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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 12 (December 15), 1999:
pp. 4307-4313
Formation of Dense Erythrocytes in SAD Mice Exposed to Chronic Hypoxia:
Evaluation of Different Therapeutic Regimens and of a Combination
of Oral Clotrimazole and Magnesium Therapies
By
Lucia De Franceschi,
Carlo Brugnara,
Philippe Rouyer-Fessard,
Helene Jouault, and
Yves Beuzard
From the Department of Internal Medicine, University of Verona,
Verona, Italy; the Experimental Laboratory of Gene Therapy, Hopital St
Louis, Paris, France; the Laboratory of Hematology, INSERM U91, Hopital
Henri Mondor, Creteil, France; and the Departments of Laboratory
Medicine and Pathology, Children's Hospital, Harvard Medical School,
Boston, MA.
 |
ABSTRACT |
We have examined the effect of hydroxyurea (HU), clotrimazole (CLT),
magnesium oxide (Mg), and combined CLT+Mg therapies on the
erythrocyte characteristics and their response to chronic hypoxia in a
transgenic sickle mouse (SAD) model. SAD mice were treated for 21 days
with 1 of the following regimens (administered by gavage): control
(n = 6), HU (200 mg/d; n = 6), CLT (80 mg/kg/d, n = 5),
Mg (1,000 mg/kg/d, n = 5), and CLT+Mg (80 and 1,000 mg/kg/d, respectively, n = 6). Nine normal mice were also treated as controls (n = 3), HU (n = 3), and CLT+Mg (n = 3). Treatment with HU
induced a significant increase in mean corpuscular volume and cell K
content and a decrease in density in SAD mice. Treatment with the CLT and Mg, either alone or in combination, also increased cell K and
reduced density in SAD mice. After 21 days of treatment, the animals
were exposed to hypoxia (48 hours at 8% O2) maintaining the same treatment. In the SAD mice, hypoxia induced significant cell
dehydration. These hypoxia-induced changes were blunted in either HU-
or Mg-treated SAD mice and were completely abolished by either CLT or
CLT+Mg treatment, suggesting a major role for the Gardos channel in
hypoxia-induced dehydration in vivo.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
A POTENTIAL THERAPEUTIC approach for
sickle cell disease involves the use of drugs that reduce or block the
dehydration of sickle erythrocytes. This strategy is based on the
extreme dependence of hemoglobin (Hb) S polymerization on Hb S
concentration and on the presence of dense dehydrated erythrocytes in
the blood of patients with homozygous sickle cell (SS)
disease.1-3 The presence of dense cells containing
polymerized Hb S has been linked to the clinical severity of various
sickle syndromes.4 Two cation transport pathways play a
prominent role in sickle cell dehydration: the K-Cl
cotransport5,6 and the Ca2+-activated K
transport (Gardos channel).7-10
The K-Cl cotransport promotes loss of K and Cl with consequent
erythrocyte dehydration when cells are exposed to pH values less than
7.4 or when the red blood cell (RBC) magnesium (Mg) content is decreased. We have demonstrated, both in the transgenic sickle (SAD) mouse model, in SS patients, and in patients with  thalassemia intermedia, that oral Mg supplementation ameliorates erythrocyte dehydration by increasing erythrocyte Mg and K contents and
reducing K-Cl cotransport activity.11-13 We have also shown that long-term (6 months) oral Mg supplementation (using Mg pidolate salts) induces a significant reduction in the incidence of acute painful crises in patients with SS disease.14
The Ca2+-activated K transport induces K loss and
erythrocyte dehydration when cytosolic free Ca2+ increases,
as occurs upon deoxygenation of sickle cells.15 We have
shown that treatment with clotrimazole (CLT), a specific inhibitor of
the Gardos channel,16,17 can prevent erythrocyte dehydration both in the SAD mouse model and in SS
patients.18,19
Although the percentage of circulating dense cells does not predict
disease severity,20 an inverse correlation has been demonstrated between the percentage of irreversibly sickled cells (ISC)
and erythrocyte survival.21  Thalassemia and an
increased cellular content of fetal Hb (Hb F) have been
shown to be associated with a reduction in the number of circulating
dense cells.3,22,23 Dense cells have also been shown to
increase before or in the very first phase of painful crises and to
decrease significantly thereafter.24,25 Dense ISC have been
shown to play an important role in the trapping of cells in
postcapillary venules26 and associated microvascular
obstructions.27
The availability of an animal model for sickle cell anemia offers a
useful tool for studying the pathophysiology of the disease and for
evaluating the effectiveness of therapeutic agents in vivo. Several
different transgenic mouse models for SS disease are
available.28-34 Many of these models show (to different
degrees) significant RBC sickling upon deoxygenation in vitro and the
presence of circulating ISC in vivo. The 2 more recent
models33,34 seem to mimic closely the clinical and
pathologic features of the human disease. The SAD mouse model has been
widely used, especially for studies on ion transport and cell
dehydration, although these mice do not have anemia, have only mild
reticulocytosis, and have normal RBC survival (C. Joiner, personal
communication, December 1998). The ion transport pathways
of SAD erythrocytes have been characterized in detail,35
and their response to either oral CLT or Mg therapies reproduces that
seen in patients with SS disease.11,18
Hydroxyurea (HU) therapy induces macrocytosis, leukopenia, and an
increase of the synthesis of the minor globin chain, with improvement of anemia in a mouse model of human thalassemia intermedia.36,37 In normal mice, 30 days of HU therapy
induce macrocytosis and leukopenia, with no changes in reticulocyte
counts.36 Because there is no clearly demonstrable
equivalent of Hb F in mice, studies with HU in SAD mice may be helpful
to identify effects that are not related to increased cellular
concentration of Hb F. Clinical studies in patients with SS disease
have identified cellular changes that are independent of Hb F levels
and may explain some of the beneficial effects of HU
therapy.38
The human and mouse studies indicate that both Gardos channel and K-Cl
cotransport are involved in the in vivo generation of dense sickle
cells, as recent in vitro studies suggest.9,10,39-42 The
objectives of this study using the SAD mouse model are to determine
whether chronic hypoxia (48 hours) induces in vivo changes in
erythrocyte features, including the formation of dense cells; what the
effects are of different pharmacological regimens, including either HU,
CLT, or Mg on the cellular changes induced by hypoxia; and what is the
added benefit of combining CLT and Mg therapies.
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MATERIALS AND METHODS |
Drugs and chemicals.
NaCl, KCl, ouabain, bumetanide, Tris (hydroxymethyl) aminomethane
(Tris), 3(N-morpholino) propanesulfonic acid (MOPS), choline chloride,
and Acationox were purchased from Sigma Chemical Co (St Louis, MO).
MgCl2, dimethylsulfoxide (DMSO), n-butyl phthalate, and all
other chemicals were purchased from Fisher Scientific Co (Fair Lawn,
NJ). Microhematocrit tubes were purchased from Drummond Scientific Co
(Bromall, PA). All solutions were prepared using double-distilled water.
Animals and experimental design.
Transgenic Hbbsingle/single SAD1 (SAD) mice were used for
the experiment, whereas the control group consisted of nontransgenic
litter mates. All of the mice were obtained from breeding performed in
the animal facility of INSERM at Henri Mondor Hopital (Creteil,
France).31 Males between 4 and 6 months of age (weight, 28 to 30 g) were used for this study. Twenty-eight SAD mice were divided
into 5 different groups: control (n = 6), HU (200 mg/d, n = 637), CLT (80 mg/kg/d, n = 5),18 Mg (1,000 mg/kg/d, n = 5),11 and CLT+Mg (80 and 1,000 mg/mg/d,
respectively, n = 6).
Nine normal control mice were divided into 3 groups, which were treated
for 21 days with 1 of the following regimens: control, HU (200 mg/d),
and CLT+Mg (80 and 1,000 mg/kg/d, respectively).
HU was suspended in water (0.2 mL). CLT was suspended in a solution
containing deoxycholate (5 mg/mL) and cellulose (0.6%) to a final
concentration of 20 mg/mL. Mg supplementation was achieved by adding an
additional 600 mg/kg body weight/d for a total Mg of 1,000±20 mg
Mg/kg body weight/d to the Mg contained in the regular mouse feed. The
Mg supplement consisted of magnesium hydroxide dissolved in water. HU
as well as CLT and Mg were administered by gavage. HU and Mg were
administrated once daily, whereas CLT was administered twice daily.
The different mouse groups were studied at baseline, at 21 days of
therapy, and after 48 hours of hypoxia. No changes in body weight were
observed during the treatments. A total of 200 µL of blood was drawn
from each animal at the specific times and used for Rb+
influx measurements, erythrocyte phthalate density distribution curves,
cell morphology, erythrocyte cation content, and other hematological
parameters. It is our experience that 200 µL of blood can be drawn
from mice without incurring significant reticulocytosis.
Hypoxia studies.
Treated and untreated SAD and control mouse groups were maintained at
8% oxygen for 48 hours. Oxygen pressure inside the enclosed cage was
monitored with an oxygen electrode. Hematological parameters, cell
morphology, RBC density patterns, Gardos channel, and erythrocyte cation content were examined before and after 48 hours of hypoxic exposure. The different therapeutic regimens were continued during the
exposure to hypoxic conditions.11,18,37,43
Hematological data and cation content.
Blood was collected from ether-anesthetized mice by retro-orbital
venipuncture into heparinized microhematocrit tubes. Hb concentration
was determined by spectroscopic measurement of the cyanmet derivative.
Hematocrit (Hct) was determined by centrifugation in a micro-Hct
centrifuge. Reticulocytes were counted on a Coulter EPICS profile II
(Coulter Electronics, Hialeah, FL) using thiazole orange staining: 2.5 µL of whole blood was incubated for 20 minutes with 0.1 mg of
thiazole orange dissolved in 1 mL of filtered phosphate-buffer saline
(PBS) buffer. The fluorescence of 50,000 erythrocytes was collected
with log amplification.44 White blood cells (WBCs) were
measured on a Coulter STK-S hematology analyzer.
Density distribution curves were obtained according to Dannon and
Marikovsky,45 using phthalate esters in microhematocrit tubes, after washing the cells 3 times with PBS solution (330 mosmol/L) at 25°C in 2-mL tubes. The remaining cells
were washed 4 additional times with choline washing solution (170 mmol/L choline, 1 mmol/L MgCl2, 10 mmol/L Tris-Mops, pH
7.4, at 4°C, 330 mosmol/L) for measurements of
internal Na and K content by atomic absorption spectrometry.
Measurements of Ca2+-activated Rb+ influx in
mouse RBCs.
Whole blood was incubated for 30 minutes at room temperature in the
presence of 1 mmol/L ouabain, 10 µmol/L bumetanide, and 20 mmol/L
Tris-Mops, pH 7.4. The ionophore A23187 was added to the mouse blood to
a final concentration of 80 µmol/L, followed by an additional 6 minutes of incubation under stirring at 22°C. At 0 time, RbCl was
added to the cell suspension to a final concentration of 10 mmol/L in
plasma and incubated at 37°C. Aliquots were removed after 0, 2, 3, and 5 minutes; transferred to a 2 mL medium containing 150 mmol/L NaCl
and 15 mmol/L EGTA, pH 7.4, at 4°C; washed 3 times at 4°C with
the same solution; and lysed in 1.5 mL of 0.02/Acationox. The lysate
was then centrifuged for 10 minutes at 3,000g. Rb+
content was measured in the supernatant by atomic absorption spectrophotometry.
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RESULTS |
Effects of HU, CLT, Mg, and CLT+Mg treatments on hematological
parameters.
HU therapy in normal control mice produced no significant changes in
Hct and Hb (data not shown). In SAD mice, HU induced an increase in Hct
(from 44.4% ± 1.1% to 46.7% ± 1.1%, P < .005), mean corpuscular volume (MCV; from 43.1 ± 0.4 fL to 45.8 ± 0.3 fL, P < .05), Hb, and reticulocyte counts
(Table 1) over their normal baseline values
and a decrease in WBC counts (Table 1). A shift in the phthalate
density distribution curve towards lower values was also observed
(Fig 1B and
Table 2).
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Table 1.
Effects of HU, CLT, Mg, and CLT + Mg Treatments Under
Ambient and Hypoxic Conditions on Hematological Parameters in SAD
Mice
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| Fig 1.
(A) The effect of chronic hypoxia (48 hours at 8%
O2) on the density distribution of erythrocytes in an SAD
mouse. This animal is representative of 6 animals. D50 was
1.099 ± 0.002 at baseline and increased after hypoxia to 1.107 ± 0.004 (P < .05, n = 6). (B) The effect of HU treatment on
the changes induced by chronic hypoxia in an SAD mouse. This animal is
representative of 6 animals. D50 was 1.101 ± 0.002 at
baseline, 1.098 ± 0.003 (P < .05, n = 6) after 3 weeks of
HU treatment, and 1.101 ± 0.004 (not significantly different
[NS] from baseline) after hypoxia. (C) The effect of
CLT treatment on the changes induced by chronic hypoxia in an SAD
mouse. This is representative of 5 animals. D50 was 1.097 ± 0.001 at baseline, 1.092 ± 0.01 (P < .005, n = 5)
after 3 weeks of CLT treatment, and 1.097 ± 0.02 (NS from baseline)
after hypoxia. (D) The effect of Mg treatment on the changes induced by
chronic hypoxia in an SAD mouse. This is representative of 5 animals.
D50 was 1.097 ± 0.001 at baseline, 1.093 ± 0.001 (P < .05, n = 5) after 3 weeks of Mg treatment, and 1.096 ± 0.002 (NS from baseline) after hypoxia. (E) The effect of CLT+Mg
treatment on the changes induced by chronic hypoxia in an SAD mouse.
This is representative of 6 animals. D50 was 1.102 ± 0.002 at baseline, 1.095 ± 0.002 (P < .005, n = 5) after 3 weeks of CLT+Mg treatment, and 1.096 ± 0.005 (P < .05 from baseline) after hypoxia.
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Table 2.
Effects of HU, CLT, Mg, and CLT + Mg Treatments Under
Ambient and Hypoxic Conditions on Erythrocyte Gardos Channel
Activity, K Content, and D50 of SAD Mice
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CLT treatment of SAD mice resulted in a significant increase in Hct
(from 43.9% ± 1.3% to 47.0% ± 1.2%, P < .05) and
a decrease in cell density (Table 2 and Fig 1C). Hb, reticulocyte, and
WBC counts were unchanged after CLT therapy (Table 1).
Mg treatment of SAD mice resulted in significant increases in Hct (from
43.6% ± 0.7% to 45.5% ± 0.2%, P < .05) and Hb
(Table 1) and decreased cell density (Table 2 and Fig 1D), as described in our previous report.11 Mg treatment did not induce
significant changes in either reticulocyte or WBC counts (Table
1).
A combination of CLT+Mg treatments in normal control mice resulted in
significant increases in Hb (from 14.1 ± 0.4 to 15.3 ± 0.3 g/dL, P < .02) and Hct (from 45.8% ± 1.3% to 48.2% ± 0.6%, P < .02). Because we have previously
demonstrated that CLT administration did not affect the hematological
parameters of normal mice,18 whereas Mg increased Hb and
Hct,11 these effects are most likely due to Mg supplementation.
Combined treatment with CLT+Mg of SAD mice resulted in significant
increases in Hct (from 44.5% ± 1.0% to 47.6% ± 1.4%,
P < .005) and Hb (Table 1) and decreased cell density (Table
2 and Fig 1E). No significant changes were observed in either
reticulocyte or WBC counts (Table 1).
Effects of HU, CLT, Mg, and CLT+Mg treatments on erythrocyte K and Mg
content and Gardos channel activity.
SAD mice have a reduced erythrocyte K content and normal activity of
the Gardos channel (Table 2 and De Franceschi et al11,18).
HU treatment did not modify the activity of the Gardos channel in
either normal control (data not shown) or SAD mice (Table 2). The
erythrocyte K content was unchanged by HU treatment in normal mice
(data not shown), whereas it increased significantly in SAD mice (Table
2).
CLT treatment of SAD mice induced marked inhibition of the Gardos
channel and significantly increased erythrocyte K content (Table 2), as
described in our previous study.18
Mg treatment of SAD mice yielded no changes in Gardos channel activity
and significantly increased erythrocyte K content (Table 2), as shown
before.11 Erythrocyte Mg content increased from 9.6 ± 0.7 mmol/kg Hb to 13.9 ± 1.1 mmol/kg Hb (n = 5, P < .05) and plasma Mg increased from 0.94 ± 0.9 mmol/L to 1.5 ± 0.7 mmol/L (n = 5, P < .05).
Combined CLT+Mg treatments of normal control mice inhibited Gardos
channel without affecting K content (data not shown). As expected, SAD
mice treated with CLT+Mg showed a reduction in the activity of the
Gardos channel and increased erythrocyte K content (Table 2). CLT+Mg
treatment resulted in an increase in both plasma and erythrocyte Mg
levels in SAD mice (plasma: 0.75 ± 0.4 mmol/L at baseline v
1.7 ± 0.6 mmol/L, n = 5, P < .05; erythrocytes: 8.7 ± 1.2 mmol/kg Hb at baseline v 12.9 ± 1.7 mmol/kg Hb after treatment, n = 5, P < .05).
Effects of hypoxia.
To evaluate the effect of the 4 therapeutic regimens on the changes
induced by hypoxia, control and transgenic mice were exposed for 48 hours to an atmosphere containing 8% O2. No significant changes in Hct or Hb were observed in normal control mice after hypoxia
(data not shown).
In untreated SAD mice, hypoxia induced a shift of the phthalate density
profiles toward higher erythrocyte density values, indicating that
hypoxia exacerbates RBC dehydration (Table 2 and Fig 1A). Erythrocyte K
content also decreased significantly with hypoxia (Table 2). No
significant changes were observed in either reticulocyte or WBC counts
after hypoxia (Table 2).
In HU-treated SAD mice, hypoxia decreased Hb levels (Table 1), MCV
(from 45.8 ± 0.3 fL to 43.4 ± 0.2 fL, P < .05), and
cell K content (Table 2), whereas cell density showed a trend toward higher values (Fig 1B), which, however, was not statistically significant (Table 2). Reticulocyte or WBC counts did not change with
hypoxia in HU-treated SAD mice (Table 1). Cell K content and density in
HU-treated SAD mice exposed to hypoxia were still significantly
different from those of untreated, hypoxic SAD mice (P < .02 and P < .05, respectively, ANOVA).
In CLT-treated mice exposed to hypoxia, discrepant results were
obtained between measured cell density, which increased significantly (Table 2 and Fig 1C), and measured cell K content, which was unchanged
(Table 2). This unexplained discrepancy does not allow us to determine
with certainty how much of the erythrocyte dehydration induced by
chronic hypoxia is mediated by the Gardos channel. It should be noted
that, with hypoxia, erythrocyte density and cation contents of
CLT-treated SAD mice were still significantly different from those of
untreated SAD mice (Table 2, ANOVA, P < .005), indicating an
effect of CLT on hypoxia-induced dehydration.
In Mg-treated mice exposed to hypoxia, a reduction in Hb (Table 1) and
cell K content and an increase in cell density were noted (Table 2 and
Fig 1D) that almost completely abolished the changes induced by 21 days
of Mg therapy. The K loss induced by chronic hypoxia was essentially
the same as that of untreated SAD mice, indicating that the K-Cl
cotransport plays a minor role in hypoxia-induced dehydration of SAD
erythrocytes, which seems to be mostly a Gardos phenomenon.
Interestingly, the density and cation content of Mg-treated mice after
hypoxia were still significantly different (P < .005 and
P < .03, respectively, ANOVA) than those of hypoxic,
untreated SAD mice (Table 2).
In SAD mice treated with CLT+Mg, hypoxia induced no significant changes
in either Hb, reticulocyte, or WBC counts (Table 1). Erythrocyte K
content and cell density did not change significantly with hypoxia and
remained significantly different from baseline values (Table 2) and
from untreated SAD mice (P < .002 and P < .005, respectively, ANOVA, Table 2). These data indicate that CLT+Mg
treatment almost completely abolished the density changes induced by
chronic hypoxia. Because Mg was ineffective in preventing hypoxia-induced dehydration, it is likely that blockade of the Gardos
channel is responsible for these effects. However, due to the
discrepancies observed in the CLT-treated group and differences in
baseline density among the various groups, the superiority of CLT+Mg
treatment compared with the other regimens cannot be convincingly demonstrated.
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DISCUSSION |
We have examined in this study the effect of 4 therapeutic regimens,
including either HU, CLT, Mg, or CLT+Mg, on the changes induced by a
short-term (48 hours) exposure to hypoxia in the SAD mouse model. These
studies were prompted by several in vitro and in vivo studies that have
identified a role for the erythrocyte Gardos channel and K-Cl
cotransporter in promoting erythrocyte dehydration.9,46
Combination treatment with CLT and Mg offers the theoretical
possibility of interfering with the dehydration of both reticulocytes
and mature erythrocytes by inhibiting the 2 major pathways for sickle
cell dehydration.
The SAD mouse has shown to be extremely valuable in assessing the
cellular effects of therapies aimed at preventing sickle cell
dehydration. SAD mouse erythrocytes resemble human sickle erythrocytes
in having a reduced K content, normal Gardos channel activity at
baseline, and increased K-Cl cotransport.11,18,35 The
response observed in SAD mice to either CLT or Mg therapies is similar
to that observed in patients with sickle cell
disease.11,18,19,35 Thus, although SAD mice are not anemic,
they exhibit significant RBC dehydration and organ damage and are a
valuable model for studies on ion transport and blockade of cell dehydration.
K-Cl cotransport plays a major role in the dehydration of sickle
erythrocytes and reticulocytes. Transferrin receptor-positive (Tfr+) dense reticulocytes have greater K-Cl cotransport
activity than Tfr+ light reticulocytes, suggesting that
K-Cl cotransport may mediate dehydration of young sickle
cells.47 K-Cl cotransport activity is modulated by the
erythrocyte Mg content, which is markedly reduced both in transgenic
SAD mouse and human sickle erythrocytes.11,12 We have shown
that oral Mg supplementation induces an increase in RBC Mg content
that, in turn, leads to a reduction in K-Cl cotransport activity and
cell dehydration.11-13 However, although the Gardos channel
has been shown to become active with deoxygenation,17,48,49 the role of K-Cl cotransport in promoting dehydration in conditions of
hypoxia is not well established.10,40,50 For these reasons, we have examined the effect of pharmacological blockade of these ion
pathways in the SAD mouse under conditions of chronic hypoxia.
The results presented here indicate that (1) hypoxia induces formation
of dense erythrocytes in SAD mice; (2) HU, CLT, Mg, or CLT+Mg therapies
improve the hydration state of erythrocytes and blunt the erythrocyte
dehydration induced by hypoxia; (3) hypoxia-induced dehydration in the
SAD mouse is mediated almost exclusively by the Gardos channel; and (4)
combination of CLT and Mg treatments may have an additive effect in
protecting from the erythrocyte dehydration induced by hypoxia, but the
results presented here are not unequivocal.
Although other studies have demonstrated formation of dense cells by
hypoxia in transgenic sickle mice, no information was available on the
mechanisms underlying the formation of dense mouse erythrocytes. Rubin
et al,29 using a mouse model expressing both human and
S Antilles (50% of total Hb), exposed the transgenic
mice for 10 days at 8.4% O2 and showed a significant
increase in irreversibly sickled cells. Similar results were obtained
by Fabry et al,28 who exposed the human aH and
S (MDD) transgenic mice for 3 or 5 days
to hypoxia (8% O2). This group also observed a significant
reduction in urine osmolarity due to compromised renal
function.28 Reilly et al30 examined 3 lines of
transgenic Hb S mice with human S contents of
approximately 30%, 50%, and 80% relative to mouse globins.
Exposure to hypoxia (7% O2) for 7 days resulted in increased Hct, Hb, and MCV and significant reticulocytosis, indicating that this level of chronic hypoxia significantly stimulated
erythropoiesis. An increase in the percentage of cells residing in the
most dense fraction was also noted.30
Duration of exposure to hypoxia seems to be a critical variable for
these studies. We have observed significant reticulocytosis both in
control and thalassemic mice after 5 days of hypoxic exposition (Y. Beuzard, unpublished data). The shorter period of hypoxia
(48 hours) allowed us to study erythrocyte changes primarily due to
polymerization of Hb SAD. With our study, we were able to evaluate the
effect of 4 different treatments on these erythrocyte changes with no
significant changes in reticulocyte counts. Recently, hypoxia has been
shown to enhance sickle cell adhesion to both macrovascular and human
microvascular endothelial cells via the adhesive receptor vascular cell
adhesion molecule-1 (VCAM-1), suggesting that
reticulocytes may be involved in the enhanced adherence to the hypoxic
endothelium.51-53 It will be of interest to determine
whether adhesion to endothelium of sickle transgenic
erythrocytes,54 in addition to being modulated by hypoxia,
can also be affected by either HU, CLT, or Mg therapies.
In this study, HU treatment of SAD mice induced a significant increase
in MCV and a decrease in WBC counts (Table 1), as observed in humans.
However, because mice do not produce Hb F, the effects of HU on
erythrocyte cation content and density of SAD mice are not easily
explained. Our data clearly indicate that HU has no effect on the
activity of the Gardos channel (Table 2). In addition, HU induced a
significant reticulocytosis in SAD mice, whereas it usually decreases
reticulocyte counts in SS patients.38,55 We have described
a marked reduction of the in vitro adherence of human sickle
erythrocytes to endothelium in the early phase of HU
therapy.38 Whether this effect is present in transgenic
sickle mice remains to be determined.
These studies provide experimental evidence for a major role of the
Gardos channel in promoting dehydration of SAD mice erythrocytes under
conditions of chronic hypoxia. They also demonstrate the beneficial
effects of HU, CLT, Mg, and CLT+Mg therapies in preventing or blunting
the hypoxia-induced dehydration. Combination therapy with CLT and Mg
could in theory be superior to single-agent therapy in preventing cell
dehydration. However, under experimental conditions that maximize
dehydration via the Gardos channel, this potential additive benefit
could not be confirmed.
| |
FOOTNOTES |
Submitted April 23, 1998; accepted August 11, 1999.
Supported by National Institutes of Health grants from the Heart, Lung
and Blood Institute (P60-HL15157 and HL 58930); from the Diabetes,
Digestive, and Kidney Diseases Institute (R01-DK50422); and from the
"Associazione Filippo Collerone," Caltanissetta, Italy.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Carlo Brugnara, MD, Department of
Laboratory Medicine, The Children's Hospital, 300 Longwood Ave, Bader
760, Boston, MA 02115; e-mail: brugnara{at}A1.TCH.HARVARD.EDU.
| |
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