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Circulation. 1995;92:904-911

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(Circulation. 1995;92:904-911.)
© 1995 American Heart Association, Inc.

Articles

Accumulation of Fetal Fibronectin mRNAs After Balloon Denudation of Rabbit Arteries

Christophe Bauters, MD; Francoise Marotte; Martial Hamon, MD; Patricia Oliviéro; Farah Farhadian; Valérie Robert; Jane Lyse Samuel, MD, PhD; Lydie Rappaport, PhD

From the Departments of Cardiology (C.B., M.H.), University of Lille, France, and Inserm U127 (F.M., P.O., F.F., V.R., J.L.S., L.R.), IFR Circulation Lariboisière, Université D. Diderot, Paris, France.

Correspondence to Lydie Rappaport, Inserm U127, 41 Blvd de la Chapelle, 75010 Paris, France.


*    Abstract
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 *Abstract
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 down arrowMethods
down arrowResults
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Background Fibronectin (FN), a component of the extracellular matrix, influences cellular migration and differentiation. It is a prominent component of the extracellular matrix of normal arteries and is thought to play an important role in the pathogenesis of restenosis after angioplasty. FN exists in multiple forms that arise from a single RNA transcript that can be alternatively spliced. EIIIA- and EIIIB-containing FN mRNAs predominate in the embryo, whereas in the adult, most of the normal tissue FN lacks these domains. Since few data were available concerning pattern of expression of the different alternatively spliced forms of FN mRNA in arteries after endoluminal injury, we analyzed the expression of EIIIA and EIIIB FN isoforms at different times after experimental angioplasty.

Methods and Results The spatial and temporal alterations in FN expression were studied in an in vivo model of endothelial denudation in the rabbit aorta and iliac artery by a combination of immunochemistry and in situ hybridization methods. Alternatively spliced forms of FN EIIIA and EIIIB were detected in the media and the adventitia of both types of vessels 24 to 48 hours after injury. Two weeks after injury, EIIIA and EIIIB mRNAs were found to accumulate within the luminal layers of the neointima. The cellular form of FN protein was not found until 2 weeks after the injury and accumulated in the inner part of the neointima.

Conclusions These data demonstrate that FN upregulation is an early and long-lasting process after arterial injury. These results suggest that the induction of the embryonic FN isoforms may be involved in the restenotic process that follows balloon denudation of arteries.


Key Words: restenosis • muscle • smooth • cells • endothelium


*    Introduction
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 up arrowAbstract
 *Introduction
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The response of the arterial wall to experimental endoluminal injury has been studied extensively during the past few years.1 2 3 Blood vessel balloon denudation evokes a series of events, restricted to the injured part of the vessels, that include permeation of platelet factors into the vessel wall, smooth muscle cell proliferation and migration to the intima, and accumulation of a large amount of collagen, elastin, fibronectin (FN), and proteoglycans.4 5 6 Such increased production of extracellular matrix materials in the vessel wall is thought to play an important role in the pathogenesis of restenosis after angioplasty.

FN, an important component of the extracellular matrix, has been shown to influence, through an interaction with specific cellular receptors known as integrins, cellular functions that include motility, differentiation, and many of the cellular responses associated with wound healing.7 It is a prominent component of the extracellular matrix of normal arteries, and its deposition is augmented in hypertension,8 9 atherosclerosis,6 and experimental arterial injury.6 10 FN exists in multiple forms that arise from a single RNA transcript that can be alternatively spliced.11 Plasma FN lacks the EIIIA and EIIIB domains, but the extracellular matrix FNs are a mixture of forms with and without these regions. EIIIA- and EIIIB-containing FN mRNAs predominate in the early embryo, whereas in the adult, most of the normal tissue FN lacks these domains.12 13 The appearance of the specific isoform containing the EIIIA domain has been described both in balloon-injured intima from rat aorta and in atherosclerotic plaques from human arteries,6 suggesting that cellular FN plays a role in the pathogenesis of atherosclerosis and angioplasty-induced restenosis. The presence of FN after arterial injury has been shown mainly by histochemical procedures,6 10 and to date, no data are available concerning the spatial and temporal pattern of expression of the different alternatively spliced forms of FN after experimental angioplasty. Knowledge of regulation of FN synthesis by vascular cells would bring new insights into the pathophysiological mechanisms that underlie restenosis and might also indicate events in this process susceptible to therapeutic intervention.

Therefore, we analyzed the expression of EIIIA and EIIIB FN isoforms 1 day, 2 days, and 15 days after balloon denudation of the rabbit aorta and iliac artery, using a combination of immunochemistry and in situ hybridization methods. Our data demonstrate an increase in EIIIA and EIIIB mRNAs in the media and adventitia of both types of vessels as early as 24 to 48 hours after injury. Two weeks after balloon denudation, when the neointima is formed, EIIIA and EIIIB mRNAs as well as the embryonic form of the protein were found to accumulate dramatically in the luminal layers of the neointima. These data demonstrate that FN upregulation is an early and long-lasting process after arterial injury.


*    Methods
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 *Methods
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Balloon Denudation
Male New Zealand White rabbits (2.5 to 2.7 kg) were anesthetized by injection of ethyl carbamate (1 g · kg-1 IV). After exposure of the right femoral artery, a 3F Fogarty balloon catheter was introduced retrogradely into the artery to a distance of 10 cm and inflated until contact was made with the endothelium. The lower abdominal aorta and the right iliac artery were deendothelialized by gentle advancement and withdrawal of the catheter three times as previously described.14 Animals were killed at various times (1, 2, and 15 days) after balloon denudation (three to five animals per time point).

Preparation of Vascular Segments
Segments 4 mm long of the denuded and nondenuded part of the abdominal aorta and iliac arteries (denuded right iliac artery and control left artery) were collected after 15 minutes of an in situ fixation with 2% paraformaldehyde in PBS (in mmol/L: NaCl 150, KCl 2.5, and phosphate buffer 10, pH 7.2) at a pressure of 110 mm Hg. After further fixation by immersion in 2% paraformaldehyde in PBS for 2 hours at 4°C, the vascular tissues were washed in PBS containing 30% sucrose for 4 hours at 4°C, embedded in OCT (RUA), and frozen in isopentane precooled with liquid nitrogen.

Antibodies
FN was immunolabeled with rabbit polyclonal antibodies directed against epitopes present in both plasmatic and cellular rat FN, referred to as total FN (Euromedex), or monoclonal antibodies directed against the cellular domain of human FN (Sigma Chemical Co). Monoclonal antibodies directed against human smooth muscle actin and von Willebrand factor (Dako) were also used.

Immunolabelings
Consecutive serial iliac and aortic cryosections (5 µm) were labeled by a double immunolabeling technique. All sections were incubated for 30 minutes at room temperature with total FN antibodies (1/200 dilution in PBS). After two washings in PBS, every third section was incubated with one of the three monoclonal antibodies. For cellular FN, sections were incubated overnight at 4°C with cellular FN antibodies diluted 1/200 in PBS containing 2% rat serum. Serial sections were incubated with antibodies directed against {alpha}-smooth muscle actin (1/50) and against von Willebrand factor (1/200), respectively, for 1 hour at 37°C. All sections were rinsed three times at room temperature in PBS and incubated for 30 minutes at room temperature with biotinylated anti-mouse IgGs (1/200 dilution in PBS, Vector Laboratories, Biosys). The biotinylated antibodies were revealed with a streptavidin–Texas red complex (1/50 dilution in PBS, Amersham). After three washing steps, sections were then incubated with a 30-fold dilution of anti-rabbit immunoglobulin antibodies conjugated to FITC fluorochrome (Amersham). Usual controls included sections incubated in the absence of primary antibodies. The adequate dilutions to be used with each specific antibody were determined in preliminary experiments on different rabbit tissues known to express or not to express these proteins.

The sections were mounted in aqueous medium (Fluoprep, BioMérieux). Fluorescence was visualized with a Leitz microscope equipped with epifluorescence optics (Leica).

By using triplicate assays, we verified that the immunolabeling pattern was independent of the distance between sections (10 to 50 µm).

Preparation of Probes for In Situ Hybridization
The vectors used in this study were either pSPT 18 or pSPT 19 from Boehringer-Mannheim. These two vectors differ only by the orientation of the multiple cloning site cassette. Subcloning and in vitro transcriptions have been described elsewhere.15 Briefly, the Sau3-Hpa II cDNA fragment encoding the ED 1 segment of human FN (FN-EIIIA) excised from the pFH111 clone,16 the Hpa II-HindIII cDNA fragment encoding the ED 2 segment of human FN (FN-EIIIB) excised from a cDNA clone kindly provided by Dr L. Zardi, and the SnaB1-Sty I cDNA fragment encoding the signal peptide, the propeptide, and the first type I homology segment of human FN (FN-I) excised from a full-length human FN cDNA expression vector17 were subcloned in either pSPT 19 or pSPT 18. For rat pro-{alpha}1 (III) collagen, the EcoRI-Pst I fragment (800 bp) was excised from a cDNA clone, kindly provided by E. Vuorio, pRGR5 containing a 2223-bp cDNA inserted in the Sma I site of pUC 13 by blunt-end ligation and subcloning in pGEMR 3Z.

FN-I was linearized with BamHI, FN-EIIIA with EcoRI, and FN-EIIIB with HindIII for cRNA synthesis, and FN-I was linearized with EcoRI for control mRNA synthesis. Rat pro-{alpha}1 (III) collagen was linearized with EcoRI for cRNA synthesis. Single-strand cRNA or RNA probes were synthesized in the presence of [35S]UTP (NEN Research Products, Dupont) and either T7 or SP6 RNA polymerases from a commercially available transcription kit (Boehringer Mannheim). The RNA probes were separated from the free ribonucleoside triphosphates by repeated ethanol precipitation and used without prior alkaline treatment. The probes were diluted to a final concentration of approximately 60 000 cpm/mL in 50% formamide, 0.3 mol/L NaCl, 20 mmol/L Tris-HCl (pH 8), 5 mmol/L EDTA, 10 mmol/L sodium phosphate (pH 8), 10% dextran sulfate, 1xDenhardt's solution, 0.5 mg/mL yeast RNA, and 20 mmol/L dithiothreitol (DTT). For some experiments, a digoxigenin-labeled probe was synthesized in the presence of digoxigenin UTP instead of 35S-UTP, under the conditions recommended by the manufacturer (Boehringer).

Prehybridization of Tissue Sections
Segments 4 mm long of parts of the abdominal aorta and of the iliac arteries were prepared as previously described.15 Serial cryosections 5 µm thick were prepared and dried a few minutes at room temperature, fixed in 4% paraformaldehyde for 5 minutes, washed in PBS (2x5 minutes), then dehydrated in ethanol and stored at -70°C with dessicant until used for in situ hybridization. Procedures before hybridization were similar to those described in Reference 15. The cryosections were allowed to thaw at room temperature for 1 hour; they were then rehydrated, fixed with 4% paraformaldehyde in PBS, digested with proteinase K, postfixed, treated with triethanolamine/acetic anhydride, washed, and dehydrated.15

Hybridization and Washing Procedures
Hybridization procedures were those described by Wilkinson et al.18 All probes were of similar specific activities and lengths (237, 213, and 257 nucleotides, respectively) and were used at the same concentrations in the hybridization mixture. Approximately 7 µL of hybridization mixture was applied onto each section, and the slides were incubated at 50°C overnight. After a washing at 50°C in 5xSSC and 10 mmol/L DTT, the sections were subjected to stringent washing at 65°C in 50% formamide, 2xSSC, and 10 mmol/L DTT. They were then washed in Tris-EDTA buffer before treatment with 20 µg/mL RNAse A for 30 minutes at 37°C. After washes in 2xSSC and 0.1xSSC at room temperature for 15 minutes, the sections were dehydrated, dipped in Kodak NTB2 nuclear track emulsion (Eastman Kodak), and exposed for 12 days in light-tight boxes with dessicant at 4°C. Sections were developed in Kodak D19, mounted, and analyzed with both light- and dark-field optics of a Leitz Dialux microscope.

Digoxigenin-labeled cRNA probes were used at a concentration of 100 pmol/mL hybridized at 37°C (conditions described above). The hybridization signals were detected according to the Boehringer procedure using alkaline phosphate–labeled Fab directed against digoxigenin revealed with nitroblue tetrazolium chloride.

The hybridization conditions and the specificity of the RNA probes were tested on adult rabbit tissues. The specificity of each FN probe was tested with sense RNA as negative control probe generating background signal (see Fig 8Down). Each series of hybridization with both sense and antisense probes included blocks at the different time points from control and pathological vessels under investigation.



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  		Figure 8. Distribution of total (A and B) and cellular (C and D) fibronectin (FN) in iliac arteries before (A and C) and 15 days after (B and D) deendothelialization. In normal artery (C), the cellular FN was undetectable throughout the entire wall, whereas it accumulated in the luminal layers of the neointimal thickening 15 days after injury (D). Total FN was present throughout the entire wall in both situations. Note the presence of cellular FN in the vicinity of internal elastic lamina (thin arrows) (x120). Large arrow indicates the lamina interna.

RNA Preparation and Northern Blot Analysis
Aortic media were isolated from rabbits that had been sham-operated or operated on and total RNAs were purified 2 and 15 days after surgery, as described in Reference 14. Denatured total RNA (20 µg) was loaded on agarose gel and transferred onto Hybond N membrane (Amersham).14 The membrane was hybridized overnight with single-strand FN cDNA probes synthesized to a specific activity of 1.5x109 dpm/µg using {alpha}-32P-dCTP and a random prime kit (Amersham) except that the random primers were replaced by a specific primer complementary to T7 (FN-EIIIA and FN-EIIIB probes) or SP6 (FN type I probe) promoter sequence. The hybridized membrane was washed under stringent-condition 2xSSC (0.30 mol/L NaCl/30 mmol/L sodium citrate, pH 7.4)/0.1% SDS for 15 minutes at 40°C, then 0.1xSSC/0.1% SDS for 10 minutes at room temperature and exposed in Phosphor-imager (Fuji). After dehybridization, the membrane was sequentially hybridized with the three FN probes, ie, FN-I, FN-EIIIA, and FN-EIIIB. Finally, the membrane was hybridized with a 24-mer oligonucleotide complementary to part of 18S RNA, 5' end-labeled in the presence of [{gamma}-32P]ATP and T4 polynucleotide kinase. After washing, the membrane was exposed to screen for Phosphor-imager, and the hybridization signal was quantified by use of MAC-BAS software (Fuji).


*    Results
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 up arrowMethods
*Results
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FN Gene Expression in Rabbit Aorta
FN mRNAs present in the vascular tissue were localized by hybridization of the sections with 35S-labeled RNA probes complementary to types I, EIIIA, and EIIIB sequences of FN (Figs 1Down and 2Down). These sequences are highly conserved in mammals15 19 ; therefore, the human probes were used to detect rabbit mRNAs under stringent conditions.



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  		Figure 1. Cellular distribution of fibronectin (FN) mRNA in aorta before and after arterial wall injury. Sections obtained from aorta before (A and D) and 2 (B) and 15 (C) days after balloon denudation were hybridized (A through C) with FN type I digoxigenin-labeled cRNA. Sections (A through D) were incubated with labeled antibodies directed against digoxigenin. In normal aorta, only endothelial cells expressed FN mRNA (A, arrow) whereas after balloon injury, smooth muscle cells, and particularly those located near the internal elastic lamina, were positively stained (B and C). Note that 2 weeks after injury, the neoendothelium expressed FN mRNA. No signal is detected when the labeled probe was omitted (D) (x250).



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  		Figure 2. Expression and distribution of fibronectin (FN)-EIIIA and -EIIIB mRNAs throughout aorta after balloon denudation. Nonconsecutive serial sections obtained from control aorta (A, B, and C) and from aorta 1 (D, E, and F), 2 (G, H, and I), and 15 (J, K, and L) days after balloon denudation were hybridized to FN-EIIIA (B, E, H, and K) or -EIIIB (C, F, I, and L) cRNA. The same sections are shown after phase-contrast (A, D, G, and J) or dark-field photomicrographs. A positive signal for EIIIA probe was observed in the adventitia 1 and 2 days after denudation. Fifteen days later, both FN-EIIIA and -EIIIB mRNAs accumulated in the luminal layers of the neointimal thickening. Small arrows delimit the media. Note that only FN-EIIIA mRNA is abundant within some smooth muscle cells of the media (x65).

To precisely assess the cellular localization of FN mRNA within the aorta, digoxigenin-labeled FN type I probes were used for some in situ hybridization experiments (Fig 1Up). In normal vessels, FN mRNAs were restricted to the endothelial cells. Soon after denudation, FN mRNA was observed in the smooth muscle cells, which were heterogeneously distributed throughout the media but preferentially accumulated along the internal elastic lamina. Two weeks after injury, a strong signal appearing as well-defined dots was still observed along the internal elastic lamina but was also present in the neointima and in the majority of the luminal cells.

Fig 2Up shows the distribution of FN-EIIIA and -EIIIB mRNAs within the aorta. In control vessels, there was no signal with either of these two probes, which hybridize to subspecies of FN mRNA. However, as early as 24 hours after deendothelialization, both EIIIA and EIIIB probes reacted with RNA present in the media and adventitia of the denuded part of the aorta. Two weeks later, FN-EIIIA and FN-EIIIB mRNAs were heterogeneously distributed throughout the aortic wall but did not exhibit a superimposable pattern of expression. Either probe gave an intense signal in the neointima, which was comparatively greater in the inner than in the outer part. The smooth muscle cells of the media, however, preferentially expressed the EIIIA form of FN mRNAs, since some of these cells were poorly labeled after hybridization to the EIIIB probe, whereas they reacted strongly with the EIIIA probe.

The localization of the protein by immunolabeling (Fig 3Down) confirmed and extended the results obtained by in situ hybridization. Total and cellular FNs were present in the adventitia and at the endothelial level in the uninjured part of the aorta but could not be detected in the media strongly reactive with {alpha}–smooth muscle actin. Fifteen days after balloon denudation, total FN was easily detected throughout the whole arterial wall, whereas cellular FN accumulated mostly in the inner part of the thickened intima but was also present in the media (Fig 3HDown). At that time, all cells of the neointima expressed {alpha}–smooth muscle actin (Fig 3GDown), whereas endothelial cells were positively stained with anti–factor VIII antibodies (Fig 3EDown).



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  		Figure 3. Distribution of total fibronectin (FN), cellular FN, {alpha}-smooth muscle actin, and von Willebrand factor in aorta before and 15 days after denudation. Nonconsecutive serial cryosections of normal (A, B, C, and D) and injured (E, F, G, and H) aorta were either double-immunolabeled with total FN (B and F) and {alpha}-smooth muscle actin antibodies (C and G) or immunolabeled with either cellular FN (D and H) or von Willebrand factor (A and E) antibodies. In normal aorta, total and cellular FNs (B and D) were present in both the endothelium and the adventitia, whereas {alpha}-smooth muscle actin was detected in the medial smooth muscle cells only (C). In injured aorta, cellular FN accumulated strongly in the luminal layers of the neointimal thickening (H), whereas total FN was abundant throughout the entire vessel wall (F). The positive staining for von Willebrand factor indicates the presence of endothelial cell layers (A and E) (x120).

To determine whether only FN mRNA or other extracellular matrix gene products were also induced, we analyzed the distribution of pro-{alpha}1 (III) collagen in these vessels (Fig 4Down). In control vessels, collagen mRNA was expressed only in the adventitia, but in denuded aortas, an intense signal that appeared to be heterogeneously distributed was observed throughout the entire wall of the vessels 15 days after balloon denudation.



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  		Figure 4. Collagen mRNA expression in aorta 1 and 15 days after angioplasty. After hybridization to collagen III cRNA, an intense signal was observed in the adventitia and throughout the entire wall of aortas 1 (A) and 15 (B) days after denudation. Open arrows delimit the media (x100).

FN Gene Expression in the Rabbit Iliac Artery
Fig 5Down shows the expression of FN type I mRNA in normal and denuded arteries. Two days after endothelial denudation, a positive signal can be found in the media, and 15 days later, FN mRNAs accumulated in the inner part of the neointima.



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  		Figure 5. Expression of total fibronectin (FN) mRNA in rabbit iliac arteries. Sections obtained from control arteries (A) and arteries 2 (B) and 15 (C) days after angioplasty were hybridized to FN type 1 cRNA. At this low magnification, FN mRNA was detected in some medial areas (B) soon after injury, whereas they accumulated strongly in the neointimal luminal layers only (C) by day 15 (x35).

The distribution of the two isoforms of FN mRNAs, EIIIA and EIIIB, was similar to that of total FN mRNA (Fig 6Down). In the left arteries (control), a positive signal was observed at the level of external elastic lamina. Two days after balloon denudation, both mRNAs accumulated and could be seen in the media and adventitia, whereas 2 weeks after injury, only FN-EIIIB mRNA was present in the luminal layers of the neointimal thickening (Fig 6Down). The specificity of FN probes was verified by use of FN-EIIIA and FN-EIIIB sense probes for iliac artery hybridization (Fig 7Down).



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  		Figure 6. Expression and distribution of fibronectin (FN)-EIIIA and -EIIIB mRNAs in iliac arteries. Nonconsecutive serial sections obtained from control arteries (A, B, and C) and from iliac arteries 2 days (D, E, and F) and 15 days (G, H, and I) after angioplasty were hybridized to FN-EIIIA (B, E, and H) or -EIIIB (C, F, and I) cRNA probes. A, D, and G, and B, E, and H are phase-contrast images and dark-field photomicrographs, respectively, of the same sections. Both the FN-EIIIA and -EIIIB probes reacted with the external elastic lamina of either control or denuded arteries. Both FN-EIIIA and -EIIIB probes gave a positive signal in the media of arteries 2 days after deendothelialization, whereas by day 15, only FN-EIIIB mRNA accumulated dramatically in the luminal layers of the neointimal thickening and along the internal elastic lamina. Note that neither FN-EIIIA nor -EIIIB mRNA was detected in the remaining part of the neointimal thickening and media 15 days after angioplasty. Arrows indicate the external and internal elastic lamina (x100).



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  		Figure 7. Specificity of fibronectin (FN) probes. Iliac arteries 15 days after balloon denudation were hybridized with FN-EIIIA and FN-EIIIB sense probes. Only a background signal is observed (x115).

We also analyzed the distribution of FN proteins after balloon denudation. Cellular FN was undetectable in normal arteries. Two weeks after injury, it accumulated primarily in the inner part of the intimal thickening, whereas total FN was present throughout the entire vessel wall (Fig 8Up).

Northern Blot Analysis of FN isomRNAs in Normal and Injured Aorta
Finally, using Northern blot analysis (Fig 9Down), we verified that FN-EIIIA and -EIIIB mRNAs were specifically upregulated in the aorta after balloon denudation. In control rabbit aorta, FN type I mRNA was expressed in the aortic media, whereas both FN-EIIIA and FN-EIIIB mRNAs were undetectable in the majority of the samples. After balloon denudation, the level of FN type I normalized to 18S rRNA increased by 80% at day 1 and remained above control level (+30%) afterward. Both FN-EIIIA and FN-EIIIB were detected in RNAs purified from injured aorta. The quantification of the hybridization signals indicated that the ratio of either FN-EIIIA/FN type I or FN-EIIIB/FN type I mRNA at day 1 was similar to that of controls but dramatically increased at days 2 and 15 (>100% at each time point).



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  		Figure 9. Northern blot analysis of fibronectin (FN) mRNA expression 1, 2, and 15 days after surgery. Total RNA (20 µg) was loaded on the agarose gel, transferred onto nylon membrane, and sequentially hybridized with FN type I, FN-EIIIA, and FN-EIIIB cDNAs and 18S RNA oligonucleotides. Note that the increases in FN-EIIIA and FN-EIIIB mRNAs occur mainly 2 and 15 days after balloon denudation (BD). C indicates control.


*    Discussion
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*Discussion
down arrowReferences
 
The combination of immunolabeling and in situ hybridization used in the present study provided an analysis of FN mRNA and protein expression within both the rabbit aorta and iliac artery in response to balloon injury. FN mRNA and protein are shown to be present in the endothelial cells of the intact vessels and to accumulate within smooth muscle cells in a specific spatial and temporal manner after balloon denudation.

It is now well established that the response of the vascular wall to balloon injury involves at least two types of cells: endothelial cells and smooth muscle cells. Endothelial cells adjacent to the denuded area migrate to reendothelialize the luminal surface, while smooth muscle cells of the media proliferate and then migrate to the neointima.1 2 20 There is now strong evidence to support a determining role of the extracellular matrix composition and organization in the specific behavior of smooth muscle and endothelial cells both in vivo and in vitro.21

Arterial smooth muscle cells exist in two distinct phenotypes, usually referred to as synthetic and contractile phenotypes.22 The synthetic cells have a fibroblast-like appearance, and their main functions are to proliferate and to produce extracellular matrix components. The contractile cells have a muscle-like appearance and contract in response to mechanical and chemical stimuli. The media of mature arteries consists of smooth muscle cells in the contractile state. However, these contractile cells retain the capability to return to a synthetic phenotype,23 24 which represents a major step in the atherogenic process. Glukhova et al6 demonstrated that in the human and rat contractile smooth muscle cells of the media, the EIIIA exon is spliced out during the FN mRNA maturation, while the same exon is included in the mature FN mRNA produced by synthetic smooth muscle cells. Balloon denudation activates medial smooth muscle cells, which respond by an immediate increase in several gene products, referred to as "primary response." This primary response has been observed in both rat25 and rabbit aorta14 and is followed 1 to 7 days later by the proliferation of a subpopulation of smooth muscle cells that undergo DNA replication.26 27 The present study demonstrates that the FN mRNAs, normally abundant in endothelium, are undetectable in the contractile smooth muscle cells of normal adult rabbit aorta and iliac artery by in situ hybridization. Furthermore, this study indicates that within 24 hours after endothelial injury, total FN mRNAs as well as the EIIIA and EIIIB isoforms become abundant in the media and/or the adventitia. Thus, the accumulation and differential splicing of FN mRNA represent an early response of vascular smooth muscle cells to endothelial injury. Increases in FN mRNAs are concomitant with DNA replication, which precedes FN accumulation in the extracellular matrix. Thus, FN mRNA accumulation probably reflects the early modulation of contractile smooth muscle cell toward a synthetic phenotype characteristic of smooth muscle cell response to endothelial denudation. It is unlikely that synthesis and secretion of FN are necessary for smooth muscle cell migration from the media to the intima,28 at least at the early stage of the response, since FN is deposited in the extracellular matrix much later, unless migration requires much smaller FN amounts than those detectable by immunocytochemical methods. Two weeks after balloon denudation, when the neointima is formed, a hybridization signal for FN probes was heterogeneously distributed in the media and the neointima (Fig 1Up). Interestingly, some cells in the close vicinity of the elastic laminae strongly expressed FN mRNA (Fig 1Up), suggesting the presence of activated smooth muscle cells at this level. At this time, at the level of the neointima, EIIIA and EIIIB mRNAs were found to accumulate dramatically in the luminal layers, and the cellular FN exhibited a distribution similar to that of the mRNAs. The localization of cellular FN in the neointima occurs in all vessels studied (this report and Reference 6). The precise distribution of FN mRNAs compared with that of {alpha}–smooth muscle actin and von Willebrand factor indicates that endothelial and some smooth muscle cells express FN during the healing process (Figs 3Up and 7Up). Two weeks after denudation, FN mRNA is specifically expressed in a subpopulation of smooth muscle cells distributed throughout the arterial wall, whereas procollagen III mRNA appears to be expressed by all smooth muscle cells.

It has been reported29 that smooth muscle cells from either minor intimal thickenings in the human child aorta or the atherosclerotic plaques produce FN-EIIIA rather than FN-EIIIB. Our results clearly demonstrate that in the rabbit aorta, both FN-EIIIA and FN-EIIIB mRNAs are increased in the media 24 to 48 hours after injury and in the luminal layers of neointima 2 weeks later. In contrast, FN-EIIIA transcripts were not detected in either layer of the injured iliac arteries at that time. These results may reflect tissue and/or species differences; they may also be related to differences in the temporal pattern of FN mRNA expression as already described during development.29

The function of each FN isoform in the injured vascular wall is unknown. Both the abundance and the reexpression of FN embryonic forms suggest a biological significance. During embryonic development, the prominent expression of FN-EIIIA and FN-EIIIB is associated with cell migration,12 30 and the reexpression of these FNs could play a similar role during restenosis, which also involves cell migration.3 Another potential function of the newly produced FN is the regulation of smooth muscle cell phenotype (review in Reference 22). Besides, FN is more efficient than laminin in promoting the transition of primary rat aortic smooth muscle cells from a contractile to a synthetic phenotype in a serum-free medium.31 However, with time in culture, the cells cultured on laminin produce more FN than those plated on FN, and then they shift toward a synthetic phenotype.31 According to Hedin et al,31 smooth muscle cells are able to adjust their biosynthetic activities according to the initial extracellular matrix composition. Thus, the de novo synthesis of the FN embryonic variants, perhaps focally initiated by rapidly responding smooth muscle cells, not only may direct a change in phenotype of neighboring cells but also may constitute a feedback mechanism to control these changes.

The factors responsible for the expression of the FN embryonic variants after vascular injury are unknown. Numerous growth32 33 and humoral factors34 have been implicated in the pathogenesis of restenosis, and some might be responsible for the changes in FN expression observed in the present study. Among all the putative candidates, transforming growth factor (TGF)-ß1 deserves special attention. TGF-ß1 is implicated not only in the regulation of smooth muscle cell proliferation35 and migration36 but also in extracellular matrix production.37 It favors the formation of FN-EIIIA by cells in culture38 and stimulates smooth muscle cell migration.36 After balloon injury of the rat carotid artery, the level of TGF-ß1 mRNA increases rapidly and remains elevated for at least 2 weeks.35 Ten weeks later, the intima displays increased staining for TGF-ß1.35 39 Lindner et al,2 using two different techniques of deendothelialization, showed that the intimal and especially the luminal smooth muscle cells are intensely stained with an antibody to TGF-ß1 2 weeks after injury. Therefore, the appearance and distribution of cellular FN mRNAs (this report) seem to parallel those of TGF-ß1.2 35 39 Taken together, these findings make plausible a role for TGF-ß1 in the regulation of FN expression after balloon denudation.

In conclusion, the present study clearly demonstrates that FN-EIIIA and -EIIIB are expressed with a specific spatial and temporal pattern after balloon denudation. The upregulation of FN mRNA that appears as an early and long-lasting response to arterial injury strongly suggests that FN is involved in the restenotic process that often follows arterial balloon dilatation.


*    Acknowledgments
 
This work was supported by INSERM, CNRS, Fondation de France, European Community (BIOMED grant), and the CH&U de Lille, CIVIS project 93-04.

Received December 6, 1994; revision received January 16, 1995; accepted February 6, 1995.


*    References
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