Which Alpha Globin Gene Is Primarily Expressed in the Vascular Endothelium?
Authors: Brooks Steven , Carhuas Carlos , Keller Alex , Isakson Brant E. , Ackerman Hans C. 
Published: June 2021

Alpha globin was recently discovered in the endothelium of resistance arteries, where it interacts with endothelial nitric oxide synthase (NOS3) to regulate the diffusion of nitric oxide. People with Sickle Cell Disease (SCD) who co‐inherit one or two deletions of the alpha globin gene (a condition called alpha thalassemia) are protected against cerebrovascular and renal complications of SCD, raising the question of how endothelial alpha globin deletion modulates vascular pathophysiology. Alpha globin is expressed by two distinct loci, HBA1 and HBA2, with HBA2 contributing the majority (~65%) of total alpha globin in human red blood cells. However, the expression profile of each locus in the vasculature is currently unknown. Determining total and relative locus‐specific expression of alpha globin in resistance arteries is critical to understanding the consequences of HBA1 or HBA2 deletion.To measure expression of vascular alpha globin, perfused vessels (conduits‐ carotid arteries, thoracic aorta; resistance vessels‐ middle cerebral arteries, gracilis arterioles, 3rd order mesenteric arteries) and whole blood were collected from C57Bl/6J mice. Total mRNA was isolated from vessels and whole blood (vessels‐ RNeasy Kit, Qiagen; blood‐ RiboPure Blood Kit, Ambion), and converted to cDNA (SuperScript IV VILO, Invitrogen). Absolute expression of Hba‐a1 (mouse homolog of HBA2), Hba‐a2 (mouse homolog of HBA1), Nos3, and Ae1 (erythrocyte anion exchanger) was quantified by digital droplet PCR (BioRad).In C57Bl/6J mice, abundant expression of Hba‐a1 and Hba‐a2 was observed in whole blood, as well as in all vascular tissues. In whole blood, the Hba‐a1/Hba‐a2 ratio was (2.55:1), consistent with human blood. However, in all vascular tissue, the Hba‐a1/Hba‐a2 ratio was inverted (0.60:1). Ae1 was highly expressed in whole blood, but not in vascular tissue; Nos3 was expressed in all vessels, but not in whole blood. These data indicate that expression of alpha globin is differentially regulated in the vasculature compared to whole blood, with Hba‐a2 being predominant within the vasculature.To further interrogate locus‐specific expression of alpha globin, absolute gene expression was quantified in Hba‐a1(−/−) global knockout (KO) and inducible endothelium‐specific Hba‐a1 KO mice. In global Hba‐a1(−/−), there was a significant reduction in the Hba‐a1/Hba‐a2 ratio in whole blood [ratio (0.069:1)] and in absolute Hba‐a1 expression in all vessels [range (80%–99%) decrease], while expression of Hba‐a2 and Nos3 remained consistent with control. In inducible endothelium‐specific Hba‐a1 KO mice, absolute Hba‐a1 expression was significantly decreased in vascular tissue; however, Hba‐a2 and Nos3 expression also decreased significantly, with Hba‐a1/Hba‐a2 ratio remaining equal to control. These findings suggest that expression of both alpha globin loci, as well as Nos3, may be co‐regulated within the vasculature.Overall, we report robust, locus‐specific expression of alpha globin in the vasculature. Further investigation into the physiologic role of endothelial alpha globin may provide insight into how deletion of alpha globin modulates vascular pathologies associated with SCD and other diseases.Support or Funding InformationNHLBI, NIAID Intramural FundingThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.