Parasitol Res (2003) 91: 187–196
DOI 10.1007/s00436-003-0937-z
O R I GI N A L P A P E R
Shankar Mukherjee Æ Thomas J. Belbin
David C. Spray Æ Dumitru A. Iacobas Æ Louis M. Weiss
Richard N. Kitsis Æ Murray Wittner Æ Linda A. Jelicks
Philip E. Scherer Æ Aihao Ding Æ Herbert B. Tanowitz
Microarray analysis of changes in gene expression in a murine
model of chronic chagasic cardiomyopathy
Received: 17 April 2003 / Accepted: 30 May 2003 / Published online: 9 August 2003
Springer-Verlag 2003
Abstract Chagas’ disease, caused by infection with
Trypanosoma cruzi, is a major cause of cardiomyopathy
in endemic regions. Infection leads to cardiac remodeling associated with congestive heart failure and dilated
cardiomyopathy. In order to study the changes in the
gene expression profile due to infection, C57BL/6·129sv
male mice were infected with 1·103 trypomastigotes of
the Brazil strain of T. cruzi. Histopathological examination of the myocardium revealed chronic inflammation, vasculitis and fibrosis 100 days post-infection.
Cardiac magnetic resonance imaging revealed a significantly dilated heart compared with uninfected mice. The
relative abundance or depletion of myocardial mRNAs
was evaluated using high-density microarrays consisting
of 27,400 mouse cDNAs, which were hybridized with
S. Mukherjee Æ T. J. Belbin Æ L. M. Weiss
M. Wittner Æ H. B. Tanowitz (&)
Department of Pathology, Albert Einstein College of Medicine,
1300 Morris Park Avenue, Bronx, NY 10461, USA
E-mail: [email protected]
Tel.: +1-718-4303342
Fax: +1-718-4308543
D. C. Spray Æ L. M. Weiss Æ R. N. Kitsis Æ H. B. Tanowitz
Department of Medicine, Albert Einstein College of Medicine,
1300 Morris Park Avenue, Bronx, NY 10461, USA
L. A. Jelicks
Department of Physiology and Biophysics,
Albert Einstein College of Medicine,
1300 Morris Park Avenue, Bronx, NY 10461, USA
D. C. Spray Æ D. A. Iacobas
Department of Neurosciences, Albert Einstein College of Medicine,
1300 Morris Park Avenue, Bronx, NY 10461, USA
P. E. Scherer
Department of Cell Biology and Medicine,
Albert Einstein College of Medicine,
1300 Morris Park Avenue, Bronx, NY 10461, USA
A. Ding
Department of Microbiology and Immunology,
Weill Medical College of Cornell University,
1300 York Avenue, New York, NY 10021, USA
fluorescent probes generated from mRNAs of T. cruzi
infected and uninfected hearts. Differentially expressed
genes were sorted according to their normalized
expression patterns and functional groups including
those involved in transcription, intracellular transport,
structure/junction/adhesion or extracellular matrix, signaling, host defense, energetics, metabolism, cell shape
and death. The regulated genes are interpreted in the
pathogenesis of chagasic heart disease.
Introduction
Trypanosoma cruzi is a protozoan kinetoplastid parasite
responsible for Chagas’ disease, a major cause of acute
myocarditis and chronic cardiomyopathy in endemic
regions. The pathogenesis of chagasic heart disease is the
result of many factors such as autoimmunity (Engman
and Leon 2002), parasite persistence (Tarleton 2001) and
vascular alterations (Tanowitz et al. 1996; Petkova et al.
2001), which lead to inflammatory and ischemic changes. Although significant advances have been made in
the identification of the genes that are altered during
T. cruzi infection, these have been primarily based on
observations from immunoblotting, polymerase chain
reaction and/or Northern blotting. These techniques
only permit the evaluation of a few pre-selected genes at
one time. Recently, there have been an increasing
number of reports on the use of microarray technology
in the study of human and experimental heart disease,
including those caused by infectious agents (Taylor et al.
2000; Cheek and Cesan 2003). DNA microarrays permit
the analysis of thousands of genes and gene products in
a single experiment.
One area in which microarrays have had an increasingly important impact is that of host-pathogen interaction (Diehn and Relman 2001). Infection by
microorganisms leads to a series of changes in the
physiology of host cells. These changes are relayed by
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signal transduction systems and are expressed by alterations in the transcription levels of various host genes.
Analysis of these transcriptional changes may elucidate
the physiological alterations in the host and also lead to
the discovery of drugs that interdict the consequences of
gene induction. Most such studies have been directed at
viral (Taylor et al. 2000) and or bacterial pathogens
(Belcher et al. 2000). There is a recent report on the
changes in the gene expression in T. cruzi-infected cultured fibroblasts (de Avalos et al. 2002).
Investigations into the pathogenesis of chagasic heart
disease have focused on cellular signaling pathways that
may contribute to remodeling of the myocardium
resulting in chronic cardiomyopathy and myocardial
dysfunction. These pathways have included those
involving cytokines, chemokines, nitric oxide synthases
(Huang et al. 1999a), mitogen activated protein kinases,
cyclins (Huang et al. 2003), and endothelin-1 (Petkova
et al. 2001). Therefore, we decided to identify genes that
could contribute to cardiac remodeling as a result of
T. cruzi infection. Detailed analyses of gene expression
were carried out by comparing gene expression changes
in infected versus uninfected hearts using cDNA
microarrays.
Materials and methods
Infection of mice
Ten 6-week-old male C57 BL/6·129sv mice with black fur were
infected with 1·103 trypomastigotes of T. cruzi (Brazil strain). Ten
age and sex matched controls were also maintained for 100 days,
housed in a similar environment in separate cages. Cardiac magnetic resonance imaging was performed on five mice at 100 days
post-infection. After cardiac magnetic resonance imaging, hearts
were used for molecular studies and histopathologic examination.
Histopathology
Hearts were obtained from infected and uninfected mice, fixed in
10% buffered formalin, paraffin-embedded and stained with
H and E.
were digested with 10 units RNase-1 for 10 min at 37C. Equimolar amounts of cDNAs (100 lg) from control (labeled with
Cy3) and infected (labeled with Cy5) hearts were mixed together
and were cleaned through Strata Clean resin (Stratagene) using
the manufacturer’s protocol. cDNAs longer than 100 nucleotides
were further selected and concentrated by passing through YM50 micron columns (Microcon). To perform reverse labeling,
equimolar amount of oppositely labeled cDNAs from the infected and uninfected hearts were mixed and hybridized to a
separate microarray slide.
DNA microarray
The concentrated cDNA probes (16.5 ll) were kept in hybridizing solution (35% formamide, 0.5% SDS, 2.5·Denhardt’s Solution, 4·SSPE) along with a blocking solution containing poly dA
(160 lg/ml), tRNA (32 lg/ml), mouse cot1 DNA (1 mg/ml) for
1 h at 50C. Microarrays containing 27,400 mouse cDNA
clones were obtained from the Albert Einstein College of Medicine Microarray Facility. The microarray slide was vapor moisturized for a second and cross-linked at 250 mJ using a
Stratalinker (Stratagene) and heat snapped for 3–5 s on a hot
plate. The slides were then successively passed through 0.1% SDS
(10 s), distilled water (10 s), boiling water (5 min) and ice-chilled
ethanol (2 s). Excess ethanol was removed by centrifugation of
slides at 1,000 g for 5 min. The slide was prehybridized with
60 ll of prehybridization solution (35% formamide, 0.5% SDS,
2.5·Denhardt’s Solution, 4·SSPE) in a hybridization chamber
for 1 h at 50C. Finally, the prehybrization solution was removed
from the slides and the hybridization solution with the labeled
probes was added. Hybridization was carried out at 50C for
16 h. For washing, the slides were passed through 2·SSC, 0.1%
SDS and washed with 0.2·SSC, 0.1% SDS for 15 min, followed
by a second washing in 0.2·SSC for 15 min (Bowtell and Sambrook 2002). The slide was spin-dried and scanned using a
GenePix 408A microarray scanner (Axon Instruments). Differential expression values were represented as a ratio of intensities
between infected and control samples. Expression data were
omitted in the regions where no signal was present or if the
signal was just above local background or derived from <40%
of the area of the printed spot. The fluorescence intensities of the
remaining spots were normalized using the Lowess normalization
method from the R statistical package (Dudoit 2000) and the
data were filtered to exclude spots with intensities less than twice
the background (150 pixels) in either channel and finally only
those spots were considered with a normalized ratio greater than
1.5 or 0.66 (in the reverse experiment).
Semiquantitative RT-PCR
Cardiac magnetic resonance imaging
Cardiac magnetic resonance imaging was performed by methods
previously described from our laboratory (Jelicks et al. 2002).
cDNA probes
Five hearts from each experimental group were pooled and RNA
was extracted with Trizol reagent as recommended by the manufacturer (Invitrogen) (Huang et al. 1999a).
To obtain sufficient mRNAs for microarray analysis, total
RNA was further purified using the RNeasy midi kit (Qiagen). A
total of 100 lg of purified RNA was annealed with 1 lg of oligo
dT (12–18) at 65C for 5 min. Fluorescent cDNA probes were
generated by reverse transcription of annealed RNA in the
presence of 2.5 nmol of Cy3 or Cy5 dUTP (Amersham) using
20 units Superscript II (Invitrogen) and 40 units RNase out at
42C for 2 h in a reaction volume of 40 ll. The remaining RNAs
The purified RNA from infected and control mouse hearts that was
used in preparation of cDNAs for microarray hybridization was
diluted and 1 lg was used to synthesize first strand cDNA by
2.5 units MuMLV reverse transcriptase and 2.5 lM oligo-dT16
primer at 42C for 15 min in a reaction volume of 20 ll. The reaction
products (cDNA) were then amplified by PCR using 2.5 units
of Ampli Taq DNA polymerase (Perkin-Elmer) and specific primer
pairs. We used the following primers, for secretory leukocyte
protease inhibitor, forward,5¢-CAGCTGAGTAACAGGAGCC
C-3¢; reverse,5¢-GCCTGCCCAGTGCCTTAAGC-3¢, for adipsin,
forward, 5¢-GGATTCCAGCCCCGAGGCCGGATTCTGG-3¢;
reverse 3¢-GAATTCTCAGGAGTCCTACAGTACAATGG-3¢,
for glyceraldehyde-3-phosphate dehydrogenase (rat), forward
5¢-TTGCCATCAACCACCCCTTC-3¢, reverse 5¢-TTGTCATGT
ATGACCTTGGC-3¢.
The mRNAs were analyzed after 35 cycles of amplification in a
thermal cycler (GeneAmp PCR System 2400, Perkin–Elmer). Each
cycle consisted of denaturation at 95C for 45 s, annealing/extension at 50C for 45 s, extension at 72C for 30 s with a final
189
Fig. 1 Representative section of the myocardium of a C57BL/
6·129sv mouse 100 days post-infection with the Brazil strain of
Trypanosoma cruzi. Note the chronic inflammation and fibrosis
extension at 72C for 7 min in a reaction volume of 100 ll. PCR
products were vacuum dried and electrophoresed in 1% agarose gel
containing ethidium bromide.
Immunoblotting
Heart lysates for immunoblotting were prepared by methods previously described (Huang et al. 1997). Thirty lg of proteins were
separated by 10% SDS-PAGE and transferred to nitrocellulose.
The primary antibodies (1:500 dilution) used were rabbit antisecretory leukocyte protease inhibitor and rabbit anti-adipsin.
Horseradish peroxidase-conjugated secondary antibodies (1:2000
dilutions) were used and subsequently the bound primary antibodies were visualized by ECL chemiluminescence (Amersham).
Results
Parasitology
As expected with this chronic murine model, none of the
mice died during the period of observation. There was a
transient parasitemia of approximately 1·104 trypomastigotes/ml 30–40 days post-infection, which then
waned over the ensuing weeks. Histopathologic examination of the myocardium revealed chronic inflammation and fibrosis as well as an intense vasculitis (Fig. 1).
This is consistent with our previous observations demonstrating that T. cruzi infection causes a chronic cardiomyopathy with inflammation, fibrosis and vascultitis
(Chandra et al. 2002).
Cardiac magnetic resonance imaging
Cardiac magnetic resonance imaging of the myocardium
was consistent with a cardiomyopathy. Consistent with
our previous observations, there was a marked dilation
of the right ventricle (Fig. 2A). The mean right ventricular internal diameter for uninfected control mice
was 1.5 mm while for infected mice it was 3.0 mm
(Fig. 2B).
Fig. 2A–C Cardiac gated magnetic resonance images of uninfected
and infected C57BL/6·129sv mice. A Uninfected mouse. B Infected
mouse. These axial images, showing the heart at mid level, were
chosen from a series of images acquired in diastole and spanning of
the heart from base to apex. Note the enlarged right ventricular
(asterisk) inner dimension (RVID) in the infected mouse heart.
C Bar graph of RVID mice determined by cardiac gated magnetic
resonance imaging. The RVID measured directly from magnetic
resonance imaging is presented as mean±SEM. The RVID is
significantly increased in infected mice compared to uninfected
controls (P<0.05)
Gene profiles
General considerations
In this study, 27,400-mouse cDNA microarray chips
were utilized. The Lowess intensity dependent normalization method was employed (Fig. 3), using the R
statistical package (Dudoit 2000), which is robust and
eliminates unnecessary bias due to incorporation efficiency, fluorescence yield and the laser power used in
the scanning. After normalization, we were left with
23,607 genes, from which data could be generated.
Since a minimum of 1.4-fold change in differential
expression can be accurately detected (Yue et al. 2001),
we selected those genes that were differentially expressed by 1.5-fold. With these criteria, we found 455
upregulated and 575 downregulated genes. From this
total of 1,030 differentially expressed genes, 337 were
identified with very high reliability with the reverse
labeling experiment. We used a single slide for labeling
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Fig. 3A, B Plots of Cy5/Cy3
ratio versus average intensity
for two replicate microarray
experiments. A Infected RNA
was labeled with Cy5 and
uninfected with Cy3. B Infected
RNA was labeled with Cy3 and
uninfected RNA with Cy5.
Intensity dependent
normalization of microarray
data eliminates any possible
bias effects in ratio
measurements due to intensity
of the signal being measured.
The dots represents gene
expression values for all of the
genes in the microarray
with both parameters from two independent experimental sets. This was done because data generated
from a reverse labeling experiment are more reliable
for reducing systematic bias than the average of two to
three independent one way labeling experiments
(Bowtell and Sambrook 2002). Out of the identifiable
genes, 65% were expressed sequence tags. We limited
this report to those genes that have clear identities and
ignored all of the expressed sequence tags. We grouped
genes that were related to transcription (tr, 23%),
intracellular transport (t2, 11%), transport across the
plasma membrane (t1, 6%), junctional, adhesive or
extracellular matrix (jae, 22%), energetics or metabolism (EnMet, 20%), cytoskeleton (cy, 4.5%), cell shape
and death (2.3%), and intracellular signaling (cs, 12%)
(Fig. 4). Table 1 lists those genes that were upregulated while Table 2 shows the genes that were downregulated due to infection.
Secretory leukocyte protease inhibitor
The inflammatory response in the myocardium is an
important aspect of the pathogenesis of chagasic heart
disease. Our microarray analysis demonstrated induction of the gene for secretory leukocyte protease inhibitor (SLPI), a protein that modulates the inflammatory
response. To confirm the microarray results, RT-PCR
and immunoblotting were performed (Fig. 5A). This
demonstrated that infection increased both the abundance of the SLPI mRNA and its protein. The induction
of SLPI has not been previously reported in T. cruzi
Fig. 4 Functional classification of upregulated genes (ratio>1.5) in
mouse hearts chronically infected with the Brazil strain of
Trypanosoma cruzi. Note that 85% of those genes affected encode
transcription factors (tr, 23%); junction, adhesion and matrix
components (jae, 22%) and enzymes and metabolites (EnMet,
20%). The other affected genes include transport across the plasma
membrane (t1, 6%); intracellular transport (t2, 11%); intracellular
signaling (cs, 12%); cytoskeleton (cy, 4%) and cell shape and death
(csd, 2%)
infection and implicates a role for this multi-faceted
immune modulator in host defense against T. cruzi.
Perhaps SLPI is involved in controlling inflammation
or/and in subsequent cardiac remodeling.
Adipsin
Adipsin gene expression was reduced in the myocardium
of these infected mice. Adipsin is a serum protease predominantly secreted from mature adipocytes, however,
low-level expression in other tissues, including heart, has
also been reported (Miner et al. 2001). RT PCR and
immunoblotting confirmed a significant decrease in the
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Table 1 Selected upregulated genes
Gene type
Gene name
Accession no.
Fold regulation
Immunoglobulins/related genes
Beta-2 microglobulin
H2 class II histocompatibility antigens
H2 class II antigen A
H2 class II locus DM
H2, complement component
H2, O region beta locus
H2, 2D region locus 1
Immunoglobulin heavy chain 1
Immunoglobulin heavy chain 3
Immunoglobulin heavy chain 4
Immunoglobulin heavy chain 6
Immunoglobulin J chain precursor
CD24a antigen
Complement component 1, q subcomponent
Cryptdin 17 or defensin
AA109951
AI323599
AA272807
AU042338
W18121
AA145469
W30548
AA182334
AU045060
AI662163
AO015491
AI323815
AU042170
AA000461
AA518520
1.5
2.0
2.2
2.2
1.5
1.5
1.4
8.3
6.2
1.9
3.2
2.8
1.6
1.6
1.5
Cytokines and growth factors
Growth differentiation factor 3
Growth factor independent I B
Insulin like growth factor binding protein –1
Growth factor receptor bound protein 7
Secretory leukocyte protease inhibitor
Small inducible cytokine subfamily B
Transforming growth factor, beta 2
C77913
W65842
AA423149
AA244622
AA200339
AA152885
AU022195
1.6
1.6
1.6
1.7
2.0
2.4
1.5
Enzymes
Galactokinase
Fructose bisphosphatase 1
Glyceraldehyde 3 phosphate dehydrogenase
GTP cyclohydrolase 1
2,3-bisphosphoglycerate mutase
ADP-ribosyltransferase
Brain creatine kinase
Fructose bisphosphatase 1
Galactokinase
Guanidinoacetate methyltransferase
Sphingosine kinase 1
AA255078
AA276043
AU043933
AI323637
AW55508
AA117045
W84226
AA276043
AA255078
AI465130
AA000819
1.7
1.7
1.8
1.6
1.6
1.5
1.5
1.7
1.7
1.5
1.6
Mitochondrial proteins
Cytochrome C oxidase, subunit VIIIa
Mitochondrial uncoupling protein (ucp)
AI326932
AA472506
1.6
1.6
Transcription factors/activators of
transcription factors
Bcl 3
GTF IIH
AI450238
AW545633
1.5
1.7
Signal transduction
cRaf, vRaf
cRas related protein
AW547875
W97276
1.5
1.5
Cell adhesion and structure
CD 44
Formin binding protein 3
Intercellular adhesion molecule 2
Laminin gamma 3
Procollagen, type I, alpha 1
CEA-related cell adhesion molecule 9
AW539558
AA178253
AA050972
AA048118
AW538541
AW545543
1.5
1.6
1.5
1.7
1.6
1.5
Cell cycle
Cdc 25 homologue
C78693
1.5
Organ morphogenesis
Cardiac morphogenesis
Gamma E crystallin
AA023480
W78478
1.6
1.6
Extra cellular transport
ABC (subfamily A)
ABC (subfamily C)
Solute carrier family
Solute carrier family
Solute carrier family
Solute carrier family
AU045475
AU018886
AA238533
AA275871
W85633
AA064246
4.5
4.0
1.6
1.5
1.6
1.6
1, member 2
2)
4
5, member alpha 1
Intra cellular transport
Adaptor protein 2 (AP-2)
Epsin 2
Kinesin heavy chain 1B
AAOO8029
AA413761
AA002775
2.0
1.6
1.5
Apoptosis
Caspase 12
AW552373
1.6
Genes implicated in vasculopathy
Clusterin (apolipoprotein J)
AA271058
1.5
Intracellular pH maintenance
Carbonic anhydrase
W59264
1.6
192
Table 1 (Contd.)
Gene type
Gene name
Accession no.
Fold regulation
Cell movement
Myosin light chain, alkali
Myosin light chain, regulatory A
Myosin Vb
Myosin-binding protein H
AA014929
AA048675
AW546331
W59642
1.5
1.6
2.0
1.5
Other important genes
Heat shock protein 84 kDa
Natriuretic peptide precursor type B
Neuromedin
Ganulin
Somatostatin
Cellular retinoic acid binding protein
C79131
AW541489
AA064128
AW551003
AI326805
AA145458
1.7
1.6
1.6
1.6
1.5
1.6
Table 2 Selected downregulated genes
Gene type
Gene name
Accession no.
Fold regulation
Mitochondrial proteins
Cytochrome P450, 7b1
Cytochrome P450, steroid inducible
AU022670
AA268120
1.5
1.8
Transcription factors/activators of
transcription factors
Metal response element binding transcription factor
Rho-associated coiled-coil forming kinase
AA545607
AA098168
1.5
2.0
Signal transduction
Mus musculus interferon-inducible GTPase
AA145136
1.5
Cell cycle
Cyclin B1 related sequence 1
AA124592
1.6
Apoptosis
Apoptosis inhibitor 3
TGF beta 1 induced anti apoptotic factor
AA097958
AA080234
2.5
3.3
Immunological markers
CD28 antigen
CD4 antigen
Interferon regulatory factor 2
AI327367
AI666534
AA200609
1.7
2.7
3.1
Other genes
Lipocalin 2
Adipisin
ESTs highly similar to caveola associated protein
Myeloblastosis oncogene
Heat shock protein, 86 kDa 1
Heterogeneous nuclear ribonucleoprotein
High mobility group protein 2
Ciliary neurotropic factor
Adrenergic receptor, beta 2
Carbon catabolite repression 4 homolog
Max dimerization protein 5
s17 protein
Treacher Collins Franceschetti syndrome
Topoisomerase (DNA) I
Thymus cell antigen 1, theta
Protein tyrosine phosphatase, non-receptor type 1
Protease, serine, 16 (thymus)
Pituitary tumor-transforming 1
Laminin, gamma 1
Keratin complex 2, basic, gene 8
AA087193
AI325224
C77256
AA267899
AW536140
AW554270
AW546306
W11161
C76960
AW556185
AW536381
AU040979
AI451115
AA170792
W13151
AI323459
AA204066
AA250500
C79482
C77408
2.0
1.5
1.8
3.0
1.6
1.5
2.1
2.5
2.0
1.5
2.1
1.7
1.5
1.5
1.8
2.2
2.9
2.3
3.1
1.7
expression of adipsin in the myocardium obtained from
infected mice (Fig. 5B).
Discussion
Chagas’ disease is a major cause of chronic cardiomyopathy. The murine model of chagasic cardiomyopathy
faithfully recapitulates many of the structural, functional and immunological alterations observed in human
chronic chagasic cardiomyopathy (Tanowitz et al. 1996;
Tarleton 2001; Petkova et al. 2001; Engman and Leon
2002). When C57BL/6·129sv mice were infected with
the myotropic Brazil strain of T. cruzi, they did not die
during the acute stage but gradually developed chronic
cardiomyopathy by day 100 post-infection The physiological findings of chronic inflammation, fibrosis and
vasculitis and a dearth of parasites is consistent with
previous observations (Chandra et al. 2002). In addition,
cardiac magnetic imaging and echocardiography
(Chandra et al. 2002) demonstrate chamber enlargement,
predominantly right ventricular, and an enlarged left
ventricle, thinning of the myocardium and a reduced
percent of fractional shortening. Therefore, we subjected
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Fig. 5 Confirmation of the microarray data by immunoblotting
and gene specific RT PCR of: A secretory leukocyte protease
inhibitor (SLPI) and B adipsin. The top row in each case shows
immunoblot for SLPI and adipsin of myocardial lysate obtained
from uninfected (Con) and infected (Inf) mouse. Second row RT
PCR for SLPI and adipsin from the uninfected (Con) and infected
(Inf) mRNAs that were used to label cDNAs for the microarray
analysis. In both the cases, RT PCR of glyceraldehydes-3phosphate dehydrogenase (GAPDH) was performed to confirm
equal loading. The molecular weight marker (M) is given on the left
hand side, that of SLPI and adipsin is shown in brackets
the hearts obtained from these chronically infected mice
to DNA microarray analysis in order to examine
whether there were important genes that were either upor downregulated at this stage. We used a 27,400-mouse
cDNA chip to examine these genes and found several
genes of interest that were differentially regulated. There
are several genes of interest, related to infection, not
reported previously. Some of these observations were
validated by RT-PCR and immunoblotting.
Inflammation and immunity
The inflammatory response in the myocardium is an
important aspect of the pathogenesis of chagasic heart
disease (Reis et al. 1993; Huang et al. 1999a, 1999b).
This is the first report, to our knowledge, to show the
upregulation of the SLPI gene (Table 1, Fig. 5A) in
chagasic cardiomyopathy. SLPI is synthesized by leukocytes, macrophages, epithelial cells in the lung, skin and
other organs (Zhu et al. 2002). This protein inhibits neutrophil function by inhibiting neutrophil elastase released
into the interstitium and to a lesser extent inhibits
cathepsin G and other proteases thereby protecting tissue
from self-degradation by these enzymes (Zhu et al. 2002).
Bacterial lipopolysaccharides, interleukin-1 and tumor
necrosis factor are known to upregulate SLPI, which may
limit neutrophil-mediated tissue injury.
Microarray analysis also demonstrated the induction
of several cytokines and important growth factor genes,
including growth differentiation factor 3 (McPherron
and Lee 1993) and insulin-like growth factor-binding
proteins (Table 1), a family of structurally homologous
secreted proteins that specifically bind and modulate the
activities of insulin-like growth factors (IGF-1 and IGF2) (Clemmons 1997), enhance cellular differentiation and
stimulate cell proliferation and muscle cell differentiation. These are important for cardiac remodeling.
Adipsin is identical to complement factor D, cleaving
complement factor B when it is complexed with activated
complement component C3. Furthermore, it has been
reported to be downregulated in the obese state (Rosen
et al.1989). Moreover, adipsin is critically involved in the
generation of acylation stimulating protein, a 76 amino
acid fragment of complement C3 that is generated by the
interaction of adipsin and factor B with C3. Acylation
stimulating protein can markedly increase triglyceride
synthesis in adipocytes. Adipsin, therefore, plays a critical
role in whole body lipid homeostasis, and its reduction or
complete absence induces resistance to obesity and
increased energy expenditure in ob/ob mice, a genetic
model of type II diabetes (Xia et al. 2002). We do not know
whether the observed reduction of adipsin in heart in
chronically infected mice (Table 2, Fig. 5B) is relevant for
systemic adipsin levels. However, the reduced expression
in heart may be indicative of reduced adipsin production
in other tissues, particularly in adipose tissue. Preliminary
data suggests that cultured 3T3-L1 adipocytes reduce
the production of adipsin in response to acute infection
(H.B. Tanowitz et al. unpublished data).
Cell adhesion and structure
We found that T. cruzi infection leads to the activation
of important cell adhesion molecules, including procollagen, CD44, ICAM 2, laminins and several cell cytoskeletal and structure related molecules like formins and
profilins (Table 1). These data verify previous observations from our laboratory regarding the increase of
vascular adhesion molecules in Chagas’ disease (Huang
et al. 1999b).This could explain the intense inflammation
seen in chagasic heart disease. Collagen is synthesized as
a procollagen precursor. Increase in the expression of
procollagen type-1 a-1 is indicative of fibrosis. Likewise,
laminins are a family of extracellular matrix glycoproteins constituting the basement membranes. The overexpression of laminin gamma, which is epithelium
specific, implies its role in cardiac remodeling.
Activation of CD44 is also important, as it is a
widely expressed plasma membrane protein involved
in cell-to-cell and cell-to-matrix interactions/adhesion.
CD44 is implicated in many diverse biological processes,
including angiogenesis, lymphogenesis, wound healing,
inflammation, and cancer metastasis (Davern et al.
2002). The over expression of ICAM-2 indicates an
upregulation of the inflammatory process as the degree
194
of inflammation due to any infection depends on
leukocyte adhesion to the inflamed region.
Immunoglobulins, histocompatibility and defensins
Genes encoding for major histocompatibility complexes
and immunoglobulins were upregulated as a result of
T. cruzi infection. In addition, the gene for b-microglobulin
is increased (Table 1). This observation is consistent with
the findings that CD8+ T cells contribute to myocardial
pathology in Chagas’ disease (Kumar and Tarleton, 1998).
This fact was further underscored by the observation that
b-microglobulin null mice had a more severe myocarditis
leading to death (Kumar and Tarleton, 1998). In addition,
CD24 (defensins) were noted to be upregulated (Table 1).
Defensins are small heavily glycosylated glycophosphatidyl
inositol anchored cell surface proteins, which are
expressed on granulocytes (Table 1). They act as broadspectrum antimicrobial peptides, with activities against
bacteria, fungi, and enveloped viruses.
Intracellular transport
We found that chronic murine chagasic cardiomyopathy
was associated with an increase in the intracellular transport of newly synthesized lysosomal enzymes from the
trans-Golgi network to the endosomes via the clathrin
coated vesicles as evidenced from the increased synthesis of
adaptor proteins, epsins and kinesins (Table 1). Clathrin
and the adaptor proteins interact with the membrane to
form the vesicle. Adaptor protein-2 is found predominantly
at the plasma membrane and its upregulation indicates
increased transport of lysosomal vesicles to the plasma
membrane. Epsins (1–3) are one of the important accessory
proteins that help in coat formation of the vesicles, while
kinesin is expressed in virtually all cells, and is important in
vesicular transport from Golgi to plasma membrane and in
Golgi to endoplasmic reticulum membrane recycling
(Kasprzak and Hajdo 2002). Increased expression of both
adaptor proteins and kinesins in the infected cardiac tissue
are indicative of an active secretary state of the cells. Apart
from increased vesicular intra-cellular transport, there is
evidence of an increase in the active transport of molecules
out of the cell, as there is increased expression of ABC
transporters (subfamily A and C), which power the translocation of the substrates by consuming ATP.
Transcription and gene regulation
Many genes encoding DNA-binding proteins and transcription factors exhibit altered expression in the T. cruzi
infected heart. Specific increases in mRNAs encoding
several transcription factors and activators of transcription factors were noted (Table 1). Infection appears to
activate transcription in general, as there is a marked
increase in the expression of auxillary general transcription factors, GTF IIH. Activation of GTF IIH helps the
cell to proceed to cell division, which may explain the
resulting cardiac remodeling associated with the disease.
We have previously reported the activation of the
NF-jB pathway in T. cruzi infection (Huang et al. 1999b,
Huang et al. 2003, 1999b). In the present report we
observed the upregulation of the Bcl 3 gene (Table 1),
which associates tightly and transactivates NF-jB (Richard et al. 1999). Bcl3 is also known to activate the transcription factor AP-1. Importantly, we have shown that in
murine Chagas’ disease there is activation of AP-1 (Huang
et al. 2003), which activates endothelin. In that regard, we
have shown that endothelin-1 contributes to the pathogenesis of chagasic cardiomyopathy (Petkova et al. 2001).
Signal transduction
Recently, we observed that T. cruzi infection leads to
the activation of ERK (phosphorylation) and endothelin-1 (Petkova et al. 2001; Huang et al. 2003). The
current study revealed that infection increases the
expression of both the Ras and the Raf genes
(Table 1). Thus, it appears that ERK activation follows
the classical Ras-Raf-MEKK pathway. This is consistent with our previous studies which showed that the
other two MAP kinase pathways are not activated due
to infection (Huang et al. 2003). Absence of upregulated genes from the JNK/SAP and p38 pathways in
our microarray also supports our previous data.
Our microarray data also demonstrated the upregulation of protein-tyrosine kinase 2. This is a cytoplasmic
kinase related to focal adhesion kinase that is regulated
by a variety of extracellular stimuli, including integrin
binding, growth factors, cytokines, chemokines, and
certain stress stimuli.
The activation of the Rho GTPase may also influence
cardiac remodeling (Table 1). For example, Rho is a
family of small GTPases that contribute to the regulation
of cellular functions including various cytoskeletonrelated events, membrane transport pathways and gene
transcription. By switching on a single GTPase, several
distinct signaling pathways can be activated in coordination. Therefore, with spatial and temporal activation of
multiple switches, Rho can activate various pathways
(Kataoka et al. 2002). We found that sphingosine kinase 1
is upregulated. It regulates the synthesis of sphingosine 1
phosphate, an agonist for several specific G protein
coupled receptors and activates the p42/p44 MAP kinase
pathway (Cho et al. 2003). Recently, Ferreira and
Burleigh (2002) demonstrated that endothelial differentiation gene-1 is up regulated in T. cruzi infected mouse
fibroblasts. Interestingly, this gene encodes a G-protein
coupled receptor for sphingosine 1 phosphate.
Cell cycle regulation and cell proliferation
Up regulation of general transcription factor (GTF IIH),
Cdc25 and septins demonstrates the proliferative nature
of the infected cardiac tissues. Cdc 25 is a dual-specificity
phosphatase and the ultimate regulator of mitosis in all
eukaryotic cells. Cdc25 catalyzes the activation of the
195
cyclin-dependent kinases, thus causing the initiation and
progression of successive phases of the cell cycle, while
septins are required for the completion of the cell cycle.
Mitochondrial bioenergetics
Various patho-physiological and diseased conditions
affect mitochondrial oxidative phosphorylation, whereby, the respiratory chain generates superoxide anions
which are then converted into hydrogen peroxide. The
decrease in cytochrome P450 expression is indicative of
less energy production from mitochondria. The upregulation of the mitochondrial uncoupling protein gene is
interesting, since it modulates the coupling between the
respiratory chain and ATP synthesis and may be involved in mitochondrial H2O2 generation (Negre-Salvayre et al. 1997). This observation could explain the
oxidative damage and inflammation associated with
chagasic heart disease (Garg et al. 2003).
Genes implicated in vasculopathy
and cardiac homeostasis
Studies from our laboratory reported the upregulation of
endothelin )1, an important gene implicated in vasculopathy in T. cruzi infection (Petkova et al. 2001). However, in the present study, we were unable to study its
expression pattern because of the absence of endothelin
cDNA in the microarray. Clusterin or apolipoprotein J,
is a secreted glycoprotein expressed by a wide variety of
tissues. It is implicated in both normal as well as in stress
induced premature senescence. The protein is upregulated in many severe physiological disturbances that relate to advanced aging, including accumulation in the
arterial wall during the development of atherosclerosis.
The increased expression of clusterin indicates the association of arteriosclerosis and chagasic heart disease. As
previously mentioned, the activation of CD44 leads to
angiogenesis, lymphogenesis and inflammation.
Alpha-sarcoglycan is a 50 kDa integral membrane
glycoprotein (alpha dystroglycan) localized to the sarcolemma of skeletal muscle. It is most abundantly expressed
in skeletal muscle, diaphragm and cardiac muscles with
a lower expression in smooth muscle cells (Roberds et al.
1993). Alpha-sarcoglycan is associated with dystrophin.
The increased gene expression of alpha-sarcoglycan in
chronic chagasic heart disease is currently not understood. Interestingly, in the acute phase of Chagas’ disease,
increased expression is not observed (H.B. Tanowitz et al.
unpublished data). This over expression may aid in the
survival of the animal in the chronic stage by keeping the
heart muscle architecture intact.
mechanisms that are responsible for the development of
congestive heart failure and cardiac remodeling have
been hindered by the technology available. To identify
factors responsible for the development of cardiac
remodeling and to ensure progress in establishing cause
and effect and possible targets of therapy, investigators
interested in the cardiovascular system now utilize
global gene profiling technology such as microarrays.
There are several papers that have identified changes in
gene clusters whose expression changed in congestive
heart failure (Friddle et al. 2000), cardiac hypertrophy
(Friddle et al. 2000), myocardial infarction (Stanton
et al. 2000) and infection (Taylor et al. 2000). These
studies have also examined pathways in cultured cells
(Peng et al. 2002). In addition, de Avalos et al. (2002)
used an in vitro model to study early invasion of
T. cruzi in human fibroblasts. Interestingly, there are
several genes that were altered both in their in vitro
and in our in vivo model, notable among them is the
induction of sphingosine phosphate kinase. The
observed differences in the expression levels of various
genes between infected and uninfected hearts in the
current study are mostly in the range of 1.4- to 2-fold.
This may reflect the fact that not every cell in the heart
is directly infected with the parasite. It is also possible
that the observed changes are a consequence of
secondary effects such as the inflammatory process. In
this study, we restricted our investigation to the global
changes in the gene expression in whole heart. In order
to look at specific cell types in tissue in vivo, future
studies will employ techniques such as laser capture, in
which it is possible to micro-dissect areas of the heart
and subject them to microarray.
The present report provides the first evidence that
sSLPI, an important modulator of the inflammatory
response, is upregulated in chronic chagasic cardiomyopathy. The observations in this report provide examples of genes whose upregulation would not have been
detected without microarray technology, illustrating
that this technique is a powerful tool for the study of
host-parasite interactions.
Acknowledgements Supported by grant number AI-12770 (HBT),
GM-61710 (AD), DK 55758 (PES), NS 42807 (DCS) and HL
07372 (HBT and DCS) from the US National Institutes of Health.
The authors wish to thank Dr. Carl Nathan, Department of
Microbiology and Immunology, Weill Medical College of Cornell
University for his support. In addition we wish to thank
Dr. Nicholas Socci, Department of Pathology and the staff of the
Albert Einstein College of Medicine Microarray Facility for
assistance with the microarrays and with data collection. The
experiments comply with the current laws of the country in which
the experiments were performed.
Conclusion
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