Cardiovascular Research (2009) 83, 140–147
doi:10.1093/cvr/cvp108
Induction of CRP3/MLP expression during vein
arterialization is dependent on stretch rather
than shear stress
Luciene Cristina Gastalho Campos, Ayumi Aurea Miyakawa*, Valerio Garrone Barauna,
Leandro Cardoso, Thaiz Ferraz Borin, Luis Alberto de Oliveira Dallan, and Jose Eduardo Krieger*
Laboratory of Genetic and Molecular Cardiology, Heart Institute (InCor), University of Sao Paulo Medical School, Av. Dr. Eneas
C. Aguiar, 44-10 andar, 05403-000 Sao Paulo SP, Brazil
Received 18 December 2008; revised 18 March 2009; accepted 25 March 2009; online publish-ahead-of-print 7 April 2009
Time for primary review: 30 days
KEYWORDS
CRP3/MLP;
Arterialized vein graft;
Myocardial revascularization;
Saphenous vein;
Stretch
Aims Cysteine- and glycine-rich protein 3/muscle LIM-domain protein (CRP3/MLP) mediates protein–
protein interaction with actin filaments in the heart and is involved in muscle differentiation and
vascular remodelling. Here, we assessed the induction of CRP3/MLP expression during arterialization
in human and rat veins.
Methods and results Vascular CRP3/MLP expression was mainly observed in arterial samples from both
human and rat. Using quantitative real time RT–PCR, we demonstrated that the CRP3/MLP expression
was 10 times higher in smooth muscle cells (SMCs) from human mammary artery (h-MA) vs. saphenous
vein (h-SV). In endothelial cells (ECs), CRP3/MLP was scarcely detected in either h-MA or h-SV. Using an
ex vivo flow through system that mimics arterial condition, we observed induction of CRP3/MLP
expression in arterialized h-SV. Interestingly, the upregulation of CRP3/MLP was primarily dependent
on stretch stimulus in SMCs, rather than shear stress in ECs. Finally, using a rat vein in vivo arterialization model, early (1–14 days) CRP3/MLP immunostaining was observed predominantly in the inner layer
and later (28–90 days) it appeared more scattered in the vessel layers.
Conclusion Here we provide evidence that CRP3/MLP is primarily expressed in arterial SMCs and that
stretch is the main stimulus for CRP3/MLP induction in veins exposed to arterial haemodynamic
conditions.
1. Introduction
Members of the cysteine- and glycine-rich protein (CRP)
family are characterized by the presence of two LIM
domains arranged in tandem and linked by short glycine
regions.1,2 These proteins are highly conserved and three
members of the CRP family have been characterized in vertebrates: CRP1, CRP2, and CRP3/MLP (muscle LIM-domain
protein).2–5 Evidence suggests that the CRP proteins regulate cell proliferation and differentiation by controlling
gene transcription processes.6,7 Moreover, they promote
protein assembly of the actin-based cytoskeleton, thus participating in cytoskeletal remodelling.2,8–11 The CRPs have a
tissue-specific distribution but appear to have similar functions in different cell types.12–14 CRP1 is expressed in a
variety of organs enriched in smooth muscle, CRP2
expression is restricted to arteries and fibroblasts, and
CRP3/MLP is predominantly expressed in organs enriched
* Corresponding author. Tel: þ55 11 3069 5068; fax: þ55 11 3069 5022;
E-mail address:
incor.usp.br (J.E.K.).
[email protected]
(A.A.M.);
krieger@
in striate muscle.8,9,15 Mice deficient in CRP3/MLP exhibit
significant alterations in the actin cytoskeleton with
defects in the architecture and function of both striate
and cardiac muscle and develop heart failure.10 Moreover,
the human CRP3/MLP mutation has been associated with
dilated cardiomyopathy.16
Remodelling of the vascular wall in response to injury
involves alterations in cell proliferation and migration, programmed cell death, and changes in production and/or
degradation of the extracellular matrix components.17,18
CRP1 and CRP2 play role in smooth muscle cell (SMC) differentiation19 and there is recent evidence that CRP3/MLP is
associated with vascular remodelling after balloon angioplasty injury in rats and mice.20 This was the first demonstration of CRP3/MLP in vascular smooth muscle raising the
possibility that, similar to observations in cardiac muscle,
this protein may also participate in the vascular response
to increased tension.
A vein graft is suddenly exposed to haemodynamic stress,
which is characterized by high pressure and flow. The adaptation of the vein to the increased haemodynamic condition
Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2009.
For permissions please email: [email protected].
CRP3 modulation in arterialized vein
141
2.4 Stretch protocol
requires reorganization of the vascular architecture to
support the increased wall tensile stress.18 In this study,
we investigated the patterns of CRP3/MLP expression in
artery, vein, and arterialized vein and demonstrate that
CRP3/MLP is present in arterial SMCs and virtually absent
in venous SMCs. Furthermore, we show that stretch is the
main stimulus for induction of CRP3/MLP expression in
SMCs during vein arterialization, rather than increased
shear stress on endothelial cells (ECs).
Primary cultures of SMCs from h-SV were stretched by using Flexercell 4000 cell stretching system (Flexcell International).24,25 1.5 105 cells were plated in Bioflex plates and the medium was
changed after 48 h, prior to initiating the stretch protocol (10%
stretch, 1 Hz, for 24 h). Control non-stretched SMCs were also cultured in Bioflex plates. At the end of the experiment, the cells
were washed with phosphate-buffered solution and lysed in 1 mL
Trizol Reagent.
2. Methods
2.5 Gene expression by RT–PCR and real
time RT–PCR
2.1 Vessel harvesting and ex vivo organ
culture system
Human saphenous vein (h-SV) and human mammary artery (h-MA)
segments were obtained from patients undergoing aortocoronary
bypass surgery in the Heart Institute (InCor), University of São
Paulo Medical School. All individuals gave informed consent to participate in the study, which was reviewed and approved by the local
Ethics Committee (SDC 2454/04/074 - CAPPesq 638/04). The investigation conforms with the principles outlined in the Declaration of
Helsink (see Cardiovascular Research 1997;35:2–4).
h-SVs were cultured in an ex vivo flow through system established
in our laboratory.21 Briefly, the organ culture system consists of a
vessel chamber, a peristaltic pump, and a pressure device, which
allows pressure and flow to be controlled independently. h-SVs
were placed in the chamber filled with Dulbecco’s modified
Eagle’s medium containing 10% foetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin. Two vein segments from the
same patient were always connected to the perfusion system and
cultured either under venous conditions (flow: 5 mL/min, no
pressure) or arterial conditions (flow: 50 mL/min, pressure:
80 mmHg), for 24 h.
2.2 Primary culture of human endothelial and SMCs
ECs were isolated by incubation of h-SV or h-MA luminal surfaces
with 1 mg/mL collagenase type II for 1 h at 378C. Then, the vessel
was flushed with phosphate buffer solution and the cell pellet was
cultured in Human Endothelial-SFM supplemented with 20% of new
born calf serum, 20 ng/mL FGF, 10 ng/mL EGF, 10 U/mL penicillin,
10 mg/mL streptomycin, and 10 U/mL heparin. ECs were characterized by their cobblestone appearance and by positive immunofluorescence staining for von Willebrand’s factor, VE-cadherin, and
PECAM-1.
h-SV and h-MA SMCs were obtained by explant protocol. Briefly,
the endothelial layer was removed by mechanical friction and
small fragments (0.09 cm2) of vessels were placed on six-well
culture plates containing 3% gelatin. The fragments were cultured
with Dulbecco’s modified Eagle’s medium containing 20% foetal
bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin.
After approximately 2 weeks, the SMCs derived from vessel fragments were isolated and expanded. SMCs were characterized by
hill-and-valley growth pattern and by immunofluorescence staining
for a-smooth muscle actin.
2.3 Shear stress protocol
Primary cultures of ECs from h-SV were submitted to controlled
shear stress as previously described.22 3 106 cells were plated in
150 mm dishes for 24 h. Before shearing, the medium was changed
to M199 medium supplemented with 1% new born calf serum for
24 h. Shear stress at 15 dyne/cm2 for 24 h was produced by using
a cone plate viscometer kindly provided by G. H. Gibbons.23 At
the end of the experiment, the cells were washed with phosphatebuffered solution and lysed in 1 mL Trizol Reagent (Invitrogen).
Total RNA was isolated with Trizol Reagent according to the manufacturer’s instructions and cDNA synthesis was performed with
random hexamers (High Capacity cDNA Archive kit-PE Applied
Biosystems).
Two hundred nanogram of cDNA was used for real time RT–PCR
reaction (SYBRw Green PCR Master Mix-PE Applied Biosystems) in
an ABI Prism 7700 Sequence Detection System (Applied Biosystems).
All samples were assayed in triplicate. The control gene 28S ribosomal RNA and Cyclophilin gene were used to normalize the results.
The comparative threshold (CT) cycle method was used for data
analyses. CT indicates the fractional cycle number at which the
amount of amplified target reaches a fixed threshold, and DCT is
the difference in threshold cycle for target (CRP3/MLP) and reference (28S or cyclophilin). The levels of CRP3/MLP gene expression
were given by 22DDCT; where DDCT is the DCT value subtracted
from DCT of heart tissue. Thus, the levels of CRP3/MLP expression
are relative to its expression in the human heart tissue, which is a
well established tissue for CRP3/MLP expression.
RT–PCR was used to determine the expression of CRP3/MLP in
samples where real time RT-PCR did not reach acceptable efficiency
of amplification. The reaction was carried out using Taq polymerase
under the following conditions: initial denaturation for 5 min at
958C followed by 40 cycles of denaturation for 15 s at 958C, annealing for 1 min at 608C, extension for 1 min at 728C, and final extension for 10 min at 728C. The PCR products were analysed by
electrophoresis on agarose gel. The bands were quantified by
using ImageJ (http://rsb.info.nih.gov/ij/).26 28S and cyclophilin
expression levels were used to normalize the results.
The following oligonucleotides primers constructions were used:
CRP1, 50 -GAGCCAGCTGCCAGAATG-30 (forward) and 50 -CCTTCGCA
CTGAACCTCTTC-30 (reverse); CRP2, 50 -GGTGAAATCTATTGTAAAGGA
TgC-30 (forward) and 50 -TACTGGGCATGAACAAGAGC-30 (reverse);
CRP3, 50 -GTGCCATCTGTGGGAAGAGT-30 (forward) and 50 -AA
GGCCTCCAAACCCAATAC-30 (reverse); 28S, 50 -TCATCAGACCCCA
GAAAAGG-30 (forward) and 50 -GATTCGGCAGGTGAGTTG-30 (reverse)
and cyclophiline, 50 - ATGGTCAACCCCACCGTGT-30 (forward) and
50 -TCTGCTGTCTTTGGGACCTTGTC-30 (reverse).
2.6 Vein graft arterialization model
in rat: surgical procedure
The arterialization in vivo model is based on the connection of rat
jugular vein to carotid artery as characterized in our laboratory.27
The investigation conforms with the Guide for the Care and Use
of Laboratory Animals published by the US National Institute of
Health (NIH Publication No. 85-23, revised 1996). This study protocol was approved by the local Ethics Committee (SDC—2253/03/047,
CAPPesq—418/03). Male Wistar rats (3 months old, 250–350 g) were
obtained from University of Sao Paulo Medical School animal facility.
Before surgical procedures, 70 UI/Kg of heparin was administered by
intraperitoneal injection followed by anaesthesia with ketalar
(50 mg/Kg) and rompum (10 mg/Kg). The right external jugular
vein was connected into the common carotid artery by an
end-to-end anastomosis with 10.0 nylon suture. After the surgery,
blood flow was re-established and the vein graft was harvested at
1, 3, 7, 14, 28, and 90 days after surgery. Vein grafts were flushed
142
L.C. Campos et al.
with heparinized saline solution, fixed by pressure perfusion with 4%
formalin, and embedded in paraffin for immunohistochemical
analysis.
2.7 Immunohistochemical analysis
The cross-sections (3 mm) were analysed starting 400 mm from the
suture. Endogenous peroxidase activity was blocked by 3 min incubation in 3% H2O2 (seven times at room temperature) and then
rinsed with phosphate-buffered solution. Non-specific reactions
were blocked in 5% BSA. The sections were incubated for 18 h at
48C with anti-CRP3, kindly provided by Silvia Arber and Pico
Caroni from University of Basel, Switzerland.4 The negative
control was maintained with BSA. Subsequently, the sections were
incubated with the solutions of kit LSAB HRP Universal (DAKO).
Immunoreactions were detected with 3,30 -diaminobenzidine (DAB)
and the sections were counterstained with aniline blue.
2.8 Western blot analyses
Pools of frozen vessels were thawed and minced into small pieces
and homogenized in cell lysis buffer (9 M urea, 2% TritonX-100,
40 mM DTT, 0.5 mM PMSF, and a mixture of protease inhibitors
(Sigma)). Insoluble tissue was removed by centrifugation at
12 000 rpm, 48C, 30 min. Samples (60 mg) were loaded and subjected to SDS–PAGE in 12% polyacrylamide gels. After electrophoresis, proteins were electro-transferred to a nitrocellulose
membrane (GE Healthcare) and transfer efficiency was monitored
by 0.5% Ponceau S staining. The membrane was incubated in a
blocking buffer (5% non-fat dry milk, 10 mM Tris–HCl, pH 7.6,
150 mM NaCl, and 0.1%Tween 20) for 2 h at room temperature and
then probed with a polyclonal antibody against CRP3 or GAPDH
(R&D) at room temperature. After incubation with peroxidaseconjugated secondary antibodies, detection was performed with
enhanced chemiluminescence reagents (GE Healthcare). Protein
levels of GAPDH were used to normalize the results.
2.9 Statistical analysis
Gene expression by RT–PCR is presented as mean + standard error
while gene expression by real time RT–PCR is presented by fold
induction and confidence interval. Comparisons between two
groups were analysed via a student’s t-test and P , 0.05 was considered significant for comparisons.
3. Results
3.1 Gene expression of the CRP family in venous
and arterial vessels
Expression of three members of the CRP family (CRP1, CRP2,
and CRP3/MLP) was evaluated by RT–PCR in both venous and
arterial vascular segments. While CRP1 and CRP2 showed
similar expression levels in h-SV and h-MA segments, CRP3/
MLP was almost absent in h-SV and presented lower
expression than CRP1 and CRP2 in h-MA (Figure 1). Interestingly, the expression of CRP3/MLP in SMC from h-MA was
greater than h-SV and no expression was detected in ECs
(Figure 1). To better characterize the expression of CRP3/
MLP, immunohistochemistry and real-time quantitative PCR
was performed. As expected, positive immunostaining was
observed for CRP3/MLP in h-MA, whereas no staining
was verified in h-SV (Figure 2A). Expression of CRP3/MLP
was 10 times higher in SMC from h-MA compared with SMC
from h-SV (Figure 2B). In both h-SV and h-MA ECs, expression
of CRP3/MLP could not be detected (Figure 2C). These
results indicate that CRP3/MLP expression is found mainly
in arterial SMC.
Figure 1 CRP1, CRP2, and CRP3/MLP mRNA expressions in human saphenous
vein and mammary artery. The amplicon sizes generated by RT–PCR reaction
were 95pb for CRP1, 95pb for CRP2, 119pb for CRP3, and 101pb for cyclophilin. Please note that all samples were resolved in the same gel, which is available as see Supplementary material online, Figure S1.
3.2 CRP3/MLP gene expression in arterialized
human saphenous vein
Since CRP3/MLP expression showed a predominantly arterial
profile, we tested whether arterialization of h-SV resulted in
induction of CRP3/MLP expression. For this purpose, h-SV
were cultured under arterial haemodynamic conditions
using an ex vivo flow-through system. Expression of CRP3/
MLP in h-SV was associated with arterialization based on
immunohistochemistry and real time RT–PCR assessments
(Figure 3A and B). Moreover, h-SV cultured under venous
condition did not modify CRP3/MLP expression.
3.3 Evaluation of shear stress and mechanical
stretch in CRP3/MLP gene expression
During the vein graft arterialization process, h-SV is exposed
to increased haemodynamic stimuli including shear stress
and stretch.17 To characterize the role of these mechanical
forces in CRP3/MLP expression, ECs and SMCs from h-SV
were submitted, respectively, to controlled shear stress
and stretch. Note that CRP3/MPL expression remained
unchanged in ECs submitted to shear stress (Figure 4A). On
the other hand, CRP3/MLP expression was induced in SMCs
by stretch (Figure 4B). These findings suggest that the
up-regulation of CRP3/MLP in arterialized h-SV is dependent
mainly on strain deformation (stretch) and not shear stress.
3.4 Temporal expression of CRP3/MLP in a rat
vein arterialization model
The time-course of CRP3/MLP expression was evaluated in
arterialized rat jugular vein from 1 up to 90 days
(Figure 5). Initially, immunohistochemical analysis showed
that expression of CRP3/MLP appears in different layers
and is mainly restricted to the carotid artery compared
with the jugular vein (Figure 5A). Upon arterialization,
there was a clear induction of CRP3/MLP in the jugular
vein especially in the inner layer after 1, 3, 7, and 14 days
of surgery. Later on (28 and 90 days after surgery), CRP3/
MLP staining decreased and appeared more scattered, in a
similar manner to that observed in carotid arteries, although
the layers are not yet well defined (Figure 5A). Similarly,
upon arterialization, protein expression, assayed by
western blotting, increased by days 1 and 3 and later on
CRP3 modulation in arterialized vein
143
Figure 2 (A) Representative sections of CRP3/MLP immunohistochemistry (stained in brown) of h-MA and h-SV. Magnification of 40 and 100. CRP3/MLP mRNA
expression in (B) SMCs and (C ) ECs of h-SV and h-MA. Each bar represents the mean value of CRP3/MLP expression, as evaluated by real time RT–PCR (n ¼ 8). The
experiment was normalized by 28S mRNA and data are represented as relative expression of CRP3/MLP present in the heart tissue. The values in brackets represent the interval of confidence. * indicates that gene expression could not be detected under the experimental conditions tested.
(day 28) decreased, but remained elevated compared with
normal jugular vein (Figure 5B). These findings were consistent with the immunohistochemistry indicating that CRP3/
MLP appears to be predominantly expressed in carotid
artery compared with native jugular vein. After vein arterialization, CRP3/MLP reaches expression levels similar to
those in arteries at early times, and then declining at later
times, remaining higher than in native veins but lower
than in arteries.
4. Discussion
In this report, we provide evidence that CRP3/MLP is
expressed mainly in arteries, but that it can be induced in
veins during the arterialization process in vitro and
in vivo. Moreover, this response is dependent on increased
stretch in SMCs, rather than increased shear stress in ECs.
Several markers for arteries and veins have been
described and well characterized in vascular beds.28–36 In
the past, the molecular and structural differences observed
between arteries and veins were often attributed to physiological factors, such as direction and magnitude of blood
flow and blood pressure levels. More recently, evidence supports the idea that there is a genetic program specifying
artery and vein identities, even before the onset of
circulation.37,38 During embryonic development, primary
vessels are pre-determined to differentiate into veins and
arteries by expression of specific markers. These molecular
pre-determinants are primarily genetically expressed and
secondarily influenced by haemodynamic forces. In the
adult vascular system, arterial and venous ECs have different phenotypic markers, as well as differences in their
ability to adapt to haemodynamic changes.35,39
In the vein graft procedure, a vein segment is submitted
to arterial haemodynamic condition and genes associated
with venous and arterial identities can be modulated as
triggers to the adaptive response. Successful vein graft
adaptation is a complex process that involves the remodelling of the vascular wall to the new biomechanical conditions.17 A reorganization of the venous architecture with
the acquisition of an artery-like structure has been demonstrated18,40–44 and there is evidence for the loss of the
venous phenotype marker, Eph-B4, during the adaptative
process, but without induction of the arterial phenotype
marker, Ephrin-B2.44 The present data indicate that the
CRP3/MLP protein may be considered as a new arterial
SMC marker, since it appears to be present mainly in
arteries (mammary in human and carotid in rat), virtually
absent in veins (saphenous vein in human and jugular vein
in rat), and it can be induced in the arterialized vein
144
L.C. Campos et al.
Figure 3 (A) Representative sections of CRP3/MLP immunohistochemistry (stained in brown) of h-SV, h-SV cultured under venous conditions (flow: 5 mL/min),
and h-SV cultured under arterial conditions (flow: 50 mL/min, pressure: 80 mmHg). Magnification 40 and 100. (B) CRP3/MLP mRNA expression in arterialized
h-SV. Each bar represents the mean value of CRP3/MLP expression of 19 experiments evaluated by real time RT–PCR. The experiment was normalized by 28S
mRNA and data are represented as relative expression of CRP3/MLP present in the heart tissue. The values in brackets represent the interval of confidence.
* indicates that gene expression could not be detected under the experimental conditions tested.
Figure 4 CRP3/MLP mRNA expression in (A) ECs submitted to shear stress and (B) stretched SMCs. Each bar represents the mean value of CRP3/MLP expression
of 12 experiments, as evaluated by real time RT–PCR in (A) or RT–PCR in (B). The experiment was normalized by 28S mRNA and data are represented as relative
expression of CRP3/MLP present in the heart tissue. The values in brackets represent the interval of confidence. * indicates that gene expression could not be
detected under the experimental conditions tested.
CRP3 modulation in arterialized vein
145
Figure 5 Temporal evaluation of the CRP3/MLP protein level in the arterialized rat jugular vein. (A) Representative sections of immunohistochemistry for CRP3/
MLP (stained in brown) of carotid artery, normal jugular vein, and jugular vein arterialized up to 90 days. Magnification 40 and 100. (B) CRP3/MLP protein
analysed by western blot in pools of vessels of carotid artery, normal jugular vein, and jugular vein arterialized for 1, 3, and 28 days. Upper panel shows the
quantification of CRP3 protein, as normalized by the GAPDH protein, and the lower panel is a representative western blot experiment for CRP3/MLP.
segment. CRP3/MLP was originally identified in cardiac and
striated muscle4 and, more recently, has been verified in
vascular smooth muscle.20 It is described to be present
exclusively in nuclei of early differentiated muscle cells
and to later accumulate in the cytoplasm.14 Nuclear
CRP3/MLP seems to interact with transcription factors and
positively regulate myogenesis, while cytoplasmic CRP3/
MLP is associated with the actin-based cytoskeleton and
may be important for the maintenance of the contractile
apparatus.10,13
Flick and Konieczny14 proposed an indirect link between
CRP3/MLP with actin filaments through the interaction of
its domain LIM1 with actinin and LIM2 with spectrin in
cardiac and skeletal muscle tissue. It may be assumed that
a similar organization could occur in vascular smooth
muscle tissue. This arrangement of the cellular cytoskeleton
enables the cell to support physical forces, such as stretching, in the SMCs. Veins normally exposed to low haemodynamic load may not require high levels of CRP3/MLP, but
when exposed to high haemodynamic stress, such as during
146
vein grafting, the induction of CRP3/MLP may contribute to
strengthen the connections of the cytoskeleton and prepare
them to support the new haemodynamic condition.
Another possible role for CRP3/MLP is in the process of cell
differentiation, it has been demonstrated that during vein
graft arterialization stem cells from adventitia and surrounding tissue migrate to the vascular wall and participate
in the remodelling process.45,46 These undifferentiated cells
are thought to be a source of SMCs that contribute to neointima formation. Nuclear CRP1 and CRP2 have been
described to act as transcriptional cofactors, facilitating
smooth muscle differentiation19 and participating in a multiprotein DNA binding complex that contains SRF, GATA
factors, and chromatin remodelling enzymes that mediate
the activation of promoters specific for differentiation of
SMC. Similarly, CRP3/MLP has also been demonstrated in
the nucleus9,12 and could influence SMC differentiation
during vein graft arterialization. In addition, the existence
of actin has been reported in the nuclear matrix47 and
nuclear CRPs can be physically associated with this microfilament/protein. Thus, nuclear CRP3/MLP could influence
transcriptional activity, since it participates in nuclear complexes that help chromatin bend or twist and optimize the
transcriptional activity.19
We and others have demonstrated that smooth muscle
a-actin decreases during early vein graft arterialization.27,48,49 Since CRP3/MLP and a-actin are closely
related to cytoarchitecture maintenance, one may speculate that modulation of these two proteins contributes to
the structural reorganization necessary to support the new
haemodynamic condition. This relationship between CRP3/
MLP and a-actin must be further investigated with regard
to the structural remodelling that takes place during vein
graft arterializations and the possible role in devising more
resistant cell architecture for veins that must sustain
greater mechanical stress.
Taken together, the results of the present study provide
evidence that CRP3/MLP is present mainly in arteries, but
that it can be induced during vein arterializations in vitro
and in vivo. Furthermore, the activation of CRP3/MLP
expression in veins is secondary to the effect of increased
stretch on SMCs, rather than increased shear stress on ECs.
Supplementary material
Supplementary Material is available at Cardiovascular
Research online.
Acknowledgements
We are grateful to Silvia Arber and Pico Caroni (Friedrich Miescher
Institute, Basel, Switzerland) for providing the anti-CRP3 antibody.
Conflict of interest: None declared.
Funding
This work was supported by Fundacao de Amparo a Pesquisa
do Estado de Sao Paulo—FAPESP [01/00009-0] and Conselho
Nacional de Desenvolvimento Cientifico e Tecnológico—
CNPq [478073/2004-6]. L.C.G.C., A.A.M., and V.G.B. are
recipient of fellowship from FAPESP—(03/14115-2, 00/
09485-7, 06/52053-7, respectively).
L.C. Campos et al.
References
1. Weiskirchen R, Pino JD, Macalma T, Bister K, Beckerle MC. The
cysteine-rich protein family of highly related LIM domain proteins.
J Biol Chem 1995;270:28946–28954.
2. Louis HA, Pino JD, Schmeichel KL, Pomiès P, Beckerle MC. Comparison of
three members of the cysteine-rich protein family reveals functional conservation and divergent patterns of gene expression. J Biol Chem 1997;
272:27484–27491.
3. Liebhaber SA, Emery JG, Urbanek M, Wang XK, Cooke NE. Characterization of a human cDNA encoding a widely expressed and highly conserved
cysteine-rich protein with an unusual zinc-finger motif. Nucleic Acids Res
1990;18:3871–3879.
4. Arber S, Halder G, Caroni P. Muscle LIM protein, a novel essential regulator of myogenesis, promotes myogenic differentiation. Cell 1994;79:
221–231.
5. Sadler I, Crawford AW, Michelsen JW, Beckerle MC. Zyxin and cCRP: two
interactive LIM domain proteins associated with the cytoskeleton. J Cell
Biol 1992;119:1573–1587.
6. Dawid IB, Breen JJ, Toyama R. LIM domains: multiple roles as adapters
and functional modifiers in protein interactions. Trends Genet 1998;14:
156–162.
7. Bach I. The LIM domain: regulation by association. Mech Dev 2000;91:
5–17.
8. Weiskirchen R, Günther K. The CRP/MLP/TLP family of LIM domain proteins: acting by connecting. Bioessays 2003;25:152–162.
9. Jain MK, Kashiki S, Hsieh CM, Layne MD, Yet SF, Sibinga NE et al. Embryonic expression suggests an important role for CRP2/SmLIM in the developing cardiovascular system. Circ Res 1998;83:980–985.
10. Arber S, Hunter JJ, Ross J Jr, Hongo M, Sansig G, Borg J et al.
MLP-deficient mice exhibit a disruption of cardiac cytoarchitectural
organization, dilated cardiomyopathy, and heart failure. Cell 1997;88:
393–403.
11. Pomiès P, Louis HA, Beckerle MC. CRP1, a LIM domain protein implicated
in muscle differentiation, interacts with alpha-actinin. J Cell Biol 1997;
139:157–168.
12. Arber S, Caroni P. Specificity of single LIM motifs in targeting and LIM/LIM
interactions in situ. Genes Dev 1996;10:289–300.
13. Kong Y, Flick MJ, Kudla AJ, Konieczny SF. Muscle LIM protein promotes
myogenesis by enhancing the activity of MyoD. Mol Cell Biol 1997;17:
4750–4760.
14. Flick MJ, Konieczny SF. The muscle regulatory and structural protein MLP
is a cytoskeletal binding partner of bI-spectrin. J Cell Sci 2000;113:
1553–1564.
15. Weiskirchen R, Bister K. Suppression in transformed avian fibroblasts of a
gene (crp) encoding a cysteine-rich protein containing LIM domains.
Oncogene 1993;8:2317–2324.
16. Geier C, Perrot A, Ozcelik C, Binner P, Counsell D, Hoffmann K et al.
Mutations in the human muscle LIM protein gene in families with hypertrophic cardiomyopathy. Circulation 2003;107:1390–1395.
17. Mehta D, Izzat MB, Bryan AJ, Angelini GD. Towards the prevention of vein
graft failure. Int J Cardiol 1997;62:S55–S63.
18. Gibbons GH, Dzau VJ. The emerging concept of vascular remodeling.
N Engl J Med 1994;330:1431–1438.
19. Chang DF, Belaguli NS, Iyer D, Roberts WB, Wu SP, Dong XR et al.
Cysteine-rich LIM-only proteins CRP1 and CRP2 are potent smooth
muscle differentiation cofactors. Dev Cell 2003;4:107–118.
20. Wang X, Li Q, Adhikari N, Hall JL. A role for muscle LIM protein (MLP) in
vascular remodeling. J Mol Cell Cardiol 2006;40:503–509.
21. Miyakawa AA, Dallan LA, Lacchini S, Borin TF, Krieger JE. Human saphenous vein organ culture under controlled hemodynamic conditions.
CLINICS 2008;63:683–688.
22. Miyakawa AA, Junqueira ML, Krieger JE. Identification of two novel shear
stress responsive elements in rat angiotensin I converting enzyme promoter. Physiol Genomics 2004;17:107–113.
23. Malek A, Izumo S. Physiological fluid shear stress causes downregulation
of endothelin-1 mRNA in bovine aortic endothelium. Am J Physiol 1992;
263:C389–C396.
24. Gilbert JA, Weinhold PS, Banes AJ, Link GW, Jones GL. Strain profile for
circular cell culture plates containing flexible surfaces employed to
mechanically deform cells in vitro. J Biomechem 1994;27:1169–1177.
25. Schaffer JL, Rizen M, L’Italien GJ, Benbrahim A, Megerman J et al. Device
for the application of a dynamic biaxially uniform and isotropic strain to a
flexible cell culture membrane. J Orthop Res 1994;12:709–719.
26. Rasband W. ImageJ: image processing and analysis in Java. http://
rsb.info.nih.gov/ij/ (2004).
CRP3 modulation in arterialized vein
27. Borin TF, Miyakawa AA, Cardoso L, Borges LF, Gonçalves GA, Krieger JE.
Apoptosis, cell proliferation and modulation of cyclin-dependent kinase
inhibitor p21cip1 in vascular remodelling during vein arterialization in
the rat. Int J Exp Pathol 2009; doi:10.1111/j.1365-2613.2009.00648.x.
28. Durbin L, Brennan C, Shiomi K, Cooke J, Barrios A, Shanmugalingam S
et al. Eph signaling is required for segmentation and differentiation of
the somites. Genes Dev 1998;12:3096–3109.
29. Liao EC, Paw BH, Oates AC, Pratt SJ, Postlethwait JH, Zon LI. SCL/Tal-1
transcription factor acts downstream of cloche to specify hematopoietic
and vascular progenitors in zebrafish. Genes Dev 1998;12:621–626.
30. Lyons MS, Bell B, Stainier D, Peters KG. Isolation of the zebrafish homologues for the tie-1 and tie-2 endothelium-specific receptor tyrosine
kinases. Dev Dyn 1998;212:133–140.
31. Herzog Y, Kalcheim C, Kahane N, Reshef R, Neufeld G. Differential
expression of neuropilin-1 and neuropilin-2 in arteries and veins. Mech
Dev 2001;109:115–119.
32. Lawson ND, Scheer N, Pham VN, Kim CH, Chitnis AB et al. Notch signaling
is required for arterial-venous differentiation during embryonic vascular
development. Development 2001;128:3675–3683.
33. Moyon D, Pardanaud L, Yuan L, Bréant C, Eichmann A. Plasticity of endothelial cells during arterial-venous differentiation in the avian embryo.
Development 2001;128:3359–3370.
34. Villa N, Walker L, Lindsell CE, Gasson J, Iruela-Arispe ML, Weinmaster G.
Vascular expression of Notch pathway receptors and ligands is restricted
to arterial vessels. Mech Dev 2001;108:161–164.
35. Wang HU, Chen ZF, Anderson DJ. Molecular distinction and angiogenic
interaction between embryonic arteries and veins revealed by
ephrin-B2 and its receptor Eph-B4. Cell 1998;93:741–753.
36. Zhong TP, Childs S, Leu JP, Fishman MC. Gridlock signalling pathway
fashions the first embryonic artery. Nature 2001;414:216–220.
37. Noble F, Moyon D, Pardanaud L, Yuan L, Djonov V, Matthijsen R et al. Flow
regulates arterial-venous differentiation in the chick embryo yolk sac.
Development 2004;131:361–375.
147
38. Torres-Vazquez J, Kamei M, Weinstein BM. Molecular distinction between
arteries and veins. Cell Tissue Res 2003;314:43–59.
39. Jones EA, le Noble F, Eichmann A. What determines blood vessel structure? Genetic prespecification vs. hemodynamics. Physiology (Bethesda)
2006;21:388–395.
40. Garret HE, Dennis EW, DeBakey ME. Aortocoronary bypass with saphenous
vein graft: seven-year follow-up. JAMA 1973;223:792–794.
41. Kwei S, Stavrakis G, Takahas M, Taylor G, Folkman MJ et al. Early adaptive responses of the vascular wall during venous arterialization in
mice. Am J Pathol 2004;164:81–89.
42. Mavromatis K, Fukai T, Tate M, Chesler N, Ku DN, Galis ZS. Early effects of
arterial hemodynamic conditions on human saphenous veins perfused ex
vivo. Arterioscler Thromb Vasc Biol 2000;20:1889–1895.
43. Westerband A, Crouse D, Richter LC, Aguirre ML, Wixon CC, James DC
et al. Vein adaptation to arterialization in an experimental model.
J Vasc Surg 2001;33:561–569.
44. Kudo FA, Muto A, Maloney SP, Pimiento JM, Bergaya S, Fitzgerald TN et al.
Venous identity is lost but arterial identity is not gained during vein graft
adaptation. Arterioscler Thromb Vasc Biol 2007;27:1562–1571.
45. Schwartz SM, deBlois D, O’Brien ER. The intima. Soil for atherosclerosis
and restenosis. Circ Res 1995;77:445–465.
46. Schwartz SM. Smooth muscle migration in atherosclerosis and restenosis.
J Clin Invest 1997;99:2814–2817.
47. Rando OJ, Zhao K, Crabtree GR. Searching for a function for nuclear
actin. Trends Cell Biol 2000;10:92–97.
48. Rodriguez E, Lambert EH, Magno MG, Mannion JD. Contractile smooth
muscle cell apoptosis early after saphenous vein grafting. Ann Thorac
Surg 2000;70:1145–1153.
49. Yamamura S, Okadome K, Onohara T, Komori K, Gugimachi K. Blood
flow and kinetics of smooth muscle cell proliferation in canine autogenous vein grafts: in vivo BrdU incorporation. J Surg Res 1994;56:
155–161.