Transcription Factor SOX18 Is Expressed in Human
Coronary Atherosclerotic Lesions and Regulates DNA
Synthesis and Vascular Cell Growth
Marta Garcı́a-Ramı́rez, José Martı́nez-González, Josep O. Juan-Babot,
Cristina Rodrı́guez, Lina Badimon
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Objective—SOX18, a member of the SOX gene family (SRY-like 3-hydroxy-3-methylglutaryl box gene), is a transcription
factor expressed in the development of blood vessels during embryogenesis. We analyzed SOX18 expression in human
coronary atherosclerotic lesions and investigated its potential function in vascular cells.
Methods and Results—In advanced human coronary atherosclerotic lesions, SOX18 immunostaining was localized in
endothelial cells (on the luminal surface, in vasa vasorum, and in intimal neovessels) and in vascular smooth muscle
cells (VSMCs) scattered in the intima, colocalizing with proliferating cell nuclear antigen. In cell cultures, SOX18 was
mainly localized in subconfluent and denuded areas. Significant SOX18 mRNA levels (by Northern blot analysis and
reverse transcription–polymerase chain reaction) were detected in cell cultures from human umbilical vein endothelial
cells and human VSMCs. Antisense SOX18 inhibited DNA synthesis ([3H]thymidine incorporation) and vascular cell
growth. Antisense SOX18 also significantly reduced VSMC regrowth after injury in an in vitro model of wound repair.
Conclusions—Our results indicate that SOX18 is involved in vascular cell growth and suggest that this transcription factor
may play a role in atherosclerosis. (Arterioscler Thromb Vasc Biol. 2005;25:2398-2403.)
Key Words: vascular biology 䡲 gene expression 䡲 endothelial function 䡲 atherosclerosis 䡲 growth factors 䡲 SOX18
T
addition, SOX18 expression has been detected in proliferating
blood vessels in adult mouse granulation tissue.14 The SOX
family comprises genes with a conserved high-mobility group
DNA-binding domain (HMG box), homologous to SRY, that
regulate cell proliferation, differentiation, and migration during embryogenesis and development.15,16 The SOX genes
specifically recognize the heptameric consensus sequence
A A
/T /TCCAA/TG and have been shown to act as activators or
repressors of gene transcription.10,17 Interestingly, analysis of
SOX18 expression in mouse mutants of VEGFRs places
SOX18 in the VEGF and Flk-1 pathways of EC
differentiation.16
Although SOX18 is not restricted to development,18 –20
there has been no information on whether it possesses any
function in adult vascular cells. In the present study, we show
that SOX18 is expressed in ECs and VSMCs both in vitro and
in vivo. Immunohistochemical analyses show that SOX18
colocalizes with a marker of cell proliferation. Inhibition of
SOX18 expression with antisense (AS) oligodeoxynucleotides (ODNs) prevents human coronary SMC proliferation
(reduce de novo DNA synthesis, VSMC growth, and VSMC
wound repair). These results indicate a role for SOX18 in
vascular activation during the progression of atherosclerosis
in humans.
he molecular mechanisms regulating migration and proliferation of adult vascular cells are critical in the
pathophysiology of atherosclerosis. The increased local secretion of growth factors and cytokines promotes vascular
smooth muscle cell (VSMC) activation and dedifferentiation.
VSMCs undergo phenotypic changes involving the transformation from contractile, resting, fully differentiated cells into
proliferative, migratory, dedifferentiated SMCs, which synthesize high amounts of extracellular matrix proteins.1,2 In
addition, during neovascularization, a process that contributes
to atherosclerotic plaque progression,3,4 endothelial cells
(ECs) migrate and proliferate in a process activated by
vascular endothelial growth factor (VEGF) and other growth
factors.5,6 These cellular processes involve the upregulation
or downregulation of multiple genes coordinately regulated
by diverse proteins that control cell cycle entry and other
cellular functions, such as cell migration or synthetic activity.7–9 However, the transcription factors controlling these
processes are not fully known.
SOX18, a member of the SOX gene family (SRY-like
3-hydroxy-3-methylglutaryl [HMG] box gene) of transcription factors, has been implicated in the developing vasculature during embryogenesis and in several adult tissues.10 –13 In
Original received October 6, 2004; final version accepted September 2, 2005.
From the Centro de Investigación Cardiovascular, CSIC/ICCC, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain.
The first 2 authors contributed equally to this work.
Correspondence to Prof Lina Badimon, Centro de Investigación Cardiovascular, Hospital de la Santa Creu i Sant Pau, Sant Antoni Maria Claret No.
167, 08025 Barcelona, Spain. E-mail [email protected]
© 2005 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org
2398
DOI: 10.1161/01.ATV.0000187464.81959.23
Garcia-Ramirez et al
Methods
Coronary Artery Sampling and Preservation
Human coronary arteries were obtained from hearts removed during
transplant operations performed at the Hospital de la Santa Creu i
Sant Pau (Barcelona, Spain). Immediately after surgical excision,
coronary arteries were dissected and processed for either immunohistochemical analysis or the isolation of VSMCs, as described
earlier.21 The specimens for immunohistochemistry were immersed
in fixative solution (4% paraformaldehyde/0.1 mol/L phosphatebuffered saline, pH 7.4). After overnight treatment, vessels were
sectioned into blocks and embedded in paraffin. The specimens for
cell culture were immersed in cell culture maintenance medium and
processed for VSMC isolation as described.21 The reviewer institutional Committee on Human Research of the Hospital of Santa Creu
i Sant Pau approved the research protocol for this study.
Immunohistochemical Analysis
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Paraffin-embedded specimens were cut into 5-␮m-thick serial sections, placed on poly-L-lysine– coated slides, deparaffinized, and
stained with Masson⬘s trichrome or processed for immunohistochemistry. Characterization of the lesions in Masson’s trichrome–
stained sections was performed according to American Heart Association criteria.22 In brief, consecutive sections were deparaffinized
and treated for nonspecific binding, and the presence of SOX18 and
other cell markers was assessed. The primary antibodies used were
as follows: anti-SOX18 (sc-20100, Santa Cruz Biotechnology Inc);
anti–␣-smooth muscle actin (␣-SMA; clone 1A4, Dako), a marker
for VSMCs; anti-proliferating cell nuclear antigen (PCNA; clone
PC10, Zymed laboratories Inc), a marker of cell proliferation;
anti-CD34 (QBEnd/10, Novocastra Laboratories Ltd) or anti-CD31
(clone JC/70A, Dako) as EC markers; and a monoclonal antibody
(clone HAM56, Dako) specific for macrophages. After incubation
with the primary antibody (1 hour; each antibody was tested in 4
sections from every lesion), sections were washed and then incubated with the appropriate biotinylated secondary antibodies (1/200,
from Vector). Finally, sections were incubated with avidin-biotin
complex (Elite, Vector), and 3,3⬘-diaminobenzidine was used as the
substrate for peroxidase, as described.21
SOX18 immunostaining was assessed by 2 investigators simultaneously using a double-headed light microscope. The extent of
staining was graded according to a semiquantitative scale of 0 to
⫹⫹⫹: 0, no staining detected; ⫹, weak staining; ⫹⫹, moderate
staining; and ⫹⫹⫹, extensive staining.
Immunofluorescence analysis of paraformaldehyde-fixed sections
was used to analyze colocalization of PCNA (rabbit polyclonal
antibody sc-7907, Santa Cruz Biotechnology Inc) with vascular cell
markers (CD31 and ␣-SMA). As secondary antibodies, fluorescein
isothiocyanate– conjugated goat anti-mouse IgG (Dako) and tetramethylrhodamine isothiocyanate– conjugated swine anti-rabbit IgG
(Dako) were used.
Cell Cultures
VSMCs were obtained by a modification of the explant technique21
from human coronary arteries of hearts removed during transplant
surgery (as indicated earlier). VSMCs were cultured in medium 199
(GIBCO) supplemented with 20% fetal calf serum (FCS, Biological
Industries), 2% human serum, 2 mmol/L L-glutamine, and antibiotics
(100 U/mL penicillin and 0.1 mg/mL streptomycin). Human umbilical vein ECs (HUVECs) were obtained as described23 and were
cultured in medium 199 supplemented with 20 mmol/L HEPES, pH
7.4 (GIBCO), 20% FCS, 30 ␮g/mL EC growth factor supplement
(Sigma), 100 ␮g/mL heparin, 2 mmol/L L-glutamine, and antibiotics.
Porcine aortic ECs (PAECs) isolated from adult normolipemic
animals as described23 were grown in medium 199 (GIBCO)
supplemented with 10% FCS, 2 mmol/L L-glutamine, and antibiotics. Cell cultures were used between passages 2 and 5.
SOX18 in Human Coronary Arteries
2399
Immunocytochemical Analysis
Cells were cultured in chamber slides (Costar), fixed with 4%
formaldehyde, and processed for SOX18 immunostaining as described.21 Horseradish peroxidase– conjugated rabbit anti-mouse antibodies (Dako) were used as secondary antibodies. In control
experiments with isotype-matched nonspecific antibodies, no immunocytochemical staining was detected. Results were evaluated with
an Olympus Vanox AHBT3 microscope, and images were digitized
by a Sony DXC-S500 camera.
Polymerase Chain Reaction
Cells seeded in 6-well plates (105 cells/well) were grown in corresponding media until confluent. Then the cells were serum deprived:
VSMCs in medium 199/0.2% FCS for 48 hours; PAECs in medium
199/0.2% FCS for 24 hours; and HUVECs in medium 199/5% FCS
for 12 hours before treatment with serum or human recombinant
VEGF-A (rhVEGF165, R&D Systems). After treatment, cells were
processed and total RNA was isolated with the Ultraspec system
(Biotex) according to the manufacturer’s recommendations. SOX18
mRNA levels were analyzed by polymerase chain reaction (PCR)
with the PCR Dig labeling mix (Roche Molecular Biochemicals) as
described.24 Specific SOX18 oligonucleotides were designed from
the human SOX18 sequence (GenBank NM018419). The oligonucleotides used to generate a 583-bp cDNA used as a probe in
Northern blot experiments were as follows: 5⬘-CCGAGTTCGACCAGTACCTC-3⬘ and 5⬘-AAGGAGCAGGTGCTTCAAAA-3⬘. The oligonucleotides used in reverse transcription
(RT)—PCR analysis were as follows: 5⬘-CCGAGTTCGACCAGTACCTC-3⬘ and 5⬘-GAGATGCACGCGCTGTAATA-3⬘. RT-PCR
analyses were performed with 28 cycles of denaturation at 94°C for
30 seconds; annealing at 61°C for 1 minute; and polymerization at
72°C for 1 minute. PCR products were resolved by electrophoresis
on agarose gels and transferred to nylon membranes (Nytran Plus,
Schleicher & Shuell) by a standard capillary technique. Blots were
UV cross-linked. Detection of digoxigenin-labeled nucleic acids was
performed with an anti-digoxigenin antibody linked to alkaline
phosphatase and disodium 3-(4-methoxy-spiro{1,2-dioxetano-3,2⬘(5⬘-chloro)tricyclo[3.3.1.13,7]decan}-4-yl) phenylphosphate used as
the substrate. Results were normalized to glyceraldehyde
3-phosphate dehydrogenase as previously described.24
SOX7 and SOX17 mRNA levels were determined by real-time
PCR. In brief, RNA was reverse-transcribed with use of a Taqman
RT kit (Applied Byosystems) with random hexamers. Assay-onDemand (Applied Biosystems) of Taqman fluorescent real-time PCR
primers and probes were used for SOX7 (Hs00846731_s1) and
SOX17 (Hs00751752_s1). Glyceraldehyde 3-phosphate dehydrogenase (4326317E) was used as an endogenous control.
Nothern Blot Analysis
Total RNA was obtained as indicated earlier and analyzed by
Northern blotting as described.23 A SOX18 583-bp cDNA obtained
by RT-PCR with specific SOX18 primers (see previous section) and
cloned into the pGEM-T easy vector (Promega) was used as a probe.
This probe recognizes a SOX18 region that shares no significant
homology with other members of the SOX family to avoid crosshybridization with SOX18-related genes. Hybridization and washes
of blots were carried out under high-stringency conditions.23 To
estimate transcript size, RNA ladders (Invitrogen) were used.
Determination of DNA Synthesis
[3H]thymidine incorporation into DNA was used as an index of cell
proliferation. HUVEC DNA synthesis was assessed as described.24
Cells under basal growth conditions (10% FCS for 12 hours) were
incubated with 10 ng/mL VEGF-A in medium containing 0.5
␮Ci/mL of [3H]thymidine and incubated for an additional 24 hours.
Similarly, arrested human coronary VSMCs (0.2% FCS for 48 hours)
were stimulated with 10% human serum in medium containing
[3H]thymidine, and DNA synthesis was determined as described.24
The effect of AS phosphorothioate ODNs against SOX18 (0.2 to
10 ␮mol/L) on cell DNA synthesis was assessed. The AS ODN used
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Arterioscler Thromb Vasc Biol.
November 2005
Figure 1. SOX18 in human coronary
arteries. A, Masson’s trichrome staining
of a human coronary artery advanced
lesion. B–E, High-power views of serial
sections (indicated in the Masson’s
trichrome section [A]) from regions corresponding to the lumen (B), adventitia (C),
neovessels in the intima (D), and VSMCs
in the intima (E). SOX18 (B1–E1); CD34
(B2–D2), and ␣-SMA (E2) are also shown.
Control-1 and -2 correspond to sections
in B and E, respectively, in which
isotype-mached nonspecific antibodies
were used. Bar⫽50 ␮m in B and C and
25 ␮m in D and E).
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was AS-SOX18 (5⬘-CGGCGATCTCTGCAT-3⬘) complementary to
nucleotides 111 to 125 of human SOX18 mRNA (GenBank accession
No. NM018419). As controls, the corresponding sense sequence
(SE-SOX18) and a random oligonucleotide (random) were used.
Cells were preincubated with ODNs for 6 hours to assess their effect
on both basal and stimulated DNA synthesis. The ODNs did not
produce any effect on cell morphology, cell apoptosis (assessed by
staining with Hoechst 33258 colorant), or cell viability as analyzed
by measuring mitochondrial dehydrogenase activity with use of a
commercial kit (XTT-based assay for cell viability, Roche).
Quantification of Cell Growth
Cell growth was evaluated by cell counting. Growth-stimulated cells
were treated with 10 ␮mol/L ODNs (AS-SOX18 or SE-SOX18), after
72 hours were dissociated with trypsin/EDTA, and then counted with
a microscope counting chamber (hemocytometer).
In Vitro SMC Injury
The ability of AS-SOX18 ODNs to inhibit VSMC wound repair after
mechanical injury was assessed in human coronary VSMCs in
culture as described.24 In brief, confluent growth-arrested human
coronary VSMCs were denuded with a scraper and incubated in
medium with 10% human serum in the presence or absence of
10 ␮mol/L AS-SOX18 ODNs for an additional 72 hours. SE-SOX18
ODNs were used as controls. Cells were fixed and stained with
methylene blue. Images were digitized by a Sony DXC-S500
camera, and cell number in the denuded zone was determined.
Statistical Analysis
Results are expressed as mean⫾SEM. The Stat View II (Abacus
Concepts) statistical package for the Macintosh computer was used
for all analysis; multiple groups were compared by 1-factor
ANOVA, followed by Fisher’s protected least significant difference
test to assess specific group differences.
vanced lesions, we detected colocalization of SOX18 expression and PCNA expression (a marker of cell proliferation) in
both endothelial luminal surface (CD31-positive cells) and
VSMC-rich areas (␣-SMA–positive cells; Figure 2; Figure
III, available online at http://atvb.ahajournals.org).
SOX18 in Vascular Cells in Culture
In ECs and VSMCs in culture, SOX18 immunostaining was
detected in subconfluent areas and in cell monolayers that had
been denuded (Figure 3). In Northern blot experiments with a
cDNA probe homologous to the human SOX18 generated by
RT-PCR and under high-stringency conditions, we detected a
single transcript corresponding to the size of SOX18 (1.6
kb)18,25 in both growing HUVECs and VSMCs (Figure 4A).
SOX18 mRNA levels were transiently induced by mitogenic
stimuli (VEGF or serum) in both HUVECs and VSMCs
(Figure 4B). SOX18 mRNA levels also were induced by
mitogenic stimulus in PAECs (data not shown).
Effect of SOX18 Inhibition on Vascular
Cell Proliferation
In VSMCs, AS-SOX18 (10 ␮mol/L) significantly reduced
SOX18 mRNA levels in both control (unstimulated) and
growth-stimulated cells (Figure 5A). SE-SOX18 or random
sequences did not produce any effect. AS-SOX18 also
strongly inhibited serum-induced VSMC DNA synthesis in a
dose-dependent manner (IC50⬍1 ␮mol/L; Figure 5B). Similarly, AS-SOX18 inhibited both SOX18 mRNA levels and
Results
SOX18 Immunohistochemical Staining in
Coronary Arteries and Vascular Cells
In human coronary artery early lesions, SOX18 immunostaining was virtually absent and rare in intermediate lesions
(Table I and Figure I, available online at http://atvb.ahajournals.
org). By contrast, in human coronary advanced atherosclerotic lesions (Figure 1A), SOX18 immunoreactivity was
detected in the luminal endothelium and in the vasa vasorum,
colocalizing with EC markers CD34 (Figure 1B and 1C) and
CD31 (data not shown). In addition, SOX18 was detected in
neovessels and in cells expressing ␣-SMA scattered in the
intima (Figure 1D and 1E). No SOX18 immunostaining was
detected in macrophage-rich areas (Figure II, available online
at http://atvb.ahajournals.org). In serial sections from ad-
Figure 2. SOX18 and PCNA in human atherosclerotic lesions.
High-power views correspond to immunostaining of PCNA (A1
and B1) and SOX18 (A2 and B2) in a neovessel (A) and in an intimal area enriched in VSMCs (B). Arrowheads indicate cells that
express both SOX18 and PCNA. Control-1 and -2 correspond
to sections in A and B, respectively, in which isotype-mached
nonspecific antibodies were used. Bar⫽25 ␮m.
Garcia-Ramirez et al
SOX18 in Human Coronary Arteries
2401
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Figure 3. SOX18 immunostaining in PAECs (A and B) and
human coronary SMCs (VSMCs, C and D) in culture. The pattern of SOX18 expression in subconfluent cultures (A and C)
and in denuded cell monolayers (B and D) is shown. Representative of 3 independent experiments performed in duplicate
Bar⫽25 ␮m.
DNA synthesis in HUVECs (Figure IV, available online at
http://atvb.ahajournals.org). AS-SOX18 also inhibited
VSMCs (30.3% inhibion versus control cells, P⬍0.005) and
EC (33.7% inhibition versus control cells, P⫽0.002) growth,
whereas SE-SOX18 did not produce any effect.
The AS-SOX18 ODNs used in these experiments were
specific for SOX18, and they did not modify mRNA levels
corresponding to SOX7 and SOX17, as determined by realtime PCR (SOX7, 100⫾17% in serum-stimulated VSMCs
versus 110⫾24% in serum-stimulated VSMCs plus ASSOX18 ODNs; SOX17, 100⫾21% in serum-stimulated
VSMCs versus 105⫾25% in serum-stimulated VSMCs plus
AS-SOX18 ODNs).
Figure 5. AS oligonucleotides against SOX18 inhibit DNA synthesis in human coronary SMCs. A, SOX18 mRNA levels in
unstimulated cells (⫺H. serum) and in cells stimulated with 10%
human serum (⫹H. serum) treated or not with oligonucleotides
(10 ␮mol/L). AS-SOX18 (10 ␮mol/L) but not SE-SOX18
(10 ␮mol/L) reduced SOX mRNA levels under both basal growth
conditions and in serum-stimulated VSMCs. B, Arrested VSMCs
were stimulated with 10% human serum (H. serum), and DNA
synthesis ([3H]thymidine incorporation) was determined in the
absence (white bars) or presence of increasing concentrations
of AS-SOX18 (0.2 to 10 ␮mol/L). The effect of SE-SOX18 and
random ODNs (10 ␮mol/L) is also shown (shaded bars) (n⫽4
experiments performed in triplicate). P⬍0.05 *vs control; †vs
serum-treated cells.
AS-SOX18 ODNs Inhibit SMC Regrowth
After Injury
In a well-established in vitro model of wound healing24
AS-SOX18 significantly inhibited serum-induced VSMC regrowth into the denuded zone by 42% (Figure 6). In contrast,
SE-SOX18 failed to modulate this process.
Discussion
Figure 4. SOX18 expression in vascular cell cultures. A, Northern blot showing the levels of SOX18 transcript (1.6 kb in size)
in HUVEC and human coronary artery VSMC (growing cells).
RNA from nonatherosclerotic human coronary arteries was used
as a negative control. Levels of ribosomal RNA 18S (rRNA) are
used as a loading control. B, RT-PCR showing SOX18 mRNA
levels in HUVEC and VSMC under basal and mitogen-stimulated
conditions (VEGF 10 ng/mL or 10% human serum; for 1, 2, 8
and 24 hours). Levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used to normalize results.
SOX18 is a transcription factor previously known to be
involved in blood vessel development and embryogenesis,12,26 which has also been related to lymphatic vessel
pathologies.27 In the present study, we show that SOX18 is
expressed in primary cultures of ECs and VSMCs and in
human coronary advanced atherosclerotic lesions. In addition, we show that in vascular cells in culture, inhibition of
SOX18 expression prevents DNA synthesis and reduces
SMC wound healing.
Recent evidence suggests that the activation of vascular
cells in processes such as intimal thickening or neovascularization requires coordinate regulation by multiple genes that
control cell cycle entry, cell migration and proliferation, and
cell synthetic activity, among other functions.7–9 Here we
show that the SRY-related HMG box factor, SOX18, a gene
previously identified in fetal brain and expressed in fetal and
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Arterioscler Thromb Vasc Biol.
November 2005
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Figure 6. AS oligonucleotides against SOX18 inhibit wound
repair after mechanical injury of human coronary VSMC monolayers. Arrested VSMCs were scraped and then incubated for 72
hours in the absence [Control(⫺)] or presence of 10% human
serum [Control(⫹)] or human serum plus 10 ␮mol/L oligonucleotides (AS-SOX18 or SE-SOX18, 10 ␮mol/L). A, Representative
photomicrograph of cells subjected to this procedure. B, Number of cells in the denuded zone were counted and represented
graphically (n⫽3 experiments performed in triplicate). P⬍0.05
*vs control(⫺); †vs control(⫹) or cells stimulated with serum and
treated with SE-SOX18.
adult tissues,18 –20 is expressed in both ECs and VSMCs from
human coronary arteries. SOX18-positive cell number increases with lesion severity. It is noteworthy that in advanced
lesions, SOX18 colocalizes with PCNA, although the proportion of PCNA-positive cells was higher and colocalization
was only partial, in agreement with previous results showing
colocalization of proteins differentially expressed through the
cell cycle.28 SOX18 is known to be involved in vascular
development13 and the induction of angiogenesis during
wound healing and repair of skin tissues.14 Indeed, SOX18 is
found in keratinocytes and in ECs from capillaries within the
granulation tissues, showing an expression pattern similar to
that of VEGFR2 (Flk-1).14 Here we show that SOX18 is
expressed in primary cultures of HUVECs and human coronary VSMCs. In fact, in both cell types, we detected significant levels of the transcript corresponding to SOX18 by
Northen blotting (a less-sensitive technique than the RTPCR– based technique used to analyze SOX18 mRNA levels
throughout the study). Although high SOX18 mRNA levels
were previously identified in ventricles and the interventricular septa of adult hearts,20 this is the first time that SOX18
has been identified in VSMCs, both in human atherosclerotic
lesions and in culture. In cell cultures, SOX18 immunostaining was mainly localized in subconfluent areas and cell
monolayers (either from ECs or VSMCs) that had undergone
denudation, suggesting its involvement in cell growth. In fact,
inhibition of SOX18 expression with specific oligonucleotides decreases both DNA synthesis and the growth of
vascular cells. In addition, inhibition of SOX18 expression by
AS oligonucleotides also reduced VSMC wound repair.24
We detected expression of SOX7 and SOX17 in human
vascular cells; however, AS-SOX18 specifically inhibited
SOX18 mRNA levels. Therefore, our results do not support
the concept of functional redundancy among these 3 SOX
family members in adult vascular cells. SOX18, together with
SOX7 and SOX17, forms group F within the SOX family.10
They share significant structural identity and similar expression patterns, in particular during embryogenesis.10,25 Based
on these similarities and in the absence of an apparent
phenotype in SOX18-deficient mice, it has been suggested
that these transcription factors could be functionally redundant.11 However, neither compensatory induction nor other
compensatory mechanisms previously described in other
genes playing overlapping functions has been demonstrated
among these SOX proteins. In fact, SOX7 and SOX17 expression levels were not analyzed in SOX18-knockout mice.11
Finally, it should be taken into account that in our model, we
modulated (not suppressed) SOX18 expression in short-term
experiments, and it is possible that the potential compensatory mechanism among SOX proteins does not work in this
early setting.
In summary, immunohistochemical studies have identified
SOX18 immunostainig in ECs and VSMCs from human
coronary artery advanced arterosclerotic lesions, colocalizing
at least in a part with a marker of cell proliferation, whereas
cell culture studies indicate that SOX18 could play an
important role in vascular cell growth. Taken together, the
results of our studies suggest that SOX18 could play a
important role in atherogenesis in those processes that
involve cell growth, such as arterial intimal thickening and
neovascularization. In fact, lesion neovascularization and
arterial intimal thickening are closely associated in advanced lesions,5,6 because neovessels are required to ensure the viability and growth of intimal cells (hyperplasia).4 Finally, Hosking et al29 recently showed that vascular
cell adhesion molecule-1 (VCAM-1), an adhesion molecule overexpressed in atherosclerotic vessels from both
human and animal models of hypercholesterolemia,30 is a
target of SOX18 in several cell lines. We did not observe
any effect of AS-SOX18 on VCAM-1 mRNA levels in
HUVECs and VSMCs (unpublished results); however,
because multiple transcription factors are involved in the
regulation of VCAM-1 in a cell-specific manner,29,31 more
focused studies are needed to elucidate the relative role of
SOX18 in the transcription rate of VCAM-1 in cells from
human adult vessels.
Transcription factors involved in development are critical in the regulation of cell proliferation, differentiation,
and migration. In recent years, some of these transcription
factors, such as the homeobox genes, have been shown to
be key players in cardiovascular system development
during embryogenesis and in disease states such as atherosclerosis.32 Furthermore, HMG box genes, transcription
factors more related to the SOX family, regulate vascular
Garcia-Ramirez et al
cell migration and proliferation.33 Recently, the SOX gene
family has emerged as a potential set of genes implicated
in diverse pathophysiologic functions.20,27,34 –36 Our results
suggest that SOX18 could play an important role in
vascular cell growth. Future experiments designed to
demonstrate the in vivo role of SOX18 in processes
involving cell proliferation, such as neovascularization and
arterial intimal thickening, will help to determine whether
this gene could be regarded as a new target to prevent
atherosclerosis progression.37
Acknowledgments
Marta Garcı́a-Ramı́rez have been a recipient of a Research contract
from the Fondo de Investigación Sanitaria (FIS), Ministry of Health,
Spain. This work was made possible by a grant funded by FIS
PI020392, PI020361, PI030355, and RECAVA C-03/01; a Freedom
to Discover Grant from Bristol-Myers Squibb; and a grant from
Fundación de Investigación Cardiovascular Catalana Occidente.
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Transcription Factor SOX18 Is Expressed in Human Coronary Atherosclerotic Lesions
and Regulates DNA Synthesis and Vascular Cell Growth
Marta García-Ramírez, José Martínez-González, Josep O. Juan-Babot, Cristina Rodríguez and
Lina Badimon
Arterioscler Thromb Vasc Biol. 2005;25:2398-2403; originally published online September 22,
2005;
doi: 10.1161/01.ATV.0000187464.81959.23
Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association, 7272
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Copyright © 2005 American Heart Association, Inc. All rights reserved.
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Online Figure I
A
B
Online Figure II
SOX18
HAM56
Online Figure III
PCNA
CD31
PCNA/CD31
PCNA
α-SMA
PCNA/α-SMA
0
100
0.2
- VEGF
1
SE-SOX18
AS-SOX18
-
*
†
*
5
10
AS-SOX18
Random
SE-SOX18
AS-SOX18
- VEGF
SE-SOX18
150
VEGF
50
Random
Control
A
SE-SOX18
B
AS-SOX18
Control
(% of Controls)
DNA synthesis
Online Figure IV
+ VEGF
SOX18
GAPDH
+ VEGF
* *
†
‡
‡
Online figure legend
Figure I. SOX18 is not present in early human atherosclerotic lesions. A, Masson
trichromic staining of an early human coronary artery lesion. B, high-power view of a
serial section immunostained with anti-SOX18 antibodies. (Bar = 100 µm).
Figure II. SOX18 immunostaining is not detected in macrophage-rich areas. High
power view of serial sections corresponding to a human coronary artery lesion
immunostained using anti-SOX18 antibodies (sc-20100) and HAM56 monoclonal
antibody (a marker of macrophages). (Bar = 50 µm).
Figure III. PCNA positive cells in the endothelial luminal surface and in smooth
muscle cell-rich areas are endothelial and smooth muscle cells. Upper panel, colocalization of PCNA (red) with CD31 (green, endothelial cell marker) in the
endothelial luminal surface. Lower panel, co-localization of PCNA (red) with α-SMA
(green, smooth muscle cell marker) in a smooth muscle cell-rich area. Arrow heads
indicate co-localization. (Bar = 25 µm).
Figure IV. Antisense oligonucleotides against SOX18 inhibit DNA synthesis in
human endothelial cells (HUVEC). A, SOX18 mRNA levels in unstimulated cells (VEGF) and cells stimulated with 10 ng/mL VEGF-A (+ VEGF) treated or not with
oligonucleotides (10 µmol/L). B, HUVEC were exposed to increasing concentration
of antisense SOX18 ODNs (AS-SOX18; 0.2 to 10 µmol/L ) and DNA synthesis
([3H]thymidine incorporation) was assessed in the absence (white bars) or presence
(black bars) of 10 ng/mL VEGF-A. The effect of AS-SOX18, sense SOX18 (SE-
SOX18) and Random ODNs (10 µmol/L) is also shown in both basal and VEGF-Astimulated conditions (n=3 experiments performed in quadruplicate). P<0.05: *, vs.
Control; †, vs. VEGF-A-treated cells; ‡, vs. Control and VEGF-A treated cells.
2
Online Figure I
A
B
Online Figure II
SOX18
HAM56
Online Figure III
PCNA
CD31
PCNA/CD31
PCNA
α-SMA
PCNA/α-SMA
0
100
0.2
- VEGF
1
SE-SOX18
AS-SOX18
-
*
†
*
5
10
AS-SOX18
Random
SE-SOX18
AS-SOX18
- VEGF
SE-SOX18
150
VEGF
50
Random
Control
A
SE-SOX18
B
AS-SOX18
Control
(% of Controls)
DNA synthesis
Online Figure IV
+ VEGF
SOX18
GAPDH
+ VEGF
* *
†
‡
‡
Online figure legend
Figure I. SOX18 is not present in early human atherosclerotic lesions. A, Masson
trichromic staining of an early human coronary artery lesion. B, high-power view of a
serial section immunostained with anti-SOX18 antibodies. (Bar = 100 µm).
Figure II. SOX18 immunostaining is not detected in macrophage-rich areas. High
power view of serial sections corresponding to a human coronary artery lesion
immunostained using anti-SOX18 antibodies (sc-20100) and HAM56 monoclonal
antibody (a marker of macrophages). (Bar = 50 µm).
Figure III. PCNA positive cells in the endothelial luminal surface and in smooth
muscle cell-rich areas are endothelial and smooth muscle cells. Upper panel, colocalization of PCNA (red) with CD31 (green, endothelial cell marker) in the
endothelial luminal surface. Lower panel, co-localization of PCNA (red) with α-SMA
(green, smooth muscle cell marker) in a smooth muscle cell-rich area. Arrow heads
indicate co-localization. (Bar = 25 µm).
Figure IV. Antisense oligonucleotides against SOX18 inhibit DNA synthesis in
human endothelial cells (HUVEC). A, SOX18 mRNA levels in unstimulated cells (VEGF) and cells stimulated with 10 ng/mL VEGF-A (+ VEGF) treated or not with
oligonucleotides (10 µmol/L). B, HUVEC were exposed to increasing concentration
of antisense SOX18 ODNs (AS-SOX18; 0.2 to 10 µmol/L ) and DNA synthesis
([3H]thymidine incorporation) was assessed in the absence (white bars) or presence
(black bars) of 10 ng/mL VEGF-A. The effect of AS-SOX18, sense SOX18 (SE-
SOX18) and Random ODNs (10 µmol/L) is also shown in both basal and VEGF-Astimulated conditions (n=3 experiments performed in quadruplicate). P<0.05: *, vs.
Control; †, vs. VEGF-A-treated cells; ‡, vs. Control and VEGF-A treated cells.
2