Antiangiogenic and antitumor effects of Trypanosoma cruzi Calreticulin.

Abstract

In Latin America, 18 million people are infected with Trypanosoma cruzi, the agent of Chagas' disease, with the greatest economic burden. Vertebrate calreticulins (CRT) are multifunctional, intra- and extracellular proteins. In the endoplasmic reticulum (ER) they bind calcium and act as chaperones. Since human CRT (HuCRT) is antiangiogenic and suppresses tumor growth, the presence of these functions in the parasite orthologue may have consequences in the host/parasite interaction. Previously, we have cloned and expressed T. cruzi calreticulin (TcCRT) and shown that TcCRT, translocated from the ER to the area of trypomastigote flagellum emergence, promotes infectivity, inactivates the complement system and inhibits angiogenesis in the chorioallantoid chicken egg membrane. Most likely, derived from these properties, TcCRT displays in vivo inhibitory effects against an experimental mammary tumor.
TcCRT (or its N-terminal vasostatin-like domain, N-TcCRT) a) Abrogates capillary growth in the ex vivo rat aortic ring assay, b) Inhibits capillary morphogenesis in a human umbilical vein endothelial cell (HUVEC) assay, c) Inhibits migration and proliferation of HUVECs and the human endothelial cell line Eahy926. In these assays TcCRT was more effective, in molar terms, than HuCRT: d) In confocal microscopy, live HUVECs and EAhy926 cells, are recognized by FITC-TcCRT, followed by its internalization and accumulation around the host cell nuclei, a phenomenon that is abrogated by Fucoidin, a specific scavenger receptor ligand and, e) Inhibits in vivo the growth of the murine mammary TA3 MTXR tumor cell line.
We describe herein antiangiogenic and antitumor properties of a parasite chaperone molecule, specifically TcCRT. Perhaps, by virtue of its capacity to inhibit angiogenesis (and the complement system), TcCRT is anti-inflammatory, thus impairing the antiparasite immune response. The TcCRT antiangiogenic effect could also explain, at least partially, the in vivo antitumor effects reported herein and the reports proposing antitumor properties for T. cruzi infection.

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Antiangiogenic and Antitumor Effects of Trypanosoma
cruzi Calreticulin
Nandy C. Lo ´pez1, Carolina Valck1, Galia Ramı ´rez1, Margarita Rodrı ´guez1, Carolina Ribeiro1, Juana
Orellana1, Ismael Maldonado1, Adriana Albini2, Daniel Anacona1, David Lemus1, Lorena Aguilar1,
Wilhelm Schwaeble3, Arturo Ferreira1*
1Institute of Biomedical Sciences, Faculty of Medicine, University of Chile, Santiago, Chile, 2Oncology Research, Science and Technology Pole, IRCCS Multimedica, Milan,
Italy, 3Department of Infection, Immunity and Inflammation, University of Leicester, Leicester, United Kingdom
Abstract
Background: In Latin America, 18 million people are infected with Trypanosoma cruzi, the agent of Chagas’ disease, with the
greatest economic burden. Vertebrate calreticulins (CRT) are multifunctional, intra- and extracellular proteins. In the
endoplasmic reticulum (ER) they bind calcium and act as chaperones. Since human CRT (HuCRT) is antiangiogenic and
suppresses tumor growth, the presence of these functions in the parasite orthologue may have consequences in the host/
parasite interaction. Previously, we have cloned and expressed T. cruzi calreticulin (TcCRT) and shown that TcCRT,
translocated from the ER to the area of trypomastigote flagellum emergence, promotes infectivity, inactivates the
complement system and inhibits angiogenesis in the chorioallantoid chicken egg membrane. Most likely, derived from
these properties, TcCRT displays in vivo inhibitory effects against an experimental mammary tumor.
Methodology and Principal Findings: TcCRT (or its N-terminal vasostatin-like domain, N-TcCRT) a) Abrogates capillary
growth in the ex vivo rat aortic ring assay, b) Inhibits capillary morphogenesis in a human umbilical vein endothelial cell
(HUVEC) assay, c) Inhibits migration and proliferation of HUVECs and the human endothelial cell line Eahy926. In these
assays TcCRT was more effective, in molar terms, than HuCRT: d) In confocal microscopy, live HUVECs and EAhy926 cells, are
recognized by FITC-TcCRT, followed by its internalization and accumulation around the host cell nuclei, a phenomenon that
is abrogated by Fucoidin, a specific scavenger receptor ligand and, e) Inhibits in vivo the growth of the murine mammary
TA3 MTXR tumor cell line.
Conclusions/Significance: We describe herein antiangiogenic and antitumor properties of a parasite chaperone molecule,
specifically TcCRT. Perhaps, by virtue of its capacity to inhibit angiogenesis (and the complement system), TcCRT is anti-
inflammatory, thus impairing the antiparasite immune response. The TcCRT antiangiogenic effect could also explain, at least
partially, the in vivo antitumor effects reported herein and the reports proposing antitumor properties for T. cruzi infection.
Citation: Lo ´pez NC, Valck C, Ramı ´rez G, Rodrı ´guez M, Ribeiro C, et al. (2010) Antiangiogenic and Antitumor Effects of Trypanosoma cruzi Calreticulin. PLoS Negl
Trop Dis 4(7): e730. doi:10.1371/journal.pntd.0000730
Editor: Ana Rodriguez, New York University School of Medicine, United States of America
Received August 4, 2009; Accepted May 11, 2010; Published July 6, 2010
Copyright: ? 2010 Lo ´pez et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This research was financed by the Bicentennial Research Project ACT 29-Chile, Bicentennial Network Research Project 07-Chile, FONDECYT-Chile
1095095 and the Associazione Italiana per la Ricerca sul Cancro (AIRC). The funders had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: aferreir@med.uchile.cl
Introduction
Chagas9 disease affects 16 million people in South America,
with 14.000 deaths per year and 0.7 million disability-adjusted life-
years [1]. T. cruzi has a variety of molecules that modulate several
effector arms of the immune system [2], calreticulin (TcCRT)
being one of them [3]. TcCRT, first isolated in our laboratory
[4,5], is highly homologous with human calreticulin (HuCRT) [6],
an exceedingly pleiotropic chaperone molecule [7]. In spite of its
primary endoplasmic reticulum (ER) location, TcCRT is also
expressed on the cell membrane [3].
Based on their capacity to bind laminin [8] and to inhibit
endothelial cell proliferation, both HuCRT and its N-terminal
fragment, vasostatin or N-TcCRT, display antiangiogenic prop-
erties in vitro and in vivo [9,10]. These HuCRT properties are
paralleled by inhibitory activities on several tumor models [11–
13]. Identifying these properties in TcCRT may define important
aspects of the host/parasite interaction.
We have recently reported that TcCRT is strongly antiangio-
genic in the chorioallantoid membrane in chicken eggs (CAM
assay) [14]. Since angiogenesis modulators behave differently
across species [15], we verified this effect in different experimental
set ups in mammals, Homo sapiens sapiens included. Thus, TcCRT
and its vasostatin-like domain, inhibit angiogenesis in the ex vivo rat
aortic ring assay. It also affects key cellular angiogenic parameters
in human endothelial cell cultures, such as proliferation,
chemotaxis and cell morphogenesis into tubular-like structures in
Matrigel. These results correlate with TcCRT binding and
internalization in these cells. Perhaps, the TcCRT antiangiogenic
(and anti-complement) properties result in anti inflammatory
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outcomes, thus inhibiting the host antiparasite immune response.
Also, at least a partial explanation for those reports [16,17]
proposing anti-tumor effects for trypanosome infection is herein
provided. Although anti-tumor effects have been reported for
several decades now, for a variety of infections with other
microbial agents [18,19], pathogen molecules mediating those
statistically based tumor resistances, have been poorly defined. In
synthesis, here we describe that a parasite chaperone molecule,
most likely by interacting with endothelial cells, and inhibiting
angiogenesis, interferes with tumor growth.
Methods
Ethics statement
Human umbilical vein endothelial cells (HUVECs) were
isolated [20], following informed patient’s written consent
(University of Chile Hospital Bioethics Committee).
Cells
The human endothelial EAhy926 cell line (kindly provided by
Dr. Gareth Owen, Pontifical Catholic University, Chile), was
maintained in Iscove’s Modified Dulbecco’s Medium (IMDM,
Invitrogen, USA) with 10% fetal bovine serum (FBS, Invitrogen,
USA) and 100 units/ml penicillin/streptomycin (Sigma, USA).
HUVECs were 80% pure by flow cytometry and immunofluores-
cence using anti CD31 monoclonal antibodies (Sigma, USA) as a
marker. The cells were cultured in M199 medium (Sigma, USA),
with 20% FBS, 2 mM glutamine (Invitrogen, USA), 100 units/ml
penicillin/streptomycin, 100 mg/ml endothelial cell growth sup-
plement (ECGS) (BD Biosciences, USA), and 10 mg/ml heparin
(Sigma, USA) in gelatin-coated flasks.
Recombinant proteins
TcCRT, its R-domain (R-TcCRT) and HuCRT were obtained
from E. coli [3,21]. N-TcCRT (amino acids 20–193, GenBank
accession no. AF162779) was amplified by PCR using Tli DNA
polymerase (Promega, USA). Primers were: (59-GGAATTC-
CACGGTGTACTTCCACGAG-39) and (59- CTCGAGCCAG-
TCTTCTTCGAGCTG-39).
N-TcCRT DNA was ligated into the EcoRI and XhoI sites of
the pET-28b (+) plasmid (Novagen, UK). Competent E. coli
TOP10F9 bacteria were transformed, plated and selected with
50 mg/ml ampicillin. E. coli BL21 (DE3)pLysS was transformed
with the plasmid and grown in the presence of 34 mg/ml
chloramphenicol with 50 mg/ml kanamycin. After adding isopro-
pyl b-D-thiogalactoside and 3 h incubation, the cells were
sonicated, centrifuged, and the supernatants filtered. The
recombinant proteins were purified using His Bind resin (Nova-
gen, UK), eluted with buffer containing 1 M imidazole, and
dialyzed against 2 mM Tris-HCl and 150 mM NaCl, pH 7.4.
Both, TcCRT and N-TcCRT were tested for endotoxin by the
Limulus Amebocyte Lysate Kinetic-QCL assay (BioWhittaker,
USA) and contained ,5 EU/10 mg protein.
The R-TcCRT domain (aa 136–281) was expressed and
purified as previously described [3].
Rat aortic ring assay
This ex vivo angiogenesis assay [22], was performed with slight
modifications. Six week old Sprague-Dawley rats, from our
Animal Facility were used in this experiment. Briefly, the animals
were sacrificed by CO2inhalation, their thoracic aortas dissected
and sliced into 1 mm thick rings. Two or three rings per well were
placed on a 24-well plate and embedded in 100 ml Matrigel (BD
Biosciences, USA), followed by 30 min incubation. Wells were
overlaid with 300 ml of FBS-supplemented M199 medium with
100 mg/ml ECGS and phosphate buffered saline (PBS) or several
TcCRT concentrations. The rings were incubated for 7 days and
visualized under phase contrast in a Nikon Eclipse E400
microscope. Fields were photographed and the length of capillaries
measured using Adobe Photoshop software. For each experiment
and in sextuplicate, 3 capillaries (shortest, medium and longest) per
ring were measured. The average length was considered as 100%.
The statistical validation of these experiments was defined by the
Student’s t-test.
Matrigel morphogenesis assay
24-microwell plates were filled with 300 ml Matrigel/well and
polymerized for 1 h at 37uC. 706103HUVECs/well were
suspended in FBS-supplemented M199 medium, with 100 mg/
ml ECGS and several TcCRT, N-TcCRT, lypopolisaccharide
(LPS), HuCRT or R-TcCRT concentrations. The cells were
layered on the gel. After 6 h incubation, morphogenesis was
assessed by phase contrast microscopy and images were imported
into the Adobe Photoshop program. Tubular capillary-like
structures were quantified by manual counting in 406 fields, in
quadruplicates, as previously described [23]. Data were analyzed
by one way ANOVA. Values are reported as means 6 SEM.
Comparison of means was performed by the Bonferroni method.
Chemotaxis assay
With HUVECs, the assays were performed in Boyden
chambers, while Transwell chambers (Costar, USA) were used
with EAhy926 cells [24]. HUVECs were pretreated for 24 h with
PBS, LPS, or variable TcCRT concentrations in FBS-supple-
mented M199 medium. EAhy926 cells were pretreated with
IMDM containing several TcCRT concentrations. 7.56104
HUVECs or 56104EAhy926 cells/chamber were washed,
resuspended in serum-free medium, and placed in the upper
compartment, with or without TcCRT or LPS. Supernatants from
NIH3T3 cells (for HUVECs) or 10% FBS (for EAhy926) were
Author Summary
In Latin America, 18 million people are infected with
Trypanosoma cruzi, a protozoan that causes Chagas’
disease. Vertebrate calreticulins (CRTs) are multifunctional,
intra- and extracellular calcium binding, chaperone pro-
teins. Since human CRT (HuCRT) inhibits capillary growth
(angiogenesis) and suppresses tumor growth, the pres-
ence of these functions in T. cruzi CRT (TcCRT) may have
interesting consequences in the host/parasite interactions.
Previously, we have cloned and expressed TcCRT and
shown that, when translocated from the endoplasmic
reticulum to the area of trypomastigote flagellum emer-
gence, it promotes infectivity, inactivates the complement
system, an innate defense arm and inhibits angiogenesis in
the chorioallantoid chicken egg membrane. TcCRT inhibits
angiogenesis, since it interferes with endothelial cell
multiplication, migration and capillary morphogenesis in
vitro, as well as angiogenesis in rat aortic rings. The
parasite molecule also displays important antitumor
effects. In these activities, TcCRT is more effective than
the human counterpart. Perhaps, by virtue of its capacity
to inhibit angiogenesis, TcCRT is anti-inflammatory, thus
impairing the antiparasite immune response. The TcCRT
antiangiogenic effect could also explain, at least partially,
the in vivo antitumor effects reported herein and the
reports proposing antitumor properties for T. cruzi
infection.
TcCRT in Angiogenesis and Tumor Growth
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used as chemo attractants in the lower chamber. After 6 h
(HUVECs) or 16 h (EAhy926) incubation, the cells on the upper
filter surface were removed, and those on the lower surface, fixed
and stained. Filters were photographed with CCD optics and a
digital analysis system (Image ProPlus, Media Cybernetics, Silver
Spring, MD) and nine fields per filter were counted (HUVECs).
EAhy926 cell migration was measured by densitometry analysis at
595 nm. All experiments were performed in triplicates. Data were
analyzed by one way ANOVA. Values are reported as means 6
SEM. Comparison of means was performed by the Bonferroni
method.
Proliferation assays
These assays were quantified using MTT (3-[4,5-dimethylthia-
zol-2-yl]2,5-diphenyltetrazoliumbromide, Calbiochem, USA) or
crystal violet reagents. Briefly, in the MTT assay, 2,500
HUVECs/well were seeded in sestuplicate in 96-well plate and
growth, in the presence of various TcCRT, N-TcCRT or HuCRT
concentrations, was assessed at 24-h periods over 4 days. Then,
MTT was added, incubated for 4.5 h, solubilized in DMSO and
the absorbance was read at 550 nm. The same assay was
performed with 2,000 VERO cells, as a negative control showing
that recombinant TcCRT did not affect the in vitro growth of an
unrelated cell line. Data were analyzed by one way ANOVA,
followed by the Bonferroni test. Values are reported as means 6
SEM. In the crystal violet assay, the same number of HUVECs
were seeded in gelatin-coated wells and treated with R-TcCRT at
different concentrations. The number of viable cells was measured
over time with the crystal violet reagent, following standard
procedures.
Protein binding and internalization assays
TcCRT was labeled with the FluoReporter FITC Protein
Labeling Kit (Molecular Probes, USA). HUVECs or EAhy926
cells were incubated with 1 mM TcCRT, FITC-TcCRT or FITC-
TcCRT plus 10 mM unlabelled TcCRT, for 1 h. After washing,
the cells were fixed with 4% paraformaldehyde, for 15 min at
room temperature, washed and mounted in 50% glycerol,
containing 49-6-diamidino-2-phenylindole (DAPI). Slides were
visualized in a Nikon Eclipse E400 epifluorescence microscope.
Protein uptake was detected by incubating the cells for 30 min, in
medium containing 1 mM FITC-TcCRT, alone or in the presence
of 25 mg/ml fucoidin (Sigma, USA). Images were collected using
the LSM510 Software system attached to a Zeiss (Oberkochen,
Germany) LSM510meta confocal microscope.
Tumor growth assay
The TcCRT and HuCRT effects on in vivo growth of the TA3
MTXR murine mammary tumor cell line was assessed in 2
independent experiments, performed 6 months apart, in adult
female A/J mice. Four animals were used in the first experiment
and 6 in the second one. In both experiments, the animals were
inoculated s.c., every other day, with 50 mg TcCRT or HuCRT or
solvent, during 25 days. At day 0, the animals were challenged
with 56105tumor cells. Tumor size was determined with a digital
caliper (Mitutoyo Corp, Japan), in a double blind procedure, as
previously described [25]. The experiments were validated by
using the Wilcoxon Signed Rank test (GraphPad Prism 4). P
values#0.05 were considered as statistically significant.
Animal welfare
Six week old New Zealand rats and adult (20–25 g) female A/J
mice were obtained from our Central Animal Facility. Experi-
ments were performed in compliance with the ‘‘Guide for the Care
and Use of Laboratory Animals’’, National Research Council,
Washington DC, USA, 2002. All procedures with these animals
were approved by the local Bioethics Committee (Bioethics
Committee, Faculty of Medicine, University of Chile). Surgeries
and sacrifices were performed by the Animal Facility Veterinary
Surgeons.
Results
TcCRT inhibits angiogenesis in the rat aortic ring assay
Two representative experiments are shown in Figure 1, A–B.
Micro vessels are observed after culturing the aortic rings for 1
week (Figure 1A, control). Incubation with 1 mM TcCRT
mediated complete capillary growth abrogation (Figure 1A,
TcCRT). A dose-dependent antiangiogenic effect is observed
(Figure 1B), until reaching complete capillary growth arrest. In
Figure 1C, quantification of this TcCRT inhibitory capacity is
shown. At concentrations of 0.1 and 1.0 mM, about 50% and
100% inhibition is respectively observed. In separate experiments,
the vasostatin like N-TcCRT also inhibits angiogenesis in this ex
vivo experimental model (data not shown).
TcCRT and its N-TcCRT inhibit endothelial cell capillary
morphogenesis
A set of representative experiments is shown in Figure 2. In a 5-
hour culture, control non-treated HUVECs generated a typical
cell network (Figure 2A). Although strong inhibitory effects were
observed with 1 mM HuCRT (Figure 2B), when N-TcCRT
(Figure 2C) and TcCRT (Figure 2D) were compared at equal
molarities with HuCRT, the effects of the parasite–derived
molecules were clearly stronger than those of the human
counterpart. Figure 2E shows the quantification of these assays.
The TcCRT inhibitory effect was dose-dependent down to
0.1 mM (data not shown), while R-TcCRT did not affect capillary
morphogenesis (Figure 2F–H).
TcCRT inhibits endothelial cell migration
HUVECs migration, as a response to the strong angiogenic
factors present in NIH/3T3 cell conditioned media, was inhibited
in a dose-dependent manner by TcCRT. LPS, at concentrations
similar to those present in the TcCRT 1 mM preparation, showed
no detectable effects (Figure 3A). Treatment with TcCRT also
significantly inhibited migration of Eahy926 cells in response to
FBS, over the same dose range (Figure 3B).
TcCRT and N-TcCRT inhibit HUVEC proliferation
Figure 4 summarize these experiments. TcCRT inhibited
endothelial cell proliferation in a dose-dependent manner, when
they were stimulated with ECGS (Figure 4A). Maximum
inhibition (60%) was observed with 1 mM TcCRT, at 96 hours
(Figure 4B). A similar activity was also observed when TcCRT or
N-TcCRT were added to HUVECs stimulated with basic
fibroblast growth factor (bFGF) (Figure 4C). R-TcCRT, up to
1 mM, had no significant effects on HUVECs proliferation
(Figure 4D). TcCRT did not affect VERO cell proliferation
(Figure 4E), used as negative control.
TcCRT binds to human endothelial cells and is
internalized
Although both HuCRT [8] and TcCRT bind to laminin, only
the former interferes with the adhesion of endothelial cells to this
molecule (data not shown). Therefore, the TcCRT antiangiogenic
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effect may be explained by other mechanisms, such as direct
interaction with endothelial cells. FITC-TcCRT binds to live
HUVECs (Figure 5C). This binding is reversed by a molar excess
of the unlabeled protein (Figure 5D). Given the similarity between
the DAPI and FITC-TcCRT mediated signals in this experiment
(Figure 5C, merge), confocal microscopy was used to test if
TcCRT was internalized after binding to the cell surface. After
30 min incubation, TcCRT accumulates around the HUVECs
nuclei, in punctuate structures (Figure 5E), a phenomenon also
observed in EAhy926 endothelial cells (data not shown). In order
Figure 2. TcCRT and N-TcCRT inhibit capillary morphogenesis.
Phase contrast images of HUVECs organization in the Matrigel
morphogenesis assay are shown. Cells were cultured on the surface
of Matrigel and incubated with: A and F. PBS (control), B. HuCRT, C.
TcCRT, D. N-TcCRT and G. R-TcCRT, all of them at 1mM, for 6 h at 37uC,
5% CO2. E and H. Tubular structures were quantified by counting at low
power fields. Data are represented as means 6 SEM, obtained from four
fields. **, p,0.01. Results are representative of 3 independent
experiments. Original magnification, 610.
doi:10.1371/journal.pntd.0000730.g002
Figure 1. TcCRT inhibits angiogenesis in an ex vivo assay. Aortic
rings were embedded in Matrigel and incubated in supplemented
media at 37uC, 5% CO2, for 7 days. A. Representative images of aortic
rings normal capillary sprouting (control) and in response to 1 mM
TcCRT. B. Dose-dependent TcCRT inhibitory effect on angiogenesis. C.
Quantitative analysis of the inner ring vessel length shown in B. Data
are shown as means 6 SEM, obtained from individual rings and are
representative of at least 3 rings in each experiment and two
independentexperiments. *, p,0.05. **, p,0.01. Original magnification,
64.
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to better substantiate the TcCRT internalization by endothelial
cells, an enlargement of a representative cell is shown (extreme
right panel in Figure 5E). TcCRT internalization seems to be
receptor-dependent, since fucoidin, a specific scavenger receptor
ligand [26,27], abrogated TcCRT uptake (Figure 5F).
TcCRT inhibits the growth of a murine mammary tumor
The TcCRT and HuCRT effects on the in vivo growth of the
TA3 MTXR murine A/J mammary tumor cell line was assessed
in adult mice, in two independent experiments, performed 6
months apart (Figure 6A–B). Under the experimental conditions
used, only the parasite chaperone molecule displayed significant
(p=0.0078) inhibitory effects on this tumor cell line, in both cases
(Figure 6A–B). In one experiment (Figure 6A), TcCRT displayed a
stronger antitumor effect, than the human orthologue (p=0.0078
vs p=0.1094). In the second experiment, HuCRT also had an
effect (Figure 6B, p=0.0078). However, again TcCRT had a
stronger antitumor effect than HuCRT (p=0.0078) (Figure 6B).
Discussion
We have shown that TcCRT strongly inhibits capillary growth
in the CAM in vivo assay [14]. Since angiogenesis modulators
behave differently, not only across species, but also depending on
the assay used [15], we studied the TcCRT antiangiogenic
properties in the rat, a natural T. cruzi host. The ex vivo rat aortic
ring assay provides a model closer to the physiologic in vivo
situation, since endothelial cells are in a quiescent state, in a
natural histological environment. In this assay, TcCRT completely
abrogates capillary growth, in a dose-dependent manner (Figure 1).
Capillary morphogenesis in Matrigel is a valid in vitro correlate of in
vivo angiogenesis. As shown in Figure 2, when TcCRT, N-TcCRT
and HuCRT were compared in their capacities to inhibit
morphogenesis, only the parasite-derived molecules significantly
interfered with this process. The relevant TcCRT aminoacid
sequence spans residues 20–191, corresponding to N-TcCRT. R-
TcCRT did not affect capillary morphogenesis, in spite of its
overlapping with N-TcCRT in aminoacids 136–191.
Chemotaxis is an essential step in capillary morphogenesis and
angiogenesis. In HUVECs and Eahy926 cells, migration was
inhibited in a dose-dependent manner by TcCRT (Figure 3). Cell
migration inhibition by TcCRT may explain (at least partly) its
potent effects on in vitro capillary morphogenesis and ex vivo
capillary formation. These results agree with those describing the
HuCRT capacity to increase cell binding to extracellular matrix,
with consequent cell migration inhibition [28,29].
As shown in Figure 4, TcCRT and N-TcCRT share the
HuCRT capacity to specifically inhibit endothelial cell prolifera-
tion, a key initial event in angiogenesis [10]. These effects were not
observed in a different cell line, like fibroblasts, used as negative
controls. In HuCRT, the smallest anti-proliferative fragment spans
aa 120–180 [10]. Since, as observed in the morphogenesis assay,
R-TcCRT had no significant effect on HUVECs proliferation,
relevant residues also map between aa 20–135. TcCRT interferes
with pro angiogenic bFGF (Figure 4C), by unknown mechanisms.
HuCRT also inhibits the proliferation of endothelial cells from
diverse origins, such as FBHE [10], BAECs [30], HUVECs [31]
and ECV304 [32], in response to bFGF and VEGF. R-TcCRT
did not affect HUVECs proliferation (Figure 4D), nor morpho-
genesis (Figure 2F–H).
HUVECs proliferation inhibition by TcCRT may imply its
involvement in the cell cycle or, alternatively, in cell death
induction. TcCRT added at different concentrations to 24, 72 and
96 h HUVECs cultures did not induce apoptosis. Therefore, in
the TcCRT-mediated inhibition of cell proliferation, a cytostatic
effect, rather than apoptosis induction, may be mediated by the
parasite molecule.
Recombinant proteins from E. coli, are normally contaminated
with LPS, an antiangiogenic molecule [33]. In all the experiments
discussed above, LPS was ineffective at concentrations equivalent
to those present in the recombinant TcCRT preparations.
Although both HuCRT and TcCRT bind laminin, only the
former interferes with endothelial cell adhesion and, as a
consequence, with angiogenesis. Thus, the antiangiogenic TcCRT
effects could be explained by other mechanisms, such as direct
TcCRT interaction with endothelial cells. Alternatively, TcCRT
Figure 3. TcCRT inhibits human endothelial cell migration. A. HUVECs or B. EAhy926 cells migration towards chemo attractants was tested in
the presence of increasing TcCRT or LPS concentrations. Serum-free medium was used as negative control. Experiments were performed in triplicates.
Data are shown as means of values 6 SEM obtained from triplicates of one representative experiment out of 3 independent ones. *, p,0.05.
**, p,0.01.
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Figure 4. TcCRT inhibits HUVECs proliferation. A. Cells were grown in ECGS and FBS supplemented media, in the presence of various TcCRT
concentrations. As negative control, the cells were grown in free growth factor and FBS media. Cell number was assessed at 24-hour periods over 4
days by the MTT method. B. Statistical analysis of the results shown in A, at 96 h. Data are representative of 3 independent experiments, performed in
sestuplicate. **, p,0.01. C. Cells were grown in bFGF supplemented media, with or without 1 mM TcCRT or N-TcCRT. Cell number was determined
after a 96-hour period by the MTT method. Data are shown as means 6 SEM obtained from sestuplicate of one representative experiment.
**, p,0.01. D. Cells were grown in ECGS and FBS supplemented media, in the presence of various R-TcCRT concentrations. Cell number was assessed
at 24-hour periods over 4 days by de Cristal Violet method. Data are shown as means 6 SEM from sestuplicates of one representative experiment.
Non significant differences were obtained by ANOVA analysis. E. VERO cells were grown in FBS supplemented RPMI media, in the presence of various
TcCRT or HuCRT concentrations. Cell number was assessed at 24-hour periods over 4 days by the MTT method. Data are shown as means 6 SD
obtained from sestuplicates of one representative experiment. Non significant differences were obtained by ANOVA analysis.
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could be internalized and fulfill other functions in the intracellular
compartments. We now show that TcCRT binds to endothelial
cells, followed by internalization. The transduction pathways
involved are unknown. SREC-I (scavenger receptor expressed by
endothelial cell-I) could be involved in these phenomena. HuCRT
binds SREC-I, is endocytosed, and delivers associated peptides for
cross presentation via MHC- I [34], [27], a fact compatible with
our observations on the fucoidin (a specific SREC-I ligand [26,27])
capacity toinhibitTcCRT
(Figure 5E). Besides being an endocytic receptor, SREC-I is an
internalization byHUVECs
interesting candidate for signal transduction. Its intracellular
domain comprises almost half of the molecule, surprisingly large
among known scavenger receptors. It also contains several
potential phosphorylation consensus sites [35,36]. These results
are compatible with the possibility that TcCRT internalization is a
requisite to mediate its antiangiogenic effects on endothelial cells.
Whether TcCRT interferes with the endothelial cell cytoskeleton,
is unknown.
Perhaps, the parasite ability to inhibit angiogenesis interferes
with immune/inflammatory responses against this aggressor. On
Figure 5. TcCRT binds to live HUVECs and is internalized. HUVECs were incubated with: A. FITC or 1 mM: B. TcCRT, C. TcCRT-FITC, D. TcCRT-
FITC + 10 mM TcCRT, E. TcCRT-FITC and F. TcCRT-FITC + 25 mg/ml fucoidin, for 1h at 37uC, 5% CO2. The cells were then washed, fixed and analyzed by
fluorescence (A–D) or confocal microscopy (E and F). Results are representative of three independent experiments. Original magnification,640 (A–D)
and 6100 (E and F).
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the other hand, the role of angiogenesis in solid tumor progression
has been long established in a variety of experimental models [37].
For six decades now, several reports have proposed a possible
growth inhibitory effects that several T. cruzi strains may have on
multiple transplanted and spontaneous tumors, in animals and
humans [16,17,38]. The induction of specific immune anti-
tumoral responses [39] and/or the secretion of ‘‘toxic substances’’
by the parasite [16,40] were invoked to explain these effects, but
no experimental evidences have been provided. Maybe, TcCRT,
by interacting with endothelial cells and preventing neoangiogen-
esis, interferes in tumor growth and metastasis. For these reasons
we tested the TcCRT and Hu-CRT capacity to inhibit in vivo the
growth of a murine mammary tumor (TA3 MTXR). Only
TcCRT displayed significant anti-tumor effects in both experi-
ments. Moreover, the parasite molecule displayed stronger effects
than HuCRT. Although maximum efforts were made to perform
the experiments under similar conditions, the tumor growth was
different by about 2-fold, in the experiments shown in Figure 6 A–
B. The cell line is maintained in our laboratories, as ascites tumor
in A/J mice, with weekly passages and the experiments were
performed six months apart. Thus, although the conclusions
drawn from both experiments are basically the same, we cannot
rule out minor variations in handling, site of inoculation or in the
cell line itself that could explain the different overall tumor growth
observed in both experiments.
While the prevalence of tumor aggressions in wild and domestic
T. cruzi hosts has not been assessed, in humans they may reach
almost epidemic dimensions (i.e. mammary, prostate, ovary and
cervix-uterine cancers, taken altogether). Thus, the TcCRT
capacity to delay tumor growth, together with its anti inflamma-
tory properties (derived from its complement inhibition capacity),
may represent an evolutionary parasite adaptation, with final
increased infectivity.
In synthesis, in this report we show that T. cruzi calreticulin has
potent antiangiogenic activities, both on rat arterial (aortic ring
assay) and human venous (HUVECs) endothelial cells. These
properties map to the N-TcCRT domain in the parasite molecule.
TcCRT plays key in vitro antiangiogenic roles, expressed as
inhibition of capillary morphogenesis, proliferation and migration
of endothelial cells. TcCRT internalization by endothelial cells is
perhaps necessary in the antiangiogenic process. These facts,
together with those previously reported by us, showing that
TcCRT is a potent in vivo inhibitor of angiogenesis in a third
vertebrate species (CAM assay), allow us to propose that the
TcCRT antiangiogenic effects may be implicated in inflammatory
and antineoplastic effects, with benefits for the parasite in its
interactions with the vertebrate host. These findings may open
interesting possibilities for the development of new antineoplastic
strategies, especially if we consider that the parasite molecule
displays stronger antiangiogenic and anti-tumor effects than its
human counterpart. Biotechnological implications of these find-
ings may be envisaged. Whether the antiangiogenic properties
were consolidated, first in the parasite chaperone molecule, and
HuCRT conserved some of these properties, as an evolutionary
relict or, alternatively, the parasite hijacked this activity from its
vertebrate host, remains an open question.
Supporting Information
Alternative Language Abstract S1
into Spanish by Arturo Ferreira
Found at: doi:10.1371/journal.pntd.0000730.s001 (0.02 MB
DOC)
Translation of the abstract
Author Contributions
Conceived and designed the experiments: NCL AA AF. Performed the
experiments: NCL JO IM DA. Analyzed the data: NCL CV GR MR CR
AA DA DL LA WS AF. Contributed reagents/materials/analysis tools:
NCL CV GR MR CR JO IM AA DL LA WS AF. Wrote the paper: NCL
CV GR AA WS AF.
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