SUPPLEMENTAL DIGITAL CONTENT
METHODS
RBC adhesion assays. Graduated-height flow chambers were used to quantify the adhesion
of RBC to HUVECs. Glass slides coated with HUVECs grown to confluence were washed with
PBS with 1.26mM Ca2+, 0.9mM Mg2+ warmed previously to 37oC and then fit into a variableheight flow chamber, as previously described (1, 2). The flow chamber was mounted on the
stage of an inverted phase contrast microscope (Diaphot, Nikon Inc., Melville, NY) connected
to a thermoplate (Tokai Hit Co., Ltd., Japan) set at 37 oC. Cells were observed using a video
camera (RS photometrics) attached to the microscope and connected to a Macintosh G4
computer. Fluorescence-labeled fresh or stored RBCs washed four times with PBS with Ca2+,
Mg2+ and suspended at 0.2% (vol/vol) in PBS with Ca2+ and Mg2+ were infused into the flow
chamber and allowed to adhere to the slide for 10 min without flow. Before exposure to flow, a
minimum of three fields at each of seven different locations along a line oriented normal to
future flow were examined for the total number of fluorescent cells. Fluid flow (PBS with Ca2+
and Mg2+) was then started using a calibrated syringe pump. After exposure to flow, the fields
were again examined and the number of adherent cells counted. The percentage of adherent
cells was presented as:
Number of cells attached after exposure to flow x100
Cells present per field before flow
The wall shear stress was calculated as:
tw = _6mQ_
wH (x)2
tw = wall shear stress (dyne/cm2); Q = volumetric flow rate (cm3/s); m is media viscosity, w is
the width of the flow channel, and H(x) is the height of the flow chamber as a function of
position along the microscope slide. Several investigators have shown that blood flow in small
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vessels may be continuous, with shear stresses of 1-2 dynes/cm2, or flow may be intermittent.
Our data were obtained using intermittent flow conditions. In some experiments, RBCs were
pretreated with glibenclamide (final concentration 10 µM), exposed to HUVECs in the presence
of apyrase (5 units/ml in PBS containing Ca2+ and Mg2+) or ATP, or pre-incubated with antiRBC adhesion receptor antibodies (below) prior to adhesion assays. In other experiments the
HUVECs were pre-incubated at 37oC with extracellular ATP or with adhesion receptor mAbs
as indicated. Where mean data at 2 dynes/cm2 are presented, the values were derived by
interpolating along a regression curve generated for each experiment, because a shear stress
of precisely 2 was not typically used. (The precise shear stresses generated vary from one
experiment to the next).
RBC perfusion of isolated mouse lungs was performed as previously described (3). Briefly,
C57BL6 mice 9-12 weeks of age were anesthetized with ketamine/xylazine, tracheostomy was
performed, and a ventilator delivered 6 µL/gm tidal volumes at a rate of 90 breaths per minute.
A midline sternotomy was performed, and following intracardiac injection of sodium heparin
(500 units), the right ventricle was incised and a cannula connected to a pressure transducer
was advanced into the main pulmonary artery (PA) and secured. A bulbed left atrial cannula
was inserted via left ventriculotomy and secured against the mitral valve. Left atrial pressure
was held constant throughout the experiment; therefore changes in PA pressure (PAP) directly
reflected changes in pulmonary vascular resistance. Modified Krebs-Henseleit buffer
(containing 4% Ficoll and 5 µM indomethacin), adjusted to pH 7.40 and bubbled with 5% CO 2 /
21% O2 / balance N2, was used to perfuse the lungs at a total rate of 1 ml/min. For RBC
perfusion (0.1 ml/min), the total rate was held constant by decreasing the perfusion rate of
coinfused buffer. Mean PAP is reported; because flow is pump-driven, there is no systolic and
diastolic pressure variation. Mean PAP was typically ~6-11 cm H20 under these conditions,
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consistent with previous reports in the isolated perfused mouse lung under similar conditions
(3, 4).
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SUPPLEMENTAL FIGURE LEGENDS
Figure S1. Influence of glibenclamide treatment of transfused RBCs on differential WBC
counts in BAL fluid (BALF). No significant differences were seen in total WBC counts, or in
the numbers of macrophages (Mac), neutrophils (PMN), lymphocytes (Lym), or eosinophils
(Eos) present. n = 6 experiments in each group. Data are mean + SEM. ANOVA with Tukey’s
was performed.
Figure S2. Influence of glibenclamide (GLIB) treatment on sequestration of transfused
RBCs in the spleen and kidney. Typical fluorescence micrographs from kidneys (A, B) and
spleens (D, E) of nude mice transfused with fresh, Dil-labeled human RBCs pretreated or not
(Con, vehicle) with the ATP-release inhibitor GLIB. Mean fluorescence intensity values,
normalized to those from kidneys of mice transfused with vehicle-exposed RBCs, are shown in
C and F (n=3 mice transfused with Con RBCs and n=4 mice with GLIB-treated RBCs). No
statistical test was applied.
Figure S3. Influence of glibenclamide and carbenoxolone on RBC deformability. Fresh
human RBCs were exposed to glibenclamide vehicle (Control), glibenclamide or
carbenoxolone, then washed twice in PBS. Deformability was assayed with a laser-assisted
optical rotational cell analyzer at the indicated shear stresses as described. n=3 independent
experiments; mean + SEM. No statistical test was applied.
Figure S4. Effect of RBC storage duration on the changes in PAP in response to
perfusion of isolated mouse lungs with human RBCs. Conventionally stored RBCs were
removed at the indicated storage times, washed, and included in the Krebs-based perfusion
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buffer. RBC-induced increases in PAP increased as storage time proceeded. Mean basal PAP
(blue, mean + SEM) is given for representative experimental groups. p<0.001 for the overall
curve; n = 4-6. ANOVA with Tukey’s was used.
Figure S5. Flow cytometric analysis of murine and human RBCs in BALF after transfusion
in nude mice. Fresh human RBCs treated or not (Control, vehicle) with glibenclamide and then
exposed or not to anti-LW mAb were transfused as described, and immediately after
euthanasia BAL was performed. BALF was incubated or not with antibodies specific for human
or murine RBCs and then analyzed in a flow cytometer. The results shown are typical of 3
independent experiments.
1.
2.
3.
4.
Xiao Y, Truskey GA: Effect of receptor-ligand affinity on the strength of
endothelial cell adhesion. Biophys J 1996. 71: 2869-2884
Zennadi R, Hines PC, De Castro LM, et al: Epinephrine acts through erythroid
signaling pathways to activate sickle cell adhesion to endothelium via LWalphavbeta3 interactions. Blood 2004. 104: 3774-3781
Doctor A, Platt R, Sheram ML, et al: Hemoglobin conformation couples
erythrocyte S-nitrosothiol content to O2 gradients. Proc Nat Acad Sci USA 2005.
102: 5709-5714
Holzmann A, Bloch KD, Sanchez LS, et al: Hyporesponsiveness to inhaled nitric
oxide in isolated, perfused lungs from endotoxin-challenged rats. Am J Physiol
1996. 271: L981-986
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