Endothelium-protective sphingosine-1-phosphate provided by HDL-associated apolipoprotein M.

Abstract

Protection of the endothelium is provided by circulating sphingosine-1-phosphate (S1P), which maintains vascular integrity. We show that HDL-associated S1P is bound specifically to both human and murine apolipoprotein M (apoM). Thus, isolated human ApoM(+) HDL contained S1P, whereas ApoM(-) HDL did not. Moreover, HDL in Apom(-/-) mice contains no S1P, whereas HDL in transgenic mice overexpressing human apoM has an increased S1P content. The 1.7-Å structure of the S1P-human apoM complex reveals that S1P interacts specifically with an amphiphilic pocket in the lipocalin fold of apoM. Human ApoM(+) HDL induced S1P(1) receptor internalization, downstream MAPK and Akt activation, endothelial cell migration, and formation of endothelial adherens junctions, whereas apoM(-) HDL did not. Importantly, lack of S1P in the HDL fraction of Apom(-/-) mice decreased basal endothelial barrier function in lung tissue. Our results demonstrate that apoM, by delivering S1P to the S1P(1) receptor on endothelial cells, is a vasculoprotective constituent of HDL.

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Endothelium-protective sphingosine-1-phosphate
provided by HDL-associated apolipoprotein M
Christina Christoffersena,1, Hideru Obinatab,1, Sunil B. Kumaraswamyc, Sylvain Galvanib, Josefin Ahnströmc,
Madhumati Sevvanad, Claudia Egerer-Sieberd, Yves A. Mullerd, Timothy Hlab,2, Lars B. Nielsena,e,2,
and Björn Dahlbäckc,2
aDepartment of Clinical Biochemistry, Rigshospitalet, 2100 Copenhagen, Denmark;bCenter for Vascular Biology, Department of Pathology and Laboratory
Medicine, Weill Cornell Medical College, Cornell University, New York, NY 10065;cWallenberg Laboratory, Department of Laboratory Medicine, Skåne
University Hospital, Lund University, SE 20502 Malmö, Sweden;dDepartment of Biology, Friedrich-Alexander-University Erlangen-Nuremberg,
D-91052 Erlangen, Germany; andeDepartment of Biomedical Sciences, University of Copenhagen, 2100 Copenhagen, Denmark
Edited* by John A. Glomset, University of Washington, Seattle, WA, and approved May 4, 2011 (received for review February 25, 2011)
Protection of the endothelium is provided by circulating sphingo-
sine-1-phosphate (S1P), which maintains vascular integrity. We
show that HDL-associated S1P is bound specifically to both human
and murine apolipoprotein M (apoM). Thus, isolated human
ApoM+HDL contained S1P, whereas ApoM−HDL did not. More-
over, HDL in Apom−/−mice contains no S1P, whereas HDL in trans-
genic mice overexpressing human apoM has an increased S1P
content. The 1.7-Å structure of the S1P–human apoM complex
reveals that S1P interacts specifically with an amphiphilic pocket
in the lipocalin fold of apoM. Human ApoM+HDL induced S1P1
receptor internalization, downstream MAPK and Akt activation,
endothelial cell migration, and formation of endothelial adherens
junctions, whereas apoM−HDL did not. Importantly, lack of S1P in
the HDL fraction of Apom−/−mice decreased basal endothelial
barrier function in lung tissue. Our results demonstrate that apoM,
by delivering S1P to the S1P1receptor on endothelial cells, is a vas-
culoprotective constituent of HDL.
endothelial function|crystal structure|sphingolipids|vascular
permeability|atherosclerosis
S
receptors (S1P1–S1P5) and regulates a plethora of biological
actions (1–6). In particular, the prototypical S1P1receptor is
essential for vascular maturation during development and pro-
motes endothelial cell migration, angiogenesis, and barrier
functions (7–9). Thus, S1P is required for maintenance of the
barrier property of the lung endothelium (10). Plasma S1P,
which is derived from several cellular sources (11, 12), is asso-
ciated with HDL (∼65%) and albumin (∼35%) (3, 5). HDL-
induced vasorelaxation as well as barrier-promoting and pro-
survival actions on the endothelium have been attributed to S1P
signaling (2, 4, 13). Hence, much of the endothelium-protective
actions of HDL may result from the actions of S1P on the en-
dothelial S1P receptors. However, the molecular nature of the
S1P binding to HDL and interaction with S1P receptors has not
been characterized.
Apolipoprotein M (apoM) is a lipocalin that resides mainly in
the plasma HDL fraction (14). The retained hydrophobic NH2-
terminal signal peptide anchors apoM in the phospholipid layer
of the lipoprotein and prevents filtration of the ∼22-kDa protein
in the kidney (15). The biological functions of apoM are un-
derstood only partly. Studies in apoM gene-modified mice sug-
gest that apoM has antiatherogenic effects, possibly related in
part to apoM’s ability to increase cholesterol efflux from mac-
rophage foam cells, to increased preβ-HDL formation, and to
antioxidative effects (16–18). The recent elucidation of the
crystal structure of human recombinant apoM (r-apoM) dem-
onstrated a typical lipocalin fold characterized by an eight-
strandedantiparallelβ-barrelenclosinganinternalbindingpocket
that probably facilitates binding of small lipophilic ligands (19).
phingosine-1-phosphate (S1P), the phosphorylated metabo-
lite of
D-sphingosine, binds to five G protein-coupled
Indeed, r-apoM expressed in Escherichia coli was found to co-
crystallize with myristic acid (19), illustrating that apoM can bind
lipid compounds with fatty acid side chains, and in vitro binding
experiments demonstrated that S1P displaced the myristic acid
with an IC50of 0.90 μM (19). We demonstrate here that apoM is
the carrier of S1P in HDL, mediating vasoprotective actions on
the endothelium.
Results and Discussion
To study the molecular basis of apoM interaction with S1P, we
determined the crystal structure of N-terminally truncated hu-
man apoM (residues 22–188) (r-apoM) in complex with S1P at
1.7-Å resolution. S1P is bound at the center of the calyx-like
ligand-binding pocket (Fig. 1 and Fig. S1). It participates in
numerous specific interactions with apoM (Fig. 1 and Fig. S2).
The phosphate moiety interacts directly with the side chains of
Arg98, Arg116, and Trp100 (Fig. 1 and Fig. S2). The S1P amino
group is hydrogen-bonded to Glu136 and to Tyr102 and Arg143
via bridging water molecules. The hydroxyl group of S1P inter-
acts via a bridging water molecule with the hydroxyl group of
Tyr147 (Fig. 1 and Fig. S2). The hydrocarbon chain of S1P points
toward the interior of the calyx. Hence, its interaction with apoM
and its location coincide with that of myristic acid in a previous
complex (Figs. S2 and S3) (19). When all these binding inter-
actions are considered together, the lipid-binding site of apoM is
highly complementary to the structure of S1P. In particular, the
recognition of the phosphate group by several arginines hints
that the S1P–apoM interaction is very specific.
To elucidate whether apoM is the physiological carrier of
HDL-associated S1P in vivo, plasma S1P was measured in apoM-
knockout (Apom−/−) mice and in two lines of human apoM
transgenic mice having either twofold (Apom-TgN) or 10-fold
(Apom-TgH) increased plasma apoM concentrations (17). Com-
pared with WT mice, plasma S1P was reduced by 46% in Apom−/−
mice (P = 0.0007) and was increased by 71% (P = 0.0005) and by
267% (P = 0.0002) in the Apom-TgNand Apom-TgHmice, re-
Author contributions: C.C., H.O., Y.A.M., T.H., L.B.N., and B.D. designed research; C.C.,
H.O., S.B.K., S.G., J.A., M.S., and C.E.-S. performed research; C.C., H.O., J.A., M.S., Y.A.M.,
T.H., L.B.N., and B.D. analyzed data; and C.C., H.O., Y.A.M., T.H., L.B.N., and B.D. wrote
the paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
Freely available online through the PNAS open access option.
Data deposition: Crystallography, atomic coordinates, and structure factors for the S1P-
apoM crystal structure have been deposited in the Protein Data Bank, www.pdb.org (PDB
ID code 2YG2).
1C.C. and H.O. contributed equally to this work.
2To whom correspondence may be addressed. E-mail: tih2002@med.cornell.edu, larsbo@
rh.dk, or bjorn.dahlback@med.lu.se.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1103187108/-/DCSupplemental.
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spectively (Fig. 2A). The plasma concentrations of HDL cho-
lesterol, HDL total phospholipids, and apoA-I were affected
only marginally in Apom−/−and Apom-TgHmice, demonstrating
that the changes in S1P concentrations are related to apoM and
not to variations in the amount of circulating HDL (17). When
lipoproteins in WT mouse plasma were separated by gel filtra-
tion, the major peak of S1P coeluted with apoM in the HDL
fractions, whereas a minor S1P peak coeluted with albumin (Fig.
2B). Apom−/−mice lacked S1P in the HDL fraction, but the S1P
peak in the albumin fractions was present (Fig. 2B). Apom-TgH
mice had increased S1P in HDL (Fig. 2B). This S1P was asso-
ciated with apoM-containing HDL, as demonstrated by a parallel
shift in S1P- and human apoM-elution profiles when a specific
monoclonal antibody against human apoM (M58) was added to
the plasma before gel filtration (Fig. S4 A and B).
Importantly, the amount of apoM in HDL is sufficient to ac-
commodate and account for all HDL-bound S1P. The average
plasma apoM concentration is similar in mice and humans, i.e.,
∼0.9 μmol/L (17). Hence, the apparent molar ratio between
HDL-bound S1P and plasma apoM is ∼1:3 in WT and Apom-TgN
mice and ∼1:6 in Apom-TgHmice. On gel filtration of human
plasma, the majority of S1P coeluted with HDL, indicating that
in humans, also, the main part of lipoprotein-bound S1P is as-
sociated with HDL (Fig. S4C). When human HDL was separated
by affinity chromatography into apoM+HDL and apoM−HDL
fractions, S1P was found exclusively in apoM+HDL (Fig. 2C).
These data indicate that S1P in HDL is bound to apoM in both
Fig. 1.
at 1.7-Å resolution. S1P is shown as green sticks together with interacting residues. Strands B–F, the N terminus (N-t), and the C terminus (C-t) are labeled in
red, and the interacting residues are labeled in black. (B) Top view of the S1P-binding site in close up. Electron density for S1P and surrounding water
molecules is contoured at 1 σ and colored blue. Water molecules are shown as red spheres in both panels, and unmodeled loops toward the N terminus are
shown as broken lines.
The structure of the ApoM–S1P complex reveals the determinants of S1P-binding specificity. (A) Stereo view of the crystal structure of apoM with S1P
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humans and mice. Intrinsic fluorescence binding studies of hu-
man r-apoM showed that apoM can bind S1P with an IC50of 0.9
μM (19). With the same experimental setup but using murine r-
apoM, an IC50for S1P of 0.95 ± 0.05 μM (n = 3) was obtained
(Fig. S5), further supporting the idea that apoM is the physio-
logical carrier of HDL-associated S1P.
The biological effects of S1P are mediated by activation of
the G protein-coupled S1P receptors, leading to activation of
downstream effectors such as p44/42, MAPK, and Akt (20). To
assess whether apoM+HDL and apoM-bound S1P can activate
the S1P1receptor, we performed a ligand-induced receptor in-
ternalization assay in HEK293 cells stably expressing the GFP-
tagged S1P1receptor (21). Both ApoM+HDL and r-apoM–
bound S1P induced robust internalization of GFP-S1P1receptor,
similarly to albumin-bound S1P that was used as a positive
control (Fig. 3A). Neither apoM−HDL nor r-apoM without S1P
caused receptor internalization. These data indicate that the
apoM–S1P complex can activate the S1P1receptor, whether it is
part of an HDL particle or not.
To test activation of endogenous S1P1 receptors and the
downstream signaling by apoM-bound S1P, human umbilical vein
endothelial cells (HUVEC) were stimulated with various carriers
complexed or not with S1P. Prominent phosphorylation of p44/42
and Akt was induced by apoM+HDL but not by apoM−HDL
(Fig. 3B). Moreover, blocking of S1P1receptors with the S1P1-
selective antagonist VPC44116 (22, 23) essentially abolished the
effect of apoM+HDL on p44/42 and Akt phosphorylation (Fig.
3B), indicating that the effects of apoM+HDL were mediated by
the S1P1receptor. Albumin-bound S1P, apoM+HDL and apoM-
bound S1P showed similar time courses and dose responses in the
activation of p44/42 and Akt (Fig. S6 A and B).
S1P is a potent chemoattractant for endothelial cells, which are
essential for wound-healing response and angiogenesis (9, 24).
ApoM+HDL stimulated chemotaxis of HUVEC, and this effect
wasabolishedbypretreatmentwiththeS1P1antagonistVPC44116
(Fig. 3C). Both albumin- and r-apoM–bound S1P worked as che-
moattractantsina concentration-dependentmanner,but r-apoM–
bound S1P showed slightly higher activity, especially at lower S1P
concentrations (Fig. S6C). S1P suppresses abnormal vascular per-
meability by inducing the assembly of vascular endothelial (VE)-
cadherin–containing adherens junctions between endothelial cells
(1, 9). As shown in Fig. 3D, HUVEC were well spread, contained
F-actin,andformedadherensjunctionswhentreatedwithapoM+
HDL and with albumin- and r-apoM–bound S1P. In contrast,
adherens junctions and F-actin were not induced efficiently by
apoM−HDL or by r-apoM without S1P.
The importance of S1P in regulating vascular integrity in vivo
has been shown in mice lacking plasma S1P (10). These mice
Fig. 2.
∼twofold (Apom-TgN) and ∼10-fold (Apom-TgH) increased plasma apoM. Each point represents data from an individual mouse; horizontal lines indicate
means. (B) Lipoproteins in pools of plasma from WT (Top), Apom−/−(Middle), or Apom-TgH(Bottom) mice were separated by gel filtration on serially con-
nected Superose 6 and 12 columns. The flow rate was 0.4 mL/min. Fractions of 275 μL were collected. Aliquots of 10 consecutive fractions were pooled before
measuring S1P (red filled symbols) and protein (dotted black line). Cholesterol concentration (solid blue line) was determined in each fraction. The scale bar
for cholesterol (mmol/L) and S1P (μmol/L) is shown on the left y axis. Protein (mg/mL) is shown on the right y axis. (C) S1P was measured with HPLC in purified
preparations of human total HDL, apoM+HDL, and apoM−HDL. Values are mean ± SEM (n = 3). S1P was not detectable in apoM−HDL. Results were confirmed
by LC-MS/MS.
ApoM gene dosage determines plasma S1P in genetically modified mice. (A) Plasma S1P in WT, Apom−/−, and apoM-transgenic female mice with
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extravasate albumin in their lungs, as demonstrated by i.v. in-
jection of Evans Blue. After i.v. injection of Evans Blue, in-
creased extravasation of Evans Blue in the lung was observed in
the Apom−/−mice compared with WT and Apom-TgHmice (Fig.
4). This observation suggests that, even though Apom−/−mice
have albumin-bound S1P in the circulation, this S1P cannot fully
maintain the endothelial barrier function in the lung. However,
the increased vascular leakage in the Apom−/−mice was not
followed by general edema, because the weight of the lung tissue
was unchanged in the different strains of mice (Fig. S7).
Taken together, our data demonstrate that apoM is the phys-
iological carrierprotein of S1Pin HDLandthat apoM candeliver
S1P to the S1P1receptor on endothelial cells. Thus, apoM-bound
S1P mediates S1P1receptor activation, resulting in downstream
(junction assembly) effects that are vasoprotective. These obser-
vations provide important information about the function of
apoM and increase the understanding of the antiatherogenic
effects of apoM demonstrated in several different mouse models
(17, 18, 25). Atherosclerosis is a chronic inflammatory disease
characterized by accumulation of oxidized lipoproteins and cho-
lesterol-filled foam cells in the arterial intima. HDL can protect
against atherosclerosis by pleiotropic mechanisms, e.g., by pro-
moting cholesterol efflux from foam cells, by attenuating LDL
oxidation, by anti-inflammatory or antiplatelet effects, and by
protecting the endothelium. Although apoM enhances the anti-
oxidant function of HDL and the ability of HDL to stimulate
cholesterol efflux from foam cells, these antiatherogenic effects
also are characteristics of apoM-free HDL (16, 17). In contrast,
the S1P-mediated vasoprotective effects of HDL are unique, be-
cause apoM is the physiological carrier protein for S1P in HDL,
andthesubsetofHDLthatcontainsapoMmediatesmultipleS1P-
dependent effects of HDL (1). However, in addition to the now-
Fig. 3.
were serum starved, stimulated for 1 h with indicated ligands, fixed, and imaged. ApoM+HDL (equivalent to ∼100 nM S1P as determined by LC/MS/MS) or
ApoM−HDL was used at 100 μg/mL Fatty acid-free BSA and r-apoM were complexed with S1P and used at afinal concentration of 100 nM (equimolar for both
protein and lipid). (Scale bar, 20 μm.) (B) HUVEC were serum starved and pretreated with 1 μM of the S1P1antagonist VPC44116 for 30 min before stimulated
with apoM+HDL (20 μg/mL protein, 20 nM S1P), apoM−HDL (20 μg/mL protein), or albumin-S1P (100 nM S1P and equimolar protein) for 10 min. (Note that
VPC44116 has no inhibitory effects on cells stimulated by FCS, because FCS can activate receptor systems other than S1P1.) Activation of p44/42 and Akt was
examined by Western blot analysis using phospho-specific antibodies. (C) HUVEC were serum starved and pretreated with 1 μM VPC44116 for 30 min where
indicated and subjected to migration assay with 10 μg/mL apoM+HDL (10 nM S1P), 10 μg/mL apoM−HDL, or 10 nM albumin-S1P. Data are mean ± SD (n = 3).
*P < 0.01. (D) Microscopy of HUVEC that were serum starved and stimulated with 100 μg/mL apoM+HDL, apoM−HDL, 100 nM albumin-S1P, r-apoM–S1P, or 100
nM S1P-free r-apoM for 1 h. After fixation, VE-cadherin (green), nuclei (blue), and F-actin (red) were visualized with confocal microscopy. (Scale bar, 20 μm.)
ApoM-bound S1P activates S1P1-mediated intracellular signaling pathways. (A) Confocal microscopy of HEK293 cells stably expressing S1P1-GFP. Cells
Fig. 4.
WT, Apom−/−, and Apom-TgHmice were injected i.v. with Evans Blue (30 μg/g
body weight). After 30 min the mice were perfused with saline; then the
lungs were removed and used for extraction of Evans Blue. Each point
represents the content of Evans Blue in the lungs of one mouse, and hori-
zontal lines represent mean values.
The apoM–S1P complex maintains lung endothelial barrier function.
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reported effects on HDL functionality, apoM also has putatively
proatherogenic effects on the metabolism of very low-density li-
poprotein (VLDL)/LDL. Hence, the concentrations of apoM and
LDL cholesterol in human plasma correlate positively (26), and
in mice the overexpression of human apoM delays the clearance
of VLDL/LDL, resulting in increased plasma concentrations of
VLDL/LDL (25). Thus, adverse effects on LDL metabolism may
counteractthebeneficialeffectsonvascularendotheliumofHDL-
associated apoM. These dual effects would agree with the lack of
association between plasma apoM levels and the risk of coronary
heart disease observed in human case-control studies (27).
Manyeffectson endothelial cellshave beenattributed toS1Pin
plasma. Recently, Camerer et al. (10) showed that plasma S1P
plays an important role in maintaining vascular barrier function.
Our in vivo data further support the conclusion that plasma S1P is
essential in maintaining vascular integrity. However, the presence
of S1P bound to albumin in plasma is not sufficient, because our
results demonstrate that S1P carried by apoM in the HDL frac-
tion has an important role in preserving vascular integrity. Thus,
even though the estimated concentration of albumin-bound S1P
is sufficient to activate the S1P receptors in the Apom−/−mice,
these mice have decreased endothelial barrier function in the
lung. Because apoM determines the S1P-binding capacity of
HDL, we propose that elevation of apoM+HDL would be vaso-
protective by preventing endothelial injury, allowing endothelial
regeneration, and maintaining vascular integrity.
Our results raise the interesting question of how S1P is de-
livered from apoM to S1P receptors. The specific anchoring of
apoM to lipoproteins and in particular to HDL by its retained
signal peptide suggests that association of apoM with HDL and
lipids in general is an important component for its function. The
structural elucidation of the apoM–S1P complex revealed several
features that possibly promote S1P uptake or release upon
membrane or even receptor associations. In the two complexes
of apoM with S1P or myristic acid, the peptide segment pre-
ceding the first β-strand of the lipocalin fold adopts multiple
conformations, thereby changing the exposure of several hy-
drophobic amino acids, partially uncovering the bottom of the
binding calyx, and possibly facilitating ligand release (Fig. S3).
Equally possible, a previously observed weak point in the apoM
barrel structure (between strands D and G) (28) could facilitate
a lateral release of the ligand from the calyx upon docking to
membranes or even to the S1P1receptors. Although these sce-
narios currently remain hypothetical, r-apoM clearly is capable
of delivering S1P to S1P1receptors at the cell surface. It remains
to be tested whether apoM is equally effective in delivering S1P
to other S1P receptors (S1P2–5) or if the S1P binding to apoM
provides receptor specificity that directs the biological effects of
HDL-associated S1P.
In summary, our studies define apoM as a carrier of S1P in HDL
and demonstrate that the HDL-associated apoM–S1P complex
mediates vasoprotective actions on the endothelium. This signaling
axis may be critical in normal vascular homeostasis and perturbed
in vascular diseases.
Materials and Methods
Detailed descriptions of materials and methods are given in SI Materials
and Methods.
Mice. Apom-TgN, Apom-TgH, and Apom−/−mice were backcrossed at least
seven times with C57B6/J mice. Mice were housed at the Panum Institute,
University of Copenhagen, Copenhagen (17, 25).
Lipoproteins and S1P. Human apoM+HDL and apoM−HDL were isolated from
human plasma with ultracentrifugation (1.063–1.21 g/L) followed by
immunoaffinity chromatography on an anti-apoM monoclonal column (16).
ApoM was quantified with ELISA (26). For gel filtration, 500-μL plasma
samples from Apom-TgH(n = 5), Apom−/−(n = 7), and WT (n = 6) mice were
separated on serially connected Superose 6 and Superose 12 10/300 GL col-
umns (16). S1P was measured with HPLC (29) or liquid chromatography
tandem MS (LC-MS/MS) (30).
Western Blotting. Western blotting was done after separation in 10 or 12%
SDS/PAGE gels with antibodies against human apoM, p44/42, phospho-p44/
42, and phospho-Akt (Cell Signaling).
Crystal Structure of r-ApoM with S1P. Human r-apoM was prepared as de-
scribed (15). The r-apoM–S1P complex was prepared by adding S1P (dry
powder) to a concentrated solution of r-apoM (13 mg/mL). Formation of the
r-apoM–S1P complex could be monitored with isoelectric focusing (Fig. S8A).
The r-apoM–S1P complex was purified by gel filtration to remove unbound
S1P before crystallization using hanging-drop vapor diffusion. Data were
collected to a resolution of 1.7 Å. The structure was solved using molecular
replacement and refined to R-work and R-free of 19.1% and 22.1%, re-
spectively (Table S1).
r-ApoM– and Albumin-Bound S1P. S1P was dissolved in methanol. After
evaporation, the S1P was redissolved by sonication in 20 mM Tris-HCl (pH 8.0)
containing equimolar amounts of r-apoM (19) or fatty acid free BSA (Sigma
A6003) and kept at 4 °C until use.
Cell Culture. Passage 4–10 HUVEC were cultured as described (31). HEK293
cells stably expressing S1P1-GFP (21) were cultured in DMEM with 10% FBS.
Confocal laser scanning microscopy was performed using a FluoView FV10i
system (Olympus). Migration assays were performed using a 96-well che-
motaxis chamber system (Neuroprobe) (32). VE-cadherin was visualized with
immunofluorescence, nuclei with TO-PRO-3 dye, and F-actin with rhodamine
phalloidin.
Vascular Permeability. Mice were injected i.v. with Evans Blue (30 μg/g body
weight). After 30 min the mice were anesthetized and perfused extensively
with saline via the right ventricle to remove intravascular Evans Blue. The
lung wet weights were measured, and Evans Blue was extracted in 1 mL
formamide at 56 °C for 16 h. Evans Blue concentration was determined from
the OD620minus OD500in the extract and a serial dilution of a standard.
Statistics. Numerical differences were analyzed with two-tailed Student’s
t test.
ACKNOWLEDGMENTS. We thank Karen Rasmussen and Charlotte Wandel
and Bernd Gardill for technical assistance, Uwe Müller for help during dif-
fraction data collection, and Dr. Kevin R. Lynch for the gift of VPC44116.
This work was supported by Grant 07143 from the Swedish Research
Council and by grants from the Söderberg’s Foundation and the Swedish
Heart-Lung Foundation (to B.D.), by Grants 09-06452/FSS (to L.B.N.) and
09-073571/FSS (to C.C.) from the Danish National Research Council, by
grants from the Rigshospitalet Research Council (to L.B.N.) and from the
Sonderfonds of the University Erlangen-Nuremberg (to Y.A.M.), and by
Grants HL-67330, HL-70694, and HL89934 from the National Institutes of
Health (to T.H.).
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