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RESEARCH ARTICLE |
Department of Pathology and Laboratory Medicine, Texas A&M University System Health Science Center, College Station, TX 77843-1114, USA
Author for correspondence (e-mail: gedavis{at}tamu.edu
Accepted April 25, 2001
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SUMMARY |
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 Top SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Endothelium, Morphogenesis, Differential gene expression, Collagen matrices
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INTRODUCTION |
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TopSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Carmeliet and Jain,
2000; Yancopoulos et al.,
2000; Conway et al.,
2001). Although considerable
work has been performed identifying factors that promote or inhibit angiogenic
responses (Folkman, 1997;
Carmeliet and Jain, 2000;
Yancopoulos et al., 2000;
Conway et al., 2001),
considerably less effort has focused on how individual and groups of ECs
assemble into capillary tubes during these events. Identifying new molecular
targets that block specific steps in EC morphogenesis may become crucial in
efforts to inhibit angiogenesis in human diseases where angiogenesis is a
pathogenic component (i.e. cancer, diabetic retinopathy, arthritis,
atherosclerosis) (Folkman,
1995; Carmeliet and Jain,
2000).
One experimental approach to address these questions has been to use in
vitro models of EC morphogenesis where many of the steps observed in vivo can
be mimicked (Montesano et al.,
1992; Vernon and Sage,
1995; Nicosia and Villaschi,
1999). The most promising
assays for elucidating relevant molecules and pathways necessary for EC
morphogenesis are those using 3D extracellular matrices (ECM) composed of
collagen type I or fibrin (Montesano and Orci,
1985; Nicosia and Ottinetti,
1990; Davis and Camarillo,
1996; Ilan et al.,
1998; Vernon and Sage,
1999; Yang et al.,
1999; Bayless et al.,
2000; Davis et al.,
2000). These matrices
represent the major matrix environments where angiogenic or vasculogenic
events take place (Vernon and Sage,
1995; Senger,
1996; Nicosia and Villaschi,
1999). In some of these
assays, particularly where ECs are suspended as individual cells in 3D
matrices, most of the ECs undergo morphogenesis simultaneously, which allows
for an analysis of differential gene expression in large numbers of ECs. This
is a critical aspect of EC morphogenic or regression microassays developed by
our laboratory (Davis and Camarillo,
1996; Bayless et al.,
2000; Davis et al.,
2000; Davis et al.,
2001). In these systems,
differential gene expression can be directly correlated with distinct events
in the EC morphogenic or regression cascade (Salazar et al.,
1999; Davis et al.,
2001).
Many studies over the years have shown that differential gene expression
controls complex biological phenomena (Brown and Botstein,
1999). Recently, the
development of gene array technology has revealed how classes of
differentially regulated genes control processes such as yeast responses to
glucose deprivation, fibroblast responses to serum mitogens and tumor
development and apoptosis (DeRisi et al.,
1997; Iyer et al.,
1999; Perou et al.,
2000; Maxwell and Davis,
2000). These approaches have
been useful to characterize the role of previously identified genes in a given
process. To identify relevant differentially expressed novel genes, additional
techniques were developed including differential display, subtraction cDNA
cloning and serial analysis of gene expression (SAGE) (Velculescu et al.,
1995; Martin and Pardee,
1999). Using this latter
technology, differentially regulated genes (known and novel) were identified
in colon carcinoma-derived endothelium versus normal colonic endothelium (St.
Croix et al., 2000).
Here, we present data using a number of the above technologies to investigate differential gene expression during human capillary morphogenesis in 3D collagen matrices. Using gene array analysis, differential display and cDNA library screening, hundreds of differentially expressed genes have been identified. We have examined the expression patterns of thousands of genes and have observed variable modes of gene upregulation as well as gene downregulation during a 48 hour time course of this process. In addition, the full length sequence of two new genes is presented. This survey analysis of differential gene expression serves as a basis for future studies concerning the molecular regulation of capillary morphogenesis in 3D extracellular matrices.
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MATERIALS AND METHODS |
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TopSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) was the kind gift
of B.D. Sanwal (University of Western Ontario, London, Canada).
Oligonucleotide primers were synthesized by Sigma-Genosys (The Woodlands, TX).
The pQE30 vector and Ni/Cd-sepharose were from Qiagen (Valencia, CA).
Antibodies to laminin were obtained from Sigma (St Louis, MO), anti-Id1 was
from Upstate Biotechnologies (Lake Placid, NY), anti-collagen type IV from
Chemicon (Temecula, CA) and anti-
2 macroglobulin from ICN (Costa Mesa,
CA). Peroxidase-conjugated rabbit anti-mouse IgG and goat anti-rabbit IgG
antibodies were from Dako (Carpinteria, CA) and chemiluminescence reagents
were from Amersham (Piscataway, NJ). Rhodamine-conjugated rabbit anti-mouse
IgG and fluorescein-conjugated goat anti-rabbit IgG were from Dako.
Capillary morphogenesis assay
Human umbilical vein endothelial cells (Clonetics, San Diego, CA) were
cultured as described (Maciag et al.,
1979). EC cultures in 3D
collagen matrices were performed as described (Davis and Camarillo,
1996; Salazar et al.,
1999) except that ECs
(passages 2-5) were seeded at 2x106 cells per ml of gel.
DNA microarray analysis
DNA microarray analysis (DeRisi et al.,
1997; Iyer et al.,
1999) was used to study
genomic-scale gene expression comparing four time points during capillary
morphogenesis. Total RNA was extracted (Chomczynski and Sacchi,
1987) from ECs in the collagen
gel, after collagenase treatment, using TRIzol reagent (Life Technologies,
Grand Island, NY) at 0, 8, 24 and 48 hour time points. Approximately 360 gels
for each time point were needed to obtain enough mRNA for this experiment.
Total RNA was passed twice through Oligotex beads to obtain mRNA (Qiagen). The
poly-A RNA was eluted in DEPC-H2O and sent to Incyte Genomics (St
Louis, MO), who performed the differential hybridization to a Unigem V chip
containing 7,075 genes comparing 0 hour with 8 hour, 24 hour and 48 hour RNA
samples. The data presented are ratios of hybridization between these time
points.
Reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA was used to create cDNA templates and was equalized between the
time points by spectrophotometry and formaldehyde agarose gel electrophoresis.
Total RNA (5 µg) was used for reverse transcription (Stratagene, La Jolla,
CA) to create random-primed cDNA at 0, 8, 24 and 48 hours of culture
progression. RT-PCR amplification parameters used were typically 94°C for
45 seconds, 60°C for 45 seconds, 72°C for 2 minutes; this was cycled
25 to 35 times, depending upon the gene, with a final extension at 72°C
for 5 minutes using an PTC-100 thermal cycler (MJ Research, Watertown, MA).
Primer sequences are shown in Table
1.
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Northern and western blot analyses
Northern blot analyses were performed using total RNA equalized by
spectrophotometry (3 µg per lane) from 0, 8, 24 and 48 hour time points as
described (Salazar et al.,
1999). Collagen gels were
removed from wells, placed directly into boiling SDS sample buffer and heated
to 100°C for 10 minutes, and stored at -20°C until use. Cell extracts
were run on standard SDS-PAGE gels or 7% Blattler SDS-PAGE gels (collagen IV,
laminin) (Blattler et al.,
1972), and blots were incubated
and developed as described previously (Salazar et al.,
1999).
Differential display analysis
Differential display protocols (GenHunter, Nashville, TN) were used to
identify genes that are differentially regulated during capillary
morphogenesis (Liang and Pardee,
1998; Martin and Pardee,
1999). A cDNA copy of the
total RNA was created using random primers from 0, 8, 24 and 48 hour time
points. This cDNA amplification was performed using combinations of three
downstream oligo dT primers, G-T11M, C-T11M and
A-T11M, and a series of random 10 mer upstream primers, AP-1 to
AP-80 (ten sets of eight). We used sets one and two of the random upstream
primers (AP-1 to AP-16) in combination with oligo dT downstream primers
(GenHunter), which represent about 20% of the total primer combinations. Forty
cycles of amplification, incorporating [
-33P]-dCTP, was used
to create the random fragments. The fragments were then run on a 6%
polyacrylamide sequencing gel using 1xTAE as the running buffer, and
resolved by autoradiography. Differentially expressed bands were excised,
boiled to extract DNA, and ethanol precipitated using glycogen as a carrier.
Individual fragments were then amplified using the appropriate differential
display primers appropriate for that band and purified for further use. DNA
fragments were TA cloned into pGEM-T-Easy (Promega, Madison, WI) and sequenced
by automated sequencing (Lonestar Laboratories, Houston, TX).
Endothelial cell morphogenic cDNA libraries
cDNA libraries were made using mRNA isolated from 8 and 24 hour cultures.
Library production required 1 mg of total RNA isolated from 13 ml of collagen
gel containing 2x106 cells/ml, which was aliquoted into 25
µl aliquots in 96-well A/2 microplates. After poly-A selection, first- and
second-strand cDNA synthesis was performed with oligo dT primers. The cDNA was
fractionated by size, and mass cloned into the ZAP-XR vector using the Uni-Zap
XR Strategene system. The cloning was performed unidirectionally, based on
opposing EcoRI and XhoI restriction sites at the 5'
and 3' ends, respectively. Each fraction was then packaged, and the
first fraction was used for amplification while the remaining fractions were
left unamplified using standard methodologies from Stratagene. These libraries
were screened using using 32P-labelled partial cDNAs to obtain
larger clones (Wahl and Berger,
1987).
Construction of recombinant adenoviruses
Recombinant adenoviral constructs were prepared essentially as described
(He et al., 1998). Full length
CMG-1 and CMG-2 were cloned into the GFP-N2 vector (Clontech) and were then
amplified as GFP fusion protein constructs and then further cloned into the
pAdShuttle-CMV vector. CMG-1 and CMG-2 were amplified and cloned into pEGFP-N2
(Clontech) using XhoI and BamHI and XhoI and
EcoRI restriction sites, respectively. The following primer sets were
used to amplify CMG-1 or CMG-2 inserts: CMG-1 5'
-AGCTCGAGACAATGGCCAGCAATCAC-3'; 5'
-AGGGATCCGGTTTCCGCTGGTGCTATG-3'. CMG-2 5'
-AGCTCGAGAGGATGGTGGCGGAGCGGT-3'; 5'
-AGGAATTCAGCAGTTAGCTCTTTCTC-3'.
For cloning of these cDNAs into the pShuttle-CMV vector,
BglII-XbaI and XhoI-XbaI were used for
CMG-1 and CMG-2, respectively. The common downstream primer 5'
-AGTCTAGATTATGATCTAGAGTCGCGGC-3' was used with the upstream primer
5' -AGAGATCTACAATGGCCAGCAATCAC-3' for CMG-1 and 5'
-AGCTCGAGAGGATGGTGGCGGAGCGGT-3' for CMG-2. These pShuttle-CMV clones
were then recombined with pAdEasy-1 and transfected into 293 cells to produce
recombinant viruses. The viruses were then amplified through three passages in
293 cells before use. Extracts of 293 cells infected with these viruses were
tested on SDS-PAGE gels and western blots with anti-GFP antibodies, showing
that the fusion proteins had a molecular weight indicative of intact CMG-1-GFP
and CMG-2-GFP fusion proteins. Endothelial cell monolayers were infected on
gelatin-coated coverslips for 4-5 hours in serum-free media and then this
media was replaced with complete growth media overnight. After 24 hours,
cultures were fixed with 3% paraformaldehyde and were either directly examined
by fluorescence microscopy or processed further for immunofluorescence
staining as described (Salazar et al.,
1999).
Recombinant CMG-2 production and extracellular matrix protein binding
assays
A portion of recombinant CMG-2 (residues 34-214) was produced in E.
coli as a recombinant His-tagged protein. A CMG-2 cDNA was
unidirectionally cloned into pQE30 through BamHI and HindIII
sites. Primers used to amplify CMG-2 were:
5'-AGGGATCCCAGGAGCAGCCCTCCTGC-3';
5'-AGAAGCTTAGAAGAATTAATTATTCC-3'. The recombinant protein was
purified using Ni/Cd-sepharose as described (Bayless and Davis,
2001) and approximately 3 mg of
protein was obtained from 400 ml of IPTG-induced bacteria. Control GFP was
produced as a His-tagged protein and purified in the same way. Both proteins
were adsorbed to plastic microwells at 10 µg/ml and, after detergent
blocking (0.1% Tween-Tris-saline, pH 7.5) for 30 minutes, biotinylated
extracellular matrix proteins were added (1 µg/ml) in 0.1%
Tween-Tris-saline containing 1% BSA for 1 hour. The biotinylated matrix
proteins were prepared as described (Davis and Camarillo,
1993). After washing, the
wells were further incubated with avidin-peroxidase at 1 µg/ml for 30
minutes in Tween-Trissaline-BSA and, after washing, were developed for
peroxidase activity and read at 490 nm.
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RESULTS |
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TopSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), was induced 11.1-fold
at 8 hours, the extracellular inorganic phosphate regulator, stanniocalcin
(Olsen et al., 1996), was
induced 9.8-fold at 24 hours, and the angiogenic regulator, angiopoietin-2
(Yancopoulos et al., 2000),
was induced 7.8-fold at 48 hours. By contrast, the EC-derived mesenchymal cell
growth factor, connective tissue growth factor, CTGF (Grotendorst,
1997), was downregulated
11.3-fold at 8 hours, the extracellular matrix gene, fibulin-3 (Giltay et al.,
1999), was downregulated
27.2-fold at 24 hours, and a member of the regulator of G-protein signaling
family, RGS5 (Seki et al.,
1998), was downregulated
34-fold at 48 hours.
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Based on the sampling of the genes represented in the microarray and additional gene expression data, the differentially regulated mRNAs appeared to fall primarily into one of five general patterns of gene expression (Fig. 2). These patterns of gene expression were confirmed for selected genes using semiquantitative RT-PCR (Fig. 3A), northern blot (Fig. 3B) and western blot analyses (Fig. 4). Patterns A-E illustrate observed trends in gene expression (Fig. 2A-E). Genes showing these patterns are listed to the right and become grouped into expression patterns, which may reflect functional importance at different stages of EC morphogenesis.
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Induction and role of basement membrane matrix and integrin genes
during endothelial cell morphogenesis in 3D collagen matrices
It is interesting that a series of genes related to basement membrane
synthesis and assembly are induced (pattern C), including the collagen type IV
1 and laminin 1 chains. These inductions were confirmed at the
protein level by marked induction of collagen type IV and laminin synthesis
and secretion during morphogenesis (Fig.
4A,C). In addition, collagen type IV deposits extracellularly
around developing capillary tubes (Fig.
4B). Also induced is the 1 integrin subunit, which forms a
heterodimer, 1ß1, with affinity for both type IV collagen and
laminin (Senger et al., 1997). The 2 integrin subunit, which forms the
heterodimer, 2ß1, is markedly upregulated (pattern B) and may
similarly contribute to the appropriate assembly of basement membrane matrix.
Blocking antibodies to the 2 integrin subunit, but not control blocking
antibodies to the 5 integrin subunit, cause complete collapse of
pre-existing tubes in the 3D collagen matrix environment (Davis et al.,
2001), suggesting a crucial
role for 2ß1 in the maintenance of forming capillary tubes. We
have previously shown that 2ß1 is required for the initiation of
lumen formation through EC intracellular vacuole formation and coalescence in
3D collagen matrices (Davis and Camarillo,
1996).
We have expanded this list of genes by performing differential expression
analysis of an additional set of extracellular matrix and integrin genes
(Fig. 5). As shown in
Fig. 5, the RT-PCR data
supports the gene array data showing inductions of the collagen type IV
1 chain, laminin 1 and the 2 and 1 integrin
subunits. Some of the genes examined show stable expression throughout the
time course, whereas others are differentially expressed. Such differentially
expressed genes include the laminin 4 subunit, thrombospondin-2, and
v integrin subunit, which are upregulated while the ß3 integrin
subunit, thrombospondin-1 and the interstitial matrix protein, fibronectin are
downregulated. Interestingly, differential expression was observed for the
1, 2, v and ß3 integrin subunits which are known to
regulate angiogenesis in vivo (Brooks et al., 1994; Senger et al., 1997). The
laminin 5 chain was expressed in a stable fashion but we have detected
little or no expression of the laminin 1 chain (not shown) during
morphogenesis. This suggests that two laminin isoforms, laminin-8 (4,
ß1, 1) and laminin-10 (5, ß1, 1), are
particularly relevant during this process. Recent studies support this
conclusion showing a role for the laminin-8 and -10 isoforms in endothelial
cell function (Miner et al.,
1998; Lefebvre et al.,
1999; Kortesmaa et al.,
2000).
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Other induced genes involved in basement membrane matrix assembly are
heparan sulfate deacetylase/sulfotransferase (i.e. an enzyme that regulates
the rate-limiting step in heparan sulfate synthesis) (Kakuta et al.,
1999), lysyl oxidase, lysyl
hydroxylase, 2-macroglobulin and melanoma-associated antigen
(Table 2;
Fig. 3;
Fig. 4). Lysyl oxidase and
lysyl hydroxylase contribute to matrix stability through protein-protein
crosslinking (Uzawa et al.,
1999), whereas
2-macroglobulin is a broad-spectrum proteinase inhibitor
with growth factor binding domains (Jensen,
1989; Borth,
1992; Gonias et al.,
2000).
2-macroglobulin may contribute to ECM stability by
inhibiting proteolysis and/or regulate EC morphogenesis by controlling growth
factor availability. It is markedly induced both at the level of mRNA
(Fig. 3A) and protein
(Fig. 4C). As shown in
Fig. 4C, two induced
2-macroglobulin bands are observed, which represent the
intact protein (upper band) and a proteolytically cleaved form (lower band)
that is consistent with proteolytic cleavage in its bait region (Jensen,
1989; Borth,
1992). Previously, we have
observed marked inductions of matrix metalloproteinase 1 (MMP-1), MMP-9,
MT1-MMP, and uPA during EC morphogenesis (Davis et al.,
2001) that may account for
this 2-macroglobulin cleavage. Melanoma-associated antigen
is a putative extracellular matrix protein with an RGD sequence and
peroxidase-like domains (Mitchell et al.,
2000)
(Fig. 3B). A highly homologous
Drosophila protein named peroxidasin contains peroxidase and
extracellular matrix motifs and has been found to incorporate into basement
membranes (Nelson et al.,
1994). Overall, these data
indicate that ECs are synthesizing basement membrane components such as
collagen type IV, laminin and heparan sulfate for the purpose of
differentiation during morphogenesis. By contrast, other ECM proteins such as
fibronectin, which are more typically expressed by mesenchymal cells, are
downregulated.
Blockade of collagen type IV synthesis interferes with endothelial
cell morphogenesis in 3D collagen matrices
To address a potential functional role for collagen type IV in EC
morphogenesis, cultures were treated with ethyl 3,4-dihydroxybenzoate (edb), a
prolyl hydroxylase inhibitor that blocks collagen synthesis (Sasaki et al.,
1987; Nandan et al.,
1990). As shown in
Fig. 6A and B, blockade of
collagen type IV induction prevents progression of tube formation.
Furthermore, edb blocks collagen type IV synthesis during capillary
morphogenesis (Fig. 6C).
Providing support for our findings are previous studies showing that blockade
of collagen or basement membrane synthesis can interfere with angiogenic
responses (Ingber and Folkman,
1988; Maragoudakis et al.,
1988; Iruela-Arispe et al.,
1991; Sephel et al.,
1996). The addition of edb to
proliferating ECs at the doses used in the above experiment on plastic
substrates had no toxic effect and previous investigators have shown no toxic
effect of edb on different cell types (Nandan et al.,
1990). These data show that
collagen type IV synthesis, which is upregulated by increases in the 1
collagen IV subunit mRNA, appears to be necessary for capillary tube formation
in collagen matrices. We are unable to rule out the possibility that other
triple-helical collagens are involved in morphogenesis, since edb can block
the synthesis of other collagen types as well.
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Upregulation of cytoskeletal proteins during endothelial cell
morphogenesis in 3D collagen matrices
Additional upregulated genes (pattern C) include cytoskeletal and
cytoskeletal regulatory proteins that mediate ECM-mediated signalling
pathways. These are myosin IC (Swanson et al.,
1999), myosin VI,
diaphanous-2, WASP-interacting protein (WIP), gelsolin and RhoGAP-5. WIP is
known to bind the actin regulatory protein, profilin (Anton et al.,
1998), a gene that shows a
similar expression pattern (Salazar et al.,
1999). We have previously
reported that gelsolin, VASP and profilin are coordinately upregulated during
capillary morphogenesis (Salazar et al.,
1999). It is interesting that
upregulation of myosin IC as well as coronins 2A and 2B
(Table 2) occur during EC
morphogenesis since they have been observed associated with phagosome
membranes (Morrissette et al.,
1999; Swanson et al.,
1999). We have previously
suggested that phagosomes show similarities with the intracellular vacuole
compartment that controls EC lumen formation (Davis and Camarillo,
1996; Bayless et al.,
2000).
Induction of genes associated with endothelial cell differentiation
and cholesterol biosynthesis occur during endothelial cell morphogenesis in 3D
collagen matrices
In addition to the induction of ECM-or cytoskeleton-related genes, there is
interesting evidence that EC differentiation genes are upregulated
(Table 2). Induced EC
differentiation markers include von Willebrand factor, angiotensin converting
enzyme (ACE), thrombomodulin, protein S and CD39 (an EC cell surface ecto
ATP/ADPase) (Saijonmaa et al.,
2000; Goepert et al., 2000).
These data suggest that phenotypic changes are occurring with increasing
expression of EC-specific genes. Other induced genes include HMG CoA
reductase, mevalonate decarboxylase, and mevalonate kinase, which are genes
associated with cholesterol biosynthesis (Olivier and Krisans,
2000). Interestingly, these
three enzymes have recently been found to associate with peroxisomes (Olivier
and Krisans, 2000). In
addition, CD26, a dipeptidyl peptidase that regulates hormone and chemokine
activities, is also upregulated (Mentlein,
1999). Additional induced
genes (pattern B) include ICAM-1, GARP (Ollendorff et al.,
1994), jagged-1 (Lindsell et
al., 1995), melanin
concentrating hormone (also identified by differential display) (Hawes et al.,
2000), the transcription
factor, egr-1 (Silvermann and Collins,
1999) and the proteinase
inhibitor, tissue factor pathway inhibitor-2 (TFPI-2) (Rao et al.,
1996)
(Table 2;
Fig. 3).
Regulated expression of growth factors, hormones and genes regulating
the JAK-STAT pathway during endothelial cell morphogenesis in 3D collagen
matrices
A series of hormones and growth factors are induced (pattern A) such as
stanniocalcin, PDGF-b, placental growth factor (P1GF) (Carmeliet and Jain,
2000; Conway et al.,
2001) and midkine, which
suggests that ECs produce a series of autocrine factors during morphogenic
events. Interestingly, a VEGF receptor with affinity for P1GF, VEGF-R1 (Park
et al., 1994), is upregulated
along with P1GF. In addition, signalling molecules in the JAK-STAT pathway are
induced including the common gp 130 subunit of a number of growth factor
receptors (e.g. oncostatin M, leukemia inhibitory factor, IL-6 receptors)
(Funamoto et al., 2000; Imada
and Leonard, 2000). Oncostatin
M and leukemia inhibitory factor have been reported to inhibit EC
proliferation (Takashima and Klagsbrun,
1996). In addition, JAK-1 and
the JAK-1 inhibitor, SOCS-1 are induced (Krebs and Hilton,
2000)
(Table 2). Interestingly, three
genes whose transcription is dependent on this pathway,
2-macroglobulin, protein S and angiotensin-converting
enzyme, are upregulated along with these genes, suggesting a functional
upregulation of the JAK-STAT signal transduction pathway (Hooper et al.,
1997; Zhang et al.,
1999b; Saijonmaa et al.,
2000). In addition, the
anti-apoptotic gene, Pim-1, which is a downstream target of STAT3 following gp
130 receptor ligation (Shirogane et al.,
1999), is upregulated
(Table 2). Two other
anti-apoptotic genes, bc12 (Nor et al.,
1999) and A20 (Lee et al.,
2000), were upregulated along
with Pim-1 (Table 2), whereas a
proapoptotic gene, Nip2, was downregulated (Brusadelli et al.,
2000)
(Table 4).
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Sprouty, a negative regulator of FGF-signalling and tyrosine kinase
receptors, is upregulated during endothelial cell morphogenesis in 3D collagen
matrices
Further upregulated genes include receptor tyrosine kinase ligands or
regulators of tyrosine kinases such as sprouty, angiopoietin-2, and IGF-2
(Pattern C). Interestingly, sprouty has previously been reported to inhibit
FGF-mediated signalling as well as other receptor tyrosine kinase-mediated
pathways (Reich et al., 1999;
Wong et al., 2000) and is
important in the regulation of EC and epithelial branching morphogenesis
(Hacohen et al., 1998; Metzger
and Krasnow, 1999; Lee et al.,
2001). In support of this
finding is the observation that the FGF-2 gene is markedly downregulated
(Fig. 3A) as well as syntenin
(Table 3), a cytoplasmic
regulator of the FGF-binding cell surface heparan sulfate proteoglycan,
syndecan (Grootjans et al.,
1997). These data suggest that
FGF-2-mediated signaling pathways are downregulated during EC morphogenesis.
Another notable downregulated growth factor related gene is frizzled-related
protein-1, which is a Wnt antagonist protein (Rattner et al.,
1997). Genes that are known to
be constitutively or heavily expressed by ECs such as caveolin-1,
endothelin-1, ICAM-2, CTGF and Cyr61 are downregulated at the 8 hour time
point, and then return to baseline or near baseline expression by 48 hours
(pattern E).
Marked downregulation of Id and RGS family genes during endothelial
cell morphogenesis in 3D collagen matrices
Additional downregulated genes (pattern D) include Id-1, 2 or 3, which are
inhibitory transcription factors of the helix-loophelix family (Norton,
2000). Recently, knockout mice
with combined defects in Id-1 and Id-3 were shown to have a defect in tumor
angiogenesis (Lyden et al.,
1999). Other downregulated
genes include RGS5, RGS4 and RGS2, which are members of the regulator of
G-protein signalling (RGS) family (Druey et al.,
1996; DeVries et al.,
2000) that inhibit G-protein
signalling through GTPase activating activity. Transglutaminase 2, which is
also referred to as G-protein Gh, is also downregulated (Vezza et
al., 1999; Zhang et al.,
1999a). Downregulation of RGS
genes suggests that activation of G-protein signaling pathways may be
occurring. Interestingly, this downregulation occurs during a period of
extensive branching morphogenesis in our system
(Fig. 1) and RGS proteins are
known to block cell migration by inhibiting G-protein-linked chemoattractant
receptors (Bowman et al.,
1998). Interestingly, a
farnesyl transferase enzyme that participates in the functional activation of
G-proteins by stimulating membrane targeting is induced during the time course
(Dietrich et al., 1996)
(Table 2).
Marked downregulation of cell cycle and ubiquitinproteasome genes
during endothelial cell morphogenesis in 3D collagen matrices
An interesting observation in the array analysis is that a large series of
genes associated with cell cycle regulation show expression changes. A series
of genes known to play a positive role in cell cycle progression such as
cyclin B2, cdc2, cyclin A1, cdc20, cdc25B, cdc46, mcm2, mad2, PRC-1, PCNA,
ran, thymidylate synthetase, transglutaminase-2 (Gh) and the Id
genes are downregulated (Nurse et al.,
1998; Jiang et al.,
1998; Tye,
1999; Heald and Weis,
2000; Howell et al.,
2000; Norton,
2000; Nilsson and Hoffmann,
2000; Rudner and Murray,
2000; Nigg,
2001)
(Table 3). In addition, a large
number of genes associated with ubiquitin-mediated protein degradation through
proteasomes are downregulated, which is consistent with this inhibition of the
cell cycle (Bounpheng et al.,
1999; Koepp et al.,
1999; Page and Hieter,
1999)
(Table 2). These data strongly
support the concept that inhibition of cell cycle progression of ECs is
occurring. It is interesting that many of the genes regulate the
G2-M checkpoint of the cell cycle by regulating the function of the
cdc2/cyclin B complex (Nurse et al.,
1998; Nigg,
2001). In support of this
conclusion is the additional finding that a cell cycle inhibitor, cdc14, a
phosphatase that inactivates cdc2 kinase (Vistin et al.,
1998), is upregulated
(Table 2). Also, the cell cycle
inhibitor, p16/INK4, which inhibits cyclin dependent kinase 4 (Serrano et al.,
1993), is upregulated
(Fig. 3A).
Isolation and identification of novel capillary morphogenesis genes
(CMGs) by differential display and cDNA library screening
Novel genes, whose messages were differentially regulated during EC
morphogenesis, were isolated using differential display and cDNA library
screening. We have termed the novel genes identified by these techniques,
capillary morphogenesis genes (CMGs), which are defined as novel genes that
are differentially expressed during the process of EC morphogenesis. A partial
list of the genes identified by this analysis are shown in
Table 4 with expression
patterns, sequence identities and functional significance, if known. A number
of known differentially regulated genes were identified in this analysis
including melanoma-associated antigen, TFPI-2, germinal center protein kinase
related kinase, melanin concentrating hormone, prothymosin-,
NADH-ubiquinone oxidoreductase-B12 subunit, sodium bicarbonate cotransporter-3
(Pushkin et al., 1999), NIP-2
(Brusadelli et al., 2000) and
Fte-1. Known functions for these genes are listed in
Table 4. Melanoma-associated
antigen (MG50) (Mitchell et al.,
2000) is markedly upregulated
(pattern C) during morphogenesis, whereas the plasmin and serine proteinase
inhibitor, TFPI-2, is strongly upregulated early in the time course (pattern
B). Interestingly, melanin concentrating hormone and another member of the
sodium bicarbonate cotransporter family (cotransporter-2 versus
cotransporter-3) were identified as being differentially regulated by DNA
microarray analysis.
To confirm the expression patterns for genes isolated by differential display analysis, RT-PCR, northern blot and western blot analyses were performed (Fig. 7). These results show that the CMGs and other genes are differentially expressed during EC morphogenesis. Note that we have currently identified the full length sequence of four novel genes, CMG-1, CMG-2, CMG-3 and CMG-4 and have small cDNA fragments of two additional genes (CMG-5 and CMG-6) that currently do not match the GenBank databases. The full-length sequences of CMG-3 and CMG-4 will be published elsewhere (A.M. et al., unpublished).
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A novel differentially expressed capillary morphogenesis gene, CMG-1,
contains coiled-coil domains and targets to an intracellular vesicular
compartment
Here, we present the full-length sequence of CMG-1, which encodes a
putative intracellular 65 kDa protein. The sequence shown in
Fig. 8A reveals a series of
coiled-coil domains (from residues 96 to 560) that, in other proteins, have
been reported to participate in protein-protein binding, protein
multimerization, vesicular fusion and other functions (Burkhard et al.,
2001). CMG-1 also contains
several consensus motifs for phosphorylation including two for tyrosine
phosphorylation at residues 96/97 and 572, one for cAMP/cGMP protein kinases
at residue 260, and multiple protein kinase C and casein kinase II sites.
Homology searches revealed the greatest similarity (24% identity, 46%
positives from residues 9-591) with a putative C. elegans protein,
C18H9.8. It also shows 24% identity from residues 104-592 (in the coiled coil
domain) with myosin heavy chain sequences from various species. The sequence
also matches human genome sequences and maps to human chromosome 9q.
Interestingly, the pattern of CMG-1 gene expression (pattern E) during EC
morphogenesis mirrors that of caveolin, and other major EC genes (Figs
2,
3). Both genes show a marked
downregulation at 8 hours of morphogenesis followed by a return to baseline by
48 hours. To examine the expression pattern of CMG-1 in adult versus fetal
human tissues, RT-PCR was performed (Fig.
8C). As shown in Fig.
8, the strongest expression was observed from adult and fetal
kidney cDNAs with detectable expression in adult heart, placenta, lung, liver
and pancreas. Minimal to no expression was observed in the adult brain or
skeletal muscle cDNA samples. Detectable expression was observed in fetal
skeletal muscle as well as fetal heart, lung and liver, while minimal to no
expression was observed from fetal brain, thymus and spleen cDNAs.
|
To reveal possible functions for CMG-1, a CMG-1-green fluorescent protein (GFP) chimera was constructed to assess where the protein targets intracellularly. Transfection of 293 epithelial tumor cells revealed targeting of the CMG-1 fusion protein to an intracellular vesicular compartment (Fig. 9A-C). To accomplish this experiment in human ECs, a recombinant adenovirus was constructed in the pAdEasy system carrying the CMG-1-GFP fusion protein. Infection of ECs resulted in an apparent intracellular distribution identical to that observed in 293 cells with targeting to multiple intracellular vesicles (data not shown). By contrast, control GFP distributes throughout 293 cells or ECs with a cytoplasmic staining pattern (Fig. 9D). Coimmunostaining of ECs expressing CMG-1-GFP by using antibodies to various known intracellular compartments such as endosomes, Weibel-Palade bodies, caveolae, mitochondria, Golgi apparatus (GM130) and lysosomes failed to reveal any colocalization. More work is necessary to identify this intracellular compartment.
|
A novel differentially expressed capillary morphogenesis gene, CMG-2,
contains a putative transmembrane domain, targets to the endoplasmic reticulum
and shows affinity for the basement membrane matrix proteins, collagen type IV
and laminin
Here, we also present the full-length sequence of a second novel gene,
CMG-2, which is markedly upregulated at 8 hours during EC morphogenesis, as
revealed by both RT-PCR and northern blots
(Fig. 8B). This gene reveals a
putative 45 kDa protein with a putative transmembrane segment and a potential
signal peptide (residues 1-33) (Fig.
8B). Polyclonal antibodies directed to recombinant CMG-2 were
prepared, affinity purified and probed on western blots of ECs undergoing
morphogenesis. As shown in Fig.
7C, induced protein bands migrating at the predicted size of 45
kDa are detected using this antibody. This antibody also specifically detects
CMG-2-GFP or CMG-2-myc epitope-tagged fusion proteins by immunoprecipitation
or immunoblotting (data not shown), demonstrating specificity for CMG-2. By
contrast, G3PDH or actin antibodies show stable expression during the time
course (not shown).
The CMG-2 gene maps to the human genome sequence and is located on
chromosome 4q. Using the PSORT II program, the protein was predicted to have a
44% probability of targeting to the endoplasmic reticulum membrane with lesser
probabilities to the Golgi apparatus or plasma membrane. Proximal to the
potential transmembrane segment, homology searches reveal a von Willebrand
Factor A domain (a matrix-binding domain) from residues 44-213. In addition,
WH-1 block homologies were detected to WASP, a cdc42-binding protein that
regulates the actin cytoskeleton (Anton et al.,
1998) (from residues 250-259
and 315-334). The human tissue distribution of CMG-2 was assessed by RT-PCR
(Fig. 8C). As shown in
Fig. 8C, CMG-2 was detected in
placenta but was not detected in the other adult or fetal tissues
examined.
To address where CMG-2 may target within ECs, the same approach described
above was performed using a recombinant adenovirus carrying a CMG-2-GFP fusion
protein. ECs were infected revealing that CMG-2-GFP primarily targets to
endoplasmic reticulum (ER) using fluorescence microscopy. This is shown in a
double staining experiment using the ER protein, Hsp47, which is a chaperone
protein for collagens type I and IV (Fig.
9E-G) (Clarke et al.,
1991; Hendershot and Bulleid,
2000; Nagai et al.,
2000). The staining pattern
does not overlap with a Golgi-specific marker (not shown). In addition, we
have observed CMG-2-GFP to be present within intracellular vesicles in some
cells, suggesting that it may be capable of cycling from the ER to
intracellular vesicles or that it can target to more than one compartment. We
have not yet observed targeting of CMG-2-GFP to the plasma membrane.
A 20 kDa portion of the CMG-2 protein with sequence homology to the Von Willebrand factor A domain was expressed in bacteria and tested for its ability to bind extracellular matrix proteins (Fig. 10A,B). The recombinantly expressed protein along with a control GFP recombinant protein were purified using their histidine tags. These proteins were adsorbed to plastic and were incubated with biotinylated collagen type IV, laminin, fibronectin, osteopontin and control albumin. As shown in Fig. 10C, the CMG-2 protein but not the control GFP protein showed strong binding to the basement membrane proteins, collagen type IV and laminin, but showed little or no binding affinity for the other ECM proteins. This data suggests that CMG-2 has affinity for matrix proteins, which implies a potential role in basement membrane matrix synthesis or assembly due to its localization within the endoplasmic reticulum of ECs.
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DISCUSSION |
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TopSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). A time course analysis
of EC morphogenesis during tube formation provides information on potentially
important genes that regulate this process. We have shown that dramatic
regulation of many genes occurs as tube formation progresses. Analysis of gene
expression revealed that jagged-1, stanniocalcin and angiopoietin-2 were the
most induced genes, whereas CTGF, fibulin-3 and RGS-5 were the most repressed
genes at 8, 24 and 48 hours, respectively. Previously, increased jagged
expression was observed during FGF-2-mediated EC morphogenesis in fibrin
matrices and was reported to inhibit invasion and tube formation, thus
implicating it as a negative regulator of the process (Zimrin et al.,
1996). A recent study
implicated a positive role for jagged-1 and its receptor, Notch-4 (which shows
a slight mRNA induction in our system) in EC morphogenesis (Uyttendaele et
al., 2000). Angiopoietin-2
has also been implicated in angiogenesis in a number of contexts (Yancopoulos
et al., 2000) and has
previously been reported to be produced by ECs (Mandriota and Pepper,
1998). It has been reported to
be an inhibitor of angiogenesis by competing with angiopoietin-1 binding to
tie-2, a receptor tyrosine kinase (Yancopoulos et al.,
2000). By contrast,
angiopoietin-2 may disrupt stable pericyte-EC interactions and initiate
angiogenesis along with VEGF (Holash et al.,
1999). Stanniocalcin, a
hormone that regulates extracellular inorganic phosphate concentrations in
vivo by affecting renal tubular reabsorption of phosphate, is known to be
upregulated in some cell types in response to elevations of extracellular
calcium (Olsen et al., 1996).
A recent report identified stanniocalcin as being upregulated during EC
morphogenesis and expressed by in situ hybridization in tumor-associated blood
vessels (Kahn et al., 2000).
In addition, stanniocalcin was found to be upregulated in terminally
differentiated neurons (Zhang et al.,
1998) and to protect neurons
from ischemic injury by decreasing the toxicity of calcium influx through
increased influx of phosphate ions (Zhang et al.,
2000). Stanniocalcin may
function similarly during EC morphogenesis to promote survival.
CTGF was originally described as an EC-derived growth factor for
mesenchymal cells and a modulator of fibroblast ECM production through
synergism with TGF-ß (Grotendorst,
1997). Recent reports show the
production of fibulin-3 by blood vessels and endothelial cells in vivo (Giltay
et al., 1999). Interestingly,
fibulin-3 was reported to bind to a growth factor, NOVH, which is in a family
of growth factors including CTGF and Cyr61 (Perbal et al.,
1999). Marked downregulation
(34-fold) of RGS-5 was observed during the time course. This gene, as well as
two others in the same family, RGS-4 and RGS-2, were also downregulated. These
genes accelerate GTPase activity of heterotrimeric G proteins, resulting in
inactivation of specific signalling pathways (DeVries et al.,
2000). Downregulation of these
genes may indicate that activation of G protein-mediated pathways may be
important during EC morphogenesis. RGS-5, and -4 have been reported to
interact with Gi family members, whereas RGS-2 and -4 also interact
with Gq (DeVries et al.,
2000). By contrast, RGS-3 was
modestly upregulated during the time course and has been reported to interact
with Gi and Gq family members and to inhibit
endothelin-1-mediated signalling (Dulin et al.,
1999).
Differential expression of basement membrane matrix and integrin
genes during EC morphogenesis in 3D collagen matrices
Our previous work and the work presented here strongly implicate the ECM-
integrin-cytoskeletal axis as being one of the key regulators of EC
morphogenesis in a 3D extracellular matrix. Previously, we have shown the
involvement of particular integrins in EC morphogenesis and the integrin(s)
involved are dictated by the ECM environment where morphogenesis occurs (Davis
and Camarillo, 1995; Davis and
Camarillo, 1996; Bayless et
al., 2000; Davis et al.,
2000). In addition, we have
previously shown that actin cytoskeletal regulatory proteins such as gelsolin,
vasodilator-stimulated phosphoprotein (VASP) and its binding partner,
profilin, are coordinately upregulated during EC morphogenesis (Salazar et
al., 1999). Here, both
2 and 1 integrin subunits were upregulated, and have affinity
for basement membrane proteins such as collagen type IV and laminin. In
addition, basement membrane matrix genes such as collagen type IV 1
chain, laminin 1 chain, laminin 4 chain and heparan sulfate
deacetylase/sulfotransferase were upregulated. The marked upregulation of
collagen type IV synthesis, which was observed during EC morphogenesis,
appears to play an important role in these events. Interference with collagen
type IV synthesis blocked EC morphogenesis. Further support of these
conclusions is the identification of a novel gene, CMG-2, which may
participate in these events. It is induced in a pattern similar to that of
2ß1 and possesses a von Willebrand factor-like A domain with
affinity for collagen type IV and laminin. Both collagen type IV and laminin
are markedly induced during EC morphogenesis and deposit around developing
tubes in a time-frame coincident with CMG-2 induction. The colocalization of
CMG-2 with Hsp47, a chaperone for collagen type IV synthesis (Hendershot and
Bulleid, 2000), within the
endoplasmic reticulum suggests that it participates in these events. More work
will need to be performed to investigate the role of CMG-2 in basement
membrane matrix synthesis/assembly and EC morphogenesis.
Interestingly, melanoma-associated antigen, which is markedly upregulated
during EC morphogenesis in a time-frame consistent with other basement
membrane-related molecules, may be a basement membrane matrix protein
(Mitchell et al., 2000). It
contains ECM-like domains and strong homology with Drosophila
peroxidasin, a protein that localizes to basement membranes (Nelson et al.,
1994). In addition, within the
melanoma antigen protein sequence, is encoded the complete sequence of IL-1
receptor antagonist, suggesting that it is the precursor protein for this
inhibitor of IL-1-mediated signalling (Mitchell et al.,
2000). Interestingly, the
peroxidase domains of the melanoma antigen protein may degrade extracellular
H2O2 and, along with IL-1 receptor antagonist protein
generation, might facilitate the development of EC quiescence during the
differentiation process (i.e. H2O2 and IL-1 are both EC
activators). It is also interesting that CD39, which degrades ATP and ADP
(Goepfert et al., 2000), and
CD26, which can inactivate biologically active peptides (Mentlein,
1999), are both markedly
upregulated in a similar fashion and might perform similar functions to
inhibit EC activation and stimulate EC quiescence.
A recent study comparing endothelial cell gene expression from tumor versus
normal colonic tissue in vivo also revealed many changes in extracellular
matrix genes (St Croix et al.,
2000). Interestingly, some of
the most prominently differentially regulated genes in tumor endothelium
compared with normal endothelium were the interstitial matrix genes, collagen
type I, 1 and 2 chains, collagen type III, 1 chain and
nidogen, a basement membrane matrix gene. This data implies that endothelial
cells may be undergoing an epithelial-mesenchymal transition during tumor
angiogenesis since the collagen proteins are more typically synthesized by
mesenchymal cells. By contrast, in our system, the ECs are inducing the
synthesis of basement membrane matrix molecules such as collagen type IV,
laminins and heparan sulfate, while they are downregulating the synthesis of
interstitial matrix proteins such as fibronectin.
Novel capillary morphogenesis genes (CMGs) are differentially
expressed during EC morphogenesis in 3D collagen matrices
One of the clear advantages of our published system (Davis and Camarillo,
1996) is its use for the
identification of differentially expressed known and novel genes in capillary
morphogenesis (Salazar et al.,
1999; Kahn et al.,
2000; Davis et al.,
2001). Other EC morphogenic
models have been used to study differential gene expression (Glienke et al.,
2000). Here, we have used a
combination of experimental approaches to screen large numbers of genes for
differential expression patterns. In addition, we present data showing our
initial characterization of a number of genes that were isolated using
differential display and cDNA library screening (see
Table 4 and
Fig. 7A).
Here, we report the full length sequences of CMG-1 and CMG-2, proteins with
coding sequences predicting proteins of 65 kDa and 45 kDa, respectively. CMG-1
is predicted to be intracellular and to contain a series of coiled-coil
domains involving 
500 amino acids of sequence. A CMG-1-GFP construct was
observed to target to an intracellular vesicular compartment
(Fig. 9A-C). Interestingly, it
has an expression pattern (pattern E; see Figs
2,
7) that mirrors that of
caveolin-1, endothelin-1 and ICAM-2. RT-PCR analysis of tissue expression
reveals its mRNA expression in a number of tissues, with the most abundant
being adult and fetal human kidney (Fig.
8C). CMG-2 contains a putative transmembrane domain and signal
peptide and was predicted to target to the endoplasmic reticulum, which was
confirmed using a CMG-2-GFP fusion protein vector
(Fig. 9E-G). As discussed
above, its affinity for basement membrane ECM proteins
(Fig. 10C) suggests a
potential role in basement membrane matrix synthesis and assembly in ECs
during morphogenesis. CMG-2 mRNA was detected in placenta and was essentially
undetectable in the other adult and fetal tissues examined
(Fig. 8C). Thu
