Loss of Alpha Globin Genes in Human Subjects Is Associated with Improved Nitric Oxide-Mediated Vascular Perfusion
Authors: Denton Christopher C , Shah Payal , Suriany Silvie , Liu Honglei , Thuptimdang Wanwara , Sunwoo John , Chalacheva Patjanaporn , Veluswamy Saranya , Kato Roberta Miyeko , Wood John C , Detterich Jon A , Khoo Michael C.K. , Coates Thomas D. 
Published: November 2020

Introduction
Absence of alpha globin genes has long been known to influence the physiology of sickle cell disease (SCD). Individuals with SCD who are missing one or two alpha globin genes have decreased rates of cerebral vasculopathy, stroke, acute chest syndrome, and leg ulcers (Bernaudin, Blood 2008; Flanagan, Blood 2011; Nolan, Br J Haematol 2006). Although there is laboratory evidence of decreased hemolytic rate in these patients (Higgs, N Engl J Med 1982), the mechanism for their improved clinical outcomes has not been identified. Recently, the alpha globin protein has been shown to be present in the endothelial wall of human arterioles, where it modulates nitric oxide (NO) scavenging during vasoconstriction (Straub, Nature 2012). In mice, pharmacological inhibition of alpha globin leads to increased endothelial NO activity, independently of NO production, and results in increased blood perfusion, reduced systemic hypertension, and increased pulmonary artery vasodilation (Keller, Hypertension 2016; Alvarez, Am J Respir Cell Mol Biol 2017). The relationship between absence of alpha globin and arterial vasodilation, and the role of alpha globin in NO-mediated vascular signaling are potential mechanisms that could explain the beneficial effect of missing alpha globin genes in SCD. Using alpha thalassemia as a naturally occurring human model of alpha globin gene knockout, we hypothesized that loss of alpha globin genes leads to improvement in microvascular blood flow in thalassemia trait subjects without hemolysis.
Methods
Alpha thalassemia trait subjects missing one or two alpha globin genes, and healthy controls were recruited to the study, which was approved by the Children's Hospital Los Angeles Institutional Review Board. Blood samples were obtained from all subjects to test for hemoglobin, mean corpuscular volume (MCV), reticulocyte count, plasma hemoglobin, lactate dehydrogenase, and alpha globin genotype. We assessed flow-mediated dilation (FMD) of the brachial artery following distal forearm occlusion (Detterich, Blood 2015) simultaneously with laser Doppler flowmetry (LDF) and photoplethysmography (PPG) in the fingertip. We also measured the increase in microvascular perfusion with a thermal stimulus. The maximal change in vascular perfusion after provocation indicates vasodilatory capacity. Statistical analysis was performed in JMP® version 14 (SAS Institute Inc., USA).
Results
Twenty-seven subjects were enrolled, including 12 controls (4 alpha globin genes), 10 patients with 3 alpha globin genes and 5 with 2. The mean MCV was lower in subjects missing alpha globin genes than in controls (p=0.0099). Importantly, hemoglobin levels and markers of hemolysis were normal in both groups. There was no detectable difference in FMD between individuals missing one and two alpha globin genes; thus, these groups were combined and labeled as alpha trait for further analyses. FMD was significantly higher in alpha trait subjects after adjusting for age (Figure 1, p=0.0357). Missing alpha globin genes had no effect on microvascular flow by LDF or PPG (data not shown).
Discussion
FMD is an established and specific predictor of NO bioavailability (Thijssen, Am J Physiol Heart Circ Physiol 2011), and, in addition to shear-mediated NO circulation in conduit vessels, it reflects the sum of flow in multiple arteriolar networks downstream of the conduit artery. Using this method, a difference in endothelial function between control and alpha thalassemia trait was easily detected (Figure 1). Because endothelial alpha globin is present in arterioles rather than conduit vessels (Butcher, Free Radic Biol Med 2014), we measured microvascular flow in a 1-mm3 volume in the skin using a laser Doppler sensor, and in the fingertip by PPG, but were unable to detect an effect of alpha trait. As none of the subjects had anemia or evidence of hemolysis, the significantly increased FMD associated with loss of alpha globin genes is most likely due to increased NO as a result of decreased scavenging by alpha globin.
The finding reported here that lower alpha globin gene number is associated with increased NO-related perfusion in humans may explain the beneficial effect of alpha thalassemia trait in SCD and suggests that the presence of alpha thalassemia trait may also play a role in other types of vascular disease.

Disclosures
Wood: BiomedInformatics: Consultancy; Imago Biosciences: Consultancy; BluebirdBio: Consultancy; Celgene: Consultancy; WorldcareClinical: Consultancy; Philips Medical Systems: Research Funding. Coates:apo pharma (Chiesi Pharma): Consultancy, Honoraria; Sangamo: Honoraria, Membership on an entity's Board of Directors or advisory committees; Agios pharma: Consultancy, Honoraria; Vifor Pharma: Consultancy, Honoraria; Celgene, BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bluebird Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees.