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Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:1958-1967

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:1958-1967.)
© 1995 American Heart Association, Inc.

Articles

Balloon Catheterization Induces Arterial Expression of Embryonic Fibronectins

Daniel Dubin; John H. Peters; Lawrence F. Brown; Barry Logan; K. Craig Kent; Brygida Berse; Sigurd Berven; Bojan Cercek; Behrooz G. Sharifi; Richard E. Pratt; Victor J. Dzau; Livingston Van De Water

From the Departments of Pathology (D.D., L.F.B., B.L., B.B., S.B., L.V.D.W.) and Surgery (K.C.K.), Beth Israel Hospital and Harvard Medical School, Boston, Mass; the Division of Cardiovascular Medicine and Falk Cardiovascular Research Center (R.E.P., V.J.D.), Stanford University School of Medicine, Stanford, Calif; and the Divisions of Pulmonary Medicine (J.H.P.) and Cardiology (B.C., B.G.S.), Department of Medicine, Cedars-Sinai Medical Center, UCLA School of Medicine, Los Angeles, Calif.

Correspondence to Livingston Van De Water, PhD, Department of Pathology, Beth Israel Hospital, 330 Brookline Ave, Boston, MA 02215.


*    Abstract
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Abstract Fibronectins (FNs) comprise a family of adhesive extracellular matrix proteins that arise by alternative splicing in three regions: V (IIICS), EIIIA (ED-A), and EIIIB (ED-B). FNs bearing the EIIIA and EIIIB segments are prevalent during embryogenesis, expressed to lesser degrees in normal adult tissues, and may be locally reexpressed at sites of adult tissue injury. RNase mapping shows that normal rat arteries express low levels of FNs that are predominantly EIIIA- and EIIIB-. Following balloon injury, arterial walls produce increased total levels of FN transcripts that preferentially include both the EIIIA and EIIIB segments. However, despite inducing increased total FN mRNA, balloon injury does not alter the relative composition of V120+, V95+, and V0 spliced forms. In situ hybridization reveals that as early as 4 days after injury medial cells express increased total FN mRNA, and by 7 days substantial neointimal and focal medial synthesis of EIIIA+, EIIIB+, and V120+ FNs occurs; macrophages do not significantly contribute to this observed vascular FN synthesis. Consistent with the mRNA data, immunofluorescence microscopic analysis reveals increased deposition of EIIIB+ and V+ FN protein forms in injured arterial walls, particularly within the neointima. Our results suggest that local synthesis of specific FN isoforms is important to the neointimal formation that ensues after balloon injury.


Key Words: artery • fibronectins • alternative splicing • neointima • macrophages


*    Introduction
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 *Introduction
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ECM is essential to the extensive tissue reorganization necessary for both embryogenesis and adult tissue repair.1 2 FN is one ECM component known to mediate functions such as cell adhesion, migration, matrix assembly, and phagocytosis.3 4 5 The FNs comprise a family of proteins whose diversity arises by alternative splicing of a single gene transcript at three sites termed the EIIIA, EIIIB, and V segments. The EIIIA and EIIIB segments are either entirely included or excluded. However, the V region may be either totally included, partially included, or excluded, thus producing three V region variants in rats: V120+ (including the V25 or CS-1 region), V95+ (excluding V25), and V0 (excluding the entire V region). Therefore, alternative splicing of the EIIIA, EIIIB, and V regions results in 12 distinct FN transcripts in rats; in humans, five V region splice variants allow for 20 distinct transcripts.

Hepatocytes produce the bulk of blood plasma FN that includes the V region but excludes the EIIIA or EIIIB regions. Cultivated cells produce a mixture of forms, termed cellular FN, that variably includes all three alternatively spliced domains.4 6 In tissues, the pattern of FN splicing appears highly regulated, with significant variations noted during embryogenesis, aging, tumorigenesis, wound healing, and fibrosis.7 8 9 10 11 12 13 14 15 For example, the embryonic FN mRNAs rich in the EIIIA and EIIIB segments that are expressed in developing skin (L. Van De Water and J.H. Peters, unpublished data, 1995) are largely absent in adult skin, yet are reexpressed during cutaneous wound healing.9 14 The increased widespread expression of EIIIA+ and EIIIB+ FNs originally observed in developing embryos suggest that these domains may influence cell migration, proliferation, and differentiation.10 16 In vitro data suggest that the EIIIA segment may influence mesenchymal cell adhesion and differentiation.15 17 In both humans and rats the V25 region is highly conserved and harbors a cell adhesion site known to bind melanoma cells, neural crest cells, and lymphocytes.18 19

FN is a prominent component of the ECM of normal adult arteries.20 During aortic development, expressed FN forms include the EIIIA and EIIIB segments (J.H. Peters, R.O. Hynes, unpublished data, 1995). As the aorta matures, EIIIA and EIIIB segment expression diminishes.21 22 However, adult arteries subjected to balloon injury, atherosclerosis, experimental hypertension, and cardiac transplantation manifest increased total expression of FN mRNAs that variably include the EIIIA and EIIIB segments.21 22 23 24 25 26 27 28 29 30 31 Experimental hypertension not only increases the expression of total FN mRNA in the aorta but also selectively induces EIIIA domain inclusion.32 Immunohistochemical studies indicate that the FNs deposited in the neointima of balloon-injured aorta and atherosclerotic plaques include the EIIIA segment; no EIIIB has been detected in human atheromas.21 22 The neointimal expression of the EIIIB 3and V regions after balloon injury has not yet been determined.

In the present study, we reexamined the balloon-injury model to analyze alternative splicing of FN in all three regions: EIIIA, EIIIB, and V. We now demonstrate that, prior to neointimal formation, balloon injury induces increased medial FN mRNA expression. Subsequently, a transition occurs in which the neointima becomes the primary site of enhanced FN mRNA synthesis. In addition to this general increase in arterial wall FN expression, balloon injury also leads to selective inclusion of both the EIIIA and EIIIB alternatively spliced segments within vascular FN transcripts. By contrast, all or part of the V region is present in nearly all arterial FN transcripts in both uninjured and injured vessels. Moreover, the pattern of V region splicing is not altered appreciably by balloon injury. Nevertheless, owing to the general increase in arterial FN mRNA expression, balloon catheterization leads to increased local expression of transcripts coding for the biologically active V25 subsegment. Consistent with this mRNA data, immunohistological analysis confirmed that balloon injury leads to increased deposition of EIIIB+ and V+ (both the V120+ and V95+ spliced forms) protein variants of FN in the neointima and to a lesser extent within the media.


*    Methods
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RNA Probes
Templates for RNase protection assays for the analysis of the rat EIIIA and EIIIB domains of FN mRNA have been described.14 33 The probes, when hybridized to tissue RNA, give rise to protected fragments that are 280 (including EIIIA) or 109 (excluding EIIIA) bases in length. The EIIIB probe yields fragments that are 350 (including EIIIB) or {approx}100 (excluding EIIIB) bases, respectively. For analysis of the V region by RNase protection assay, we prepared a template (Fig 1Down) from a BstEII digest of a plasmid containing 1.1 kb of FN cDNA (clone 74T; gift of R.O. Hynes). Digested DNA was blunted by incubation with the Klenow fragment of DNA polymerase and gel purified, and a 510-bp insert was subcloned into the HincII site of pGEM 3Zf(+) (Promega Corp) by using standard procedures.34 The V region probe yields fragments that are 510 (V120), 435 (V95), or 152 (V0) bases in length, respectively (Fig 1Down).



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  		Figure 1. a, Map showing alternative splicing pattern of V region and adjacent type III repeat (III-15) for orientation within FN; b, RNase protection (RPA) V region probe (arrow) indicating the lengths (bars) of protected fragments generated: 510 (V120), 435 (V95), or 152 (V0) bases; and c, in situ hybridization (ISH) probe employed to detect the V25 region. This probe has the capacity to recognize V120+ but not V95+ FN transcripts. Comparable maps of EIIIA and EIIIB are available.9 33

Probes for in situ hybridization included those reacting with all forms of rat FN (FN-C, 270 bases) or those specific to the EIIIA (FN-A, 213 bases) or EIIIB (FN-B, 209 bases) regions.9 Probes for collagen type I (600 bases) and lysozyme (438 bases) have been described.14 To prepare a probe to the V25 portion of rat FN with recognition for V120+ but not V95+ FN transcripts (75 bases; Fig 1Up), we performed reverse transcription of cutaneous wound RNA followed by amplification with the polymerase chain reaction by using sense (CTCCCCACTGGCA) and anti-sense (CGTGGCAGAAACAGATG) synthetic oligonucleotides. A thermal cycler (Perkin-Elmer/Cetus) was programmed for 30 cycles at 95°C for 1 minute, 55°C for 1 minute, and 72°C for 2 minutes. The resulting product was blunted with the Klenow fragment, purified on agarose gels, and subcloned into the HincII site of pGEM-3 (Promega Corp) by using established procedures.34

Antibodies
Immunopurified rabbit anti-V95 antibodies and rabbit antiserum to rat cellular FN were generous gifts from R.O. Hynes (Center for Cancer Research, MIT, Cambridge, Mass). The anti-V95 antibodies were raised to a ß-galactosidase fusion protein containing the V95 segment of rat FN that is common to both of the two V+ isoforms (V95+ and V120+) that are produced in the rat.35 Anti-V95 antibodies were immunopurified from the anti–fusion protein antiserum by passage over a column of rat plasma FN Sepharose (J.H. Peters, R.O. Hynes, manuscript in preparation, 1995). Rabbit antiserum to rat cellular FN was prepared36 that recognizes all spliced isoforms of FN (total FN). Immunopurified rabbit anti-EIIIB antibodies were raised to a glutathione S–transferase fusion protein containing the rat EIIIB segment and immunopurified on an EIIIB-maltose–binding protein column.37

Balloon Injury
Balloon injury was performed by a modification of published procedures.38 Adult male Sprague-Dawley rats (450 to 500 g) were anesthetized with sodium pentobarbital (30 mg/kg IP). A 2F embolectomy balloon catheter was passed via the femoral artery into either the suprarenal aorta or the iliac arteries, inflated to a pressure of 25 psi, and withdrawn slowly three times. To harvest tissue, rats were anesthetized with pentobarbital and perfused at physiological pressures through the ascending aorta with 200 mL freshly prepared PBS, pH 7.2, containing 4% paraformaldehyde. Animals were killed, and the midportion of aortic lesions was removed, fixed in PBS/4% paraformaldehyde, and embedded in paraffin.

Alternatively, the catheter was introduced via the left carotid artery into a femoral artery and pulled from the femoral artery to the aorta three times. Following intervals of 1, 7, and 14 days, the iliofemoral arteries were perfusion fixed with PBS/2% paraformaldehyde at physiological pressures, harvested, immersion fixed in PBS/4% paraformaldehyde for 3 hours at 4°C, transferred into 15% sucrose in PBS for 1 hour at 4°C, and embedded in OCT medium over liquid nitrogen. Iliofemoral arteries were obtained from uninjured rats for comparison with injured arteries. Seven-micrometer sections were cut and stored at -70°C pending immunostaining.

In control animals both Evans blue staining and routine histopathology were used to validate the efficacy of the methods described above to denude the aortic and iliofemoral endothelia. All procedures using animals were approved by the Institutional Animal Care and Use Committee at Beth Israel Hospital, Stanford University School of Medicine, and Cedars-Sinai Medical Center.

RNase Protection Assays
Total RNA extractions from fresh blood vessels39 and RNase protection assays14 33 were performed. In brief, uniformly labeled RNA probes were prepared by in vitro transcription from linearized templates (EIIIA, EIIIB, or V regions) by using T7 polymerase with [32P]UTP (800 Ci/mmol) included in the reaction mixture. Transcribed probes were purified on denaturing polyacrylamide gels, and a molar excess of probe was added to a solution containing carrier yeast RNA (40 µg) and RNA from aorta or iliac arteries. These mixtures were heated (60°C) to denature both mRNAs and the probe; hybridization was carried out overnight at 37°C. Unhybridized probe was removed by digestion with a mixture of RNases A and T1 for 30 minutes at 37°C. The samples were then phenol/chloroform extracted and analyzed on a denaturing 6% polyacrylamide gel. Autoradiographic images were quantified by using an Avec 2400 scanner and analyzed on a 6100/66 Power Macintosh personal computer by using ADOPE PHOTOSHOP 2.5.1, ARTISCAN 3.26R, and IMAGE 1.5.0 software.

In Situ Hybridization
Single-stranded RNA probes of either the sense or anti-sense orientation were prepared by transcription of the relevant plasmid in the presence of [35S]UTP followed by purification on polyacrylamide gels.9 14 This procedure resulted in probes with specific activities of {approx}108 cpm/µg; these were used without reduction in length. In situ hybridization was performed as described previously.9 14

Immunofluorescence Microscopy
All sections were subjected to three 5-minute washes in PBS to remove embedding medium. Sections to be stained with anti-EIIIB antibodies were then incubated overnight at 37°C with recombinant N-glycanase at 50 000 U/mL in 1x N-glycanase buffer (New England Biolabs) or 1x buffer alone. Consistent with the finding that N-linked carbohydrate interferes with direct antibody recognition of the EIIIB segment,37 enhanced staining was observed for N-glycanase–treated sections compared with closely adjacent sections treated with buffer alone, which showed negligible staining. All sections were then blocked with 2% ovalbumin (Sigma) in PBS for 60 minutes at 37°C, after which primary antibodies diluted in 2% ovalbumin were applied for 120 to 150 minutes at 37°C. Anti–total FN (anti-rat cellular FN) antiserum was diluted 1:900. After three more PBS washes, secondary fluorescein isothiocyanate–conjugated antibodies to rabbit IgG (Cappel) diluted 1:100 in ovalbumin/PBS were applied for 60 to 90 minutes at 37°C. After three more PBS washes, coverslips were mounted on a drop of gelvatol. The slides were examined and photographed with a BH-2 Olympus microscope equipped with epifluorescence optics.


*    Results
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 *Results
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Balloon-Injured Arteries Express Alternatively Spliced FN mRNAs
In agreement with the observations of Clowes et al38 on the kinetics of neointimal proliferation after arterial balloon injury, we observed in the abdominal aorta and iliac arteries neointimal development first by day 7 then more substantially by day 14 (see below). To determine the types of FN expressed after injury, we performed RNase protection assays on total RNA extracts from either uninjured rat abdominal aortas or aortas harvested from animals with extensive (14-day) neointima formation. Balloon injury induced an apparent increase in total FN mRNA (in Fig 2ADown, compare both bands in lane 2 with those in lane 1 and lane 5 with lane 4; in Fig 2BDown, compare lane 3 with 2). Densitometric analysis revealed 3.5-fold greater aortic expression of both EIIIA+ and EIIIB+ FN mRNAs in injured versus uninjured controls (compare upper positive bands [EIIIA or EIIIB inclusion] with lower negative bands [EIIIA or EIIIB exclusion] in Fig 2ADown). Similar results were obtained with injured iliac arteries (not shown). However, nearly equivalent ratios of V120+-to-V95+ FN mRNA variants were detected in control (1.1:1) and injured (1.3:1) aortas (Fig 2BDown). Negligible V0 FN mRNA was detected in either injured or control arteries, thus demonstrating that virtually all arterial FNs contain at least the V95 region. Therefore, in addition to a general increase in FN expression, aortic balloon injury induced a marked inclusion of the EIIIA and EIIIB domains without detectable alteration in the pattern of V region splicing.



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  		Figure 2. Autoradiographs showing expression of alternatively spliced FNs by rat aorta. RNase protection assays were performed on RNA (10 µg/sample) isolated from rat aortas. A, Uninjured (Uninj) aorta RNA both includes (+) and excludes (-) the EIIIA (lane 2) and EIIIB (lane 5) domains. RNA from aortas 14 days after aortic balloon injury (Inj) shows selective inclusion of both EIIIA (lane 1) and EIIIB (lane 4) containing FN forms. Yeast RNA contains no FN mRNA and is included as a negative control (lanes 3 and 6). B, Uninjured (lane 3) and injured (lane 2) aortas express both V120+ and V95+ FN mRNAs yet minimal V0 forms. Balloon injury enhanced overall V region expression, but a specific effect on V25 inclusion into V120+ forms was not noted. Yeast RNA serves as a negative control (lane 1). Note that the uppermost bands present in lanes 1 through 3 represent small amounts of residual 32P-labeled probe.

In Situ Hybridization Reveals Low-Level FN Expression by Normal Arteries
To determine the source of FN mRNAs in aortas prior to injury, in situ hybridization was performed with probes for total (FN-C labels all variants of FN) and alternatively spliced FN mRNAs in conjunction with probes for type I collagen and lysozyme. In uninjured arteries we observed a level of FN-C hybridization in the medial layer that was near background levels (Fig 3ADown and 3BDown). No detectable labeling with probes for the EIIIA, EIIIB, and V25 segments was noted within the media, intima, or adventitia of control vessels (not shown). In situ hybridization with a probe for type I collagen demonstrated clear labeling of scattered cells within the adventitia of uninjured vessels but minimal labeling of the medial layer (Fig 3CDown and 3DDown). In situ hybridization with a lysozyme probe (used as a marker for macrophages) failed to reveal these phagocytic cells within uninjured vascular walls (not shown). The results shown in Figs 2Up and 3Down demonstrate that FN mRNA is expressed at only low levels by medial cells in normal aorta.



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  		Figure 3. Photomicrographs showing distribution of FN and collagen type I mRNA by normal rat aorta. Paraffin sections of uninjured abdominal aorta were hybridized with probes for FN-C (A and B) or type I collagen (C and D) and visualized by bright-field (A and C) or dark-field (B and D) microscopy. Vessel lumen, intima (small arrows), media, and adventitia are displayed in a top-to-bottom orientation. Birefringent elastic lamina are visible by dark-field illumination (B and D). Sparse labeling with silver grains, localized primarily to the medial layer of uninjured aorta, was achieved with the FN-C probe (A and B). Focal labeling of adventitial cells was observed with the type I collagen probe (arrowheads in C) (magnification x350).

In Situ Hybridization Localizes Vascular FN mRNA Variant Synthesis Induced by Balloon Injury
To assess the changes in FN expression occurring as a consequence of balloon injury, we performed in situ hybridization on abdominal aortas 2 weeks after injury, a time at which neointimal development was substantial. Fourteen days after injury the aortic neointima labeled strongly with the FN-C probe (Fig 4ADown and 4BDown). Significant neointimal labeling was also observed with probes specific for the EIIIA (Fig 4CDown and 4DDown), EIIIB (Fig 4EDown and 4FDown), and V25 (Fig 4GDown and 4HDown) segments of FN. This latter probe specifically recognizes V120+ but not V95+ FN transcripts. Focal, but weak, labeling of medial cells with the FN-C probe and, to a lesser extent with the FN-A, FN-B, and FN-V25 probes, was also observed. Injured vessels demonstrated scattered, weak FN-C labeling within the adventitia. Hence, at day 14 after balloon injury, neointimal cells were the source of most of the FN mRNA variant transcripts expressed.



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  		Figure 4. Photomicrographs showing expression of alternatively spliced FN mRNAs by balloon-injured rat aortas. Closely adjacent paraffin sections of aortas were hybridized 14 days after injury with probes for FN-C, EIIIA, EIIIB, V25, type I collagen, and lysozyme and viewed by both bright- (A, C, E, G, I, and K) and dark- (B, D, F, H, J, and L) field microscopy. Vessel lumens, intima, media, and adventitia are displayed in a top-to-bottom orientation. Internal elastic lamina is denoted by black arrow (A, C, E, G, I, and K). Marked neointimal FN-C (A and B), EIIIA (C and D), EIIIB (E and F), and V25 (G and H) labeling was observed (white arrows). Sparse FN-C (A and B) but no collagen (I and J) label was present within the medial layer. In contrast, marked collagen (white arrowhead in I) yet minimal FN (black arrowheads in A, C, and G) labeling was observed in the adventitia. Rare neointimal cells were labeled with lysozyme probe (white arrow in K and L) (magnification x350).

The type I collagen probe strongly labeled nearly all neointimal cells 14 days after injury; focal, dense adventitial collagen mRNA labeling was also noted (Fig 4IUp and 4JUp). Compared with neointimal and adventitial labeling, far lower levels of type I collagen labeling were observed in injured aortic media. At 14 days after injury a lysozyme RNA probe, employed as a marker for identifying macrophages,14 labeled only scattered, rare neointimal cells (Fig 4KUp and 4LUp). A comparison of the patterns of expression of collagen, lysozyme, and FN mRNA indicated that macrophages did not contribute significantly to balloon injury–induced FN mRNA synthesis.

While medial cells of normal, uninjured aortas expressed low levels of FN mRNAs that appeared to predominantly lack the EIIIA and EIIIB segments, neointimal cells 14 days after injury expressed FN mRNAs that largely included these segments. Thus, alterations in the amounts, types, and localization of FN mRNAs occurred in response to arterial balloon injury. To gain further information about this transition, we studied the temporal and spatial sequence of FN mRNA expression during neointimal development.

In situ hybridization with an FN-C probe was performed on iliac arteries either prior to day 0, or 1, 4, or 7 days after balloon injury (Fig 5Down). Although there was little if any increase in medial cell labeling by 1 day (not shown) after injury, there was a marked increase in labeling of medial cells by 4 days (Fig 5BDown); neointima was not evident by day 4. By 7 days after injury, some regions of the iliac arterial lumen were lined by neointimal formations that exhibited marked FN-C expression (Fig 5CDown). Note that at this time neointimal labeling was increased over medial labeling. To determine the earliest interval at which "embryonic" FNs were expressed, we probed sections for EIIIA and EIIIB. In the case of EIIIA, we found labeling of scattered medial cells as early as 4 days after injury, a time before which neointima was evident (Fig 5DDown). By contrast, no specific label was noted in the adventitial layer. EIIIB was not detectable in the medial layer by day 4 but was evident in the neointima at day 7 (Fig 5EDown). Little specific labeling for EIIIB was observed in the medial layer at day 7. To assess the level of nonspecific hybridization, we hybridized a section from a 7-day post-injury biopsy with a sense-oriented FN-C probe and observed low-level, nonspecific labeling (compare Fig 5FDown with 5C).




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  		Figure 5. Photomicrographs showing temporal expression of FN after injury. Sections were taken from samples of rat iliac arteries either before (A) or 4 (B and D) or 7 (C, E, and F) days after balloon catheterization. Sections were hybridized with an anti-sense probe for FN (FN-C; A through C), a control sense FN-C probe (F), or anti-sense probes for either the EIIIA (D) or EIIIB (E) regions. Vessel lumen, intima, media, and adventitia are displayed in a top-to-bottom orientation. Black arrows indicate internal elastic lamina; white arrows, external elastic lamina (magnification x320).

Macrophages were not detected by lysozyme probing at either 4 or 7 days after balloon injury (data not shown). At 1 to 4 days after injury, increased medial labeling with a probe for type I collagen was evident. By 7 days after balloon injury, marked labeling with type I collagen was present in the forming neointima and adventitia; significantly less type I collagen labeling was noted within the medial layer (data not shown).

Arterial Distribution of FN Protein Variants After Balloon Injury
Although deposition of FNs that include the EIIIA segment have been observed within the aortic neointima after balloon injury in the rat,22 examination of the neointimal distribution of the V+ and EIIIB+ isoforms has not been performed. We therefore wished to evaluate the protein expression of these latter two segments in iliofemoral arteries under baseline conditions and after balloon injury.

Consistent with the in situ hybridization data, anti–total FN antibodies produced low levels of fibrillar staining of the media of control iliofemoral arteries (Fig 6ADown). These antibodies also produced sparse staining of advential tissue. In comparison to anti–total FN staining, relatively less medial and advential staining of uninjured arteries was obtained with anti-V95 antibodies (Fig 6DDown). Although overall staining of control arteries by anti-EIIIB antibodies was negligible (Fig 6GDown), an occasional patch of N-glycanase–enhanced fluorescence could be detected in the media of uninjured arteries. In addition, control iliofemoral arteries typically showed a bright "scalloped" intimal staining pattern with both the anti-total and anti-V95 antibodies (Fig 6ADown and 6DDown). This staining pattern was not a result of autofluorescence of the internal elastic lamina, since it was absent from closely adjacent sections that were subjected to the same staining procedure in the absence of primary antibodies (not shown). Also, examination under higher magnification showed that this material was located primarily on the lumenal side of the internal elastic lamina, not within it (not shown). No such intimal staining was evident with anti-EIIIB antibodies (Fig 6GDown). By day 1 after injury, there was little change in the iliofemoral arterial staining patterns produced by the three antibodies (Fig 6BDown, 6EDown, and 6HDown), except that the intimal staining produced with the anti–total FN and anti-V95 antibodies appeared to be disrupted in some areas (Fig 6BDown and 6EDown). By contrast, at 14 days after injury, large neointimal formations were microscopically evident, and each of the three antibodies stained these formations brightly in a pattern consistent with pericellular matrix (Fig 6CDown, 6FDown, and 6IDown). At an intermediate time point (7 days), the newly formed neointima stained brightly with anti-V95, anti-EIIIB, and anti–total FN antibodies (Fig 7Down). Medial staining with each of the three antibodies was increased above that of uninjured artery at this time (compare Fig 6ADown, 6DDown, and 6GDown with Fig 7ADown through 7C) but was generally less intense than the neointimal staining. The specificity of the anti-EIIIB staining was established by comparison of sections either deglycosylated (Fig 7CDown) or not deglycosylated (Fig 7DDown) before immunostaining.



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  		Figure 6. Photomicrographs showing deposition of FN protein isoforms in iliofemoral arterial walls before and after balloon injury. Sections from uninjured (A, D, and G) and balloon-injured iliofemoral arteries at 1 day (B, E, and H) and 14 days (C, F, and I) were immunostained for total FN (A through C), V95+ FN (D through F), or EIIIB+ FN (G through I). EIIIB staining was performed after pretreatment of sections with N-glycanase; untreated sections gave no staining (not shown). Note that under baseline conditions both anti–total FN and anti-V95 antibodies brightly stain the intimal region near the internal elastic lamina (arrows in A, D, and G), whereas medial staining by the anti-V95 antibodies is somewhat less intense than for anti–total FN antibodies. In contrast, staining by anti-EIIIB antibodies is absent in the intimal region, and only very faintly evident in patches in the uninjured media. Little change in staining with any of the three antibodies is apparent at 1 day after injury (B, E, and H). However, by 14 days, a prominent neointima is present (small arrows in C, F, and I), much of which has become separated from the media during fixation. Neointimal cells are embedded in ECM rich in V95+ (the subsequence common to the V120+ and V95+ forms) and EIIIB+ FNs. Note that each of the three anti-FN antibodies gave increased staining of the media at this time (large arrows in C, F, and I) compared with control arteries and arteries 1 day after injury (magnification x125).



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  		Figure 7. Photomicrographs showing deposition of FN protein isoforms in arterial walls 7 days after balloon injury. Sections from an iliofemoral artery were stained with anti–total FN antibodies (A), anti-V95 antibodies (B), or anti-EIIIB antibodies with (C) and without (D) pretreatment of the section with N-glycanase to remove interfering linked carbohydrate. Small arrows in A through C indicate neointima, whereas the point of the single large arrow lies within the media (magnification x125).


*    Discussion
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 up arrowMethods
 up arrowResults
 *Discussion
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We report here that balloon injury triggers increases in the levels of arterial wall FN mRNA and protein content. Increased medial cell FN mRNA synthesis occurs as early as 4 days after injury, a time at which neointimal formation is not yet microscopically evident. The spatial pattern of FN mRNA expression changes from medial synthesis on day 4 to predominantly neointimal production by 2 weeks after injury. Balloon injury induces a 3.5-fold increase in the inclusion of the EIIIA and EIIIB segments within FN transcripts. Even under baseline conditions, all or part of the V region is included in virtually all arterial FN mRNAs as a mixture of V120+ and V95+ spliced forms; V region splicing does not change significantly in response to injury. Although injury does not selectively induce inclusion of the integrin-binding V25 subsegment, the local arterial expression of V25+ mRNA variants is enhanced as a consequence of increased total FN mRNA synthesis. Consistent with the mRNA data, immunohistological analysis reveals increased protein deposition of total FNs, the EIIIB segment, and the V95 segment (common to V120+ and V95+ FNs) in the neointima and, to a lesser extent, in the media at 7 and 14 days after balloon injury.

Balloon injury initiates a cascade of events that could alter vascular FN gene expression.40 41 42 Deendothelialization leads to exposure of the underlying media to platelet-derived, locally expressed, and humoral SMC growth factors (eg, transforming growth factor–ß, platelet-derived growth factor, angiotensin II, and endothelin).40 43 44 45 Furthermore, cellular deformation alone can induce significant changes in gene expression.46 47 In concert, these signals may promote the transition of quiescent medial SMCs from a "contractile" to a "synthetic" phenotype that manifests increased expression of structural proteins as well as an enhanced capacity to migrate into the intimal compartment.41 48 49 50 Once the neointima is established, paracrine/autocrine stimulation by transforming growth factor–ß,51 platelet-derived growth factor,52 53 or angiotensin II43 54 may provide further regulation of SMC FN expression.

Using differential cloning methodology, Giachelli et al55 56 and Majesky et al57 have recently demonstrated that in comparison with normal adult medial SMCs, rat pup medial and adult neointimal SMCs selectively express specific ECM genes including elastin, type I collagen, and osteopontin. This similarity in ECM gene expression between neointimal and pup SMCs suggests that gene expression during adult vascular remodeling at least partially recapitulates an embryonic pattern. Our in situ hybridization data show that as early as 4 days after balloon injury, medial SMCs begin to increase FN synthesis. Abundant neointimal FN synthesis is detectable in injured 7-day iliac arteries and 14-day aortas. By day 4, medial SMCs express EIIIA+ FNs, and by day 7, EIIIB expression becomes apparent within the neointima. This gradual and eventually marked expression of "embryonic" FNs, first by medial and then by neointimal cells, further supports the hypothesis that adult vascular remodeling involves the recapitulation of embryonic gene expression patterns.

Our results indicate that synthetic SMCs are the likely source of FN in the developing neointima. Type I collagen expression is enhanced in synthetic SMCs, and we observed a close association between neointimal embryonic FN expression and collagen type I synthesis. In contrast, very few macrophages populated the neointima of balloon-injured rat aorta or iliac artery. The paucity of macrophages within these early neointimal proliferations is consistent with studies of balloon injury in other normolipemic animals.26 58 While macrophages are the important local producers of FNs in inflammatory lung disease,59 experimental glomerular nephritis,60 and early cutaneous wound healing,14 macrophages appear to have little role in directing vascular FN expression within 2 weeks of balloon injury. In human atheromas macrophages are abundant and may promote, but are not directly responsible for, FN mRNA expression.26 44 58

We observed that balloon injury induces local vascular EIIIA+ FN mRNA expression. This is in concordance with a prior report demonstrating increased neointimal EIIIA immunostaining after balloon injury.22 We now report that balloon-injured arteries show increased expression of EIIIB+ and V+ FN mRNAs. The latter transcripts include both V120+ (as shown by RNase protection and in situ hybridization using a V25 probe) and V95+ (as shown by RNase protection) transcripts. Our findings add to the accumulating evidence that increases in local expression of FN mRNAs accompany adult tissue remodeling processes including growing tumors,7 11 cutaneous wounds,9 14 liver fibrosis,15 glomerular nephritis,60 renal allografts,61 inflammatory lung disease59 atherosclerotic plaques,21 22 26 systemic hypertension,24 27 28 29 experimental coronary artery hypertension,25 and coronary allografts.30 31 The intracellular mechanism governing alternative splicing of FN involves both cis sequences and trans-acting factors.62 63 64 Extracellular signals that may alter FN mRNA splicing during embryogenesis or tissue repair include growth factors such as transforming growth factor–ß.65 Tight regulation of alternative splicing likely accounts for the regional and cellular heterogeneity of FN variant expression during both embryonic and adult tissue remodeling processes.9 14 15 32 66

Our findings also contrast with the pattern of splicing noted in hypertensive rat aortas in which a selective increase in EIIIA+ FN but not EIIIB+ has been observed.32 However, our findings that injured arteries temporally express EIIIA+ before EIIIB+ FNs parallels the sequence noted in adult tissues undergoing fibrosis.15 The specific signals that account for these differences in the pattern of FN splicing remain to be elucidated.

Generally, our data concerning the spatial and temporal distribution of FN mRNAs and protein variants confirm that the EIIIB and V region splice variants detected by in situ hybridization are translated and incorporated in the vascular ECM. One discrepancy involved the uninjured arterial intima, which was brightly stained by anti-V95 and anti–total FN antibodies but not by anti-EIIIB antibodies. This staining was localized primarily to the lumenal side of the internal elastic lamina. In contrast, our in situ hybridization studies failed to show any such intimal localization of V+ or total FN mRNAs. A possible clue to this discrepancy is found in the V+B- splicing composition of the intimal FN protein forms. The plasma pool of FNs contains approximately 50% V+ isoforms6 but less than 1% EIIIB+ isoforms.37 Therefore, the intimal localization of V+B- FNs likely represents the deposition of liver-derived plasma FNs. Such deposition has been observed by Oh et al,67 who have shown that soluble FNs in the blood are in equilibrium with insoluble FNs in the walls of blood vessels.

Interestingly, while we found that EIIIB expression occurred in balloon-injured neointima, earlier immunohistological studies have not observed EIIIB deposition in human atheromas.21 This may reflect significant mechanistic differences in the evolution of atheromatous versus balloon injury–induced neointimal proliferations. Alternatively, EIIIB segment expression may decrease as neointimal proliferations develop into atheromas; indeed, a temporal loss of EIIIB expression occurs during normal ontogeny.10 21 Finally, our anti-EIIIB antibodies recognized the EIIIB segment directly37 and consequently might be expected to show more sensitive and specific recognition of B+ FN isoforms than the previously used antibody (BC-1), which recognizes a conformation-dependent epitope in the constantly expressed seventh type III repeat of FN in an EIIIB-dependent manner.68 Such an epitope might be responsive to additional factors, such as the inclusion/exclusion status of the other two spliced regions (EIIIA and V) of FN.

Balloon injury induces local expression of alternatively spliced FNs that supplement the ample plasma FN deposited shortly after endothelial denudation.69 70 Plasma FN can modulate the phenotype of SMCs from contractile to synthetic,71 and SMCs express FN receptors that mediate adherence to FN-rich matrices.72 73 Recent data also provide clues about the function of EIIIA. EIIIA+ FNs can promote the transition of rat liver lipocytes into myofibroblasts in vitro.15 The marker used in these studies was SMC-{alpha} actin. Thus, it is intriguing to speculate that neointimal and medial deposition of embryonic EIIIA+ FNs influences vascular SMC differentiation in injured arteries. Furthermore, endothelial cells express FN receptors,74 75 76 77 and in vitro data suggest that V25+ FNs (V120+) can influence endothelial cell migration.78 Thus, the mixture of plasma FN and embryonic FNs present within the neointima could regulate SMC migration and differentiation and affect the extent of reendothelialization in balloon-injured arteries.69 70

In summary, we have established that the FNs expressed in response to balloon injury include not only the EIIIA segment but also the EIIIB and V (including V25) regions. The early expression of FN mRNA shortly after balloon injury but before neointimal migration suggests that these FNs may promote the differentiation of vascular SMCs from a contractile to a synthetic, migratory phenotype. The expression of "embryonic" FNs in the vascular response to balloon injury further advances the hypothesis that adult tissue remodeling recapitulates cellular behavior during development. Establishment of the precise pattern of FN splicing after balloon injury provides a framework in which targeted intervention can be systematically designed to interfere with neointimal hyperplasia.


*    Selected Abbreviations and Acronyms
 
ECM = extracellular matrix
FN = fibronectin
PBS = phosphate-buffered saline
SMC = smooth muscle cell


*    Acknowledgments
 
This work was supported by National Institutes of Health grants HL 35252 (Dr Dzau), HL 42663 (Dr Pratt), and GM 36812 (Dr Van De Water), by American Heart Association grant GIA 92-758 and a Whitaker Health Sciences Fund grant (both to Dr Van De Water), and in part by the BI Surgical Trust, the BI Pathology Foundation, Inc, and the Charles B See Foundation (Dr Peters).

Received December 29, 1994; accepted August 29, 1995.


*    References
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  1. Clark RAF, Henson PM, eds. The Molecular and Cellular Biology of Wound Repair. New York, NY: Plenum Press; 1988:597.
  2. Hay ED, ed. Cell Biology of the Extracellular Matrix. 2nd ed. New York, NY: Plenum Press; 1991:468.
  3. Mosher DF, ed. Fibronectin. New York, NY: Academic Press; 1989:474.
  4. Hynes RO. Fibronectins. New York, NY: Springer-Verlag; 1990:546.
  5. Yamada KM. Fibronectin and other cell interactive glycoproteins. In: Hay ED, ed. Cell Biology of Extracellular Matrix. New York, NY: Plenum Press; 1991:111-148.
  6. Schwarzbauer JE. Alternative splicing of fibronectin: three variants, three functions. Bioessays.. 1991;13:527-533. [Medline] [Order article via Infotrieve]
  7. Borsi L, Carnemolla B, Castellani P, Rosellini C, Vecchio D, Allemanni G, Chang SE, Taylor-Papadimitriou J, Pande H, Zardi L. Monoclonal antibodies in the analysis of fibronectin isoforms generated by alternative splicing of mRNA precursors in normal and transformed human cells. J Cell Biol.. 1987;104:595-600. [Abstract/Free Full Text]
  8. Norton PA, Hynes RO. Alternative splicing of chicken fibronectin in embryos and in normal and transformed cells. Mol Cell Biol.. 1987;7:4297-4307. [Abstract/Free Full Text]
  9. ffrench-Constant C, Van De Water L, Dvorak HF, Hynes RO. Reappearance of an embryonic pattern of fibronectin splicing during wound healing in the adult rat. J Cell Biol.. 1989;109:903-914. [Abstract/Free Full Text]
  10. ffrench-Constant C, Hynes RO. Alternative splicing of fibronectin is temporally and spatially regulated in the chicken embryo. Development.. 1989;106:375-388. [Abstract]
  11. Oyama F, Hirohashi S, Shimosato Y, Titani K, Sekiguchi K. Oncodevelopmental regulation of the alternative splicing of fibronectin pre-messenger RNA in human lung tissues. Cancer Res.. 1990;50:1075-1078. [Abstract/Free Full Text]
  12. Magnuson VL, Young M, Schattenberg DG, Mancini MA, Chen D, Steffensen B, Klebe RJ. The alternative splicing of fibronectin pre-mRNA is altered during aging and in response to growth factors. J Biol Chem.. 1991;266:14654-14662. [Abstract/Free Full Text]
  13. Pagani F, Zagato L, Vergani C, Casari G, Sidoli A, Baralle FE. Tissue-specific splicing pattern of fibronectin messenger RNA precursor during development and aging in rat. J Cell Biol.. 1991;113:1223-1229. [Abstract/Free Full Text]
  14. Brown LF, Dubin D, Lavigne L, Logan B, Dvorak HF, Van De Water L. Macrophages and fibroblasts express `embryonic' fibronectins during cutaneous wound healing. Am J Pathol.. 1993;142:793-801. [Abstract]
  15. Jarnagin WR, Rockey DC, Koteliansky VE, Wang S-S, Bissell DM. Expression of variant fibronectins in wound-healing: cellular source and biological activity of the EIIIA segment in rat hepatic fibrogenesis. J Cell Biol.. 1994;127:2037-2048. [Abstract/Free Full Text]
  16. George EL, Georges-Labouesse EN, Patel-King RS, Rayburn H, Hynes RO. Defects in mesoderm, neural tube and vascular developments in mouse embryos lacking fibronectin. Development.. 1993;119:1079-1091. [Abstract]
  17. Xia P, Culp LA. Adhesion activity in fibronectins alternatively spliced domain EDa (EIIIA) and its neighboring type III repeats: oncogene-dependent regulation. Exp Cell Res.. 1994;213:253-265. [Medline] [Order article via Infotrieve]
  18. Humphries MJ, Akiyama SK, Komoriya A, Olden K, Yamada KM. Identification of an alternatively spliced site in human plasma fibronectin that mediates cell type-specific adhesion. J Cell Biol.. 1986;103:2637-2647. [Abstract/Free Full Text]
  19. Humphries MJ. Peptide recognition motifs involved in the binding of integrins to their ligands. Kidney Int.. 1992;41:645-649. [Medline] [Order article via Infotrieve]
  20. Stenman S, Vaheri A. Distribution of a major connective tissue protein, fibronectin, in normal human tissue. J Exp Med. 1978;147:1054-1064. [Abstract/Free Full Text]
  21. Glukhova M, Frid M, Shekhonin B, Balabanov Y, Koteliansky V. Expression of fibronectin variants in vascular and visceral smooth muscle cells in development. Dev Biol.. 1990;141:193-202. [Medline] [Order article via Infotrieve]
  22. Glukhova M, Frid M, Shekhonin B, Vasilevskaya T, Grunwald J. Expression of extra domain A fibronectin sequence in vascular smooth muscle cells is phenotype dependent. J Cell Biol.. 1989;109:357-366. [Abstract/Free Full Text]
  23. Jensen BA, Holund B, Clemmensen I. Demonstration of fibronectin in normal and injured aorta by an indirect immunoperoxidase technique. Histochemistry.. 1983;77:395-403. [Medline] [Order article via Infotrieve]
  24. Takasaki I, Chobanian AV, Sarzani R, Brecher P. Effect of hypertension on fibronectin expression in the rat aorta. J Biol Chem.. 1990;265:21935-21939. [Abstract/Free Full Text]
  25. Sanuel JL, Barrieux A, Dufour S, Dubus I, Contard F, Koteliansky V, Farhadian F, Marotte F, Thiery J-P, Rappaport L. Accumulation of fetal fibronectin mRNAs during the development of rat cardiac hypertrophy induced by pressure overload. J Clin Invest.. 1991;88:1737-1746.
  26. Liptay MJ, Parks WC, Mecham RP, Roby J, Kaiser LR, Cooper JD, Botney MD. Neointimal macrophages colocalize with extracellular matrix gene expression in human atherosclerotic pulmonary arteries. J Clin Invest.. 1993;91:588-594.
  27. Saouf R, Takasaki I, Eastman E, Chobanian AV, Brecher P. Fibronectin biosynthesis in the rat aorta in vitro: changes due to experimental hypertension. J Clin Invest.. 1991;88:1182-1189.
  28. Mamuya WS, Brecher P. Fibronectin expression in the normal and hypertrophic rat heart. J Clin Invest.. 1992;89:392-401.
  29. Villarreal FJ, Dillmann WH. Cardiac hypertrophy-induced changes in mRNA levels for TGF-ß1, fibronectin and collagen. Am J Physiol.. 1992;262:H1861-H1866. [Abstract/Free Full Text]
  30. Clausell N, Rabinovitch M. Upregulation of fibronectin synthesis by interleukin-1 beta in coronary artery smooth muscle cells in associated with the development of the post-cardiac transplant arteriopathy in piglets. J Clin Invest.. 1993;92:1850-1858.
  31. Coito AJ, Binder J, Brown LF, de Sousa M, Van De Water L, Kupiec-Weglinski JW. Anti-TNF-{alpha} treatment downregulates the expression of fibronectin and decreases cellular infiltration of cardiac allografts in rats. J Immunol.. 1995;154:2949-2958. [Abstract]
  32. Takasaki I, Chobanian AV, Mamuya WS, Brecher P. Hypertension induces alternatively spliced forms of fibronectin in rat aorta. Hypertension.. 1992;20:20-25. [Abstract/Free Full Text]
  33. Schwarzbauer JE, Patel RS, Fonda D, Hynes RO. Multiple sites of alternative splicing of the rat fibronectin gene transcript. EMBO J.. 1987;6:2573-2580. [Medline] [Order article via Infotrieve]
  34. Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1989.
  35. Schwarzbauer JE, Paul JI, Hynes RO. On the origin of species of fibronectin. Proc Natl Acad Sci U S A.. 1985;82:1424-1428. [Abstract/Free Full Text]
  36. Mautner VM, Hynes RO. Surface distribution of LETS protein in relation to the cytoskeleton of normal and transformed cells. J Cell Biol.. 1977;75:743-768. [Abstract/Free Full Text]
  37. Peters JH, Trevithick JE, Johnson P, Hynes RO. Expression of the alternatively spliced EIIIB segment of FN. Cell Adh Comm.. 1995;3:67-89. [Medline] [Order article via Infotrieve]
  38. Clowes A, Reidy M, Clowes M. Kinetics of cellular proliferation after arterial injury, I: smooth muscle growth in the absence of endothelium. Lab Invest.. 1983;49:327-333. [Medline] [Order article via Infotrieve]
  39. Chirgwin J, Przybyla A, MacDonald R, Rutter W. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry.. 1979;18:5294-5299. [Medline] [Order article via Infotrieve]
  40. Clowes AW. Prevention and management of recurrent disease after arterial reconstruction: new prospects for pharmacological control. Thromb Haemost.. 1991;66:62-66. [Medline] [Order article via Infotrieve]
  41. Schwartz SM, Heimark RL, Majesky MW. Developmental mechanisms underlying the pathology of arteries. Physiol Rev.. 1990;70:1177-1209. [Abstract/Free Full Text]
  42. Gibbons GH, Dzau VJ. The emerging concepts of vascular remodeling. N Engl J Med.. 1994;330:1431-1437. [Free Full Text]
  43. Dzau VJ, Gibbons GH, Pratt RE. Molecular mechanisms of vascular renin-angiotensin system in myointimal hyperplasia. Hypertension.. 1991;18:100-105.
  44. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature.. 1994;362:801-809.
  45. Dubin D, Pratt R, Dzau V. Endothelin, a potent vasoconstrictor, is a vascular smooth muscle mitogen. J Vasc Med Biol.. 1989;1:150-154.
  46. Vandenburg HH. Mechanical forces and their second messengers in stimulating growth in vitro. Am J Physiol.. 1992;62:R350-R355.
  47. Watson PA. Function follows form: generation of intracellular signals by cell deformation. FASEB J.. 1991;5:2013-2019. [Abstract]
  48. Campbell GR, Campbell JH, Manderson JA, Horrigan S, Rennick RE. Arterial smooth muscle: a multifunctional mesenchymal cell. Arch Pathol Lab Med.. 1988;112:977-986. [Medline] [Order article via Infotrieve]
  49. Hassler O. The origin of the cells constituting arterial intima thickening: an experimental autoradiographic study. Lab Invest.. 1970;22:286-295. [Medline] [Order article via Infotrieve]
  50. Clowes AW, Schwartz SM. Significance of quiescent smooth muscle migration in the injured rat carotid artery. Circ Res.. 1985;56:139-145. [Abstract/Free Full Text]
  51. Gibbons GH, Pratt RE, Dzau VJ. Vascular smooth muscle cell hypertrophy vs. hyperplasia: autocrine transforming growth factor-ß1 expression determines growth response to angiotensin II. J Clin Invest.. 1992;90:456-461.
  52. Wilcox J, Smith K, Williams L, Schwartz S. Platelet-derived growth factor mRNA detection in human atherosclerotic plaques by in situ hybridization. J Clin Invest.. 1988;82:1134-1143.
  53. Naftilan AJ, Pratt RE, Dzau VJ. Induction of platelet-derived growth factor A-chain and c-myc gene expressions by angiotensin II in cultured rat vascular smooth muscle cells. J Clin Invest.. 1989;83:1419-1424.
  54. Sadoshima J, Xu Y, Slayter HS, Izumo S. Autocrine release of angiotensin II mediated stretch-induced hypertrophy of cardiac myocytes, in vitro. Cell.. 1993;75:977-984. [Medline] [Order article via Infotrieve]
  55. Giachelli C, Bae N, Lombardi D, Majesky M, Schwartz S. Molecular cloning and characterization of 2B7, a rat mRNA which distinguishes smooth muscle cell phenotypes in vitro and is identical to osteopontin (secreted phosphoprotein I, 2ar). Biochem Biophys Res Commun.. 1991;177:867-873. [Medline] [Order article via Infotrieve]
  56. Giachelli CM, Bae N, Almeida M, Donhardt D, Alpers CF, Schwartz SM. Osteopontin is elevated during neointima formation in rat arteries and is a novel component of human atherosclerotic plaques. J Clin Invest.. 1993;92:1686-1696.
  57. Majesky MW, Giachelli CM, Reidy MA, Schwartz SM. Rat carotid neointimal smooth muscle cells re-express a developmentally regulated mRNA phenotype during repair of arterial injury. Circ Res.. 1992;71:759-768. [Abstract/Free Full Text]
  58. Weidinger FF, McLenachan JM, Cybulsky MI, Fallon JT, Hollenberg NK, Cooke JP, Ganz P. Hypercholesterolemia enhances macrophage recruitment and dysfunction of regenerated endothelium after balloon injury of the rabbit iliac artery. Circulation. 1991;84:755-767. [Abstract/Free Full Text]
  59. Yamauchi K, Martinet Y, Crystal RG. Modulation of fibronectin gene expression in human mononuclear phagocytes. J Clin Invest.. 1987;80:1720-1727.
  60. Barnes JL, Hastings RR, De la Garza MA. Sequential expression of cellular fibronectin by platelets, macrophages and mesangial cells in proliferative glomerulonephritis. Am J Pathol.. 1994;145:585-597. [Abstract]
  61. Gould VE, Martinez-Lacabe V, Virtanen I, Sahlin MK, Schwartz MM. Differential distribution of tenascin and cellular fibronectins in acute and chronic renal allograft rejection. Lab Invest.. 1992;67:71-79. [Medline] [Order article via Infotrieve]
  62. Barone MV, Henchcliffe C, Baralle FE, Paolella G. Cell type specific trans-acting factors are involved in alternative splicing of human fibronectin pre-mRNA. EMBO J.. 1989;8:1079-1085. [Medline] [Order article via Infotrieve]
  63. Norton PA, Hynes RO. In vitro splicing of fibronectin pre-mRNAs. Nucleic Acids Res.. 1990;18:4089-4097. [Abstract/Free Full Text]
  64. Huh GS, Hynes RO. Elements regulating an alternatively spliced exon of the rat fibronectin gene. Mol Cell Biol.. 1993;13:5301-5314. [Abstract/Free Full Text]
  65. Balza E, Borsi L, Allemanni G, Zardi L. Transforming growth factor B regulates the levels of different fibronectin isoforms in normal human cultured fibroblasts. FEBS Lett.. 1988;228:42-44. [Medline] [Order article via Infotrieve]
  66. Van De Water L, ffrench-Constant C, Brown L. Fibronectin expression during cutaneous wound healing. In: Adzick N, Longaker M, eds. Fetal Wound Healing. New York, NY: Elsevier; 1992:281-302.
  67. Oh E, Pierschbacher M, Ruoslahti E. Deposition of plasma fibronectin in tissues. Proc Natl Acad Sci U S A.. 1981;78:3218-3221. [Abstract/Free Full Text]
  68. Carnemolla B, Leprini A, Allemanni G, Saginati M, Zardi L. The inclusion of the type III repeat ED-B in the fibronectin molecule generates conformational modifications that unmask a cryptic sequence. J Biol Chem.. 1992;267:24689-24692. [Abstract/Free Full Text]
  69. Madri JA, Reidy MA, Kocher O, Bell L. Endothelial cell behavior after denudation injury is modulated by transforming growth factor-ß and fibronectin. Lab Invest.. 1989;60:755-765. [Medline] [Order article via Infotrieve]
  70. Lindner V, Reidy MA, Fingerle J. Regrowth of arterial endothelium. Lab Invest.. 1989;61:556-563. [Medline] [Order article via Infotrieve]
  71. Hedin U, Thyberg J. Plasma FN promotes modulation of arterial smooth muscle cells from contractile to synthetic phenotype. Differentiation.. 1987;33:239-246. [Medline] [Order article via Infotrieve]
  72. Clyman RI, McDonald KA, Kramer RH. Integrin receptors on aortic smooth muscle cells mediate adhesion to fibronectin, laminin, and collagen. Circ Res.. 1990;67:175-186. [Abstract/Free Full Text]
  73. Clyman RI, Mauray F, Kramer RH. ß1 and ß3 integrins have different roles in the adhesion and migration of vascular smooth muscle cells on extracellular matrix. Exp Cell Res.. 1992;200:272-284. [Medline] [Order article via Infotrieve]
  74. Dejana E, Colella S, Conforti G, Abbadini M, Gaboli M, Marchisio PC. Fibronectin and vitronectin regulate the organization of their respective Arg-Gly-Asp adhesion receptors in cultured human endothelial cells. J Cell Biol.. 1988;107:1215-1223. [Abstract/Free Full Text]
  75. Albelda SM, Buck CA. Integrins and other cell adhesion molecules. FASEB J.. 1990;4:2868-2880. [Abstract]
  76. Massia SP, Hubbell JA. Vascular endothelial cell adhesion and spreading promoted by the peptide REDV of the IIICS region of plasma fibronectin is mediated by integrin {alpha}4ß1. J Biol Chem.. 1992;267:14019-14026. [Abstract/Free Full Text]
  77. Luscinskas FW, Lawler J. Integrins as dynamic regulators of vascular function. FASEB J.. 1994;8:929-938. [Abstract]
  78. Kocher O, Kennedy SP, Madri JA. Alternative splicing of endothelial cell fibronectin mRNA in the IIICS region. Am J Pathol.. 1990;137:1509-1524.[Abstract]



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