© 1997 American Heart Association, Inc.
Articles |
Calreticulin, a Potential Vascular Regulatory Protein, Reduces Intimal Hyperplasia After Arterial Injury
From the Cardiovascular Disease Research Group and the Department of Medicine, University of Alberta, and Vascular Biology Group, Robarts Research Institute and Department of Medicine, University of Western Ontario (E.D., D.K., M.L.H., L.Y.L., A.L.); the MRC Group in Molecular Biology of Membranes and Department of Biochemistry, University of Alberta (N.M., M.M.); the Department of Laboratory Animal Medicine, University of Alberta (N.N.); the Division of Hematology and Department of Medicine, University of Alberta (B.R., S.R.); and the Department of Pathology, University of Alberta (M.S., W.E.).
Correspondence to Alexandra Lucas, MD, Vascular Biology Group, The John P. Robarts Research Institute, PO Box 5015, 100 Perth Dr, London, Ontario N6A 5K8. E-mail arl{at}rri.on.ca
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Abstract Both thrombotic and inflammatory responses to arterial injury have been implicated in atherosclerotic plaque growth. Calreticulin is a ubiquitous calcium-binding protein with antithrombotic activity and, in addition, is associated with leukocyte activation. We are investigating calreticulin as a potential vascular regulatory protein. The development of intimal hyperplasia was studied at sites of balloon injury in iliofemoral arteries from 91 rats. Calreticulin was infused directly into the artery immediately before balloon injury, and plaque growth was then assessed at 4 weeks' follow-up. Parallel studies of the effects of each calreticulin domain as well as a related calcium-binding protein, calsequestrin, were examined. The effects of calreticulin on platelet activation, clot formation, and mononuclear cell migration were also studied. When infused before balloon injury in rat iliofemoral arteries, calreticulin, or its high-capacity Ca2+-binding C domain, significantly reduces plaque development, whereas calsequestrin, a related calcium-binding protein that lacks the multifunctional nature of calreticulin, does not decrease plaque area (saline: 0.037±0.007 mm2, calsequestrin: 0.042±0.021 mm2, calreticulin: 0.003±0.002 mm2, n=46, P<.04). The N domain and more specifically the P domain, a low-capacity, high-affinity calcium-binding domain in calreticulin, do not reduce intimal hyperplasia (N+P domain: 0.038±0.012 mm2, C domain: 0.003±0.002 mm2, n=45 rats, P<.0001). Calreticulin reduces macrophage and T cell staining in the arterial wall after injury but has no direct effect on monocyte migration in vitro (percent medial area staining positive for macrophage 24 hours after injury (N+P: 4.06±1.42, calreticulin: 0.273±0.02; n=26, P<.009). Calreticulin does, however, reduce platelet-dependent whole blood clotting time, in vitro (baseline: 78.23±2.04 seconds, calreticulin: 113.5±1.95 seconds; n=5, P<.002). We conclude that calreticulin significantly reduces intimal hyperplasia after arterial injury, potentially acting as a vascular regulatory protein.
Key Words: atherosclerosis • thrombosis • calreticulin • monocytes
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Introduction |
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Atherosclerotic plaque is believed to develop in response to damage to the arterial wall induced by hypertension, diabetes, hyperlipidemia, smoking,1–3 and angioplasty techniques designed to open occluded arteries.4,5 Injury to the intima activates thrombotic and inflammatory cascades with release of signaling factors and mitogens6–10 that induce cellular invasion and proliferation at the site of injury to seal the inner arterial layers. These events reflect an ongoing balance between tissue breakdown and healing, which can in some cases produce an excessive response with accelerated buildup of tissue or atherosclerotic plaque.1–5,9–13 Inhibition of multiple levels of the coagulation cascade,8,9,14–16 platelet activation,17–18 Ca2+ transport,19,20 and growth factors21,22 have been reported to diminish restenosis in animal models of atherosclerosis, but therapy has been only partially successful in clinical trials,9 a result indicating that understanding of these events is as yet limited.
Calreticulin is a 60-kDa, ubiquitous Ca2+-binding
protein of the endoplasmic reticulum (ER)
membrane23,24 with two classes of
Ca2+-binding sites (Fig 1
): a high-capacity, low-affinity binding
site (>25 mol/mole of protein) in the acidic C domain and a
high-affinity, low-capacity Ca2+-binding site
(Kd 
1 µM) in the P
domain.25,26 The protein plays an important role
in calcium homeostasis, including Ca2+ storage in
the ER and regulation of Ca2+
influx.27–31 The N terminal domain of
calreticulin does not bind Ca,2+ but it is a site
for calreticulin interaction with other
proteins.32–36
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Furthermore, calreticulin has been found in extracellular locations.28,37,38 An exciting finding is that calreticulin has been reported to play a role in both the thrombotic and inflammatory responses of circulating blood. Kuwabara et al24 demonstrated that calreticulin, specifically the C domain, binds vitamin K–dependent coagulation factors and inhibits experimentally induced coronary thrombosis in a canine model of acute arterial occlusion.24 Low levels of calreticulin have been detected in bovine serum,39 are secreted in ixodid tick saliva,40 and interact with fibrinogen,38 findings suggesting that this protein may regulate hemostasis. Calreticulin has also been associated with regulation of immune responses; it is concentrated around phagocytosed particles in neutrophils,41 has NH2-terminal amino acid sequence identity with the C1q receptor,28 and has been reported to alter cellular adhesion34 and migration.37
On the basis of the capacity of calreticulin to interact with both the thrombotic and inflammatory cascades, we have studied calreticulin as a potential key regulatory protein for modulation of acute responses to vascular injury. We have investigated the effect of calreticulin infusion on plaque development, mononuclear cell invasion, and platelet-dependent coagulation after balloon-induced injury.
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Methods |
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Rat Model of Intimal Proliferation
In all, 91 Sprague Dawley rats were studied in a series of three studies; 10 rats for Study 1 (pilot study of calreticulin efficacy), 36 rats for Study 2 (titration of calreticulin and calsequestrin), and 45 rats for Study 3 (analysis of calreticulin domains). Each rat had introduction of a 1.5-mm-diameter, 20-mm-long angioplasty balloon (USCI) retrograde, via femoral arteriotomy, to the iliac arterial ostium at the iliac bifurcation under general pentobarbital anesthetic (6.5 mg per 100 g weight, IM injection, Somnotrol, MTC Pharmaceuticals, Cambridge, Ontario). Infusions (1.0 mL volume) were given immediately before balloon inflation through the distal central lumen of the angioplasty balloon catheter into the arterial circulation, upstream of the injury site. After infusion of calreticulin and each of the other proteins and protein fragments tested, the balloon was positioned just below the iliac bifurcation across the left iliac and femoral artery branches and inflated to 4 bars pressure and then advanced and withdrawn 4 to 5 mm for three passes. The infusion doses for each study protocol were as follows: In Study 1, 0.01 mg of calreticulin was given to 5 rats and saline was given to 5 rats (Fig 2a
, 2b).
In Study 2, doses of 0.0001 to 0.1 mg of calreticulin were given to 12
rats (0.0001 to 2 rats, 0.001 to 2 rats, 0.01 mg to 2 rats, and 0.1 mg
to 6 rats), and doses of 0.0001 to 0.1 mg of calsequestrin were given
to 11 rats (0.0001 to 2 rats, 0.001 to 2 rats, 0.01mg to 2 rats, and
0.1mg to 5 rats). Saline control infusions were given to 13 rats (Figs 2a, 3a, and 3b). In Study 3, 0.1 mg of C (9 rats), N (4 rats), P (4
rats), or combined N and P (4 rats) domains as well as human (4 rats)
calreticulin were given (Figs 3c, 3d and
Fig 4). Higher doses of 1.0 mg of C
domain (5 rats), combined N and P domains (4 rats), or saline (5 rats)
were later given to test for effects of higher doses of the individual
calreticulin domains on intimal hyperplasia in Study 3. The five
saline-treated rats from Study 3 were added to the data from the
control saline rats for the titration curve (Fig 3a and 3b) in Study 2.
After infusion and angioplasty, the femoral artery was tied off with
3-0 silk (Study 1), or the arteriotomy site was sealed with local
application of n-butyl cyanoacrylate monomer (Nexaband, Veterinary
Products Laboratories, Phoenix, Arizona) (Studies 2 and 3). Each
rat was maintained on a normal rat diet and was followed for 4 weeks
post surgery until sacrifice with 1.0 mL of euthanyl per kilogram. The
research protocols and animal care conform to the Guiding Principles
for Animal Experimentation of the Canadian Council on Animal Care.
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In addition, in Study 3, three arterial sections from each
of six rats had infusion of either calreticulin or saline, immediately
before balloon angioplasty as described above, again under general
anesthetic. These rats were sacrificed early at 24 hours after balloon
injury to examine the effects of the infusions on leukocyte invasion
(macrophage and T cell) and ICAM expression by
immunohistochemical analysis (Figs 6 and 7).
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Calreticulin: Source and Purification
Calreticulin (rabbit and human) was purified by the ammonium
sulfate precipitation procedures as described
earlier.26,29 Canine cardiac calsequestrin was
prepared from whole tissue homogenates in the presence of
protease inhibitors by a previously described
procedure.26,40 Recombinant full-length
calreticulin was expressed in Escherichia coli as GST fusion
proteins and purified.26,29 The following domains
of calreticulin and recombinant GST were expressed in E.
coli and purified: N-domain (amino acid residues 1 to 182), the
P-domain (amino acid residues 139 to 273), N and P-domain (amino acids
1 to 273), and the C-domain (amino acid residues 270 to
401).26 Protein was determined by the method of
Lowry et al or Bradford as previously
described.26,29
Histology, Immunohistochemistry, and Morphometric Analysis
Each specimen was fixed in 10% sodium phosphate–buffered
formalin, processed, impregnated, and embedded in paraffin and cut into
5-µm sections by microtome as previously
described.42 The iliac and femoral
arterial branches were removed and divided into three
sections (proximal, mid, and distal) and stained with hematoxylin and
eosin for light microscopic and morphometric examination. The section
with the largest detectable area of atherosclerotic plaque was outlined
by using a Nikon Optiphot drawing device attachment connected to a
Nikon Model Labophot-2 light microscope (Nikon, Nippon Kogaku K.K.,
Tokyo, Japan). Plaque area and thickness were measured by using a
Jandel Scientific Sigma Scan program and Summagraphics digitizing Summa
sketch pad coupled to a Mac IIcx computer.42 More
recent specimens were measured with a Sony Power HAD 3CCD color
videocamera attached to a Zeiss Axioskop connected to the Empix
Northern Eclipse trace application program (Empix Imaging Inc.,
Mississauga, Ontario). Each system was calibrated to the microscopic
objective used. To avoid variability or differences inherent in
individual morphometric systems, only one system was used for each
series of measurements made in a study. All histologic sections were
independently assessed by a veterinary pathologist.
Macrophage, smooth muscle cell, T cell populations, and intercellular adhesion molecule (ICAM) in the intimal and medial layers of the arterial wall at 24 hours (6 rats) and 4 weeks (20 rats) after balloon injury and peptide infusions were examined by immunohistochemical staining of adjacent formalin-fixed, paraffin-embedded sections taken from the balloon injury sites as has been previously described.43 In brief, 5-µm tissue sections were incubated with primary antibodies specific for each cell type to be examined or ICAM and then immunostained by using the indirect peroxidase-labeled antibody technique.44,45 The primary antibody for arterial smooth muscle cells was mouse monoclonal anti-alpha smooth muscle actin, diluted 1/400 (Sigma, St. Louis, Missouri, USA). For macrophage and ICAM staining, mouse monoclonal anti-rat macrophage and anti-rat CD54 antibodies, respectively, were used at 1/200 dilutions as primary antibody (Pharmingen, Mississauga, Ontario). For T cell staining, mouse monoclonal anti-rat thymocyte and T lymphocyte antibody, diluted 1/200 (Vector Laboratories, California) was used as primary antibody. After incubation with primary antibody, sections were incubated for 30 minutes with biotinylated anti-mouse IgG-diluted 1/200 (Vector Laboratories California) and avidin-biotin-peroxidase complex for 40 minutes and developed with 3'3-diaminobenzidine for 5 minutes. Control stains lacking the primary or secondary antibodies or using irrelevant primary antibody (IgG1 antibody to the cytoplasmic tail of vesicular stomatitis virus G protein from Dr. T. Kresi) were performed for each new antibody examined.
Whole Blood Clotting Time and Platelet Activation
Assays
Whole blood from five rats and four human samples was
collected into one-tenth volume 3.8% trisodium citrate; 25 µL of
citrated whole blood was mixed in the cuvette used for the clotting
assay (WBC) with 25 µL of tris-buffered saline (0.14 M NaCl, 0.01
mol/L tris, pH 7.2), 5 µL of bovine type I acid soluble
collagen (Helena Laboratories, Beaumont, Texas), 5 µL of
calreticulin, calreticulin domains (N, P, or C), or calsequestrin (14
µg/mL, 22 µg/mL, 54 µg/mL, 129
µg/mL, or 244 µg/mL). The reactants were incubated at
37°C for 60 seconds. The ST4 coagulation analyzer
(Diagnostica Stago, Asnieres, France) was used to monitor
whole blood clotting. Ca2+-dependent WBC time was
titrated by the addition of calcium at concentrations of 1.5 to
58.9 mmol/L to reaction mixtures treated with calreticulin
or calsequestrin. For the platelet-free plasma studies, whole human
blood was spun at 3000g, and the supernatant
platelet-poor plasma was removed and passed through a 0.2-µm
filter to remove residual platelets. A separate aliquot of whole
blood was spun at 200g, and the resulting platelet-rich
plasma and buffy coat layer were removed. The packed red cells from
this second tube were then resuspended in an equivalent volume of
platelet-poor plasma.
Platelet activation was assessed by measuring ATP release after activation on vWf-coated beads.46 Platelet-rich plasma (PRP) prepared from citrate-buffered whole blood from four normal donors was incubated with calreticulin or calsequestrin. Parallel PRP plasma specimens were assessed in the absence of either protein. Platelet counts were adjusted to 200 to 300x 109/L with autologous plasma. ATP release was measured as previously described.46 B5m polystyrene beads (100=B5L) were coated with pure vWf under alkaline conditions to approximately 1 U of vWf per 108 beads (a unit of vWf is defined as the amount found in 1 mL of pooled normal plasma). One hour before testing, the beads were blocked with 1% bovine serum albumin (Sigma) in normal saline. Blocking solution was removed by centrifugation, and the beads were resuspended to the original volume. Calreticulin or calsequestrin diluted in saline was added to PRP and incubated at 37°C for 2 minutes. Ten B5L vWf beads prepared as above and 15 mL of B5L chronolume reagent were added to the cuvettes, and the suspension was stirred at 500 rpm. The response was monitored for 15 minutes or until aggregation and/or ATP release had plateaued (expressed as pmol per 108 platelets). Platelet response to the vWf beads was monitored by using a Chronolog 560 VS lumi-aggregometer linked to a model 810 Aggro/Link data reduction system. ATP release was quantitated using a luciferin/luciferase assay kit (Chrono-Log Corp, Havertown, Pennsylvania), standardized to known ATP standards.
Monocyte Migration Through Modified Boyden Chambers
Blood from three normal donors was drawn into 2 x 5 mL
EDTA vacutainer tubes (Collaborative Biomedical Products, Bedford,
Massachusetts). Blood was diluted 1:1 in PBS and layered over 15 mL of
Ficoll (Pharmacia Biotech, Uppsala, Sweden) in a 50-mL
polycarbonate conical centrifuge tube (Fisher, Edmonton) and
spun for 15 minutes at 2200 rpm (880g) in a Sorvall H1000-8
rotor in a Sorvall Instruments GLC-4 general laboratory
centrifuge at room temperature (brake off). Mononuclear cells
were collected from the interface and washed three times in 15 mL of
PBS in a 15-mL polystyrene conical centrifuge tube at 1850 rpm
for 10 minutes at 4°C (720g) and resuspended in
unsupplemented DMEM. Cells (3x205) in 200 µL of medium
were preincubated with calreticulin or calsequestrin for 15 minutes on
ice and placed in the upper well of transwell plates (Costar) with
8-µm pores, thin coated with 1/50 DMEM diluted matrigel
(Collaborative Biomedical Products) and incubated for 2 hours at
37°C in a 5% CO2 incubator. Media were
aspirated out of the upper well, and cells on the upper filter were
scraped off the surface.47 Filters were then
fixed for 15 minutes in 3% glutaraldehyde
(Polysciences), incubated for 3 minutes in 0.5% Triton X-100, stained
for 15 minutes in Gill's Hematoxylin number 2, and washed three times
in deionized H2O. Monocytes were counted in 10
high-power fields and averaged; three wells were used for each
condition.
Statistics
Correlations between the measured plaque area with infusion of
calreticulin, domains, calsequestrin, or saline were assessed both by
ANOVA and Student's t test. The mean value for all measured
plaque areas in arterial specimens from one experimental
animal was used to determine the significance of any differences after
treatment with each of the test substances.
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Results |
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Reduced Intimal Hyperplasia After Calreticulin Infusion at Sites of Balloon Injury
Of the 18 saline infused control rats (Studies 1 to 3), all had either intimal proliferation, macrophage infiltration, or collateral development 4 weeks after injury (Fig 2a
). Areas of plaque
development were characterized by cellular proliferation and fibrosis
with varying degrees of lumen obstruction (Fig 2a). Macrophage
infiltration was seen in some specimens, but there was no apparent
fatty, thrombotic, or complex plaque development. One specimen had
minimal intimal hyperplasia but aneurysmal dilatation with
macrophage infiltration and collateral development. The
contralateral arteries that did not have surgical intervention were
normal in all but one rat (Fig 2b). Ten of the 21 calreticulin-treated
rats had plaque development after balloon injury (P<.001).
Calreticulin significantly inhibited intimal hyperplasia in rat
iliofemoral arterial branches (Fig 2c).
Dose-dependent inhibition of intima hyperplasia was also investigated.
Histologic specimens had graded decreases in the measured plaque area
and thickness at 0.001- to 0.1-mg doses of calreticulin (Fig 3a, 3b)
but minimal decrease to no decrease in plaque growth after 0.0001-mg
dose infusion (Fig 3a, 3b), respectively. Plaque growth was not seen in
histologic sections taken from rats that received infusions of human
calreticulin (Fig 3c, 3d) and was comparable to the reduction seen with
rabbit calreticulin (P<.0004 on comparison with saline
infusion) (0.1-mg dose in Fig 3a, 3b).
Calsequestrin, a Ca2+-binding protein that has
physiochemical properties and intracellular localization similar to
those of calreticulin,47 was infused into the
femoral artery to assess the capacity of a similar
Ca2+-binding protein for prevention of plaque
growth after balloon injury. Calsequestrin has twice as high a capacity
for Ca2+ binding as calreticulin has, and at the
doses given, it has comparable or greater
Ca2+-binding properties in comparison with
calreticulin. The plaque-inhibitory activity of
calreticulin was not reproduced by calsequestrin (Figs 2d, 3a, 3b) at
any of the infusion doses given, 0.0001 to 0. 1 mg (Figs 2d, 3a, 3b;
P<.03 by ANOVA). The plaque that developed in the
calsequestrin-treated rats was similar to that seen with low-dose
calreticulin or saline infusion.
Analysis of the Calreticulin Domains for
Antiproliferative Activity
To assess which individual domain of calreticulin has
antiproliferative activity, the N, P, and C domains of the protein were
expressed, isolated, and individually infused after injury. Infusion of
the purified C domain of calreticulin, but not the N or P domains,
decreased the measured plaque area and thickness (Figs 3c, 3d). Two of
nine of the specimens taken from rats that had C domain infusion had
evidence of intimal hyperplasia, while 14 of 16 of P, N, or combined
N+P domain–infused rats had evidence of plaque growth. Intimal
hyperplasia was significantly reduced after C domain infusion (Fig 4a) in comparison with the saline and N
domain (Fig 4b) or P domain infusions (Fig 4c). Human and rabbit
calreticulin and C domain infusions significantly reduced plaque on
comparison with the N or the P domains and on comparison with the
saline controls (P<.0001) at 0.1-mg infusion dose. To
assess a possible titrable effect of higher-dose N or P domain
infusions, the effects of 1.0-mg infusions of N+P domain fragments were
compared with those of 1.0-mg infusions of calreticulin. Again there
was a significant reduction in plaque area with C domain infusion in
comparison with N+P domain infusion (mean plaque area for N+P domain
infusion: 0.035±0.012, that for C domain infusion: 0.001±0.001;
P<.03). No significant difference was detectable in a
comparison of rabbit (Figs 3c, 3d) and human (Figs 3c, 3d) calreticulin
infusion.
Effects of Calreticulin on Thrombosis
To examine a potential effect of calreticulin on
platelet-dependent clot formation, whole blood clotting times (WBC)
were measured after individual addition of calreticulin, C domain, N
and P domains, and calsequestrin. As was previously reported by
Kuwabara et al,24 there was no change in the PT
or PTT at 2 hours after calreticulin infusion (data not shown).
Calreticulin, however, prolonged WBC time significantly for both human
blood (Fig 5a) and rat blood (Fig 5b).
This inhibitory activity was also seen after addition of
the C domain but not the N or P domains both in human (Fig 5c) and rat
blood (Fig 5c).
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Clot formation was assessed after stimulation with thrombofax or activated partial thromboplastin in samples of platelet-free plasma after addition of calreticulin. Calreticulin had no effect on clotting time when mixed with platelet-poor plasma, but clotting time was again prolonged after addition of the platelets back to a red cell free and platelet-free plasma suspension. Optimum prolongation of clotting time was seen at 4.4 to 14.7 mmol/L Ca2+ concentrations in this assay, but the Ca2+ effects on prolongation of WBC time were equivalent for calreticulin and calsequestrin (data not shown).
A significant decrease in platelet activation as measured by ATP
release was also seen after addition of calreticulin and calsequestrin
to activated platelets in the presence of von
Willebrand's factor bead induced platelet activation (Fig 5d).48 There was, however, no detectable
significant difference in ATP release in this assay between
calreticulin and calsequestrin.
Effects of Calreticulin on Monocyte and T Cell
Migration
Mononuclear cell invasion into sites of intimal hyperplasia 4
weeks after balloon injury was reduced significantly (Figs 6a, 6b) after calreticulin infusion. A
reduction in detectable T cell, macrophage, and ICAM staining
was seen in the media of arterial sections 4 weeks after
treatment with calreticulin but not after treatment with saline or
combined N+P domain infusion. T cell and ICAM staining was also reduced
in the intima, but macrophage staining in the intima was not
significantly reduced (Fig 6a). Rats that were sacrificed at 24 hours
after balloon injury and calreticulin infusion demonstrated a
significant reduction in visible staining for macrophage (Fig 6c, Figs 7a, 7b) and T cell invasion into
the medial layer as well as ICAM (Figs 7c, 7d) staining in comparison
with sections treated with saline. Smooth muscle cell staining 24 hours
after arterial injury was not affected by calreticulin
infusion.
To determine whether calreticulin was exerting a direct effect on the
monocyte reactions to the balloon induced arterial injury
or whether the observed reduction in leukocyte invasion was a secondary
effect, the effect of calreticulin on purified
monocyte/macrophage migration was examined in a modified Boyden
chamber assay using matrigel-coated membranes.44
(Fig 6d). No significant difference in activity of cells treated with
saline, calreticulin, or calsequestrin was detectable, a finding
suggesting that the observed reductions in mononuclear cell staining in
the arterial wall after calreticulin infusion were not the
result of a direct inhibitory activity on monocyte
migration but rather secondary to some other inhibitory
activity. However, this result does not remove the possibility that the
observed inhibition of ICAM expression or T cell activation is the
result of a direct and specific effect of calreticulin on either T cell
activation or ICAM expression that in turn limits monocyte tissue
invasion.
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Discussion |
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In this study, we have demonstrated that calreticulin, a naturally occurring high-capacity Ca2+-binding protein, inhibits intimal hyperplasia at sites of balloon-induced endothelial injury in rat iliofemoral arteries. Reduced plaque growth was observed in the presence of calreticulin and the high-capacity Ca2+-binding C domain of calreticulin but not in the presence of either the N or the P domains of the protein. Calsequestrin, a related, high-capacity Ca2+-binding protein with similar physicochemical properties did not inhibit plaque growth, a result indicating that observed effects of calreticulin are not solely the result of Ca2+ chelation. Both calreticulin and calsequestrin prolonged WBC through platelet-dependent mechanisms and reduced platelet activation–associated release of ATP in vitro. We propose that the complementary effects of calreticulin on inhibition of platelet activation and plaque development are the result of local binding of Ca2+ and Ca2+-dependent clotting factors, factors IX and X and prothrombin. However, this mechanism can represent only a partial explanation for the decrease in intimal hyperplasia observed in these studies, as calsequestrin did not reduce plaque growth. Calreticulin did decrease the acute inflammatory cell responses in sections taken from the rat arterial wall both early and late after balloon injury but did not directly inhibit monocyte migration in vitro in a Boyden chamber assay. The precise role of these calreticulin associated antithrombotic and anti-inflammatory actions in vascular responses to injury has not yet been defined.
Calreticulin belongs to a family of KDEL resident proteins of the ER; therefore, this protein is not expected to be found outside of cells. However, Sueyoshi et al39 found that calreticulin was present at low levels in human plasma, despite the presence of the ER calreticulin retrieval sequence. There may therefore be physiological conditions in which calreticulin is secreted.48 In response to stress, ER luminal proteins are overexpressed and frequently found secreted from the cell.48,49 The presence of surface calreticulin has recently been reported;37,38 furthermore, it was reported that protein disulfide isomerase, another KDEL protein, is found on the cell surface and associated with platelets.48 We have recently shown that calreticulin and protein disulfide isomerase interact.50 It is conceivable that the two proteins may be secreted as a complex. Importantly, calreticulin can interact and bind to cell surface via its lectinlike activity.37,38 Local release of calreticulin may have synergistic effects with inhibition of platelet-dependent factors that initiate intimal hyperplasia and associated inhibition or activation of inflammatory responses to injury.
Other researchers have found that Ca2+ channel blockers decrease smooth muscle cellular proliferation in several rat models of hypertension and in vitamin D toxicosis.51 Furthermore, several clinical trials have suggested that Ca2+ channel blockers decrease the risk of recurrent ischemic episodes in acute ischemic syndromes, unstable angina, and acute myocardial infarction, in which the pulmonary pressure is not elevated.52 All this suggests that Ca2+ and Ca2+-binding proteins may play an important role in the control of these processes. However, the experimental results in rats have not translated into a clear-cut benefit in clinical studies in which follow-up quantitative angiography has been used to follow plaque growth with Ca2+ channel blocker therapy.53 Our results with both calreticulin and calsequestrin are consistent with a partially calcium-dependent inhibitory process. However, the fact that calsequestrin had no plaque-inhibitory efficacy at the highest infusion dose indicates that calreticulin reduces plaque by a calcium-independent mechanism as well.
Recently, Kuwabara et al24 showed that
calreticulin or the C domain of calreticulin has a potent
antithrombotic activity, in that it binds to vitamin K–dependent
coagulation factors, stimulates endothelial nitric
oxide production, and limits thrombosis in canine
coronary arteries. Furthermore, they showed that calreticulin
binds to endothelial cells with a high affinity
(Kd 7.37 nM).24 In
vivo clearance studies indicated that infused calreticulin became
rapidly associated with endothelial cells in the vessel
wall and was associated with altered nitric oxide
release.24 However, other researchers have
reported a nitric oxide–independent effect of calreticulin on
endothelial cell relaxation. Calreticulin is a
Ca2+-binding protein, and several steps in the
coagulation process require Ca2+ as a cofactor.
For example, the binding of the clotting factors IX and X and
prothrombin with Ca2+ on the platelet surface
is believed to accelerate the activation of platelets, thrombus
formation, and acute inflammatory responses to injury. As calreticulin
binds to the surface of endothelial
cells24 with a high affinity, it is conceivable
that the protein might also chelate Ca2+ locally
in the area of balloon injury and, as a consequence, displace
coagulation factors from the endothelial cell surface
membrane and alter their abilities to be activated by
Ca2+. This again indicates that the reduction in
plaque and the prolongation of clotting time after calreticulin
infusion is not based on a Ca2+-dependent process
alone, or on a mechanism based on inhibition of platelet activation
alone, but rather requires a precise localization of the protein to the
area of injury. Calsequestrin, although it binds large amounts of
Ca,2+ has not been demonstrated to bind to
vascular wall and therefore it is unlikely to produce the local effects
observed with calreticulin, in vivo. Furthermore, during injury to a
blood vessel, calreticulin may be released from the
endothelial cells or other cells. It will be important
to test whether there are detectable elevated levels of calreticulin in
the blood after vascular injury.
In arterial sections taken from the sites of balloon injury, a marked decrease in monocyte and T cell invasion with an associated reduction in ICAM staining was detected after calreticulin infusion in comparison with saline and combined N+P domain infusions. This reduction in inflammatory response was detectable both early (24 hours) and late (4 weeks) after injury and infusion of calreticulin. The reduced inflammatory cell response was accompanied by a reduction in detected ICAM stain in the medial layer. However, there was no direct inhibitory activity on monocyte migration or invasion through matrigel-coated filters. These results would suggest that the observed reduction in monocyte invasion at sites of injury is not directly altered by calreticulin. Rather, the decrease in cellular invasion at sites of arterial injury may well be the result of either a reduction in platelet activation and activation of the clotting cascade or a decrease in ICAM expression in damaged endothelium. Our own bias is that, in keeping with the known wide range of activities of calreticulin, the reduction in plaque growth seen in these experiments after calreticulin infusion may well be the result of inhibitory effects of calreticulin on both the thrombotic and the inflammatory responses to vascular injury.
In summary, we have documented a profound inhibitory effect of calreticulin on intimal hyperplasia in rat iliofemoral arteries after balloon injury in vivo. The C domain of calreticulin is a high capacity Ca2+-binding area of the protein that binds factors IX and X and prothrombin in the coagulation cascade. This plaque-inhibitory activity appears to be partially dependent on inhibition of Ca2+-dependent platelet activation and clot formation but cannot be entirely explained by a mechanism based on inhibition of platelet activation and thrombosis, as calsequestrin did not effectively reduce plaque growth. Calsequestrin also has no known effects on nitric oxide release or leukocyte adhesion, activation, and phagocytosis. We postulate that the antiatherosclerotic activity of calreticulin is the result of the multifunctional nature of calreticulin, which allows calreticulin to exert local effects at sites of endothelial injury through binding to the cell surface and synergistic inhibition of platelet activation and thrombosis, enhanced nitric oxide release, and as yet undefined effects on inflammation.
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Acknowledgments |
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E.D. is currently a research fellow and N.M. was a fellow of the Heart and Stroke Foundation of Canada for the duration of this work. M.M. is an MRC Scientist and Senior Scholar of the Alberta Heritage Foundation for Medical Research. A.L. was a Clinical Investigator for the Alberta Heritage Foundation for Medical Research for the duration of this work and is now a Scientist in the Vascular Biology group at the John P. Robarts Research Institute at the University of Western Ontario. This research was funded by grants from the Medical Research Council of Canada (M.M.) and the Alberta Heart and Stroke Foundation (M.M. and A.L.) as well as a grant from Spectral diagnostics (M.M. and A.L.). We would like to thank Dr Wei-dong Yan for his contributions to this study and his helpful discussions. We would also like to thank Fran Plumb for her help in typing this manuscript.
Received July 22, 1996; accepted August 27, 1997.
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