpha globin is expressed by two distinct loci, HBA1 and HBA2, with HBA2 expression predominating in red cell progenitors. However, gene expression of each specific locus in the vasculature, as well as the cellular localization and abundance of alpha globin in human vascular endothelium, is currently unknown.MethodsSmall arteries from the omentum and subcutaneous adipose were collected from patients during clinically indicated surgery at NIH Clinical Center (protocol 13‐C‐0176) and perfused to remove residual blood. To determine locus specific gene expression, RNA was extracted from 8–10 small arteries dissected from omentum and subcutaneous adipose of each patient. Expression of HBA1, HBA2, NOS3 (endothelial Nitric Oxide Synthase, eNOS), and AE1 (an erythrocyte gene) was quantified by digital droplet PCR. To interrogate vascular localization and abundance, dissected and perfused arteries were stained with fluorescent antibodies for alpha globin and eNOS and were imaged by multiphoton microscopy (Leica DMi8). Nuclear staining was performed with DAPI. The abundance of alpha globin was calculated as the ratio between alpha globin positive punctates and the number of endothelial nuclei measured in each vessel.ResultsRobust expression of HBA1 and HBA2 was observed in freshly dissected small arteries from both the omentum and subcutaneous adipose of human research subjects. While HBA2 is expressed 10‐fold higher than HBA1 in whole blood, HBA2 and HBA1 are expressed only 1.6:1 in the vasculature. AE1 was highly expressed in blood but not in arteries, confirming complete perfusion of vessels. NOS3 was expressed in all vessels, but not blood. These data indicate that alpha globin is expressed in human small arteries, but locus‐specific expression is significantly different in vessels than in blood, suggesting tissue‐specific regulation of HBA transcription. Multiphoton microscopy of intact small arteries from the omentum showed robust localization of alpha globin on the basolateral side of the vascular endothelium, co‐localizing with eNOS. Structural analysis shows alpha globin primarily localized to the junctions of the endothelium and vascular smooth muscle in small protrusions through the internal elastic lamina. In both omentum and subcutaneous adipose arteries, imaging revealed a ratio of 1.2 alpha globin positive punctates per nucleus, suggesting that the alpha globin within each cell localizes in a single point along the internal elastic lamina.ConclusionWe found strong expression of HBA1 and HBA2 in human arterial tissue, with an expression ratio distinct from expression in whole blood. We also found that alpha globin protein localizes with eNOS at junctures between the vascular endothelium and smooth muscle. Building on these results, we plan to investigate the mechanisms of tissue‐specific enhancement of HBA expression, as well as to further elucidate the function of endothelial alpha globin in arterial vasoregulation.Support or Funding InformationFunding from NIH Division of Intramural Research.