Alpha globin and vascular reactivity in humans: loss of alpha globin genes in human subjects is associated with improved nitric oxide-mediated vascular perfusion

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Content ALPHA GLOBIN AND VASCULAR REACTIVITY IN HUMANS:
LOSS OF ALPHA GLOBIN GENES IN HUMAN SUBJECTS IS ASSOCIATED
WITH IMPROVED NITRIC OXIDE-MEDIATED VASCULAR PERFUSION
by
Christopher Clement Denton, MD
A Thesis Presented to the
FACULTY OF THE USC KECK SCHOOL OF MEDICINE
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(Clinical, Biomedical and Translational Investigation)
May 202 1
Copyright 2020 Christopher Clement Denton, MD
ii

ACKNOWLEDGMENTS
This project was completed under the mentorship of Thomas D. Coates, MD
1
and the scholarly
oversight of the other members of my Master’s Committee: Jon A. Detterich, MD
1
, Yves DeClerck, MD
1
,
and Michael C.K. Khoo, PhD
2
. Contributing authors also included Payal Shah, MS
3
, Silvie Suriany, MS
3
,
Honglei Liu, BS
3
, Wanwara Thuptimdang, PhD
2
, John Sunwoo, PhD
2
, Patjanaporn Chalacheva, PhD
2
,
Saranya Veluswamy, MD
1
, Roberta Kato, MD
1
, and John C. Wood, MD PhD
1
.
C.C.D. performed the experiments, analyzed and interpreted the data, performed statistical
analyses, and wrote the paper. P.S. assisted with experiments, analyzed the data, and assisted with
statistical analyses. S.S. and H.L. managed serum samples and analyzed the data. T.W., J.S., P.C. and S.V.
analyzed and interpreted the data. R.K., J.C.W. and M.C.K.K. designed the experiments and analyzed the
data. J.A.D. designed and assisted with the experiments and analyzed and interpreted data. T.D.C.
conceived and designed the experiments, analyzed and interpreted the data, and assisted in writing the
paper. All authors reviewed and revised the manuscript.
The authors thank Martine Torres, PhD for critical reading of the manuscript and editorial
assistance.

1
Department of Pediatrics, Keck School of Medicine, USC
2
Department of Biomedical Engineering, Viterbi School of Engineering, USC
3
Department of Pediatrics, Children’s Hospital Los Angeles
iii

TABLE OF CONTENTS

Acknowledgments.........................................................................................................................................ii
List of Tables & Figures................................................................................................................................iv
Abstract…......................................................................................................................................................v
Introduction............................…...................................................................................................................1
Methods….....................................................................................................................................................3
Results…........................................................................................................................................................5
Discussion…..................................................................................................................................................7
References....................................................................................................................................................9
Appendix.....................................................................................................................................................12

iv

LIST OF TABLES & FIGURES

Table I…………………………………………………………………………………………………………………………………………....Page 5
Figure 1……………………………………………………………………………………………………………………………………….....Page 6
Supplemental Table I and Supplemental Figure 1..............................................................................Page 12
Supplemental Table II and Supplemental Figure 2.............................................................................Page 13
Supplemental Table III and Supplemental Figure 3............................................................................Page 14

v

ABSTRACT
Alpha thalassemia is a hemoglobinopathy due to decreased production of alpha globin protein from loss
of up to four alpha globin genes, with one or two affected in trait phenotype. Co-inherited alpha
thalassemia trait reduces the risk of morbid outcomes in sickle cell disease, in which the
pathophysiology is largely driven by blood perfusion. The alpha globin protein is present in the
endothelial wall of human arterioles and participates in nitric oxide scavenging during vasoconstriction.
Decreased production of alpha globin due to alpha thalassemia trait may thereby limit nitric oxide
scavenging and promote vasodilation. To evaluate this mechanism, we performed flow-mediated
dilation, microvascular post-occlusive reactive hyperemia, and local thermal hyperemia in 27 subjects
(15 alpha thalassemia trait and 12 healthy controls). Flow-mediated dilation was significantly higher in
subjects with alpha thalassemia trait after controlling for age (P=0.0357), but the latter studies were not
different between groups. As subjects had no anemia or hemolysis, the improvement in vascular
function can be attributed to the difference in alpha globin gene status. This may explain the beneficial
effect of alpha thalassemia trait in sickle cell disease and suggests that alpha globin gene status may play
a role in other vascular diseases.
1

INTRODUCTION
Humans normally have four α-globin genes encoding the α-globin protein. Individuals who are
missing one α-globin gene have no clinical phenotype and are called silent carriers. Those missing two α-
globin genes are commonly referred to as α-thalassemia trait and have microcytosis, while individuals
missing three or four α-globin genes have moderate (hemoglobin H) or profound anemia (homozygous
α-thalassemia), respectively (Piel & Weatherall, 2014). Individuals with sickle cell disease (SCD) can co-
inherit loss of one or two α-globin genes, which has long been known to influence SCD physiology
(Steinberg & Sebastiani, 2012). SCD patients missing one or two α-globin genes have higher hemoglobin,
and lower bilirubin and reticulocyte count (Higgs et al., 1982). Interestingly, absence of α-globin genes in
SCD is associated with decreased rates of stroke (Flanagan et al., 2011), cerebral vasculopathy
(Bernaudin et al., 2008; Joly et al., 2016), acute chest syndrome and leg ulcers (Nolan et al., 2006), with
risk reductions ranging from 34 to 71%. These effects are thought to be related to higher nitric oxide
(NO) bioavailability (Nolan et al., 2006) due to the associated lower hemolytic rate (Higgs et al., 1982)
and therefore lower NO consumption, although the exact mechanism is not known.
Recently, studies showed that the α-globin protein is present in the endothelial wall of
myoendothelial junctions of human arterioles, where it interacts with endothelial NO synthase to
modulate NO scavenging during vasoconstriction (Butcher, Johnson, Beers, Columbus, & Isakson, 2014;
Straub et al., 2012). In a mouse model, disruption of this interaction using an α-globin mimetic peptide
increases endothelial NO activity, independently of NO production, and reduces systemic hypertension
(Straub et al., 2014). Treatment with the α-globin mimetic also increases blood perfusion in mice, and
similarly ameliorates vasoconstriction in biopsied human arterioles ex vivo (Keller et al., 2016). Thus,
there appears to be a relationship between absence of α-globin and arterial vasodilation, but studies of
vasodilation in humans have not been reported.
2

The association between α-globin and NO-mediated vascular signaling suggests a mechanism
that could explain the clinical effect of missing α-globin genes in SCD, where sickling crises are related to
changes in blood perfusion as well as deformability of red cells (Eaton, Hofrichter, & Ross, 1976).
Chronic hemolysis in SCD impairs NO bioavailability through release of plasma hemoglobin and arginase,
which scavenge NO and eliminate NO precursors, respectively (Morris et al., 2005). However, given the
fact that α-globin is present in the arteriolar endothelium, it would seem that reduced number of α-
globin genes, and consequently reduced α-globin protein, would decrease local NO scavenging in the
arteriole and improve microvascular perfusion, perhaps explaining the effect of missing α-globin genes
on SCD complications. Therefore, to isolate the effects of α-globin from those of hemolysis in humans,
we investigated whether loss of α-globin genes improves blood perfusion in non-SCD subjects with α-
thalassemia trait, a naturally occurring human model of α-globin gene knockout.

3

METHODS
Subjects missing one or two α-globin genes and healthy controls were recruited from the
Hematology Clinic at the Children’s Hospital Los Angeles (CHLA). Informed consent was obtained from all
patients as approved by the CHLA Institutional Review Board. Blood samples were obtained from all
subjects for determination of hemoglobin, mean corpuscular volume (MCV), reticulocyte count, plasma
hemoglobin, lactate dehydrogenase (LDH), and α-globin gene status, which was determined using the α-
Globin StripAssay® (ViennaLab Diagnostics GmbH, Austria). Patients with hemoglobin H and α-
thalassemia major were excluded due to potential confounding effects of anemia and transfusion
therapy on blood perfusion (Porter & James, 1953).
We performed flow-mediated dilation (FMD) using Doppler ultrasound (Vivid Gi, GE Healthcare,
USA) and the Brachial Analyzer software (Medical Imaging Applications, USA) to measure brachial artery
diameter before and seven minutes after a three-minute cuff occlusion of the forearm to 30 torr above
baseline systolic blood pressure (Detterich et al., 2015). Microvascular post-occlusive reactive
hyperemia was measured simultaneously using laser Doppler flowmetry (LDF; Periflux 5000, Perimed
AB, Sweden) and photoplethysmography (PPG; Nonin Medical Inc., USA) in the fingertip (Morales et al.,
2005). We also used a local heating unit integrated with the LDF probe to measure local thermal
hyperemia in the forearm by warming the skin to 42°C (Minson, 2010). LDF uses light scatter to
determine the concentration and velocity of moving red cells, assessing blood perfusion in a 1-mm
3

region just below the skin surface (Braverman, Keh, & Goldminz, 1990). PPG similarly measures
volumetric microvascular perfusion (Allen, 2007).
Blood pressure was also measured in the finger at a sampling rate of 200 Hz (Nexfin,
Netherlands). All signals were recorded continuously using the Acqknowledge data acquisition system
(Biopac Systems Inc., Goleta, CA, USA) and exported to a specially designed browser in MATLAB for
processing and analysis (Chalacheva et al., 2019; Shah et al., 2020). Vascular response was quantified by
4

maximal percent change from baseline in brachial artery diameter, LDF, or PPG. We also calculated
vascular compliance by the instantaneous ratio of the change in fingertip blood volume to the change in
pressure, using PPG and blood pressure, respectively.
Statistical analysis was performed with JMP® version 14 (SAS Institute Inc., USA). Univariate
analyses used Student’s t test or Wilcoxon non-parametric test for continuous variables, and Pearson’s
χ
2
test or Fisher’s exact test for dichotomous variables. Skewed continuous variables were transformed
to most closely approximate normality for regression analyses. Stepwise multivariate linear regression
was used to assess vascular response in each experiment, including analysis of variable interaction and
confounders. A p<0.05 was considered significant for the final model.

5

RESULTS
The 27 subjects enrolled in the study included 12 controls (four α-globin genes) and 15 α-trait
subjects, with 10 missing one α-globin gene and 5 missing two. The α-trait subjects had lower mean
MCV than controls (p=0.0099), but hemoglobin levels and markers of hemolysis were normal in both
groups (Table I).

Table I. Subject characteristics
Control
(n=12)
α-trait
(n=15)
P
Age, years 28.7 [±13.4] 40.3 [±16.3] 0.0603*
Sex
Male
Female

5 [42%]
7 [58%]

7 [47%]
8 [53%]

0.7950†
Race/ethnicity
Asian
Other (Black, White)

1 [8%]
11 [92%]

9 [60%]
6 [40%]

0.0140‡
BMI, kg/m
2
25.6 [±7.5] 26.4 [±5.4] 0.7829§
Laboratory values
Hemoglobin, g/dL
MCV, fL
Absolute Reticulocyte, K/µL
LDH, U/L
Plasma hemoglobin, mg/dL

13.1 [±1.4]
85 [±7.3]
63.0 [±24.6]
478 [±106]
9.8 [±9.1]

12.7 [±1.4]
76.6 [±8.4]
67.6 [±18.9]
570 [±91]
7.9 [±6.3]

0.5113§
0.0099§
0.5417*
0.0266§
0.4011§
Values expressed as mean [± standard deviation] for continuous variables, and as n [%] for categorical
variables. P determined by *Wilcoxon rank sum test, †Pearson’s χ
2
test, ‡Fisher’s exact test or
§Student’s t test. Bold P values indicate significant differences, P<0.05.
BMI=Body mass index; MCV=mean corpuscular volume; LDH=lactate dehydrogenase.

There was no detectable difference in FMD between individuals missing one and two α-globin genes;
thus, these subjects were combined into one group referred herein as α-trait. FMD was not associated
with α-trait on univariate analysis (see Appendix, Supplemental Table 1 and Supplemental Figure 1), but
FMD was significantly higher in α-trait subjects (least squares mean ± standard error; 5.5 ± 0.5) than in
controls (3.6 ± 0.5) after adjusting for age (Figure 1A, P=0.036). The known decrease in FMD with age
(Skaug et al., 2013) (Figure 1B) was the same in control and α-trait (P=0.963).

6

Figure 1. Multivariate analysis of flow-mediated dilation (FMD) vs. α-trait and age. (A) Least squares
means of FMD in Control and α-trait. FMD is significantly higher in α-trait (P=0.0357) after controlling for
age. (B) Multivariate regression plot. Regression lines are shown with Control indicated by red points
and α-trait indicated in blue. Model R
2
=0.22 (F 2,24=3.407, P=0.0498).

Although the α-trait and control groups were not ethnicity-matched, ethnicity was not a determinant of
FMD on univariate or multivariate analysis (Supplemental Table I).
We did not detect an effect of α-trait on microvascular perfusion by LDF or PPG after cuff
occlusion (Supplemental Table II, Supplemental Figure 2), or by LDF after thermal stimulus
(Supplemental Table III, Supplemental Figure 3). Multiple subjects were excluded from assessment of
calculated compliance due to incomplete blood pressure measurements, therefore that data is not
shown.

7

DISCUSSION
FMD is thought to reflect NO-mediated vasodilatory effects due to shear in conduit vessels
(Belhassen et al., 2001; Raitakari & Celermajer, 2000; Thijssen et al., 2011), and the extent of arterial
dilatation after cuff release largely depends on the amount and duration of shear stress (Pyke &
Tschakovsky, 2005). Conduit arterial flow, and hence shear stress, is increased by vasodilation of
downstream arteriolar networks (Mullen et al., 2001), which is due to greater NO bioavailability in
individuals with α-trait through decreased α-globin. This is consistent with the approximately 50% higher
FMD that we observed in α-trait subjects.
Endothelial α-globin resides in myoendothelial junctions, which are in greater number in small
resistance arteries and arterioles, but are also present to a lesser extent in larger vessels (Heberlein,
Straub, & Isakson, 2009). We had therefore expected to see an effect of α-trait at the microvascular
level, but we were unable to detect changes in microvascular perfusion (LDF or PPG) or compliance in α-
trait individuals. Multiple factors including NO regulate the microvascular response in post-occlusive
reactive hyperemia (Roustit & Cracowski, 2012), but the relative importance of NO in this vascular
function test has been debated (Cracowski, Minson, Salvat-Melis, & Halliwill, 2006). Local thermal
hyperemia is initially neurally mediated before NO becomes the more prominent vasodilator (Minson,
Berry, & Joyner, 2001); our thermal stimulus was applied for a shorter than typical duration, so again we
may not have captured NO-mediated vasodilation with this test. FMD, on the other hand, has been
demonstrated to be strongly NO-mediated (Green, Dawson, Groenewoud, Jones, & Thijssen, 2014),
therefore it was the most sensitive of these methods to detect vascular changes due to α-trait.
As none of the subjects had evidence of anemia or hemolysis (Table 1), the difference in FMD
between the control and α-trait groups could be best explained by the increased vasodilation due to the
loss of α-globin genes resulting in lower α-globin protein levels (Galanello & Cao, 2011). Whereas one
recent study found no effect of α-thalassemia trait on blood pressure or arterial stiffness (Etyang et al.,
8

2017), this study is the first to employ vascular function methodologies to evaluate α-thalassemia trait in
humans. These findings provide a plausible explanation for the beneficial effect of α-thalassemia trait in
SCD and suggest that exploring α-globin gene status in other vascular diseases may point to a role for
NO in the pathophysiology of these disorders.

9

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12

APPENDIX

Supplemental Table I. Univariate and multivariate analyses of flow-mediated dilation (FMD) vs. subject
characteristics. None of the univariate analyses were significant, but FMD is significantly higher in α-trait
(P=0.0357) after controlling for age. Model R
2
=0.22 (F 2,24=3.407, P=0.0498).
Subject characteristic Univariate analysis: FMD vs. x
Parameter estimate (P)
Multivariate model
Parameter estimate (P)
Age -0.0341 (0.2165) -0.0563 (0.0473)
Sex* ---- (0.2617)
Asian* ---- (0.7252)
BMI -0.0770 (0.2699)
Hemoglobin 0.2802 (0.3786)
MCV -0.0189 (0.7070)
Absolute Reticulocyte -0.0060 (0.7733)
Log 10[LDH] 5.765 (0.2526)
Log 10[Plasma hemoglobin] 2.336 (0.0740)
α-trait* ---- (0.1367) 0.9408 (0.0357)
*Wilcoxon rank sum test. LDH and Plasma hemoglobin were log-transformed to better approximate
normality.

Supplemental Figure 1. Univariate analysis of FMD vs. α-trait, P=0.1367.

13

Supplemental Table II. Univariate analyses of microvascular post-occlusive reactive hyperemia (PORH)
measured by laser Doppler flowmetry (LDF) and photoplethysmography (PPG) vs. subject characteristics.
Asian subjects had lower PORH by LDF than non-Asians (P=0.0184); Asians trended toward higher PORH
by PPG than in non-Asians, but the difference was not significant (p=0.0669). There were no other
significant associations.
Subject characteristic PORH by LDF vs. x
Parameter estimate (P)
PORH by PPG vs. x
Parameter estimate (P)
Age -21.46 (0.4718) 2.643 (0.1764)
Sex* ---- (0.5654) ---- (0.5165)
Asian* ---- (0.0184) ---- (0.0669)
BMI -99.74 (0.2311) -7.972 (0.0917)
Hemoglobin -242.6 (0.4864) 17.18 (0.4336)
MCV -46.90 (0.3700) 0.5648 (0.8724)
Absolute Reticulocyte -18.99 (0.3946) -1.548 (0.2790)
Log 10[LDH] -1925 (0.7474) -131.9 (0.7210)
Log 10[Plasma hemoglobin] 2701 (0.1000) 16.39 (0.8728)
α-trait* ---- (0.2673) ---- (0.1649)
*Wilcoxon rank sum test. LDH and Plasma hemoglobin were log-transformed to better approximate
normality.

Supplemental Figure 2. Univariate analyses of (A) PORH by LDF vs. α-trait, P=0.2673; and (B) PORH by
PPG vs. α-trait, P=0.1649. Note that outliers were excluded in these boxplots, and the difference in scale
reflecting greater relative change in LDF compared to PPG.

14

Supplemental Table III. Univariate analyses of local thermal hyperemia (LTH) vs. subject characteristics.
There were no significant associations.
Subject characteristic LTH vs. x
Parameter estimate (P)
Age 4.298 (0.4124)
Sex* ---- (0.5101)
Asian* ---- (0.2586)
BMI 10.54 (0.4254)
Hemoglobin -16.92 (0.7791)
MCV 2.766 (0.7698)
Absolute Reticulocyte -5.083 (0.1900)
Log 10[LDH] -540.6 (0.5728)
Log 10[Plasma hemoglobin] -417.5 (0.0960)
α-trait* ---- (0.6256)
*Wilcoxon rank sum test. LDH and Plasma hemoglobin were log-transformed to better approximate
normality.

Supplemental Figure 3. Univariate analysis of LTH vs. α-trait, P=0.6256.

Abstract (if available)

Abstract Alpha thalassemia is a hemoglobinopathy due to decreased production of alpha globin protein from loss of up to four alpha globin genes, with one or two affected in trait phenotype. Co-inherited alpha thalassemia trait reduces the risk of morbid outcomes in sickle cell disease, in which the pathophysiology is largely driven by blood perfusion. The alpha globin protein is present in the endothelial wall of human arterioles and participates in nitric oxide scavenging during vasoconstriction. Decreased production of alpha globin due to alpha thalassemia trait may thereby limit nitric oxide scavenging and promote vasodilation. To evaluate this mechanism, we performed flow-mediated dilation, microvascular post-occlusive reactive hyperemia, and local thermal hyperemia in 27 subjects (15 alpha thalassemia trait and 12 healthy controls). Flow-mediated dilation was significantly higher in subjects with alpha thalassemia trait after controlling for age (P=0.0357), but the latter studies were not different between groups. As subjects had no anemia or hemolysis, the improvement in vascular function can be attributed to the difference in alpha globin gene status. This may explain the beneficial effect of alpha thalassemia trait in sickle cell disease and suggests that alpha globin gene status may play a role in other vascular diseases.

Linked assets

University of Southern California Dissertations and Theses

Asset Metadata

Creator Denton, Christopher Clement (author)

Core Title Alpha globin and vascular reactivity in humans: loss of alpha globin genes in human subjects is associated with improved nitric oxide-mediated vascular perfusion

School Keck School of Medicine

Degree Master of Science

Degree Program Clinical, Biomedical and Translational Investigations

Publication Date 12/14/2020

Defense Date 12/14/2020

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag alpha globin,alpha thalassemia trait,flow-mediated dilation,local thermal hyperemia,OAI-PMH Harvest,post-occlusive reactive hyperemia,sickle cell disease,vascular biology

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Coates, Thomas D. (committee chair), DeClerck, Yves (committee member), Detterich, Jon A. (committee member), Khoo, Michael C.K. (committee member)

Creator Email chdenton@chla.usc.edu,dentonius@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c89-413408

Unique identifier UC11666639

Identifier etd-DentonChri-9222.pdf (filename),usctheses-c89-413408 (legacy record id)

Legacy Identifier etd-DentonChri-9222.pdf

Dmrecord 413408

Document Type Thesis

Rights Denton, Christopher Clement

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA