© 1995 American Heart Association, Inc.
Articles |
Balloon Catheterization Induces Arterial Expression of Embryonic Fibronectins
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.
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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
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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.
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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
100 (excluding EIIIB) bases,
respectively. For analysis of the V region by RNase protection
assay, we prepared a template (Fig 1
) 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 1).
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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 1
), 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
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.
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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 2A
, compare both bands in lane 2 with those in
lane 1 and lane 5 with lane 4; in Fig 2B, 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 2A). 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 2B). 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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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 3A and 3B). 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 3C and 3D). 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 2 and 3
demonstrate that FN mRNA is expressed at only low levels by medial
cells in normal aorta.
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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 4A and 4B). Significant neointimal labeling was
also observed with probes specific for the EIIIA (Fig 4C and 4D), EIIIB
(Fig 4E and 4F), and V25 (Fig 4G and 4H) 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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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 4I and 4J).
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 4K and 4L). 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 5). 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 5B);
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 5C). 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 5D). 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 5E). 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 5F with 5C).
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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 6A). 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 6D). Although overall staining of control arteries by anti-EIIIB
antibodies was negligible (Fig 6G), 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 6A and 6D). 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 6G). By day 1 after injury,
there was little change in the iliofemoral arterial
staining patterns produced by the three antibodies (Fig 6B, 6E, and 6H), except that the intimal staining produced with the anti–total
FN and anti-V95 antibodies appeared to be disrupted in some areas (Fig 6B and 6E). 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 6C, 6F, and 6I). At an intermediate time point (7 days), the newly formed
neointima stained brightly with anti-V95, anti-EIIIB, and
anti–total FN antibodies (Fig 7). Medial staining
with each of the three antibodies was increased above that of uninjured
artery at this time (compare Fig 6A, 6D, and 6G with Fig 7A 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 7C) or not
deglycosylated (Fig 7D) before immunostaining.
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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-
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.
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Acknowledgments |
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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.
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References |
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