Activation of mitogenic and antimitogenic pathways in cyclically stretched arterial smooth muscle

Paul R. Standley, Melinda A. Stanley, and P. Senechal

Department of Physiology, Midwestern University, Glendale, Arizona 85308


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Biophysical forces regulate vascular smooth muscle cell (VSMC) physiology and evoke vascular remodeling. Two VSMC autocrine molecules, insulin-like growth factor I (IGF-I) and nitric oxide (NO), are implicated in remodeling attributable to VSMC hyperplasia. We investigated the role of in vitro cyclic stretch on rat VSMC IGF-I, NO, and cellular growth. Cyclic stretch (1 Hz at 120% resting length for 48 h) stimulated VSMC proliferation 2.5-fold vs. unstretched cells and was accompanied by a 1.8-fold increase in VSMC IGF-I secretion. Despite activation of this proliferative pathway, cyclic stretch induced inducible (i) nitric oxide synthase (NOS) expression and a twofold increase in NO secretion, a molecule with documented antiproliferative effects. Cytokine treatment enhanced iNOS expression and NO secretion while inhibiting vascular growth by approx 50% in static cells. Cytokine treatment of stretched VSMC enhanced NO secretion 2.5-fold while inhibiting growth by approx 80%. Exogenous IGF-I increased NOS activity 1.5-fold and NO secretion 8.5-fold in static cells. In turn, iNOS inhibition increased IGF-I secretion 1.6-fold and enhanced VSMC growth 1.6-fold in stretched cells. An NO donor (sodium nitroprusside) similarly inhibited VSMC proliferation in static (24%) and stretched (50%) VSMC while also inhibiting IGF-I secretion from stretched cells by approx 35%. Thus cyclic stretch stimulates mitogenic (IGF-I) and antimitogenic (NO) pathways in VSMC. These two molecules regulate each other's secretory rates, providing tight regulation of VSMC proliferation. These data may have profound implications in understanding vascular growth alterations in vascular injury and hypertension.

vascular smooth muscle; insulin-like growth factor; nitric oxide; hyperplasia


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE VASCULAR WALL is subjected to a variety of mechanical forces in the form of stretch due to blood pressure and shear stress due to blood flow. Alterations in either of these forces are known to result in vascular remodeling, an adaptation characterized by modified morphology and function of the blood vessels. Such remodeling allows the vessels to cope with physiological or pathological conditions such as restenosis and hypertension. The processes involved in vascular remodeling include cellular hypertrophy, hyperplasia, and enhanced protein synthesis or extracellular matrix protein reorganization. Studies using vascular smooth muscle cells (VSMC) have attempted to identify potential mechanisms underlying such structural alterations. Possible pathways include ion channels, integrin interaction between cells and the extracellular matrix, activation of tyrosine kinases, and autocrine production and release of growth factors (14, 25).

Two candidate autocrine molecules involved in biophysical force-induced vascular remodeling and vascular resistance are insulin-like growth factor I (IGF-I) and nitric oxide (NO). We have previously shown that in vitro cyclic stretch increases VSMC growth via the autocrine action of IGF-I (25). These studies parallel observations showing enhanced IGF-I expression in vessel segments subjected to elevated pressures (4) and enhanced IGF-I protein levels seen postballoon angioplasty (23). That IGF-I has been found to be secreted by VSMC and that VSMC express IGF-I receptors imply that this hormone is involved in an autocrine feedback loop by which VSMC hyperplasia is regulated at the local tissue level.

NO is antiproliferative and therefore may be vasculoprotective in states defined by hyperproliferation (20). The observed effects can be elicited either by exogenous NO added as a gas released from NO donors (6, 31), or by endogenous stimulation of VSMC inducible (i) nitric oxide synthase (NOS) via cytokines such as lipopolysaccharides, interferon, tumor necrosis factor, and interleukin. Specifically, NO inhibition of VSMC proliferation is associated with the following two distinct and reversible cell cycle arrests: an immediate cGMP-independent S-phase block, followed by a shiftback in the cell cycle from the G1-S boundary to a quiescent G0-like state (8, 22). Recently, we and others have published data showing that the vasodilatory effects of IGF-I are mediated by NO (19, 30). Specifically, IGF-I increases NOS activity/expression in isolated aorta, and the vasodilatory properties of IGF-I are attenuated when iNOS is inhibited. Together, these data suggest that proliferative (e.g., IGF-I) and antiproliferative (e.g., NO) pathways cross talk with one another in VSMC.

The current study therefore investigates the role of cyclic stretch-induced regulation of IGF-I and its potential regulation of VSMC NO expression and action. We hypothesize that stretch, via IGF-I and NO, activates reciprocal pathways involved in proliferation and antiproliferation, respectively. Furthermore, we anticipate that these two molecules regulate each other's activity/expression to provide integrated regulation of vascular growth and repair.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Culturing of aortic VSMC. Studies were carried out using A7r5 aortic smooth muscle cells from DB1X rats purchased from the American Type Culture Collection (Rockville, MD). These cells have been used extensively by our group and others in studying VSMC growth, ion transport mechanisms, contractility, etc. (25). VSMC were cultured in DMEM supplemented with 9% FBS and a 1% solution of penicillin/streptomycin at 37°C, 5% CO2, and 100% humidity. Cells were fed every other day with fresh medium and were passaged upon confluence (usually 3-7 days). For each experiment, cell passage number was matched such that all experimental groups originated from a single VSMC plate of passages 4 through 15.

Stretch apparatus. The Flexercell FX-2000 apparatus (Flexcell, Hillsboro, NC) is a computer-controlled device designed to deliver preprogrammed stretch regimens to wells attached to a vacuum-assisted gasketed baseplate. The baseplate remains in a tissue culture incubator throughout the duration of the stretch paradigm. The user programs the 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 while control wells remain unstretched (i.e., static). Many investigators have used this apparatus to study a variety of vascular cell functions (12, 25, 27). It has been determined that force on the attached cells is uniaxial (17).

Stretch paradigms. VSMC were seeded (~50,000 cells/well) on six-well collagen I-coated FlexI plates in DMEM-9% FBS. These plates have an elastomeric surface that allows the cells grown on them to stretch under vacuum. Once cells were ~50-60% confluent (usually 24-48 h postseeding), growth medium was replaced with serum-free medium (SFM) for 24 h, and on the day of the experiment, fresh SFM was substituted. This period of quiescence results in a mitotic index of <10% (13). With the use of the 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 to the collagen matrix were determined in subsets of VSMC grown in the presence or absence of stretch, inhibitors, and other treatments. Representative wells were viewed with phase-contrast optics (×40-200) to determine the potential gross areas of cell removal poststretch. Additionally, cell viability was determined via trypan blue exclusion, as we have described elsewhere (25), and by the cell's ability to bioreduce MTS (Owen's reagent) into formazan (Cell Titer 96 Proliferation Assay kit; Promega, Madison, WI).

Proliferation indexes. Hyperplastic growth responses were estimated by measuring [3H]thymidine incorporation and/or by directly quantifying dsDNA [fluorometrically utilizing (N,N'-tetramethyl-1,3-propane diamino)propylthiazole orange iodide, methanesulfonate] after terminating the stretch regimens. Briefly, 1 µCi/ml [3H]thymidine was added to prewashed wells, and treatment (stretch, inhibitors, etc.) was continued for the required duration. After 4 h, the media were aspirated and saved at -80°C for IGF-I and NO determinations. Wells were then washed three times with PBS supplemented with BSA. Next, ice-cold 15% 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 were then removed, added to 10 ml of scintillation cocktail, and counted in a liquid scintillation counter (model LS6500; Beckman Instruments, Fullerton, CA). Alternatively, when assessing and verifying the proliferation index fluorometrically, the Fluorescent Cell Titer 96 Cell Number Assay (Promega) was used. All wells were first prewashed with physiological salt solution followed by cell lysis. The fluorescent signal produced via the kit's reagent was measured with a Lab Systems (Helsinki, Finland) Ascent microtiter plate spectrofluorometer and was analyzed as relative fluorescent units.

Western blotting. After the appropriate duration of stretch or static conditions and treatment with vehicle or inhibitor, cell lysates were obtained immediately by incubating cells in ice-cold lysis buffer (30 µl/well) containing 10% protease inhibitor cocktail (Sigma Chemical) and gently scraping cells with a cell scraper. The protein content of lysates was determined using the Pierce BCA Protein Assay (Pierce Chemical, Rockford, IL), and aliquots of lysates (1-10 µg/well) were diluted 1:1 in 2× gel-loading buffer followed by boiling for 5 min. Volumes of lysates were adjusted for each gel so that total protein content was equalized among all wells. The gel (7.5% acrylamide) was run at 30 mA for 40 min. Proteins were transferred (Bio-Rad Mini-PROTEAN II Electrophoresis Cell and Mini Trans-Blot Electrophoretic Transfer Cell) to nitrocellulose (Nitrobind 0.45 µm; Fisher Scientific) for 1 h at 185 mA. After being blocked with dry milk at 37°C for 30 min, the nitrocellulose was incubated with goat anti-rabbit iNOS polyclonal antibody (1:15,000) for 45 min at 37°C, washed repeatedly, and then incubated with anti-rabbit IgG-horseradish peroxidase conjugate (1:80,000) at 37°C for 45 min. Protein bands were visualized with the Pierce Super Signal West Femto Kit.

Detection of immunoreactive IGF-I in conditioned medium. Specific IGF-I immunoreactivity was determined as we have described previously (25). 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. Samples were then centrifuged (5 min at 10,000 g), and the supernatant was removed and neutralized with 1 M Tris base. Samples were then diluted in assay buffer (PBS with 0.1% gelatin, 0.05% Tween 20, and 0.01% thimersal; pH 7.4) and subjected to IGF-I RIA. Typical assay performance for this RIA is as follows: sensitivity = 5 ng/ml; intra-assay precision = 3.7%; interassay precision = 4.9%.

NOS activity. NOS activity was assessed by measuring the conversion of L-arginine to citrulline plus NO as described previously (9). Briefly, cells were washed with Hanks' balanced salts (HBS) supplemented with BSA, 2 µCi/ml L-[3H]arginine, and aprotinin. After various times (0-60 min), conversion to citrulline was terminated by removal of incubation solution and replacement with fresh HBS supplemented with 5 mM unlabeled L-arginine and EDTA. Cells were washed and lysed with 20 mM Tris buffer and sonicated for 30 s, and cellular debris was collected quantitatively. After centrifugation, an aliquot of supernatant was applied to a 1:1 slurry of Dowex AG50WX8 resin in water. This slurry was added to a Bio-Rad EconoColumn, washed, and collected in vials. Only [3H]citrulline passes through the resin, and radioactivity of each sample was determined by liquid scintillation counting and was corrected on a per proliferation index basis.

Replicates and statistical analysis. Each experiment was performed a minimum of three times. For each experimental trial, triplicate wells from each group (i.e., control, stretch, stretch + inhibitor, etc.) were assayed. All data are expressed as means ± SE. Replicate experiments all showed equivalent changes in the assayed variable and therefore were pooled and expressed as a percentage of the respective control. Outliers, when present, were identified and removed via Dixon's gap test. Student's t-test or ANOVA with the post hoc Bonferonni correction assessed differences in population means. Multiple comparisons, when required, were performed using the Tukey-Kramer multiple-comparisons test. Population means were considered significantly different at P < 0.05. In Figs. 1-8, different lowercase letters above each bar denote significant differences in population means among the respective groups. All data were analyzed using the InStat software suite (GraphPad Software, San Diego, CA).


View larger version (23K):
[in this window]
[in a new window]
 
Fig. 1.   Effects of cyclic stretch (1 Hz at 120% original length for 48 h), cytomix (10 µg/ml lipopolysaccharide, 400 U/ml interferon, 1,000 U/ml tumor necrosis factor, and 100 U/ml interleukin-1beta ), and sodium nitroprusside (SNP; 100 nM) on vascular smooth muscle cell (VSMC) proliferation as measured fluorometrically with a probe quantitating dsDNA (different letters represent P < 0. 01; n = 6).



View larger version (17K):
[in this window]
[in a new window]
 
Fig. 2.   Effects of cyclic stretch and cytomix on VSMC nitric oxide secretion measured fluorometrically as total nitrite + nitrate in conditioned media samples (different letters represent P < 0.01; n = 6 experiments). Brackets denote concentration.



View larger version (31K):
[in this window]
[in a new window]
 
Fig. 3.   A: representative examples of Western blots showing the effects of cytomix treatment (top) and cyclic stretch (bottom; both treatments as described for Fig. 1) on inducible (i) nitric oxide synthase (NOS) expression in VSMC. Arrows indicate placement of 130-kDa molecular weight marker. B: densitometric data obtained from 3 separate Western blots showing change in iNOS content in static vs. stretched VSMC (P < 0.001 vs. static group).



View larger version (17K):
[in this window]
[in a new window]
 
Fig. 4.   Effects of cyclic stretch and NOS blockade [100 µM NG-monomethyl-L-arginine (L-NMMA)] on VSMC proliferation index (different letters represent P < 0.05; n = 4).



View larger version (22K):
[in this window]
[in a new window]
 
Fig. 5.   Effects of cyclic stretch, 100 µM L-NMMA, and 100 nM SNP on VSMC immunoreactive (IR) IGF-I secretion expressed per proliferative index [relative fluorometric units (rfu)] to account for changes in cell number among treatment groups (different letters represent P < 0.05; n = 3).



View larger version (10K):
[in this window]
[in a new window]
 
Fig. 6.   Effects of exogenous IGF-I (10-7 M) on NOS activity in static VSMC. NOS activity was measured by assessing conversion rates of radiolabeled arginine to radiolabeled citrulline as described in MATERIALS AND METHODS (n = 3).



View larger version (15K):
[in this window]
[in a new window]
 
Fig. 7.   Effects of exogenous IGF-I (10-7 M) on NO secretion from static VSMC. NO was measured fluorometrically in conditioned media samples (*P < 0.001 vs. respective timed controls, n = 4).



View larger version (15K):
[in this window]
[in a new window]
 
Fig. 8.   Schematic representation of major findings in this study. Cyclic stretch simultaneously stimulates IGF-I secretion and NOS activity and expression in VSMC. IGF-I is necessary for cyclic stretch-induced proliferation, as we have reported previously (25). IGF-I effects are attenuated by the concomitant increase in NO secretion, which serves to attenuate the proliferative response. Citrulline, another product of NOS, may also add to such antiproliferative effects.

Chemicals, kits, and antibodies. Human recombinant IGF-I and IGF-I RIA components were kind gifts from Genentech (San Francisco, CA). Recombinant mouse interferon-gamma was purchased from PBL Laboratories (New Brunswick, NJ); lipopolysaccharide, mouse recombinant tumor necrosis factor-alpha , NG-monomethyl-L-arginine (L-NMMA), sodium nitroprusside (SNP), and rat recombinant interleukin (IL)-1beta were purchased from Sigma Chemical (St. Louis, MO). Goat anti-iNOS polyclonal antibody and anti-rabbit IgG HRPO conjugate were both from Transduction Laboratories (Lexington, KY). The Cell Titer 96 Cell Number Assay and The Cell Titer 96 Cell Proliferation Assay kits were from Promega. The fluorometric nitrate/nitrite assay kit was from Cayman Chemical (Ann Arbor, MI).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cyclic stretch does not affect cell viability. Before the start of stretch, 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 (i.e., static) cells or stretched groups. Furthermore, cell viability was >= 95% in stretched and static wells, as determined by the Cell Titer 96 kit, which tests the cell's ability to bioreduce MTS into formazan.

Cyclic stretch enhances VSMC proliferative index, iNOS expression, and NO secretion. We confirmed our previous studies (25) showing that VSMC stretched on Flexwells at 1 Hz and 120% Lo for 48 h respond by inducing a 2.5-fold increase in DNA synthesis (Fig. 1). Microscopic examination revealed that cell detachment from the elastomeric support in the stretch and nonstretch groups was insignificant. In addition, cell viablility in all groups was >95% as determined by trypan blue exclusion and the Cell Titer 96 assay. When conditioned media from stretched cells were assayed, they displayed increased levels of NO compared with static control cells (Fig. 2). Furthermore, iNOS expression was induced in cyclically stretched VSMC (Fig. 3).

Cytokine induction of NOS increases iNOS expression and NO secretion but dramatically inhibits proliferation in stretched cells. We sought to ascertain whether our cell model expressed iNOS using a proven stimulator of the enzyme and, if so, the effects of its induction on proliferation and NO secretion. VSMC were pretreated with "cytomix" (containing 10 µg/ml lipopolysaccharide, 400 U/ml interferon, 1,000 U/ml tumor necrosis factor, and 100 U/ml interleukin; see Ref. 5) at the beginning of the 48-h stretch (or static) regimen. This standardized treatment dosage was used to test for the ability of these cell's iNOS cascade to be induced and therefore served as a convenient positive control. Cytomix inhibited proliferation (Fig. 1) while inducing iNOS expression (Fig. 3) and NO secretion (Fig. 2) in static and stretched VSMC.

NOS inhibition enhances and NO donor inhibits stretch-induced proliferation. Inhibition of NOS activity by pretreating VSMC with 100 µM L-NMMA (a dose known inhibit IGF-I-induced vasodilation; see Ref. 30) at the beginning of the 48-h stretch regimen resulted in enhancement of the stretch-induced proliferative response. Equivalent treatment in static cells had similar effects (Fig. 4). Furthermore, treatment with 100 nM SNP (an NO donor) inhibited the VSMC proliferation index in both static and stretched VSMC (Fig. 1).

NOS inhibition increases and NO donor decreased IGF-I secretion from stretched VSMC. In previous studies, we have shown that cyclic stretch of VSMC increases immunoreactive IGF-I secretion (25). Because we postulated cross talk between the IGF-I and NO pathways, we assessed the effects of NOS inhibition and an NO donor on IGF-I secretion in static and stretched VSMC. In both static and stretched cells, 100 µM L-NMMA enhanced immunoreactive IGF-I secretion on a per-cell basis (Fig. 5). This exaggerated IGF-I secretion paralleled the increased proliferative response seen in equivalently treated cells (Fig. 4). Furthermore, 100 nM SNP inhibited stretch-induced IGF-I secretion by ~35% (Fig. 5).

Exogenous IGF-I stimulates NOS activity and NO secretion in static VSMC. To further explore the cross talk between the IGF-I and NO pathways, we treated static VSMC with 10-7 M IGF-I and then assessed NOS activity by measuring the conversion of L-[3H]arginine to [3H]citrulline. This concentration has been shown by us to dramatically enhance the proliferative response in static, serum-starved cells (25). Figure 6 shows that NOS activity is increased by ~30-35% over control values within 30 min of IGF-I addition (each time point is expressed as a percentage of control cells not treated with IGF-I; time 0 value of 94 ± 6% is not statistically different from 100%). This increase seemed to be reflective of maximal IGF-I stimulatory activity, since NOS activity did not increase further at the 60-min time point. Figure 7 confirms that identical treatment with 10-7 M IGF-I for 30 min, 60 min, and 24 h results in greater NO secretion (vs. nontreated cells), as detected fluorometrically in conditioned medium.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Our studies show that cyclic stretch of VSMC activates both proliferative and antiproliferative pathways. Specifically, cyclic stretch increases secretion of IGF-I, a process that we have previously shown is requisite for stretch-induced hyperplasia (25). Second, we have shown that the identical stretch paradigm increases NO secretion from VSMC. Our preliminary data suggest that this occurs, in part, via an increase in iNOS activity and upregulation of its expression. Furthermore, inhibition of NOS (with L-NMMA) increases, whereas stimulation of iNOS (with cytokine mix) or addition of an NO donor decreases, the VSMC proliferative responses. Finally, we have shown that the proliferative and antiproliferative pathways appear to cross talk: inhibition of NOS (with L-NMMA) further stimulates IGF-I secretion, whereas an NO donor inhibits it. Therefore, our data suggest that IGF-I and NO reciprocally regulate cyclic stretch-induced proliferation and cross talk by a yet to be defined mechanism.

Figure 1 confirms data that we have published previously (25) showing that cyclic stretch enhances VSMC proliferation. This enhanced growth is dependent on autocrine IGF-I, since anti-IGF-I treatment completely blocks this response (25). Cyclic stretch has been shown to induce Egr-1 (early growth response gene) and c-jun mRNA in VSMC (18), suggesting a plausible mechanism by which stretch-induced IGF-I (and possibly platelet-derived growth factor and autocrine ANG II; Ref. 15) asserts these effects. Furthermore, antisense targeting of the IGF-I receptor (IGF-IR) in VSMC demonstrates that a decrease in IGF-IR density attenuates VSMC proliferation (16). Therefore, autocrine IGF-I seems necessary and sufficient to produce stretch-induced growth of VSMC in a serum-free environment. Figure 1 also shows that 100 nM SNP for 48 h also decreased the VSMC proliferation index in static and stretched VSMC. This SNP concentration has been shown to significantly increase NO in media for sustained periods of time (see Ref. 8). These experiments confirmed that an exogenous NO donor is capable of decreasing the proliferative index of static VSMC (10).

Novel to this study is the fact that cyclic stretch of VSMC concomitantly increases NO secretion (Fig. 2). Previous studies have shown that other biophysical forces such as shear stress enhance vascular endothelial cell constitutive NOS (cNOS) expression by altering transcriptional rates of the enzyme, whereas oscillatory shear stress and cyclic stretch appear to control cNOS expression through posttranscriptional regulatory events (32). Others have shown that cyclic stretch upregulates cNOS expression in aortic endothelial cells, thus emphasizing the importance of hemodynamic forces in the regulation of NOS in vivo (1).

In regard to the effects of cyclic stretch regulation of NO in VSMC, our NO secretion and iNOS expression data seemingly contradict a previous report showing that cyclic stretch failed to induce iNOS mRNA (vs. protein expression) in rat aortic VSMC (29). However, those studies used a stretch magnitude of 110% Lo (compared with 120% Lo in our study) for a period of 24 h (compared with 48 h in our study). Additionally, those authors did not measure other NOS endpoints such as NOS activity, NOS protein content, or NO secretion. Therefore, the stated differences in stretch parameters coupled with a possible short half-life of the iNOS mRNA transcript may explain, in part, this contradiction.

Depicted in Figs. 1 and 2 is the ability of cytomix to regulate VSMC growth and NO secretion. Although cytomix treatment was originally intended to serve simply as a positive control in our studies, several interesting conclusions can be drawn from our data. First, cytomix enhanced NO secretion in static cells (Fig. 2). This effect likely underlies decreased proliferation in cytokine-treated cells (Fig. 1), as previously suggested by similar studies describing the antiproliferative activity of NO (10). Second, cytomix had a much more dramatic effect on cyclically stretched VSMC growth and NO secretion: cytomix enhanced NO secretion twofold over nontreated stretched cells (Fig. 2), whereas it depressed proliferation by 80% vs. nontreated stretched cells. One interpretation of these data is that superinduction of NOS, by a combination of cytomix, stretch, and stretch-induced IGF-I action, resulted in the dramatic growth suppression.

Western blotting data show that static cells express no detectable iNOS protein (Fig. 3A), despite displaying a basal level of NO secretion (Figs. 2 and 7). Immunoblotting VSMC lysates 30 min poststretch with anti-iNOS also showed no detectable expression (data not shown). Similarly, immunoblotting attempts with several cNOS antibodies also resulted in no detectable cNOS expression. However, others have reported that VSMC express cNOS mRNA and that insulin (having high IGF-I homology and affinity for IGF-IR; see Ref. 26) stimulates cNOS activity 3.5-fold within 30 min (28). Therefore, basal cNOS expression may underlie basal and early induction of NOS activity (Fig. 6) and NO secretion (Fig. 7; 30- and 60-min time points), whereas stretch induction of iNOS is likely responsible, at least in part, for later (24 h; Fig. 3A) increases in NO.

Pretreatment of VSMC with an NOS inhibitor (L-NMMA) before beginning stretch results in significant enhancements of stretch-induced growth (Fig. 4) and IGF-I secretion (Fig. 5). Similarly, an NO donor (SNP) also inhibits VSMC proliferation (Fig. 1) and IGF-I secretion (Fig. 5). There are several interpretations of these data. First, the enhanced proliferative response in L-NMMA-treated stretched cells resulted from enhanced IGF-I secretion/action. That SNP decreased IGF-I secretion supports this conclusion. Second, stretch-induced NO secretion (Fig. 2) normally attenuates IGF-I secretion, but when NOS is inhibited, such suppression is removed. Finally, the described antiproliferative effects of basal NO action (9) in these cells 1) is exemplified in our studies using SNP and 2) is blunted (via L-NMMA), leading to further proliferative ability. Thus there appears to be a stretch-induced IGF-I-dependent fraction and an NO-dependent fraction of the total proliferative response. In vivo data seem to support this idea, as exemplified by the observation that decreased NO may be necessary, but not sufficient, to induce VSMC proliferation in response to a decrease in blood flow since decreased VSMC proliferation (induced by flow switching) is accompanied by declines in NO production, cNOS mRNA, and cNOS protein (16).

Figures 6 and 7 show that 100 nM exogenous IGF-I, a concentration known to dramatically increase VSMC growth (25) and NO secretion from endothelial-denuded aorta (19), increases both NOS activity and NO secretion from VSMC. This dose of IGF-I is also equivalent to published rat serum IGF-I concentrations of ~90 nM (2). Recent reports addressing the effects of IGF-I on NO release are contradictory. For example, it has been reported that IGF-I decreases NOS activity in cytokine-treated rat aortic VSMC but has no effect on untreated VSMC (23). However, the highest IGF-I concentration (30 ng/ml = 4 nM) tested in that study 1) was 28-fold less than that tested in the present study, 2) was less than that shown to attenuate vascular contractility (30), 3) was less than that which increased NO release from endothelial-denuded rat aorta (19), and 4) may well be less than the local tissue IGF-I concentrations attained in vivo. Thus the IGF-I concentration differences may underlie this discrepancy.

Our data lend support to the theory that the IGF-I and NO pathways may cross talk with one another, possibly by regulating each other's production. Specifically, IGF-I enhances NOS activity and NO secretion, whereas NO is likely poised to inhibit IGF-I secretion (Fig. 5). Previous data suggest that IL-1beta induction of iNOS expression involves potentiation of cytokine-induced activation of mitogen-activated protein kinase (MAPK; see Ref. 11). IGF-I also stimulates MAPK, (26) so despite NO inhibition of further IGF-I secretion, it may well enhance IGF-I signaling at the level of MAPK. This potential cross talk mechanism is an area of investigation we are actively pursuing.

In conclusion, Fig. 8 depicts a simplified view of stretch regulation of VSMC growth based on data reported thus far. Stretch concomitantly activates a mitogenic (IGF-I) and an antimitogenic (iNOS) pathway in VSMC. In an autocrine fashion, IGF-I stimulates a series of growth genes that induce cell cycle progression, whereas NO stimulates antimitogenic/apoptotic signaling cascades. L-Citrulline (an additional product of iNOS action on arginine), which significantly decreases aortic VSMC proliferation (21), may also underlie a portion of this antiproliferative effect. Furthermore, cyclic stretch may stimulate iNOS activity in an IGF-I-dependent manner, whereas autocrine NO appears to blunt IGF-I secretion and its proliferative effects. A potential point of signaling overlap between these two molecules that might explain such cross talk is the p44/42 MAPK cascade, an area we are currently investigating.


    ACKNOWLEDGEMENTS

This study was supported by a Scientist Development Grant from the American Heart Association National Center (P. R. Standley), a Grant-in-Aid from the American Heart Association Desert-Mountain Affiliate (P. R. Standley), and a grant from Midwestern University's Office of Research and Sponsored Programs (P. R. Standley).


    FOOTNOTES

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).

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. Section 1734 solely to indicate this fact.

Received 27 November 2000; accepted in final form 16 July 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Awolesi, MA, Sessa WC, and Sumpio BE. Cyclic strain upregulates nitric oxide synthase in cultured bovine aortic endothelial cells. J Clin Invest 96: 1449-1954, 1995[ISI][Medline].

2.   Bouvy, ND, Margaret RL, Tseng LN, Steyerberg EW, Lamberts SW, Jeekel H, and Bonjer HJ. Laproscopic versus conventional bowel resection in the rat. Earlier restoration of serum IGF-I levels. Surg Endosc 12: 412-415, 1998[ISI][Medline].

3.   Cercek, B, Sharifi B, Barath P, Bailey L, and Forrester JS. Growth factors in pathogenesis of coronary arterial restenosis. Am J Cardiol 68: 24C-33C, 1991[Medline].

4.   Chen, Y, Bornfeldt KE, Arner A, Jennische E, Malmqvist U, Uvelius B, and Arnqvist HJ. Increase in insulin-like growth factor I in hypertrophying smooth muscle. Am J Physiol Endocrinol Metab 266: E224-E229, 1994[Abstract/Free Full Text].

5.   Chester, AH, Borland JA, Buttery LD, Mitchell JA, Cunningham DA, Hafizi S, Hoare GS, Springall DR, Polack JM, and Yacoub MH. Induction of nitric oxide synthase in human vascular smooth muscle: interactions between proinflammatory cytokines. Cardiovasc Res 38: 814-821, 1998[ISI][Medline].

6.   Cornwell, TL, Arnold E, Boerth NJ, and Lincoln TM. Inhibition of smooth muscle growth by nitric oxide and activation of cAMP-dependent protein kinase by cGMP. Am J Physiol Cell Physiol 267: C1405-C1413, 1994[Abstract/Free Full Text].

7.   Du, J, and Delafontaine P. Inhibition of VSMC growth through antisense transcription of rat IGF-I receptor DNA. Circ Res 76: 963-969, 1995[Abstract/Free Full Text].

8.   Garg, UC, and Hassid A. NO generating vasodilators inhibit mitogenesis and proliferation of Balb/C 3T3 fibroblasts by a cGMP-independent mechanism. Biochem Biophys Res Commun 171: 474-479, 1990[ISI][Medline].

9.   Hayakawa, H, and Raij L. The link among nitric oxide synthase activity, endothelial function, and aortic and ventricular hypertrophy in hypertension. Hypertension 29: 235-241, 1997[Abstract/Free Full Text].

10.   Hecker, M, Cattaruzza M, and Wagner AH. Regulation of inducible nitric oxide synthase gene expression in vascular smooth muscle cells. Gen Pharmacol 32: 9-16, 1999[Medline].

11.   Jiang, B, and Brecher P. N-acetyl-L-cysteine potentiates interleukin-1 beta induction of nitric oxide synthase: role of p44/42 mitogen-activated protein kinases. Hypertension 35: 914-918, 2000[Abstract/Free Full Text].

12.   Krishnankutty, S, Wilson E, Chatterjee K, and Ives HE. Mechanical strain and collagen potentiates mitogenic activity of angiotensin II in rat vascular smooth muscle cells. J Clin Invest 92: 3003-3007, 1993[ISI][Medline].

13.   Krug, LM, and Berk BC. Na+,K+-adenosine triphosphatase regulation in hypertrophied vascular smooth muscle cells. Hypertension 20: 144-150, 1992[Abstract].

14.   Lehoux, S, and Tedgui A. Signal transduction of mechanical stress in the vascular wall. Hypertension 32: 338-345, 1998[Abstract/Free Full Text].

15.   Li, Q, Muragaki Y, Ueno H, and Ooshima A. Stretch-induced proliferation of cultured vascular smooth muscle cells and a possible involvement of local renin-angiotensin system and platelet-derived growth factor (PDGF). Hypertens Res 20: 217-223, 1997[Medline].

16.   Mattsson, EJ, Geary RL, Kraiss LW, Vergel S, Liao JK, Corson MA, Au YP, Hanson SR, and Clowes AW. Is smooth muscle growth in primate arteries regulated by endothelial nitric oxide synthase? J Vasc Surg 28: 514-521, 1998[ISI][Medline].

17.   Mills, I, Letsou G, Rabban J, Sumpio B, and Gewirtz H. Mechanosensitive adenylate cyclase activity in coronary vascular smooth muscle cells. Biochem Biophys Res Commun 171: 143-147, 1990[ISI][Medline].

18.   Morawicz, H, Ma YH, Vives F, Wilson E, Sukhatme VP, Holtz J, and Ives HE. Rapid induction and translocation of Egr-1 in response to mechanical strain in vascular smooth muscle cells. Circ Res 84: 678-687, 1999[Abstract/Free Full Text].

19.   Muniyappa, R, Walsh MF, Rangi JS, Zayas RM, Standley PR, Ram JL, and Sowers JR. IGF-I increases vascular smooth muscle NO production. Life Sci 61: 925-931, 1997[ISI][Medline].

20.   Ravil, S, Szabolcs M, Barbone A, Albala A, Michler RE, and Cannon PJ. iNOS is upregulated in human transplant coronary artery disease. Transplant Proc 29: 2579-2580, 1997[ISI][Medline].

21.   Ruiz, E, Del Rio M, Somoza B, Ganado P, Sanz M, and Tejerina T. L-Citrulline, the by-product of nitric oxide synthase, decreases vascular smooth muscle proliferation. J Pharmacol Exp Ther 290: 310-3213, 1999[Abstract/Free Full Text].

22.   Sarkar, R, Gordon D, Stanley JC, and Webb RC. Dual cell cycle-specific mechanisms mediate the antimitogenic effects of nitric oxide in vascular smooth muscle cells. J Hypertens 15: 275-283, 1997[ISI][Medline].

23.   Schini, VB, Catovsky S, Schray-Utx B, Busse R, and Vanhoutte PM. IGF-I inhibits induction of nitric oxide synthase in vascular smooth muscle cells. Circ Res 74: 24-32, 1994[Abstract].

24.   Sidway, AN, Hakim FS, Jones BA, Norberto JM, Neville RF, and Korman LY. IGF-I binding in injury-induced hyperplasia in rabbit aorta. J Vasc Surg 23: 308-313, 1996[ISI][Medline].

25.   Standley, PR, Obards TJ, and Martina CL. Cyclic stretch regulates autocrine IGF-I in vascular smooth muscle cells: implications in vascular hyperplasia. Am J Physiol Endocrinol Metab 276: E697-E705, 1999[Abstract/Free Full Text].

26.   Standley, PR, Ram JL, and Sowers JR. The vasculature as an insulin sensitive tissue: Implications of insulin and IGF-I in hypertension, diabetes, atherosclerosis and arterial smooth muscle cell growth. In: Calcium Regulating Hormones and Cardiovascular Disease, edited by Crass BF.. Boca Raton: CRC, 1994, p. 275-293.

27.   Sumpio, B, and Banes AJ. Response of porcine aortic smooth muscle cells to cyclic tensional deformation in culture. J Surg Res 44: 696-701, 1988[ISI][Medline].

28.   Trovati, M, Massucco P, Mattiello L, Costamagna C, Aldieri C, Cavalot F, Anfossi G, Busia A, and Ghigo D. Human VSMC express a constituitive nitric oxide synthase that insulin rapidly activates, thus increasing guanosine 3':5'-cyclic monophosphate and adenosine 3':5'-cyclic monophosphate concentrations. Diabetologia 42: 831-839, 1999[ISI][Medline].

29.   Wagner, CT, Durante W, Christodoulldes N, Hellums JD, and Schaefer AI. Hemodynamic forces induce the expression of heme oxygenase in cultured vascular smooth muscle cells. J Clin Invest 100: 585-596, 1997.

30.   Walsh, MF, Barazi M, Pete G, Muniyappa R, Dunbar JC, and Sowers JR. Insulin-like growth factor I diminishes in vivo and in vitro vascular contractility: role of vascular nitric oxide. Endocrinology 137: 1798-1803, 1996[Abstract].

31.   Yang, W, Ando J, Korenaga R, Toyo-oka T, and Kamiya A. Exogenous NO inhibits proliferation of cultured vascular endothelial cells. Biochem Biophys Res Commun 203: 1160-1167, 1994[ISI][Medline].

32.   Ziegler, T, Silacci P, Harrison VJ, and Hayoz D. Nitric oxide synthase expression in endothelial cells exposed to mechanical forces. Hypertension 32: 352-355, 1998.


Am J Physiol Endocrinol Metab 281(6):E1165-E1171
0193-1849/01 $5.00 Copyright © 2001 the American Physiological Society