ARTICLE
Auteur(s) : Dhananjay K Kaul
Department of Medicine, Albert Einstein College of Medicine,
Bronx, NY, USA
The goal of this review is to highlight new insights into
vascular pathobiology of sickle cell disease derived from recent
studies in sickle patients and transgenic sickle mouse models.
A single amino acid substitution in hemoglobin A (β6: Glu
→Val) leads to pleiotropic (multiple) effects of the mutant
βS gene. This single point mutation leads to
polymerization of the mutant hemoglobin (sickle hemoglobin or HbS)
under deoxygenated conditions resulting in morphological
deformation, rigidity and dehydration of red blood cells. The
events originating from red blood cell abnormalities lead to
multiple pathologies including excessive destruction of sickle red
cells (hemolysis), reticulocytosis, anemia, vaso-occlusive events
(both transient and clinical), reperfusion injury, adhesion of
sickle red cells to endothelium, and red cell oxidative stress.
These abnormalities result in excessive oxidant generation, reduced
nitric oxide (NO) bioavailability and endothelial dysfunction,
which adversely affect vascular homeostasis and result in altered
and instable steady state. In fact, recent studies indicate that
the interaction between oxidative stress and the extent of NO
bioavailability is a major source of vaso-occlusive pathophysiology
including altered microvascular function, abnormal blood
cell-endothelium interactions, inflammation and end organ
damage.
This review will focus on etiology and effects of oxidative
stress and NO deficiency, the interaction between these two
factors, and the role of non-NO vasoregulatory mechanisms in sickle
cell disease. This review will also draw attention to the
intravital findings in transgenic sickle mouse models that have
contributed to a better understanding of the human sickle
disease.
Oxidative stress
Figure 1
represent the proposed paradigm of major events originating from
the primary defect in sickle cell disease (SCD), i.e. hemoglobin S
(HbS) polymerization and intravascular sickling, and leading to
oxidative stress and reduced NO bioavailability. Oxidative stress
is a critical factor in endothelial activation and organ-specific
complications in sickle cell disease as noted for other ischemic
diseases [1, 2]. Both sickle cell disease patients and
transgenic-knockout sickle mice (BERK model) expressing exclusively
human α- and βS-globins show increased superoxide
(O2-) production [3, 4]. Increased production
of reactive oxygen species (ROS) in this disease will have
two-pronged adverse effects: i) it will result in endothelial
activation, up-regulation of endothelial adhesion molecules,
leukocyte recruitment (inflammation) and red cell adhesion to
activated endothelium, reduced microvascular perfusion and
increased red cell transit times, which will promote hypoxia,
sickling and vaso-occlusion; ii) it will result in compromised
microvascular function and altered microvascular responses to
NO-mediated vasoactive stimuli secondary to NO inactivation by
oxidative stress and hemolysis. On the other hand, oxidative stress
is modulated by NO bioavailability. Two factors that are persistent
source of oxidant generation in this disease are reperfusion injury
and Hb autoxidation.
Reperfusion injury
Sickle cell disease is characterized by reperfusion injury
physiology as a result of recurring vaso-occlusive events.
Vaso-occlusive events, both clinical (painful vaso-occlusive
crisis) and sub-clinical (transient) episodes, will contribute to
hypoxia during the ischemic phase and oxidant generation during
reperfusion. Intermittent vaso-occlusive events, involving
transient blockage of microvascular bed by rheological abnormal
sickle red cells, are likely to be more prevalent than the painful
vaso-occlusive crisis. Effects of reperfusion injury physiology
were demonstrated in transgenic sickle mice subjected to
hypoxia-reoxygenation. Osarogiagbon et al. described increased
lipid peroxidation, ethane generation and hydroxyl (OH−)
radical generation in transgenic sickle mice after
hypoxia-reoxygenation [5]. Studies by Kaul and Hebbel [6] showed
that hypoxia-induced sickling is etiologic in oxidant generation
following reoxygenation in transgenic sickle mice. These studies
demonstrated markedly greater endothelial oxidant (e.g.
H2O2) generation following the onset of
reperfusion in sickle mice (figure 2). Reperfusion
injury occurs during the period when molecular oxygen is
reintroduced into the tissue after ischemia that results in
depletion of ATP and accumulation of its metabolites such as
hypoxanthine and the conversion of the enzyme xanthine
dehydrogenase to xanthine oxidase (XO). Reperfusion allows XO to
act on hypoxanthine with the resulting generation of superoxide
(O2-) that is converted to
H2O2 by superoxide dismutase (SOD). In
addition, increased levels of O2- are
generated by NADPH oxidase and “uncoupling” of endothelial nitric
oxide synthase (eNOS); the latter is likely due to depleted
substrate arginine and cofactor tetrahydrobiopterin (BH4) as
discussed later. Microvascular endothelium is a prominent target of
reperfusion injury, although reperfusion injury is not limited to
endothelium. In sickle cell disease, oxidant generation may affect
vascular homeostasis by causing endothelial dysfunction/activation,
altered vascular responses and inflammatory effects in
postcapillary venules, the sites of inflammatory response. In
transgenic sickle mice, reperfusion leads to exaggerated
leukocyte-endothelium interactions compared with control mice
followed by a marked leukocyte infiltration (emigration or
diapedesis) into the interstitium [6, 7], suggesting that
reoxygenation-induced oxidant production leads to endothelial
activation and inflammation. The inflammatory effects of
reperfusion injury were significantly attenuated by antioxidant
enzymes, SOD and catalase [7]. Catalase (reduces
H2O2 to water) was found to be more effective
compared to SOD and allopurinol, an inhibitor of xanthine oxidase
activity. As shown in figure 3A and B, the
anti-inflammatory effect of these antioxidants clearly paralleled
the fluorescent intensity levels (ΔI) of endothelial oxidant
generation in postcapillary venules as determined using
dihydrorhodamine (DHR), an
H2O2- sensitive fluorochrome
[6]. Thus, targeting endothelial oxidant generation may constitute
an anti-inflammatory therapeutic approach.
While elevated leukocyte counts in sickle patients and sickle
transgenic mice models [6, 8] suggest a systemic effect of
oxidative stress and inflammation, direct evidence of organ
oxidative stress has been obtained in transgenic sickle mice [9].
Homozygous sickle mice show increased peroxidation of membrane
lipids and a depletion of antioxidants, i.e. reduced glutathione
(GSH), superoxide dismutase (SOD), catalase and glutathione
peroxidase (GPx) [9]. In sickle cell disease, depleted levels of
antioxidants likely result in increased susceptibility to lipid
peroxidation [10, 11]. Lipid peroxidation causes damage to the cell
and mitochondrial membranes, and contributes to multiple organ
damage. Moreover, degradation and consumption of antioxidants by
the release of ROS during reperfusion has been recognized [12]. The
reduced GSH levels in sickle cell disease could be in response to
oxidative stress and other pathophysiological conditions such as
vaso-occlusive events and hemolysis. Morris et al. [13] have
implicated decreased GSH and glutamine content in red cell
integrity, hemolysis and reduced NO bioavailability, the factors
that may participate in pathogenesis of pulmonary hypertension in
sickle cell disease. Notably, arginine supplementation of
transgenic sickle mice significantly increases NO metabolites
accompanied by enhanced level/activity of antioxidants and reduced
lipid peroxidation [14]. The results support the notion that
oxidative stress is modulated by NO bioavailability and vice versa
[15-17].
HbS autoxidation
Oxidative stress in the microcirculation is not exclusive to
vascular endothelium, but also occurs in red cells as a consequence
of HbS autoxidation. Autoxidation of HbS within red cells or after
it is released from hemolysing red cells can potentially activate
endothelium by generating O2- and
H2O2. Electron transfer from iron to
heme-bound O2 induces O2-
production that readily dismutates to H2O2.
Recent studies of Kiefmann et al. [18] have shown that
oxidants produced by hypoxic mouse red cells diffuse to endothelial
cells of adjoining vessel wall resulting in increased microvascular
oxidants and up-regulation of P-selectin and probably other
adhesive molecules involved in red cell and leukocyte adhesion.
Notably, infusion of red cells from hemizygous BERK mice (express
15% HbS) resulted in a greater inflammatory response. Although
these studies were carried out in artificially perfused mouse
lungs, we believe that these results are quite relevant to
understanding the potential role of HbS autoxidation in sickle
vasculopathy. In this context, it is relevant to recall the
insightful work of Hebbel and co-workers [19] who showed that SS
RBCs generate excessive amounts of reactive oxygen species due to
the presence of unstable HbS and spontaneous autoxidation of iron
in heme. Also, enhanced sickle red cell adhesion induces oxidant
stress in cultured endothelium as evidenced by increased
peroxidation, activation of the transcription factor nuclear
factor-κB (NF-κB), and increased expression of intercellular
adhesion molecule-1 (ICAM-1), vascular cell adhesion
molecule-1 (VCAM-1) and E-selectin [20]. Future studies will
be needed to evaluate the efficacy of specific oxidants and
inhibitors of Hb autoxidation in endothelial activation and
adhesive interactions.
Reduced NO bioavailability
Vascular endothelial cells constitutively generate NO, which acts
on the adjacent smooth muscle cells to produce vasorelaxation via
activation of guanylate cyclase. Catalytic action of endothelial NO
synthase (eNOS) on the substrates arginine and oxygen results in
the generation of NO and citrulline. NO has diverse effects,
including regulation of blood pressure, dynamic modulations of the
vascular tone (vasomotion), blood flow and down-regulation of
endothelial adhesion molecules [21-23]. In sickle cell disease,
oxidative stress and hemolysis are two major factors that
contribute to consumption/inactivation of NO. Contribution of
oxidative stress to NO consumption will be significant and even
independent of hemolysis particularly during episodes of transient
vaso-occlusive events and reperfusion injury. NO inactivation by
plasma ferrous hemoglobin, as shown by Gladwin, Schechter and
coworkers [24], is a major contributor to oxidative stress. NOS
itself can be decoupled by oxidation of the essential cofactor BH4
[25, 26], and decoupled NOS produces superoxide in place of NO.
Hemolysis
Both sickle patients and sickle mouse models show depleted levels
of L-arginine and NO metabolites (NOx) [27-30]. Arginine depletion
may occur as a result of initial increase in NOS activity and
excessive NO generation in response to NO consumption/inactivation
by cell-free plasma heme and/or oxygen radicals [24, 31, 32]. In
sickle cell disease, red cell sickling, red cell membrane fragility
and ISCs contribute to hemolysis. Furthermore, oxygen radicals have
been implicated in hemolysis of red cells in several pathological
states including β-thalassemia and sickle cell anemia [19, 33].
Arginine therapy of sickle patients (0.1 g/kg TID for
1 month) has been reported to increase red cell glutathione
(GSH), a red cell anti-oxidant that may reduce red cell oxidative
stress and hemolysis [28]. These observations further support the
notion that oxidative stress is modulated by NO bioavailability and
vice versa [15-17].
Persistent hemolysis in this disease results in the release of
large amount of cell-free hemoglobin in plasma, which would
exacerbate NO inactivation. Gladwin, Schechter et al. have
elegantly demonstrated that NO in sickle patients is rapidly
destroyed by its reaction with the iron-containing heme group of
oxyhemoglobin (ferrous hemoglobin) present in the plasma [5, 10,
12]. NO reacts with cell-free oxyhemoglobin to form methemoglobin
and inactive nitrate. NO can also be inactivated by its reaction
with deoxygenated Hb to form iron nitrosyl Hb. In humans, hemolysis
also releases red blood cell arginase-1 into plasma.
Conversion of arginine to ornithine by the enzyme arginase further
reduces the required substrate and limits NO bioavailability in
sickle cell disease [16, 28]. Importantly, plasma heme
concentrations in sickle patients during steady state (“crisis
free”) are ~ 10-15-fold higher than in normal controls, rising
during vaso-occlusive crisis leading to further depletion of NO
(figure 4A)
[24]. Furthermore, Reiter et al. [24] have shown a correlation
between plasma hemoglobin levels and NO consumption in sickle
patients (figure
4B). The rate of intravascular hemolysis is a major risk
factor in pulmonary hypertension, a serious complication in sickle
patients [34].
Oxidative stress, NO depletion and eNOS uncoupling
Increased vascular O2- production in human
patients and transgenic sickle mouse models [4] is implicated in NO
consumption and formation of peroxynitrite (ONOO-) that
results in enhanced nitration of tyrosine residues (nitrotyrosine
formation) as demonstrated in transgenic sickle mice [31, 32, 35].
Because the rate of reaction of NO with superoxide is 100-fold
greater than its reaction with plasma heme [24], future studies are
needed to explore the relative contribution of hemolysis and
oxidants in NO consumption, although both pathways have the
potential to limit NO bioavailability [36].
In sickle cell disease, consumption of NO by plasma heme and
superoxide may first lead to excessive production of NO followed by
depletion of arginine (a substrate) and BH4 (a
cofactor), both required for NO synthesis. Chronic deficiency of
arginine and oxidation of BH4 may lead to dysfunctional eNOS.
Increased O2- generation by eNOS under
certain pathophysiological conditions may be a consequence of
“uncoupling” of electron flow from L-arginine oxidation and NO
production [25]. Reduced levels of BH4 could lead to
uncoupling of NADPH oxidation and NO synthesis, with oxygen as
terminal electron receptor instead of arginine, leading to
O2- generation by NOS. Excessive
ONOO- formation can reduce eNOS activity and disrupt
eNOS dimers [25]. Elevated nitrotyrosine levels, first reported by
us in transgenic sickle mice [32], have been validated by Gladwin
and co-workers [31], who also showed that increased
ONOO- levels are associated with impaired eNOS activity
with a loss of eNOS dimerization in homozygous sickle mice. Future
studies should evaluate the role of BH4 in microvascular tone
and reactivity in sickle cell disease.
Endothelial activation
In sickle cell disease, vascular endothelium is likely to be in a
perpetually activated state because of chronic oxidative stress,
hemolysis and reduced NO. Inhibition of NO production causes
endothelial oxidant generation [37], endothelial activation and
increased leukocyte-endothelial interaction [21], while NO
replenishment has a protective effect on oxidative stress and
reperfusion injury [9, 35, 38]. As described earlier, another
source of endothelial activation is ROS generated by sickle red
cells due to the presence of unstable HbS and spontaneous
autoxidation of heme iron [19]. Endothelial oxidant generation
caused by reperfusion and depleted NO can lead to increased
peroxidation and up-regulation of the redox-sensitive transcription
factor nuclear factor (NF)-κB that can activate genes for adhesion
molecules such as vascular cell adhesion molecule-1 (VCAM-1),
P-selectin, E-selectin and intercellular adhesion
molecule-1 (ICAM-1) [20]. Also, in this disease, elevated
levels of inflammatory cytokines such as tumor necrosis factor-α
(TNF-α), interleukin-1β and platelet-activating factor (PAF) induce
up-regulation of endothelial adhesion molecules, probably via
endothelial oxidant generation and activation of NF-κB [39, 40].
Activated phenotype of circulating (detached) endothelial cells in
sickle cell disease is suggested by increased expression of cell
adhesion markers [41].
The well-recognized markers of endothelial activation are
soluble VCAM-1, P-selectin, E-selectin and ICAM-1. VCAM-1 is only
expressed on activated endothelium and mediates red cell adhesion
via α4β1 integrin expressed on sickle
reticulocytes.42 P-selectin is induced in activated
endothelium and mediates sickle red cell adhesion, as well as
transient adhesive interaction (rolling) of leukocytes with
endothelium [43]. E-selectin is expressed in cytokine-stimulated
endothelium and is thought to be responsible for slow rolling
interaction of leukocytes and possibly the initiation of firm
adhesion [43]. ICAM-1 is constitutively expressed on
endothelium and is responsible for firm adhesion of leukocytes
[43]. Soluble forms of these molecules released from activated
endothelium are increased in transgenic sickle mouse models, as
well as in sickle patients [8, 44]. Adhesion molecules are reliable
markers of endothelial dysfunction and increased blood cell (red
cells and leukocyte) – endothelial interactions. In fact, the
levels of soluble adhesion molecules (sVCAM-1,
sP-selectin,sE-selectin and sICAM-1) increase with the disease
severity and represent the degree of oxidative stress and hemolysis
[44-46]. A decrease in hemolysis and oxidative stress augments
anti-inflammatory NO, down-regulates endothelial activation markers
and ameliorates the disease severity (i.e. vaso-occlusive episodes)
as noted in patients on hydroxyurea therapy [45, 47, 48]. Moreover,
as shown by Stuart and Setty, in patients with the acute chest
syndrome, plasma sVCAM level is markedly elevated [49]. Thus,
monitoring endothelial activation markers is a useful indicator of
endothelial activation and disease severity.
Blood cell-endothelium interactions
In sickle cell disease, adhesive interactions of red cells and
leukocytes with vascular endothelium are implicated in the
initiation of vaso-occlusive processes [6, 50, 51]. These
interactions are facilitated by activation of endothelium and
up-regulation of adhesion molecules (recently reviewed in [8, 52,
53]). In an ex vivo microvascular bed, adhesion of sickle red cells
in postcapillary venules is followed by trapping of dense sickle
red cells that can result in vaso-occlusion [42, 51]. Sickle red
cell adhesion is particularly enhanced when endothelium is exposed
to increased cytokine levels in sickle cell disease [54].
Additionally, generation of reactive oxygen species (oxidants)
induced by inflammatory cytokines and/or reperfusion events results
in enhanced red cell adhesion and recruitment of leukocytes in
postcapillary venules, which will lead to increased microvascular
transit time, red cell sickling and vaso-occlusion as recently
reviewed by Kaul et al. [42]. These observations indicate that
endothelial activation and enhanced red cell and leukocyte adhesion
can potentially initiate postcapillary vaso-occlusion. Future
studies are warranted to determine the validity and significance of
these observations in patients with sickle cell disease.
Vascular dysfunction in sickle cell disease
The seminal finding by Hebbel et al. [50] that sickle red
cells adhered abnormally to vascular endothelium led to the
recognition that vascular factors played an indispensable role in
the pathophysiology of sickle cell disease. The vascular tone
adaptations in sickle cell disease are in response to anemia,
hemolysis and intravascular sickling. Anemia combined with
intravascular sickling and hemolysis may exacerbate tissue hypoxia.
Also, oxidant generation during transient vaso-occlusive events is
likely to affect vasoregulatory function of vascular endothelium as
in other inflammatory diseases [32]. In sickle patients, chronic
anemia is compensated by hyperperfusion to maintain optimal oxygen
delivery to tissues [55, 56]. Hyperperfusion in large conduit
arteries characterizes sickle patients, and is also observed in the
microcirculation of transgenic-knockout sickle (BERK) mice that
express exclusively HbS [32]. Also, sickle patients and
transgenic-knockout sickle mice exhibit a lower systemic blood
pressure [32, 57], a likely consequence of the dilation of
resistance vessels (arterioles). Sickle patients show 50-60%
decrease in the peripheral resistance [55]. NO
consumption/inactivation by cell-free plasma heme and oxidative
stress significantly influence vascular function in this disease.
Resistance to NO
Blunted vascular (arterioles) responses to acetylcholine (ACh; an
endothelial-dependent vasodilator) and sodium nitroprusside (SNP; a
NO donor) were first described by Kaul and coworkers [58] in a
transgenic sickle mouse model expressing HbS and
HbS-Antilles (S+S-Antilles mice). Studies by Nath
et al. validated the attenuated vascular responses in the same
mouse model, and also showed a marked increase in oxidative stress
(lipid peroxidation) in these mice [59]. Because hemolytic rate is
low in S+S-Antilles mice, consumption of NO by oxidants is a likely
explanation for the attenuated vascular responses in these mice.
However, the response to a NOS inhibitor in S+S-Antilles mice was
not significantly different as compared to normal mice. On the
other hand, in human sickle cell disease and transgenic-knockout
sickle mice, inactivation of NO is accelerated due to synergistic
effects of chronic oxidative stress and persistent hemolysis.
A synergistic effect of these factors is evidenced by
attenuated responses to NO-mediated vasodilators and NOS
antagonists in transgenic-knockout sickle mice [4, 32, 60].
Similarly, sickle patients with high concentrations of plasma
hemoglobin show attenuated blood flow responses to NO synthase
inhibitor L-NMMA and sodium nitroprusside (SNP), a NO donor [45,
61]. During steady state, the concentration of hemoglobin
(4 μM) in sickle patients is sufficient to deplete almost all
endothelial-derived NO, thereby attenuating vasodilation [62].
During vaso-occlusive crisis, the release of free hemoglobin
decreases NO bioavailability to the level that may be insufficient
for activation of soluble guanylate cyclase in vascular smooth
muscle cells [63]. Also, the reported increase in the
endothelial-bound XO could catalyze the increased generation of
O2- and H2O2, and
interfere with vessel responses to NO-mediated vasodilators [4].
Hemolysis and depleted NO are two major factors implicated in
vaso-constriction and pulmonary hypertension in sickle patients and
BERK model of sickle cell disease [34, 36, 60]. Patients with
higher rates of hemolysis suffer from a range of complications that
include pulmonary hypertension, priapism, cutaneous leg ulcers, and
a correlation may exist between pulmonary hypertension and
cerebrovascular disease in sickle cell patients [34]. Phenotypic
differences may result from variations in vascular responses
depending on intrinsic differences in hemolytic rates and oxidative
stress [64], and organ to organ and gender based differences exist
in susceptibility to hemolysis and NO depletion [33, 45, 64].
NO-independent vasoregulatory factors
Although hemolysis and oxidative stress limit NO bioavailability
and affect vascular responses, sufficient evidence exists that
favors a role of NO-independent factors in vascular responses in
sickle cell disease. In transgenic sickle mice, NO-independent
vasodilatory responses elicited by forskolin are intact, suggesting
that cAMP-mediated vasodilatory mechanism remains functional [58].
Belhassen et al. [56] showed that vasodilator response to ACh
in sickle patients was considerably stronger than in normal
individuals, suggesting a wall shear stress-induced endothelial
release of NO and/or prostacyclin. Flow-mediated wall shear stress
may play an important role in vascular regulation in sickle cell
disease as first demonstrated by vasodilatory responses to
hyperoxia in a transgenic sickle mouse [65] and later in sickle
patients [56], suggesting a role of red cell rheological component
(HbS depolymerization).
Induction of Non-NO vasodilators
Under steady state conditions, vasodilatory mechanisms predominate
to maintain optimal systemic blood flow in sickle patients and
transgenic-knockout sickle mice. In sickle patients, higher levels
of PGE2 and induction of HO-1 will contribute to
vasodilation, hyperpefusion and lower peripheral resistance
[66-68]. The induction of cyclooxygenase-2 (COX-2) enzyme in
transgenic-knockout sickle (BERK) mice [32], is the source of
elevated plasma levels of PGE2. COX-2 is induced under
conditions of increased oxidative stress (i.e., increased
O2- generation and ONOO-
formation) and chronic hypoxia (i.e. intravascular sickling and low
hematocrit), the conditions prevailing in BERK mice [32, 69, 70].
In contrast, the expression of COX-1, a constitutively expressed
enzyme, in these mice is not different from that observed in
control mice, suggesting that increased PGE2 levels in BERK
mice are due to COX-2 activity [35]. Studies are required to
ascertain if increased PGE2 levels [66] in sickle patients is
a consequence of COX-2 induction. The second non-NO
vasodilator HO-1 produces CO, a vasodilator and
anti-inflammatory molecule [32, 35, 71]. HO-1 is induced in
response to excessive plasma heme, hypoxia and oxidative stress,
and catalyzes degradation of heme to biliverdin/bilirubin and CO
[72, 73]. Hence, HO-1 is also a marker of hemolysis.
Additionally, during ischemic/reperfusion events, endothelial
generation of H2O2, a vasodilator and
hyperpolarizing factor [74], may contribute to vasodilation. Very
likely, the expression of non-NO vasodilators will contribute to
altered vascular responses to NO-mediated vasoactive stimuli [24,
32, 35, 45]. The induction of non-NO vasodilator enzymes
COX-2 and HO-1 is likely to mediate the observed
vasodilation, hypotension and hyperperfusion required for adequate
oxygen delivery in the face of chronic anemia. While vasodilator
mechanisms exert predominant effect during crisis-free steady
state, vaso-occlusive events may involve excess production of
vasoconstrictors such as endothelin and isoprostanes [68, 75].
Modulators of sickling, hemolysis and oxidative stress
Intravital studies in transgenic-knockout sickle (BERK) mice have
yielded novel insights into the relationship between sickling,
oxidative stress, hemolysis and NO bioavailability. These studies
show that reducing hemolysis and oxidative stress improves vascular
reactivity NO donors, which is accompanied by reduced expression of
non-NO vasodilators. BERK mice were used to determine the effect of
anti-sickling fetal hemoglobin and arginine supplementation [32].
In BERK mice, arteriolar diameter response to NO donor SNP shows
a strong relationship with the extent of hemolysis, which is
consistent with the ability of plasma heme to consume NO; the
latter determined by plasma NO metabolites (NOx) levels. The
greater plasma heme level in BERK mice caused blunted diameter
response. While low plasma heme levels in controls C57BL mice were
associated with maximal arteriolar dilation, other variants of BERK
mice (i.e. BERK-hemizygous [~ 15% HbS], and BERK+γ mice expressing
fetal hemoglobin [HbF]), with intermediate levels of the plasma
heme, showed improved diameter response to SNP as compared with
BERK mice (figure
5). Importantly, BERK+γ mice expressing 20% HbF exhibit an
improved response to NO-mediated vasoactive stimuli (ACh, SNP and
L-NAME), as well as to a vasoconstrictor (norepinephrine),
indicating a trend towards normalization of vascular reactivity
[32]. Some pathology is almost completely corrected by the
introduction of HbF (e.g. vessel diameters, COX-2 and
nitrotyrosine expression). However, the response to vasoactive
stimuli such as ACh and SNP is only partially corrected, suggesting
that, even when the vessel diameter is normal, the signaling
pathways for the maintenance of vascular tone are still disrupted
in BERK+γ mice, perhaps due to higher than normal oxidant
generation and hemolytic rates in these mice [32].
In sickle cell disease, nitric oxide (NO) depletion leads to
arginine deficiency, impaired NO bioavailability and chronic
oxidative stress. Arginine supplementation was used to evaluate if
the substrate replenishment will enhance NO bioavailability in BERK
mice and inhibit expression of non-NO vasodilators, improve
vascular reactivity, and reduce hemolysis and oxidative stress
[35]. Arginine supplementation of BERK mice significantly
alleviates several discernible abnormalities. First, arginine
decreases the expression of non-NO vasodilator enzymes
COX-2 and HO-1. Arginine reduces COX-2 expression and
PGE2 levels (figure
6), probably by decreasing oxidative stress (i.e. by
reduced ONOO- formation). Interestingly,
COX-1 expression remains unaffected, confirming the role of
COX-2 in enhanced PGE2 generation. The markedly reduced
HO-1 expression in arginine-supplemented BERK mice is
associated with decreased hemolysis. Second, the decreased
expression of non-NO vasodilators is associated with reduced
vasodilation and improved vascular reactivity to NO-mediated
vasoactive stimuli (ACh, SNP and L-NAME). Third, arginine reduces
hemolysis and oxidative stress both of which are implicated in
consumption/inactivation of NO and altered microvascular reactivity
to NO-mediated vasoactive stimuli.
Notably, arginine causes a pronounced > 50% decrease in
cell-free plasma hemoglobin in BERK mice [35]. Arginine decreases
oxidative stress as evidenced by reduced expression of
nitrotyrosine. Oxygen radicals have been implicated in hemolysis of
red cells in several pathological states including β-thalassemia
and sickle cell anemia [19, 33]. Arginine therapy of sickle
patients at a comparable dose level (0.1 g/kg TID) has been
reported to increase red cell GSH, a key red cell anti-oxidant that
reduce red cell oxidative stress and hemolysis [76]. Another
potentially concurring mechanism may involve inhibition of red cell
Gardos channel by arginine as reported by Romero et al. in
transgenic sickle mice [30], which will reduce red cell
dehydration, improve red cell deformability and decrease
intravascular red cell lysis. Arginine treatment of sickle
transgenic mice reduces plasma endothelin-1 (ET-1) levels
[77]. Because ET-1 has been shown to stimulate the Gardos
channel [30], reduction of ET-1 may partially account for
reduced Gardos channel activity in arginine supplemented sickle
mice.
We analyzed our data whether hemolysis and/or oxidative stress
affected the observed arteriolar diameter response (figure 7A, B) to SNP, a NO
donor, as a measure of NO bioactivity. Linear regression of the
data showed a strong relationship between plasma hemoglobin levels
and the mean arteriolar diameter increase (%) in response to SNP
(figure 7A).
A strong relationship was also evident when arteriolar
diameter response to SNP was plotted against levels of
nitrotyrosine expression (figure 7B). Future studies
will be required to differentiate the relative contribution of
oxidative stress and cell-free plasma hemoglobin in the consumption
of NO and vascular responses in sickle cell disease.
These studies showed that arginine exerts a range of protective
effects in sickle mice, which include improved microvascular
function, reduced expression of non-NO vasodilators, decreased
hemolysis and oxidative stress, and increased NO bioavailability.
Thus, increasing NO bioavailability by therapeutic agents such as
arginine is a promising strategy in the management of SCA.
Conclusions and future directions
Although the primary defect of sickle cell disease is HbS
polymerization and intravascular sickling, the events originating
from this defect lead to multiple pathologies including reperfusion
injury, HbS autoxidation and hemolysis leading to excessive
oxidative stress and reduced NO bioavailability. Recent
observations in sickle patients and intravital studies in
transgenic sickle mouse models suggest that vascular pathobiology
involves interaction between oxidative stress and reduced NO
bioavailability that results in endothelial activation/dysfunction,
altered microvascular function, abnormal blood cell-endothelium
interaction, inflammation and multiple organ damage.
Notwithstanding the significant recent developments in the
exposition of the role of hemolysis, reduced NO bioavailability and
oxidative stress in NO resistance to vascular responses in sickle
patients and transgenic sickle mouse models, it is important that
the role of other factors including NO-independent mechanisms (e.g.
cAMP-mediated vascular responses and wall shear stress-induced
vasodilation) be analyzed without overemphasizing the role of given
factor. Finally, therapeutic strategies that address the underlying
defects (i.e. intravascular sickling, reperfusion injury, oxidative
stress and hemolysis) need to be developed and refined to alleviate
the vascular dysfunction and end organ damage.
Acknowledgments
Supported by a grant from National Institutes of Health, USA
(HL070047) to D.K.K.
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