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(Circulation. 1996;93:99-105.)
© 1996 American Heart Association, Inc.

Articles

Induction of DNA Synthesis by a Single Transient Mechanical Stimulus of Human Vascular Smooth Muscle Cells

Role of Fibroblast Growth Factor–2

George C. Cheng, MS; Peter Libby, MD; Alan J. Grodzinsky, PhD; Richard T. Lee, MD

From the Cardiovascular Division (G.C.C., P.L., R.T.L.), Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston; and Division of Health Sciences and Technology (G.C.C., A.J.G., R.T.L.) and Department of Mechanical Engineering (A.J.G., R.T.L.), Massachusetts Institute of Technology, Cambridge, Mass.

Correspondence to Richard T. Lee, MD, Brigham and Women's Hospital, 75 Francis St, Boston, MA 02115.

	Abstract

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Background Although mechanical vascular injury leads to smooth muscle cell proliferation that contributes to restenosis after balloon angioplasty, the role of the single transient mechanical stimulation of smooth muscle cells in this process is unknown.

Methods and Results To test the hypothesis that a single transient mechanical stimulus can increase DNA synthesis, human vascular smooth muscle cells cultured in a three-dimensional collagen gel system were subjected to transient compression. Transient compression (5-minute duration) of smooth muscle cell–collagen gel cultures in defined serum-free conditions led to delayed increases in [³H]thymidine incorporation. At 12 to 24 hours after compression, there was a 3.3±0.5-fold (P<.001 versus control) and 3.0±0.6-fold (P<.002 versus control) increase for 60% and 80% strain, respectively; at 24 to 36 hours after compression, there was a 1.8±0.5-fold (P<.05 versus control) and 4.3±0.8-fold (P<.001 versus control) increase. Also, serum-free media conditioned by transiently compressed gel cultures induced DNA synthesis in control, unstimulated smooth muscle cell cultures, suggesting the release of growth factors by transient compression. Although neutralizing antibodies against platelet-derived growth factor did not affect the mechanical induction of DNA synthesis, a neutralizing monoclonal antibody against fibroblast growth factor–2 (FGF-2) decreased this induction by 89% and completely blocked the increase in DNA synthesis caused by media conditioned by transiently compressed gels. Media conditioned by transient compression contained elevated levels of FGF-2 (17±5 versus 2±2 pg/mL for control, P<.005) with no increase in lactate dehydrogenase activity, suggesting release of FGF-2 with sublethal cellular injury.

Conclusions A single transient mechanical stimulus increases DNA synthesis in human vascular smooth muscle cells, in part by autocrine or paracrine FGF-2 release.

Key Words: arteriosclerosis • stress • growth substances

	Introduction

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Although percutaneous transluminal coronary angioplasty is widely used in the treatment of coronary atheroma, the success of this procedure is often limited by late restenosis. Within months after angioplasty, 20% to 40% of patients develop recurrent neointimal lesions consisting of abundant extracellular matrix and hyperplastic vascular smooth muscle cells.¹ A cascade of pathways involving vascular smooth muscle cells, endothelial cells, platelets, and macrophages may mediate neointimal growth through cytokines and growth factors.¹ The vascular responses to the direct mechanical strain of balloon angioplasty, as well as to acute local thrombosis, may be important early events in this cascade.

Mechanical forces may influence biological responses through various signal transduction mechanisms to alter structure and function at the cellular and molecular levels.^{2 3 4} Vascular smooth muscle cells in vivo undergo dynamic and static mechanical strains from superimposed pulsatile and mean pressure loads of the cardiac cycle, and these strains may be altered in pathological conditions such as hypertension.^{5 6 7 8} Several in vitro studies have assessed potential biological responses of vascular smooth muscle cells to physiologically relevant mechanical conditions. For example, dynamic mechanical strain can modulate cellular orientation,^{9 10} matrix synthesis,^{11 12} and proliferation^{5 9 13 14} of cultured vascular smooth muscle cells, and continuous static strain increases proto-oncogene expression and phosphoinositide turnover.¹³

During balloon angioplasty, smooth muscle cells and other vascular cells are exposed to a transient mechanical strain of relatively large magnitude, as the vessel is dilated briefly and then allowed to relax. In vivo studies of balloon injury demonstrate intimal smooth muscle cell hyperplasia caused by both migration from the media and increased proliferation,^{15 16} and these processes may be mediated by PDGF and FGF-2. PDGF is both a mitogen and a chemoattractant for vascular smooth muscle cells¹⁷ and may be synthesized and released by platelets, smooth muscle cells, and endothelial cells.¹⁸ In animal models, vascular injury induces expression of mRNA for the PDGF-A chain and the PDGF receptor and ß subunits in the neointima.^{19 20} Also, intimal thickening induced by injury can be inhibited by a polyclonal antibody against PDGF.²¹ Under some conditions, PDGF can be mechanically induced in cultured vascular smooth muscle cells.⁵

FGF-2, also known as basic fibroblast growth factor, may be essential for the early smooth muscle cell proliferative response in injured rat arteries. Like PDGF, FGF-2 is mitogenic and chemotactic for vascular smooth muscle cells in vitro and can be synthesized by both vascular smooth muscle cells and endothelial cells.^{22 23} This growth factor lacks a signal sequence for classic secretion²⁴ and may be released on cell injury.^{25 26 27} In rat arteries, injury-induced smooth muscle cell proliferation is significantly greater with combined injury to endothelial and smooth muscle cells than with endothelial denudation alone,²⁸ can be inhibited by a neutralizing antibody against FGF-2,²⁹ and does not require circulating platelets.³⁰ In addition, balloon injury leads to increased expression of mRNA for both FGF-2 and its receptor by smooth muscle cells and endothelial cells.³¹

Although animal models of restenosis have revealed potential mechanisms of smooth muscle cell hyperplasia, they have not readily dissected the specific role of the transient mechanical strain on smooth muscle cell responses. This study was performed to test the hypothesis that a single transient mechanical strain can induce DNA synthesis in human vascular smooth muscle cells, potentially mediated by autocrine growth factors.

	Methods

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Cell Culture
Human vascular smooth muscle cells were derived from explants of discarded portions of saphenous veins obtained during coronary bypass surgery performed at Brigham and Women's Hospital. Monolayer smooth muscle cells were cultured in DMEM (Whittaker Bioproducts, Inc) with 10% FCS at 37°C and 5% CO₂. These conditions are selective for growth of smooth muscle cells over endothelial cells.³² The explant and culture technique was identical to the protocol used in previous studies of cultured vascular smooth muscle cells.^{33 34 35} Cells were cultured through passages 3 through 5 before transfer to a three-dimensional collagen gel culture system for use in transient compression experiments.

Cell culture in hydrated collagen gels was performed with Vitrogen 100 collagen (Celtrix Pharmaceutical) as previously described.³⁶ Cells (2.5 to 3.0x10⁵) were suspended in 1.25 mL collagen (2.8 mg/mL) and cultured in standard 24-well (16-mm diameter) culture plates (Costar Corporation). Gel cultures were maintained in DMEM supplemented with 10% FCS and 0.07 mmol/L ascorbate-2-phosphate (Wako Pure Chemical Industries) and received a change of culture medium every 2 days. The contraction of collagen gel matrices by these cells may be resisted by local adhesions to the culture plate wells; thus, to facilitate uniform, axisymmetrical gel contraction, on day 5 the gel cultures were gently detached from the sides of the wells. Over the course of 10 days in culture, smooth muscle cell gels contracted uniformly to disks with nearly identical dimensions in a given experiment; the standard deviation of gel thicknesses, measured by micrometer before compression, was less than 5% of the mean value. Final contracted gel diameter ranged from 4.5 to 7.0 mm, and thickness ranged from 1.0 to 2.5 mm in different experiments.

Mechanical Stimulation
Smooth muscle cell–gel cultures received a final media change on day 10 and were subjected to a uniaxial, unconfined, transient compression consisting of a brief fixed-displacement static compression, followed by release of compression. This mechanical stimulus was selected to simulate the radial compressive strain experienced by the vessel wall during balloon angioplasty. For serum-free conditions, smooth muscle cell gels were washed six times on day 9 with defined, serum-free IT medium (equal volumes of DMEM and Ham's F-12 supplemented with 1 µmol/L insulin and 5 µg/mL transferrin) at 37°C and further incubated for 24 hours in this medium before the transient compression. The compression apparatus consisted of a base to hold the culture plate and a rigid top compression plate (Fig 1). Compression platens and spacer posts, machined from autoclavable polysulfone rod stock (Patriot Plastics & Supply, Inc) to a uniform length (19.05 mm), were affixed to this top plate and concentrically aligned with culture plate wells. By lowering the top plate assembly, static compression was applied to the gels by the compression platens at a desired strain determined by the thickness of Teflon disks positioned beneath the spacer posts within vacant adjacent wells; this imposed strain ranged from 50% to 80% of the initial gel thickness in all experiments. For most experiments, a moderate strain of 50% to 65% was used; the 80% strain was used for evaluating the effect of higher amplitudes of initial strain. The duration of transient compression ranged from 0.2 to 5.0 minutes; by 2 hours after removal of compression, the gels were observed to reswell to a state of <10% strain. Parallel control smooth muscle cell gels were not compressed but were otherwise handled in identical fashions.

View larger version (18K): [in this window] [in a new window]   Figure 1. Diagram of apparatus used to apply transient mechanical compression to three-dimensional vascular smooth muscle cell cultures (SMC-Gel). A compression plate was lowered onto the gel cultures, and strain was limited by Teflon disks. After a brief period (usually 5 minutes), the compression plate was removed.

[³H]Thymidine Incorporation
After compression, culture media were supplemented with methyl-[³H]thymidine (6.7 mCi/mmol, DuPont-NEN) to measure DNA synthesis. To assess time course, designated smooth muscle cell–gels were radiolabeled during consecutive 12-hour periods; additional control gels were radiolabeled during the 12 hours preceding the transient compression and served as an initial baseline. After radiolabeling, smooth muscle cell–gel matrices were solubilized overnight in 4 mol/L urea and 0.5% sodium dodecyl sulfate and precipitated with 10% trichloroacetic acid at 4°C. Precipitates were vacuum-collected on 0.45-µm filters (Millipore Corp) and washed with 10% trichloroacetic acid at 4°C. Filters were air-dried, mixed with Ecolume (ICN Biomedicals) scintillation cocktail, and counted with a scintillation counter (Pharmacia LKB Nuclear). To assess nonspecific radiolabeling of the gel matrices, parallel smooth muscle cell–gel cultures in some experiments were subjected to three cycles of freezing at -80°C and thawing at 37°C before radiolabeling. The nonspecific counts were generally less than 5% of those in the live smooth muscle cell–gel cultures.

Conditioned-Media Experiments
To test the hypothesis that transient compression of the smooth muscle cell–gel cultures triggers release of mitogens from the cells, conditioned-media experiments were performed. Smooth muscle cell–gel cultures were either compressed for 5 minutes (60% strain) or were not compressed, and then further incubated for 24 hours. Culture media were then exchanged between the two groups and supplemented with 4 µCi/mL methyl-[³H]thymidine for 24 hours of radiolabeling. Uncompressed control gels were incubated for 24 hours, and the media were removed and immediately returned to the identical wells before radiolabeling.

Antibody Neutralization Experiments
Smooth muscle cell–gel cultures, made quiescent under defined serum-free conditions as described above, were subjected to transient compression (60% strain, 5 minutes). In separate experiments, neutralizing polyclonal rabbit antibodies against either human PDGF-A (10 µg/mL) or PDGF-B (5 µg/mL) (Genzyme Corporation) chain were added to designated culture media immediately after release of compression, and the gel cultures were further incubated for either 12 or 24 hours. Media were then supplemented with 4 µCi methyl-[³H]thymidine for 12 hours of radiolabeling. For positive controls, exogenous recombinant human PDGF-AA (2 ng/mL) or PDGF-BB (4 ng/mL) (Genzyme Corporation) was added to the culture media of control (unstimulated) smooth muscle cell gels in either the presence or absence of the appropriate neutralizing anti-PDGF antibody.

In similar experiments, we assessed the ability of a neutralizing monoclonal antibody against human FGF-2 (0.1, 1.0, or 10 µg/mL, Upstate Biotechnology Inc) to inhibit the induction of [³H]thymidine incorporation by transient compression (65% strain, 5 minutes). A nonimmune mouse IgG antibody (10 µg/mL, Sigma Chemical Co) was added to parallel cultures to assess the specificity of the anti–FGF-2 antibody. As a positive control, exogenous human recombinant FGF-2 (10 ng/mL, Upstate Biotechnology Inc) was added to the culture media of control smooth muscle cell gels in either the presence or absence of the anti–FGF-2 antibody.

Measurement of FGF-2 and LDH Activity
Serum-free media conditioned for 12 hours by transiently compressed and control gels were harvested and assayed for FGF-2 with a quantitative enzyme immunoassay (Quantikine Human FGF basic Immunoassay, R&D Systems). To assess cell injury, LDH activity in the media was measured with a quantitative, colorimetric assay (Sigma). The lower limit sensitivity of this assay was 40 U/mL. Parallel media, conditioned by gel cultures subjected to three cycles of freeze/thaw, were used as positive controls.

Measurement of Total DNA Content
To further assess cell injury, total DNA in smooth muscle cell–gel cultures was measured at 12 hours after transient compression (65%) and compared with that in uncompressed gel cultures. Smooth muscle cell–gel cultures were frozen, lyophilized, and digested in 1 mL/gel papain (Sigma, 125 µg/mL) for 24 hours at 60°C. Hoechst 33258 dye solution was added to digest aliquots, and DNA content was measured fluorometrically, using calf thymus DNA as a standard.³⁷

Measurement of FGF-2 mRNA
Total cellular RNA in smooth muscle cell–gel cultures was isolated by guanidine isothiocyanate extraction at 0, 0.5, 1, 2, 3, and 12 hours after compression; RNA extracted from smooth muscle cells treated for 30 minutes with either interleukin-1 or 10% FCS was used as a positive control. Total RNA (7.5 µg) of each sample was electrophoresed in the presence of ethidium bromide on 1.4% agarose gels containing formaldehyde and transferred to nylon hybridization membranes. Blots were photographed under UV illumination to verify evenness of loading and transfer, UV immobilized, and then hybridized to a 1.2-kb cDNA probe (gift of Prof M. Klagsbrun, Children's Hospital, Boston, Mass) for FGF-2 that was labeled with [³²P]dCTP using the random primers method. Blots were washed and autoradiographed with x-ray film for 3 days at -80°C.

Statistical Analysis
All data are presented as mean±1 SD for three or four measurements. For comparison between specific groups of continuous variables, the two-sample Student's t test was used. A value of P<.05 was considered statistically significant. The findings are representative of results of at least two independent experiments.

	Results

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DNA Synthesis After Transient Strain
In serum-supplemented conditions, transient compression (5 minutes) led to an increase in [³H]thymidine incorporation by vascular smooth muscle cells in collagen gels after an initial period of decreased incorporation (Fig 2). From 12 to 24 hours after release of compression of 60% or 80% strain, there were no differences in [³H]thymidine incorporation relative to control; from 24 to 36 hours, there was a 1.6±0.4-fold (P<.05 versus control) and a 2.4±0.6-fold (P<.005 versus control) increase in [³H]thymidine incorporation for 60% and 80% strain, respectively.

View larger version (15K): [in this window] [in a new window]   Figure 2. Plot showing increase in [3H]thymidine incorporation by human vascular smooth muscle cells after a 5-minute compression at time 0 in the presence of 10% FCS at strains of 80% or 60%. Control 12-hour incorporated counts were 710±220, 560±210, 1290±207, and 930±140 cpm at 0, 12, 24, and 36 hours after compression, respectively, for three or four measurements; error bars denote 1 SD.

Exogenous serum-associated growth factors were not required for the mechanical induction of DNA synthesis. In defined serum-free medium, transient compression of gel cultures again led to a delayed increase in [³H]thymidine incorporation (Fig 3). From 12 to 24 hours after release of compression, there was a 3.3±0.5-fold (P<.001 versus control) and 3.0±0.6-fold (P<.002 versus control) increase in [³H]thymidine incorporation for 60% or 80% strain, respectively; from 24 to 36 hours, there was a 1.8±0.5-fold (P<.05 versus control) and a 4.3±0.8-fold (P<.001 versus control) increase in [³H]thymidine incorporation for 60% and 80% strain, respectively. Cumulative incorporated counts during the 36-hour period after compression were 1670±120, 2730±440, and 3960±470 cpm for 0%, 60%, and 80% compressions, respectively (P<.001 by ANOVA). Maximum inductions of [³H]thymidine incorporation by strains of 60% to 65% in different experiments ranged from 1.6- to 3.0-fold in the presence of serum (five experiments) and from 1.8- to 10-fold in the absence of serum (seven experiments).

View larger version (17K): [in this window] [in a new window]   Figure 3. Plot showing increase in [3H]thymidine incorporation by human vascular smooth muscle cells after a 5-minute compression at time 0 in defined serum-free conditions at strains of 80% or 60%. Control 12-hour incorporated counts were 730±200, 350±70, and 590±120 cpm at 12, 24, and 36 hours after compression, respectively, for four measurements; error bars denote 1 SD.

To minimize potential effects of exogenous mitogens and other serum constituents, further characterization of this induction of [³H]thymidine incorporation was performed under serum-free conditions. Defined serum-free media conditioned for 12 hours by transiently compressed gels increased [³H]thymidine incorporation in target control (unstimulated) gels by 2.5±0.5-fold more than control media (P<.005). Conversely, transiently compressed gels whose media were replaced with control media demonstrated no induction in DNA synthesis. These data suggested the release of autocrine growth factor(s) by transient compression of the smooth muscle cell gels.

Autocrine Growth Factor Studies
Antibody neutralization experiments were performed under serum-free conditions to study the possible role of specific endogenous growth factors. Neutralizing antibodies against either PDGF-A (Fig 4A) or PDGF-B (Fig 4B) chain did not inhibit induction of [³H]thymidine incorporation by transient compression. In parallel control experiments, neutralizing antibodies to PDGF were able to block the induction of [³H]thymidine incorporation by the appropriate exogenous PDGF homodimers. However, a monoclonal anti–FGF-2 neutralizing antibody (10 µg/mL) inhibited strain-induced [³H]thymidine incorporation by 89% (P<.005 versus compressed) (Fig 5), whereas the mouse nonimmune IgG antibody at that concentration had no effect. In control experiments, the monoclonal anti–FGF-2 antibody also completely blocked the induction of [³H]thymidine incorporation by exogenous FGF-2. Although nearly complete inhibition of compression-induced [³H]thymidine incorporation was achieved in this experiment, inhibition by the monoclonal anti–FGF-2 antibody (10 µg/mL) ranged from 65% to 89% in four independent experiments using a strain of 60% to 65%. Also, compression-conditioned media significantly induced [³H]thymidine incorporation in target gel cultures by 1.7±0.2-fold more than control-conditioned media (P<.001), and this induction was completely blocked by the monoclonal anti–FGF-2 antibody (Fig 6).

View larger version (15K): [in this window] [in a new window]   Figure 4. Bar graphs showing [3H]thymidine incorporation by human vascular smooth muscle cells 12 to 24 hours after a 5-minute 60% compression (Comp) in serum-free conditions. Control cell gels received no stimulation. A, Addition of a neutralizing polyclonal antibody to PDGF-A immediately after compression (Comp + anti-AA) did not inhibit the effect of transient compression (Comp) (P=NS). Addition of PDGF-AA in the absence of compression led to an increase in [3H]thymidine incorporation (PDGF-AA) that was inhibited by the polyclonal antibody (AA + anti-AA). B, Addition of a neutralizing polyclonal antibody to PDGF-B immediately after compression (Comp + anti-BB) did not inhibit the effect of transient compression (Comp) (P=NS). Addition of PDGF-BB in the absence of compression led to an increase in [3H]thymidine incorporation (PDGF-BB) that was inhibited by the polyclonal antibody (BB + anti-BB). Values are based on three or four measurements; error bars denote 1 SD.

View larger version (21K): [in this window] [in a new window]   Figure 5. Bar graph showing [3H]thymidine incorporation by human vascular smooth muscle cells 12 to 24 hours after a 5-minute 65% compression (Comp) in serum-free conditions. Control cell gels received no stimulation. The effect of transient compression was inhibited by a neutralizing monoclonal antibody to FGF-2 (10 µg/mL, P<.005) added immediately after compression (Comp + antiF10) but was unaffected by a mouse nonimmune IgG (10 µg/mL) (Comp + IgG10). Nonsignificant inhibitory effects of lower concentrations of antibody to FGF-2 are shown (Comp + antiF1, 1 µg/mL; Comp + antiF.1, 0.1 µg/mL). Addition of FGF-2 in the absence of compression led to an increase in [3H]thymidine incorporation (FGF-2) that was inhibited by the monoclonal antibody (FGF-2 + anti-F10). Values are based on three or four measurements; error bars denote 1 SD.

View larger version (12K): [in this window] [in a new window]   Figure 6. Bar graph showing ability of conditioned media from transiently compressed vascular smooth muscle cell–gel cultures to increase [3H]thymidine incorporation by unstimulated gel cultures. Media harvested 12 hours after a 5-minute 60% compression (Comp) or no compression (Control) were transferred to unstimulated gel cultures, and [3H]thymidine incorporation was measured from 12 to 36 hours later. The stimulatory effect of the compression-conditioned media was reversed by a neutralizing monoclonal antibody to FGF-2 (Comp + anti-FGF-2) (P<.001 vs Comp). Values are based on four measurements; error bars denote 1 SD.

To assess the release of FGF-2 in response to transient compression, culture media were analyzed by a quantitative immunoassay. Increased levels of FGF-2 were found in serum-free media 12 hours after transient compression (Table). Analysis of media after freeze-thawing of the gel cultures demonstrated that the released FGF-2 represented only a small component (<3%) of total available FGF-2. There was no significant increase in LDH release after the transient compression stimulus. In another experiment, DNA content in compressed gels (3.71±0.08 µg/gel) was no different from that in control gels (3.63±0.07 µg/gel) at 12 hours after mechanical stimulation, suggesting that the FGF-2 release occurs without widespread cell death. In addition, Northern blot analysis demonstrated no differences in FGF-2 mRNA levels at 0, 0.5, 1, 2, 3, and 12 hours after compression, and these levels were low compared with those in cells stimulated with interleukin-1 or serum (data not shown).

View this table: [in this window] [in a new window]   Table 1. FGF-2 and LDH Release

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
A cascade model for restenosis is emerging in which mechanical strain, as well as acute local thrombosis, may trigger the early expression of cytokines and growth factors by smooth muscle cells and macrophages.¹ These mediators stimulate multiple cell types in paracrine and autocrine fashions to regulate their own expression or induce one another. Amplification feedback loops and redundant pathways may be ushered in, circumventing therapeutic modalities intended to block a specific growth factor or cytokine. Thus, an understanding of the initial cellular responses to mechanical strain may be important for identifying rational therapeutic targets early in the cascade.

In this study, we tested the hypothesis that a single transient mechanical stimulus induces DNA synthesis in cultured vascular smooth muscle cells. Transient compression of human vascular smooth muscle cell–collagen gel cultures increased DNA synthesis in a delayed, strain-dependent fashion, without exogenous serum-associated mitogens. Conditioned-media experiments demonstrated that DNA synthetic induction was not solely mediated by an intracellular mechanism. Although antibodies against PDGF-A or PDGF-B chain had no effect on the DNA synthetic response, a monoclonal antibody against FGF-2 inhibited the mechanical induction of DNA synthesis. In addition, increased levels of FGF-2 in the culture media after compression were measured by a quantitative immunoassay. Taken together, these data strongly suggest that transient mechanical strain of in vitro vascular smooth muscle cells stimulates DNA synthesis, in part by autocrine release of FGF-2.

In a series of experimental restenosis studies, Reidy¹⁵ made the important observation that early smooth muscle cell proliferation in response to vascular injury is probably mediated not by PDGF from platelets but instead by FGF-2 released from injured medial smooth muscle cells. However, given the potential dual roles of FGF-2 and PDGF in promoting migration and proliferation of smooth muscle cells^{17 38 39 40 41} and the multiple sources of these factors,^{18 22 23} in vivo experiments have not directly assessed the ability of isolated vascular smooth muscle cells to proliferate in direct response to a transient mechanical strain. The present study offers direct evidence for autocrine FGF-2 in mediating the induction of DNA synthesis by transient mechanical stimulation of these cells.

After transient compression, FGF-2 was released, presumably from preexisting stores without increased FGF-2 gene expression; although FGF-2 mRNA levels in the compressed gel cultures did not change during the 12-hour period after compression, FGF-2 levels in conditioned media increased by severalfold. FGF-2 lacks a leader sequence for the classic secretory pathway and is believed to be stored in the cytoplasm or nucleus and released on disruption of the cell membrane or by a novel uncharacterized secretory mechanism.^{26 42 43} In our experimental model, FGF-2 release was not accompanied by lethal smooth muscle cell injury, as assessed by LDH activity and total gel DNA content; however, these parameters may be insensitive to minor cell injury. Such injury might account for the observed initial period of decreased DNA synthesis after compression. Although the amount of FGF-2 released was less than 3% of the total presynthesized pool, it should be noted that measurements of FGF-2 in the media may underestimate local concentrations since FGF-2 is selectively bound to heparan sulfate proteoglycans and sequestered from the high-affinity cell surface receptors.⁴⁴ Thus, we do not know whether FGF-2 is being released from smooth muscle cells undergoing minor focal injury, from extracellular matrix sites disrupted by the mechanical strain, or from both; further study with this system may elucidate mechanisms of strain-induced FGF-2 release.

Although autocrine FGF-2 may be critical to the mechanical induction of DNA synthesis, additional mechanisms are likely. First, inhibition of strain-induced DNA synthesis by the neutralizing anti–FGF-2 monoclonal antibody was incomplete, suggesting the existence of FGF-2–independent autocrine and/or intracellular mechanisms. Second, although conditioned-media treatment led to increased DNA synthesis that could be blocked by the neutralizing antibody, the concentration of FGF-2 measured in this conditioned media was lower than exogenous FGF-2 concentrations (>1 ng/mL) needed to induce DNA synthesis in these serum-deprived gel cultures. Thus, mechanically induced DNA synthesis may depend on the release of additional factors. Under certain conditions, the mitogenic effects of FGF-2 on vascular smooth muscle cells can be potentiated by other growth factors such as PDGF,⁴⁵ and an autocrine PDGF-dependent growth response to continuous cyclic strain has been described in cultured neonatal rat vascular smooth muscle cells.⁵ However, a similar role for PDGF in our transient strain system was not observed, reflecting possible dependence of cellular responses on the nature of the mechanical strain (transient versus dynamic) or on culture configuration (three-dimensional versus monolayer).

The DNA synthetic response we observed may have implications not only for restenosis but also for development and progression of atherosclerosis in general. Although atherosclerosis is multifactorial,⁴⁶ vascular insult from mechanical and/or biochemical sources is believed to be a major predisposing factor.⁴⁷ These data indicate that vascular smooth muscle cells may be sensitive to brief, sublethal variations in mechanical strain and further emphasize the potential influence of mechanical forces on vascular cell biology. Additional study of the early smooth muscle cell responses to mechanical stimuli may reveal promising therapeutic targets for vascular diseases.

	Selected Abbreviations and Acronyms

 

FCS = fetal calf serum FGF-2 = fibroblast growth factor–2 LDH = lactate dehydrogenase PDGF = platelet-derived growth factor

	Acknowledgments

 
This work was supported in part by a Grant-in-Aid from the American Heart Association, Massachusetts Affiliate; by US Public Health Service NRSA award 2-T32-GM07753-12; by grants from the National Heart, Lung, and Blood Institute (HL-47840, HL-48743); and by a grant from the Milton Fund.

Received March 16, 1995; revision received August 8, 1995; accepted August 16, 1995.

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