© 1995 American Heart Association, Inc.
Articles |
Osteopontin Is Not a Marker for Proliferating Human Vascular Smooth Muscle Cells
From the Department of Medicine (C.M.N., B.C.B., P.K.M., C.M.S., P.L.W.), University of Cambridge, Addenbrooke's Hospital, Cambridge, UK, and the Department of Surgery (K.E.P.), University of Leicester, Leicester Royal Infirmary, Leicester, UK.
Correspondence to Dr C. Newman, Section of Cardiology, Clinical Sciences Centre, Northern General Hospital, Sheffield S5 7AU, UK. E-mail c.newman@sheffield.ac.uk.
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Abstract Osteopontin (OP) is a secreted glycoprotein that contains the Arg-Gly-Asp (RGD) cell-binding sequence that binds calcium and is chemotactic and adhesive for rat vascular smooth muscle cells (VSMCs). OP gene expression is upregulated in cultured rat VSMCs in vitro and after balloon carotid injury in vivo, suggesting that OP may be a marker for proliferating VSMCs in vivo and in vitro. Our in situ hybridization studies of human atherosclerotic coronary vessels, however, have shown OP mRNA expression in plaque macrophages but not in VSMCs. The current study investigated OP mRNA expression in cultured human VSMCs and macrophages and in an organ culture model of neointima formation in human saphenous vein. OP mRNA expression was not detected by Northern blot analysis of total RNA from subconfluent or confluent cultures of human VSMCs of any passage maintained in normal growth medium or after stimulation with TGFß1 (20 ng/mL), angiotensin II (1 µmol/L), or basic fibroblast growth factor (10 ng/mL) but was just detectable after stimulation with activated vitamin D3 (1 µmol/L). In contrast, cultured human macrophages expressed high levels of OP mRNA that were not dependent on lipid loading. OP mRNA was detected in isolated foci in all layers of saphenous veins maintained in organ culture for 14 days, including <2% of neointimal cells, a distribution that paralleled that of tissue macrophages. These results suggest that OP gene expression is not a marker for proliferation of human VSMCs in vitro and highlight a fundamental difference in the biology of human and rodent VSMCs.
Key Words: osteopontin • atherosclerosis • cells, vascular smooth muscle • macrophages • proliferation
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Dedifferentiation, proliferation, and migration of VSMCs are important features of many vascular pathologies, including atherosclerosis, restenosis after percutaneous angioplasty, and accelerated coronary vascular disease after cardiac transplantation.1 2 3 The molecular events that initiate, maintain, and accompany these processes have, therefore, become the focus of intense investigation.
OP is a highly acidic, secreted glycoprotein that binds calcium and contains the Arg-Gly-Asp (RGD) consensus motif for interaction with the integrin family of cell-adhesion molecules.4 Although originally isolated from bone calcified matrix,5 differential screening of a rat VSMC cDNA library subsequently identified OP as one of several genes upregulated in rat neointimal and pup medial VSMCs compared with uninjured adult medial VSMCs.6 7 With the use of a similar technique, we have shown that OP mRNA expression is upregulated in adult rat VSMCs that have proliferated in culture.8 This link with VSMC proliferation was confirmed by Gadeau et al,9 who demonstrated that OP is a delayed early (late G1) gene in rat VSMCs in culture. OP gene expression is also elevated in the neointima of the rat carotid artery after injury, with a time course and pattern that parallels VSMC proliferation.7 Consequently, OP gene expression has been used as a marker of VSMC activation in vivo.10
More recently, we have shown by in situ hybridization and immunocytochemistry that OP is also highly expressed in human coronary atherosclerotic plaque but not in the surrounding normal media,11 a finding that has subsequently been confirmed by others.12 Our data, however, showed that OP gene expression in these lesions largely colocalized with cells immunocytochemically identified as macrophages rather than VSMCs. This observed paucity of OP gene expression in plaque VSMCs may reflect the low frequency of VSMC proliferation in established lesions.13 Therefore in the present study we examined OP gene expression in human VSMCs under conditions more directly comparable with those in which rat VSMCs have been shown to express OP mRNA at very high levels.6 7 8 9 In particular, we have studied OP gene expression in human aortic VSMCs in culture, including the effects of a number of hormones and cytokines known to upregulate OP gene expression in rat VSMCs. We also examined OP gene and protein expression in a model of neointima formation in human saphenous vein in vitro, since this system shares many of the kinetic and cellular features of the response of the rat carotid artery to experimental balloon injury in vivo.14 15 Given our observations in human atherosclerotic plaques,11 we have also investigated OP gene expression in cultured human monocyte-derived macrophages, including the effects of lipid loading with modified LDL in vitro.
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VSMC Cultures
Primary human VSMC cultures were established from the aortic media of transplant organ donors by an explant method. Segments of intact abdominal aorta were first deendothelialized by rubbing with a sterile cotton bud. Visible atherosclerotic plaques were avoided or removed before strips of the underlying medial layer were taken and cut into 2-mm cubes, leaving the adventitial layer behind. Approximately 10 such cubes were placed into each well of a 12-well tissue culture plate (Nunc) and covered with 1 mL of M199 medium (Gibco BRL) supplemented with 20% (vol/vol) FCS, penicillin (100 U/mL), streptomycin (100 µg/mL), and amphotericin B (250 ng/mL). The medium was changed infrequently (approximately every 4 to 5 days) to maximize contact of each cube with the plastic surface. Migrating VSMCs appeared after
7
days, and cultures were passaged for the first time after 3 to 4
weeks. Established secondary cultures were maintained in the same
medium, and their identity as VSMCs was established by
immunocytochemistry for smooth muscle 
-actin using a mouse
monoclonal antibody (Clone 1A4, Sigma Chemical Co; dilution, 1:200) and
an FITC-conjugated anti-mouse secondary antibody (F2266, Sigma;
dilution, 1:1000) for detection. RNA was isolated from fully confluent
cultures or during logarithmic growth (50% to 75% confluent
cultures). When we studied the effects of TGFß1 (20
ng/mL, Austral Biologicals), angiotensin II (1 µmol/L,
Sigma), bFGF (10 ng/mL, R & D Systems), and 1,25-dihydroxyvitamin
D3 (1 µmol/L, Calbiochem) on OP mRNA levels, confluent
human VSMCs were incubated in serum-free M199 medium supplemented
with the appropriate factor for 24 hours before RNA isolation. For
comparative purposes, a number of human VSMC cultures were established
by enzymatic dispersion of 2-mm cubes of human aortic media,
essentially as described previously for dispersion of rat
aortae.16
Monocyte Purification and Culture
Human circulating monocytes were prepared by Percoll gradient
centrifugation with the use of a modification of the
method of Johnson et al.17 Approximately 200 mL of blood
was drawn from healthy volunteers into four 50-mL sterile plastic
centrifuge tubes (Falcon, Becton Dickinson Labware) containing
5 mL 3.8% (wt/vol) sodium citrate solution. The tubes were spun at
300g for 20 minutes at room temperature and the plasma
supernatant was removed, avoiding the buffy coat. One half of the
plasma was converted to serum by the addition of 75 mg of sterile
calcium chloride per 25 mL plasma. The remainder was spun at
2000g for 10 minutes at room temperature to generate
platelet-poor plasma. The volume of each erythrocyte/buffy coat
mixture was restored to 45 mL by the addition of sterile 0.9% (wt/vol)
sodium chloride solution, to which was added 5 mL of sterile 6%
(wt/vol) dextran in 0.9% (wt/vol) sodium chloride (dextran MW 500000,
Sigma). Once the erythrocytes had settled (30 to 40 minutes) the
supernatant containing the leukocytes was removed and spun at
300g for 7 minutes at room temperature. The leukocyte pellet
was resuspended in 50 mL Hanks' balanced salt solution (HBSS,
Sigma) and spun at 300g for 7 minutes. The four leukocyte
pellets were resuspended in a total volume of 2 mL of
platelet-poor plasma. This suspension was carefully applied to
the surface of a Percoll (Sigma) cushion consisting of 2 mL 51%
(vol/vol) Percoll in platelet-poor plasma overlain with 2 mL
42% (vol/vol) Percoll in platelet-poor plasma in a 10 mL
sterile centrifuge tube (Falcon), which was then spun at
300g for 10 minutes at room temperature. Under these
conditions, the leukocyte suspension separated into two broad bands,
with the upper being monocyte rich and the lower primarily
polymorphonuclear cells. The upper layer was aspirated and washed
twice in HBSS by centrifugation at 300g for
7 minutes at room temperature. The final pellet was resuspended in
Iscove's modification of Dulbecco's medium (Gibco BRL) supplemented
with 10% (vol/vol) autologous serum, penicillin (100 U/mL),
streptomycin (100 µg/mL), and human insulin (8 µg/mL), and the
cells were then plated into 24-well tissue culture plates (Falcon) at a
seeding density of 106 cells/well. Each culture
was washed with fresh medium after 3 hours of incubation at 37°C to
remove nonadherent cells. Cultures were maintained for at least 9 days
before analysis to allow differentiation into
macrophages. More than 95% of these cells were positive for
the CD68 macrophage marker as determined by immunocytochemistry
using a mouse monoclonal antibody (Clone EBM11, Dako; dilution 1:200)
and a horseradish peroxidase–based detection system
(StreptABComplex/HRPDuet, Dako) according to the manufacturer's
instructions. Human LDL, acetylated human LDL, and
lipoprotein-deficient human serum were prepared as previously
described.18 19
RNA Isolation
After trypsinization of cultured VSMCs, total cytoplasmic RNA
was isolated by lysis in 150 mmol/L NaCl, 10 mmol/L Tris (pH 7.4), 1
mmol/L MgCl2, and 0.5% (vol/vol) Nonidet P-40
(Sigma). The nuclei were pelleted by centrifugation,
and the supernatant was supplemented with 20% (wt/vol) sodium
dodecyl sulfate (SDS) to a final concentration of 1.5%
(wt/vol) before extracting twice with Tris-equilibrated phenol. The
RNA was precipitated with 10% (vol/vol) 3 mol/L sodium acetate (pH
5.2) and 250% (vol/vol) ethanol, and the RNA pellet was resuspended in
water. An identical procedure was used to isolate RNA from cultured
macrophages, except that the cells were scraped directly into
lysis buffer that was supplemented with 10 mmol/L ribonucleoside
vanadyl complexes (Sigma) to inhibit RNAse activity.
Northern Blot Analysis
Total cytoplasmic RNA (10 to 15 µg per lane) was
electrophoresed in 1.5% agarose gels containing 2.2 mol/L
formaldehyde in a buffer containing 20 mmol/L MOPS, 1 mmol/L
EDTA, 5 mmol/L sodium acetate, and 0.5 µg/mL ethidium bromide
(EtBr). The integrity of the RNA was visualized by UV illumination of
gels before and after transfer to Hybond-N (Amersham International) as
specified by the manufacturer. Filters were hybridized as described
previously.20 32P-labeled cDNA probes were
generated from purified insert DNA using an oligolabeling kit
(Pharmacia). Filters were washed three times at 65°C (10, 30, and 60
minutes) in 0.1xSSC (SSC contains 150 mmol/L NaCl and 15 mmol/L sodium
citrate)/0.1% (wt/vol) SDS before exposure to Fuji RX x-ray film.
The human OP probe was generated from a full-length cDNA
clone of 1.4 kb that was obtained from the ATCC/NIH repository
(Rockville, MD). The SM22 probe was generated from a 1.0-kb rat cDNA
clone (3RF10), which has been shown to cross-hybridize with human
mRNA under the conditions described above.11 Total
cytoplasmic RNA isolated from passage 12 rat VSMCs was used as a
positive control for OP mRNA expression.8 Total RNA from
human abdominal aortic media was prepared after enzymatic dispersion of
deendothelialized donor aorta from which the
adventitia had also been stripped; visible atherosclerotic material was
also removed before enzymatic digestion.
RT-PCR Analysis
Total cellular RNA (2 µg) was reverse-transcribed in a
20-µL reaction containing 30 U of AMV reverse transcriptase (Super
RT, HT Biotechnology Ltd) and 50 µg/mL oligo (dT)12-18 primer
(Pharmacia). The reaction mix also contained 50 mmol/L Tris-HCl (pH
8.3), 50 mmol/L KCl, 4 mmol/L DTT, 10 mmol/L MgCl2,
1 mmol/L each of dGTP, dATP, dCTP, and dTTP, and RNAsin (1 U/µL)
(Promega). Control reactions without reverse transcriptase were
performed for each RNA analyzed. A further control reaction
without RNA was also performed. Reactions were performed at 41°C for
1 hour, followed by 10 minutes at 80°C before being placed on ice.
Each completed reaction was diluted with water to yield a 100-µL cDNA
stock.
For PCR amplification, each 25-µL reaction mix contained 3-µL diluted cDNA stock, 250 ng of each primer, 1 U Thermus aquaticus DNA polymerase (Promega), 10 mmol/L Tris-HCl (pH 9.0), 50 mmol/L KCl, 1 mmol/L MgCl2, and 0.1% (vol/vol) Triton X-100. The cycling parameters were 94°C for 2 minutes, 55°C for 90 seconds, and extension at 72°C for 2 minutes for 30 cycles, with a final extension period of 6 minutes at 72°C. A 5-µL aliquot of each reaction was electrophoresed through a 1% (wt/vol) agarose gel, and the DNA was visualized by EtBr staining under ultraviolet light transillumination. The oligonucleotide primers for human OP were 5' ACTGATTTTCCCACGGAC 3' (forward, nucleotides 466 to 483 of cDNA21 ) and 5' ATGGCTGTGGAATTCACG 3' (reverse, nucleotides 877 to 894 of cDNA), giving a predicted product size of 428 bp. The identity of the PCR fragment was confirmed by Southern blot analysis using a 32P-labeled full-length human OP probe (data not shown). Control primers were designed to human ß2- microglobulin, 5' GATTCAGGTTTACTCACG 3' (forward, nucleotides 51 through 68 of cDNA sequence, exon 2,22 23 ) and 5' CCATGATGCTGCTTACATG 3' (reverse, nucleotides 327 to 345 of cDNA sequence, exon 3), giving a predicted product size of 294 bp.
Organ Culture of Human Saphenous Vein
Surplus saphenous vein was obtained from patients undergoing
coronary artery bypass surgery and was prepared and cultured
essentially as described by Soyombo et al.14 Briefly,
excess fat and adventitial tissue were removed, and the extreme ends of
the vein were excised with a scalpel blade and discarded. The remainder
of the sample (usually 2 to 3 cm in length) was cut into 0.5-cm rings
with a scalpel blade. Each ring was then opened by cutting along its
length with fine scissors. One segment was incubated for 1 minute at
room temperature in phosphate-buffered saline (PBS), pH 7.4,
containing 0.2% (wt/vol) trypan blue (Sigma) to visually assess
endothelial integrity. The remaining segments were
prepared for organ culture if endothelial coverage was
estimated to be at least 60%. Individual vein segments,
endothelial surface uppermost, were placed on top of a
2-cmx1-cm square polyester cloth (P500, Henry Simon) and were pinned
into a layer of set Sylgard resin (Sylgard 184, Dow Corning) in 55-mm
glass Petri dishes using minuten pins (size A1, Watkins and Doncaster).
Segments were cultured in RPMI 1640 tissue culture medium containing 2
g/L sodium bicarbonate, penicillin (100 U/mL), streptomycin (100
µg/mL), amphotericin B (250 ng/mL), and 30% (vol/vol) FCS. Cultures
were conducted at 37°C in a humidified incubator equilibrated with
5% (vol/vol) CO2 for 2 weeks. The medium was replaced
every 2 to 3 days.
In Situ Hybridization
Segments of human saphenous vein were mounted in
Tissue-Tek OCT embedding compound (Miles Ames Division, Inc),
snap-frozen in liquid nitrogen, and subsequently stored at
-70°C before sectioning. Sections were cut (8 to 10 µm) and
mounted onto gelatinized slides, refrozen, and stored at -70°C
until use. The sections were processed for in situ hybridization as
previously described.24 35S-UTP-labeled sense
and antisense OP riboprobes were generated by in vitro transcription
from the T7 and T3 promoters of a Bluescript SK–plasmid containing the
same full-length OP cDNA that was used for Northern blot
analysis.
Immunocytochemistry
The studies shown in Figs 6
and 7 were performed with the
use of 5 µm transverse sections of saphenous vein that had been fixed
for 4 to 18 hours in freshly prepared 4% (wt/vol)
paraformaldehyde in PBS before paraffin embedding.
Sections were stained with hematoxylin and eosin to identify basic
tissue morphology and with elastic Ponceau S to identify elastic and
collagen fibers. Tissue macrophages were identified with the
use of a monoclonal anti-human CD68 antibody (Dako PG-M1) and VSMCs
with the use of a monoclonal anti–human smooth muscle actin
antibody (Dako 1A4). Both were visualized with the use of a horseradish
peroxidase–based detection system (ABComplex/HRP, Dako) according
to the manufacturer's instructions. Controls were performed
substituting the primary antibody with PBS.
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Characteristics of Human VSMCs in Culture
Explant cultures of human VSMCs were established from organ donors ranging in age from 2 to 26 years and were studied between passages 5 and 16. All cultures exhibited a typical hill and valley morphology, stained positively for
–smooth muscle actin, and
exhibited a cell doubling time of 48 hours. Many cultures also
formed nodules.
Expression of OP mRNA in Cultured Human VSMCs as Seen on Northern
Blot Analysis
OP mRNA was consistently detectable by Northern blot
analysis of total RNA from rat VSMCs, despite the use of a
32P-labeled probe generated from the human cDNA and after
washing the filters at high stringency (Fig 1). In contrast, OP mRNA expression was
not detected in RNA isolated from subconfluent or confluent human
explant VSMC cultures of any passage from any donor grown in medium
containing 20% (vol/vol) FCS throughout (Fig 1).
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We considered the possibility that this failure to detect OP
mRNA in human VSMCs was related to the means by which our primary
cultures were generated, because the rat VSMCs had been obtained by
enzyme dispersion, whereas the human cells were prepared by an
explant method. OP mRNA was just detectable in freshly
dispersed human aortic media, as we have previously
reported,11 but not in RNA from secondary human VSMC
cultures, whether initially isolated by enzyme dispersion or by explant
(Fig 2). Each culture expressed the mRNA
for SM22, a smooth muscle–specific gene that we have previously
shown to be expressed by all rat VSMCs, whether freshly dispersed or
passaged in culture.8
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To investigate whether human VSMCs in culture can be induced to
express OP mRNA, confluent VSMCs isolated from a 2-year-old donor
(passage 6) were exposed to a number of factors known to upregulate OP
mRNA in cultured rat VSMCs7 and/or other cell
types.4 OP mRNA was not detectable by Northern blot
analysis of RNA isolated 24 hours after treatment with bFGF,
angiotensin II, or TGFß1 (Fig 3, lanes b through d). OP mRNA was just detectable after
treatment with 1,25-dihydroxyvitamin D3 (Fig 3, lane a), which has been shown to upregulate OP mRNA
expression in a number of cell types.4
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Detection of OP mRNA by RT-PCR
To increase the sensitivity of OP mRNA detection, further
aliquots of the RNA extracted from the VSMCs of the 2-year-old
donor (passage 6, confluent) were reverse-transcribed into cDNA and
amplified using specific primers for human OP. cDNAs prepared by
reverse transcription of RNA prepared from cultured
monocyte–derived human macrophages and human kidney (the
latter a kind gift of Dr Fiona Karet, Addenbrooke's Hospital) were
used as positive controls. OP mRNA was again detected in the VSMC
culture treated with 1,25-dihydroxyvitamin D3 (lane c, Fig 4) but was also detected in control cells incubated in
20% (vol/vol) FCS throughout (lane a, Fig 4). In a
separate experiment, aliquots of each PCR reaction were removed after
15, 20, 25, and 30 cycles. The amplified product was detectable
after 15 cycles with the use of cDNA from VSMCs treated with
1,25-dihydroxyvitamin D3, macrophages, and
kidney but was detectable after 20 cycles with the use of cDNA from
VSMCs maintained in 20% (vol/vol) FCS alone (data not shown). Control
reactions with the use of primers for human
ß2-microglobulin indicated equal cDNA loading in each
tube.
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Expression of OP mRNA in Cultured Human
Macrophages
We have previously shown that macrophage-derived
foam cells in human atherosclerotic plaque express high levels of OP
mRNA.11 Hirota et al25 also detected OP mRNA
in association with plaque macrophages but not in human Kupffer
cells or alveolar macrophages. We therefore investigated
whether OP mRNA expression in human macrophages is dependent on
lipid loading, as occurs in the foam cells of atherosclerotic lesions.
To do this, human monocyte-derived macrophages were
cultured in the presence or absence of acetylated human LDL,
which has been used to generate foam cells in vitro.26 OP
mRNA was highly expressed in cultured human macrophages
maintained in 10% (vol/vol) autologous serum for 14 days (Fig 5). Expression was not substantially
affected by incubation in 10% (vol/vol) lipoprotein-deficient
human serum for the last 5 days before RNA isolation or by the addition
of acetylated human LDL (50 µg/mL) to the standard growth
medium for the same period. Similarly, incubation of human
macrophages in medium supplemented with 50 µg/mL native human
LDL had no effect on OP mRNA expression (data not shown). Similar
results were obtained when macrophages were harvested after 9
days in culture (data not shown). These data suggest that in culture at
least the high level of OP mRNA expression by human
monocyte-derived macrophages is not dependent on the lipid
content of the growth medium.
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OP mRNA and Protein Expression in Human Saphenous Vein in
Culture
Transverse sections of saphenous vein segments cultured for 14
days showed the development of a neointima, 10 to 70 µm
in depth, which was hypercellular (Fig 6D
) but devoid of
collagen or elastic fibers (Fig 6E). As previously
described14 most of the cells within the
neointima were immunocytochemically identified as VSMCs
(Fig 7A). A small proportion (<2%) of the
neointimal cells, however, stained positively for the CD68
macrophage marker (Fig 7B). Furthermore,
scattered cells throughout the media and adventitia of both freshly
isolated and cultured veins also stained positively for the
macrophage marker (Fig 6C and 6F). The
distribution of OP mRNA expression in cultured veins was strikingly
similar in that isolated foci of hybridization were seen throughout the
media and adventitia, with positive cells occasionally within the
neointima (Fig 8D). No specific
hybridization was seen in freshly isolated veins (Fig 8B) or when the control (sense) probe was used (data
not shown). In situ hybridization for OP mRNA was also performed using
human kidney as a positive control. Positive hybridization was
localized to distal convoluted tubules (data not shown). Specific
hybridization to glomeruli or blood vessels was not
detected.
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Frozen sections of cultured human saphenous vein and human kidney were also stained for the presence of OP protein with the use of a goat anti-human urinary OP polyclonal antiserum (OP189) kindly provided by Dr Cecilia Giachelli, Department of Pathology, University of Washington, Seattle. No specific staining of cultured saphenous vein was observed over a wide range of primary antibody concentrations, despite clear specific staining of human kidney in a distribution similar to that observed for OP mRNA in human kidney (data not shown). Identical results were obtained using sections of human saphenous vein and kidney that had been fixed in paraformaldehyde and paraffin embedded (data not shown).
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This study highlights a major difference between the behavior of human and rat VSMCs in culture. OP mRNA is barely detectable in freshly isolated rat aortic medial VSMCs but is dramatically upregulated after proliferation in culture.8 In contrast, we have shown that OP mRNA is barely detectable in cultured human VSMCs. TGFß1, bFGF, and angiotensin II, all of which upregulate OP gene expression in cultured rat VSMCs7 and which may influence neointimal development in vivo, had no effect on OP gene expression in cultured human VSMCs. 1,25-dihydroxyvitamin D3 had a small stimulatory effect compatible with previous evidence that OP expression is positively regulated at the transcriptional level by this hormone.27 In contrast, in culture we have shown that human monocyte-derived macrophages express high levels of OP mRNA that are not dependent on lipid loading in vitro. OP is also expressed by osteoclasts, cells that are derived from the same progenitor lineage as circulating monocytes, and is essential for bone resorption and remodeling.28 The function of OP in the vasculature is less clearly understood, but possible roles in several aspects of atherogenesis have been suggested (see References 11, 12 and 2911 12 29 , and below).
Detailed analysis of OP gene expression in synchronized cultures of rat VSMCs has shown that OP is a "delayed early" cell cycle gene in these cells.9 OP gene expression is also detectable at high levels in asynchronously cycling,9 confluent,6 8 and postconfluent7 cultures of rat VSMCs. Taken together these data suggest that there is an important relationship between current or recent proliferation and OP gene expression in rat VSMCs. We have investigated OP gene expression in cultured human VSMCs under very similar conditions, including subconfluent cultures during logarthmic growth and cultures that have reached confluence. Explant-derived human VSMC cultures were used for these experiments because unlike enzyme-dispersed human VSMCS these cells proliferate readily in culture.30 Despite this, OP gene expression was barely detectable in explant-derived human VSMCs from any donor or at any passage.
Our data suggest that upregulation of OP gene expression in human VSMCs
does not accompany proliferation in culture and hence is not a useful
marker thereof. Indeed, the observation that OP mRNA is just detectable
by Northern blot analysis of RNA from freshly
enzyme-dispersed human aortic media suggests that OP gene
expression by human VSMCs may be downregulated rather than upregulated
during proliferation in culture. It is possible, however, that freshly
enzyme-digested human donor aorta may have contained a number of
OP-expressing macrophages, giving rise to the weak signal shown
in Fig 2, lane a. Detailed investigation of OP gene
expression throughout the cell cycle of pure populations of cultured
human VSMCs would be required to distinguish between these
possibilities.
Proliferation of rat VSMCs in culture is accompanied by
significant downregulation of smooth muscle–specific proteins (eg,
smooth muscle -actin and myosin heavy
chain8 31 32 33 ), suggesting that such cells dedifferentiate
under these conditions. It is therefore conceivable that upregulation
of OP gene expression in cultured rat VSMCs is dependent on
dedifferentiation as well as proliferation. Indeed, we have shown that
OP gene expression is maximal in late passage (passage 12) cultured rat
VSMCs, which are also maximally dedifferentiated as assessed by smooth
muscle–specific gene expression. Furthermore, the cell-cycle
experiments performed by Gadeau et al9 used rat VSMCs that
had reached passage 17 in culture.
It has recently been shown that enzyme-dispersed human aortic VSMCs
maintain expression of smooth muscle–specific -actin and
myosin at high levels over many passages in culture,30
suggesting that such cells remain highly differentiated. In contrast,
explant-derived human VSMCs express these differentiation markers
at low levels and proliferate readily in culture,30
features similar to those observed in rat VSMCs under similar
conditions. It was for this reason that explant-derived human VSMC
cultures were used in the present studies. Nevertheless it is
conceivable that the failure of human VSMCs to upregulate OP gene
expression in culture may relate to a difference in the pattern of
dedifferentiation in association with proliferation in rat and human
VSMCs under these conditions. We therefore went on to investigate OP
gene expression in a separate model of human VSMC proliferation and
dedifferentiation, namely neointima formation in human
saphenous vein maintained in organ culture for 14
days.14 15 The neointima that forms under
these conditions is largely composed of VSMCs embedded in an
extracellular matrix, which is rich in
glycosaminoglycans (K.E. Porter, unpublished
observations) but does not contain collagen or elastic fibers (see
References 14 and 1514 15 and Fig 6 of the present
study). The proliferative index of these cells is maximal after 14
days, reaching 40% to 50%.14 15 Furthermore, the
neointimal VSMCs contain abundant secretory organelles and
rough endoplasmic reticulum, (K.E. Porter, unpublished observations)
suggestive of a dedifferentiated "secretory"
phenotype.34 We therefore felt that any changes in
VSMC differentiation and proliferative status that accompany
neointima formation in the balloon-injured rat carotid
artery were also likely to have occurred during neointima
formation in the human saphenous vein. Despite this, OP gene expression
was detected in <2% of neointimal cells in cultured human
saphenous vein, with scattered cells throughout the media and
adventitia also showing positive hybridization. Interestingly, this
pattern of OP gene expression paralleled that of tissue
macrophages, although we cannot exclude the possibility of OP
mRNA expression by a small subpopulation of VSMCs. OP gene expression
was not detectable in freshly isolated veins, suggesting that the cells
that express OP mRNA must have become activated to express OP
mRNA during the 14-day culture period. OP protein was completely
undetectable in cultured saphenous vein. This may reflect a lag between
activation of gene transcription and translation or expression at
levels undetectable with the use of the available antiserum. Overall,
it is clear that neither OP gene nor protein expression is a marker of
human VSMC proliferation in vitro.
The observed paucity of OP gene expression by cultured human VSMCs is in agreement with our previous in situ hybridization data showing high levels of OP mRNA expression by macrophages rather than VSMCs in the established human coronary atherosclerotic plaque.11 However, with the use of in situ hybridization Giachelli et al12 have recently reported detectable OP mRNA in human atherectomy specimens. As in our own study most mRNA expression was detected in areas of lipid accumulation and calcium deposits. Their detection of OP mRNA in association with VSMCs in such lesions contrasts with our own data, although both groups agree that OP protein is detectable in areas of VSMC accumulation. The reason for this apparent discrepancy is not clear. However, since human VSMCs can be induced to express OP mRNA, and since we could detect low levels of expression in unstimulated cultured human medial VSMCs with the use of RT-PCR, it is likely that our results differ quantitatively rather than qualitatively. More importantly, it is possible that VSMCs within established lesions exhibit a pattern of OP gene expression different from those within the media or cultured in vitro, and we are therefore currently studying OP gene expression in human VSMCs cultured directly from atherosclerotic plaque.
Does OP play a role in the pathogenesis of human vascular lesions in
vivo? One hypothesis would be that OP secreted by macrophages
in early vascular lesions promotes VSMC migration into the developing
neointima and encourages residency of both VSMCs and
macrophages therein through its adhesive
properties.35 OP may also be involved in other aspects of
atherogenesis, specifically in the progression and/or regulation of
plaque calcification.11 12 29 Definitive evidence for such
suggestions will require experiments in which OP is either
overexpressed or inhibited in models of vascular disease. In this
regard, it is interesting to note that recent data indicate that
administration of an antiplatelet glycoprotein
GpIIb/IIIa (IIBß3 integrin) antibody fragment reduces the
incidence of clinical restenosis after coronary
intervention.36 While designed to act as a platelet
fibrinogen receptor antagonist, it is intriguing to
speculate that this antibody may also affect the function of OP by
inhibiting interaction with the vß3 integrin with which it
cross-reacts.37
The differences between the behavior of rat and human VSMCs in culture add to previous concerns that animal models of vascular disease may bear little relation to the clinical problem, particularly restenosis after angioplasty.38 Indeed, many agents known to have beneficial effects in such models have proved ineffective in clinical trials in humans.39 40 Studies of atherogenesis and other vascular pathologies should therefore be carried out using human cells and tissues wherever possible.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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Dr Newman is supported by a Clinician Scientist Fellowship from the Medical Research Council. Drs Porter, Shanahan, and Prof Weissberg are supported by the British Heart Foundation. We would like to acknowledge the cooperation of the transplant team at Addenbrooke's and Papworth hospitals. We thank Jo Horsley and Carolyn Belcher for their excellent technical assistance and Dr Nat Cary for his helpful advice. Dr Newman is now at the University of Sheffield, Sheffield, UK.
Received April 25, 1995; accepted August 29, 1995.
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References |
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