Reinjury of Arterial Lesions Induces Intimal Smooth Muscle Cell Replication That Is Not Controlled by Fibroblast Growth Factor 2
In this study we have examined the response of rat carotid arteries with intimal lesions to an angioplasty injury. Rat carotid arteries were subjected to injury with a 2F Fogarty catheter (first injury), and 28 days later the same arteries were subjected to reinjury with a 1.5-mm-diameter coronary dilation catheter (second injury) or a sham operation. After the second injury, the injured arterial surfaces were covered by a platelet monolayer, with occasional small thrombi. The size of the intimal area was significantly increased 28 days after the second injury, although the luminal area was not changed at this time. Intimal and medial cell replication, measured by 5-bromo-2 prime-deoxyuridine labeling, was significantly increased at 2 days after the second injury but was markedly reduced by 7 days. Addition of fibroblast growth factor-2 (FGF2, 60 micro g IV) did not increase smooth muscle cell (SMC) replication in arteries subjected to the second injury, and replication was not inhibited with an antibody against FGF2 (120 mg IV). Both these reagents, however, did significantly affect SMC replication in normal carotid arteries subjected to Fogarty catheter injury. In a similar manner, heparin (888 UPS units/kg body wt IV) did not inhibit cell replication after second injury, although it did suppress SMC replication after a single injury. One conclusion is that rat intimal cells in vivo are different from medial SMCs and that other, as-yet-unknown, factors are important for their proliferation.
Intimal hyperplasia and arterial remodeling have been suggested to be important for restenosis, [1-6] but the cellular processes involved in restenosis are not well understood. One commonly held belief is that animal models of vascular injury, which have been studied in detail, mimic the processes of restenosis in humans. Although it is likely that there are similarities in the formation of these lesions, there are some fundamental differences that might account for the lack of success in transposing the data obtained in experimental animal models to the clinical setting. [7-9]
The most widely used model of intimal lesion development has been that of balloon injury to arteries of small mammals, and this has been mainly focused on how normal arteries respond and develop intimal lesions. [10-14] This approach has been critical in allowing us to define the cellular mechanisms involved in the development of new intimal lesions, but it is clearly different from restenosis after angioplasty in human arteries. One obvious difference between human and small mammal arteries is that the animal arteries do not possess intimal SMCs, and so most experimental studies are focused on the response of medial SMCs to injury. [10,14] Intimal cells have unique properties not found in medial cells, [15-17] and one possibility is that the data derived from studies of injury to a normal artery may not be directly applicable to an understanding of continued lesion growth in an artery with an existing intimal lesion.
The purpose of the present study, therefore, was to determine the response of a rat artery with a preexisting intimal lesion to an angioplasty injury. Our data showed that the SMCs of existing intimal lesions were stimulated to replicate by angioplasty injury and that this response was not controlled by FGF2.
Materials and Methods
The Animal Model
Male Sprague-Dawley rats (B&K Universal), aged 3 to 4 months, were used in all experiments. Rats were anesthetized by intraperitoneal injection of xylazine (Xyla-ject, 4.6 mg/kg body wt, Phoenix Pharmaceutical Inc) and ketamine (Ketaject, 70 mg/kg body wt, Phoenix Pharmaceutical Inc). A 2F Fogarty balloon catheter (Baxter Healthcare Co) was introduced through the right femoral artery and passed into the left common carotid artery. A small midline neck incision was made to confirm position of the balloon catheter. The balloon was inflated at 1.5 atm and passed through the common carotid three times with constant rotation (first injury). After the catheter was removed, the right femoral artery was ligated and wounds were closed. At 28 days after the first injury, a second injury or sham operation was performed. Under the same anesthesia, a midline incision in the neck was made to expose left carotid bifurcation and the external carotid artery. A 1.5-mm-diameter coronary dilation catheter (ACS TEN; balloon length, 20 mm; Advanced Cardiovascular Systems Inc) was introduced into the left common carotid artery through the left external carotid. The proximal edge of the balloon segment was positioned at carotid bifurcation. The injury consisted of two 1-minute inflations of the balloon at 4 atm with a 30-second interval (second injury). During this procedure, rotation of the catheter was not performed, and expansion of the carotid was confirmed visually to ensure effective dilation by the angioplasty catheter. The catheter was then removed, the left external carotid was ligated, and the wound was closed. In a sham operation, the left external carotid artery was exposed and ligated without dilation of the common carotid artery.
Rats were killed at 2, 7, 14, and 28 days after the second injury (Figure 1). One group of rats was killed at 28 days after the first injury (control). Ten minutes before death by overdose injection of sodium pentobarbital (intravenous Nembutal, Abbott Laboratories), rats received an injection of Evans blue (200 mL of a 5% solution) to mark the deendothelialized area. Lactated Ringer's solution (Baxter Healthcare Co) was infused at a pressure of 120 mm Hg in retrograde fashion from the abdominal aorta, and blood was drained from both sides of the jugular vein. After the drained solution began to be clear, 4% phosphate-buffered paraformaldehyde (0.1 mol/L PO sub 4 buffer, pH 7.3) was perfused for 5 minutes under 120 mm Hg through the same route. The whole length of the left carotid arteries was excised and immersed in the same fixative for one additional hour. Connective tissue around the carotid was removed, and two segments of carotid were cut out: between 3 and 7 mm from carotid bifurcation for histological study and between 7 and 12 mm from the bifurcation for scanning electron microscopy.
Scanning Electron Microscopy
In addition to the time course samples described above, rats were killed at 15 minutes (n=3) and 1 hour (n=3) after the second injury to assess platelet adhesion and thrombus formation. After the perfusion/fixation described above, the arteries were fixed overnight in phosphate-buffered 1% glutaraldehyde/2% paraformaldehyde solution. The vessels were cut open longitudinally, pinned flat onto polytetrafluoroethylene (Teflon) sheets, and dehydrated through a series of increasing concentrations of ethanol. Tissues were dried with a critical point dryer, mounted on stubs with colloidal silver paste, and coated with gold/palladium by a sputter coater. The luminal surfaces of specimens were examined by a Jeol 35C microscope at 15 kV.
Transmission Electron Microscopy
Rats were killed at 15 minutes (n=3) and 1 hour (n=3) after the second injury. Lactated Ringer's solution was infused as described above, and phosphate-buffered 2.5% glutaraldehyde/2% paraformaldehyde fixative was perfused at 120 mm Hg for 5 minutes. Specimens were immersed overnight in the same fixative after excision, postfixed in 1% OsO4 for 1 hour, stained en bloc in 2% uranyl acetate for 30 minutes, and embedded in Medcast (Ted Pella Inc). Sections were cut, stained with 6% uranyl acetate and Reynolds lead citrate, and examined by a Jeol JEM 1200EXII microscope at 80 kV.
Histology and Morphometry
Fixed tissues were embedded in paraffin, and three 4-micro m sections (each >or=to100 micro m apart) were cut per animal and stained with hematoxylin. Photomicrographs of each stained section were taken at a magnification of x13.2, scanned with a Polaroid Sprint Scan 35 slide scanner, and analyzed with NIH Image software (version 1.55) run on a Power Macintosh 8100/80 AV Computer. Luminal area and EEL area were measured by tracing around the inside edge and EEL of the vessel and quantifying the area inside the circles. EEL area was used as an index of arterial size. The area encompassed by IEL was measured in the same way, and intimal area was calculated as the value of IEL area minus luminal area. Mean values for each area were determined by averaging values from three cross sections per animal and used for statistical analysis.
To identify the cellular composition of the arterial wall, these paraffin sections were immunostained for alpha-smooth muscle actin and macrophages. The monoclonal antibody against alpha-smooth muscle actin (1A4, DAKO Co) was applied at a 1:500 dilution after blocking by 1% normal horse serum.  Subsequent incubation with biotinylated horse anti-mouse IgG (1.2 micro g/mL, rat-adsorbed, Vector Laboratories) and an ABC Elite kit (Vector Laboratories) was performed. A monoclonal antibody to rat monocyte-macrophage (ED-1, Chemicon) was used, and a protocol was followed as described previously. [19,20] Control slides of both immunostains were stained with matching concentrations of nonimmune mouse IgG instead of these primary antibodies.
At 1, 9, and 17 hours before they were killed, each rat was injected subcutaneously with BrdU (25 mg/kg body wt, Boehringer-Mannheim). Replicating cells were identified by monoclonal antibody against BrdU (Bu 20a, DAKO Co), as previously described,  and counterstained with hematoxylin. The number of labeled nuclei and the total number of nuclei were counted using a square-shaped reticule in a x10 eyepiece with a x40 objective. BrdU labeling index (cell replication index) was calculated as the number of labeled nuclei[divide by]total nuclei x100 on each section. Intimal cell number was determined as the total number of intimal nuclei on each section, and intimal cell density was calculated as the ratio of intimal cell number to intimal area. The average of each value from three sections (each >or=to100 micro m apart) per animal was used for statistical analysis.
Western Blot Analysis
Carotid arteries were briefly flushed with lactated Ringer's solution at physiological pressure to remove blood and excised, and connective tissue around the carotid was stripped. The excised carotid arteries, except normal carotid arteries, were opened longitudinally, and the neointima was stripped at the IEL from media. All specimens were then snap-frozen in liquid nitrogen and stored at -80 degrees C. Frozen tissue was ground to a fine powder under liquid nitrogen and suspended in cell lysis solution (1% SDS, 100 mmol/L NaCl, 62.5 mmol/L Tris [pH 7.6], 1 mmol/L phenylmethylsulfonyl fluoride, and 10 micro g/mL leupeptin), and insoluble matter was removed by centrifugation after 30 minutes of incubation at 4 degrees C. The protein concentration was measured by bicinchonic acid assay (Pierce). Equal amounts (10 micro g of total protein) of each lysate were boiled for 5 minutes with sample buffer (1% SDS, 10% glycerol, 50 mmol/L Tris [pH 6.8], 0.01% bromophenol blue, and 2.5% 2-mercaptoethanol [final concentration]), separated on SDS-PAGE, and transferred to nitrocellulose membrane. The blots were blocked with 5% nonfat milk in Tris-buffered saline (pH 7.6) containing 1% Tween 20 (TBS-T) for 3 hours and incubated with anti-FGF2 monoclonal antibody (1.2 micro g/mL, DE-6, a kind gift from Dr T.M. Reilly, DuPont Merck Co)  or rabbit anti-FGFR1 polyclonal antibody (1:1000, Flg 1A#3, a kind gift from Dr M. Jaye, Rhone-Poulenc Rorer)  diluted in 2.5% nonfat milk for 1 hour at room temperature. The anti-FGFR1 antibody was against type 1 carboxyl terminal. After incubation, the nitrocelluloses were washed three times with TBS-T and incubated for 1 hour with appropriate horseradish peroxidase-linked secondary antibody (Amersham) at room temperature. After they were washed, the blots were processed for enhanced chemiluminescence (Amersham).
Stimulation Experiments With Exogenous FGF2 Injection
Human recombinant FGF2 (60 micro g per rat in 1 mL of Ringer's solution, a gift from Scios Nova) was added immediately after the second injury via the tail vein; vehicle (1 mL of Ringer's solution) was injected into control rats in the same manner. These rats were killed at 2 days after the second injury (Figure 2). FGF2 was also injected into animals whose carotid arteries had been subjected to a conventional balloon injury. Briefly, left carotid artery of each rat was denuded with a 2F Fogarty balloon catheter, the same volume of FGF2 or vehicle was injected, and these rats were killed after 2 days. Before the study, BrdU was injected into all rats according to the protocol described above. Tissue preparation and BrdU index calculation were also the same as above.
Neutralizing Anti-FGF2 Antibody
A goat was immunized intradermally with 100 micro g of human recombinant FGF2 and Freund's adjuvant (Sigma Chemical Co). A booster immunization (100 micro g of FGF2 in Freund's incomplete adjuvant) was given 14 days later. Plasma from the goat was obtained at 28 days after first immunization. The IgG fraction of the immune plasma was prepared by the caprilic acid method. Briefly, caprilic acid (5.5 mL/100 mL plasma, Sigma) was added to plasma, after lowering the pH to 5.4, and centrifuged at 8000 rpm for 30 minutes, and the supernatant was filtered through diatomaceous earth. The solution obtained was then dialyzed at room temperature after raising the pH to 7.0. An IgG fraction of nonimmune goat plasma was also prepared in a similar manner and used as control IgG. The neutralizing ability and specificity of the goat anti-FGF2 antibody were examined on mouse 3T3-D1 cells stimulated with 5 ng of human recombinant FGF2, FGF1 (R&D Systems Inc), PDGF-BB (Zymogenetics), EGF (R&D Systems Inc), and calf serum (10%). 3T3-D1 cells were plated at a density of 4x104 cells per well in a 24-well tray in DMEM supplemented with 10% calf serum. After the cells reached confluence, the cells were made quiescent by incubation in medium containing 1% calf serum for 4 days. The growth factors described above were preincubated (30 minutes at 37 degrees C) with three different volumes (50, 100, and 500 micro g) of either anti-FGF2 antibody or nonimmune IgG and incubated with the prepared 3T3 cells for 20 hours. One microcurie of [sup 3 H]thymidine (6.7 mCi/mmol, DuPont/New England Nuclear) was added to the cells. After 2 hours, incorporation into DNA of the cells was measured by a liquid scintillation counter, as described previously. 
Blocking Experiment With Antibody Against FGF2
To assess the role of FGF2 in cell replication after the second injury, the neutralizing antibody against FGF2 or nonimmune IgG was injected intravenously just before and at 1 day after the second injury (total, 120 mg). The rats were killed at 2 days after the second injury (Figure 2). To validate the effectiveness of this antibody in vivo, the antibody or nonimmune IgG was also added to normal rats whose carotid arteries had been subjected to balloon injury, and rats were killed at 2 days after the injury (control). The BrdU index for all rats was quantified as described. The localization of the antibody in the artery was determined by staining of the tissue with anti-goat IgG antibody. Biotinylated rabbit anti-goat IgG antibody (Vector Laboratories) was applied at a 5 micro g/mL concentration after blocking by 1% normal rabbit serum, and an ABC Elite kit was used. Cross sections from reinjured rats that did not receive the goat IgG were stained in an identical manner as a negative control for nonspecific binding of the rabbit anti-goat IgG.
Inhibition of Replication With Heparin
Heparin (Elkins-Sinn Inc) was administered to rats before the second injury. Rats received either a bolus injection of standard heparin (888 UPS units/kg body wt) or an equivalent volume of saline via the tail vein 10 minutes before the second injury and were killed at 2 days (Figure 2). Heparin or saline was injected into normal rats after conventional balloon injury to evaluate the ability to block cell replication at 2 days after the injury. The BrdU index for all rats was calculated in the manner described above.
To analyze the differences in controls and at each time point after the second injury, ANOVA followed by Fisher's protected least significant difference test was used for studies involving the intimal area, luminal area, EEL area, intimal cell number, and intimal cell density. The Kruskal-Wallis test followed by Scheffe's F test was used for studies involving the intimal and medial BrdU index. To aid especially in the analysis of luminal area, EEL area, and intimal cell density, unpaired Student's t test was performed. Differences between each time point after the second injury and sham operation, as well as comparisons between treatment values and their matched control, were analyzed by unpaired Student's t test. All data were considered significant at P<.05.
Morphology of Carotid Artery
Scanning electron microscopy showed that lumens of arteries, after the second (angioplasty) injury, were covered mainly with platelet monolayer (Figure 3A and Figure 3B). A platelet monolayer was seen 15 minutes, 1 hour, and 2 days after the second injury but had disappeared by 7 days (data not shown). Small thrombi were occasionally observed in these arteries both 15 minutes and 1 hour after the second injury (Figure 3C) but were not present at later times. Transmission electron microscopy confirmed the presence of fibrin in these thrombi 15 minutes after the second injury (Figure 3D), although this finding was rare.
There was no loss of intimal mass after angioplasty (Figure 4B), although occasionally arteries did show widespread loss of intimal cells at day 2 (data not shown).
The size of the intimal area was significantly increased 28 days after angioplasty injury compared with control arteries, arteries at days 2 and 7 after the second injury, and the shamoperated arteries (Figure 4C and Figure 5A). No increase in intimal size, however, was observed at earlier times (Figure 4B and Figure 5A). In general, the luminal area in these arteries did not change significantly, although at day 14 a difference was observed (Figure 5B). The total area of the artery, as measured by the area enclosed by the EEL, was increased after 28 days (Figure 5C).
Immunohistochemistry revealed that the majority of intimal cells were alpha-smooth muscle actin positive and that very few ED-1-positive cells were observed in intima and media at all time points after the second injury (data not shown).
Cell Replication After Second Injury
Intimal cell replication, measured by the BrdU labeling index, increased significantly at 2 days after angioplasty injury, but no increase was observed at other time points (Figure 4 and Figure 6A). A significant increase in intimal cell number was detected at day 7, although no additional increase in intimal cell number was observed at later times (Figure 6B). The BrdU index of medial SMCs was increased significantly 2 days after the second injury, but not at other times (Figure 6C).
Changes in Density of Intimal Cells
Intimal cell density was calculated from intimal area and cell number. Although ANOVA revealed no significant change in the control condition and at all time points after the second injury (P=.051), the intimal cell density at day 28 was significantly smaller than control arteries, arteries at days 7 and 14 after the second injury, and sham-operated arteries by Student's t test (Figure 6D).
Western Blot Analysis of FGF2 and FGFR1
FGF2 could be detected in all arteries, although compared with normal control arteries, less protein was found in arteries both before and after angioplasty (Figure 7A). The dominant species of FGF2 had a molecular mass of 23 kD, which we have observed previously in rat arteries. 
The antibody raised against the type 1 carboxyl terminal of FGFR1 recognized three variants of 123, 135, and 145 kD. In the three variants, the 123-kD form was dominant and was expressed abundantly in normal carotid arteries and intima at 28 days after balloon denudation (Figure 7B).
Effect of Goat Anti-FGF2 Antibody
Fifty micrograms of the IgG fraction from the immune plasma neutralized the mitogenic effect of human recombinant FGF2 on 3T3-D1 cells (Figure 8), and increasing the volume of the antibody (100 micro g of 500 micro g) almost totally blocked the effect of the added FGF2 (data not shown). However, the 3T3-D1 cells responses to FGF1, PDGF-BB, EGF, and calf serum were not reduced with the anti-FGF2 antibody at volumes of 50 micro g (Figure 8), 100 micro g, and 500 micro g (data not shown).
Stimulation With Exogenous FGF2 and Blocking With Antibody Against FGF2 After the Second Injury
FGF2 (60 micro g IV) administered immediately after the second injury did not increase cell replication in intima or media at 2 days after the injury (Figure 9A). To demonstrate the efficacy of the FGF2, a similar experiment was carried out with a normal rat carotid subjected to Fogarty balloon denudation. In this case, FGF2 significantly increased medial BrdU index at 2 days after balloon denudation in a manner that we have previously observed (Figure 9A). 
To determine if the rapid increase in cell replication seen at 2 days after angioplasty was stimulated by FGF2, we attempted to block this replication with an anti-FGF2 antibody. Injection of the antibody (120 mg IV) had no effect on BrdU labeling of either intimal or medial SMCs 2 days after the second injury, although the same antibody significantly blocked medial cell replication of normal carotid arteries injured by balloon catheter (Figure 9B).  The absence of any inhibition after the second injury was surprising, and one possible explanation was that the antibody did not penetrate the thickened intima. To evaluate the penetration of the antibody into the intima, sections of these arteries were stained with an anti-goat IgG antibody. The intima showed both diffuse and punctate staining when the animals had been injected with the goat antibody (Figure 10A). This staining was not present in animals that did not receive the goat antibody (Figure 10B).
Inhibition of SMC Replication With Heparin
Bolus injection of standard heparin (888 UPS units/kg body wt) has been shown to effectively block the replication of SMCs in rat arteries.  Therefore, we treated the rats in this experiment with a similar injection of heparin immediately before angioplasty. This treatment had no effect on the replication of SMCs 2 days after the second injury, although it did significantly inhibit SMC replication in a control rat carotid artery subjected to conventional balloon injury (Figure 9C).
The rationale for the present study was to determine whether the SMCs of an established intimal lesion behave differently from the medial cells in response to balloon catheter injury. There were several reasons for thinking that the response of these cells could be different. One is that, in vitro, rat intimal cells have been shown to have different phenotypes than medial cells, as evidenced by their ability to replicate without the need of exogenous mitogens and to express certain genes. [27-29] Another more compelling reason was our observation that FGF2 was relatively weak as a mitogen for the intimal cells of a preexisting lesion compared with the induced replication of medial cells immediately after balloon injury.  Since, in our experience, FGF2 is the most potent mitogen for rat SMCs in vivo, [25,26] these data might suggest that intimal cells of an established rat arterial lesion are incapable of any sustained replication. That lesion SMCs are not capable of replicating is in part suggested by data from a recent study on human lesions after angioplasty, in which almost no cell replication was observed in these restenosing lesions.  In this study, we wanted to determine if the SMCs of rat intimal lesions could be stimulated by the use of a mechanical injury, which is perhaps the best in vivo procedure to initiate cell replication. The results of this study showed that after an angioplasty injury, intimal cell replication was significantly increased. The duration of this replicative response was similar to that seen in medial cells after a single balloon injury, in that replication was high by 2 days, markedly reduced by 7 days, and equal to background levels by 14 days.  In the present study, the increase in replication was followed by an increase in intimal cell number by day 7, and by day 28 the area of the intima was significantly increased, although there was no appreciable change in the luminal area at this time. Thus, these data clearly demonstrate that rat intimal SMCs are capable of proliferation over a short period of time but that the increase in lesion size is apparently unrelated to this early cell replication.
These data are of interest for several reasons, one being that the time course of replication by these intimal cells is relatively brief compared with the replication of intimal cells in an artery subjected to a single balloon injury.  In this latter situation, the replication of the intimal cells is high, exceeding 70%, and continues to be significantly elevated for several weeks.  We are currently unsure of what is responsible for the shortened period of replication seen in the present study, but this result may offer an explanation for the lack of cell replication seen in human lesions after angioplasty.  If proliferation of the cells in human lesions after angioplasty is as transient as in the rat lesions described here, then the likelihood of detecting cell replication in human samples is remote because of infrequency of obtaining human samples within 4 days after angioplasty. Indeed, O'Brien et al  reported almost no replicating cells in human lesions after angioplasty, but very few of these samples were obtained within the first week. Furthermore, the same author has found that higher rates of cell replication in human tissue can be detected, but only in those specimens obtained within days after angioplasty (E.R. O'Brien, personal communications, 1996). Thus, in light of these data, the apparent absence of replicating cells in human lesions is not surprising, but care must be taken in extrapolating data from experimental animal models to human lesions. It should be noted that cell replication has been reported to occur for longer times in other animal models of reinjury. [31,32] In these studies, however, animals had been fed a cholesterol-rich diet, and so the cellular composition was very different from the rat arteries, which are almost completely composed of SMCs.
The second interesting finding of the present study is that the increase in intimal area was not accompanied by changes in intimal cell number. Intimal cell number increased and reached a maximum at day 7, yet an increase in intimal area was detected only after 28 days. Further, intimal cell density, a ratio of intimal cell number to intimal area, was reduced at 28 days after the second injury compared with control, days 7 and 14 after the second injury, and sham operation. These data would therefore suggest that the change in intimal size might be attributed to an increase in matrix synthesis or in cell hypertrophy. Neither of these parameters was examined in the present study, but Strauss et al  quantified matrix synthesis in a model of reinjury in rabbit arteries and noted an increase in extracellular matrix present in these arteries. In animal models of balloon injury, a decrease in cell density has also been noted at later times in the developed lesions. 
Another interesting aspect of our data is that the increase in cell mass in the intima did not lead to any decrease in luminal area. Normal rat arteries do not have developed intimas, but after balloon catheter injury, the increase in growth of the neointima is often associated with a reduction in lumen size.  In the present study, injury to an established intimal lesion increased the size of the intima, but this was accompanied with a compensatory enlargement of the artery, and no reduction of the lumen size was detected. This ability of arteries to maintain lumen size has been previously documented. Glagov et al  noted that large intimal lesions can develop in humans with no narrowing of the lumen. A similar pattern of remodeling was noted by Jamal et al  in balloon-injured rabbit arteries, where despite a significant increase in intimal thickening, no change in vessel diameter was observed.
The most surprising finding of the present study was that the intimal cell replication induced by angioplasty was not controlled by FGF2. This conclusion was based on the fact that the blocking antibody to FGF2 (120 mg) had no effect on the replication rate of SMCs after the second injury. We have previously proposed that mechanical injury and subsequent cell death permitted FGF2 to be released from injured SMCs and so stimulate cell replication. [25,26] Cell death would appear to be widespread after angioplasty, yet neither anti-FGF2 antibody (120 mg) nor heparin (888 UPS units/kg body wt) inhibited the induced cell replication. These treatments, however, did significantly reduce SMC replication in animals subjected to a single balloon injury. [21,26] These data again highlight differences between SMCs of rat arterial lesions and those of normal healthy arteries, strongly suggesting that a mitogen(s) other than FGF2 is responsible for the replication of these cells. The identity of this growth factor(s) will be the subject of future studies.
Our previous studies have also shown that medial SMCs in balloon-injured rat arteries were very responsive to FGF2, and a single bolus injection (60 micro g) caused a 3- to 30-fold increase in cell replication of injured arteries.  In the present study, however, the addition of FGF2 (60 micro g) had no effect on the replication of the intimal cells. To ensure that this lack of response was not attributed to some unforeseen experimental difficulty, the same studies were repeated using normal rat carotid arteries subjected to acute balloon catheter injury. In this case, the addition of FGF2 (60 micro g) caused a highly significant increase in medial cell replication, and as noted above, the infusion of the blocking antibody significantly reduced the replication of medial cells. This concurs with our previously reported findings. [25,26] We believe that these data clearly show that SMCs of an established rat intimal lesion respond differently to FGF2 than do the medial cells of a previously uninjured artery. The reason for this finding in not clear, although we do have preliminary data showing that spliced or truncated variants of FGF receptor are present in the intimas of injured arteries (data not shown).
In conclusion, the present study showed that injury to a preexisting rat intimal lesion caused a significant increase in SMC replication, but only at 2 days. The replication of these cells was not increased by the addition of FGF2 (60 micro g) or inhibited with a blocking antibody made against this mitogen (120 mg). After 28 days, the lesion was significantly larger than the controls, with an increase in SMC number, although because of an increase in artery size, no decrease in lumen size was detected. These data suggest that rat intimal SMCs in vivo are stimulated by different processes than are medial cells, although at this time the identity of this factor(s) is not known.
This study was supported by National Institutes of Health grants HL-03174 and HL-41103.
Received September 16, 1996; accepted January 9, 1997.
- Selected Abbreviations and Acronyms
- 5-bromo-2 prime-deoxyuridine
- external elastic lamina
- epidermal growth factor
- fibroblast growth factor
- fibroblast growth factor receptor 1
- internal elastic lamina
- platelet-derived growth factor
- smooth muscle cell
- Tris-buffered saline with Tween 20
- © 1997 American Heart Association, Inc.
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