|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 96 No. 1 (July 1), 2000:
pp. 314-320
RED CELLS
Reperfusion injury pathophysiology in sickle
transgenic mice
U. Raymond Osarogiagbon,
Stephana Choong,
John D. Belcher,
Gregory M. Vercellotti,
Mark S. Paller, and
Robert P. Hebbel
From the Department of Medicine, University of Minnesota Medical
School, Minneapolis, MN.
 |
Abstract |
Reperfusion of tissues after interruption of their vascular supply
causes free-radical generation that leads to tissue damage, a scenario
referred to as "reperfusion injury." Because sickle disease
involves repeated transient ischemic episodes, we sought evidence for
excessive free-radical generation in sickle transgenic mice. Compared
with normal mice, sickle mice at ambient air had a higher ethane
excretion (marker of lipid peroxidation) and greater conversion of
salicylic acid to 2,3-dihydroxybenzoic acid (marker of hydroxyl radical
generation). During hypoxia (11% O2), only sickle mice
converted tissue xanthine dehydrogenase to oxidase. Only the sickle
mice exhibited a further increase in ethane excretion during
restitution of normal oxygen tension after 2 hours of hypoxia. Only the
sickle mice showed abnormal activation of nuclear factor- B after
exposure to hypoxia-reoxygenation. Allopurinol, a potential therapeutic
agent, decreased ethane excretion in the sickle mice. Thus, sickle
transgenic mice exhibit biochemical footprints consistent with
excessive free-radical generation even at ambient air and following a
transient induction of enhanced sickling. We suggest that reperfusion
injury physiology may contribute to the evolution of the chronic organ
damage characteristic of sickle cell disease. If so, novel therapeutic
approaches might be of value.
(Blood. 2000;96:314-320)
© 2000 by The American Society of Hematology.
| |
Introduction |
A significant proportion of the tissue damage resulting
from interruption of vascular flow can be attributed to oxygen free radicals that are generated after restitution of blood flow and, with
it, oxygen supply. This concept is referred to as "reperfusion injury." The occurrence of oxidant-mediated damage resulting from such ischemia-reperfusion cycles has been demonstrated in several different animal organ and tissue models.1-3
Among the biochemical changes that occur under low oxygen tension after
tissues lose their blood supply are catabolism of adenosine
triphosphage to hypoxanthine and conversion of xanthine dehydrogenase
(XD) to xanthine oxidase (XO).2-4 Unlike XD, in the
presence of oxygen XO generates superoxide when it catalyzes the
conversion of xanthine or hypoxanthine to uric acid. Therefore, restitution of blood flow and oxygen supply allows XO to rapidly generate superoxide and ultimately, through iron-dependent chemistry, generate the highly reactive and damaging hydroxyl
radical.5,6 In some reperfusion injury models, other
oxidant-generating mechanisms seem to predominate in the free-radical
generation that is common to all reperfusion injury situations.
Examples include oxidants derived from activated inflammatory
cells,7 the mitochondrial electron transport
chain,8 and peroxynitrite metabolism.9-11 The
many consequences of this initial free-radical generation include
peroxidation of polyunsaturated membrane fatty acids1-11 and activation of nuclear factor-B (NF-B).12,13
Induction of cytokines during this process is an additional mechanism
that activates NF-B.8,9,14,15 This transcription factor
up-regulates expression of a number of proinflammatory molecules such
as endothelial cell adhesion molecules. Its activation thereby promotes
neutrophil recruitment and a generalized endothelial prothrombotic
state.14-16 Thus, reperfusion injury states are
characterized by vigorous inflammatory responses.8,9
Considering these characteristics of reperfusion injury physiology, we
hypothesized that sickle cell anemia represents an archetypal
reperfusion injury scenario. Insofar as the vasoocclusive character of
sickle cell disease is caused by reversible red blood cell (RBC)
sickling, perfect opportunity should be provided for transient ischemia
or hypoxia, followed by reperfusion. In fact, no other human disease is
characterized by lifelong, and perhaps near-constant,17,18
development of transient vasoocclusion. While no direct evidence
for reperfusion injury in sickle cell disease has been reported yet,
several aspects of the disease are consistent with it (see
"Discussion"). Therefore, as a partial test of our hypothesis
that reperfusion injury physiology contributes to the vascular
pathobiology of sickle disease, in particular to chronic organ
deterioration, we investigated several markers of reperfusion injury in
sickle transgenic mice.
| |
Materials and methods |
Mice
We established a colony of sickle transgenic mice using founders
kindly provided by Dr Mary Fabry. These mice have linked human  and
 S globin transgenes on the C57Bl/6J background
homozygous for absence of murine major
globin.19 Hematologic characteristics, the organ damage
that these mice undergo at ambient air, their response to hypoxia, and
other features of this mouse model have been reported
elsewhere.19-21 Normal control mice were C57Bl/6J, the same
genetic background as the sickle mice.
Study protocol
We studied mice subjected to a sequential hypoxia-reoxygenation
(H/R) protocol and mice at ambient air. The H/R protocol involved a
30-minute prehypoxia period for baseline measurements at ambient air,
followed by 120 minutes of hypoxia, and then a 50-minute reoxygenation
period during which ambient air conditions were restored. We used 2 different methods to induce the hypoxia.
For studies that required collection of expired gas, the mice were
anesthetized with intraperitoneal pentobarbital (10 mL/kg of a 0.5%
solution) and intubated with a Silastic tracheostomy tube that was
connected to a low-resistance unidirectional flow valve (provided by
Hans Rudolph, Kansas City, MO). Inflow gases (ultrapure
oxygen and nitrogen) passed through a flow meter, mixing chamber, and
humidifier before passing into the mouse, which breathed the regulated,
nonpressurized mixture spontaneously on demand. Outflow gases passed
into a collection tube (see "Measurement of expired ethane"). To
induce hypoxia, the flow of gases was adjusted to supply
11% ± 1% oxygen, which we found to correspond to a
PaO2 of 70 ± 2 mmHg, a level at which the sickle mice
developed a significant increase in proportion of sickled cells. During the reoxygenation period, the flow of gases was adjusted to
restore 21% oxygen.
For studies not requiring collection of expired gas, unanesthetized
mice were placed in a closed chamber, and the mixture of gas
flow was adjusted to supply 11% ± 1% oxygen for the duration of
the hypoxia period. For reoxygenation and for the ambient air controls,
mice were left to breathe room air. Thus, this protocol entailed
neither surgical manipulation nor anesthesia.
Measurement of induced sickling
To measure red cell sickling, we drew blood via a carotid artery
catheter directly into a syringe containing 10% glutaraldehyde. A drop
of blood was viewed by light microscopy to assess the proportion of
sickled cells out of 500 randomly counted cells. Sickled cells were
defined as having a length-to-width ratio of at least 3:1.
Measurement of expired ethane
As an index of whole-body lipid peroxidation, we measured ethane in
expired gas,22 a method that has previously been applied successfully to study of oxidant stress in the mouse.23-28
Expired gas was passed through a Carbotrap 200 desorption tube (Supelco Inc, Bellefonte, PA), which adsorbs short-chain hydrocarbons at room
temperature.29 Expired gas was thus collected during the 30-minute prehypoxia baseline period and the 50-minute reoxygenation period. After collection, the Carbotrap tube was immediately
transferred to a ballistic thermal desorber (Thermal Tube Desorber
Model 890; Dynatherm Analytical Instruments, Kelton, PA),
which transferred the trapped hydrocarbons to a gas chromatograph
(Hewlett-Packard 5890A; Hewlett Packard, Palo Alto, CA)
with a flame ionization detector. The amount of expired ethane was
calculated by comparison of a sample signal with that from known
standard gases.
To evaluate the effect of allopurinol on ethane excretion, we studied
animals at ambient air. A 30-minute gas collection at baseline was
followed by intraperitoneal injection of 40 mg/kg of a 0.4% (wt/vol)
solution of allopurinol (Sigma, St Louis, MO) or vehicle
control. Ambient air conditions were continued for 4 hours, during the
last 30 minutes of which another postallopurinol gas collection was made.
Measurement of 2,3-dihydroxybenzoic acid
To indirectly detect hydroxyl radical generation, we measured
hydroxylation of salicylic acid (SA) (Sigma) to form
2,3-dihydroxybenzoic acid (2,3-DHBA)30,31 using a
modification of the method of Kim and Wells.32 This method
has been previously applied successfully to the study of oxidant stress
in the mouse.33,34 Thirty minutes after intraperitoneal
injection of 0.1 g/kg of a 10-mg/mL solution of SA, 100 to 200 µL of
blood was drawn and centrifuged for 10 minutes. Then, 50 µL of plasma
was extracted and vortexed with 50 µL of 8-µM 3,4-DHBA (added as an
internal standard), 50 µL of 12-N HCl, and 500 µL of ethyl acetate.
This was centrifuged for 2 minutes at 5000 rpm, and 200 to 400 µL of
supernatant was extracted. The ethyl acetate was evaporated under
nitrogen gas, and the residue was dissolved in 50µL of the mobile
phase solution, centrifuged for 10 minutes, and injected in aliquots of
10 µL into a high-performance liquid chromatographer
(Hewlett-Packard Series II 1080; Vydac 201HS RP-18C analytical column;
flow rate 0.8 mL/minute, running time 60 minutes; mobile phase
solution: 0.03-M sodium citrate and 0.03-M sodium acetate, pH 4.0).
Peaks for 2,3-DHBA, 3,4-DHBA, and SA were recorded on Chemstation
software (Hewlett-Packard) and the molar amounts calculated using
standard curves recorded daily. Levels of 2,3-DHBA (µM) and SA (mM)
were corrected using the 3,4-DHBA internal standard and the final level of 2,3-DHBA expressed as a ratio to level of SA.30
The differing half-lives of SA and 2,3-DHBA, coupled with the wide
variation in rate of clearance of SA from animal to animal, made it
impossible to apply this assay to animals run through our H/R protocol,
which would have required more than 1 injection of SA. Therefore, this
assay was applied only to animals breathing ambient air.
Measurement of NF-B activation
Nuclear protein extraction.35
From mice rendered unconscious by a 20-second exposure to
CO2, liver and kidney were rapidly harvested and
immediately frozen in liquid nitrogen.
A total of 500 mg of tissue was ground in a mortar while still immersed
in liquid nitrogen, crushed with the tight pestle of a Dounce
homogenizer in 5 mL of a cell lysis buffer (0.6% Nonidet P-40
[NP-40]; 150-mmol/L NaCl; 10-mmol/L HEPES, pH 7.9; 0.5-mmol/L phenylmethylsulfonyl fluoride [PMSF]) and centrifuged for 30 seconds at 2000 rpm. The supernatant was incubated on ice for 5 minutes and
recentrifuged at 5000 rpm for 5 minutes. The nuclear pellet was
resuspended in 500 µL of a nuclear extraction buffer (25% glycerol;
20-mmol/L HEPES, pH 7.9; 420-mmol/L NaCl; 1.2-mmol/L MgCl2;
0.2-mmol/L ethylenediaminetetraacetic acid [EDTA]; 0.5-mmol/L dithiothreitol (DTT); 0.5-mmol/L PMSF; 2-mmol/L
benzamidine, and 0.5 µg/mL each of pepstatin, leupeptin and
aprotinin), incubated on ice for 20 minutes, and centrifuged for 20 seconds at 14 000 rpm. Aliquots of the supernatant were stored at
 70°C until use. Sample protein content was assayed using a
BCA Protein Assay kit (Pierce, Rockford, IL); after incubation
(37°C for 30 minutes), sample absorbance at 600 nm was compared
with an albumin standard.
NF-B
probes.36
A 35-base-long oligonucleotide probe, designed from the
NF-B binding site
(5'-TCTCAACAGAGGGGACTTTCCGAGAGGCCATCTGG-3'), was radioactively labeled randomly using 32P-labeled
deoxycytidine triphosphate and deoxyguanosine triphosphate (Amersham
Corp, Buckinghamshire, England) and Klenow fragment.
The probe was centrifuged for 5 minutes at 2300 rpm in a Sephadex G-50
Quick Spin Column (Boehringer Mannheim, Indianapolis, IN).
Manipulations
To verify specificity of bands for NF-B, we used an excess of
unlabeled probe to compete for binding ("cold competition" assay). To obtain positive control cells for NF-B assays, we used
microvascular endothelial cells treated with tissue necrosis factor in
vitro, and we used mice given Escherichia coli
lipopolysaccharide and D-galactosamine.37
To identify which NF-B subunits were being visualized, we
conducted supershift assays in which antibodies to p50, p52, p65, or
cRel were included. Activity of each of these antibodies had previously been verified in other systems.
Polyacrylamide gel
A 4% polyacrylamide gel was pre-run for 1 hour in at 4°C prior
to loading samples. Each gel lane was loaded with a mixture of 10 µg
of nuclear protein extract, 5 µL of Markman's buffer (50-mM KCl;
20-mM HEPES, pH 7.9; 0.5-mmol/L EDTA; 5% glycerol, 1-mmol/L DTT;
0.5-mmol/L PMSF, 1-mg/mL bovine serum albumin, 0.1% NP-40), 1.5 µL
of labeled probe, 1 µL of poly(dIdC), 1 µL of bromphenol blue (if
needed, 1 µL of unlabeled probe or antibody), and distilled water to
a total volume of 26 µL. Electrophoresis was done for 1.25 hours in a
cold room.
Tissue XD-XO conversion
Hypoxia-induced conversion of tissue XD to XO was measured by
comparing XO activity with total XD-plus-XO activity38 in hypoxic and nonhypoxic tissue. This method has previously been used
successfully in mice.39,40 Hypoxic tissue was harvested after 2 hours of hypoxia with no reoxygenation period. Nonhypoxic tissue was harvested at ambient air.
From mice anesthetized with diethyl ether, liver and kidney were
harvested and immediately frozen in liquid nitrogen; 0.3 to 1 g of
tissue was crushed to a powder with a mortar and pestle while
continuously immersed in liquid nitrogen. The crushed tissue was then
transferred to a Dounce homogenizer containing 10 mL of "XD-XO
buffer" (0.25-mol/L sucrose, 100-mmol/L DTT, 100-mmol/L PMSF,
50-mmol/L EDTA, 50-mmol/L KH2PO4, pH 7.4) per
gram of tissue, homogenized with 10 strokes of the tight pestle, and
centrifuged for 30 minutes at 40 000g. The supernatant was
frozen in liquid nitrogen and stored at 70°C until use. A
total of 5 µL of the supernatant was diluted in 2 mL of a
"standard buffer" (50-mmol/L KH2PO4;
0.1-mmol/L EDTA, pH 7.4) in a glass cuvette at 37°C and read in an
LS-5B Luminescence Spectrophotometer (Perkin-Elmer Corp, Norwalk, CT)
set to 345-nm excitation, 390-nm emission, and 5-nm bandwidth slit. The
rate of pterin oxidation to isoxanthopterin, indicating XO activity,
was determined by adding 20 µL of 1-mM pterin and measuring the
fluorescence change over 5 minutes. Then, 20 µL of 1-mM methylene
blue was added as an electron acceptor to measure combined XD and XO
activity and the fluorescence change measured over 5 minutes. After
inhibiting the reaction with 20 µL of 10-mmol/L allopurinol, 5 µL
of 1-mM isoxanthopterine was added and the immediate increase in
fluorescence measured as an internal standard for calculating enzyme
activity using the formula: U = { F × [IXPT]/FIXPT} × 0.001 × Vc/(Vs × T), where U is the enzyme
activity in micromolars per minute per grams of tissue; F is the change in fluorescence intensity observed per minute; [IXPT] is the concentration of isoxanthopterin added at the end of
assay; FIXPT is the immediate increase in fluorescence
produced by addition of 5 µL of 1-mM isoxanthopterin; Vc
is the cuvette volume in milliliters; Vs is the volume of
sample added to the cuvette in milliliters; and T is the amount of
tissue, in grams per milliliter of homogenate.
Statistical analysis
Results are expressed as mean ± SD. ANOVA was done, and study
groups were compared using the Student t test. Instat software (GraphPad Software, San Diego, CA) was used for all
statistical analyses.
| |
Results |
Reversible sickling
Blood obtained from sickle mice that had been exposed to 2 hours of
hypoxia (11% ± 1% oxygen, reducing PaO2 from
98 ± 2 to 70 ± 2 mmHg) developed an increased proportion of
sickled RBCs (Figure 1). This was reversed
during the reoxygenation period, although there was some persistence of
misshapen, but not sickled, forms. There was no significant difference
in proportion of sickled cells between the prehypoxia and the
reoxygenation periods. Normal mice, as expected, did not show any
evidence of sickling with hypoxia (data not shown).
View larger version (36K):
[in this window]
[in a new window]
| Fig 1.
Proportion of sickled cells in sickle transgenic mice
before, during, and after induction of hypoxia.
The photomicrographs show representative sections of blood smears taken
from a single mouse during each period. There was no significant
difference between the prehypoxia and reoxygenation periods. Data shown
as mean ± SD (n = 9).
|
|
Unlike previously published models of reperfusion injury that entailed
induction of complete ischemia by completely occluding the blood supply
to a specific organ, our experimental design used exposure to moderate
systemic hypoxia to increase the likelihood that sickling-induced
occlusion would occur. Thus, we used the reversible RBC sickling as our
method of temporarily obstructing blood flow in the whole animal. The
disadvantage of this approach is that the degree to which hypoxia
occurs undoubtedly varies from organ to organ and from animal to
animal, thus increasing variability in the data. The advantage,
however, is that this manipulation simulates what happens
pathophysiologically in sickle disease. Moreover, this allows normal
mice subjected to the same conditions to be used as hypoxia-only
controls. Normal mice, which cannot show RBC sickling, would be exposed
only to transient hypoxia, while the sickle mice would possibly develop
reversible vasoocclusion and thereby develop actual ischemia and
reperfusion injury physiology.
Ethane excretion
Peroxidation of polyunsaturated membrane fatty acids during periods
of oxidant stress leads to formation of the volatile alkanes, which are
then excreted through the lungs. Thus, measurement of the levels of
ethane in the expired gases can identify a state of
increased oxidant stress.22 As shown in Figure
2, the sickle mice subjected to
hypoxia-reoxygenation had a significantly higher rate of ethane
excretion during the reoxygenation period than during the baseline,
prehypoxia period (P < .018). Normal mice, on the other
hand, exhibited no significant difference between the 2 periods. Of
additional interest was the fact that, compared with normal mice, the
sickle mice had a significantly (P < .03) higher rate of
ethane excretion, even under ambient air, prior to induction of
hypoxia. This was shown in separate experiments (not shown) but is also
evident in Figure 2.
View larger version (30K):
[in this window]
[in a new window]
| Fig 2.
Rate of ethane excretion in respiratory gases from normal
and transgenic mice studied prehypoxia and posthypoxia.
There was a significant difference between the baseline and
reoxygenation periods for the sickle mice but not for the normal mice.
Data shown as mean ± SD (n = 5 for the normal mice, n = 13 for
the sickle mice).
|
|
Activation of NF-B
When activated, NF-B moves from cytoplasm to the nucleus in
response to multiple potentially injurious environmental stimuli, including various cytokines and oxygen-free
radicals.12,13,15 Examining nuclear proteins extracted from
liver and kidney, we compared levels of nuclear translocation of
NF-B in sickle and normal mice, with and without exposure to H/R.
Figure 3 shows a gel representative of our
findings. There was no consistent evidence of NF-B activation at
ambient air in kidneys (lane 2) or liver (lane 9) of sickle mice.
However, after H/R, there was intense activation of NF-B in both the
kidneys (lane 4) and the liver (lane 11) of the sickle mice. In
contrast, there was no activation of NF-B in the normal mice either
at ambient air (lanes 7 and 13) or after H/R (lanes 8 and 14).
View larger version (27K):
[in this window]
[in a new window]
| Fig 3.
Electrophoretic mobility shift assay for NF-B.
Lane 1, positive control with TNF-treated microvascular endothelial
cells. Lanes 3 and 10 show a weak NF-B band in mice pretreated with
Escherichia coli LPS and D-galactosamine, a combination that
stimulates murine NF-B activation. The sickle mouse kidney reveals a
strong NF-B band after H/R (lane 4), which is not present in the
sickle mouse at ambient air (lane 2). The band on lane 4 is identified
as NF-B by the ability to competitively inhibit it with unlabeled
NF-B consensus oligonucleotide (lane 5). Sickle mouse liver at
ambient air shows a faint NF-B band (lane 9) that is much more
prominent in the mouse liver after H/R (lane 11). This band is
competitively inhibited by unlabeled NF-B consensus oligonucleotide
(data not shown). Antibody to the cRel subunit of NF-B does not
cause a supershift in sickle mouse liver or kidney. There is no NF-B
band demonstrable in the normal mouse liver or kidney with or without
H/R. Hb SS indicates sickle mouse; LPS, Escherichia coli
lipopolysaccharide plus D-galactosamine; cold wt comp, unlabeled
NF-B consensus oligonucleotide probe; anti-cRel, antibody to cRel
subunit of NF-B; MVEC, microvascular endothelial cell; TNF, tumor
necrosis factor; amb air, ambient air; H/R, hypoxia and
reoxygenation.
|
|
To confirm band specificity for NF-B, we conducted supershift
experiments. As shown in Figure 4, the
activated NF-B in the sickle mice consists of the p65 (lanes 6 and
11) and the p50 (lanes 8 and 13) subunits. There was no gel retardation
in response to antibodies to either the cRel subunit (lanes 6 and 12 in
Figure 3 and lanes 7 and 12 in Figure 4) or the p52 subunit (lanes 9 and 14 in Figure 4) of NF-B.
View larger version (38K):
[in this window]
[in a new window]
| Fig 4.
Electrophoretic mobility shift assay with antibody
supershift to identify the subunits of NF-B in the kidney and liver
of sickle mice exposed to H/R. Lanes 1 to 4, TNF-treated MVEC
controls; lanes 5 to 9, sickle mouse kidneys after H/R; lanes 10 to 14, sickle mouse liver after H/R. The supershift assay shows the presence
of the p65 (lanes 6 and 11) and p50 (lanes 8 and 13) subunits of
NF-B in the kidney and liver of the sickle mice after H/R. There is
no retardation with antibodies to p52 and cRel, suggesting that these
subunits are not involved in the H/R-induced activation of NF-B.
This experiment also confirms that the identified bands are indeed
caused by the presence of NF-B. MVEC indicates microvascular
endothelial cells; TNF, tumor necrosis factor; H/R, hypoxia and
reoxygenation; anti-p65/50/52/cRel, antibodies to these NF-B
subunits.
|
|
Therapeutic effect of allopurinol
Because excessive ethane generation (Figure 2) was evident in the
sickle mouse even at ambient air, we shifted our focus to the ambient
situation. To test the involvement of the XO pathway in the increased
ethane excretion, we administered the XO inhibitor allopurinol to
sickle and normal mice under ambient air conditions. As shown in Figure
5, preadministration of allopurinol
significantly reduced the rate of ethane excretion in both sickle
(P < .03) and normal mice (P < .02).
View larger version (36K):
[in this window]
[in a new window]
| Fig 5.
Rate of ethane excretion at ambient air in normal and
transgenic mice before and after administration of allopurinol.
Data shown as mean ± SD (n = 5 for both groups).
|
|
Salicylic acid hydroxylation
Hydroxyl radical attacks SA, generating the salicylate hydroxylation
products 2,5-DHBA and 2,3-DHBA. Unlike 2,5-DHBA, the only known route
of in vivo production of 2,3-DHBA is by hydroxyl radical attack on
SA.30,31 Therefore, we measured the level of 2,3-DHBA
production from SA in both sickle and normal mice at ambient air. As
shown in Figure 6, we observed
significantly higher levels of 2,3-DHBA in sickle mice than normal mice
(P = .0018), indicating a higher level of hydroxyl radical
generation at ambient air in the sickle mice. As noted previously,
technical constraints precluded reliable application of this test to
mice run through our H/R protocol.
View larger version (37K):
[in this window]
[in a new window]
| Fig 6.
Generation of hydroxyl radical in normal and transgenic
mice at ambient air, as measured by production of 2,3-DHBA from SA.
Data shown as mean ± SD (n = 9 for both groups).
|
|
Conversion of XD to XO
Because one mechanism of generation of free radicals involves
conversion of XD to XO during periods of interruption of tissue oxygen
supply, we compared the levels of XD in the liver and kidneys of mice
at ambient air and at the end of a 2-hour period of hypoxia. The liver
of sickle mice that were subjected to hypoxia (Figure 7) showed a significantly higher proportion
of XO than either hypoxic normal mice (P < .03) or sickle
mice at ambient air (P < .04). Results were similar in
kidney tissue (Figure 8).
View larger version (34K):
[in this window]
[in a new window]
| Fig 7.
Proportion of XO relative to XD in the liver of sickle
and normal mice at ambient air and after 2 hours of hypoxia only
(without reoxygenation).
Only the sickle mice show a significantly higher proportion of XO after
exposure to hypoxia. Data shown as mean ± SD (n = 4 for both
groups).
|
|
View larger version (31K):
[in this window]
[in a new window]
| Fig 8.
Proportion of XO relative to XD in the kidneys of sickle
and normal mice at ambient air and after 2 hours of hypoxia only
(without reoxygenation).
Only the sickle mice show a significantly higher proportion of XO after
exposure to hypoxia. Data shown as mean ± SD (n = 6) for both
groups.
|
|
| |
Discussion |
Sickle cell disease is characterized by such remarkable clinical and
hematologic heterogeneity, even within specific genotypes, that there
is a great deal of interest in the influence of acquired and
environmental factors in modulating the phenotypic appearance of
disease.41-43 Studies showing discordance between levels of intracellular hemoglobin polymer formation and the clinical course of
sickle cell disease44 argue that the wide variability in morbidity in patients with sickle cell anemia cannot be explained solely by the physical sickling of their red blood cells. Therefore, the chronic organ damage that occurs in these patients irrespective of
the frequency of vasoocclusive crises45 perhaps suggests the existence of a more subtle, yet inexorable, mechanism of injury that continues beyond the dramatic bouts of clinically evident severe
vasoocclusion. Mechanisms underlying the progression of this chronic
organ injury need to be identified.
Notably, sickle cell anemia patients manifest many features that are
consistent with the presence of ongoing reperfusion injury physiology
(described in "Introduction"). These include, for example, transient vascular occlusion, the iron-replete condition of patients, and an apparent influence of white blood cell counts on
mortality.46 Sickle patients tend to have elevated levels
of cytokines and other biological modifiers.17 Indeed, they
have elevated levels of various acute phase reactants even when not
experiencing acute painful episodes of sufficient severity to draw
clinical attention.17,47 This is believed to reflect
occurrence of subclinical ischemic episodes. It is also known that
endothelial cells react to reperfusion injury with a repertoire of
responses, including up-regulation of expression of cell adhesion
molecules, interleukins, von Willebrand factor, tissue factor, and
neutrophil recruitment.8,9,48,49 These features are also
believed to be part of the endothelial biology of sickle
disease.50-54 Indeed, it recently has been suggested that
sickle cell patients are in a near-constant inflammatory
state,51 which could result from ongoing reperfusion injury.
Thus, we propose that the repeated transient interruption of vascular
luminal patency, which occurs life-long in sickle patients, comprises a
state of ongoing ischemia-reperfusion injury physiology. In the present
report, we have conducted a partial test of this hypothesis by taking
advantage of the availability of the sickle transgenic mouse. We used a
model that has a phenotypically mild form of disease yet slowly
develops evidence of organ damage at ambient air,19-21
potentially allowing us to demonstrate the influence of subtle and
insidious contributors to chronic organ damage. We tested for the
presence of several typical footprints of reperfusion injury:
conversion of XD to XO, expired ethane to indicate lipid peroxidation,
conversion of plasma SA to 2,3-DHBA to indicate hydroxyl radical
generation, and activation of NF-B in liver and kidney as a
downstream effect of oxidant stress or evolution of cytokines. While
none of these individual markers is specific for reperfusion injury per
se, their concurrent development should be expected if reperfusion
injury is occurring and, therefore, provides evidence for presence of
this unique physiology.
Our results demonstrate an increase in these markers of reperfusion
injury in sickle mice subjected to hypoxia followed by reoxygenation.
The absence of such changes in normal mice subjected to the same
protocol clearly indicates that something different occurs in the
sickle mice. This is supported, in particular, by the fact that only
sickle mice converted XD to XO during hypoxia (without subsequent
reoxygenation). Because only the sickle mice could develop RBC sickling
and vascular occlusion during hypoxia, these results provide evidence
for occurrence of reperfusion injury physiology in the sickle mouse. Of
great additional interest, we found that the markers of oxidative
stress (expired ethane and salicylate hydroxylation) also were
expressed to a significantly higher level in sickle mice even under
ambient air conditions. This suggests a state of chronic reperfusion
injury physiology in the unmanipulated mouse at ambient air that could
explain the slow development of chronic organ damage.21
These observations also reveal why we could not provide
"hypoxia-only" control measurements in the sickle model. Unlike
traditional reperfusion injury models in which blood supply is
completely cut off for a defined period, the very nature of sickle
disease dictates that a period of modest hypoxia would itself include
some degree of concurrent reoxygenation, as the transient and
unpredictable occlusions develop and resolve.
Extant questions pertain to how directly these observations can be
extrapolated to humans with sickle disease. Four issues are
particularly important.
1. It is not yet known how to gauge the magnitude of an oxidant stress
measured in this manner in terms of whether it truly represents risk
for developing chronic organ damage, the predicted consequence of
ongoing reperfusion injury physiology. Neither our results nor the
literature are helpful in this regard. We are hopeful that future
application of preventive strategies to the sickle mouse model may be
able to answer this question.
2. Even though the oxidation detection systems we employed have been
validated previously in mice (per "Materials and methods"), there
is no way for us to know if our model generates a great deal of oxidant
stress or only a modest amount. The reason is that the endpoints
(expired ethane and salicylate hydroxylation) are systemic detectors
that, therefore, report averaged (diluted) oxidation from the whole
mouse. Yet the relevant damaging stress is likely to be taking place
only in certain organs or even suborgan regions. There can be no
"positive control" for our specific experimental model, because
our preliminary exploration of this model showed that more severe
hypoxia resulted in animal death, presumably from irreversible vascular
occlusion. We do note, however, that the -fold increases in our
measured endpoints (comparing sickle with normal animals at ambient air
and comparing sickle animals with versus without hypoxia-reoxygenation)
are consistent with those seen in previously described models of
oxidant stress. For example, we earlier described a 2-fold increase in
ethane excretion in the rat subjected to complete unilateral renal
ischemia followed by restoration of blood flow.55 Others
have similarly observed 2-fold ethane changes in mice that are
acutely23 or chronically25 iron-loaded, or that
are subjected to acute ethanol toxicity,24 or that develop
paracetamol26 or paraquat27 toxicity. For salicylate hydroxylation, no models as subtle as ours have been reported in mice, but severe concussive injury33 and
bilateral carotid occlusion followed by
reperfusion34 are reported to produce, respectively, 4-fold
and 1.5-fold increases in salicylate hydroxylation within the brain
tissue itself. Severe systemic oxidant stress in mice caused by
paraquat induced a 10-fold increase in plasma hydroxylation
products.32 In terms of XD/XO, the degree of XD-to-XO
conversion reported in the reperfusion injury literature varies widely
from tissue to tissue and model to model.
3. The extent to which sickle mouse physiology parallels human sickle
disease physiology remains to be defined.
4. Rodents seem to have higher levels of tissue XD/XO than
other animals,56 perhaps increasing the likelihood that we
would detect an effect of XD/XO in this model. Therefore, the apparent therapeutic benefit of allopurinol seen here might not hold up in
sickle humans if other mechanisms of oxidant generation predominate.
These issues will require future study. Nevertheless, our demonstration
that sickle mice exhibit biochemical footprints that are typical of
well-defined models of reperfusion injury physiology supports the
hypothesis that this is a feature of sickle disease. In separate
studies, we have preliminarily used this model to also observe abnormal
leukocyte endothelial attachment and emigration in sickle
mice.57 Thus, our findings could help explain the chronic
organ damage, the vascular injury and endothelial
activation,51,52 and the apparent inflammatory state that
are characteristic of sickle cell anemia. Indeed, there are striking
parallels between the vascular pathobiologies of reperfusion
injury58 and of sickle disease. Additionally, we have
furnished preliminary evidence for a potential therapy in sickle cell
disease: allopurinol. Various antioxidants and antiinflammatory
strategies would be expected to have therapeutic efficacy in this model
but remain to be tested. Our results argue for a general exploration of
the role of antioxidants and inhibitors of the various ischemia and
reperfusion mechanisms in the therapy of sickle cell disease. If our
results can be corroborated in human studies, it will provide the
rationale for initiation of completely novel therapy for patients with
sickle cell disease in an attempt to prevent chronic organ damage.
| |
Acknowledgments |
We thank Kuan Sheng, Marsha Patten, and Troy Decker for technical assistance.
| |
Footnotes |
Submitted October 22, 1999; accepted February 17, 2000.
Supported by the National Institutes of Health (PO1-HL55552).
Reprints: Robert P. Hebbel, Box 480 UMHC, 420 Delaware St, SE,
Minneapolis, MN 55455.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
References |
1.
McCord JM.
Oxygen-derived free radicals in postischemic tissue injury.
N Engl J Med.
1985;312:159[Abstract].
2.
Southorn PA, Powis G.
Free radicals in medicine, I & II.
Mayo Clin Proc.
1988;63:381[Medline]
[Order article via Infotrieve].
3.
Zimmerman BJ, Granger DN.
Mechanisms of reperfusion injury.
Am J Med Sci.
1994;307:284[Medline]
[Order article via Infotrieve].
4.
Freeman BA, Crapo JD.
Biology of disease: free radicals and tissue injury.
Lab Invest.
1982;47:412[Medline]
[Order article via Infotrieve].
5.
Voogd A, Sluiter W, van Eijk HG, Koster JF.
Low molecular weight iron and the oxygen paradox in isolated rat hearts.
J Clin Invest.
1992;90:2050.
6.
Kirschner RE, Fantini GA.
Role of iron and oxygen-derived free radicals in ischemia-reperfusion injury.
J Am Coll of Surg.
1994;179:103[Medline]
[Order article via Infotrieve].
7.
Ramos CL, Pou S, Britigan BE, Cohen MS, Rosen GM.
Spin trapping evidence for myeloperoxidase-dependent hydroxyl radical formation by human neutrophils and monocytes.
J Biol Chem.
1992;267:8307[Abstract/Free Full Text].
8.
Gute DC, Ishida T, Yarimizu K, Korthuis RJ.
Inflammatory responses to ischemia and reperfusion in skeletal muscle.
Mol Cell Biochem.
1998;179:169[Medline]
[Order article via Infotrieve].
9.
Granger DN, Korthuis RJ.
Physiologic mechanisms of postischemic tissue injury.
Annu Rev Physiol.
1995;57:311[Medline]
[Order article via Infotrieve].
10.
Grisham MB, Granger DN, Lefer DJ.
Modulation of leukocyte-endothelial interactions by reactive metabolites of oxygen and nitrogen: relevance to ischemic heart disease.
Free Radic Biol Med.
1998;25:404[Medline]
[Order article via Infotrieve].
11.
Humphries KM, Yoo Y, Szweda LI.
Inhibition of NADH-linked mitochondrial respiration by 4-hydroxy-2-nonenal.
Biochemistry.
1998;37:552[Medline]
[Order article via Infotrieve].
12.
Sen CK, Packer LP.
Antioxidant and redox regulation of gene transcription.
FASEB J.
1996;10:709[Abstract].
13.
Helenius M, Hanninen M, Lehtinen SK, Salminen AJ.
Aging-induced up-regulation of nuclear binding activities of oxidative stress responsive NF-B transcription factor in mouse cardiac muscle.
J Mol Cell Cardiol.
1996;28:487[Medline]
[Order article via Infotrieve].
14.
Collins T, Read MA, Neish AS, Whitley MZ, Thanos D, Maniatis T.
Transcriptional regulation of endothelial cell adhesion molecules: NF-B and cytokine-inducible enhancers.
FASEB J.
1995;9:899[Abstract].
15.
Barnes PJ, Karin M.
Nuclear factor-kappa B: a pivotal transcription factor in chronic inflammatory diseases.
N Engl J Med.
1997;336:1066[Free Full Text].
16.
Oeth PA, Parry GC, Kunsch C, Nantermet P, Rosen CA, Machman N.
Lipopolysaccharide induction of tissue factor gene expression in monocytic cells is mediated by binding of c-Rel/p65 heterodimers to a B-like site.
Mol Cell Biol.
1994;14:3772[Abstract/Free Full Text].
17.
Embury SH, Hebbel RP, Steinberg MH, Mohandas N.
Pathogenesis of vasoocclusion. In:
Embury SH,Hebbel RP,Mohandas N,Steinberg MH, eds.
Sickle Cell Disease: Basic Principles and Clinical Practice. New York, NY: Raven Press; 1994:311.
18.
Faller DV.
Vascular modulation. In:
Embury SH,Hebbel RP,Mohandas N,Steinberg MH, eds.
Sickle Cell Disease: Basic Principles and Clinical Practice. New York, NY: Raven Press; 1994:235.
19.
Fabry ME, Nagel RL, Pachnis A, Suzuka SM, Costantini F.
High expression of human s- and -globins in transgenic mice: hemoglobin composition and hematological consequences.
Proc Natl Acad Sci U S A.
1992;89:12150[Abstract/Free Full Text].
20.
Fabry ME, Costantini F, Pachnis A, et al.
High expression of human s- and -globins in transgenic mice: erythrocyte abnormalities, organ damage, and the effect of hypoxia.
Proc Natl Acad Sci U S A.
1992;89:12155[Abstract/Free Full Text].
21.
Fabry M.
Transgenic animal models. In:
Embury SH,Hebbel RP,Mohandas N,Steinberg MH, eds.
Sickle Cell Disease: Basic Principles and Clinical Practice. New York, NY: Raven Press; 1994:105.
22.
Kneepkens CMF, Lepage G, Roy CC.
The potential of the hydrocarbon breath test as a measure of lipid peroxidation.
Free Rad Biol Med.
1994;17:127[Medline]
[Order article via Infotrieve].
23.
Preece NE, Evans PF, King LJ, Parke DV.
Effects of glutathione depletion, chelation and diuresis on iron nitrilotriacetate-induced lipid peroxidation in rats and mice.
Xenobiotica.
1990;20:879[Medline]
[Order article via Infotrieve].
24.
Letteron P, Fromenty B, Terris B, Degott C, Pessayre D.
Acute and chronic hepatic steatosis lead to in vivo lipid peroxidation in mice.
J Hepatol.
1996;24:200[Medline]
[Order article via Infotrieve].
25.
Kawabata T, Ogino T, Awai M.
Protective effects of glutathione against lipid peroxidation in chronically iron-loaded mice.
Biochim Biophys Acta.
1989;1004:89[Medline]
[Order article via Infotrieve].
26.
Younes M, Siegers C-P.
The role of iron in the paracetamol- and CCl4-induced lipid peroxidation and hepatotoxicity.
Chem Biol Interact.
1985;55:327[Medline]
[Order article via Infotrieve].
27.
Younes M, Cornelius S, Siegers C-P.
Fe2+-supported in vivo lipid peroxidation induced by compounds undergoing redox cycling.
Chem Biol Interact.
1985;54:97[Medline]
[Order article via Infotrieve].
28.
Lawrence GD, Cohen G.
Ethane exhalation as an index of in vivo lipid peroxidation: concentrating ethane from a breath collection chamber.
Analyt Biochem.
1982;122:283.
29.
Massias L, Postaire E, Renault C, Hazebroucq G.
Thermal desorption-gas chromatographic determination of ethane and pentane in breath as potential markers of lipid peroxidation.
Biomed Chromatogr.
1993;7:200[Medline]
[Order article via Infotrieve].
30.
Coudray C, Talla M, Martin S, Fatome M, Favier A.
High-performance liquid chromatography-electrochemical determination of salicylate hydroxylation products as in vivo marker of oxidative stress.
Analyt Biochem.
1995;227:101.
31.
Halliwell B, Kaur H, Ingelman-Sundberg M.
Hydroxylation of salicylate as an assay for hydroxyl radicals: a cautionary note.
Free Rad Biol Med.
1991;10:439[Medline]
[Order article via Infotrieve].
32.
Kim PH, Wells PG.
Phenytoin-initiated hydroxyl radical formation: characterization by enhanced salicylate formation.
Mol Pharmacol.
1996;49:172[Abstract].
33.
Hall ED, Andrus PK, Yonkers PA.
Brain hydroxyl radical generation in acute experimental head injury.
J Neurochem.
1993;60:588[Medline]
[Order article via Infotrieve].
34.
Althaus JS, Andrus PK, Williams CM, Von Voigtlander PF, Cazers AR, Hall ED.
The use of salicylate hydroxylation to detect hydroxyl radical generation in ischemic and traumatic brain injury.
Mol Chem Neuropathol.
1993;20:147[Medline]
[Order article via Infotrieve].
35.
Deryckere F, Gannon F.
A one-hour minipreparation technique for extraction of DNA-binding proteins from animal tissues.
Biotechniques.
1994;16:405[Medline]
[Order article via Infotrieve].
36.
Bierhaus A, Zhang Y, Deng Y, et al.
Mechanism of the tumor necrosis factor -mediated induction of endothelial tissue factor.
J Biol Chem.
1995;270:26419[Abstract/Free Full Text].
37.
Bohrer H, Qiu F, Zimmerman T, et al.
Role of NF B in the mortality of sepsis.
J Clin Invest.
1997;100:972[Medline]
[Order article via Infotrieve].
38.
Beckman JS, Parks DA, Pearson JD, Marshall PA, Freeman BA.
A sensitive fluorometric assay for measuring xanthine dehydrogenase and oxidase in tissues.
Free Rad Biol Med.
1989;6:607[Medline]
[Order article via Infotrieve].
39.
Siddiqi NJ, Pandey VC.
Studies on hepatic oxidative stress and antioxidant defence systems during arteether treatment of Plasmodium yoelii nigeriensis infected mice.
Mol Cell Biochem.
1999;196:169[Medline]
[Order article via Infotrieve].
40.
Srivastava M, Kale RK.
Effect of radiation on the xanthine oxidoreductase system in the liver of mice.
Rad Res.
1999;152:257[Medline]
[Order article via Infotrieve].
41.
Hebbel RP.
Beyond hemoglobin polymerization: the red blood cell membrane and sickle disease pathophysiology.
Blood.
1991;77:214[Free Full Text].
42.
Nagel RL.
Severity, pathobiology, epistatic effects, and genetic markers in sickle cell anemia.
Semin Hematol.
1991;28:180[Medline]
[Order article via Infotrieve].
43.
Embury SE, Steinberg MH.
Genetic modulators of disease. In:
Embury SH,Hebbel RP,Mohandas N,Steinberg MH, eds.
Sickle Cell Disease: Basic Principles and Clinical Practice. New York, NY: Raven Press; 1994:299.
44.
Poillon WN, Kim BC, Castro O.
Intracellular hemoglobin S polymerization and the clinical severity of sickle cell anemia.
Blood.
1998;91:1777[Abstract/Free Full Text].
45.
Powars DR, Elliott-Mills DD, Chan L, et al.
Chronic renal failure in sickle cell disease: risk factors, clinical course and mortality.
Ann Intern Med.
1991;115:614.
46.
Platt OS, Brambilla DJ, Rosse WF, et al.
Mortality in sickle cell disease: life expectancy and risk factors for early death.
N Engl J Med.
1994;330:1639[Abstract/Free Full Text].
47.
Akinola NO, Stevens SME, Franklin IM, Nash GB, Stuart J.
Subclinical ischaemic episodes during the steady state of sickle cell anaemia.
J Clin Pathol.
1992;45:902[Abstract/Free Full Text].
48.
Collins T.
Endothelial nuclear factor-B and the initiation of the atherosclerotic lesion.
Lab Invest.
1993;68:499[Medline]
[Order article via Infotrieve].
49.
Pinsky DJ, Naka Y, Liao H, et al.
Hypoxia-induced exocytosis of endothelial cell Weibel-Palade bodies.
J Clin Invest.
1996;97:493[Medline]
[Order article via Infotrieve].
50.
Francis RB, Hebbel RP.
Hemostasis. In:
Embury SH,Hebbel RP,Mohandas N,Steinberg MH, eds.
Sickle Cell Disease: Basic Principles and Clinical Practice. New York, NY: Raven Press; 1994:299.
51.
Solovey A, Lin Y, Browne P, Choong S, Wayner E, Hebbel RP.
Circulating endothelial cells in sickle cell anemia.
N Engl J Med.
1997;337:1162[Free Full Text].
52.
Solovey A, Gui L, Key NS, Hebbel RP.
Tissue factor expression by endothelial cells in sickle cell anemia.
J Clin Invest.
1998;101:1899[Medline]
[Order article via Infotrieve].
53.
Hebbel RP.
Adhesive interactions of sickle erythrocytes with endothelium.
J Clin Invest.
1997;99:2561[Medline]
[Order article via Infotrieve].
54.
Hebbel RP, Vercellotti GM.
The endothelial biology of sickle cell disease.
J Lab Clin Med.
1997;129:288[Medline]
[Order article via Infotrieve].
55.
Paller MS, Hebbel RP.
Ethane production as a measure of lipid peroxidation after renal ischemia.
Am J Physiol.
1986;251:F839.
56.
Greene EL, Paller MS.
Oxygen free radicals in acute renal failure.
Miner Electrolyte Metab.
1991;17:124[Medline]
[Order article via Infotrieve].
57.
Kaul DK, Hebbel RP.
Leukocyte-endothelial interactions in transgenic sickle mice during steady-state and after hypoxia-reoxygenation [abstract].
Blood.
1999;94(suppl):676a.
58.
Granger DN.
Ischemia-reperfusion: mechanisms of microvascular dysfunction and the influence of risk factors for cardiovascular disease.
Microcirculation.
1999;6:167[Medline]
[Order article via Infotrieve].
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
Y.-S. Kim, E. Nur, E. J. van Beers, J. Truijen, S. C.A.T. Davis, B. J. Biemond, and J. J. van Lieshout
Dynamic Cerebral Autoregulation in Homozygous Sickle Cell Disease
Stroke,
March 1, 2009;
40(3):
808 - 814.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. T. Gladwin and E. Vichinsky
Pulmonary Complications of Sickle Cell Disease
N. Engl. J. Med.,
November 20, 2008;
359(21):
2254 - 2265.
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. R. Morris, J. H. Suh, W. Hagar, S. Larkin, D. A. Bland, M. H. Steinberg, E. P. Vichinsky, M. Shigenaga, B. Ames, F. A. Kuypers, et al.
Erythrocyte glutamine depletion, altered redox environment, and pulmonary hypertension in sickle cell disease
Blood,
January 1, 2008;
111(1):
402 - 410.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. J. Telen
Role of Adhesion Molecules and Vascular Endothelium in the Pathogenesis of Sickle Cell Disease
Hematology,
January 1, 2007;
2007(1):
84 - 90.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. K. Kaul, X.-d. Liu, X. Zhang, L. Ma, C. J. C. Hsia, and R. L. Nagel
Inhibition of sickle red cell adhesion and vasoocclusion in the microcirculation by antioxidants
Am J Physiol Heart Circ Physiol,
July 1, 2006;
291(1):
H167 - H175.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. Pacher, A. Nivorozhkin, and C. Szabo
Therapeutic effects of xanthine oxidase inhibitors: renaissance half a century after the discovery of allopurinol.
Pharmacol. Rev.,
March 1, 2006;
58(1):
87 - 114.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. H. Adewoye, L. McMahon, J. C. Mavropoulos, T. A. Fields, S. V. Pizzo, Y. Levy, A. Gorshtein, A. J.L. Macario, and E. C. de Macario
Chaperones and Disease
N. Engl. J. Med.,
December 29, 2005;
353(26):
2821 - 2822.
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. D. Belcher, H. Mahaseth, T. E. Welch, A. E. Vilback, K. M. Sonbol, V. S. Kalambur, P. R. Bowlin, J. C. Bischof, R. P. Hebbel, and G. M. Vercellotti
Critical role of endothelial cell activation in hypoxia-induced vasoocclusion in transgenic sickle mice
Am J Physiol Heart Circ Physiol,
June 1, 2005;
288(6):
H2715 - H2725.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. A. Nath, J. P. Grande, A. J. Croatt, E. Frank, N. M. Caplice, R. P. Hebbel, and Z. S. Katusic
Transgenic Sickle Mice Are Markedly Sensitive to Renal Ischemia-Reperfusion Injury
Am. J. Pathol.,
April 1, 2005;
166(4):
963 - 972.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Solovey, R. Kollander, A. Shet, L. C. Milbauer, S. Choong, A. Panoskaltsis-Mortari, B. R. Blazar, R. J. Kelm Jr, and R. P. Hebbel
Endothelial cell expression of tissue factor in sickle mice is augmented by hypoxia/reoxygenation and inhibited by lovastatin
Blood,
August 1, 2004;
104(3):
840 - 846.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. K. Kaul, X.-d. Liu, S. Choong, J. D. Belcher, G. M. Vercellotti, and R. P. Hebbel
Anti-inflammatory therapy ameliorates leukocyte adhesion and microvascular flow abnormalities in transgenic sickle mice
Am J Physiol Heart Circ Physiol,
July 1, 2004;
287(1):
H293 - H301.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. L. Jison, P. J. Munson, J. J. Barb, A. F. Suffredini, S. Talwar, C. Logun, N. Raghavachari, J. H. Beigel, J. H. Shelhamer, R. L. Danner, et al.
Blood mononuclear cell gene expression profiles characterize the oxidant, hemolytic, and inflammatory stress of sickle cell disease
Blood,
July 1, 2004;
104(1):
270 - 280.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. C. Wood, R. P. Hebbel, and D. N. Granger
Endothelial cell P-selectin mediates a proinflammatory and prothrombogenic phenotype in cerebral venules of sickle cell transgenic mice
Am J Physiol Heart Circ Physiol,
May 1, 2004;
286(5):
H1608 - H1614.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. A. Pritchard Jr., J. Ou, Z. Ou, Y. Shi, J. P. Franciosi, P. Signorino, S. Kaul, C. Ackland-Berglund, K. Witte, S. Holzhauer, et al.
Hypoxia-induced acute lung injury in murine models of sickle cell disease
Am J Physiol Lung Cell Mol Physiol,
April 1, 2004;
286(4):
L705 - L714.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. de Franceschi, A. Baron, A. Scarpa, C. Adrie, A. Janin, S. Barbi, J. Kister, P. Rouyer-Fessard, R. Corrocher, P. Leboulch, et al.
Inhaled nitric oxide protects transgenic SAD mice from sickle cell disease-specific lung injury induced by hypoxia/reoxygenation
Blood,
August 1, 2003;
102(3):
1087 - 1096.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. D. Belcher, C. J. Bryant, J. Nguyen, P. R. Bowlin, M. C. Kielbik, J. C. Bischof, R. P. Hebbel, and G. M. Vercellotti
Transgenic sickle mice have vascular inflammation
Blood,
May 15, 2003;
101(10):
3953 - 3959.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Aslan, T. M. Ryan, T. M. Townes, L. Coward, M. C. Kirk, S. Barnes, C. B. Alexander, S. S. Rosenfeld, and B. A. Freeman
Nitric Oxide-dependent Generation of Reactive Species in Sickle Cell Disease. ACTIN TYROSINE NITRATION INDUCES DEFECTIVE CYTOSKELETAL POLYMERIZATION
J. Biol. Chem.,
January 31, 2003;
278(6):
4194 - 4204.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. T. Gladwin, A. N. Schechter, F. P. Ognibene, W. A. Coles, C. D. Reiter, W. H. Schenke, G. Csako, M. A. Waclawiw, J. A. Panza, and R. O. Cannon III
Divergent Nitric Oxide Bioavailability in Men and Women With Sickle Cell Disease
Circulation,
January 21, 2003;
107(2):
271 - 278.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Wyllie, P. Seu, F. Q. Gao, P. Gros, and J. A. Goss
Disruption of the Nramp1 (also known as Slc11a1) gene in Kupffer cells attenuates early-phase, warm ischemia-reperfusion injury in the mouse liver
J. Leukoc. Biol.,
November 1, 2002;
72(5):
885 - 897.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Aessopos, D. Farmakis, and D. Loukopoulos
Elastic tissue abnormalities resembling pseudoxanthoma elasticum in beta thalassemia and the sickling syndromes
Blood,
January 1, 2002;
99(1):
30 - 35.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. A. Solovey, A. N. Solovey, J. Harkness, and R. P. Hebbel
Modulation of endothelial cell activation in sickle cell disease: a pilot study
Blood,
April 1, 2001;
97(7):
1937 - 1941.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
W. C. Mentzer and Y. W. Kan
Prospects for Research in Hematologic Disorders: Sickle Cell Disease and Thalassemia
JAMA,
February 7, 2001;
285(5):
640 - 642.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
Top
Abstract
Introduction