Am J Physiol Lung Cell Mol Physiol 308: L314–L324, 2015.
First published November 14, 2014; doi:10.1152/ajplung.00252.2014.
Physiology In Medicine
Vasculopathy and pulmonary hypertension in sickle cell disease
Karin P. Potoka1,2 and Mark T. Gladwin1,3
1
Pittsburgh Heart, Lung, Blood and Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, Pennsylvania; 2Division
of Newborn Medicine, Department of Pediatrics, University of Pittsburgh, Pittsburgh, Pennsylvania; and 3Division
of Pulmonary, Allergy and Critical Care Medicine, Department of Medicine, University of Pittsburgh, Pittsburgh,
Pennsylvania
Submitted 2 September 2014; accepted in final form 11 November 2014
sickle cell disease; pulmonary hypertension; nitric oxide; cell free hemoglobin
is an autosomal recessive disease caused by
point mutations in the gene that encodes the ␤-globin chains of
hemoglobin. There are two alleles coding for ␤-globin, and two
␤-globin molecules combine with two ␣-globins (encoded by 4
alleles) to form the normal hemoglobin tetramer. In ⬃75% of
patients with sickle cell disease, a single nucleotide substitution (A to T) in the codon for amino acid 6 causes a substitution
of glutamine for a valine producing hemoglobin S (HbS). The
valine is only exposed in the deoxygenated T-state tetramer
and produces a contact site between ␤-chains leading to polymerization of the hemoglobin in deoxygenated erythrocytes.
The other common form of sickle cell disease is HbSC disease,
caused by dual mutations in the two ␤-globin alleles, one
coding for the glutamic acid-to-valine substitution and the
other for a valine-to-lysine substitution. Other compound heterozygote inheritance patterns can lead to sickle cell disease,
such as HbS-beta thalassemic mutations, where the thalassemia
SICKLE CELL DISEASE
Address for reprint requests and other correspondence: M. T. Gladwin, Div.
of Pulmonary, Allergy, and Critical Care Medicine, Univ. of Pittsburgh, NW
160 Montefiore Hospital, 354 Fifth Ave., Pittsburgh, PA 15213 (e-mail:
[email protected]).
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mutation reduces the expression of a normal ␤-globin protein
and therefore enhances the relative expression of the mutant
␤-sickle globin and HbS.
Of relevance to disease pathogenesis and treatment, hemoglobin comes in various forms, which are expressed at different
stages of development in fetuses, infants, and adults. In early
fetal life until early infancy ␥-globin is expressed instead of the
␤-globin and combines with ␣-globins to form fetal hemoglobin (HbF) tetramers. Because the HbF hemoglobin does not
contain the HbS mutation, sickle cell disease does not become
manifest until ⬃6 mo of life, with fevers, irritability, poor food
intake, splenomegaly, and swelling and inflammation of the
fingers, called dactylitis. Genetic variability in adult HbF
expression explains much of the disease phenotypic variability,
with patients with high HbF levels, often resulting from reduced expression of the transcriptional repressor BCL11A,
exhibiting milder disease severity (12, 61, 88). The FDAapproved medication hydroxyurea acts by increasing the expression of HbF in children and adults.
The pathogenesis of sickle cell disease is driven by the
principles of hemoglobin S polymerization within red blood
cells. This is a biophysical process obeying liquid crystalliza-
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Potoka KP, Gladwin MT. Vasculopathy and pulmonary hypertension in sickle
cell disease. Am J Physiol Lung Cell Mol Physiol 308: L314 –L324, 2015. First
published November 14, 2014; doi:10.1152/ajplung.00252.2014.—Sickle cell disease (SCD) is an autosomal recessive disorder in the gene encoding the ␤-chain of
hemoglobin. Deoxygenation causes the mutant hemoglobin S to polymerize,
resulting in rigid, adherent red blood cells that are entrapped in the microcirculation
and hemolyze. Cardinal features include severe painful crises and episodic acute
lung injury, called acute chest syndrome. This population, with age, develops
chronic organ injury, such as chronic kidney disease and pulmonary hypertension.
A major risk factor for developing chronic organ injury is hemolytic anemia, which
releases red blood cell contents into the circulation. Cell free plasma hemoglobin,
heme, and arginase 1 disrupt endothelial function, drive oxidative and inflammatory
stress, and have recently been referred to as erythrocyte damage-associated molecular pattern molecules (eDAMPs). Studies suggest that in addition to effects of cell
free plasma hemoglobin on scavenging nitric oxide (NO) and generating reactive
oxygen species (ROS), heme released from plasma hemoglobin can bind to the
toll-like receptor 4 to activate the innate immune system. Persistent intravascular
hemolysis over decades leads to chronic vasculopathy, with ⬃10% of patients
developing pulmonary hypertension. Progressive obstruction of small pulmonary
arterioles, increase in pulmonary vascular resistance, decreased cardiac output, and
eventual right heart failure causes death in many patients with this complication.
This review provides an overview of the pathobiology of hemolysis-mediated
endothelial dysfunction and eDAMPs and a summary of our present understanding
of diagnosis and management of pulmonary hypertension in sickle cell disease,
including a review of recent American Thoracic Society (ATS) consensus guidelines for risk stratification and management.
Physiology In Medicine
PULMONARY HYPERTENSION AND SCD
Hemolytic Anemia, Erythrocyte-DAMPs, and Vasculopathy
Although the spleen is the natural site of RBC removal from
the circulation, this system is impaired in patients with sickle
cell disease (SCD). Over time, patient’s spleens are subject to
recurrent vaso-occlusion and infarction, resulting in autosplenectomy. This functional asplenia perpetuates and likely increases the rates of intravascular hemolysis with the sustained
release of red blood cell microparticles and intracellular contents into the circulation. From a clinical perspective, it is
increasingly clear that the severity of hemolytic anemia, as
measured directly by products of intravascular hemolysis (cell
free plasma hemoglobin and red blood cell microparticles) and
by indirect measures (low plasma haptoglobin levels, reticulocyte counts, bilirubin levels, red blood cell aspartate aminotransferase, and lactate dehydrogenase enzymes) is associated
with increased risk of certain vascular complications of sickle
cell disease (68). These include low oxygen saturations and
pulmonary hypertension, increased systemic systolic artery
pressures and pulse pressures, chronic kidney disease and
proteinuria, cutaneous leg ulcerations, and stroke in children.
In numerous clinical cohorts the severity of both anemia and
measured indexes of hemolytic rate are risk factors for developing pulmonary hypertension in sickle cell patients (6, 23, 36,
37, 40, 46, 48, 49, 54, 63). Interestingly, this is in contrast to
the risk factors for other major complications, such as vasoocclusive painful crisis and the acute chest syndrome, which
are associated with higher steady-state hemoglobin levels
(lower hemolysis) and high leukocyte counts (more inflammation) (73). These associations suggest the existence of two
major disease mechanisms that underlie sickle cell disease
pathogenesis and lead to discrete phenotypes: 1) hemolysisendothelial dysfunction leading to vasculopathy, and 2) inflammation and hyperviscosity leading to vaso-occlusion (39).
While the epidemiology links risk of disease phenotype to
these mechanisms, most patients do not present at the extreme
end of the continuum, and significant overlapping features
exist. In addition, during acute vaso-occlusive crisis the red
blood cells can hyperhemolyze, likely leading to the activation
of both mechanisms during acute severe disease exacerbations.
There are a number of mechanisms of hemolysis that lead to
vasculopathy and are caused by the release of molecules that
are typically compartmentalized within the red blood cell,
which shields the endothelium from their pathological effects.
With increased studies characterizing the pathogenic properties
of these intracellular molecules when released into the plasma,
these have now been collectively referred to as erythrocyte
danger-associated molecular pattern molecules (eDAMPs),
which similar to other intracellular DAMPs, are released during massive tissue injury and can activate innate immunity
pathways via the toll-like receptors (TLRs) and the nucleotidebinding oligomerization domain (NOD)-like receptors of the
inflammasome (35).
Three major effects of red blood cell hemolysis have been
characterized that can lead to vascular dysfunction, injury, and
inflammation (Fig. 1).
1) Alteration of the balance between nitric oxide (NO) and
reactive oxygen species (ROS) leading to impaired endothelial
function. The red blood cell releases significant concentrations
of hemoglobin into the plasma when lysed. Hemoglobin is a
potent NO scavenger via the NO dioxygenation reaction and
iron-nitrosylation reactions (69). As little as 6 ␮M cell free
plasma hemoglobin completely impairs NO signaling in endothelium (25). The levels of cell free hemoglobin in patients
with sickle cell disease are tightly correlated to an intrinsic
resistance to NO signaling, as measured experimentally by
impaired blood flow responses to infusions of NO donor
medications, in both humans (78) and transgenic mouse models of sickle cell disease (44, 51, 52, 66). In addition to plasma
hemoglobin, the red blood cell contains significant concentrations of the enzyme arginase 1, which can metabolize L-arginine to ornithine. This reduces L-arginine availability, which is
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tion kinetics, with the extent of HbS polymerization proportional to the degree of hemoglobin deoxygenation, time, and
the concentration of HbS to the 15th power (19). From a
clinical standpoint, factors that modulate these factors will
increase red blood cell HbS polymerization and cause flares of
disease. These factors include hypoxia and high pH (reducing
HbS oxygen affinity), lag time of red blood cells in the
microcirculation caused by inflammation and cellular adhesive
events, and an increase in the intraerythrocytic HbS concentration, often mediated by the activation of membrane ion
channels like the Gardos channel that deplete intracellular
water. Red blood cells with high HbS polymer content become
rigid and become entrapped in the microcirculation, leading to
ischemia and reperfusion events. This is amplified by primary
or secondary inflammation, which increases the expression of
critical adhesion molecules on red blood cells, endothelium,
leukocytes, and platelets that lead to cellular aggregates in the
postcapillary venules. This process is called vaso-occlusive
painful crisis and leads to severe tissue ischemia, reperfusion
injury, and infarction of virtually any organ system. Bone
marrow and periosteal infarction results in severe pain. Vasoocclusion in the brain leads to stroke, in the lung leads to a lung
injury syndrome called the acute chest syndrome, and in the
spleen, kidney, heart, and skeletal muscle leads to organspecific dysfunction. We direct the reviewer to recent comprehensive reviews that summarize in more detail the genetics,
epidemiology, and clinical manifestations of this complex and
common monogenetic disease (19, 39, 77).
Intracellular HbS polymerization also leads to red blood cell
hemolysis, secondary to mechanical shearing of the red blood
cell membrane by the intracellular HbS crystal polymers and
also secondary to intracellular oxidative stress and metabolic
stress, which reduce membrane fluidity and increase the expression of surface phosphatidylserine residues. Hemolysis
causes chronic anemia with a compensatory high cardiac output state, necessary to sustain oxygen delivery, and hyperbilirubinemia with an increase in the formation of bilirubin gallstones and causing jaundice. It is now appreciated that intravascular hemolysis also releases toxic red blood cell products
that impair endothelial function and drive oxidative and inflammatory stress, ultimately leading to chronic vasculopathy
and pulmonary hypertension (37, 39, 45, 59, 64, 78, 82). In the
current review we will focus on this aspect of sickle cell
disease pathogenesis, with a summary of the mechanisms and
effects of hemolysis on vascular function and a review of the
risk factors, prevalence, and impact of pulmonary hypertension
in patients with sickle cell disease. We will also summarize
new therapies targeting sickle cell vasculopathy and available
animal models used to study sickle cell lung disease.
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PULMONARY HYPERTENSION AND SCD
A
B
Cell proliferation
L-Arg
Cell proliferation
NO
L-Arg
L-Cit
eNOS
NO
NO
Platelets
Arginase
ADP
NO3
O2
NO
Cell free zone
NO
NO
SMC
cGMP
GTP
sGC
Vasorelaxation
Vasoconstriction
Cell proliferation
C
L-Arg
eNOS
Uncoupled eNOS
Cell proliferation
D
TLR4
BH2
O2–/H2O2
NO
Heme
Purines
O2–/H2O2
DNA nets
Heme
ONOO–
PMN
O2–/H2O2
O2
XO
NADPH oxidase
NLRs
Uric acid
IL1-β
sGC
Vasoconstriction
Vasoconstriction
Fig. 1. A: normal vascular conditions. Red blood cells (RBC) circulate avoiding the cell free zone under laminar flow. Platelets accompany RBC and are inhibited
by endothelial derived nitric oxide (NO). Endothelial NO synthase (eNOS) produces NO from substrate L-arginine (L-Arg), making L-citrulline (L-Cit) and NO.
NO has a paracrine effect diffusing into smooth muscle cells (SMC), which then activates soluble guanylate cyclase (sGC) forming cyclic guanosine
monophosphate (cGMP) from guanosine triphosphate (GTP), leading to vasodilation. B: sickled cells undergo intravascular hemolysis releasing arginase, ADP,
and hemoglobin tetramers into the vascular space. Cell free hemoglobin (CFH) enters the cell free zone scavenging NO and forming reactive oxygen species
(ROS). Arginase consumes substrate L-Arg. ADP activates platelets via P2Y receptors. C: hypoxia/reoxygenation/hemolysis activates oxidase activity.
Ischemia-reperfusion activates endothelial xanthine oxidase (XO) in addition to mobilizing soluble hepatic XO which binds to vascular endothelial cells and
forms ROS from excess purine nucleotides released from increased RBC formation/enucleation. Leukocyte and endothelium-derived NADPH oxidase (NOX)
forms ROS. ROS inhibits sGC via oxidation, additional superoxide (O2·⫺) formation by eNOS uncoupling and O2·⫺ scavenging of NO within the cell free zone
to form peroxynitrite (ONOO⫺). D: intravascular hemolysis activates the inflammasome. Uric acid formed by XO binds to the intracellular nucleotide-binding
oligomerization domain-like receptor (NLRs) which promotes release of IL1-␤. Cell free heme (CFH) activates the TLR-4 receptors. Both mechanisms lead to
sterile inflammation. ROS and cell free heme activate polymorphonuclear cells (PMN), which release DNA NETs. Products of intravascular hemolysis, such as
hemoglobin, cell free heme, uric acid, and ATP are thus considered danger-associated molecular pattern molecules (DAMPs).
required for de novo NO synthesis by endothelial NO synthase
enzyme. Both arginase 1 enzyme activity and the ratio of the
levels of arginine/ornithine are reduced in patients with
sickle cell disease with higher rates of hemolysis, and
predict both the development of pulmonary hypertension
and the risk of death (64).
In addition to inhibition of NO, cell free hemoglobin can
redox cycle and undergo fenton and peroxidase reactions to
form ROS, which further promote endothelial dysfunction. A
number of studies have suggested that hemoglobin deposition
in tissues, such as the kidney, will oxidize to form ferric and
ferryl hemoglobin species that can react with lipids to generate
injurious lipid peroxide radicals. This has been clearly demonstrated in the case of myoglobinuria during skeletal muscle
injury (16, 76). Hemoglobinuria has been shown to activate a
cascade of compensatory antioxidant and stress responses in
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Endothelium
Physiology In Medicine
PULMONARY HYPERTENSION AND SCD
Pulmonary Vascular Disease in SCD
Pulmonary hypertension (PH) is a disease in which the blood
pressure within the pulmonary system is elevated. It is characterized by endothelial dysfunction and imbalance between
vasodilators and vasoconstrictors within the pulmonary circulation, followed by progressive smooth muscle and neointimal
hyperplasia and physical obliteration of the arteriolar circulation. Progressive increases in pulmonary vascular resistance
(PVR) lead to right ventricle (RV) failure and reduced cardiac
output. Most of the symptoms and signs of PH relate to this
decrease in cardiac output, especially with exertion. Symptoms
include dyspnea on exertion, pre-syncope and syncope, and
peripheral edema with weight gain. The diagnostic evaluation
of a patient with suspected PH includes a history, physical
examination, Doppler-echocardiography, CAT scan, and ventilation-perfusion imaging to exclude thromboembolic disease
and advanced lung disease, laboratory screenings, functional
assessments of exercise tolerance such as the 6-min walk
distance, and right heart catheterization for definitive diagnosis. A distinction between the classifications of PH requires
right heart catheterization (RHC) and is required for the diagnosis of pulmonary arterial hypertension (PAH) and for approval of specific therapy (53). When the mean pulmonary
arterial pressure (mPAP) is ⱖ25 mmHg, this is considered PH.
To further distinguish if the disease is precapillary or postcapillary, the left ventricular end-diastolic pressure must be estimated with an indirect assessment of the pulmonary artery
occlusion pressure (PAOP) or directly with left heart catheterization. If the “wedge” pressure or PAOP is ⱕ15 mmHg this is
defined as precapillary disease and termed PAH, and often
classified as Group 1 PAH (53). If the PAOP is ⬎15 mmHg, it
is considered postcapillary pulmonary venous hypertension
(PVH) or Group 2 PH.
PH has become increasingly recognized as a chronic complication of SCD and other hemolytic anemias. Using Dopplerechocardiographic screening, up to 30% of adults with SCD
have borderline elevations of their estimated pulmonary artery
systolic pressures (20, 37). Transthoracic echocardiography
(Echo) is an efficient tool to estimate elevation in pulmonary
artery systolic pressure by measuring the tricuspid regurgitant
jet velocity (TRV) and using the modified Bernoulli equation
(P ⫽ 4 ⫻ V2 ⫹ right atrial pressure). A value of 2.5–2.9 m/s
is considered borderline and is about 2–3 SDs above the
normal population mean. Despite the fact that only 25% of
these patients will have PH confirmed by right heart catheterization, this borderline group remains at high risk of death and
for progression to frank PH. Patients with borderline values for
TRV who also have a low exercise capacity (6-min walk
distance of ⬍330 m or who have a high plasma level of
NH2-terminal pronatriuretic peptide, a pre-pro-hormone released from the cardiomyocyte under pressure stress) are at
higher risk of having true PH diagnosed at right heart catheterization. A value of ⬎2.9 m/s suggests a 67–75% risk of
having PH identified at right heart catheterization and is associated with a greater than 10-fold increased risk of death in
multiple studies. Additionally, the presence of right heart
dilation or a pericardial effusion on Echo is associated with a
higher probability of having PH at right heart catheterization
and more severe PH with increased mortality (23). The use of
TRV alone does not equate to a clinical definition of PH, which
requires a right heart catheterization, but it does define mortality risk and identifies patients at higher risk of having PH
(20, 37).
Three major studies have screened patients with sickle cell
disease for PH and then have confirmed with right heart
catheterization. These studies indicate that 6 –10% of adult
SCD patient population has a mean pulmonary artery pressure ⱖ
25 mmHg, defining PH (28, 59, 70a). About half of these
patients have a component of diastolic LV dysfunction indi-
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the kidney, including a strong induction of the heme metabolizing enzyme heme oxygenase-1 (8). It is likely that the
formation of ROS by cell free hemoglobin activates downstream oxidases, such as xanthine oxidase and NADPH oxidases that promote vascular oxidative stress (2, 3, 91).
2) Alteration of the balance between NO and ROS, leading
to platelet activation, hemostatic activation, and oxidative
burden. The inhibition of NO signaling by cell free plasma
hemoglobin in platelets also leads to platelet activation and
activation of the hemostatic system (4, 5, 74, 89). In addition,
red blood cells contain high levels of ADP, which can activate
platelets via the P2Y receptors (43). Plasma free hemoglobin at
the level similar to what is observed in SCD patients, ⬃5 ␮M,
causes extravascular macrophage and neutrophil localization
within the lung parenchyma. It also has the ability to activate
the endothelium and increase proinflammatory effects.
VCAM-1 levels have been shown to correlate with both the
rate of intravascular hemolysis and with in vivo measures of
endothelial dysfunction (38, 47, 51). Buehler and colleagues
(17) infused hemoglobin into a rat model and found a modest
increase in plasma oxidative stress markers (malondialdehyde
and 4-hydroxynonenal) and increased lung ICAM protein expression, suggesting an oxidative and inflammatory effect. Cell
free hemoglobin redox cycling from the oxidized ferric hemoglobin to reduced ferrous hemoglobin occurs at a slower rate,
due to the oxidative environment seen in intravascular hemolysis or hemoglobin infusion (17).
3) Heme and ATP-mediated activation of the innate immune
system, leading to sterile inflammation. Recent studies have
shown that intravascular oxidation of cell free plasma hemoglobin generates heme in plasma. Heme has been found to
activate the TLR4 receptor and downstream NF␬B-mediated
inflammation (13, 33). Heme has also recently been shown to
increase ROS and induce neutrophils to release DNA neutrophil extracellular traps (NETs) (22), as well as stimulate the
release of angiogenic placenta growth factor, which drives
pulmonary vascular cellular proliferation (90). Interestingly, a
high rate of hemolysis is also associated with a high rate of red
blood cell production. New red blood cells release their nuclei
and present significant DNA for metabolism, leading to increasing concentrations of uric acid. Uric acid crystals can bind
to the NOD-like receptors to potentially activate the NALP3
inflammasome and increase IL-1␤ production (57). ATP released during hemolysis will also activate this pathway. Hemolysis-dependent elaboration of heme, ATP, and uric acid
will all drive sterile inflammation and can be considered a
new form of danger-associated molecular pattern molecule
(eDAMPs) (35).
Figure 1 summarizes these pathways in the vasculature and
how they inhibit NO signaling in the vasculature and activate
the hemostatic system.
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PULMONARY HYPERTENSION AND SCD
Summary of Recent Guidelines for Screening and Treatment
A consensus guideline has recently been published by the
American Thoracic Society and endorsed by the Pulmonary
Hypertension Association and the American College of Chest
Physicians (53). These guidelines summarize that an increased
risk for mortality is associated with increased TRV ⱖ 2.5 m/s,
right heart catheterization confirmation of PH with a mean
pulmonary artery pressure ⱖ 25 mmHg (either PAH or PVH),
and an elevated NT-BNP ⱖ 160 pg/ml. Stable SCD patients
should be screened with Echo and/or NT-BNP every 1–3 yr to
assess this mortality risk. If patients are found to have TRV ⬎
2.5 m/s and or NT-BNP ⬎ 160 pg/ml, suggesting an increased
risk of PH and higher mortality, they should undergo right
heart catheterization to confirm the diagnosis of PH and guide
specific therapy (53). Patients identified at high risk of death
should be screened for comorbid factors that are treatable,
including low oxygen, iron overload, and thromboembolic
disease. The underlying sickle cell disease should be treated
more aggressively as well, with initiation of hydroxyurea
therapy or consideration of chronic transfusion therapy. Patients diagnosed with PAH by right heart catheterization are at
the highest risk and should be referred to PAH specialists for
consideration of FDA-approved PAH therapies (summarized
below).
Mouse Models of Sickle Cell Disease
A number of transgenic humanized mouse models of SCD
have been developed to better understand the pathophysiology
of the disease and evaluate potential therapies. In the early
nineties, mouse models of SCD were developed by transgenic
expression of sickle human hemoglobin in mice still expressing
mouse hemoglobin or bred with beta-thalassemic mice to
reduce mouse ␤-globin levels. However, these mice failed to
fully model sickle cell disease phenotype due to the persistent
expression of murine adult hemoglobin. To overcome this
problem, knockout mice for both adult mouse ␤-globin genes
and both adult mouse ␣-globin genes were generated, through
targeted deletion. The double knockout mice provided the
adequate genetic background for mice expressing only human
hemoglobin in mature red blood cells. In 1997, the Berkeley
(BERK) mouse was generated by developing transgenic mice
expressing human, ␣1, ␥, ␤s globin and bred with knockout
mice for the murine ␣-globin and ␤-globin genes (24, 71, 72).
This sickle cell mouse develops irreversibly sickled red blood
cells, anemia, and multiorgan pathology similar to that found in
humans with sickle cell disease. However, the BERK mouse
has an unbalanced chain synthesis, ␣/␤ ratio of ⬃1.25, resulting in a ␤-thalassemia-like condition with microcytosis (71). In
addition, there is no expression of ␥-globin in the BERK adult
mice, leading to a more severe phenotype with more challenging breeding.
Control mice for the BERK SCD model were generated with
the same transgenic construct containing the human ␣-globin,
␥-globin, and the human ␤s-globin genes in a heterozygous
mouse for the murine ␤-globin deletion (wild type/␤s), and
they have corrected sickle cell phenotype. These are referred to
as hemizygote mice and are often used as controls, with some
notable limitations (67).
An additional SCD mouse model is the humanized transgenic “Townes” mouse. These mice were created using the
same knockout-transgenic approach described above to replace
the endogenous murine ␣-globin and major and minor ␤-globin
genes with a human hemoglobin transgene construct. The
transgene contains the human ␣-globin gene and both the
human ␥- and ␤s-globin genes. Control mice were generated by
knock-in of a construct containing the human ␥-globin and the
human wild-type ␤-globin gene to obtain a mouse producing
human ␣-globin and ␤-globin (wild type) that have corrected
sickle cell phenotype (84). The Townes mouse model also
develops severe anemia with reduced hematocrit and high
reticulocyte counts. This mouse expresses more fetal hemoglobin at birth, does not have a thalassemic phenotype, and is
easier to breed. Additionally, both of these transgenic models
show multiorgan pathology, including pulmonary vascular
congestion and alveolar thickening, liver infarction, and fibrosis, glomerular congestion and afferent vessel dilation, splenic
congestion and thrombosis, and microvascular congestion in
the cortex of the brain (24, 71, 84). Despite the severe anemia
in these mice, they survive up to 15 mo of age, which provides
an effective model for testing therapeutic approaches for this
disease.
Mouse models have been developed with special emphasis
to analyze the effect of HbF expression in SCD. Patients with
SCD are protected from the complications of the disease at
birth and up to 6 mo of age because of the predominance of
HbF during this period of time. Rational design of therapeutic
strategies for ␤-globin disorders, such as SCD, include targets
that mediate HbF induction. One of these mouse models is the
NY1KO mouse. This mouse model is a knockout of both
murine ␣- and ␤-globins, and expresses exclusively human
sickle hemoglobin by transgene insertion of human ␣-globin,
human ␤s-globin, and human ␥-globin. Minor differences in
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cated by the finding of PAOP ⬎ 15 mmHg (postcapillary PH
or pulmonary venous hypertension) and half have PAH. Interestingly, even in patients with an elevated PAOP the transpulmonary gradient (mean pulmonary artery pressure minus the
PAOP) and the PVR define the risk of death, suggesting that
pulmonary vascular disease defines risk of death in this population (58, 59). Patients diagnosed with PH by RHC with PAH,
evident by elevated mPAP, PVR, and transpulmonary gradient,
have an associated higher mortality rate compared with those
diagnosed as postcapillary PH (53). Consistent with a role for
hemolytic anemia in the pathogenesis, SCD patients diagnosed
with PAH have increased severity of their hemolytic anemia as
indicated by lower hematocrit, higher reticulocyte counts,
higher lactate dehydrogenase, higher red blood cell aspartate
aminotransferase, and higher bilirubin levels (68). In addition,
they have poorer functional capacity evidenced by reduced
6-min walk distance and higher levels of NH2-terminal probrain natriuretic peptide (NT-BNP) (54). Other risk factors
independent of hemolytic anemia have also been identified,
including markers of iron overload, cholestatic liver dysfunction, systolic systemic hypertension, and renal insufficiency
and proteinuria (37, 41, 85). It is likely that functional asplenia
and vascular thrombosis also contribute. Clinical evidence of
vascular dysfunction and PH include lower oxygen saturations, dyspnea, and reduced 6-min walk distance (40, 63). In
addition to age, these clinical characteristics can be used as
tools in order to better determine the need for screening in
patients with SCD.
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Pulmonary Hypertension in Transgenic Humanized Sickle
Cell Mice
Mouse models of SCD provide the opportunity to study the
pathophysiological mechanisms underlying the development
of PH and potential therapeutic strategies. The double knockout, transgenic mouse has been used to demonstrate development of PH as a complication of chronic intravascular hemolysis. In 2006 indirect evidence of PH was shown by histological examination finding pulmonary artery thickening in mice
1– 6 mo old that also had low hematocrit and high reticulocytosis (56a). The use of intact-chest catheterization of BERK
mice 3–5 mo old was able to demonstrate PAH with an average
systolic pulmonary arterial pressure of 23 mmHg compared
with 12.5 mmHg in littermate controls (44). It is important to
note that this PH was correlated with increased NO consumption and eNOS uncoupling in the sickle mice (44). The Townes
sickle cell mouse has been used to interrogate downstream
mechanisms of development of PH. Recently PPAR␥ was
shown to decrease as endothelin-1 was increased in pulmonary
artery endothelial cells, extracted from Townes mice, and
exposed to hemin (a component of cell-free hemoglobin) (18).
A tissue factor inhibition strategy was shown to decrease
vascular injury in the Townes sickle mouse model, a potential
target for therapy (21a). Using the mouse model of sickle cell
disease is a strategy to guide future therapeutic studies in PH.
PAH Therapies and Application to Sickle Cell DiseaseAssociated PH
Treatment of underlying sickle cell disease. SCD patients
with PH have to adapt to both severe hemolytic anemia and
increased pulmonary vascular resistance. They present with a
chronically elevated cardiac output, which likely contributes to
pulmonary vascular shear injury over time and increases the
demands on the right ventricle in the setting of rising pulmonary vascular resistance (1). A central goal of therapy is to
reduce the severity of hemolytic anemia with treatment with
hydroxyurea, which increases the concentration of fetal hemoglobin, and in more severe cases or in patients who cannot
tolerate hydroxyurea, through chronic red blood cell transfusions regimens. Proper transfusion therapy can improve tissue
oxygen delivery and decrease the amount of sickle RBCs being
synthesized. Cardiovascular complications in other hemolytic diseases have been improved with these therapeutic
strategies (87). In addition, therapies that reduce HbS polymerization will reduce vaso-occlusive crises and acute
chest syndrome events. It has been shown that during
episodes of the acute chest syndrome the pulmonary pressures can rise and patients can develop acute right heart
failure (60). These patients suffer a high risk of death after
the acute chest syndrome event, suggesting that the prevention of acute chest syndrome may reduce mortality in
patients with sickle cell disease and PH (53).
Table 1. Transgenic murine models of sickle cell anemia
Mouse Model
Mouse
␣-Globin
Knockout
Mouse
␤-Globin
Knockout
Wild- type
BERK
No
Yes
No
Yes
Townes
Yes
Yes
NY1KO
Yes
(Deleted)
Sickle-Antilles
No
(Deleted)
Transgenic Mutations
None
Human-miniLCR⫹; humanA␥G␥human␣1 human␦
human␤S
Knock-in: inserted downstream of erythroidspecific, murine deoxyribonuclease I superhypersensitive sites; human␣1
humanA␥human␤S
Human-miniLCR⫹human␣2;
human-miniLCR⫹human␤S;
human-miniLCR⫹␥L/M/H
human␣2human␤S; human␣2 human␤S-Antilles
Adult ␥⫺Globin
Expression
Average Adult Mouse Hb
or Hct/Retic, %
None
Human ␤S
0%
0%
Hb 13.5/4–5%
Hct 21–28.7/26.8–48%
Human ␤S
3.2–7.7% ␥/␥⫹␤S
Hb 5.5/58.4%
Human ␤S
␥L ⬍3%
␥M 20%
␥H 40%
0%
␥L Hct 22.4/63%
␥M Hct 34/30%
␥H Hct 41/12.9%
Hct 37/10%
␤S Mutation
Human ␤S/human
␤S-Antilles
See Refs. 24, 27, 56a, 71, 72, 84, 92, 93.
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the ␥-globin construct guide production of three levels of HbF
(low, medium. or high) (24, 72, 92, 93). The increase of HbF
expression from less than 3% to 20% and 40% correlated with
escalating improvement of hematocrit, diminution in reticulocyte
counts, and reduction of severity of disease and tissue damage.
Another model was developed using a transgenic ␤s
mouse and adding a transgene of the ␤-globin sickleAntilles (␤S-Antilles), a mutation originating in a family in the
Caribbean. The ␤S transgene contains the human locus control
region, human ␣-globin, and human ␤s-globin on a mouse
background of murine ␤-globin major deletion. The S-Antilles
transgene contains the human ␤S-Antilles mutation, which occurs
at ␤23 valine-to-isoleucine. This bitransgenic variant makes
HbS plus HbS-Antilles, which enhances the deoxygenationrelated polymerization of hemoglobin tetramers within the red
blood cells (27). Mice have a spontaneous ⬃3.7-kb deletion of
murine ␤-globin major in addition to an insertional disruption
of the murine ␤-globin minor (27, 92). They continue to
express murine ␣-globin, and it must be noted that murine
␣-globin prevents polymerization of Hb tetramers nearly as
effectively as ␥-globin (27, 72). These mice express ⬃60%
human ␣-globin, but under hypoxic conditions develop red blood
cell sickle morphology, severe hemolytic anemia, and organ
pathology similar to what is seen in SCD patients (27).
The array of mouse models for SCD (Table 1) recapitulate
many of the features of sickle cell disease pathophysiology,
including hyperhemolysis, adhesion molecule expression with
vaso-occlusion, endothelial dysfunction, activation of oxidases
such as xanthine oxidase and NADPH oxidases, inflammation,
and ischemia-reperfusion injury (2, 13, 50 –52, 66, 67, 70, 71).
They provide an opportunity to invasively study the complex
pathobiology of vaso-occlusion and vasculopathy and provide
an avenue to probe new potential therapies.
Physiology In Medicine
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PULMONARY HYPERTENSION AND SCD
Vasoconstriction/
Cell proliferation
Oral L-Arginine
Sildenafil
Ambrisentan
PDE5
cGMP
GTP BH4
S -Arg
cGMP
O
L
N
GTP e
ETA-R
L-C
sGC
Inhaled
NO/Nitrite
Endothelin
pathway
it
NO-sGC
pathway
Normal
blood flow
Epoprostenol
Endothelin-1
NO
Restricted
blood flow
IP-R
AC
ETB-R
ATP
cAMP
Selexipag
Bosentan
Prostacyclins
PGE
Prostacyclin
pathway
COX
Arach acid
Vasodilation
The development of therapies to treat pulmonary hypertension has evolved over the past twenty years with drugs that
target the canonical pathways that regulate pulmonary vasodilation and vasoconstriction (Figs. 2 and 3).
Therapies in PAH and SCD targeting L-arginine-NO-sGCcGMP-PDE5-I signaling nexus. Nitric oxide is an important
regulator of vascular function due to its activation of the
smooth muscle cell soluble guanylate cyclase, which in turn
converts GTP to cGMP. As described earlier, in sickle cell
disease the NO signaling axis is disrupted at multiple steps,
suggesting that therapies to enhance this pathway may improve
vascular function. The supplementation of oral L-arginine has
been evaluated in 10 subjects with SCD and was shown to
improve pulmonary artery systolic pressure in patients with
Intravascular space
Fig. 3. New therapies that target the NO
signaling pathway. Soluble guanylate cyclase (sGC) within smooth muscle cells
(SMC) can be stimulated by NO, which
binds to the heme moiety within the heterodimer, activating its cyclase function,
producing cyclic guanosine monophosphate
(cGMP) and promoting vasodilation. sGC
can be oxidized by reactive oxygen species
(ROS) such as superoxide (O2·⫺). The oxidized form does not respond to NO and is
quickly degraded by the proteosome. sGC
activator targets the oxidized (ferric or
heme-free) form of sGC and salvages its
degradation while activating the cyclase
and inducing production of cGMP promoting vasodilation.
eNOS eNOS
BH4
eNOS
uncoupling
eNOS
eNOS
BH2
O2• –
NO
Endothelial cell
sGC activator
sGC stimulator
GTP
cGMP
Fe+2
Fe+3
Oxidized/heme loss
Reduced
(native) sGC
Oxidized
(heme-free) sGC
Smooth muscle cell
Vasorelaxation
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Fig. 2. Vasoconstriction and vasodilation pathways and therapies for pulmonary hypertension (PH). Activation of soluble guanylate
cyclase (sGC) by nitric oxide (NO) increases
cyclic guanosine monophosphate (cGMP) levels and leads to vasodilation and inhibition of
cellular proliferation in smooth muscle cells
(SMC) through the activation of sGC by NO.
Inhaled NO, oral nitrite and nitrate, oral L-arginine or tetrahydrobiopterin (BH4), and phosphodiesterase 5 (PDE5) inhibitors (sildenafil/tadalafil) modulate PH through the NO-sGC-cGMP
pathway. Prostacyclins, produced from arachidonic acid by cyclooxygenase (COX) in endothelial cells, target the prostanoid receptor (IP)
on smooth muscle cells leading to the activation
of adenylate cyclase, which forms cyclic adenosine monophosphate (cAMP) from adenosine
triphosphate (ATP). cAMP promotes vasodilation and inhibits cellular proliferation in SMC.
Epoprostenol is a prostanoid derivative and selexipag is a new IP receptor agonist that can treat
PH through the prostacyclin pathway. Endothelin-1 (ET-1) promotes vasoconstriction and vascular remodeling through binding to ET-A and
-B receptors of the SMC. Ambrisentan binds to
ET-A and bosentan binds to both ET-A and B.
Both ET-1 blockers have been used to treat PH
in the endothelin pathway.
Physiology In Medicine
PULMONARY HYPERTENSION AND SCD
caused by systolic left ventricular dysfunction was supported
by a phase IIb double-blind, randomized, placebo-controlled,
dose-ranging hemodynamic study (15).
In a disease like SCD a profound amount of hemolysis
exists, leading to low NO bioavailability (38), through NO
scavenging by the hemoglobin-dioxygenation reaction. The
oxidative stress within the vasculature renders much of the
smooth muscle cell sGC oxidized and inactive. The ability of
these new sGC activator medications to bypass the NO pathway and activate sGC directly and increase cGMP and induce
vasorelaxation may offer a potential therapeutic advantage for
PH in hemolytic diseases. Raat and colleagues (75) recently
demonstrated that plasma free heme-associated vasoconstriction, due to scavenging of NO, had little improvement using
both sodium nitroprusside and sildenafil. However, the vasoconstriction could be reversed with sGC modulators working
independently of NO, and restoring cGMP mediated vasodilation in rats.
Endothelin 1 pathway. The inflammatory cytokine endothelin-1 (ET-1) induces vasoconstriction, inflammation, fibrosis,
and cellular proliferation when it binds to its receptors ET-A
and ET-B (62). Levels of ET-1 are elevated in patients with
SCD suffering from vaso-occlusive crisis and PH (42). In
erythrocytes when ET-1 binds to ET-B it activates the membrane Gardos channels, which causes dehydration of the RBCs
and increases hemolysis (80, 81). A recent small retrospective
case series showed safety and efficacy of ET receptor blockers
in SCD associated PH, with significant improvement in 6-min
walk distance (6MWD) and a trend towards significant decrease in NT-BNP and LDH indicating its usefulness in this
population (62). A placebo-controlled clinical trial of bosentan
was stopped early secondary to withdrawal of support by the
sponsor, but did report safety of the drug in this patient
population (10).
Epoprostenol is an extremely short acting, but potent, pulmonary vasodilator. This therapy was proven effective with 12
wk of continuous infusion in patients with primary PH, with
improved 6MWD, mPAP, and PVR (11). The use of this
intravenous prostacyclin has been shown to acutely decrease
PVR in SCD patients with PH diagnosed during RHC (20).
Further development of the prostanoids has produced subcutaneous, inhaled, and oral formulations that have been effective
in improving symptoms and exercise performance in clinical
trials (30). There are no data with SCD patients with associated
PH using these agents.
New medications in development. ACT-293987, otherwise
known as selexipag, is a non-prostanoid-selective IP agonist
and has potential for potent vasodilation without as many side
effects as prostacyclins, due to its target selectivity. It is
delivered in an oral form, which has an easier delivery than
many other prostanoids. This drug has only been tested in
primary PAH, and not in SCD patients. In a Phase II trial in
patients with PAH, there was a significant decrease in PVR
compared with patients treated with placebo (86).
Conclusions
Patients with SCD in the developed world are now living
long into adulthood. This change in the survival of patients
with this disease results in the presentation of new long-term
vasculopathic complications such as systemic hypertension and
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SCD-associated PAH treated for 10 days; however, this has not
been tested in a larger clinical trial (65). The inhalation of NO
was approved by the FDA for the treatment of persistent fetal
circulation in newborns, and has been shown to be effective in
PAH patients (21). While inhaled NO gas has not been studied
in patients with SCD and PH, a multicenter trial of inhaled NO
for SCD patients admitted for veno-occlusive crisis showed no
efficacy (34).
Sildenafil is a phosphodiesterase 5 inhibitor that increases
the levels of the downstream amplified second messenger
cGMP and promotes pulmonary vasodilation. A trial of sildenafil for the treatment of PAH demonstrated improved exercise
capacity, World Health Organization functional class, and
hemodynamics in patients with symptomatic pulmonary arterial hypertension that were treated for 12 wk (29). Machado
and colleagues (56) tested sildenafil in a pilot study of 12
patients with SCD treated for up to 6 mo. They observed
improved PH and exercise capacity. This small pilot study lead
to the multicenter, double-blind, placebo-controlled Phase 2
trial evaluating the effects of sildenafil in subjects with SCD
(55). The study’s primary endpoint was improved exercise
capacity; however, the study was stopped early by the data
safety and monitoring board based on the observation of an
increased rate of hospitalizations secondary to pain episodes
compared with placebo (55). It is important to note that the first
trial using sildenafil to treat PAH (idiopathic, congenital systemic to pulmonary shunt and associated with connective tissue
disease) also had an increased incidence of myalgias, back
pain, and limb pain compared with placebo (29). In the longterm follow-up study, the same was observed, with treatmentrelated myalgias, back pain, limb pain, and also arthralgias
noted in the treatment group (83). After 6 mo of treatment with
tadalafil (a long-acting phosphodiesterase 5 inhibitor) for erectile dysfunction there was an observed increase in myalgias,
back pain, and general pain compared with placebo in both the
10-mg and 20-mg treatment groups (42a). These data suggest
that there is a class effect of the phosphodiesterase 5 inhibitors
on chronic back and limb pain that may have increased hospitalization rates for pain in SCD patients in the Walk-PHaSST
(Pulmonary Hypertension and Sickle Cell Disease with Sildenafil Therapy) trial. Future studies will be required to ascertain
whether newer agents that increase cGMP via sGC activation
will have the same complications. However, these do not
appear to cause myalgia or back pain in PAH or chronic
thromboembolic PH (CTEPH) patients (31, 32).
The potential to treat conditions in which nitric oxide levels
are insufficient would have great therapeutic potential. A new
class of drugs has been developed that are able to bypass nitric
oxide bioavailability. These drugs are able to target soluble
guanylate cyclase within smooth muscle cells directly, increasing intracellular levels of cyclic guanosine monophosphate and
inducing vasodilation (Fig. 3). One compound stimulates the
reduced form of the cyclase enzyme’s sensitivity to even low
amounts of NO that is available (26). The other compound can
activate the oxidized form of guanylate cyclase without NO
being present. Three recent studies were able to demonstrate
the effectiveness of the stimulator form for different types of
pulmonary arterial hypertension. First, riociguat for the treatment of idiopathic pulmonary arterial hypertension was shown
to be effective (32). Second, it has demonstrated improvement
of CTEPH (31). Additionally, riociguat for patients with PH
L321
Physiology In Medicine
L322
PULMONARY HYPERTENSION AND SCD
PH, which are now recognized as major risk factors for death
in adults with SCD. A major risk factor for development of
chronic vasculopathy and PH in patients with SCD is hemolytic anemia (37), caused by the pathological effects of plasma
cell free hemoglobin and other eDAMPs. Screening patients
with SCD for PH for early diagnosis and disease modifying
therapy is recommended.
DISCLOSURES
M. T. Gladwin is a coinventor on a National Institutes of Health government patent for the use of sodium nitrite for the treatment of cardiovascular
diseases.
AUTHOR CONTRIBUTIONS
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