Transcript Abstract #2245, "A Novel Selectin Antagonist, GMI

A Novel Selectin Antagonist, GMI-1070, Prevents Vaso-Occlusion in Sickle Cell
Mice by Inhibiting Leukocyte Adhesion and Activation
Jungshan Chang, John Patton*, Arun Sarkar*, John L. Magnani*, Paul S. Frenette
Mount Sinai School of Medicine, New York, NY, USA; GlycoMimetics Inc., Gaithersburg, MD, USA*
Results
Abstract
Summary
RBC Interactions/WBC/min
Number WBCs / mm2
Rolling Flux Fraction(%)
Previous studies using intravital microscopy in a sickle cell disease mouse model suggest that
Figure 4. Effect of selectin inhibitors, GMI-1070 and GMI-1077, on
Figure 1. Schema of intravital microscopy protocol
adherent leukocytes play a key role in vaso-occlusion by capturing circulating erythrocytes in
leukocyte behavior and RBC capture in TNF-α treated sickle mice.
1
st
cremasteric venules . In addition, mice deficient in both P-and E-selectins are protected from Carotid artery 1 dose (GMI-1077,GMI-1070, anti-P-/E-sel antibodies or PBS i.c. )
vaso-occlusion (VOC) induced by surgical trauma and TNF-a stimulation1, suggesting that cannulation
TNF- a 0.5 mg i.p.
2nd dose
targeting selectins or their ligands represents a potentially useful strategy. Selectins bind to
A.
B.
C.
specific sialylated and fucosylated carbohydrate structures presented by glycoprotein or glycolipid -60 -50
-20
0
70
90
150
X
***
ligands. Here, we tested the effect of novel small glycomimetic selectin inhibitors, GMI-1070 and
Recording
Time of death
GMI-1077, on leukocyte behavior and sickle cell VOC. Fully engrafted male SCD mice were Tracheotomy Cremaster
surgery
treated with TNF-a and prepared for intravital microscopy examination of the cremaster muscle
***
***
**
90 min later. GMI-1070, GMI-1077, or vehicle (PBS) were administered immediately prior to
Table 1. Hemodynamic parameters in TNF-α primed sickle cell
cytokine stimulation (t= 0 min), and an additional dose was given at t= 70 min. Another group of
*
***
mice treated with selectin antagonists, antibodies and PBS.
mice was injected with antibodies against P-and E-selectins (1 mg/kg) as positive control. Several
12
1.5
post-capillary and collecting venules were examined between t= 90min and t= 150 min. Antibody
2500
Treatments
Mice (N) Weight (g) Venules (N)
Venular Diameter( m )
Centerline velocity (mm/s)
**
blockade of endothelial selectins completely ablated leukocyte rolling, whereas GMI-1070 and
10
1.2
2000
GMI-1077 significantly increased the rolling flux fractions. Furthermore GMI-1070 and GMI-1077 PBS-control
10
42
25.9±0.8
21.3±0.5
1.4±0.1
significantly reduced the recruitment of adherent leukocytes compared to sickle mice injected with
8
GMI-1077
4
49
25.7±0.9
20.7±0.5
1.7±0.1
0.9
1500
PBS control. Although the reduction in leukocyte adhesion was not as marked as with anti-P and
6
GMI-1070
5
51
26.9±0.3
20.9±0.4
3.1±0.2*
E-selectins, GMI-1070, in particular, dramatically inhibited the capture of sickle RBCs by adherent
0.6
1000
Anti-Psel +
leukocytes and markedly improved the blood flow in venules to levels observed in non-sickle
4
20.7 ± 0.4
3.1 ± 0.3*
5
58
26.3±0.7
Anti-Esel
mice. The increased leukocyte rolling fluxes by these glycomimetics suggest that they inhibit E0.3
500
2
selectin > P-selectin. Since the hallmark of E-selectin-mediated adhesion is the slow leukocyte Data are presented as mean ± SEM.*P < 0.05 compared to PBS - control or GMI-1076.
rolling, we analyzed leukocyte rolling velocities in the various group and indeed found a near 20
0.0
0
Experimental
sickle
cell
mice
weight
ranged
from
22
to
29
g.
During
observation
under
fold increase in rolling velocities in sickle mice treated with GMI-1070 compared to PBS control.
These studies suggest that E-selectin-mediated adhesion/signaling may play a more important brightfield intravital microscopy, venular size from 17 to 25 μm were recorded for future
role than previously appreciated in the pathophysiology of SCD, and suggest that GMI-1070 may analyses. The mean centerline velocity (Vrbc) in sickle mice treated with GMI-1077 was
slightly higher than PBS-treated animals but the difference was not significant. Vrbc in
be beneficial for the treatment of sickle cell vaso-occlusion.
mice treated with GMI-1070 or antibodies was > 2-fold increased of compared to PBS (A) Both GMI-1076 and GMI-1070 dramatically increased leukocyte rolling flux fraction by nearly
control. *p < 0.05.
2-fold. (B) Average number of leukocytes adherent to endothelium was significantly reduced in
sickle cell mice treated with either GMI-1077 or GMI-1070. (C) Both small molecule inhibitors
Figure 2. Mean calculated blood flow rate
reduced the capture rates of erythrocytes per adherent leukocytes, but only GMI-1070 displayed
a statistically significant inhibitory effect. *p<0.05; **p<0.01 and ***p<0.001
***
Animals
Material & Methods
A.
500
20
The blood flow rate in GMI-1070- and P-/E-sel
antibodies-treated mice was significantly higher than
in PBS control or GMI-1077-treated animals. This
difference was not due to venular size as the average
venular diameter was nearly identical ( ～ 21μm)
among the three groups. **p<0.01 and ***p<0.001
250
0
15
1
PBS control (n=193)
Vmean=20.9±1.1 mm/s
10
5
B.
0
Figure 3 Representative images of venules from sickle mice treated
with PBS, GMI-1070, GMI-1077, or anti- P and E selectin antibodies.
10 m GMI-1077
20
15
Vmean=25.5±1.1 mm/s
10
C.
0
25
GMI-1070 (n=481)
15
Vmean=37.8±1.2 mm/s***
10
Anti-P/Esel
Each still frame was taken at the 30 min timepoint after TNF-α injection. Both small
Statistical analyses
molecule selectin antagonists and anti-P/E selectin antibodies significantly reduced the
All data are displayed as mean ± SEM. Parametric data were analyzed using ANOVA. Statistical significance for
non-parametric distributions (RBC-WBC interactions) was assessed using the Mann-Whitney test. A value of p less number of adherent leukocytes (yellow circles), and RBCs interacting with adherent
leukocytes (yellow arrows). The white arrows indicate the direction of blood flow.
than
0.05 was deemed significant.
.
0.6
GMI-1077 Vmedian=20 mm/s
0.4
GMI-1070 Vmedian=32.5 mm/s
0.2
0
20
40
60
80
100 120 140 160 180 200 220
GMI-1077 (n=467)
20
GMI-1070
PBS Vmedian=16 mm/s
Leukocyte rolling velocity ( mm/s)
5
PBS
0.8
0
25
Image analyses for brightfield intravital microscopy
All analyses were made using playback assessment of videotapes as previously described. Briefly, rolling WBCs
were defined as those moving at a velocity less than that of free-flowing erythrocytes in a given vessel and counted
over 1-min intervals per venule. Adherent WBCs were defined as those remaining stationary for at least 30 s over a
100 µm venular segment. RBCs were identified by their size and shape (discoid and sickle-shaped cells). An
interaction between RBCs and adherent WBCs was defined as the arrest of an RBC on an adherent WBC for more
than 2 video frames (> 0.07 s). This time interval corresponds to a readily discernable adhesion event when the
videotape is played in real time. Leukocyte rolling velocity (Vwbc) was calculated by dividing the traveled distance by
the tracking time or as the average translation over 2 seconds for 10 WBCs per venule, and expressed as μm / s.
Leukocyte transit time was calculated as 100 μm / Vwbc. The flux fraction (F) of rolling leukocytes, corresponding to the
percentage of leukocytes that are rolling per min, was calculated by F = WBCr / (0.25πd2 Vrbc60 [WBC]), where WBCr r
is the number of leukocytes rolling past a fixed reference point in the venule per minute, d is venule diameter, Vrbc is
centerline velocity, and [WBC] is the systemic leukocyte count.
25
Cumulated frequency
We used the protocol shown on Fig. 1 in which sickle cell mice were anesthetized by i.p. injection of a mixture of
2% chloralose and 10% α-urethane in PBS (6 mL/kg). A polyethylene tube was inserted into the trachea in order to
facilitate spontaneous respiration and the right carotid artery was cannulated for administration of selectin antagonists
(GMI-1070 and GMI-1077), mixture of antibodies against P- and E- selectins, or vehicle PBS control. The cremaster
muscle was gently exteriorized and then continuously superfused throughout the experiment with warmed (37°C)
bicarbonate-buffered (pH 7.4) saline aerated with a mixture of 95% N2 and 5% CO2. Each experimental mouse
received 2 doses of each antagonist, PBS saline containing antibodies or equal volume of PBS control. The first
injection was performed immediately after TNF–a injection (T0) and the second injection was made at 70 minutes (T70)
after TNF-a administration. Twenty minutes after exposure to antagonists, PBS or the antibody cocktail, 8-12 venules
of each mouse were videotaped over a period of 60 min, with each venule recorded continuously for at least 2 min.
Venules were visualized with a custom-designed intravital microscope (MM-40, Nikon), using a 60X water immersion
objective (Nikon). Images were recorded using a charge-coupled device video camera (Hamamatsu, Bridgewater, NJ)
and video recorder (Sony SVHS, SVO-9500). Venular diameter was measured with a video caliper. Centerline red
cell velocity (VRBC) was measured for each venule in real time using an optical Doppler velocitometer (Texas A&M,
College Station, TX). Wall shear rate (g) was calculated based on Poiseuille’s law for a Newtonian fluid, g = 2.12
(8Vmean) / Dv, where Dv is the venule diameter, Vmean is estimated as VRBC / 1.6, and 2.12 is a median empirical
correction factor obtained from actual velocity profiles measured in microvessels in vivo. Blood flow rate (Q) was
calculated as Q= Vmean πd2/4, where d is venule diameter, and is expressed as nL/s. Blood samples taken immediately
after recording, through a cardiac puncture, were used to determine periphery white blood cell counts by using
Hemocytometer methods.
750
frequency (%)
Intravital experimental protocol
Figure 5. Leukocyte rolling velocity histograms
**
1000
Blood flow rate (nL/s)
Berkeley SCD mouse bone marrow was transplanted into lethally irradiated C57BL/6 animals to generate age- and
gender-matched genetically identical cohorts of SCD mice. Fully chimeric male sickle cell mice (expressing >97%
human globin, including betaS) were subjected to intravital microscopy 3-5 months after bone marrow transplantation1.
5
0
0
10 20 30 40 50 60 70 80 90 100
Leukocyte rolling velocity ( mm/s)
The velocity of leukocyte rolling was evaluated from (A)
193 leukocytes in 42 venules of PBS-treated sickle cell
mice (n=10). Leukocytes in sickle cell mouse venules
treated with PBS rolled at average velocity of 20.9 ± 1.1
µm/s, ranging from 0.3 to 90 µm /s. (B) Leukocyte
rolling velocities in GMI-1077-treated sickle mouse
venules were 25.5 ± 1.1 µm/s, ranging from 2.5 to 250
µm/s (analyses derived from 467 leukocytes in 51
venules of 4 GMI-1077-treated mice). (C) In contrast,
leukocyte rolling velocity in GMI-1070-treated mice were
significantly greater than PBS-treated controls and GMI1077 treated animals, with an average rolling velocity of
37.8 ± 1.2µm/s, ranging from 2.5 to 250 µm/s (data from
481 leukocytes in 58 venules of 5 GMI-1070-treated
mice) *** p<0.001. (D) A cumulative frequency
histogram for these three groups demonstrated that
300 GMI-1070 shifted leukocyte rolling from slower to faster
with ~2-fold higher median rolling velocities than PBS.
 GMI-1070 markedly improves blood flow rates in sickle
cell mice challenged with surgical trauma and TNF-a.
 GMI-1070, but not GMI-1077, dramatically inhibited the
ability of adherent leukocytes to capture circulating sickle
RBCs.
 Both selectin antagonists, GMI-1070 and GMI-1077 can
increase rolling flux fraction and reduce the recruitment of
adherent leukocytes in cremasteric venules in sickle cell
mice.
 Leukocytes of mice treated with GMI-1070 rolled
significantly faster than controls on cremasteric
endothelium. In contrast, GMI-1077 had a marginal effect
on rolling velocities.
Discussion
Here, we demonstrate that the administration of a pan-selectin antagonist GMI-1070
can profoundly alter the course of acute vaso-occlusive episodes in sickle cell mice. We
show that GMI-1070 dramatically improves flow rates in microvessels of sickle cell mice
that have been challenged with a lethal crisis. This increase in flow may prolong survival of
sickle cell animals since a strong positive correlation between blood flow rate and survival
has been demonstrated in sickle cell mice2.
GMI-1070 exhibits the capability to increase leukocyte rolling flux fraction and leukocyte
rolling velocities, resulting in reduced numbers of adherent leukocytes. These attributes
suggest that GMI-1070 effectively interrupted E-selectin-mediated rolling machinery since
slow rolling is controlled by E-selectin. Furthermore, GMI-1070 markedly affected the
capture of circulating erythrocytes by adherents leukocytes, suggesting an association
between RBC-WBC interactions and signals emerging from E-selectin ligands.
These in vivo studies thus suggest that GMI-1070 or similar compounds may be
beneficial in the treatment or prevention of sickle cell disease manifestations. Although
further studies are needed to understand further its mechanisms and evaluate the
conditions that would benefit from selectin inhibition, these data indicate that GMI-1070
should be assessed in a clinical trial to treat acute painful crises.
References
1. Turhan A, Weiss LA, Mohandas N, Coller BS, Frenette PS. Primary role for adherent
leukocytes in sickle cell vascular occlusion: a new paradigm. Proc Natl Acad Sci U S A.
2002;99:3047-3051.
2. Jungshan Chang, Patricia A. Shi, Elaine Y Chiang, and Paul S Frenette
Intravenous immunoglobulins reverse acute vaso-occlusive crises in sickle cell mice
through rapid inhibition of neutrophil adhesion. Blood, Oct 2007; doi:10.1182/blood2007-04-084061.