Blood, Vol. 95 No. 2 (January 15), 2000:
pp. 365-367
FOCUS ON HEMATOLOGY
Introduction: anti-adhesion therapy in sickle cell disease
John M. Harlan
From the Division of Hematology, University of Washington, Seattle,
WA.
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Article |
In this issue of Blood, Kaul et
al1 report that stimulation by platelet-activating factor
(PAF) of artificially perfused rat mesocecum ex vivo promotes the
adhesion of human sickle red blood cells (SS RBC) to postcapillary
endothelium. Notably, this adhesive interaction was blocked by two
different monoclonal antibodies (MoAbs) to endothelial 
V
3
integrin receptor, with resulting improvement in microvascular
hemodynamics. This study is an elegant and important contribution to
our understanding of SS RBC interactions with the vessel wall and,
hence, the mechanisms of vasoocclusion. Vasoocclusion of small and
sometimes large vessels is the hallmark of sickle cell disease,
accounting for much of its morbidity and mortality. The pathophysiology
of the vasoocclusive episodes is complex, involving not only the
polymerization of the mutant hemoglobin, but also interactions between
SS RBC, endothelium, platelets, leukocytes, and plasma constituents.
Intracapillary sickling and vasoocclusion occur when transit time
through capillaries is longer than the lag time for
deoxygenation-induced polymerization of sickle hemoglobin. Thus,
processes that delay passage of SS RBC through the microvasculature may
participate in the initiation and propagation of vasoocclusion.
In particular, an increase in SS RBC adhesion to postcapillary
endothelium could initiate vasoocclusion by impairing flow, thereby
delaying transit time of less deformable SS RBC and propagating
intracapillary sickling.2 Factors such as inflammatory
mediators that activate endothelial cells3 and
enhance endothelial adhesivity for SS RBC might, therefore, promote
vasoocclusion. Conversely, anti-adhesive or anti-inflammatory therapies
might attenuate vasoocclusion.
Seminal studies by Hebbel et al4 and Hoover et
al5 two decades ago first demonstrated that SS RBC showed
increased adherence to endothelial cells in vitro. Moreover, Hebbel et
al6 showed that vasoocclusive severity correlated with
adhesivity of SS RBC in vitro. Subsequently, these and many other
investigators have defined adhesion pathways involved in SS RBC
adhesion to cultured endothelium under static and flow conditions
(Table; also reviewed in references 2 and
7). Adhesion receptors on SS RBC include 41 integrin8,9 and CD368,10 whose expression is
increased on sickle versus normal reticulocytes8-10 and is
down-regulated by treatment with hydroxyurea.11 Aggregated,
membranous band 312 and sulfated glycolipids exposed on the
surface of damaged RBC also have been implicated. On the endothelial
side, cytokine-induced VCAM-1,9,13,14 a ligand for
41, and v3 integrin,10 which binds von
Willebrand factor (vWf) and thrombospondin (TSP), have been
demonstrated to mediate SS RBC adhesion. GpIb and CD36 are also
potential endothelial adhesion receptors. The adhesive protein
TSP,15,16 released by platelets, and vWf,17
released by endothelium, promote SS RBC adhesion to cultured
endothelium, serving as bridging molecules between endothelial and SS
RBC receptors.
SS RBC interactions with the vessel wall also may involve interactions
with subendothelial matrix components such as laminin (LN),18,19 TSP,18 vWf, or
fibronectin20 (Table). Matrix components may be exposed by
vascular injury or by endothelial retraction induced by stimuli such as
thrombin.21 A sulfated glycolipid isolated from SS RBC was
shown to bind to LN and TSP,18 and sulfated glycolipids
also have been reported to bind to vWf.22 B-CAM/LU (basal
cell adhesion molecule/lutheran protein) was recently shown to be a
major LN receptor on SS RBC.19
The majority of studies of SS RBC interactions with endothelium or
subendothelial matrix have used in vitro approaches. Studies with
cultured cells or purified matrix components, under static or flow
conditions, have been invaluable in identifying potential adhesive
interactions. Ultimately, however, it is necessary to validate concepts
and pathways in animal models. Fabry et al23 used an intact
rat model with gamma camera imaging and visualization of the
microvasculature by silicone injection. They demonstrated that
desmopressin, possibly by releasing vWf, increased the retention of
arterially injected deformable SS discocytes without producing overt obstruction, and suggested that narrowing of postcapillary venules by the adhesion of deformable SS RBC facilitated trapping of
less deformable SS RBC, triggering vasoocclusion.
The century-old technique of intravital microscopy, coupled with modern
video image analysis, allows precise quantification of blood cell
interactions with the vessel wall. This approach has proven to be a
powerful tool in defining leukocyte-endothelial interactions, leading
to the formulation of the multistep model of leukocyte emigration with
selectin-mediated tethering/rolling and integrin-dependent
sticking/transmigration.24 Kaul and colleagues have been
leaders in applying intravital microscopy to elucidate SS RBC
interactions with the vessel wall, using the rat mesocecum ex
vivo25 and the sickle transgenic mouse in
vivo.26 Their studies demonstrated that deformable SS RBC
are more likely to adhere than dense SS RBC25 and that
adhesion was limited to postcapillary venules.25,26 They
also showed that desmopressin-stimulated human SS RBC adhesion to
postcapillary venules in the ex vivo rat mesocolon was inhibited by
anti-vWf
but not anti-TSPantibody, providing the first trial of
adhesion blockade with specific reagents in an animal model of sickle
disease.27
Previous studies in vitro10,28 suggested that V3
integrin receptor, which is expressed on the luminal surface of
endothelial cells,29 may be involved in SS RBC adhesion to
endothelium. Kumar et al28 found that a conformationally
constrained RGD-containing peptide that inhibits V3 significantly
reduced plasma-induced SS RBC adhesion to cultured endothelial cells
under flow. Sugihara et al10 reported that the
anti-V3 MoAb LM609 and anti-IIb3 MoAb 7E3, which
cross-reacts with V3 but not the non-cross-reactive anti-IIb3 MoAb 10E5, reduced plasma-dependent SS RBC static adhesion to cultured human endothelial cells. In the current study, Kaul and colleagues used the same reagents in the ex vivo rat model to
address the role of V3 integrin. Platelet-activating factor, a
mediator that is increased in plasma of sickle cell patients, was used
to stimulate SS RBC adhesion to postcapillary venules, possibly by
releasing vWf or by provoking endothelial cell retraction. As shown in
the dramatic videomicroscopy (vide infra), treatment with the
V3-blocking MoAb 7E3 largely abolished PAF-stimulated SS RBC
adhesion to the vessel wall. MoAb 7E3 and the V3-specific MoAb
LM609, but not the IIb3-specific MoAb 10E5, also improved
hemodynamics in the PAF-treated vessels.
This study provides compelling evidence for a role of endothelial
V3 integrin in SS RBC adhesion to PAF-stimulated postcapillary venules in the rat. There are, of course, a number of obvious cautions
in extrapolating these exciting observations in the animal model to
sickle cell vasoocclusive crises. First, this is an ex vivo model with
artificial perfusion of isolated cells in a plasma-free medium,
certainly not reflecting the complex hemodynamics and rheology that
occur in the microcirculation during inflammation and hypoxia. Second,
although the in vitro studies with large vessel human endothelial cells
are consistent with the ex vivo results in the rat regarding the role
of V3 integrin, the interaction of human SS RBC with human
microvascular endothelium in vivo may involve adhesion pathways
different from those observed in the rat microvasculature. Third, the
model examines only a single inflammatory stimulus, PAF, in the absence
of other blood cells, whereas vasoocclusion occurs in a complex milieu
of platelets, leukocytes, plasma, and multiple inflammatory mediators
that may markedly alter the adhesion pathways used.
Despite these caveats, the current study by Kaul et al suggests that
blockade of endothelial V3 integrin might be beneficial in
treatment of vasoocclusion in sickle cell disease. Integrin receptors
have emerged as important therapeutic targets. Anti-adhesion therapies
directed to platelet IIb3 integrin receptor have been proven to
be of considerable benefit in acute coronary syndromes.30 These drugs include abciximab, a derivative of MoAb 7E3 that
cross-reacts with V3 integrin.31 Antagonists of
leukocyte 41 and 2 integrin receptors have demonstrated
efficacy in diverse animal models32 and are now in clinical
trial in several acute inflammatory disorders. Small molecule and
antibody-based specific inhibitors of V3 integrin are being
developed as anti-angiogenic agents.33 If V3
antagonists continue to prove safe in other indications, what
additional studies would be required before testing them for the
prevention or acute treatment of vasoocclusive episodes in sickle cell
disease? Certainly, confirmatory results in other animal models of
sickle cell disease would be encouraging. However, given the strong
rationale, the supporting ex vivo and in vitro evidence, and the
limitations of any of the animal models, it would seem reasonable even
now to consider a clinical trial.
| |
Footnotes |
Reprints: John M. Harlan, Division of Hematology, Box 359756, Harborview Medical Center, 325 Ninth Avenue, Seattle, WA 98104.
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.
| |
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