Department of Physiology, Midwestern University, Glendale, Arizona 85308
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ABSTRACT |
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Vascular smooth muscle cells (VSMC) subjected to
acute or chronic stretch display enhanced growth rates in vitro and in
vivo. Clinical examples of vascular hyperplasia (e.g., systolic
hypertension and postinjury restenosis) suggest that local insulin-like
growth factor I (IGF-I) expression is enhanced. Therefore, we
investigated the role of in vitro cyclic stretch on rat VSMC IGF-I
secretion and cellular growth. In serum-free medium, cyclic stretch (1 Hz at 120% resting length for 48 h) stimulated thymidine incorporation ~40% above that seen in nonstretched cells. Graded stretch magnitude (100-125% resting length) yielded graded increases in VSMC
growth. Exogenous IGF-I increased growth of serum-starved, nonstretched VSMC in a dose-dependent manner, with maximal growth seen with 107 M. IGF-I secretion from
stretched cells was 20- to 30-fold greater than from those cells
cultured in a static environment. Stretch-induced increases in growth
were completely blocked on addition of anti-IGF-I and partially blocked
with platelet-derived growth factor (PDGF) antibodies and with a
tyrosine kinase inhibitor (tyrphostin-1). Finally, blockade of
stretch-activated cation channels with
GdCl3 profoundly inhibited
stretch-induced growth. We conclude that stretch increases VSMC IGF-I
secretion and that such autocrine IGF-I is required for stretch-induced
growth. PDGF and stretch-sensitive cation channels are likely
additional components of a complex pathway that regulates
stretch-induced VSMC seen in systolic hypertension and postinjury restenosis.
insulin-like growth factor I; restenosis; platelet-derived growth factor; tyrosine kinase
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INTRODUCTION |
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THE PULSATILE NATURE of blood flow, through its pressure waveform acting on the compliance of the arterial wall, produces circumferential stretching of the wall and underlying vascular smooth muscle cells (VSMC). Despite the fact that vascular cells reside in a dynamic state in vivo and are constantly subjected to pulsatile hydrostatic pressures and shear stress, much of our knowledge regarding VSMC growth has come from studies in vitro utilizing a static tissue culture environment. For cells such as VSMC, which normally reside in such an active environment, this passive environment may be suboptimal or artifactual (1).
Vessels obtained from hypertensive humans and animals as well as
restenotic vessels manifest medial thickening of the vessel wall (2).
Although a topic of continued debate, it is becoming clear that medial
thickening in these two states is defined by 1) VSMC hypertrophy (i.e., increase
in cell size; Ref. 2); 2) VSMC
hyperplasia (i.e., increase in cell number; Ref. 3); and
3) increased mass of extracellular
matrix proteins (26). A preliminary event in postinjury vascular growth
is platelet adherence and degranulation resulting in release of
-granule products. However, VSMC growth continues at an accelerated
rate well past the disappearance of platelets (and associated growth factors) from an injured site. These data suggest that the vessel itself is a site of growth factor production, which may be necessary and sufficient for such intimal growth.
In vivo, increased transmural pressure after portal vein ligation leads to a fourfold increase in VSMC insulin-like growth factor I (IGF-I) mRNA (6) and increased medial mass. This phenomenon is seen only in vessel segments proximal to the ligation where stretch of the vessel is apparent. In addition, VSMC IGF-I mRNA and protein increase ninefold in rapidly growing vessel segments 1 wk after balloon angioplasty (5). The involvement of growth hormone (the most common physiological regulator of IGF-I expression and/or release in vivo) has been ruled out because equivalent increases are seen in hypophysectomized rats. Therefore, these studies specifically suggest that local production of IGF-I may be involved in stretch-induced medial growth. Because balloon angioplasty as well as increased transmural pressure evokes stretch stimuli on VSMC, we investigated whether 1) cellular stretch regulates IGF-I secretion, 2) secreted IGF-I is involved in stretch-induced cellular growth, and 3) platelet-derived growth factor (PDGF) and stretch-sensitive cation channels play roles in such stretch-regulated events.
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MATERIALS AND METHODS |
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Culturing of aortic VSMC. Studies were carried out with A7r5 aortic smooth muscle cells from DB1X rats purchased from American Type Culture Collection (Rockville, MD). These cells have been utilized extensively by our group and others (48, 49) in studying VSMC growth, ion transport mechanisms, and contractility. VSMC were cultured in Dulbecco's modified Eagle's medium supplemented with 9% fetal bovine serum and antibiotics at 37°C, 5% CO2, and 100% humidity (46). Cells were fed every other day with fresh medium and passaged upon confluence (usually 3-5 days). Cells from passages 3-9 were used for these studies. For each experiment, cell passage number was matched such that all experimental groups originated from a single VSMC plate of passages 3-9.
Stretch apparatus. The Flexercell apparatus (Flexcell; McKeesport, PA) is a computer-controlled device designed to deliver preprogrammed stretch regimens to wells attached to a vacuum-assisted gasketed baseplate (Flexwells). The baseplate remains in a tissue culture incubator throughout the duration of the stretch paradigm. The user programs rate and duration of both stretch and relaxation phases for an infinite number of stretches. Valves located in the baseplate allow selected culture plates to be stretched, whereas control wells remain unstretched. Many investigators have utilized this apparatus to study a variety of vascular cell functions (24, 36, 50). It has been determined that force on the attached cells is uniaxial (32). An important aspect of the stretch paradigm imparted by this device is that stretch is nonuniform across the well (1). Data from the Flexercell apparatus has been principally analyzed as integrated responses of the entire population of cells subjected to a continuum of stretch magnitudes. In selected studies, we took advantage of this stretch nonuniformity by assessing physiological variables in cells obtained by "punch biopsies" from various regions of the elastomeric culture support. Specifically, 5-mm punches were obtained (with a leather punch and mallet) from selected, carefully measured coordinates on the well that represent percent strains from 0 to 25%. We then correlated our physiological end points with the calculated strain magnitude for each punch.
Stretch paradigms. VSMC were seeded (~10,000 cells/ml) onto six-well collagen I-coated FlexI plates in DMEM-9% FBS. Once cells were ~50-60% confluent (usually ~48 h postseeding), growth medium was replaced with serum-free medium (SFM) for 48 h, and on the day of the experiment, fresh SFM was substituted. This period of quiescence results in a mitotic index of <10% (25). With the use of Flexercell apparatus, cells were stretched at 1 Hz to an average of 120% of their initial resting length (Lo) for various time periods.
Cell viability. Cell viability and adherence were determined in subsets of VSMC grown in the presence or absence of stretch, antibodies, and other treatments. Representative wells were viewed with phase contrast optics (×40-200) to determine potential gross areas of cell removal poststretch. Additionally, cell viability was determined via trypan blue exclusion, as we have described elsewhere (44), as well as by the ability of the cells to bioreduce MTS (Owen's reagent) into formazan (CellTiter 96 kit, Promega, Madison, WI).
Detection of IGF-I in conditioned medium. Specific IGF-I immunoreactivity was determined with modifications of verified methods (3, 15, 37). An aliquot of conditioned medium from stretched and control cells (100 µl retained from each well) was treated at a 1:5 ratio with acid-ethanol (12.5% 2 N HCl-87.5% ethanol) at 4°C for 30 min to remove high-molecular-weight binding proteins (13). Samples were then centrifuged (5 min × 10,000 g), and the supernatant was removed and neutralized with 1 M Tris base. Samples were then diluted in assay buffer (phosphate-buffered saline with 0.1% gelatin, 0.05% Tween 20, and 0.01% thimerosal; pH 7.4) and were subjected to IGF-I radioimmunoassay. Typical assay performances for this RIA are sensitivity (5 ng/ml), intra-assay precision (3.7%), and interassay precision (4.9%).
Growth assays. Hyperplastic growth
responses were estimated by measuring
[3H]thymidine
incorporation (as a measure of DNA synthesis rate; Ref. 55) at various
times during and after the stretch regimens. Briefly, at each time
point tested, 1 µCi/ml
[3H]thymidine was
added to prewashed wells, and treatment (stretch, antibodies) was
continued for the required duration. After 4 h, the media were
aspirated and saved at 80°C for IGF-I determinations. Wells
were then washed three times with phosphate-buffered saline supplemented with bovine serum albumin. Next, ice-cold 15%
trichloroacetic acid (TCA) was added and incubated at 4°C for 30 min. TCA was removed via aspiration, and the Flexwells were allowed to
dry at room temperature for 2 h. The flexible well bottoms (or punch biopsies obtained from them) were then removed, added to 10 ml of
scintillation cocktail, and counted in a liquid scintillation counter
(Beckman Instruments model LS6500, Fullerton, CA).
Replicates and statistical analysis. Each experiment was performed a minimum of four times. For each experimental trial, triplicate wells from each group (i.e., control, stretch, stretch + inhibitor) were assayed. When the punch biopsy technique was utilized, triplicate wells were still utilized and the following replicate punches from each well were obtained: one from the center of the wells and three each from the middle and peripheral regions of the wells. All data are expressed as means ± SE. Replicate experiments all showed equivalent changes in the assayed variable compared with the representative graphs illustrated. Outliers, when present, were identified and removed via Dixon's gap test. Student's t-test or analysis of variance with post hoc Bonferroni correction assessed differences in population means. Population means were considered significantly different if P < 0.05. All data were analyzed with the InStat software suite (GraphPad Software, San Diego, CA).
Chemicals. Goat polyclonal anti-mouse IGF-I was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Goat polyclonal anti-human PDGF was purchased from Upstate Biotechnology (Lake Placid, NY). [3H]thymidine (~7 Ci/mM) was obtained from Amersham (Arlington Heights, IL). Tyrphostins, GdCl3, and other chemicals were purchased from Sigma (St. Louis, MO). Human recombinant IGF-I and IGF-I radioimmunoassay components were kind gifts from Genentech (San Francisco, CA).
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RESULTS |
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Cyclic stretch does not affect cell
viability. Before stretching was begun, and after 24 and 48 h of cyclic stretch, cell viability was assessed. Microscopic
evaluation revealed no gross areas of cell detachment from the
elastomeric substrate in nonstretched control cells or stretched
groups. Furthermore, cell viability was 95% in stretched as well as
nonstretched wells as determined by the CellTiter 96 kit that tests the
ability of the cells to bioreduce MTS into formazan.
Cyclic stretch enhances VSMC thymidine
incorporation. We sought to determine whether cyclic
stretch, as would be experienced by cells in vivo (1), stimulates VSMC
growth. Figure 1 illustrates that VSMC
stretched on Flexwells at 1 Hz and 120%
Lo for 48 h respond by inducing a 40% increase in DNA synthesis. Microscopic examination revealed that cell detachment from the elastomeric support
in the stretch and nonstretch groups was insignificant. In addition,
cell viability in all groups was >95% as determined by trypan blue
exclusion and the CellTiter 96 assay.
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Exogenous IGF-I stimulates VSMC DNA synthesis in
nonstretched cells. Pure populations of static, aortic
VSMC responded to exogenous IGF-I by displaying increased DNA synthesis
rates as measured by
[3H]thymidine
incorporation (Fig. 2). When VSMC were
quiesced in SFM for 24 h followed by several washes and reincubation in
fresh SFM plus exogenous human recombinant IGF-I, DNA synthesis
increased in a dose-dependent manner (Fig. 2). At concentrations
>107 M, IGF-I profoundly
inhibited growth.
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Cyclic stretch stimulates VSMC IGF-I secretion in a
time-dependent manner. To determine the extent of IGF-I
secretion from stretched VSMC, we assayed immunoreactive IGF-I levels
in conditioned medium samples obtained after our routine stretch
paradigm (1 Hz at 120%
Lo). Figure
3 illustrates that media obtained from VSMC
stretched for 1-3 days displayed 20-fold greater immunoreactive IGF-I than nonstretched cells. This immunoreactivity increased further
if stretch was continued for 4-6 days. IGF-I immunoreactivity in
media samples from nonstretched cells was barely detectable with our
radioimmunoassay.
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Antibodies to IGF-I completely block cyclic
stretch-induced DNA synthesis. Once we determined that
stretch induces IGF-I release and DNA synthesis rate, we sought to
determine whether autocrine production of IGF-I was, at least in part,
responsible for this growth effect. Figure
4 illustrates that addition of 10 µg/ml polyclonal anti-IGF-I antibody attenuated basal and
completely eliminated stretch (1 Hz at 120%
Lo × 48 h)-induced increases in DNA synthesis. Equivalent dilution of preimmune serum was without effect on the growth response (data not shown). These data suggest that
secreted IGF-I is required for cyclic stretch-induced growth of VSMC.
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Graded cyclic stretch induces graded growth
responses. As previously mentioned, we developed a
punch biopsy technique to assess if graded stretch magnitude resulted
in graded thymidine incorporation. Figure 5
illustrates that cells from the center of the wells (an area equivalent
to 0-4% strain) displayed thymidine incorporation rates
significantly less than cells obtained from the middle (4-10% strain) or periphery (10-25% strain) of the wells. Similarly
obtained punches from nonstretched cells displayed no such differences.
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Antibodies to platelet-derived growth factor partially
block stretch-induced VSMC growth. Several cell types
respond to platelet-derived growth factor (PDGF) by displaying
increased expression of IGF-I and/or its mRNA (37). Figure
6 illustrates that addition of 25 µg/ml
polyclonal anti-PDGF resulted in attenuation of stretch-induced thymidine incorporation in middle and periphery punches. Nonstretched cells receiving equivalent anti-PDGF treatment displayed no such attenuation (data not shown).
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Tyrosine kinase blockade attenuates stretch-induced
growth. We utilized the tyrosine kinase inhibitor
tyrphostin-1 ([4-methoxybenzylidene]-malononitrile; Ref.
23) to determine the involvement of tyrosine kinase activity on
stretch-induced growth. Figure 7
illustrates that 10 µM tyrphostin-1 significantly inhibited
stretch-induced thymidine incorporation as measured in punches obtained
from central and peripheral locations throughout the wells.
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Role of stretch-activated cation
channels. To assess whether our cyclic stretch paradigm
confers enhanced growth by activating such channels, we treated cells
with and without 30 µM GdCl3. Figure 8 illustrates that
GdCl3 attenuated stretch-induced
thymidine incorporation in all areas of the wells. Such treatment had
no effect in nonstretched cells (data not shown).
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DISCUSSION |
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Recent data demonstrate that VSMC cultured in a static (nonstretch) environment transcribe IGF-I and secrete IGF-I into culture medium (3, 17). However, the direct effects of stretch on VSMC IGF-I expression and release have not been investigated. In this study, we have shown for the first time that both acute (48 h) and chronic (6 days) cyclic stretch stimulates IGF-I secretion from arterial smooth muscle cells. Furthermore, cyclic stretch-induced VSMC growth depends, at least in part, on autocrine IGF-I action. IGF-I is necessary, but likely not sufficient, as antibodies to PDGF as well as inhibitors of stretch-activated cation channels also attenuate stretch-induced growth. Despite the likelihood of several involved signaling pathways, activation of tyrosine kinase activity plays a key role in stretch-induced VSMC growth. Taken together, these studies suggest vascular hyperplasia is controlled, at least in part, by biomechanical forces that transduce information via IGF-I and several other cellular signaling pathways. These observations may shed light on treatment modalities for such diseases as systolic hypertension and postinjury restenosis.
Many investigators have utilized the Flexercell apparatus to study a host of vascular cell physiological responses, including autocrine action of PDGF (55), angiotensin II (ANG II)-induced mitogenesis (24), stretch regulation of VSMC adenylate cyclase-G protein regulation (32, 53), myosin phosphorylation (32), and parathyroid hormone gene expression (36). Force analyses of the strain on the Flex-I wells during stretch at various vacuum levels have been calculated by finite element analysis and empirically measured with a micrometer. It has been determined that force on the attached cells is uniaxial (21, 32). An important limitation of this device, however, is that stretch is nonuniform across the well (55). Data from the Flexercell apparatus described above and elsewhere have been principally analyzed as integrated responses of the entire population of cells being subjected to a continuum of stretch magnitudes. Our punch biopsy technique exploits this limitation by permitting analyses of multiple cell populations exposed to various stretch magnitudes.
Our data illustrate that cyclic stretch stimulates VSMC hyperplasia in SFM (Fig. 1). These data are in concert with reports showing that cultured VSMC stretched on a flexible substratum display increased rates of DNA and collagen synthesis (26). A similar phenomenon associated with pulsatile stretch that VSMC normally encounter (i.e., variations in systolic-pulse pressure) was also investigated (58). DNA synthesis and cell number were elevated after 24 h in cultured VSMC stretched to an average of 125% of initial length at 1 Hz. In vivo, the severity of stretch (by graded balloon inflation) parallels the degree of intimal hyperplasia (41), indicating an equivalent clinical response to stretch. Cellular growth in our studies did not depend on the presence of added serum, suggesting that 1) serum mitogens other than IGF-I need not be present to stimulate VSMC growth or 2) other required mitogens are produced by VSMC that may act in concert with IGF-I to manifest cellular growth.
It is clear from our in vitro studies that stretch magnitude (0-25% strain) correlates with VSMC growth rate (see Fig. 5). In vivo, stretch magnitude is determined by vessel distensibility, capacitance, vessel size, and pulse pressure. Pathologically, such a stretch stimulus is abnormal. For example, stretch magnitude during angioplasty is altered acutely but drastically impacts the relative loss of endothelium and subsequent extent of vascular regrowth (5). Furthermore, enhancing the degree of balloon inflation results in more profound restenosis. In hypertensive patients, vessels are less distensible because of, in part, increased cell growth and increased masses of extracellular matrix proteins (26). Whether these examples of vascular remodeling are causes of or responses to increased vascular load is still debated, but these findings implicate that stretch magnitude is a variable to be considered when addressing the issue of mechanical stimuli that may be abnormal in some forms of hypertension. As hypertension worsens, the degree of vascular proliferation increases, and as hypertension is successfully treated, vascular hyperplasia is reduced. In vitro studies support these observations and implicate several signaling pathways. For example, increased stretch (as elicited by raising Flexwell pressure from 10 to 15 and 20 kPa) decreases adenylate cyclase activity in a stepwise manner in cultured VSMC (32), suggesting that stretch magnitude is an important determinant in regulating signal transduction and subsequent regulation of gene expression. Stretch magnitude also appears to be the major determinant regulating stretch-activated calcium channel opening in VSMC (14) as well as phospholipase C (PLC) activation in rabbit aortic muscle (31). Therefore, stretch magnitude clearly has an impact on vascular growth in vivo and in vitro.
We have been investigating for several years the role of IGF-I in regulating vascular hyperplasia and tone, with emphasis on the ability of the hormone to regulate glucose transport and Na-K-ATPase activity and gene expression. We have reported that IGF-I is a potent stimulator of VSMC glucose transport (47). In addition, we have shown that IGF-I stimulates Na-K-ATPase activity via a mechanism involving regulation of the Na/H exchanger (45). Interestingly, Liu et al. (30) have recently reported that cyclic stretch increases intracellular Na+ and subsequent activation of Na-K-ATPase activity and expression. Increases in ambient glucose stimulate cellular hyperplasia (20), and cellular growth responses are associated with increases in Na-K-ATPase activity (33, 35, 36). Therefore, these are two candidate transport proteins that are regulated by autocrine IGF-I and are implicated in VSMC growth.
It is clear from our antibody experiments that IGF-I is required for stretch-induced growth. More than likely, the stretch-induced IGF-I that is released from our cells (Fig. 3) is the component blocked by our antibody. Although it may be possible that intracellular IGF-I may be affecting VSMC growth rates, IGF-I binding to its extracellular receptor subunit has been shown to be the primary mechanism by which the effects of the hormone are signaled (4, 12, 15, 17). Surprisingly, we also observed a decrease in thymidine incorporation in static cultures of VSMC treated with equivalent anti-IGF-I titers (Fig. 4). These data suggest that serum-deprived VSMC rely on autocrine IGF-I for basal growth as well. This notion is consistent with data showing that static VSMC express IGF-I in response to ANG II stimulation (17).
Others have shown that IGF-I induces VSMC c-myc (2), stimulates VSMC mitogenesis (9, 38), and increases synthesis of vascular extracellular matrix proteins (1, 28). For example, IGF-I enhances VSMC elastin (22) and collagen (26) gene expression, suggesting that IGF-I-induced intimal hyperplasia results from enhanced matrix protein production as well as vascular growth. That hyperplastic rat aortas postangioplasty display enhanced IGF-I binding (43) also suggests a pivotal role for IGF-I in vascular hyperplasia. Given that anti-IGF-I blocks reactive oxygen species (e.g., H2O2, xanthines)-induced vascular hyperplasia, this suggests that effects of IGF-I on vascular growth are far reaching (16).
Because addition of exogenous IGF-I to nonstretched cells is sufficient to induce a similar growth profile as that seen in stretched cells (Fig. 2 and Ref. 42), we can conclude that 1) mitogens other than IGF-I are not required for stretch-induced growth or 2) mitogens other than IGF-I that are required for growth are produced by (and perhaps dependent on) IGF-I. Indeed, there are several reports suggesting that growth factors other than IGF-I are likely involved in stretch-induced hyperplasia. Most often cited is PDGF.
Cyclic stretch in vitro increases PDGF expression after 48 h in
cultured VSMC (55), a phenomenon possibly implicating a local renin-ANG
II system (29). This correlates with stretch-induced expression of a
PDGF-A promoter (56). Antisense oligonucleotides to PDGF-A attenuate
arterial proliferation in spontaneously hypertensive rats, underscoring
at least a permissive effect of PDGF on vascular growth. Antibodies to
PDGF attenuate, but do not abolish, stretch-induced growth (Fig. 6 and
Ref. 55). Some reports suggest that PDGF may be required for
IGF-I-induced growth (40, 55). These data are consistent with the fact
that growth of most tissues requires a competence factor (such as PDGF)
for cells to enter the G1 phase of
the cell cycle and that a progression factor (such as IGF-I) is
required for cells to traverse the
G1-to-S phase of the cell cycle
(27). For example, IGF-I, by itself, induces certain genes such as
ribosomal DNA, c-fos, and
c-jun but does not induce the DNA
synthesis genes. This is exemplified by observations that these
synthesis genes are induced only when cells are primed with other
growth factors such as PDGF (40). IGF-I alone modestly stimulates VSMC
growth via a protein kinase C
(PKC
)-dependent fashion, whereas further addition of PDGF-AA dramatically enhances growth (51). It is thought that this synergistic effect results from
the ability of PDGF to stimulate mitogen-activated protein kinase and
PKC
. Moreover,
PDGF has been shown to increase IGF-I receptor number and activity (52)
in static VSMC. Therefore, autocrine IGF-I may likely be regulated by
and/or required for PDGF-induced VSMC growth.
Fibroblast growth factor (FGF) has also been implicated as an important player in stretch-induced proliferation. The magnitude of cyclic stretch parallels VSMC FGF-2 release from both cytoplasmic and nuclear stores (7). These observations likely implicate synergistic effects of FGF and vascular endothelial growth factor (11). When antibodies to FGF are added to cyclically stretched cells, proliferative rates fall by 89% (8). How FGF potentially regulates VSMC IGF-I expression and/or activity is unknown but is an important area of research for future studies.
Activation of tyrosine kinase activity is a proximal postreceptor
binding event for IGF-I, PDGF, and several other mitogens. Cyclic
stretch stimulates ANG II VSMC expression, and subsequent ANG
II-induced growth is tyrosine kinase activity (TKA) and PDGF-B dependent (29). Not only does ANG II stimulate TKA, but it also results
in phosphorylation of the IGF-I receptor -chain and insulin receptor
substrate-1, the latter providing binding sites for proteins with
src homology-2 domains (18). It is
becoming increasingly clear that many mitogens other than classical
growth factors (e.g., thrombin, serotonin, ANG II) involve tyrosine
kinase activation in coordinating VSMC growth. In fact, it is likely
that many such G protein-regulated pathways cross talk with tyrosine
kinase pathways to coordinate a variety of cellular events (15).
Previous reports indicate that VSMC express gadolinium-sensitive, stretch-activated cation channels (30). It is thought that such channels primarily conduct inward calcium current, but may likely conduct inward sodium current as well. These channels have been implicated in the classical myogenic response and autoregulation of arterial blood flow. In VSMC preparations, cyclic stretch stimulates Na entry (30) and subsequent enhancement of Na-K-ATPase activity and gene expression. Gadolinium blocks such changes in intracellular Na and gene expression (30). Additional potential mechanisms of such stretch-activated channels come from data showing that gadolinium decreases stretch-activated PLC in rabbit VSMC (31). Interestingly, PDGF has been shown to increase Ca2+ channel currents in rabbit arterial smooth muscle; this action was inhibited by TKA antagonists (54). Because a host of growth-promoting signals are transduced via PLC, the overall signaling cascade is indeed quite complex and likely includes Ca2+ channel activation, tyrosine kinase activation, upregulation of PLC, and induction of growth factor expression.
We have recently reported that exogenously applied IGF-I dramatically
increases nitric oxide (NO) secretion from cultured VSMC (34).
Additional studies with endothelium-denuded aortic preparations
revealed that aminoguanidine (an inhibitor of inducible NO synthase)
blocks IGF-I-induced increase in NO production. These data support the
notion that the vasodilatory properties of IGF-I are due, at least in
part, to NO production (19). Further evidence has shown that NO is
antiproliferative and therefore may be vasculoprotective in states
defined by hyperproliferation (39). The observed antiproliferative effects can be elicited either by exogenous NO added as a gas, released
from NO donors, or by endogenous stimulation of VSMC inducible nitric
oxide synthase (10, 57). These studies provide further interpretation
of our data: although stretch-induced IGF-I certainly is the underlying
mechanism of enhanced proliferation seen in stretched cells, the
resultant IGF-I secretion also likely stimulates NO release from VSMC.
If this is the case, IGF-I-induced growth as shown in Fig. 2 (for
109-10
7M)
may be underestimated because of simultaneous stimulation of mitogenic
(i.e., IGF-I) and antimitogenic (or apoptotic; i.e., NO) pathways. It
is therefore possible to speculate that the growth inhibitory effects
seen with the addition of
10
6 M exogenous IGF-I (Fig.
2) are a result of overwhelming NO-mediated antiproliferation. We are
currently investigating these possibilities in related studies.
In conclusion, cyclic stretch enhances vascular growth in a magnitude-
and IGF-I-dependent manner. Further involvement of PDGF, TKA, and
stretch-sensitive cation channels provides for a coordinated cascade of
signaling events that underlie stretch-induced growth responses. Figure
9 depicts a simplified overview of some of
these key events. Stretch opens a subset of gadolinium-sensitive calcium channels, causing transient increases in intracellular Ca2+ concentration. Such
biophysical forces also stimulate expression and action of multiple
growth factors, including IGF-I, PDGF, and FGF. Whether such expression
is a result of stretch-induced rises in intracellular
Ca2+ concentration is uncertain.
These growth factors act synergistically to regulate each other's
expression and action, as well as to modulate vascular growth directly.
The Na-K-ATPase and membrane glucose transporter are both stimulated by
IGF-I and have both been ascribed key roles in the pathogenesis of
vascular hyperplasia. IGF-I induction of other autocrine substances
(e.g., prostanoids, other growth inhibitors, mitogens) may also be
implicated in such a scheme. Cyclic stretch also stimulates expression
and release of elastin and collagen, resulting in enhanced
extracellular matrix protein density. Such changes in extracellular
matrix constituents and density likely impact on the ability of the
cells to respond to subsequent biophysical stimuli. These observations
may likely provide insight into new treatment modalities for vascular
hyperproliferation seen in systolic hypertension and postinjury
restenosis.
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ACKNOWLEDGEMENTS |
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The authors acknowledge the technical assistance of David Ward, Huan Le, and Mike Dyre. Facilities were made possible by Drs. M. Saffo and M. Grober (Arizona State University-West, Glendale, AZ) and Dr. C. Johnston (Arizona State University, Tempe, AZ). Preliminary IGF-I measurements and technical assistance from Dr. N. Levin, Genentech (San Francisco, CA) were greatly appreciated. The Flexwell apparatus is on kind loan from Dr. R. Schiebinger, Wayne State University (Detroit, MI).
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FOOTNOTES |
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These studies were supported by a grant from the American Heart Association Southwest Affiliate (P. R. Standley) and an intramural grant from Midwestern University (P. R. Standley).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: P. R. Standley, Dept. of Physiology, Midwestern Univ., 19555 N. 59th Ave., Glendale, AZ 85308 (E-mail: pstand{at}arizona.midwestern.edu.).
Received 17 August 1998; accepted in final form 21 December 1998.
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