© 1999 American Heart Association, Inc.
Vascular Biology |
Subendothelial Cells From Normal Bovine Arteries Exhibit Autonomous Growth and Constitutively Activated Intracellular Signaling
From the Developmental Lung Biology Research Laboratory, Department of Pediatrics (M.G.F., A.A.A., K.R.S.), and the Division of Renal Diseases and Hypertension, Department of Medicine (R.A.N., R.H.), University of Colorado Health Sciences Center, Denver; the Institute of Molecular Medicine (A.A.A.), National Center for Cardiology and Internal Medicine, Bishkek, Kyrgyzstan; and the Department of Medicine (J.Y.W.), National Jewish Medical and Research Center, Denver, Colo.
Correspondence to Maria G. Frid, PhD, Developmental Lung Biology Research Laboratory, University of Colorado Health Sciences Center, Box B131, 4200 E 9th Ave, Denver, CO 80262. E-mail Maria.Frid{at}UCHSC.edu
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Abstract—The arterial media is comprised of heterogeneous smooth muscle cell (SMC) subpopulations with markedly different growth responses to pathophysiological stimuli. Little information exists regarding the intracellular signaling pathways that contribute to these differences. Therefore, we investigated the growth-related signaling pathways in a unique subset of subendothelial SMCs (L1 cells) from normal, mature, bovine arteries and compared them with those in "traditional" SMCs derived from the middle media (L2 SMCs). Subendothelial L1 cells exhibited serum-independent autonomous growth, not observed in L2 SMCs. Autonomous growth of L1 cells was driven largely by the constitutively activated extracellular signal–regulated kinase (ERK-1/2) cascade. Inhibition of upstream activators of ERKs (MAP kinase kinase-1, p21ras, receptor tyrosine kinases, and Gi protein–coupled receptors) led to suppression of autonomous growth in these cells. L1 cells also exhibited constitutive activation of important downstream targets of ERKs (cytosolic phospholipase A2, cyclooxygenase-2) and secreted large amounts of prostaglandins. Importantly, L1 cells secreted potent mitogenic factor(s), which could potentially contribute in an autocrine fashion to the constitutive activation of these cells. Our data suggest that unique arterial cells with autonomous growth potential and constitutively activated signaling pathways exist in normal arteries and may contribute selectively to the pathogenesis of vascular diseases.
Key Words: smooth muscle cell proliferation • heterogeneity • mitogen-activated protein kinase • G proteins • prostaglandins
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Introduction |
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The pathogenesis of many cardiovascular diseases such as arteriosclerosis, postangioplasty restenosis, pulmonary hypertension, and diabetic vasculopathy is characterized by abnormal proliferation of arterial smooth muscle cells (SMCs), resulting in intimal and/or medial thickening. Numerous studies in animal models and in humans have demonstrated that cells in the neointima differ markedly from SMCs in the tunica media and that many of the biochemical and proliferative characteristics of neointimal cells are similar to those displayed by embryonic, fetal, and/or neonatal vascular SMCs (reviewed in Reference 11 ). It has been suggested that the accumulation of nonmuscle-like cells in the neointima may not simply be the result of a "dedifferentiation" of mature SMCs migrating from the tunica media but is potentially the result of expansion of a phenotypically unique subset of vascular cells.2 3 4 5 This hypothesis is supported by both in vivo and in vitro studies demonstrating that relatively undifferentiated SMCs with enhanced growth potential exist in the uninjured vascular media.6 7 8 9 10 We have previously reported that a unique subset of SMCs could be isolated from the subendothelial aspect of normal, mature, bovine arterial media.11 These phenotypically unique subendothelial cells had the ability for serum-independent growth and exhibited remarkably augmented growth in response to various growth-modulating stimuli, including hypoxia, compared with SMCs derived from adjacent middle media. The mechanisms responsible for the augmented growth potential of subendothelial cells are presently unknown; however, a better understanding of these mechanisms could facilitate the design of more specific treatment strategies.
Proliferation in most cell types is driven by activation of the mitogen-activated protein kinases (MAPKs) p44mapk and p42mapk, also known as extracellular signal–regulated kinases (ERKs), ERK-1 and ERK-2.12 Activation of the ERK pathway is initiated by ligand binding to cell-surface receptors and is mediated largely through 2 major receptor-mediated signaling pathways: receptor tyrosine kinases (RTKs) and G protein–coupled receptors (GPCRs).13 14 One pathway involves the activation of RTKs by traditional polypeptide growth factors, such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and basic fibroblast growth factor (bFGF).13 A second signaling pathway acts through 7 membrane-spanning receptors coupled to heterotrimeric GTP-binding proteins (G proteins).14 Several ligands that signal via distinct G-protein subtypes have been shown to elicit mitogenic responses. Thrombin and lysophosphatidic acid have been shown to stimulate growth via pertussis toxin–sensitive GiPCRs.15 16 In some but not all cells, vasoactive agents such as angiotensin II and endothelin, which act through GqPCRs, have also been shown to initiate proliferation.17 There is accumulating evidence that activation of 1 particular receptor-mediated signaling pathway can amplify intracellular signaling within a parallel but separate pathway.18 Additionally, continuous or "constitutive" activation of the MAPK pathway, as might occur in a cell secreting autocrine growth factors acting via either GPCRs or RTKs, could lead to a "priming" effect on the cell, rendering it more susceptible to activation by other mitogenic stimuli and resulting in subsequent proliferation.18
Studies in established cell lines and transformed cells from tumors have dissected in great detail the intracellular signaling mechanisms regulating enhanced growth of these cells. However, the signaling pathways contributing to the enhanced proliferative capacity of unique subsets of SMCs in the arterial wall remain obscure. The purpose of the present study, therefore, was to investigate the growth-related ERK pathway(s) in a unique subset of subendothelial SMCs (termed L1 cells) derived from normal, mature, bovine arteries, which exhibit serum-independent, self-driven growth. We also assessed the members of the eicosanoid production pathway, previously shown to be important in controlling SMC proliferation, viz, cytosolic phospholipase A2 (cPLA2), the inducible form of cyclooxygenase (COX-2), and prostaglandins (PGE2 and PGI2).19 20 For comparison purposes, we used SMCs isolated from the middle media (termed L2 SMCs), which exhibit a relatively slow, serum-dependent growth pattern traditionally described for vascular SMCs from the conducting, large, elastic arteries of large mammalian species.
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Isolation of Cell Subpopulations
Main pulmonary arteries and aortic arches were obtained from adult (2-year-old) cows at a local slaughterhouse. Vessels were cut open, and the endothelium was removed by gentle scraping of the luminal surface of the vessel with a scalpel blade. The tunica media was then mechanically dissected into a very thin subendothelial layer and a middle layer, and tissue explants from each medial layer were incubated in complete Dulbecco’s modified Eagle’s medium (DMEM, Sigma Chemical Co) with either 10% calf serum (HyClone Laboratories) or 10% plasma-derived serum (Cocalico Biologicals, Inc). Because previous in vivo studies had demonstrated the presence of nonmuscle-like cells in the subendothelial compartment of the mature, bovine arterial media,21 our objective was to selectively isolate these cells in culture. In primary cultures of subendothelial tissue explants incubated in 10% calf serum, 2 morphologically distinct cell subpopulations were observed. Cells of 1 subpopulation (hereafter termed L1 cells), comprising <10% of all cell colonies, appeared small and rhomboidal and at confluence, formed a dense, multilayered network. Cells of the other subpopulation appeared bipolar or spindle-shaped and at confluence, formed the "hill-and-valley" pattern traditionally described for SMCs. To selectively isolate cell colonies with a rhomboidal appearance (L1 cell colonies), we used "ring-based" isolation techniques. In brief, to isolate individual colonies of cells with a distinct rhomboidal appearance, plastic or Teflon rings (5 to 10 mm in diameter; greased on the bottom) were placed over each cell colony of interest. Cells within the ring were trypsinized and transferred to a 24-multiwell plate for expansion. Because small rhomboidal cells represented only a minority of cell colonies in primary cultures from subendothelial media grown in 10% serum, attempts were made to selectively increase the number of this cell type in primary culture. Because use of plasma instead of serum had previously been shown to allow selective growth of unique SMC subpopulations,7 we incubated subendothelial tissue explants in medium supplemented with 10% plasma-containing DMEM. Under these conditions, rhomboidal cell colonies were observed to predominate (>90%). When rhomboidal-appearing L1 cells initially grown in 10% plasma were subsequently subcultured in 10% serum-containing medium, their characteristic rhomboidal appearance did not change. Thus, utilizing selective isolation techniques and/or culture medium supplemented with 10% plasma, we were able to isolate and expand in culture a subpopulation of nonmuscle-like cells. Virtually all cell colonies isolated from the middle media (L2) explants grown in 10% serum exhibited uniform morphological characteristics. These cells (hereafter termed L2 SMCs) exhibited a spindle-shaped, bipolar appearance and at confluence, showed the hill-and-valley pattern traditionally described for vascular SMCs. No cell growth was observed when middle-media explants were incubated in 10% plasma.
All experiments were carried out on cells at passages 3 to 7. Cell cultures were tested for Mycoplasma contamination by using a Gen-Probe Mycoplasma TC rapid detection system (Gen-Probe Inc) and were negative for such contamination.
Cell Growth Assays
Cells were plated onto 24-multiwell plates at a density of 10 or
20x103 cells per well in DMEM supplemented with
10% calf serum. On day 1, cells were rinsed with PBS and DMEM
supplemented with 0.1% calf serum, 10% calf serum, 10% plasma, or
10% medium conditioned by L1 cells for 72 hours (termed L1-conditioned
medium). Cells (4 wells for each condition) were trypsinized and
counted at different time points in a standard SPotlite hemacytometer
(Baxter). Data were expressed as cell numberx1000 per well.
DNA Synthesis Assay
DNA synthesis in distinct cell subpopulations in response to
various mitogens, inhibitors, serum, or L1-conditioned
medium (see description above) was determined by measuring
[3H]thymidine incorporation as described
elsewhere.11 The following purified peptide mitogens were
used in this study: PDGF-BB (10 ng/mL, Bachem Fine Chemicals), bFGF (30
ng/mL, Bachem), EGF (100 nmol/L, UBI), thrombin (0.01 U/mL, Sigma),
endothelin-1 (500 nmol/L, Sigma), and lysophosphatidic acid (10
µmol/L, Sigma). The mitogen doses used in this study had been
previously shown to elicit maximal responses in bovine SMCs (data not
shown). The following inhibitors were used in the study:
pertussis toxin (potent inhibitor of the GiPCR pathway; 100
ng/mL 3 hours before the addition of mitogens; List Biologicals),
genistein (inhibitor of RTKs; 100 nmol/L 15 minutes
before the addition of mitogens; UBI), PD98059 (a specific MAP kinase
kinase-1 [MEK-1] inhibitor; 10 µmol/L; Biomol
Research), BZA-5B (a farnesyl transferase inhibitor that
blocks activation of p21ras; 100 µmol/L;
Genentech), indomethacin and sulindac sulfide (both
nonselective COX inhibitors; both at 10 µmol/L;
Sigma), and nordihydroguaiaretic acid (a nonselective
lipoxygenase inhibitor; 5 µmol/L;
Sigma).
Conditioned Medium and Coculture Experiments
To determine whether L1 cells secrete growth-modulating
activity, both live coculture experiments as well as conditioned medium
experiments were performed. Coculture experiments with L1 cells as the
source cells and growth-arrested L2 SMCs as the target cells were
performed as previously described by Majack and colleagues (Cook et
al).22 For conditioned medium experiments, source L1 cells
at 90% confluence were rinsed with PBS and maintained in
serum-deprived (0.1% calf serum) medium for 72 hours, and then this
conditioned medium was collected and either added fresh to
growth-arrested target L2 SMCs, along with
[3H]thymidine (0.5 µCi/mL) for 24 hours to
assess DNA synthesis, or divided into aliquots and snap-frozen in
LN2. Freezing/thawing of the conditioned medium
did not change its growth-promoting effect. Both live coculture and
conditioned medium experiments yielded similar results.
Measurement of MAPK Activity
MAPK activity was analyzed by an immunocomplex kinase
assay as described previously.23 Subconfluent cell
cultures in 6-well plates were serum deprived (0.1% calf serum) for 72
hours and then stimulated for 5 minutes with 10% calf serum or
individual mitogens. Cells were then washed with ice-cold PBS and lysed
in 0.3 mL of lysis buffer (25 mmol/L Tris-HCl, pH 7.2; 50
mmol/L ß-glycerophosphate; 0.5% Triton X-100; 1 mmol/L EGTA;
100 µmol/L Na3VO4;
1 mmol/L DTT; 2 mmol/L MgCl2; 0.06 U/mL
aprotinin; 0.1 mmol/L PMSF; and 20 µmol/L leupeptin).
Insoluble material was pelleted by centrifugation
(14 000g for 10 minutes), and supernatants were quantitated
by the Bio-Rad protein assay according to Bradford (Bio-Rad
Laboratories). ERK-1 and ERK-2 were immunoprecipitated by incubation
for 2 hours with anti–ERK-1 and anti–ERK-2 antibodies (Santa Cruz
Biotechnology) conjugated to protein A–Sepharose beads. The beads were
washed in lysis buffer, and kinase activity was determined by using an
EGF-receptor peptide as previously described.23
Results were expressed as picomoles of
[32P]phosphate incorporated into substrate per
minute per milligram of extracted protein and are
representative of at least 3 independent experiments
performed in duplicate.
Measurement of cPLA2 Activity and PG
Production
Measurement of cPLA2 activity was
performed as previously described.24 In brief, cells were
minced with a Dounce homogenizer in
homogenization buffer, pH 7.4 (250 mmol/L
sucrose, 50 mmol/L HEPES, 1 mmol/L EDTA, and 1 mmol/L
EGTA) with protease inhibitors (20 µmol/L pepstatin,
20 µmol/L leupeptin, 0.1 mmol/L PMSF, and 1000 kU/mL
aprotinin). Extracts were centrifuged at 100 000g
for 1 hour. Supernatants were incubated with 15 µmol/L
[14C]arachidonylphosphatidylcholine (Amersham
Corp) in the presence of 4 µmol/L CaCl2
for 30 minutes at 37°C. The reaction was quenched with the addition
of 40 mL of ethanol:acetic acid (98:2) containing 500 µg/mL
arachidonic acid. Samples were analyzed by
thin-layer chromatography by using the organic phase of
an ethyl acetate:isooctane:acetic acid:water (55:75:8:100) mixture.
Plates were stained with I2 vapor, and bands
corresponding to arachidonic acid and
phosphatidylcholine peaks were scraped and counted by liquid
scintillation counting. Results are expressed as picomoles per minute
per milligram protein.
For the PG production assay, cells were grown in 24-well plates
to subconfluence and then incubated in serum-depleted (0.1% calf
serum) medium for 72 hours, which was replaced with fresh, serum-free
medium for 30 minutes. PGE2 and
PGI2 (the latter measured as its stable analogue
6-keto-PGF2
) released into the medium were
determined by using an ELISA kit (Cayman Chemical, Inc) and following
the manufacturer’s protocol. Results are presented as
picograms of PG secreted per cell number per hour.
Immunoblotting of cPLA2 and COX
Isoforms
Extracts matched for protein content were analyzed by
8% SDS–polyacrylamide gel electrophoresis and Western
blotting with specific antibodies (monoclonal
anti-cPLA2 antibody and goat polyclonal
anti–COX-2 antibodies, both purchased from Santa Cruz, Inc, and both
used at a dilution of 1:200). Secondary antibodies were conjugated with
horseradish peroxidase and used according to the manufacturer’s (Santa
Cruz) protocol. Detection was performed with an ECL-Plus (Amersham
Corp) according to the manufacturer’s directions.
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Results |
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Phenotypically Unique Subendothelial L1 Cells Exhibit Augmented Growth Potential
Cells isolated from the subendothelial aspect of the vessel wall (L1 cells) were small and rhomboidal with long, lamellar extensions and formed a dense, multilayered network at confluence. In contrast, cells isolated from the middle media (L2 SMCs) were larger and spindle-shaped and at confluence, exhibited a hill-and-valley appearance traditionally described for vascular SMCs (Figure 1
). As previously
described,11 subendothelial L1 cells
expressed little -SM actin and no SM myosin, whereas middle-media L2
SMCs expressed high levels of both -SM actin and SM myosin. These 2
phenotypically distinct cell subpopulations exhibited markedly
different growth capabilities when tested under similar conditions.
After a prolonged period of serum deprivation (72 hours in
0.1% calf serum), basal levels of DNA synthesis (as measured by
[3H]thymidine incorporation) in
subendothelial L1 cells was 6- to 9-fold higher than
that in middle-media L2 SMCs (Figure 2A).
In response to serum stimulation (10% calf serum, 24 hours),
subendothelial L1 cells exhibited much higher rates of
[3H]thymidine incorporation than did
middle-media L2 SMCs (Figure 2A). Similarly, L1 cells exhibited
a higher rate of proliferation (as defined by an increase in cell
number) and a higher saturation density (number of
cells/cm2 at plateau of the growth curve) in 10%
calf serum compared with L2 SMCs (Figure 2B). Most
interestingly, L1 cells demonstrated an ability for autonomous or
self-driven growth, as defined by significant increases in cell number
when grown under serum-deprived (0.1% calf serum) conditions (Figure 2C). L2 SMCs remained quiescent in 0.1% calf serum (Figure 2C).
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L1 Cells Exhibit Constitutively Activated MAPK and
Eicosanoid Production Pathways Under Serum-Deprived
Conditions
Cell proliferation is known to be regulated mainly through 2
major receptor-mediated signaling pathways: the RTK-mediated and
the GPCR pathways. Therefore, we first tested whether activation of
either pathway was contributing to the autonomous, self-driven growth
of subendothelial L1 cells. We utilized
antagonists of both pathways: pertussis toxin, a potent
inhibitor of the Gi/Go pathway, and genistein, an
inhibitor of RTKs. We found that treatment with
either pertussis toxin or genistein significantly decreased basal
(0.1% calf serum, 72 hours) levels of
[3H]thymidine incorporation in
subendothelial L1 cells (Figure 3A), suggesting that both RTK and GiPCR
pathways were constitutively activated in L1 cells and
contributed to their autonomous growth. In contrast, pertussis toxin
and genistein had no effect on basal levels of DNA synthesis in
middle-media L2 SMCs (Figure 3B).
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Because proliferation in most cell types has been shown to be
correlated with activation of MAPK (p44mapk and
p42mapk, also known as ERK-1 and ERK-2,
respectively) and because both RTK-mediated and GiPCR pathways have
been shown to converge on activation of ERKs, we evaluated basal (after
72 hours in 0.1% calf serum) ERK-1/ERK-2 activity in both cell types
and the effects of the inhibitors of GiPCR and RTK pathways
(pertussis toxin and genistein) on basal ERK activity. As shown in
Figures 3C
and 3D, basal ERK activity (open bars) in L1 cells
was 2.5-fold higher than that in middle-media L2 SMCs. Both pertussis
toxin and genistein dramatically suppressed basal ERK activity in L1
cells but had no effect on basal ERK activity in L2 SMCs.
High basal levels of ERK activity suggested that constitutively
activated ERKs were playing an essential role in the
self-driven growth of subendothelial L1 cells. To test
this hypothesis, we employed PD98059, a highly selective
inhibitor of MEK-1 activation that has been shown to
effectively inhibit activation of ERKs.20 25 Treatment of
L1 cells with 10 µmol/L PD98059 markedly (by 80%)
inhibited autonomous, self-driven growth (assessed by an increase in
cell number in serum-deprived medium; Figure 4). In contrast, PD98059 exerted minimal
effect on the low, basal levels of DNA synthesis in L2 SMCs (data not
shown). We also assessed the role of the small G protein Ras
(p21ras), an upstream activator of
MAPK, in mediating MAPK-dependent autonomous growth of
subendothelial L1 cells. We used the farnesyl
transferase inhibitor BZA-5B, which had been previously
demonstrated to effectively block activation of Ras.26
BZA-5B (100 µmol/L) only partially (43%) inhibited growth of L1
cells under serum-deprived conditions (Figure 4), suggesting
that the self-driven growth of L1 cells is mediated through both
Ras-dependent and Ras-independent signaling pathways.
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It has recently been shown that differential growth responses of
vascular SMCs may be the result of differences in the availability of
downstream targets for MAPK, including
cPLA2.20 cPLA2
is known to be phosphorylated and activated by
MAPK, and cPLA2 expression is at least partially
driven by activation of Ras.26 27 28
PLA2 is a key enzyme in the eicosanoid
production pathway, a pathway with many important biological
actions, including regulation of vascular SMC
growth.19 20 26 We therefore sought to determine whether
the elevated levels of MAPK activity in autonomously growing L1 cells
under basal conditions would be associated with increased expression of
cPLA2 and other members of eicosanoid
production pathways. Under basal (serum-free) conditions,
cPLA2 protein expression and activity were
markedly elevated in subendothelial L1 cells compared
with middle-media L2 SMCs (Figures 5A and 5B). Arachidonic acid, liberated by
cPLA2, is metabolized by constitutive (COX-1) or
inducible (COX-2) COXs to PGs. We observed high levels of expression of
inducible COX-2 in L1 cells under serum-deprived conditions (Figure 6A). COX-2 was not detectable in L2 SMCs
under identical conditions (Figure 6A). L1 cells also
demonstrated remarkably high basal levels of PGE2
and PGI2 (the latter measured by its stable
analog 6-keto-PGF2) released into the culture
medium compared with L2 SMCs (Figure 6B).
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To assess the contribution of eicosanoids to the autonomous growth of L1 cells, we examined the effect of 2 types of inhibitors of eicosanoid production: nonselective COX inhibitors, which suppress PG production (indomethacin and sulindac sulfide), and a nonselective lipoxygenase inhibitor, nordihydroguaiaretic acid, which suppresses hydroxyeicosatetraenoic acid production. Sulindac sulfide and indomethacin inhibited PG production at the concentrations used but did not have any effect on basal DNA synthesis of L1 cells, whereas nordihydroguaiaretic acid significantly (by 50%) inhibited basal DNA synthesis in L1 cells grown in serum-deprived medium (data not shown).
L1 Cells Are More Sensitive to Mitogenic Signals via
the GiPCR Pathway Than Are L2 SMCs (Agonist and Antagonist
Studies)
As shown above, subendothelial L1 cells grow
faster than do middle-media L2 SMCs not only under conditions of serum
deprivation but also under serum-stimulated conditions (see
Figure 2B). Experiments in other cell systems have shown that
constitutive activation of a particular signaling pathway can
significantly affect the magnitude and/or responsiveness of a cell to
other mitogenic stimuli.18 These observations
suggested that the augmented growth of L1 cells in response to serum
could be due to an enhanced ability of L1 cells, compared with L2 SMCs,
to amplify exogenous growth signals acting through multiple
receptor-mediated signaling pathways. Therefore, we assessed the
contribution of both GiPCR and RTK-mediated signaling pathways to the
enhanced serum-stimulated growth of L1 cells by utilizing agonists and
antagonists of both pathways. The data presented in
Table 1 show that specific agonists of
RTK, viz, PDGF-BB and bFGF, increased DNA synthesis in both L1 cells
and L2 SMCs. Interestingly, EGF, another well-known RTK agonist, did
not significantly stimulate growth in either cell type (data not
shown). On the other hand, thrombin, an agonist of
GiPCRs,15 was a potent stimulator of DNA synthesis in L1
cells, whereas it had almost no effect on L2 SMCs (Table 1).
Similarly, lysophosphatidic acid, which has been shown to signal
through a Gi-coupled pathway in certain cell types,16
stimulated DNA synthesis in L1 cells but not in L2 SMCs (Table 1). Endothelin-1 and angiotensin II, which have been
shown to signal primarily through activation of Gq rather than Gi, did
not stimulate DNA synthesis in either L1 or L2 SMCs under the
conditions tested (0.1% calf serum for 72 hours before addition of
these agonists; data not shown).
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To further evaluate the contribution of RTK-mediated and GiPCR
signaling pathways to the augmented serum-stimulated growth of
subendothelial L1 cells, we examined the effects of
antagonists of these pathways on serum- and peptide
mitogen–stimulated growth of L1 cells compared with middle-media L2
SMCs. Data presented in Table 2
demonstrate that pretreatment with pertussis toxin reduced the
stimulatory effect of 10% serum on the growth of L1 cells by 80% and
reduced the thrombin-stimulated increase in
[3H]thymidine uptake by 87.7%. In middle-media
L2 SMCs, however, pertussis toxin pretreatment reduced the
mitogenic effect of 10% serum by 28.8%. (As shown above,
there was no stimulatory effect of thrombin on DNA synthesis in L2
SMCs.) As expected, pertussis toxin did not have any effect on
stimulation by PDGF in either cell type (data not shown). Genistein
markedly attenuated the mitogenic effect of 10% serum and
PDGF in both L1 and L2 SMCs and the mitogenic effect of
thrombin in L1 cells (data not shown). The latter findings are
compatible with previous observations demonstrating that genistein not
only inhibits RTK signaling but can also inhibit signaling via
Gi-coupled pathway(s) in some cell types.29
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Some but Not All Colonies of Subendothelial L1
Cells Secrete a Paracrine Mitogenic Factor(s)
The observations that subendothelial L1 cells
exhibited autonomous, serum-independent growth and demonstrated a
constitutively activated MAPK cascade, which was effectively
blocked by RTK and GiPCR inhibitors, raised the possibility
that these cells could be producing mitogenic factor(s). To
test this hypothesis, we assessed the effect of serum-free medium
conditioned by subendothelial L1 cells for 72 hours
(L1-conditioned medium) on DNA synthesis of quiescent (72 hours of
serum deprivation) L2 SMCs. We performed both live
coculture and conditioned medium experiments (see Methods) and found
that both methods yielded similar data with regard to stimulation of
DNA synthesis in growth-arrested L2 SMCs. (Therefore, only the data on
conditioned medium experiments are presented here.) We found
that in some colonies of L1 cells, serum-free medium conditioned by
these cells for 72 hours dramatically (11-fold) increased
[3H]thymidine uptake in quiescent medial L2
SMCs (Figure 7). Conditioned medium from
other colonies of L1 cells, which exhibited all of the characteristics
described above (autonomous growth, constitutively activated
ERKs, markedly high levels of cPLA2 and COX-2
expression, and PG production), did not exert any
mitogenic effect on either L2 SMCs or NIH 3T3 fibroblasts
(data not shown). We also evaluated the effect of serum-deprived medium
conditioned by L2 SMCs on 3T3 fibroblasts and observed no detectable
paracrine mitogenic activity (data not shown). Preliminary
data from our laboratory demonstrate that the paracrine
mitogenic factors present in L1-conditioned medium from
certain L1 cell colonies belong to the heparin-binding family of growth
factors. When L1-conditioned medium was passed over a heparin-Sepharose
column, all mitogenic activity in the run-through fraction
was abolished (data not shown). Elution of the fraction bound to the
heparin-Sepharose column by various concentrations of NaCl demonstrated
that the peak of mitogenic activity was present in the
fraction eluting with 0.75 mol/L NaCl (data not shown).
High-performance liquid chromatography
purification of the fraction with peak mitogenic activity
and further analysis by SDS–polyacrylamide gel
electrophoresis as well as amino acid sequencing of the proteins in
this fraction demonstrated that there are at least 2 unique members of
the heparin-binding family of growth factors (manuscript in
preparation).
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Effect of L1-Conditioned Medium, Containing Paracrine
Mitogenic Factors, on MAPK and Eicosanoid
Production Pathways in L2 SMCs
Because we observed high levels of intrinsic ERK activity as well
as high levels of cPLA2 and COX-2 expression in
L1 but not in L2 SMCs, we wanted to assess whether the conditioned
medium from those L1 cell colonies that secreted paracrine
mitogenic factor(s) would increase ERK-1/ERK-2 activity and
the levels of cPLA2 and COX-2 expression in
middle-media L2 SMCs to the levels observed in L1 cells. A 5-minute
exposure of quiescent L2 SMCs to 10% L1-conditioned medium increased
ERK-1/ERK-2 activity by 4.6-fold (Table 3), as might have been expected from the
L1-conditioned medium effect on L2 SMC proliferation (as shown in
Figure 7). Interestingly, however, addition of L1-conditioned
medium to quiescent L2 SMCs for 24, 48, or 72 hours did not
significantly affect the low level of cPLA2 or
COX-2 expression as determined by Western blotting assay (data not
shown). The effect of L1-conditioned medium on PG production in
L2 SMCs could not be tested because L1-conditioned medium itself
contains very large amounts of PGE2 and
PGI2 (see data above). However, we tested the
ability of L2 SMCs to secrete these prostanoids in response to
stimulation with arachidonic acid (10 µmol/L,
added for 30 minutes). PG production in L2 SMCs remained low
even under stimulation with arachidonic acid (Table 3). Compared with control (30 minutes in serum-free medium),
there was a moderate increase in PGE2 and
PGI2 (the latter measured by
6-keto-PGF2) production by L2 SMCs
(Table 3); however, these levels were still dramatically (80%)
lower than those in unstimulated L1 cells (cf Figure 5D).
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In the present study, we began to evaluate the pro-proliferative intracellular signaling pathways in bovine arterial subendothelial SMCs (L1 cells) that, in contrast to traditional vascular SMCs, exhibit the capacity for autonomous, self-driven growth. Under conditions of prolonged serum deprivation, subendothelial L1 cells were found to exhibit constitutively activated MAPK (ERK-1/ERK-2) and eicosanoid production (cPLA2/COX-2) pathways. In contrast, SMCs isolated from the adjacent middle media (L2 SMCs), which exhibited slow growth under serum-stimulated conditions and remained quiescent under serum deprivation, had low basal levels of ERK-1/ERK-2 and very low levels of cPLA2 and COX-2 expression. Both agonist and antagonist studies performed on L1 cells indicated the critical role of the GiPCR pathway in their augmented growth capabilities. In contrast, the middle-media L2 SMCs appeared to mediate mitogenic signals predominantly via RTK-mediated pathways. We found that some colonies of L1 cells secreted paracrine mitogenic factors, whereas other L1 cell colonies did not secrete any detectable paracrine mitogenic activity. Both secreting and nonsecreting L1 cells were indistinguishable on the basis of all other characteristics described in the present study (autonomous growth, constitutively activated MAPK, and eicosanoid production pathways), which suggested that secretion of the paracrine mitogenic factors is not a prerequisite for the autonomous growth capability of L1 cells. Moreover, when conditioned medium from L1 cells secreting paracrine mitogenic factor(s) was added to L2 SMCs, it did not affect cPLA2 and/or COX-2 expression in these latter SMCs. In other words, the conditioned medium from L1 cells did not convert the L2 SMCs into those of an L1 phenotype. Taken together, these data suggest that there are intrinsic functional differences between distinct subsets of vascular cells residing in close proximity in the arterial wall and specifically, that the growth of a unique subset of subendothelial cells is controlled by mechanisms distinct from those mediating growth in a population of more traditional SMCs in the middle media.
Autonomous growth capability has been described for mesenchymal cells derived from patients with diseases such as atherosclerosis as well as pulmonary and renal interstitial fibrosis.30 31 32 33 The possibility has been raised that the presence of a cell type with an enhanced proliferative phenotype in the diseased organ could explain the progression of the fibroproliferative response without further exogenous signaling. Studies in experimental animals have described autonomous growth capabilities in SMCs derived from embryonic, normal adult, and injured arteries.22 34 35 Based on these previous observations, the question was raised as to whether expression of an autonomous growth phenotype is a potential of all medial SMCs in response to injury or whether a unique subset of cells with autonomous growth potential normally exists in the uninjured arterial wall and in response to injury, is selectively recruited into the vascular repair process. Recent studies in several species have demonstrated that the arterial media is comprised of heterogeneous populations of SMCs that exhibit distinctly different proliferative capabilities.2 3 4 5 6 21 36 37 38 These data, together with our finding of a unique subset of cells (L1 cells) with autonomous growth potential and derived from the subendothelial compartment of normal tunica media, support the hypothesis that specific subsets of vascular cells can contribute in unique ways to the process of neointimal thickening after vascular injury. However, the concept of selective contribution of such cells to the disease process does not rule out the possibility that other more differentiated SMCs in the arterial media modulate their phenotype in response to injury and are also recruited, in different ways, into the vascular remodeling process.
Little is known of the signaling pathways utilized by nontransformed mesenchymal cells with autonomous growth potential.22 30 31 32 33 34 35 In the present study, we began to evaluate the intracellular mechanisms regulating the autonomous growth of subendothelial L1 cells isolated from normal, mature, arterial media presuming that this information might provide insight into understanding neointimal thickening after vascular injury. We found that under conditions of prolonged serum deprivation, L1 cells exhibited constitutively activated ERK-1/ERK-2. Based on experiments with inhibitors of upstream activators of ERKs (such as inhibitors of MEK-1, Ras, RTK, and GiPCR pathways), the constitutively activated MAPK pathway was found to be critical for autonomous growth of L1 cells. The finding that self-driven growth of L1 cells is markedly inhibited by pertussis toxin, a potent inhibitor of the GiPCR pathway, emphasized a crucial role of Gi-coupled events in contributing to autonomous growth potential. This finding, together with the observation that DNA synthesis in L1 cells can be readily activated by agonists of the GiPCR pathway, such as thrombin and lysophosphatidic acid, suggested a heightened activity and/or sensitivity of the GiPCR pathway in L1 cells. Several possibilities may be considered in explaining these findings. First is the production by L1 cells of an autocrine factor that acts through a GiPCR. This factor(s) could be acting in a juxtacrine fashion or could be secreted in very low quantity, not sufficient to stimulate mitogenesis in other cell types. Importantly, such a hypothetical GiPCR agonist could also contribute to the enhanced growth capabilities of L1 cells by interacting with other growth-promoting pathways. There is accumulating experimental evidence that activation of the GPCR signaling pathway can markedly amplify the responses produced by a separate coincident activation of other receptors.18 A second possibility is that of mutation-independent constitutive activation of a heptaspanning receptor in the absence of a ligand, as has been recently demonstrated in the nonmalignant human colon epithelial cell line NCM460.39 The third most unlikely possibility is that of a constitutively active Gi, as has been described for many tumor cells with autonomous growth potential and bearing activating mutations in G-protein subunits.40
In addition to constitutive activation of ERKs, remarkably high levels
of cPLA2 and COX-2 protein expression and
activity were observed in L1 cells. A critical role for
cPLA2 in proliferation of human vascular SMCs has
recently been proposed.19 Constitutively elevated MAPK
activity in subendothelial L1 cells could potentially
serve as an intracellular "motor" in maintaining elevated levels of
cPLA2 activity in these cells, since
cPLA2 is known to be
phosphorylated and activated by
MAPK.27 28 Interestingly, previous experiments showed that
overexpression of H-Ras in rat vascular SMCs induced elevated levels of
cPLA2 expression and activity.41
Concomitantly, these SMCs exhibited marked downregulation of -SM
actin compared with wild-type SMCs, a characteristic similar to that
observed in L1 cells. It is possible, therefore, that elevated
cPLA2 activity contributes to both the
proliferative phenotype and the differentiation status of L1
cells.
Increased activity of cPLA2 is associated with increases in the levels of free arachidonic acid, which are then metabolized to a variety of bioactive eicosanoids, including PGs, through the action of COXs. We found that L1 cells express markedly elevated levels of the inducible form of COXs, COX-2, and they secrete large amounts of PGs. Several studies have demonstrated a pronounced inhibitory effect of prostanoids on vascular SMC growth.42 43 20 In contrast, we found that inhibition of PG production had no effect on autonomous growth of L1 cells. These data are consistent with a previous report by Morisaki et al44 demonstrating a lack of inhibition of DNA synthesis by endogenous and exogenous PGs in cultured neointimal compared with medial SMCs from injured rabbit aortas. Available data from clinical trials, though conflicting, in general also do not support the concept that PGs can be successfully used to suppress excessive SMC proliferation in restenosis after percutaneous transluminal coronary angioplasty.45 46 Moreover, elevated production of PGs by a number of rapidly growing cancer and transformed cells has been suggested as an important contributor to enhanced cell proliferation.26 Taken together, these data suggest that activation of prostanoid production pathways can have divergent effects in different subsets of vascular SMCs and support the idea that growth may be controlled differently in distinct subsets of vascular SMCs.
In contrast to our observations that inhibition of COX activity in L1 cells did not affect their growth rate, we found that inhibition of lipoxygenase production by the nonselective lipoxygenase inhibitor nordihydroguaiaretic acid markedly suppressed basal DNA synthesis. These observations are in agreement with studies by Rao et al47 on rat aortic SMCs, which showed that 15-hydroxyeicosatetraenoic acid, a 15-lipoxygenase product of arachidonic acid, accounts for some of the arachidonic acid–induced activation of ERKs Future studies are essential to elucidate the role of the lipoxygenase system in the autonomous, self-driven growth of subendothelial L1 cells.
Our data with observations by other investigators suggest that phenotypically and functionally unique vascular cells with high proliferative potential (similar to L1 cells described herein) normally exist in the subendothelial region of large, elastic arteries, raising the possibility of their selective contribution to the pathogenesis of vascular diseases. The basis for the differences in growth capabilities of distinct subsets of vascular cells might lie in our finding that different SMC subpopulations use distinctly different intracellular signaling mechanisms to control growth. These observations suggest that pharmacological therapies aimed at inhibiting vascular remodeling should consider the specific growth control pathways utilized by the cells involved.
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Acknowledgments |
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This work was supported by grants PPG HL-14985 (to K.R.S.) and SCOR HL-57144 (to M.G.F. and K.R.S.) from the National Institutes of Health, Bethesda, Md. The authors gratefully acknowledge Steve Hofmeister and Marcia McGowan for help in preparation of the manuscript and Dr John T. Reeves for valuable discussion of the manuscript.
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Footnotes |
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M.G. Frid and A.A. Aldashev contributed equally to this work.
Received December 28, 1998; accepted May 27, 1999.
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References |
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