© 2000 American Heart Association, Inc.
Molecular Medicine |
Novel Embryonic Genes Are Preferentially Expressed by Autonomously Replicating Rat Embryonic and Neointimal Smooth Muscle Cells
From the Department of Pediatrics (M.C.M.W.-E., P.E.S., N.A.G., B.E.Q., S.S.G., P.M.M., R.A.M., K.R.S.), University of Colorado Health Sciences Center, Denver, Colo; and Large Animal Surgery (J.K.B.), Auburn University, Auburn, Ala.
Correspondence to Dr Mary C.M. Weiser-Evans, Department of Pediatrics, Campus Box B131, 4200 East Ninth Ave, University of Colorado Health Sciences Center, Denver, CO 80262. E-mail Mary.Weiser{at}UCHSC.edu
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Abstract |
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Abstract—We sought to identify and characterize the expression pattern of genes expressed by smooth muscle cells (SMCs) during periods of self-driven replication during vascular development and after vascular injury. Primary screening of a rat embryonic aortic SMC–specific cDNA library was accomplished with an autonomous embryonic SMC–enriched, nonautonomous adult SMC–subtracted cDNA probe. Positive clones were rescreened in parallel with embryonic SMC–specific and adult SMC–specific cDNA probes. We identified 14 clones that hybridized only with the embryonic cDNA ("emb" clones), 11 of which did not share significant homology with sequences in any of the databases. Five of these novel emb genes (emb7, emb8, emb20, emb37, and emb41) were selectively and only transiently reexpressed in vivo by neointimal SMCs during periods of rapid replication. The emb8:embryonic growth–associated protein (EGAP), which was studied the most extensively, was expressed at high levels by cultured, autonomously replicating embryonic and neointimal SMCs but was detected only at low levels even in mitogenically stimulated adult SMCs. Finally, the administration of antisense EGAP oligonucleotides markedly attenuated embryonic and neointimal SMC replication rates. We suggest that autonomous replication of SMCs may be essential for normal vascular morphogenesis and for the vascular response to injury and that these newly identified "embryonic" genes may be part of the molecular machinery that drives this unique growth phenotype.
Key Words: arteries • vasculature • restenosis • muscle, smooth • clones
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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During vascular development, aortic smooth muscle cells (SMCs) in vivo undergo a distinct phase of rapid proliferation, a time period during which the vessel wall acquires its complement of SMCs, followed by a period of extensive extracellular matrix production that contributes to the structural maturation of the vessel wall.1 2 3 4 5 We have documented in the rat that aortic SMCs replicate at a high rate (>80%/d) throughout the embryonic period of life, demonstrate dramatic decreases in replication at the embryonic-to-fetal transition of intrauterine life (rates decrease to <40%/d), and gradually acquire a quiescent phenotype, reaching replication rates of <0.06%/d in the adult.1 Significant proliferation of SMCs in the adult animal is observed only during certain stages in the development of vascular fibroproliferative diseases such as atherosclerosis, in restenosis after angioplasty, and in transplant arteriopathies.6 7 8 9 For instance, Clowes et al10 have shown that after experimental arterial injury, neointimal SMCs demonstrate large increases in replication rates that reach levels similar to those observed during embryonic life. Furthermore, data from this laboratory and others suggest that after vascular injury, as during development, adult SMCs proceed through a period of rapid cell division followed by a period of extracellular matrix production.4 11 12 13 However, the factors that regulate SMC proliferation during vascular development and neointimal SMC replication after vascular injury to the adult blood vessel remain poorly defined.
Corresponding to the in vivo observations of high replication indices, embryonic aortic SMCs in culture exhibit a distinct growth phenotype characterized by their ability to replicate in an autonomous, mitogen-independent manner.1 The capacity for self-driven replication appears to be lost by fetal life, as demonstrated by the mitogen-dependent growth phenotype exhibited by fetal, neonatal, and adult rat aortic SMCs. The loss of autonomous growth potential suggests that important changes in gene expression and phenotype occur in developing SMCs between the embryonic and fetal periods of intrauterine life. In addition, we have demonstrated that adult SMCs that replicate in experimentally injured arteries transiently exhibit an autonomous, mitogen-independent growth phenotype.11 This observation is consistent with the hypothesis that SMCs reiterate a pattern of gene expression during injury repair similar to that expressed by SMCs during early vascular development.
Because we found that the capacity for self-driven, autonomous replication contributes to high rates of SMC replication during vascular development and during early neointima formation after vascular injury, the purpose of the present study was to identify and characterize molecules capable of conferring autonomous growth capabilities to SMCs. Previous studies that cloned and identified genes expressed by vascular SMCs used animals from postnatal or later postinjury periods, stages at which changes in the vascular wall cells are characterized more by matrix synthesis than by extremely high rates of cell replication.14 15 16 17 Therefore, we sought to identify genes that are preferentially expressed during vascular development when SMCs express autonomous growth capabilities and to determine whether these same genes would be reexpressed in SMCs after injury to the adult blood vessel in the period characterized by high rates of intimal SMC proliferation. We used a subtractive hybridization approach with a cDNA library prepared from RNA isolated from cultured, autonomously replicating embryonic aortic SMCs and an embryonic SMC–enriched, adult nonautonomous SMC–subtracted cDNA probe. We report the cloning and expression patterns of 5 novel genes (referred to as "emb" genes) that are highly expressed in vivo and in vitro by SMCs during periods of vascular growth that are characterized by autonomous replication and suggest that these clones may be part of the molecular machinery that participates in autonomous replication of SMCs. The emb genes, therefore, may represent an entirely new class of genes reexpressed in the setting of vascular diseases characterized by excessive SMC replication.
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Materials and Methods |
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An expanded Materials and Methods section can be found in an online data supplement available at http://www.circresaha.org.
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Results |
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Using a subtractive hybridization cloning technique, we sought to identify novel genes expressed by SMCs during periods of self-driven growth observed during development and after vascular injury to the adult blood vessel. Primary screening of an embryonic SMC–specific library was accomplished with an embryonic SMC–enriched, adult SMC–subtracted cDNA probe as described in Materials and Methods (available in an online-only data supplement at http://www.circresaha.org). Approximately 9x103 clones, or 3% of the library, hybridized with the probe. Twenty clones (based on relative signal intensity) were picked for secondary screening. Of 76 positive secondary clones, 43 represented unique classes based on restriction digest patterns (data not shown). The 43 positive clones were pooled, replated, and lifted in duplicate as an embryo-enriched library. This embryonic SMC–enriched library was then screened in parallel with embryo-specific and adult-specific cDNAs with the use of hybridization probes prepared from autonomously replicating embryonic SMCs and from mitogenically stimulated adult SMCs. A total of 14 (33%) apparently independent clones of this group hybridized only with embryonic cDNA and hereafter are referred to as embryonic, or emb genes (emb1, emb2, emb3, emb7, emb8, emb12, emb15, emb20, emb22, emb29, emb36, emb37, emb39, and emb41).
These emb clones were sequenced at their 3' and 5' ends, and the sequences were compared against the GenBank database (data not shown). Of the 14 independent clones, 3 were found to exhibit significant homology to previously described genes, including the transcription factor pendulin (emb2), the S6 ribosomal protein (emb22), and rat cdc25A (emb39). The remaining 11 clones did not share significant homology in their 3' and 5' ends with any sequences in the databases and therefore represent potentially novel genes.
The 14 positive emb genes were simultaneously screened on
tissue sections by in situ hybridizations for expression in embryonic
aortas, in adult aortas, and in balloon-injured adult carotid
arteries 9 days after injury. The relative amounts of each emb gene at
the different time points were quantified as described in Materials and
Methods (available in an online-only data supplement at
http://www.circresaha.org). Five of the putative novel emb clones
(emb7, emb8:embryonic growth–associated protein [EGAP], emb20,
emb37, and emb41), as well as 2 previously identified clones
(emb2:pendulin and emb22:S6 ribosomal protein), were highly expressed
in the embryonic aorta, were essentially absent in the adult aorta, but
were significantly reexpressed in the day 9 neointima
(>4-fold over adult) (Figure 1
).
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The emb clones were also screened by in situ hybridization for
expression in adult testis, epidermis, brain, lung, skeletal muscle,
kidney, intestines, and liver to assess the tissue specificity of
expression and to determine whether emb genes are expressed in highly
replicative adult tissues. The emb8:EGAP (Figure 2), which was widely distributed in the
embryo, continued to be expressed at high levels in the adult in
primary spermatocytes, in the basal epidermis, and in
interstitial fibroblasts of skeletal muscle and in
intestinal crypts. In contrast, the expression of emb37 (Figure 2) was more restricted in the embryo and, in the adult, was
undetectable in primary spermatocytes, intestinal crypts, and
bronchiolar epithelium, remaining detectable only in the basal
epidermis and Purkinje cells of the brain. The characteristics and
expression patterns of all 14 emb clones are outlined in Table 1 online
(available in an online-only data supplement at
http://www.circresaha.org). Although considerable variability obviously
exists in the spatial pattern of expression of the mRNAs
represented by these clones, all are expressed in SMCs
specifically during periods of autonomous replication in the developing
aorta, and 7 of the 14 (5 novel emb genes) are significantly
reexpressed in the injured carotid artery during periods of autonomous
growth. We chose to study these 5 novel clones in more detail,
concentrating on emb8:EGAP. The emb clones that shared homology with
sequences in the databases or were determined, through in situ
hybridization, to be only moderately regulated between embryonic and
adult developmental stages or to not be reexpressed after vascular
injury were not analyzed further.
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A complete developmental analysis of emb gene expression was
performed on tissue sections obtained from whole embryos or fetuses
(embryonic/fetal days 12 through 21) and from aortic tissues (postnatal
days 1, 7, 16, 30, and 90). The typical pattern of expression in the
developing aorta of the 5 clones is illustrated in Figure 3A, with emb8:EGAP given as an example.
The emb8:EGAP mRNA was first detected in the embryonic aorta as early
as embryonic day 12 (data not shown) and showed high levels of
expression throughout embryonic life. There was a dramatic loss of
signal intensity by fetal day 19, and expression of emb8:EGAP mRNA was
essentially absent throughout postnatal development. We quantified the
in situ signal for both emb8:EGAP and emb37 and plotted the results in
conjunction with in vivo aortic SMC replication rates. As previously
reported1 and as illustrated in Figure 3B, aortic
SMCs showed high rates of replication during embryonic life but
exhibited dramatic decreases in replication by fetal life. The
emb8:EGAP and emb37 mRNAs were expressed by 
70% to 80% aortic SMCs
during the embryonic period, demonstrated marked decreases by embryonic
day 18 (25% positive SMCs), and were essentially undetectable by
fetal day 19 (<10% positive SMCs). Because the embryo-to-fetus
transition (embryonic day 17 to embryonic day 19) marks a period during
which cultured SMCs lose the capacity to replicate
autonomously,1 the loss of emb8:EGAP mRNA by fetal day 19
suggests that emb8:EGAP (and other emb clones) may play a functional
role in the regulation of autonomous replication.
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Previous data from our laboratory demonstrated that pulmonary
artery and airway SMC and airway epithelial cell replication rates
during development parallel those observed for aortic
SMCs,18 suggesting that an intrinsic, developmentally
timed mechanism controls the replication of a variety of cell types. We
examined the expression of emb8:EGAP mRNA in the developing rat lung, a
tissue known to undergo extensive remodeling throughout intrauterine
and early postnatal life. Intense expression of emb8:EGAP mRNA was
observed in embryonic extraparenchymal pulmonary arteries and
airways, there was a marked decrease in signal intensity in all lung
structures by fetal life, and virtually no emb8:EGAP signal was
detected in pulmonary arteries or airways during early
postnatal life (Figure 4). The decrease
in signal intensity was typical for most tissues that express emb8:EGAP
(data not shown), suggesting that along with its potential for
regulating vascular SMC autonomous growth, emb8:EGAP may, in general,
play an integral role in driving early developmental growth
programs.
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Our previous data demonstrated that adult SMCs isolated from
experimentally injured arteries during periods of peak in vivo
replication exhibit an autonomous growth phenotype similar to
that expressed by embryonic SMCs.11 We next
analyzed a complete series of balloon-injured rat carotid
arteries to determine the temporal pattern of expression of emb8:EGAP
mRNA after vascular injury (Figure 5).
The emb8:EGAP mRNA was undetectable in uninjured carotid arteries and
was first detected at high levels in the arterial media 4
days after injury, and expression remained high throughout the
developing neointima at 7 and 9 days after injury. By 14
days after injury, emb8:EGAP mRNA remained detectable only on the
growing, luminal edge of the neointima. By 3 weeks after
injury, emb8:EGAP mRNA expression was undetectable in the
arterial media and neointima. A similar pattern
of expression was observed for emb37 (not shown). Therefore,
reexpression of emb8:EGAP (and emb37) mRNA after vascular injury
correlated temporally with large in vivo increases in
neointimal SMC replication and with the reexpression of an
autonomous growth phenotype by neointimal SMCs.
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These data clearly demonstrate that emb8:EGAP mRNA is temporally
expressed in vivo during periods of significant SMC replication.
Northern blot analysis was used to examine the expression of
emb8:EGAP mRNA in cultured embryonic, adult, and neointimal
SMCs. RNA was isolated from highly replicative, autonomous embryonic
and neointimal SMCs maintained in serum-free conditions and
from highly replicative, serum-stimulated adult SMCs. As shown in
Figure 6, emb8:EGAP mRNA was readily
detectable in serum-deprived, but growing, embryonic and
neointimal SMC cultures; was detected only at low levels in
serum-stimulated, replicating adult SMCs; and was essentially
undetectable in serum-deprived adult SMC cultures (not shown),
consistent with the in vivo in situ hybridization data and
consistent with its potential role in the regulation of
autonomous replication of SMCs.
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Northern blot analysis revealed a single emb8:EGAP transcript
of 2.6 kb, and this clone has been sequenced in its entirety (Figure 1
online; available in an online-only data supplement at
http://www.circresaha.org). Translation analysis of the cDNA
showed that the emb8:EGAP gene product contains an open reading
frame encoding a 725–amino acid protein. The translated protein
contains a consensus translation initiation signal sequence, a
potential nuclear translocation signal, and putative tyrosine and
serine phosphorylation sites but lacks a signal
sequence for sorting through the secretory pathway. DNA and protein
sequence analyses showed no significant homologies to known
sequences in the GenBank database, suggesting that emb8:EGAP is a novel
protein. However, a blast search of all deposited expressed
sequence tags (updated monthly) showed significant homology to human
clone RP11-379P1 located on chromosome 9. Homologies extended from
bases 901 through 2278 and identities ranged from 83% to 100% over
this region. Similarly, there was significant homology to Mus
musculus clone MNCb-6493 extending from base 985 through 1833 and
exhibiting 93% identity in this region. There also were significant
homologies with Drosophila and Caenorhabditis
elegans expressed sequence tags of unknown function
(71% and 63%, respectively). Although the biological function of
emb8:EGAP and its homologs is unknown, these data suggest that
emb8:EGAP is a highly conserved gene.
To determine whether inhibition of the expression of emb8:EGAP could
affect autonomous growth potential, we administered antisense emb8:EGAP
oligodeoxynucleotides (ODNs) to embryonic and
neointimal SMC cultures. Using FITC-labeled
oligonucleotides, we found that the efficiency of
uptake of ODNs by embryonic, neointimal, and adult SMCs was
essentially equal and that ODNs were localized to the cytoplasm (Figure 7C). As shown in Figure 7A, under
serum-free conditions, untreated and sense ODN-treated SMC cultures
continued to demonstrate high rates of bromodeoxyuridine (BrdU)
incorporation (replication index >70%). In contrast, a single
administration of antisense emb8:EGAP ODNs (5 µmol/L) resulted
in a significant decrease in BrdU-labeled embryonic and
neointimal SMCs (replication index 59±0.5% and
30.5±3.5%, respectively), suggesting that emb8:EGAP may play a
functional role in the regulation of SMC autonomous growth. We detected
no difference in results in parallel experiments with an
interferon-
–neutralizing antibody, suggesting that antisense ODN
treatment did not result in nonspecific inhibition of SMC growth
through double-strand DNA induction of interferon- (not shown). In
contrast, emb8:EGAP sense and antisense ODN (10 µmol/L)
administration had no effect on serum-stimulated adult SMC growth
(Figure 7B), suggesting that emb8:EGAP is uniquely involved in
the regulation of SMC self-driven replication.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study, we sought to identify genes preferentially expressed by embryonic and neointimal SMCs with autonomous growth potential, with the ultimate goal of elucidating the molecular mechanisms responsible for conferring this unique and important growth property to SMCs. We hypothesized that this growth phenotype may involve the biologically timed expression of specific growth-associated genes controlled by a mechanisms inherent to the SMCs themselves. Using a subtractive hybridization technique that was aimed at specifically identifying only those genes expressed by cells with autonomous growth potential, we identified 14 clones in embryonic SMCs that were not expressed by highly proliferative adult SMCs. Eleven emb clones are presumably novel genes, because no significant sequence homologies to known genes were found in database screenings.
The expression pattern of each of the 14 genes was not exclusive to vascular structures, because 10 emb genes were ubiquitously expressed in all embryonic tissues evaluated. Importantly, however, the vast majority of emb genes were not expressed in any of a variety of adult tissues and specifically were never expressed by any cells in adult uninjured blood vessels. When detected in adult tissues, these genes were expressed only by rapidly replicating cells in tissues that undergo continuous renewal (eg, intestinal crypts, spermatocytes, and basal epidermis), a process that occurs via mechanisms as yet largely unknown. We found that 7 emb genes were specifically, but only transiently, upregulated in SMCs in response to balloon catheter–induced injury. The expression patterns of these 7 embryonic genes correlated to extremely high rates of in vivo replication during development and after injury and to autonomous growth capabilities of SMCs in vitro. However, the existence of 7 genes in embryonic cells that are not reexpressed after vascular injury suggests that the reexpression of embryo-specific genes is not all inclusive and that certain unique properties of embryonic SMCs are not observed or reexpressed after balloon catheter injury. Finally, antisense constructs to emb8:EGAP specifically reduced the autonomous proliferative potential of embryonic and neointimal SMCs. Thus, our data suggest that unique gene products may be necessary to confer autonomous growth potential to SMCs. Elucidation of the function of these genes could provide important insight into the mechanisms that control SMC growth during critical periods of development and in response to injury.
Both emb2:pendulin and emb22:S6 ribosomal protein, reexpressed by autonomously growing cells in the setting of vascular injury, have been shown in other cell systems to play integral roles in processes related to cell growth. For example, Drosophila strains defective in ribosomal protein synthesis exhibit a delayed developmental program manifested by slower rates of cell growth and division.19 The mammalian S6 ribosomal protein, rapidly phosphorylated when cells are stimulated to grow or divide, is essential for normal development.20 Likewise, pendulin, a member of a superfamily of proteins that contain Armadillo repeats, is required for the nuclear import of DNA-binding proteins involved in cellular proliferation.21 Furthermore, emb39:cdc25A was highly expressed in embryonic, autonomously replicating SMCs but not in adult SMCs or in injured arteries. Cdc25 phosphatases act during G1/S or G2/M as critical links between various developmental signals and cell cycle control.22 23 Although emb39:cdc25 was not significantly reexpressed in injured arteries, our observation that emb39:cdc25A is highly expressed in embryonic, autonomously replicating SMCs and not in adult SMCs is strongly suggestive of a functional role for this gene product in self-driven replication and again supports the idea that not all embryonic processes are reiterated in adult SMCs that respond to injury. Furthermore, that fact that the protein products of these known emb genes are regulators of cell growth is supportive evidence that our cloning method yielded genes that are functionally involved in the regulation of certain aspects of self-driven replication rather than merely being markers of an embryonic SMC.
The expression patterns of at least 7 genes in our study were similar
to those of another gene, EVEC, that was recently described by Kowal et
al.24 The authors cloned a novel calcium-binding,
EGF-repeat–like protein from PAC-1 cells, a highly differentiated SMC
line, but suggested that its structure and expression pattern are
consistent with a role for EVEC in the regulation of vascular
growth during development and in lesions in injured vessels. In our
study, the expression pattern and signal intensity of the most
extensively studied emb gene, emb8:EGAP, were also highly suggestive of
a functional role for EGAP in SMC growth during development and in
response to injury. However, the structure of the emb8:EGAP-translated
product did not lend insight into the specific functions of this
protein We therefore used an antisense approach to determine whether
emb8:EGAP was participating in the autonomous growth process.
Inhibition of emb8:EGAP in embryonic and neointimal SMCs
decreased the autonomous growth capacity of these cells. However, the
loss of function of only 1 emb gene in embryonic and
neointimal SMCs did not result in the same degree of growth
arrest as observed in adult SMCs under mitogen-free conditions,
suggesting that 
2 emb gene products may coordinately interact to
determine an autonomous growth phenotype. These results are not
surprising given that the development of a mature vascular system or
the formation of a neointima after injury likely involves
the integration of multiple intracellular signals. Inasmuch, future
studies have been designed to analyze the interactions of
multiple emb genes on the autonomous growth potential of SMCs.
Interestingly, the degree of growth inhibition by emb8:EGAP ODN was consistently greater in neointimal SMCs than in embryonic SMCs, suggesting that although the autonomous growth characteristics of embryonic and neointimal SMCs are similar, the factors that ultimately regulate the growth phenotypes of these 2 cell types might differ slightly. Based on our fluorescent-labeled ODN experiments, it is unlikely that these results are due to more efficient uptake of antisense ODNs by the neointimal SMC populations used in this study. It is currently unclear whether all SMCs in the vascular media are capable of expressing emb genes and the autonomous growth phenotype or whether this is the unique capacity of only certain SMC subpopulations. Several investigations not only point out the heterogenous nature of SMCs in the adult vessel wall but also have suggested that only certain unique cell populations contribute to the intimal thickening process.25 26 27 The finding that SMCs may originate from sources other than traditional mesoderm-derived cells is highly suggestive of the possible existence of several distinct subtypes of SMCs.28 29 Nevertheless, the transient reexpression of an autonomous growth phenotype after injury11 in conjunction with the reexpression of several embryo-specific genes suggests that at least some SMC populations exhibit plasticity in relation to their growth properties.30 Our data therefore suggest that although the self-driven growth of neointimal SMCs is similar to that embryonic SMCs, it is unlikely that an entire developmental growth program is reexpressed after vascular injury.
The treatment of vascular diseases characterized by excessive SMC proliferation has in large part been unsuccessful and continues to be an important clinical issue. However, strategies designed to inhibit the growth of SMCs and decrease the formation of the neointima after experimental injury have been only variably successful,31 32 33 supporting the idea that neointimal SMCs use unique growth pathways that have not yet been fully defined. This could explain the failure to identify an exogenous factor responsible for neointimal SMC replication despite intense efforts to find one. The findings of the present study point to the possibility that a combination of novel genes may be used to drive SMC proliferation during critical early periods of embryonic growth and neointima formation. Studies are ongoing in our laboratory to further characterize the structure and function of these emb gene products, and future studies aimed at mutational deletion of these genes could confirm their roles in proliferation, thereby providing potential alternative treatment strategies for fibroproliferative vascular diseases.
Note Added in Proof
After submission and acceptance of this manuscript, another
group (Yi XJ, Li XF, Yu FS. A novel epithelial wound-related gene is
abundantly expressed in developing rat cornea and skin. Curr Eye
Res. 2000;20:430–440) described the cloning and expression of a
novel corneal wound healing–related protein from rat healing corneal
epithelium. Sequence analysis showed that clone T4a is identical to
emb8:EGAP. Furthermore, the authors found that the clone T4a was
abundantly expressed in rapidly replicating corneal epithelium as well
as in the epidermis, similar to the pattern of expression of EGAP,
strengthening the possibility that this gene plays a role in cellular
development and wound repair.
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Acknowledgments |
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This work was supported by grant P50-HL-57144-04 from the National Institutes of Health and by Grant 95008610 from the American Heart Association.
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Footnotes |
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1 Deceased.
Received July 3, 2000; revision received August 2, 2000; accepted August 2, 2000.
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References |
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