ORIGINAL ARTICLE

Endothelial activation and inflammation biomarkers in childrenand adolescents with sickle cell disease

Emmanuil S. Hatzipantelis • Zoe Dorothea Pana • N. Gombakis •

A. Taparkou • V. Tzimouli • D. Kleta • D. J. Zafeiriou • V. Garipidou •

F. Kanakoudi • M. Athanassiou

Received: 29 September 2012 / Revised: 12 June 2013 / Accepted: 17 June 2013 / Published online: 27 June 2013

� The Japanese Society of Hematology 2013

Abstract Sickle cell disease pathogenesis is a complex

interplay of multiple factors associated with vascular

endothelial activation, intense oxidative stress, and

increased sickle cell adhesion. The aim of this study was to

determine and compare three panels of plasma circulating

biomarkers at ‘steady state’ and during veno-occlusive

crises (VOC) in a cohort of children and adolescents with

SCD and healthy controls. The following biomarkers were

assessed: acute phase reactants, endothelial factors, and

adhesion molecules. Forty-one SCD pediatric patients and

28 healthy children were enrolled. Patients at ‘steady state’

presented significantly elevated plasma levels of endothe-

lin-1 (ET-1), soluble-VCAM-1 (sVCAM-1), soluble

P-selectin (sP-selectin), and d-dimers compared to the

control group. ET-1, sP-selectin, platelet-derived growth

factor (PDGF), von Willebrand factor (vWf), d-dimers,

erythrocyte sedimentation rate (ESR), and C-reactive

protein (CRP) seems to represent additional, but not

independent, prognostic markers of VOC crisis. Elevated

plasma levels of sP-selectin, ET-1, and sVCAM-1 were

associated with VOC frequency. The present study pro-

vides preliminary evidence of a possible association

between these biomarkers and the endothelial activation at

steady state and VOC in childhood SCD. Further pro-

spective studies are required to confirm the potential

independent prognostic value of these markers in different

stages of pediatric SCD.

Keywords Sickle cell disease � Childhood � ET-1 �sP-selectin � Endothelial dysfunction

Introduction

Sickle cell disease (SCD) is an inherited disorder of

hemoglobin (Hb) synthesis, caused by a single nucleotide

substitution (GTG[GAG) at the sixth codon of the beta-

globin gene that presents a worldwide distribution with

long-term health implications for a significant number of

SCD pediatric patients [1]. For many years, SCD patho-

genesis was attributed predominantly to the polymerization

of a defective form of Hb, HbS, which leads not only to

substantial alterations in red cell morphology but also in its

function, shortening red cell life span and inducing vascular

occlusion phenomena. Though, the rapidly expanding field

of vasculopathy research has recently revealed that SCD is

the final result of more complex multifactorial microva-

sculopathy phenomena [2–4]. Among various pathological

mechanisms attributed to SCD, the red sickle cell–endo-

thelium interaction, the endothelial activation and finally

the adhesion of white and red blood cells on the vessel wall

can induce the formation of a heterocellular aggregate.

E. S. Hatzipantelis � Z. D. Pana (&)

2nd Pediatric Department of Aristotle University of

Thessaloniki, AHEPA University Hospital, St. Kyriakidi 1,

54639 Thessalonı́ki, Greece

e-mail: panazoi@gmail.com

E. S. Hatzipantelis

e-mail: chatzipa@med.auth.gr

N. Gombakis � A. Taparkou � V. Tzimouli �D. J. Zafeiriou � F. Kanakoudi � M. Athanassiou

1st Pediatric Department of Aristotle University of Thessaloniki,

Hippokration General Hospital, Thessalonı́ki, Greece

D. Kleta

General Haematology Laboratory, Hippokration General

Hospital, Thessalonı́ki, Greece

V. Garipidou

2nd Propedeutic Department of Internal Medicine,

Hippokration General Hospital, Thessalonı́ki, Greece

123

Int J Hematol (2013) 98:158–163

DOI 10.1007/s12185-013-1392-y

These phenomena may increase blood viscosity leading

eventually to the obstruction of micro-circulation [2, 4].

Prior studies have shown that increased production of

adhesion molecules have been implicated in SCD patho-

genesis and progression, but the quantitative comparison of

a large panel of plasma circulating biomarkers in the SCD

pediatric setting between acute sickle cell crises and

steady-state conditions is not sufficiently evaluated. The

goal of this study was to quantitatively assess and compare

three categories of circulating biomarkers at ‘steady state’

condition and during VOC crises in a cohort of children

and adolescents with SCD and healthy controls.

Materials and methods

We studied 41 children and adolescents with the diagnosis

of SCD (22 boys and 19 girls) aged 2–24 years (mean age

14.3 years). Thirty-seven children were double heterozy-

gotes of SCD and thalassemia (S/b-thal) and 4 children

SCD homozygotes (S/S). Among the S/b-thal group, 2

children presented with S/Hb Lepore hemoglobinopathy, 1

with S/a-thal, 29 with S/b?-MA and 5 with S/b0-MA,

respectively. A second control group consisted of 28

healthy children (mean age 6.4 years, range 2.5–14). In all

41 SCD patients, blood samples were obtained during an

‘‘asymptomatic state’’ condition and additionally during a

VOC episode.

From the peripheral blood samples of the patient and

control groups the following parameters were determined.

(a) Acute phase reactants: erythrocyte sedimentation rate

(ESR), C-reactive protein (CRP), and interleukin 6 (IL-6);

(b) endothelial factors: nitric oxide (NO), endothelin-1

(ET-1), platelet-derived growth factor (PDGF), von

Willebrand factor (vWF), thrombomodulin (TM) and

D-dimers; and (c) adhesion molecules: soluble P-selectin,

vascular cell adhesion molecule-1 (VCAM-1), and b2-

integrins (LFA-1, LFA-2, and LFA-3). Voluntary informed

consent was obtained from each parent as appropriate.

We also assessed the presence of significant quantitative

differences in the above-mentioned laboratory parameters

between subgroups stratified according to: age (2–14 and

15–22 years) and sex (male/female). Data from the medical

records of the 41 SCD patients were collected, concerning

the number of thrombotic crisis/year (\5 episodes, [5

episodes), presence of silent (tacit) cerebral infarction and

presence of osteonecrosis in femoral head. These data were

further compared with the laboratories parameters during

‘‘steady state’’, respectively.

Venous blood samples (10–15 ml) were obtained from

peripheral veins. Blood was drawn in plastic tubes con-

taining 3.2 % trisodium citrate as anticoagulant. EDTA and

plain tubes were used to perform baseline hematological

and biochemical investigations, as well as for the mea-

surement of ESR and levels of CRP and IL-6. Platelet-poor

plasma was prepared by centrifugation at 2000g for 10 min

at room temperature and then the plasma aliquots were

stored at -70 �C until use. vWF was assayed using a Dade

Behring kit (BCS Dade Behring analyzer). TM, PDGF,

soluble P-selectin, VCAM-1, ET-1, and IL-6 levels were

determined by ELISA. Regarding NO, a quantitative

method (R&D Systems’ Total Nitric Oxide Assay) that

counts NO2- was used. The levels of the b2-integrins

LFA1, LFA2, and LFA3 were measured by flow cytometry.

Statistical analysis

Differences between groups were assessed with analysis of

variance (ANOVA), and post hoc comparisons were per-

formed with Dunnett’s multiple comparison and Scheffe’s

tests. In all cases, P \ 0.05 was considered statistically

significant. All analyses were carried out with SPSS Sta-

tistical package 10.1 for Windows.

Results

Comparison of age and sex

The stratification of our patient and control groups

according to age, contributed to the comparison of the

biomarkers values among the different age subgroups,

which revealed no significant statistical differences

between patient and control group. The comparison of the

different age subgroups among patients with SCD showed

that patients aged \14 years presented elevated levels of

ET-1, sP-selectin, TM and HbF and decreased levels of

fibrinogen, D-dimers and Hb compared to children with

older age ([14 years) (Table 1). As far as sex is concerned,

differences in the circulating levels of ET-1, sP-selectin,

VCAM-1 and TM in the patient SCD group (41 pts) were

observed. In particular, male patients with SCD presented

in general higher mean levels of the above-mentioned

biomarkers compared to the females (ET-1, sP-selectin,

VCAM-1 and TM mean levels in males were 19.5 ± 3,

398 ± 21, 570 ± 23 and 12.8 ± 1.6, respectively, versus

18.1 ± 5.2, 386.3 ± 42, 565 ± 19 and 12.3 ± 2 in

females), but none of these markers’ differences reached

statistical significance (Table 1).

Asymptomatic state

In the patient group during asymptomatic state the fol-

lowing results were observed: normal values of acute phase

markers, such as CRP, ESR and IL-6, and statistical sig-

nificant elevated levels of D-dimers, compared to the

Biomarkers in SCD 159

123

healthy control group. Pathological values were also

reported for the endothelial activation markers. In partic-

ular, statistical significant elevated levels of ET-1

(P \ 0.001), sVCAM-1 and sP-selectin were observed. On

the other hand, all 3 integrins LFA-1, LFA-2, LFA-3 levels

were not statistically elevated compared to the control

group. The mean values and the differences in all patient

and control groups are shown in Table 2. Furthermore, the

Table 1 Laboratory parameters

between male/female patients

and between 2 different age

groups

Laboratory parameters Male patients (N = 22) Female patients (N = 19) P value

ET-1 19.5 ± 3 18.1 ± 5.2 NS

sP-selectin 398 ± 21 386.3 ± 42 NS

VCAM-1 570 ± 23 565 ± 19 NS

TM 12.8 ± 1.6 12.3 ± 2 NS

Laboratory parameters Age group I: 2–14

years (N = 16)

Age group II: 15–22 years

(N = 25)

P value

ET-1 18.4 ± 2.3 15.15 ± 2.71 NS

sP-selectin 370 ± 59 268 ± 17 NS

TM 15.84 ± 4.8 10.23 ± 3.55 0.04

HbF 14.2 ± 1.83 7.78 ± 1.79 0.02

Fibrinogen 274.2 ± 23.5 398 ± 58.2 NS

D-Dimers 298.2 ± 62.5 367.3 ± 3.5 NS

Hb 8.2 ± 0.22 9.4 ± 0.28 NS

Table 2 Acute phase reactants, endothelial factors, adhesion molecules and blood cell count in SCD patients and controls

Laboratory parameters

(normal value range)

SCD patients in ‘‘steady state’’

(N = 41) (mean ± SE)

SCD patients during VOC

(N = 41) (mean ± SE)

Healthy controls

(N = 28) (mean ± SE)

P1 value P2 value

I. Acute phase reactants

ESR (1–20 mm/h) 8.4 ± 2.3 38.2 ± 7.8 5.8 ± 1.9 NS 0.001

CRP (\0.5 mg/dl) 0.3 ± 0.0 5.6 ± 2.0 0.2 ± 0.0 NS 0.02

Fibrinogen (180–350 mg/dl) 339.4 ± 34.7 554.1 ± 38.1 266.2 ± 24.3 NS 0.05

IL-6 (0–12 pg/ml) 4.1 ± 0.7 6.0 ± 1.6 4.3 ± 0.6 NS NS

II. Endothelial factors

ET-1 (0.3–0.9 pg/ml) 18.1 ± 3.8 39.4 ± 9.2 0.6 ± 0.1 0.002 0.01

D-dimers (50–228 lg/l) 341.4 ± 75.2 721.8 ± 164.2 153.3 ± 55.6 0.04 0.03

vWF (50–150 %) 105.4 ± 9.5 177.6 ± 24.7 99.7 ± 9.1 NS 0.04

Trombomodulin (TM)

(1–12 ng/ml)

12.1 ± 3.1 12.7 ± 4.6 10 ± 3.6 NS NS

PDGF (14–44 ng/ml) 32.6 ± 2.9 59.1 ± 8.1 28.8 ± 1.8 NS 0.04

III. Adhesion molecules

sP-selectin (50–150 ng/ml) 247.8 ± 34.9 398.8 ± 47.9 81.1 ± 7.0 0.002 0.03

LFA-1 (\75 %) 76.4 ± 3.6 80.7 ± 1.6 74.7 ± 5.0 NS NS

LFA-2 (\75 %) 78.9 ± 2.9 84.5 ± 1 77.2 ± 3.6 NS NS

LFA-3 (\75 %) 78.9 ± 3.6 79.8 ± 1.2 76.3 ± 5.5 NS NS

sVCAM-1 (400–700 pg/ml) 787.6 ± 53.2 799.6 ± 38 510.9 ± 19.8 0.03 NS

IV. Blood cell count

White blood cells

(5000–10500/mm3)

10532.26 ± 558.36 12207.14 ± 561.83 7030.4 ± 202.3 NS NS

Platelets (150–350 9 103/ll) 375.6 ± 24.2 487.1 ± 130.9 243.2 ± 24.1 NS NS

Hemoglobin (11–13 g/dl) 8.7 ± 2.1 7.2 ± 1.47 13.83 ± 1.32 0.05 NS

P1 values refer to the comparison of SCD patients in ‘‘steady state’’ and healthy controls, while P2 values to the comparison of SCD patients in

‘‘steady state’’ and during VOC episode

160 E.S. Hatzipantelis et al.

123

values of the complete blood cell count for the patient and

control group were recorded in Table 2.

VOC

The patient group during VOC has shown pathological

values as follows: significantly higher values of acute

phase reactants, such as ESR and CRP compared to

‘‘steady state’’ patient group and elevated levels of IL-6

that did not reach statistical significance. As far as the

endothelial activation markers is concerned, the patient

group during VOC crisis presented significantly elevated

levels of ET-1, sP-selectin, and PDGF compared to both

the patient group during asymptomatic state and the control

group, while the integrins LFA-1, LFA-2, LFA-3 levels

were increased during VOC, but not significantly when

compared to both controls and ‘‘steady state’’ patients.

Finally, our data revealed an increase of NO levels during

VOC compared to steady-state patients, but this change did

not reach statistical significance (Table 2).

VOC frequency

Furthermore, we collected the number of VOC crisis per

year in our patient group, which was for that reason strat-

ified into two subgroups: in particular, 19 (46.34 %)

patients with SCD presented [5 VOC episodes per year,

while the remaining 22 (53.6 %) patients \5 for the same

period, respectively. Among patients with an increased

frequency of VOC/year, the biomarkers observed in sig-

nificantly higher levels were only sP-selectin, sVCAM-1

and ET-1. The mean values of the significantly elevated

biomarkers between these two subgroups are shown in

Table 3.

MRA findings

Magnetic resonance angiography imaging (MRA) has

revealed 8 SCD patient cases (19.5 %) with silent cerebral

infarction. In this subgroup, elevated levels of sP-selectin,

ET-1, and D-dimers were observed, but not statistically

significant. Further imaging evaluation with magnetic

resonance imaging (MRI), showed femoral osteonecrosis

in 10 cases (24.3 %), which presented elevated levels of

D-dimers, ET-1, sP-selectin that did not reach statistical

significance.

Discussion

Accumulating data have shown that the vascular endothe-

lial cell activation in SCD induces the expression of cir-

culating inflammatory cytokines mediating the chronic

inflammatory state implicated in the disease pathogenesis

and progression [3]. The pathological adherence of sickle

cell erythrocytes and white blood cells to the vascular

endothelial cells is mainly induced by an increased pro-

duction of adhesion molecules. In the recent years there has

been an increased interest in evaluating various sensitive

biomarkers associated with outcome, risk of vaso-occlu-

sion phenomena, cardiovascular disease, pulmonary

hypertension and in general SCD disease burden [5, 6].

Interacting factors include endothelial adhesion proteins,

such as vascular cell adhesion molecule-1 (VCAM1),

intercellular adhesion molecule-1 (ICAM-1), E-selectin

and P-selectin, and a long list of plasma proteins that could

serve as intermediaries in adhesion phenomena, such as

fibrinogen, von Willebrand factor, and thrombomodulin.

Pathways that activate adhesive components potentially

include inflammatory cytokines, NO deficiency, and oxi-

dant stress. Likewise, increased exposure to inflammatory

cytokines such as interleukin-6 and angiogenic factors have

been also associated with pulmonary hypertension and

altered cardiopulmonary function in the pediatric SCD

setting [5, 6]. In our study, we quantitatively assessed and

compared three categories of circulating biomarkers at

‘steady state’ and during VOC crises in a cohort of children

and adolescents with SCD and healthy controls. To our

knowledge, this is the first study conducted in Greece in

pediatric SCD patients evaluating together in a more

holistic approach biomarkers of all three possible patho-

genetic mechanisms implicated in SCD: (I) acute phase

reactants (ESR, CRP, IL-6), (II) endothelial factors (ET-1,

PDGF, vWF, TM) and (III) adhesion molecules (sP-

selectin, LFA-1, LFA-2, LFA-3, sVCAM-1).

In particular, inflammatory markers, such as ESR and

CRP were strongly associated with VOC crisis in our

pediatric SCD patients, while IL-6 levels presented dif-

ferences between patient group with VOC and in steady

state or with controls, but did not reach statistical signifi-

cance. These results are partially consistent with a recent

study published by Krishnan et al., in which high sensi-

tivity CRP (hs-CRP) level was the most significant corre-

late of hospitalizations for painful episodes. Furthermore,

hs-CRP was the only inflammatory marker positively

Table 3 Comparison of laboratory parameters and report of signifi-

cant differences in patients with SCD based on the number of

thrombotic crisis/year (\5 episodes, [5 episodes)

Laboratory

parameters

[5 episodes of

thrombotic crisis/

year (n = 19)

\5 episodes of

thrombotic crisis/

year (n = 22)

P value

ET-1 43.47 ± 3.84 27.43 ± 2.44 0.03

sVCAM-1 815 ± 2.47 720 ± 12.0 0.05

sP-selectin 269.71 ± 5.22 186.71 ± 4.15 0.04

Biomarkers in SCD 161

123

correlated with veno-occlusive crisis in a regression model

[7]. In another study, conducted by Naprawa, IL-6 levels

were entered in a binary recursive partitioning model as a

possible prognostic marker for the diagnosis of VOC and

acute chest syndrome (ACS) in patients with SCD.

According to their results, IL-6 was not an accurate marker

in identifying present or incipient ACS in young SCD

patients [8].

Among the endothelial activation biomarkers and the

adhesion molecules, it is important to emphasize that

serum levels of ET-1 presented significantly higher levels

in patients during steady state compared to controls and

additionally higher levels during VOC crisis compared to

patients during steady state, reaching in both comparisons

statistical significance (mean plasma values of ET-1 during

steady state 18.1 ± 3.8 and VOC 39.4 ± 9.2, respectively,

while in healthy controls ET-1 levels were 0.6 ± 0.1).

SP-selectin levels were increased in steady-state patients

compared to controls. Additionally, in patients during the

acute crisis, sP-selectin levels were over fourfold the level

of normal volunteers. Both significantly elevated levels of

ET-1 and sP-selectin were additionally associated with a

higher frequency of VOC per year in our patient group. On

the other hand, sVCAM-1 levels were increased in SCD

patients compared to controls, but the differences between

steady state and VOC were not significant. The above-

mentioned endothelial dysfunction in patients with sickle

cell anemia during and after sickle cell crises was recently

also presented by both brachial artery assessment and

increased serum levels of adhesion molecules in a study

conducted by Blum et al. [9].

Although not statistically significant, the mean values of

vWf were higher in SCD patients with VOC crisis than in

those with steady state, while D-dimers levels reached

statistical significance difference between asymptomatic

and during VOC crisis in SCD patients. Similarly, Ataga

et al. have observed that patients with SCD had elevated

levels of similar biomarkers, such as thrombin–antithrom-

bin complex, prothrombin fragment F1?2 and D-dimers,

while measures of hemolytic rate correlated with indices of

hypercoagulability [10].

Platelet activation has also been proposed as significant

contributing factor to the pathogenesis of and outcome of

SCD patients. In our cohort study, the levels of the

angiogenic marker PDGF, implicated in the PTL activa-

tion, was statistically significantly elevated during VOC

compared to controls and elevated but not significantly

during the steady state. The role of PDGF in SCD pro-

gression was also present in a study of Niu et al. [11], in

which PDGF-BB was independently associated with

increased risk of developing worsening pulmonary hyper-

tension as it was correlated with an elevated tricuspid

regurgitation velocity in a logistic regression model.

Akinsheye et al. [12] has recently reviewed the role of

the pathological NO bioavailability in SCD expressed by

decreased production or increased consumption or due to

NO resistance. Though, data regarding clinical studies on

NO levels in SCD are rather controversial. The majority of

them focused on evaluating plasma NO metabolite levels,

nitrite and nitrate (NOx), and plasma arginine levels.

A number of them report that patients with SCD present

high levels of NO, while others support NOx levels

and L-arginine are depressed, particularly during vaso-

occlusive crisis and the acute chest syndrome (ACS), and

that these levels vary inversely with pain symptomatology

[13–18]. A more recent study by Radhakrishnan et al. [19]

has shown that lower airways NO level is increased in

children with SCD. In our study, plasma NO was assessed

by a quantitative method that counts NO2-. Our data

revealed an increase of NO levels during VOC compared to

steady-state patients, but this change did not reach statis-

tical significance.

Furthermore, Gladwin et al. [20] reported sex differ-

ences in sickle cell disease morbidity and mortality among

adult patients, which was attributed to an apparent pro-

tective effect of ovarian estrogens on endothelial function.

According to the authors, estrogens increase endothelial

NO synthase expression and basal endothelial NO pro-

duction and appear to prevent endothelial dysfunction. In

our cohort study, it is interesting to note that male patients

with SCD presented in general higher mean levels of

several plasma circulating biomarkers, such as ET-1,

sP-selectin, VCAM-1 and TM, compared to the female

ones, but a statistical significance was not reached. This

observation needs further evaluation especially in older

pediatric and adolescent patients to clarify the possible

protective role ovarian estrogens have on endothelial

function in sickle cell disease.

One of the limitations in our study was the absence of a

multiple regression model analysis which could reveal

highly sensitive independent prognostic factors during VOC

crisis in children with SCD. Though the aim of our study

was to assess the differences in a large panel of biomarkers

during steady state and VOC in pediatric SCD patients, our

results may serve as preliminary data of evidence that could

possibly help in the future as a basis to select the appropriate

biomarkers for a regression model in larger cohort studies

and furthermore to determine valuable prognostic cut off

values. These data could help in a better clinical classifi-

cation of these children, to a prompt diagnosis of SCD

complications and finally to a better selection of the group

that could benefit the most from potential future anti-

adhesion and anti-inflammatory therapeutic interventions.

To summarize, endothelial dysfunction, as well as

inflammation processes, was apparent in both ‘‘steady

state’’ and during VOC in SCD patients. From our analysis,

162 E.S. Hatzipantelis et al.

123

SCD patients during ‘steady state’ have presented signifi-

cantly elevated plasma levels of ET-1, sVCAM-1, sP-

selectin and d-dimers compared to the control group.

Furthermore, ET-1, sP-selectin, PDGF, vWf, D-dimers,

ESR and CRP might represent additional, but not inde-

pendent, prognostic markers of VOC crisis in childhood

SCD, since their plasma values were significantly different

from those in control and steady state group. Further

evaluation has shown possible association of elevated

levels of sP-selectin, ET-1 and sVCAM-1 with the fre-

quency of VOC crisis. Additional prospective studies are

required to confirm the potential independent prognostic

value of these markers in children with SCD.

Conflict of interest The authors declare that they have no conflict

of interest.

References

1. Stuart MJ, Nagel RL. Sickle-cell disease. Lancet. 2004;364:

1343–60.

2. Conran N, Franco-Penteado CF, Costa FF. Newer aspects of the

pathophysiology of sickle cell disease vaso-occlusion. Hemo-

globin. 2009;33:1–16.

3. Kaul DK, Finnegan E, Barabino GA. Sickle red cell–endothelium

interactions. Microcirculation. 2009;16:97–111.

4. Kato GJ, Hebbel RP, Steinberg MH, et al. Vasculopathy in sickle

cell disease: biology, pathophysiology, genetics, translational

medicine, and new research directions. Am J Hematol. 2009;84:

618–25.

5. Villagra J, Shiva S, Hunter LA, et al. Platelet activation in

patients with sickle disease, hemolysis-associated pulmonary

hypertension and nitric oxide scavenging by cell-free hemoglo-

bin. Blood. 2007;110:2166–72.

6. Haywood LJ. Cardiovascular function and dysfunction in sickle

cell anemia. J Natl Med Assoc. 2009;101:24–30.

7. Krishnan S, Setty Y, Betal SG, et al. Increased levels of the

inflammatory biomarker C-reactive protein at baseline are

associated with childhood sickle cell vasocclusive crises. Br J

Haematol. 2010;148:797–804.

8. Naprawa JT, Bonsu BK, Goodman DG, et al. Serum biomarkers

for identifying acute chest syndrome among patients who have

sickle cell disease and present to the emergency department.

Pediatrics. 2005;116:e420–5.

9. Blum A, Yeganeh S, Peleg A, et al. Endothelial function in

patients with sickle cell anemia during and after sickle cell crises.

J Thromb Thrombolysis. 2005;19(2):83–6.

10. Ataga KI, Moore CG, Hillery CA, et al. Coagulation activation

and inflammation in sickle cell disease-associated pulmonary

hypertension. Haematologica. 2008;93:20–6.

11. Niu X, Nouraie M, Campbell A, et al. Angiogenic and inflam-

matory markers of cardiopulmonary changes in children and

adolescents with sickle cell disease. PLoS ONE. 2009;4:e7956.

12. Akinsheye I, Klings ES. Sickle cell anemia and vascular dys-

function: the nitric oxide connection. J Cell Physiol. 2010;224:

620–5.

13. Rees DC, Cervi P, Grimwade D, et al. The metabolites of nitric

oxide in sickle-cell disease. Br J Haematol. 1995;91:834–7.

14. Morris CR, Kuypers FA, Larkin S, et al. Patterns of arginine and

nitric oxide in patients with sickle cell disease with vaso-occlu-

sive crisis and acute chest syndrome. J Pediatr Hematol Oncol.

2000;22:515–20.

15. Enwonwu CO, Xu XX, Turner E. Nitrogen metabolism in sickle

cell anemia: free amino acids in plasma and urine. Am J Med Sci.

1990;300:366–71.

16. Eberhardt R, McMahon L, Duffy S, Steinberg MH. Sickle cell

anemia is associated with reduced nitric oxide bioactivity in

peripheral conduit and resistance vessels. Am J Hematol.

2003;74:104–11.

17. Wood KC, Hsu LL, Gladwin MT. Sickle cell disease vasculop-

athy: a state of nitric oxide resistance. Free Radic Biol Med.

2008;44:1506–28.

18. Gladwin MT, Vichinsky E. Pulmonary complications of sickle

cell disease. N Engl J Med. 2008;359:2254–65.

19. Radhakrishnan DK, Bendiak GN, Mateos-Corral D, et al. Lower

airway nitric oxide is increased in children with sickle cell dis-

ease. J Pediatr. 2012;160(1):93–7.

20. Gladwin MT, Schechter AN, Ognibene FP, et al. Divergent nitric

oxide bioavailability in men and women with sickle cell disease.

Circulation. 2003;107:271–8.

Biomarkers in SCD 163

123