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Prepublished online as a Blood First Edition Paper on July 5, 2002; DOI 10.1182/blood-2002-04-1080.
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Blood, 1 December 2002, Vol. 100, No. 12, pp. 4026-4032
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Human -defensin regulates smooth muscle cell contraction: a
role for low-density lipoprotein receptor-related
protein/ 2-macroglobulin receptor
Taher Nassar,
Sa'ed Akkawi,
Rachel Bar-Shavit,
Abdullah Haj-Yehia,
Khalil Bdeir,
Abu-Bakr Al-Mehdi,
Mark Tarshis, and
Abd Al-Roof Higazi
From the Departments of Clinical Biochemistry,
Oncology, Interdepartmental Unit, and School of Pharmacy, Hadassah
University Hospital and Hebrew University-Hadassah Medical School,
Jerusalem, Israel; and the Department of Pathology and
Laboratory Medicine and Department of Environmental Medicine,
University of Pennsylvania, Philadelphia.
 |
Abstract |
We have previously identified -defensin in association with
medial smooth muscle cells (SMCs) in human coronary arteries. In the
present paper we report that -defensin, at concentrations below
those found in pathological conditions, inhibits phenylephrine (PE)-induced contraction of rat aortic rings. Addition of 1 µM -defensin increased the half-maximal effective concentration (EC50) of PE on denuded aortic rings from 32 to 630 nM. The
effect of -defensin was dose dependent and saturable, with a
half-maximal effect at 1 µM. -Defensin binds to human umbilical
vein SMCs in a specific manner. The presence of 1 µM -defensin
inhibited the PE-mediated Ca++ mobilization in SMCs by more
than 80%. The inhibitory effect of -defensin on contraction of
aortic rings and Ca++ mobilization was completely
abolished by anti-low-density lipoprotein receptor-related
protein/ 2-macroglobulin receptor (LRP) antibodies as
well as by the antagonist receptor-associated protein (RAP). -Defensin binds directly to isolated LRP in a specific and
dose-dependent manner; the binding was inhibited by RAP as well as by
anti-LRP antibodies. -Defensin is internalized by SMCs and interacts
with 2 intracellular subtypes of protein kinase C (PKC) involved in muscle contraction, and . RAP and anti-LRP antibodies inhibited the binding and internalization of -defensin by SMCs and its interaction with intracellular PKCs. These observations suggest that
binding of -defensin to LRP expressed in SMCs leads to its internalization; internalized -defensin binds to PKC and inhibits its enzymatic activity, leading to decreased Ca++
mobilization and SMC contraction in response to PE.
(Blood. 2002;100:4026-4032)
© 2002 by The American Society of Hematology.
 |
Introduction |
Inflammation has been implicated as a risk factor
in the development of atherosclerosis (reviewed by
Tracy1); -defensins may provide one link between the 2 processes. -Defensins are composed of 3 closely related gene
products, also referred to as human neutrophil peptides 1-3 (HNPs-1-3).
-Defensins are cationic peptides composed of 29 to 32 amino acids
arranged in 3 antiparallel sheets that are stabilized by 3 canonical intramolecular disulfide bonds.2,3
-Defensins are normally sequestered in the granules of neutrophils,
where they constitute 5% of the total cellular protein and are
involved in the intracellular killing of prokaryotic organisms.4 However, -defensins are released
extracellularly during inflammatory reactions, which could exert
deleterious effects on host cells. Increased plasma concentrations of
-defensins have been described in several inflammatory diseases. For
example, defensin concentration approaches 30 µM in the blood of
patients with bacterial septicemia or meningitis.5 Even
higher levels are found in sputum from cystic fibrosis (CF)
patients6 or in pleural fluid in patients with
empyema.7
In previous studies, we reported that -defensins inhibit the
fibrinolytic system,8 a process that favors the
development of atherosclerotic lesions.9,10 We also
observed that -defensins regulate lipoprotein metabolism by
stimulating the binding of lipoprotein (a) (Lp(a))11 and
low-density lipoprotein (LDL)12 to vascular cells and cell
matrix.13 The biologic relevance of these observations is
supported by the presence of -defensins in normal-appearing adult
epicardial coronary arteries as well as in vessels with minimal intimal
thickening; staining was particularly intense in and around vascular
smooth muscle cells (SMCs).14 -Defensins were also
found in association with intimal and medial SMCs in
atherosclerotic carotid arteries.11
Considering that -defensins are found in human blood vessels, in and
around vascular SMCs, we asked if -defensins had any effect on SMC
activity that may be involved in the evolution of vascular disease. The
results of the present study indicate that -defensins inhibit the
contraction of SMCs in aortic rings. We show that low-density
lipoprotein receptor-related protein/ 2-macroglobulin receptor (LRP) mediates the internalization of -defensin.
Internalized -defensin binds to protein kinase C (PKC) and , inhibiting their activity and causing decreased
Ca++ mobilization.
 |
Materials and methods |
Materials
-Defensin (human neutrophil peptide 3) and murine monoclonal
antidefensin antibodies were the kind gift from Dr T. Ganz (Department of Medicine, Univiversity of California at Los Angeles) and were prepared and characterized as previously described15; the
protein migrated as a single band on sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
LRP-antagonist receptor-associated protein (rRAP), soluble LRP, and
anti-LRP were kindly provided by Dr D. Strickland (American Red Cross,
Rockville, MD). Anti-LRP antibodies were also purchased from American
Diagnostica (Greenwich, CT). Human umbilical vein vascular SMCs were
the kind gift from Dr D. Cines (Department of Pathology and Laboratory
Medicine, University of Pennsylvania) and were prepared as described by Grobmyer et al.16 Protein G-agarose was purchased from
GIBCO (Bethesda, MD). Rabbit anti-PKC antibodies and calphostin C were purchased from Sigma (St Louis, MO).
Contraction response
Experiments were performed as described by Haj-Yehia et
al.17 Briefly, male Sprague Dawley rats (250-275 g) were
killed by exsanguination. The thoracic aorta was removed, dissected
free of fat and connective tissue, and cut into transverse rings 5 mm
in length.18,19 The rings were gently rotated on a
stainless steel rod to remove the endothelium (denuded aorta). The
tissues were kept in an oxygenated (95% O2, 5%
CO2) solution of Krebs-Henseleit (KH) buffer (144 mM NaCl,
5.9 mM KCl, 1.6 mM CaCl2, 1.2 mM MgSO4, 1.2 mM
KH2PO4, 25 mM NaHCO3, and 11.1 mM
D-glucose). The rings were mounted to record isometric tension in a
10-mL bath containing KH solution under continuous aeration. The rings
were equilibrated for 1.5 hours at 37°C and maintained under a
resting tension of 2 g throughout the experiment. Each aortic
ring was then contracted by adding phenylephrine (PE) in stepwise
increments (from 10 10 M to 10 5 M). In other
experiments, various concentrations of -defensin were added 5 minutes before adding PE. In each experiment, rings exposed to KH
buffer alone were analyzed in parallel. Isometric tension was measured
with a force displacement transducer and recorded online using a
computerized system (ExperimentiaÆ, Budapest, Hungary). The maximal
contraction of the rings (100%) was determined by adding 20 to 40 mM KCl.
The half-maximal effective concentration (EC50) was
calculated by measuring the response of aortic rings (y-axis) to
increasing concentrations of PE (x-axis). The lines that intersect with
the y- and x-axes were drawn to determine the concentration of PE that
induces 50% of its maximal effect. Each EC50 was
calculated as the mean ± SD of 3 separate experiments.
Binding and internalization of -defensin
125I- -defensin was radiolabeled and characterized
as described previously.20 Cells were grown to confluence
in 48-well Falcon multiwell tissue culture plates (Becton Dickinson,
Lincoln Park, NJ) to a final density of approximately
5 × 104 cells per well in Dulbecco modified Eagle
medium (DMEM) (Bet Haemek Israel) supplemented with
10% fetal calf serum (FCS). The cells were prechilled to 4°C for 30 minutes and washed twice with KH buffer. The cells were then incubated
with KH buffer containing varying concentrations of
125I-defensin for 1 hour at 4°C. Washing the cells 4 times with binding buffer to remove unbound ligand terminated the
incubation. Radiolabeled ligand bound to the cell surface was released
by adding 50 µM glycine HCl, pH 2.8, as described by Higazi et
al.21 Nonspecific binding was determined by measuring
cell-associated radioactivity in the presence of 100 µM unlabeled
-defensin. Specific binding was defined as the difference between
total and nonspecific binding.
In one set of experiments, after the cells were incubated with
125I-defensin for 1 hour at 4°C and washed 4 times with
binding buffer to remove unbound ligand, the cells were warmed to
37°C for another hour, and radiolabeled ligand bound to the cell
surface was released with glycine HCl, pH 2.8. After releasing cell
surface-bound ligand, internalized -defensin was determined by
solubilizing the cells with 0.1 N NaOH and measuring the
cell-associated radioactivity.
To determine the incorporation of radiolabeled -defensin into
isolated aortic rings, denuded aortic rings were incubated in KH buffer
with 1.5 µM 125I-defensin (the concentration that gave
50% of the maximal inhibitory effect) at 37°C for 30 minutes in the
absence or presence of 100 µM unlabeled -defensin. The incubation
was terminated by washing the aortic rings 4 times with binding buffer
to remove unbound ligand. Radiolabeled ligand incorporated into the
aortic rings was measured using a -counter. Nonspecific binding was
determined by measuring cell-associated radioactivity in the presence
of 100 µM unlabeled -defensin. When indicated, 100 nM anti-LRP
antibodies, 20 nM rRAP, or 100 nM anti-LDL receptor antibodies (as
irrelevant antibodies) were added 5 minutes before the radiolabeled
-defensin.
Binding of -defensin to isolated LRP
Binding of 125I-defensin to LRP-coated wells was
determined as described by Higazi et al.21 Varying
concentrations of 125I-defensin were added to LRP-coated
wells in the presence or absence of excess unlabeled -defensin, and
the total and specific binding was measured as in the previous
paragraph. Specific binding was defined as the difference between total
and nonspecific binding.
Measurement of intracellular calcium
The effect of -defensin on PE-induced intracellular calcium
(Ca++i) in human umbilical vein SMCs
was measured as described by Haj-Yehia et al,17 Tozawa et
al,22 and Ikeda et al.23 SMCs were incubated
for 30 minutes at 37°C in media in the presence or absence of 1 µM
fluo-3 acetomethyl ester (Fluo-3; Molecular Probes, Eugene,
OR).24 The cells were washed 3 times with Krebs-Ringer
bicarbonate solution that contained 2 µM Ca++. One
milliliter of Ca++-containing medium was then added to each
well. Baseline pictures were taken at 490 nm excitation and 520 nm
emission using a Hamamatsu ORCA-100 cooled CCD digital camera and an
inverted Nikon-TMD Diaphot epifluorescence microscope. PE (0.1 µM) was then added, and Fluo-3 emission was measured at 5 minutes.
-Defensin had no effect on dye uptake.
To quantify calcium concentration, at least 5 individual cells in 3 or
more plates for each condition were outlined, the average pixel
intensity per cell was calculated, and the background emission was
subtracted. Dye loading was uniform among plates and cells and did not
contribute to these analyses. The mean fluorescence pixel intensity in
control cells was designated as 100%. Images representative of a
minimum of 3 experiments are shown.
In the set of experiments used to determine the rapid transient peak in
intracellular Ca++, a 1-mM stock solution of Fluo-4
(Molecular Probes) was mixed with an equal volume of a 20% (wt/vol)
solution of the nonionic detergent pluronic acid F-127 in dimethyl
sulfoxide (DMSO) (Molecular Probes) and then added to washed
cells to a final concentration of 5 µM. Cells were incubated with
Fluo-4 for 20 minutes at 37°C in a 5% CO2 atmosphere.
Cells were then washed with phosphate-buffered saline (PBS) to
remove any dye that was nonspecifically associated with the cell
membrane and then incubated for another 30 minutes to allow complete
de-esterification of intracellular acetoxymethyl (AM) esters.
The Fluo-4 fluorescence was determined by employing a 20 ×/0.6 plan
Neofluor lens (Zeiss) in a Zeiss LSM 410 confocal system with an
Axiovert 135 inverted microscope. Fluorescence excitation was
induced by an argon ion laser with a 515-nm emission filter and
measured every 10 seconds for 8 minutes.
Several random fields for each experiment were taken and scored.
Confocal TIF images were transferred to a Zeiss imaging workstation for
fluorescence intensity analysis. The results were expressed numerically
in terms of arbitrary fluorescence units of 0 to 250, where white is
the highest amount of fluorescence above background per area.
Radiolabeled Ca++ uptake
Experiments were performed as previously
described.25 Human umbilical vein SMCs were grown in 60-mm
dishes. 45Ca++ uptake was determined by
overlaying the cells with KH buffer containing 25 µM
45Ca++ at 37°C. PE (0.1 µM) was then added.
When indicated, 1 µM -defensin, alone or together with rRAP or
anti-LRP antibodies, was added 5 minutes before PE. Fifteen minute
after adding PE with or without the additives, the cells were washed to
remove free 45Ca++, solubilized, and
45Ca++ determined.
Immunoprecipitation
A total of 108 human SMCs were incubated in KH
buffer with 1 µM 125I- -defensin at 37°C for 30 minutes in the presence or absence of anti-LRP antibodies (100 nM) or
irrelevant immunoglobulin G IgG (100 nM). In another set of
experiments the cells were incubated with 125I- -defensin
and a 50-fold excess of unlabeled -defensin. Washing the cells 4 times with binding buffer to remove unbound ligand terminated the
incubation. The cells were then lysed by adding 1 mL of 10 mM Tris
(tris(hydroxymethyl)aminomethane) HCl buffer containing 100 mM NaCl, 1 mM EDTA (ethylenediaminetetraacetic acid), and 1% Triton
X-100 for 15 minutes. The mixture was added to 3 mL protein G-agarose
in KH buffer containing 100 nM anti-PKC antibodies or irrelevant
antibodies and incubated for 6 hours at 4°C. The beads were
centrifuged at 5000 rpm for 10 minutes, and the supernatant was
decanted. The precipitate was washed 4 times with KH buffer, and the
radioactivity precipitated by protein G-agarose was measured.
Nonspecific binding of -defensin was determined by measuring the
precipitation of 125I- -defensin in the presence of an
excess of unlabeled -defensin or when irrelevant IgG was used
instead of anti-PKC antibodies. Both methods used to determine
nonspecific binding gave very close results.
Confocal microscopy
Cells grown on coverslips were incubated in DMEM
containing 1 µM defensin and supplemented with 10% FCS for 30 minutes at 37°C. The cells were washed 3 times with PBS, fixed for 10 minutes with 4% formaldehyde in PBS, and permeabilized with 0.2%
Triton X-100 in PBS-bovine serum albumin (BSA) buffer for 3 minutes. The coverslips were overlaid for 20 minutes with 2% normal
horse serum and then incubated for 40 minutes with antidefensin
antibodies diluted 1:500 with PBS. After 4 washes with PBS, the cells
were stained for 40 minutes with Alexo 488-labeled goat antirabbit serum (Molecular Probes), washed 4 times with PBS, and mounted in 80%
glycerol-20% PBS supplemented with 3% DABCO
(1,4-diazabicyclo-[2,2]-octane) as antibleaching agent. No staining
was seen when either antidefensin or the secondary antibody was omitted.
Confocal microscopy was performed using a Zeiss LSM 410 confocal laser
scanning system attached to the Zeiss Axiovert 135M inverted
microscope 40 ×/1.3 plain oil immersion lens. The system was equipped
with a 25-mW argon laser (488-nm excitation line with 515-nm low-pass
barrier filter) for the excitation of Alexa 488 green
fluorescence. The differential interference contrast (DIC) images
according to Nomarski were collected simultaneously using a transmitted
light detector. Autofluorescence of the specimen was set to background
level. To reduce the visual noise, all optical sections were performed
in the fast line-scan acquisition mode (512 pixels per line) by the
averaging of 8 images before the final image was produced on the
monitor. In each experiment, background level, exciting light
intensity, photomultiplier, imaging filters, aperture (pinhole)
contrast, and electronic zoom size were monitored at the same level.
Confocal images were converted to a TIF format and transferred to a
Zeiss imaging workstation to provide pseudocolor representation. Z
series of optical sections were acquired at 0.7-µm intervals from the
surface through the vertical axis of the specimen, assembled in an
image processor, and projected on a monitor into a single image using
image analysis software (Zeiss). The display of the images in
color-coded mode (depth coding) was used to determine the depth at
which the features of interest lay.
All experiments were performed in triplicate and were repeated a
minimum of 3 times. All data are presented as mean ± SD of the 3 experiments.
 |
Results |
Because -defensin is present in human blood
vessels11 in and around arterial SMCs,14 it
was of interest to see whether -defensin affects activity of
vascular SMCs. To address this question, we examined the effect of
-defensin on aortic rings denuded of endothelium. Figure
1A shows that at concentrations up to 10 µM -defensin alone had no effect on the contraction of rat aortic
rings. However, the presence of 1 µM -defensin inhibited the
phenylephrine (PE)-induced contraction of denuded rat aortic rings
(Figure 1A); 1 µM -defensin increased the EC50 of PE
almost 20-fold, from 32 to 630 nM (Figure 1B). As shown in Figure 1B,
the effect of -defensin on PE-induced contraction was completely
abolished by antidefensin antibodies. The inhibitory effect of
-defensin was dose dependent and saturable, with a half-maximal
effect observed at a concentration of about 1 µM (not
shown).

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| Figure 1.
Effect of -defensin on contraction of aortic rings.
(A) The effect of increasing concentrations of -defensin on the
contraction of denuded rat aortic rings ( ). The effect of
increasing concentrations of PE was measured in the absence ( ) or
presence ( ) of 1 µM -defensin. (B) The effect of
anti- -defensin antibodies: EC50 of PE was determined in
the absence (Cont) or presence of 1 µM -defensin (Def),
-defensin and specific anti- -defensin antibody (Def+sAb), or
-defensin and irrelevant antibodies (Def+irAb). All experiments,
unless otherwise indicated, were performed in triplicate and were
repeated at least 3 times. In this and in all graphs, the data are
presented as means ± SD of 3 experiments.
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To examine the reversibility of the inhibitory effect of
-defensin, we incubated aortic rings with or without 1 µM
-defensin for 30 minutes. After the incubation, the rings were
washed and the response to PE was determined at different time points.
Figure 2 shows that the inhibitory effect
of -defensin began to decrease after 30 minutes of washing and was
undetectable after 120 minutes.

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| Figure 2.
The reversibility of the inhibitory effect of
-defensin.
Aortic rings were incubated with ( ) or without ( ) 1 µM
-defensin for 30 minutes. After washing, the response to PE was
determined immediately (0 minute) or after 15, 30, 60, 75, or 120 minutes. The EC50 was determined as described in
"Materials and methods."
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To elucidate the mechanism through which -defensin inhibited SMCs
contractility, we examined the binding of -defensin to SMCs. Figure
3 shows that 125I-defensin
binds in a dose-dependent, saturable, and specific fashion to human
vascular SMCs, with a calculated dissociation constant
(Kd) of about 1.5 µM, close to the
concentration that induced half-maximal inhibition of SMC
contraction.

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| Figure 3.
Specific binding of 125I-defensin to SMCs.
Human umbilical vein SMCs were incubated with the indicated
concentrations of 125I- -defensin in the presence or
absence of 100 µM unlabeled -defensin. Specific binding was
determined as described in "Materials and methods."
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Studies were then performed to identify the receptor involved in
the -defensin-induced inhibitory effect. Although we have previously observed12 that -defensin binds to the LDL
receptor (LDL-R), anti-LDL-R antibodies had no effect on
-defensin-induced inhibition of aortic contractility (Figure
4). We then turned our attention to the
low-density lipoprotein receptor-related protein/ 2-macroglobulin receptor (LRP), another member
of the LDL-R family26,27; Bacskai et al reported that
binding of 2-macroglobulin to LRP increased
Ca++ influx into cultured neurons.28
Therefore, we examined the effect of LRP inhibitors on the activity of
-defensin. Figure 4 shows that the effect of -defensin on
contractility of aortic ring was inhibited by anti-LRP antibodies and
was abolished by the LRP-antagonist receptor-associated protein
(rRAP).29,30

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| Figure 4.
Role of LRP in -defensin-mediated vasocontractility.
The contraction of aortic rings was induced by PE in the presence of 1 µM -defensin (Def), -defensin and 100 nM anti-LDL receptor
antibody (anti-LDLR), -defensin and 20 nM anti-LRP antibody
(anti-LRP), -defensin and 20 nM rRAP (rRAP), or PE with no
additives (Cont).
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The interaction between LRP and -defensin was examined in further
detail: Figure 5A shows that
125I- -defensin bound specifically to LRP-coated wells
with half-maximal binding at about 2 µM. Binding of -defensin to
LRP was inhibited by rRAP as well as by anti-LRP but not by irrelevant
antibodies (Figure 5B).

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| Figure 5.
Binding of radiolabeled -defensin to isolated LRP.
(A) Binding of 125I- -defensin to LRP-coated wells.
Varying concentrations of 125I- -defensin were added to
LRP-coated wells in the presence or absence of excess unlabeled
-defensin, and the total ( ) and specific ( ) binding were
measured. (B) Inhibition of -defensin binding by rRAP and anti-LRP
antibodies. 125I-defensin (2 µM) was added to LRP-coated
wells in the presence of medium alone (Cont) or medium supplemented
with 20 nM rRAP (rRAP), anti-LRP antibody (anti-LRP), or irrelevant
antibody (irAntib).
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On the basis of the known capacity of LRP to mediate the
internalization of diverse ligands and to characterize the interaction between -defensin and cell-associated LRP, we asked whether
-defensin is internalized by SMCs. To address this question, we
first examined the capacity of cell-bound -defensin to be
internalized. SMCs were incubated with 125I-defensin at
4°C for 30 minutes, and the cells were washed to remove unbound
-defensin and then warmed to 37°C. Under these conditions most of
the bound -defensin was internalized (Figure 6A). Figure 6B shows that RAP and
anti-LRP inhibited the binding and internalization of -defensin by
SMCs, whereas irrelevant IgG had no effect on either parameter (not
shown). Anti-LRP antibodies (Figure 6C) also inhibited the binding and
internalization of -defensin, as seen directly by confocal
microscopy; again, irrelevant antibodies had no effect (not shown).

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| Figure 6.
Internalization of -defensin by SMCs.
(A) Human umbilical vein SMCs were incubated for 1 hour at 4°C with 2 µM 125I- -defensin. Washing of the cells terminated
incubation. Bound 125I-defensin released immediately from
the surface of SMCs (a) or after subsequent warming to 37°C
for 30 minutes (b2). Internalized 125I- -defensin at
4°C (c) or 37°C (d). (B) The role of LRP in binding and
internalization of -defensin by SMCs. Experiments were performed as
in panel A. Binding (a,b,c) and internalization (d,e,f) of
125I-defensin were determined at 37°C in the absence of
additives (a,d) or in the presence of 20 nM anti-LRP antibody (b,e) or
20 nM rRAP (c,f). (C) Confocal fluorescence images of defensin binding
and internalization by SMCs. Alexo 488-labeled antigens are depicted
in red, and DIC images are shown in green. (i) SMCs incubated with
antidefensin IgG (rabbit), followed by labeled goat antirabbit IgG, in
the absence of -defensin. (ii) SMCs incubated with 1 µM
-defensin for 30 minutes and later treated as in panel i. (iii)
Cells preincubated for 10 minutes with anti-LRP antibodies (20 nM)
before addition of 1 µM -defensin and later treated as in
panel B. (iv) Image reconstruction of 16 single sections presented in
the color-coding scale. The upper section is shown in red, and the
bottom section is shown in blue. Original
magnification × 400.
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To investigate the mechanism by which the interaction of -defensin
with LRP inhibits SMC contractility, we examined the effect of
-defensin on PE-induced Ca++ mobilization. As seen in
Figure 7A, 100 nM PE induced a 162% increase in intracellular Ca++, which was markedly
inhibited by 1 µM -defensin. Moreover, 1 µM -defensin almost
completely inhibited PE-induced 45Ca++
internalization (Figure 7B). We then examined the involvement of LRP in
the -defensin effect on Ca++ mobilization. Figure 7B
shows that rRAP and anti-LRP antibodies completely inhibited the effect
of -defensin, whereas irrelevant antibodies had no effect (not
shown). Phenylephrine stimulation produces a rapid transient peak in
intracellular Ca++. To examine the effect of -defensin
on this transient peak of intracellular Ca++, we determined
the time course of the increase in Ca++ concentration using
consecutive confocal microscopy images. Figure 8A shows that PE induced the appearance
of a transient peak, followed by more permanent ones, and that
-defensin inhibited the appearance of both peaks of Ca++
(Figure 8B). In the experiment shown in Figure 8, -defensin was
added immediately after PE. In other experiments, -defensin was
added before PE and the same result was obtained.

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| Figure 7.
Effect of -defensin on PE-induced Ca++
mobilization in human umbilical vein SMCs.
(A) SMCs were incubated in media with no additives (Cont.), or in the
presence of 100 nM PE (PE) or 100 nM PE and 1 µM -defensin
(PE+Defensin) and photographed at 490 nm excitation and 520 nm
emission. Original magnification × 40. (B) SMCs
were incubated with buffer containing 45Ca++
and 100 nM PE (PE); 100 nM PE and 1 µM -defensin (PE+Def.);
45Ca++ with no additives (cont); 100 nM PE, 1 µM defensin, and 20 nM rRAP (PE+Def.+rRAP); or 100 nM anti-LRP
antibodies instead of rRAP (PE+Def.+Ab.).
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| Figure 8.
Effect of -defensin on temporal changes of
intracellular Ca++.
The change of fluorescence over time from selected areas containing the
same number of cells is illustrated using arbitrary units. Cells were
treated with PE (100 nM) alone (A) or 100 nM PE with 2 µM
-defensin (B).
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Our data show that -defensin exerts its inhibitory effect on
PE-induced SMC contraction and Ca++ mobilization by
interacting with LRP. At the same time, -defensin alone has no
effect on cell contraction. These data suggest that -defensin may
inhibit some of the mechanisms that are activated during SMC
contraction. Charp et al found that -defensin inhibits the activity
of PKC.31 It is widely accepted that PKC plays an
important role in the contraction of SMCs; in the case of cerebral vasospasm, PKC is considered to have a significant role by
phosphorylating various ion channels and augmenting voltage-dependent
Ca++ channels, which leads to vessel
contraction.32 PKC has also been reported to enhance
CaV1.2b channel currents in various SMC preparations.33-37 A number of different agonists,
including norepinephrine, endothelin, angiotensin II, and serotonin,
have been suggested to stimulate CaV1.2b channels via a
PKC-dependent mechanism (reviewed by Gollasch and
Nelson38). Yang et al39 show that inhibitors of and PKC inhibit the ethanol-induced contraction of cerebral arterial smooth muscle as well as the ethanol-induced increase in
intracellular Ca++. To determine if -defensin has any
direct interaction with PKC present in the SMCs, we incubated the cells
with radiolabeled -defensin at 37°C for 30 minutes. At the end of
incubation the cells were washed and lysed. Anti- or -
PKC antibodies were added and later precipitated by protein G
conjugated to beads, as described by Higazi et al.40
Figure 9 shows that radiolabeled -defensin is immunoprecipitated by anti-PKC antibodies, whereas irrelevant IgG had no effect. These data indicate that -defensin added to SMCs interacts with intracellular PKC and . Figure 9
shows that the presence of anti-LRP antibodies during incubation of the
cells with radiolabeled -defensin inhibited the
coimmunoprecipitation of -defensin with PKC. In contrast, anti-LRP
antibodies added after cell lysis had no effect on the precipitation of
radiolabeled -defensin (not shown).

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| Figure 9.
Coimmunoprecipitation of -defensin with PKC.
SMCs were incubated with radiolabeled -defensin at 37°C for 30 minutes. At the end of incubation the cells were washed and lysed.
Antibodies against PKC (Anti-a), PKC (Anti-b), or irrelevant
antibodies (ir Ab) were added to the lysed cells and later precipitated
by protein G conjugated to beads. In another set of experiments, the
anti-LRP antibodies were added to the cells before the -defensin and
anti- PKC antibodies (Anti-LRP-a) or anti- PKC antibodies
(Anti-LRP-b).
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Our data show that -defensin interacts with intracellular PKC; the
data of Charp et al31 indicate that this interaction leads
to inhibition of the enzyme. To confirm that inhibition of PKC is
sufficient to induce inhibition of PE-induced SMC contraction, we
examined the effect of other known inhibitors of PKC. Figure 10 shows that calphostin C at a
concentration of 1 µM inhibited the PE-induced contraction of denuded
aorta. In contrast to its inhibitory effect on the PE-induced
vasoconstriction and as in the case of -defensin, 1 µM calphostin
C alone had no effect on SMC contraction (not shown). These results
support our conclusions concerning the mechanism of the inhibitory
effect of -defensin. Neither anti-LRP antibodies nor rRAP affected
the inhibitory effect of calphostin C (Figure 10).

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| Figure 10.
The effect of calphostin C on PE-induced contraction of
aortic rings.
Contraction of rat's aortic rings was induced by PE alone (Cont) or in
the presence of 1 µM calphostin C (Cal), calphostin C and anti-LRP
antibodies (Cal+ Anti-LRP), or calphostin C and rRAP (Cal+rRAP).
|
|
 |
Discussion |
We have previously reported that -defensin is found in human
arteries11 in and around vascular SMCs.14 The
present study shows that -defensin, at concentrations below those
reached during pathological conditions,5 inhibits the
contractility of vascular SMCs. The inhibitory effect of -defensin
on the contractility of SMCs is exerted by blocking the increase in
intracellular Ca++ induced by PE.
The effect of -defensin on SMCs is mediated through LRP; this
conclusion is based on results of several experiments in which anti-LRP
antibodies or rRAP inhibited (1) the -defensin effect on contraction
of aortic rings and on Ca++ mobilization induced by PE in
SMCs, (2) binding and internalization of -defensin by SMCs, and (3)
the interaction of -defensin with intracellular PKC. Furthermore,
isolated LRP binds -defensin in a specific fashion, and anti-LRP
antibodies or rRAP inhibit the binding.
The involvement of LRP in the regulation of intracellular
Ca++ by -defensin is consistent with the emerging
literature on the signal-transducing properties of this receptor; LRP
has been shown to mediate the mitogenic effect
2-macroglobulin41 in SMCs and, more
recently, Bacskai et al reported that binding of
2-macroglobulin to LRP increased Ca++ influx
into cultured neurons28 by inducing receptor dimerization.
However, in contrast to other known LRP ligands reported to increase
intracellular Ca++, binding of -defensin alone has no
direct effect on Ca++ mobilization or on SMC contraction.
Rather, binding of -defensin to LRP modulates the responsiveness of
cell to the agonists PE, that bind to other receptors to induce
Ca++ mobilization and SMC contraction. The
possibility that defensin induces its effect by interfering with PKC
activity could explain the observation that it inhibits PE-induced
contraction, although it has no intrinsic effects. PKC is inactive in
resting cells; therefore, addition of an inhibitor (such as
-defensin or calphostin C) should induce no response.
Apparently it seems difficult to reconcile our findings about the LRP
as a mediator of signal transduction with the notion of an endocytic
receptor that is delivered to lysosomes in other words, how defensin,
which is probably internalized into endosomes with LRP, gains access to
PKC in the cytoplasm. This question has indeed been asked with respect
to the HIV-Tat protein that binds to LRP, is internalized into
endosomes, and is later translocated by a process that is poorly
understood to the nucleus where it stimulates
transcription.42 Furthermore, Pseudomonas
exotoxin A, which mediates adenosine diphosphate (ADP)
ribosylation of elongation factor 2 in the cytoplasm, enters the cell
exclusively via LRP.43 In the case of exotoxin A, the
toxin is composed of a single-chain polypeptide that harbors a
fusogenic domain, which mediates the translocation of the toxin into
the cytoplasm.43
Another observation that is pertinent to the role of LRP in signal
transduction is the increasing number of cytoplasmic proteins that have
been found to interact with the receptor. A search for such proteins
was initiated because it became impossible to reconcile a bewildering
spectrum of experimental observations relating to various functions of
LRP and other members of the LDL receptor gene family with a simple
role as an endocytic receptor or cellular transporter of extracellular
ligands.42
When all data are taken together, it appears likely that the
-defensin regulation of SMC contraction and intracellular
Ca++ concentrations is mediated by LRP. Taking into
consideration that LRP can interact with several intracellular
proteins,44 it could be expected that different ligands
may induce different responses. It is feasible that some of the LRP
ligands inhibit PKC activity, and it will not come as a surprise if
other LRP ligands will stimulate the activity of PKC. There is no doubt that additional studies will have to be performed to clarify the details of the signaling pathway that mediates the effect of
-defensin and LRP on SMC contraction.
 |
Footnotes |
Submitted April 10, 2002; accepted June 5, 2002.
Prepublished online as
Blood First Edition Paper, July 5, 2002; DOI
10.1182/blood-2002-04-1080.
Supported in part by grants from the National Institutes of
Health HL 67381-01, HL60169, and HL58107.
T.N. and S.A. contributed equally to this work.
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.
Reprints: Abd Al-Roof Higazi, Department of Pathology
and Laboratory Medicine, University of Pennsylvania, 513A
Stellar-Chance, 422 Curie Blvd, Philadelphia, PA 19104; e-mail:
higazi{at}mail.med.upenn.edu.
 |
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