© 1999 American Heart Association, Inc.
Original Contribution |
EVEC, a Novel Epidermal Growth Factor–Like Repeat-Containing Protein Upregulated in Embryonic and Diseased Adult Vasculature
From the Departments of Internal Medicine (R.C.K.), Molecular Biology and Oncology (R.C.K., E.N.O.), and Pathology (J.A.R.), University of Texas Southwestern Medical Center, Dallas, Tex, and Department of Physiology (J.M.M.), Cardiovascular Research Center, Medical College of Wisconsin, Milwaukee, Wis.
Correspondence to Eric N. Olson, Chairman, Department of Molecular Biology and Oncology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75235-9148. E-mail eolson{at}hamon.swmed.edu
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Abstract |
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Abstract—A hallmark of vascular lesions is the phenotypic modulation of vascular smooth muscle cells (VSMCs) from a quiescent, contractile state to a more primitive, proliferative phenotype with a more fetal pattern of gene expression. Using subtraction hybridization to identify genes that may regulate this transition, we cloned a novel gene named EVEC, an acronym for its expression in the embryonic vasculature and the presence of Ca2+ binding epidermal growth factor–like repeats contained in the predicted protein structure. Although these repeats are characteristic of the extracellular matrix proteins, fibrillin, fibulin, and the latent transforming growth factor-ß binding proteins, EVEC most closely resembles the H411 and T16/S1-5 gene products, the latter of which are believed to regulate DNA synthesis in quiescent fibroblasts. Using in situ hybridization, we demonstrated that EVEC is expressed predominantly in the VSMCs of developing arteries in E11.5 through E16.5 mouse embryos. Lower levels of expression are also observed in endothelial cells, perichondrium, intestine, and mesenchyme of the face and kidney. EVEC mRNA expression is dramatically downregulated in adult arteries, except in the uterus, where cyclic angiogenesis continues; however, EVEC expression is reactivated in 2 independent rodent models of vascular injury. EVEC mRNA is observed in cellular elements of atherosclerotic plaques of LDL receptor–deficient, human apolipoprotein B transgenic mice and in VSMCs of the media and neointima of balloon-injured rat carotid arteries. These data suggest that EVEC may play an important role in the regulation of vascular growth and maturation during development and in lesions of injured vessels.
Key Words: EVEC • vascular smooth muscle cell • atherosclerosis • restenosis
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Introduction |
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Treatment of both atherosclerosis and restenosis after angioplasty and bypass graft surgery remains a major issue in clinical cardiology. A hallmark of these vascular lesions is the dysregulated migration and proliferation of vascular smooth muscle cells (VSMCs) of the arterial media into the neointima stimulated by dysfunction and injury of the overlying endothelium.1 2 VSMCs within these lesions undergo phenotypic modulation from a quiescent, contractile state within the adult arterial media to a phenotype reminiscent of fetal VSMCs, marked by cellular proliferation and expression of extracellular matrix molecules, as well as an alteration in the elaborated repertoire of growth factors and receptors.3 4
Studies with cultured vascular cells and animal models using vascular injury and gene disruption techniques have implicated a cadre of growth factors, coagulation factors, and matrix proteins in the maturation of the interactions between VSMCs and overlying endothelial cells (ECs) in both developing and injured vessels.1 5 6 For example, platelet-derived growth factor, transforming growth factor-ß1 (TGF-ß1), and heparin binding (HB) epidermal growth factor (EGF) play essential roles in early vascular development by stimulating the formation of an organized periendothelial cell layer from surrounding mesenchyme.7 8 These factors also function in the phenotypic modulation of VSMCs in injured vessels.9 10 11
Members of the coagulation cascade, including tissue factor and thrombin, appear to have similar functions during development.12 13 14 Furthermore, treatment of balloon-injured vessels with anti-thrombin receptor antibodies minimizes the extent of neointimal formation at the site of injury.15 In contrast, recent studies of mice lacking elastin demonstrate the role of this microfibril matrix protein in regulating VSMC growth.16 Studies such as these demonstrate that the complex extracellular environment of VSMCs (and ECs) profoundly influences their growth and contractile phenotypes.
To identify novel regulators of vascular growth, we used subtractive hybridization to isolate transcripts enriched in a differentiated pulmonary artery VSMC line that we and others have characterized.17 18 We report the cloning and expression pattern of a novel gene that encodes a predicted protein designated EVEC (embryonic vascular EGF-like repeat-containing protein). The EVEC sequence contains a signal peptide for translocation into the lumen of the endoplasmic reticulum but lacks a transmembrane domain and is characterized by a tandem array of 6 Ca2+ binding EGF (CB-EGF)–like repeats. EVEC shows a high degree of similarity to S1-5, a secreted protein that has been shown to have growth-stimulatory properties in fibroblasts.19 EVEC expression is most pronounced in the VSMCs of the fetal arterial vasculature and is downregulated in most adult vascular beds. EVEC mRNA expression is reactivated in plaques of a mouse model of atherosclerosis and is dramatically upregulated early in the course of neointimal formation after balloon injury of the rat carotid artery. Together, these data suggest that EVEC may be a novel regulator of VSMC growth and phenotype during development and in vascular pathology.
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Materials and Methods |
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Cell Culture, Preparation of RNA, and Northern Blotting
PAC1 cells17 were maintained in DMEM with 10% FBS, penicillin, and streptomycin (medium A) at 37°C in a 6% CO2 incubator and grown to confluence before harvest. Primary neonatal rat cardiac myocytes were prepared as described20 and harvested for RNA within 48 hours of preparation. Total RNA was prepared from cultured cells and mouse tissues using Trizol reagent (GIBCO) according to the manufacturer's recommendations. Poly(A+) RNA was isolated by 2 sequential applications of total RNA to oligo-(dT) cellulose (Pharmacia) according to the manufacturer's protocol. Northern blot analysis of EVEC mRNA was performed as described21 using a 32P-labeled probe corresponding to nucleotides 835 to 1768 of the EVEC cDNA (see below).
Subtraction Hybridization and Cloning of EVEC
Suppressive subtractive hybridization22 was
performed using a subtraction kit (PCR-Select cDNA Subtraction Kit,
Clontech) according to supplied protocols, without modification.
Briefly, cDNA was prepared using 2 µg of
poly(A+) RNA from PAC1 cells and rat neonatal
primary cardiac myocytes. Both cDNA pools were digested with
RsaI. The digested PAC1 "tester" cDNA was divided and
ligated to the supplied adapter sets 1 and 2R. After separate melting
and hybridization of these pools with "driver" cDNA from cardiac
myocytes, the 2 tester pools were annealed and subjected to
adapter-specific polymerase chain reaction (PCR) to yield
"forward"-subtracted cDNA. The subtraction was also performed using
cardiac myocyte cDNA as tester to produce a "reverse"-subtracted
cDNA pool. The forward-subtracted cDNA was subcloned into pGEM TA
cloning vector (T-Easy, Promega), and recombinant colonies were
arrayed on filters and hybridized with
32P-labeled probes derived from the forward- and
reverse-subtracted pools by standard techniques.23
Colonies that hybridized specifically and solely with the
forward-subtracted probes were picked and sequenced. A partial cDNA of
EVEC corresponding to nucleotides 835 to 1768 (an internal
RsaI fragment) was identified.
EVEC cDNA clones were isolated from a PAC1 cDNA library prepared in the Lambda Zap XR vector (Stratagene) according to supplied protocols and screened with the probe described above. All clones were sequenced using an ABI-PRISM 377 sequencer. Sequence-specific oligonucleotides were obtained from Operon Technologies.
A carboxyl-terminal myc/6His epitope–tagged EVEC fusion construct (EVEC/myc/his) was prepared by high-fidelity PCR using a cDNA PCR polymerase mix (Advantage, Clontech) with oligonucleotides that introduced an EcoRI site in the 5' untranslated region and an in-frame HindIII site in place of the termination codon. The sequence of the oligonucleotides, with restriction sites underlined, are as follows: upstream, 5'-GGGAATTCTGCTGAATTACTGAATTAACTGAAGGGGGT-3', and downstream, 5'-GGGAAGCTTGAACGGATACTGGGACACG-3'. The fragment was cloned into the EcoRI/HindIII sites of pcDNA 3.1 myc/his(–) A (Invitrogen) and harvested using QIAGEN Maxi columns.
EVEC Transcription/Translation and Expression in Cultured
Cells
The EVEC cDNA was used to program the TnT reticulocyte lysate
system (Promega) in the presence of
35S-Translabel (ICN). Translated
product was visualized by 12% SDS-PAGE followed by treatment of
the gels with ENHANCE (NEN Life Sciences) and
autoradiography.
Both COS and 293 cells were grown to 40% to 50% confluence on coverslips in medium A and transfected with 0.5 µg of EVEC/myc/his or the parent vector using 3 µL of Fugene 6 transfection reagent (Boehringer Mannheim) according to the manufacturer's protocol. After incubation at 37°C for 48 hours, cells were washed twice with PBS, fixed at 4°C for 15 minutes in 4% paraformaldehyde in PBS, and washed 3 times for 5 minutes with PBS. For permeabilization, cells were treated for 3 minutes at 4°C with PBS containing 0.1% Triton X-100. The cells were incubated for 15 minutes at room temperature with blocking buffer (PBS with 0.5% fraction V BSA) and exposed to an anti-myc antibody (Santa Cruz Biochemicals) at 0.2 µg/mL in blocking buffer for 30 minutes at room temperature followed by 3 washes with PBS. Cells were subsequently incubated with FITC-conjugated anti-rabbit IgG (Sigma) for 15 minutes at room temperature followed by 3 washes with PBS.
Models of Neointimal Formation
All animal procedures were conducted in accordance with NIH and
local institutional guidelines. The
LDLR-/-/Tg:apoB+/+
model of atherosclerosis was kindly provided by Dr
Helen Hobbs (University of Texas Southwestern Medical Center,
Dallas).24 To assess EVEC expression in an
independent model of neointimal formation, we performed
balloon withdrawal injury of the rat carotid artery as
described.25 Sham-operated contralateral arteries served
as controls. Animals were sacrificed at the indicated time after injury
by sodium pentobarbital overdose and cardiac exsanguination. The
vasculature was sequentially perfused under
physiological pressure with PBS containing 4%
paraformaldehyde (pH 8.0). Carotid arteries were
carefully excised, postfixed for 6 hours, and embedded in paraffin.
In Situ Hybridization of Animal Tissue Sections
RNA probes corresponding to the sense and antisense strands of
the EVEC cDNA were prepared using the MaxiScript kit (Ambion). In situ
hybridization was performed as previously described.26
Briefly, sections were deparaffinized with xylene and hydrated through
graded ethanols. Tissue sections were postfixed,
permeabilized, and acetylated overnight before
hybridization. After hybridization, unbound riboprobe was washed from
the slides in a series of SSC/formamide washes and RNase A treatment.
The slides were coated with Ilford K.5 nuclear emulsion, and EVEC
signal was resolved after 21 to 28 days of exposure at 4°C with
standard photographic developing.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Identification of EVEC by Subtraction Cloning
In an attempt to identify factors that regulate development and phenotypic modulation of VSMCs, we used subtraction hybridization with cDNA from PAC1 cells as the tester pool and neonatal primary cardiac myocyte cDNA as driver. PAC1 is a subcloned cell line, derived from adult rat proximal pulmonary artery, that expresses markers of the contractile phenotype through multiple passages.17 18 Neonatal primary cardiac myocytes were chosen as the source for driver cDNA because of their myogenic lineage and their recent transition from a fetal pattern of gene expression, which includes many smooth muscle markers, such as SM22
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smooth-muscle -actin and calponin,3 to a more adult
pattern of cardiac-specific transcription. Twenty-five differentially
expressed clones were identified, of which 5 were not
represented in the GenBank database of published sequences.
One of these novel clones, designated EVEC, was used as a probe to
screen a cDNA library derived from PAC1 cells. Twelve overlapping
partial cDNA clones were isolated, as well as 2 2.4-kb cDNAs, which
appear to encode the entire EVEC open reading frame (Figure 1
). These clones each contain an in-frame
stop codon upstream of the codon encoding the proposed initiator
methionine. 5'-RACE (rapid amplification of cDNA ends) failed to
reveal additional transcribed sequence upstream of the reported 5'
untranslated region (data not shown).
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EVEC Expression in Cultured Cells and Adult Tissues
Northern blot hybridization revealed a single EVEC transcript of
2.4 kb in PAC1 cells but not in neonatal cardiac myocytes (Figure 2A), confirming the success of the
initial cDNA subtraction. Blotting of total RNA from multiple adult
mouse tissues revealed most abundant expression of EVEC mRNA in the
lung, uterus, and kidney, with slightly lower levels of expression in
the heart (Figure 2B). EVEC mRNA was also observed in aorta at
levels similar to those observed in the kidney; by comparison, these
levels are at least 5-fold lower per µg total RNA than the expression
level observed in PAC-1 cells (data not shown).
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EVEC, a Novel Protein Containing EGF-Like Repeats
The predicted EVEC protein contains 448 amino acids and can be
subdivided into several domains (Figure 1
). The amino terminus
encodes a putative signal sequence for sorting via the secretory
pathway,27 with a predicted cleavage occurring between
amino acids 23 and 24.28 The region encompassing
amino acids 24 to 341 contains 6 EGF-like repeats, all containing
consensus sequences for Ca2+
binding.29 Five of the repeats also contain consensus
sequences for ß-hydroxylation of Asn and Asp residues.
EGF-like repeats 2 to 5 have the characteristic array of 6 cysteine
residues, which likely fold with the pattern of intramolecular
disulfide bonds characteristic of this motif (Figure 1); repeat
6 has an additional 2 cysteine residues at the amino-terminal portion
of the repeat. The first EGF-like repeat contains the
Ca2+ binding consensus and 6 cysteines but
deviates from the standard spacing of these residues. The first repeat
is also separated from the others by a short proline- and tyrosine-rich
region (PYR domain). The carboxyl-terminal 107 amino acids are free of
cysteines. EVEC also contains an RGD sequence embedded in the first
EGF-like repeat which, in numerous other extracellular proteins,
mediates interaction with integrins.30 Two potential
N-linked glycosylation sites lie in the fifth and sixth
EGF-like repeats. No potential transmembrane domain or
sorting/retention signals can be identified, suggesting that EVEC is
likely a peripheral membrane or secreted factor.
The predicted EVEC sequence shows striking similarity to both mouse
S1-5/rat T1619 31 and hamster H411 (GenBank accession No.
AF046870) proteins (Figure 3),
each of which contains a predicted signal sequence and shares a similar
array of 6 CB-EGF–like repeats. Overall, the sequences share 45%
amino acid identity. The region of greatest similarity, however, is the
cysteine-free carboxyl terminus with amino acid identities of 50% to
55%. The spacer sequence between CB-EGF–like repeats 1 and 2 (the PYR
domain in EVEC) is the one region lacking conservation between these
three proteins.
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indicate the positions of the N-glycosylation
sequences, and •, the RGD sequence (which is unique to EVEC). B,
Alignment of EVEC and the related proteins T16/S1-5 and H411. Letters
with a dark gray background indicate amino acid identity; light gray
background, conservative substitutions.The mouse S1-5 and rat T16 proteins share 93% amino acid identity; given their lower degree of identity, EVEC and H411 are not likely to be orthologues of S1-5/T16, but rather paralogous members of this novel gene family. To lend further support to this hypothesis, we identified human and mouse expressed sequence tags (ESTs) with >90% nucleotide identity to the rat EVEC sequence and aligned their translated products. The portions of EVEC represented in the EST database were 95% to 99% identical across species lines (at the amino acid level). In addition, chromosomal localization of human EVEC by radiation hybrid mapping places the gene on the distal portion of chromosome 14q (R.C.K., E.N.O., R. Schultz, unpublished data, 1998). The human homologue of S1-5/T16 has previously been mapped to chromosome 2p16,32 further suggesting that EVEC, S1-5/T16, and H411 are products of different but related genes.
The next most closely related proteins are the C and D isoforms of
fibulin-1 (Figure 3), which contains 8 CB-EGF–like repeats and
shares 33% to 38% similarity with EVEC in overlapping repeats. The C
and D isoforms differ in the carboxyl terminus via alternative
splicing; the D isoform has 32% similarity with EVEC in this domain
(data not shown).
EVEC Translocates Through the Secretory Pathway
Combined in vitro transcription/translation of the longest EVEC
cDNA yields a protein with a molecular mass of 51 kDa, slightly
greater than predicted from the sequence (Figure 4A). Translation in the presence of
pancreatic microsomes produced a protein with a larger apparent
molecular weight, suggesting that EVEC undergoes translocation into
microsomes and subsequent modification. Consistent with this
finding were the results of immunofluorescence
performed on cultured COS and 293 cells transfected with the
EVEC/myc/his expression construct (Figure 4B and 4C). Expressed
protein was identified with an anti-myc epitope antibody and revealed a
punctate pattern of fluorescence, most intense in a perinuclear
distribution, characteristic of proteins translocated via the secretory
pathway. Staining of nonpermeabilized cells revealed a
punctate pattern over the plasma membrane, demonstrating that a portion
of the myc/his-tagged EVEC reaches the cell surface and suggesting that
EVEC may interact with specific membrane-associated proteins (Figure 4D). In addition, immunoblotting of the medium
from transfected cells revealed a small fraction of soluble EVEC that
was not cell associated (data not shown).
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EVEC Expression During Embryogenesis
We used in situ hybridization to characterize the developmental
and cell type–specific pattern of EVEC mRNA expression. Figure 5 shows dark-field microscopy of
transverse thoracic sections from mouse embryos at E11.5 through E16.5
hybridized with 35S-labeled cRNA. EVEC expression
was not observed at E8.5 to E9.5. We first detect significant EVEC
expression as early as E11.5 (Figure 5A) in the developing,
paired dorsal aortae. Signal is also noted over the small segment of
cardiac outflow tract present in this section. By E13.5, the
predominant signal is noted in the VSMCs of the developing great
vessels (Figure 5B). Higher-power magnification reveals silver
grains over ECs in addition to VSMCs (data not shown). By E16.5, robust
expression is observed in the outflow tracts, great vessels, and distal
branches of the pulmonary and internal mammary arteries (Figure 5C). Higher magnification of an adjacent E16.5 section (Figure 5D) reveals expression in the coronary arteries as well.
In addition, hybridization is reduced in cells of the developing
semilunar valves of the great vessels, which no longer express smooth
muscle markers as they convert to a more fibroconnective
phenotype.33
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Expression of EVEC appears restricted to the arterial
vasculature (Figure 5A and 5C). A similar pattern of
arterial-enriched expression is observed in the descending
aorta and its mesenteric branches on abdominal sections (data not
shown). Longer exposures of hybridized sections reveal weaker
expression in other tissues of mesodermal origin, including facial and
renal mesenchyme, perichondrium (note expression in the tracheal
perichondrium in Figure 5C), pericardium, intestinal lamina
propria, and nonneural cells in dorsal root ganglia (data not shown).
Interestingly, this pattern of embryonic expression has significant
overlap with that of latent TGF-ß binding protein-2
(LTBP-2)34 (see Discussion).
EVEC Expression in Normal and Diseased Adult Arteries
In adult mice, EVEC mRNA is greatly diminished in large and
medium-sized arteries. Figures 6C and 6D
show in situ hybridization of sections through the normal adult aorta.
Negligible signal is noted compared with that observed on embryonic
sections. Similar downregulation of EVEC mRNA expression occurs in the
vasculature of the adult kidney, lung, heart, and gastrointestinal
tract (data not shown). Significant expression is still observed,
however, in myometrial arteries of the uterus (Figure 6A and 6B); interestingly, these vessels undergo cyclic remodeling in contrast
to the aorta and its proximal branches. As during embryogenesis, no
expression is observed in the neighboring veins. In addition to the
uterus, weaker EVEC expression is seen in cells with the histologic
appearance of tissue histiocytes in the interstitium of the
adult lung, renal tubular parenchyma, heart, and lamina propria of the
gastrointestinal tract (data not shown).
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The downregulation of EVEC mRNA in adult arteries by in situ hybridization appears to contradict the finding of EVEC mRNA in the normal adult aorta by Northern blotting (see above). This apparent discrepancy likely stems from the technical inability to compare EVEC mRNA in adult aorta with isolated fetal aorta by Northern analysis. As a result, in situ hybridization appears to be a better method to compare levels of EVEC mRNA expression during development. Furthermore, EVEC mRNA levels in the adult aorta by Northern blotting mirror levels seen in the kidney and lung, which by in situ hybridization analysis are significantly lower than levels observed in the fetal vasculature.
To determine whether the fetal pattern of EVEC expression is
reactivated in diseased arteries, we assessed the expression of
EVEC in the LDL receptor–/–/Tg:apoB+/+
mouse model of atherosclerosis.24
These animals have a similar lipoprotein profile to humans and develop
marked atherosclerosis of large vessels, which
recapitulates many features of human plaques. Figures 6E and 6F
demonstrate in situ hybridization with these lesions, revealing
reactivation of EVEC in ECs and VSMCs constituting the underlying
plaque.
To assess the change in EVEC expression in an independent model of
phenotypically modulated VSMCs, we used the well-characterized
balloon-injured rat carotid artery model of vascular
injury,25 in which the neointima formed after
arterial injury is composed of primarily migrating and
proliferating VSMCs originating from the vessel media. Figure 7 shows a time course of EVEC mRNA
expression in injured carotid arteries. Elastin staining of adjacent
sections is presented to highlight the vessel media. Uninjured
vessels have minimal signal consistent with the data obtained
from mouse aorta (Figure 7A and 7B). EVEC expression is focally
upregulated in medial smooth muscle as early as 2 days after injury
(Figure 7C and 7D), a time when medial SMC are focally
undergoing mitosis and migration through the internal elastic
lamina.25 Signal is more prominent at day 4 in the media
and forming neointima (Figure 7E and 7F). At day 14,
robust signal is restricted to the neointima, most
prominently in the luminal aspect of these lesions. Less prominent
upregulation of EVEC mRNA is also observed in the adventitia (data not
shown). Prior studies have demonstrated that at this time point both
medial and intimal VSMC replication is reduced, but residual
proliferation of phenotypically modulated neointimal smooth
muscle cells occurs primarily in the luminal aspect of the
vessels.35
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have cloned a novel CB-EGF–like repeat-containing protein, EVEC, that is expressed predominantly in developing and injured arteries. CB-EGF–like repeats are characteristic of a variety of extracellular and transmembrane proteins including the fibrillin,36 fibulin,37 38 and LTBP families of matrix proteins34 39 ; the developmental regulatory protein Notch40 ; its ligand, Serrate29 ; and a variety of coagulation factors including proteins C and S.41 The importance of these repeats has been well established by the identification of mutations that affect their structural integrity in both human disease and developmental abnormalities. Multiple alleles of the Marfan disease gene fibrillin-1 have been identified that affect one or more of the 43 CB-EGF–like repeats,42 and a mutation in this domain has been reported in the protein C gene of a patient with a hereditary hypercoagulable state.41 Two CB-EGF–like repeats in the Notch protein are essential for interaction with its ligand, Serrate, and subsequent signal transduction. The combination of the carboxyl-terminal–most CB-EGF–like repeats and the cysteine-free region of fibulin-1C is essential for interaction with nidogen in vitro.43 These findings, in addition to structural studies demonstrating the role of these repeats in the formation of rigid rodlike structures, implicates this motif in mediating protein:protein interactions, for processes ranging from structural homeostasis to signal transduction. As yet, no proteins interacting with EVEC have been identified.
The striking similarity between EVEC and the predicted protein sequences of hamster H411 and the proteins T16 (from rat) and S1-5 (from mouse) implies that they are members of a novel protein family and may share similar functions. The more distant similarity of S1-5 to isoforms of fibulin-1 has previously been noted.44 No functional properties of H411 have been reported. In contrast, T16 was identified as a second ligand for the surface receptor for the DAN tumor suppressor gene product, which, when added to cultured neuroblastoma lines, can suppress cell division.45 Its mouse homolog, S1-5, was identified via subtractive screening using dividing and senescent fibroblasts. S1-5 mRNA can stimulate DNA synthesis in host and neighboring cells when introduced into cultured fibroblasts by microinjection.19 These data suggest that EVEC and H411 may also regulate cell proliferation in an autocrine or paracrine fashion. Furthermore, it is unclear whether these related proteins will interact with the same ligand or whether the subtle differences in structure, particularly in the PYR domain, will delineate interactions specific to each family member.
The presence of an RGD sequence within the first EGF-like repeat in EVEC (which is not present in S1-5/T16 or H411) may suggest a mechanism for EVEC action. This sequence is critical for binding of integrins to their ligands.30 Several proteins, including Entactin46 and BA46,47 contain functional RGD sequences embedded within EGF-like motifs. In these proteins, the RGD lies in a proposed peptide loop between adjacent antiparallel ß strands. Because repeat 1 of EVEC has an atypical pattern of cysteines, it is unclear whether the RGD motif resides within a similar structure and will be functional for interaction with cell-surface integrins.
The in vitro translation and cultured-cell expression studies suggest that EVEC is translocated through the endoplasmic reticulum/Golgi network and undergoes subsequent modification. Despite the absence of a traditional membrane-spanning domain, only small amounts of EVEC were identified in the medium of transfected cells (data not shown). It is unclear whether EVEC remains cell associated as a peripheral membrane protein or is incorporated into matrix or, alternatively, whether a cofactor is needed for efficient folding and sorting in transfected cells.
The expression pattern of EVEC in embryonic, adult, and abnormal vasculature is consistent with a role as a regulator of vascular growth and/or maturation. EVEC expression is noted as early as E10.5 (data not shown) and is robust in the developing medial layer and EC of the latter-stage embryo. Such timing appears to exclude EVEC from a role in the determination of either VSMCs or ECs, since many smooth muscle-specific markers are expressed earlier in angiogenesis.3 Rather, EVEC expression occurs during this period of continued VSMC proliferation and maturation of both the vascular matrix and the interaction between ECs and VSMCs.35 The high degree of similarity of the pattern of EVEC expression to that of LTBP-2 suggests that possible interaction with this protein and a role in regulating TGF-ß signaling must be explored in future studies. By in situ hybridization, VSMCs of the adult contractile phenotype express reduced levels of EVEC, as do the overlying ECs in these vessels, compared with the VSMCs of embryonic and diseased arteries. The only vessels examined that express significant levels of EVEC mRNA in the adult are the myometrial arteries, which are continually undergoing angiogenesis and apoptosis in contradistinction to other adult vascular beds.
The reactivation of EVEC expression in vascular lesions of a mouse model for atherosclerosis and balloon-injured rat carotid arteries is also consistent with a possible role in growth regulation, matrix maturation, or signaling between VSMCs and EC. Several other proteins with similar patterns of fetal expression and reactivation in injured vessels have been identified and are often either components of the extracellular matrix or regulators of cell proliferation and migration.1 The time course of activation of these 2 classes of proteins after balloon injury varies. HB-EGF is rapidly upregulated within hours after balloon vessel injury and persists for at least 14 days.11 In contrast, many of the upregulated extracellular matrix components, including tropoelastin and perlican, are transcriptionally activated during the weeks to months after injury, either after the cessation of neointimal proliferation48 or in nonreplicating cells.35 EVEC expression in balloon-injured vessels more closely resembles the former pattern and parallels the time course of activation described for HB-EGF by immunohistochemistry.11 Thus, EVEC represents a novel gene the expression of which corresponds to periods of vascular growth, maturation, and response to injury. Understanding the function of EVEC in normal development, atherosclerosis, and neointimal proliferation may shed light on alternative approaches to control the progression of vascular disease.
Note Added in Proof
After this manuscript was prepared, two sequences denoted UP50 and
UPH1 were submitted to GenBank (accession Nos. AF093118 and AF093119).
Sequence analysis reveals that UP50 and UPH1 correspond to the
human homologs of rat EVEC and hamster H411, respectively.
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Acknowledgments |
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This work was supported by grants from the NIH and Muscular Dystrophy Association (to E.N.O.). We thank M. Kathy Kunkle, John Shelton, Alisha Tizenor, Robert Webb, Jeff Stark, and Luanne Kelly for excellent technical assistance. Helen Hobbs kindly provided us with the mouse model of atherosclerosis.
Received December 9, 1998; accepted March 4, 1999.
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