Cloning of a Unique Lipase from Endothelial Cells Extends the
Lipase Gene Family*
Ken-ichi
Hirata
,
Helén L.
Dichek§,
Joseph A.
Cioffi¶,
Sungshin Y.
Choi
,
Nicholas J.
Leeper
,
Leah
Quintana¶,
Gregory S.
Kronmal¶,
Allen D.
Cooper**
, and
Thomas
Quertermous

From the
Division of Cardiology, Stanford University
Medical School, Stanford, California, 94305, § Children's
Hospital Oakland Research Institute, Oakland, California 94609 and
Department of Pediatrics, University of California at San Francisco,
California 94143, ¶ Progenitor, Inc., Menlo Park, California,
94025,
Palo Alto Medical Foundation, Palo Alto, California,
94301, and ** Division of Gastroenterology, Stanford University Medical
School, Stanford, California, 94305
 |
ABSTRACT |
A new lipoprotein lipase-like gene has been
cloned from endothelial cells through a subtraction methodology aimed
at characterizing genes that are expressed with in vitro
differentiation of this cell type. The conceptual endothelial
cell-derived lipase protein contains 500 amino acids, including an
18-amino acid hydrophobic signal sequence, and is 44% identical to
lipoprotein lipase and 41% identical to hepatic lipase. Comparison of
primary sequence to that of lipoprotein and hepatic lipase reveals
conservation of the serine, aspartic acid, and histidine catalytic
residues as well as the 10 cysteine residues involved in disulfide bond formation. Expression was identified in cultured human umbilical vein
endothelial cells, human coronary artery endothelial cells, and murine
endothelial-like yolk sac cells by Northern blot. In addition, Northern
blot and in situ hybridization analysis revealed expression
of the endothelial-derived lipase in placenta, liver, lung, ovary,
thyroid gland, and testis. A c-Myc-tagged protein secreted from
transfected COS7 cells had phospholipase A1 activity but no
triglyceride lipase activity. Its tissue-restricted pattern of
expression and its ability to be expressed by endothelial cells, suggests that endothelial cell-derived lipase may have unique functions
in lipoprotein metabolism and in vascular disease.
 |
INTRODUCTION |
The process of angiogenesis, the formation of new blood vessels,
is central to physiological and pathophysiological conditions including
placental development, wound healing, diabetic retinopathy, and tumor
growth (1-3). Because of the great potential for treatment of human
disease through manipulation of angiogenesis, fundamental information
regarding the molecular pathways that regulate endothelial cell
differentiation is of central importance. To study the cellular and
molecular basis of angiogenesis, a number of in vitro models of vascular formation have been derived, whereby cultured endothelial cells produce a capillary-like network on various extracellular matrices. In particular, a tumor-derived basement membrane preparation composed primarily of laminin, collagen, and growth factors has been
shown to reproducibly induce human umbilical vein endothelial cells
(HUVEC)1 to form a
3-dimensional capillary network (4, 5).
Through their functional activities, the proteins encoded by the lipase
gene family are intimately linked to the endothelium. Lipoprotein
lipase (LPL) is synthesized by several different types of parenchymal
cells, including adipocytes, muscle cells, and macrophages. However,
LPL protein released by these cells translocates to functional binding
sites on the surface of vascular endothelial cells, where it
participates in lipoprotein metabolism and uptake (6). Hepatic lipase
(HL) is synthesized and secreted by hepatocytes where some protein is
bound to the cell surface. In addition, HL translocates from the
hepatocyte surface to the endothelial surfaces of liver sinusoids.
Despite the association of both LPL and HL with vascular endothelium,
extensive previous studies have failed to detect production of either
lipase in endothelial cells (7). The critical association between
lipids and vascular disease coupled with the central roles of LPL and
HL as modulators of lipid levels suggests an intimate relationship
between these enzymes and the vasculature (8-12). One of these
lipases, LPL, has been implicated in the genesis and progression of
atherosclerotic disease in the blood vessel wall through local
expression (13). Further information regarding the role of the
endothelial cell in lipid metabolism would thus be of great significance.
We have used the in vitro model of endothelial cell tube
formation on the basement membrane material Matrigel coupled with a
polymerase chain reaction-based suppression subtractive hybridization cloning strategy to identify genes that may be activated during vascular formation (14). One of these genes was found to encode a new
member of the lipase gene family, endothelial cell-derived lipase
(EDL). Despite a high sequence similarity to LPL, EDL was found to be
expressed in a distinct and complementary tissue-restricted fashion,
with high level expression in the liver, placenta, lung, and steroid
hormone-producing organs. The cloning and characterization of EDL
provides the first evidence for lipase production directly by the
endothelial cell. Production of a lipase in the vessel wall would
provide a mechanism for local regulation of lipolytic activity,
allowing the vessel to participate in lipid metabolic processes that
are related to atherosclerosis and other vascular diseases, as well as
those that are related to angiogenesis.
 |
EXPERIMENTAL PROCEDURES |
Cell Culture--
HUVEC and human coronary artery endothelial
cells were obtained from Clonetics, Inc. (San Diego, CA). Namalwa
(human B-cell malignancy), MOLT4 (human T-cell malignancy), JEG3 (human
choriocarcinoma cells), HeLa (human epithelial cell tumor), 143B (human
osteosarcoma cells), A549 (human lung epithelial cells), MEL (mouse
erythroleukemia cell line), NIH3T3 (mouse embryonic fibroblast cell
line), C2C12 (mouse myoblast cell line), RAW 264.7 (mouse
monocyte/macrophage cell line), HepG2 (human hepatoma cell line), Hepa
1-6 (mouse hepatoma cell line), and COS7 cells were obtained from
American Type Culture Collection (Manassa, VA). These cells were
cultured under recommended conditions. Yolk sac cells were derived and cultured as described previously (15, 16). To facilitate in vitro tube formation, HUVECs (3.9 × 104
cell/cm2) were plated onto Matrigel (Becton Dickinson,
Bedford, MA) at 480 µl/35-mm dish and incubated at 37 °C for
3 h. The medium was removed, the cells were rinsed three times
with cold phosphate-buffered saline, 2 ml of MatriSperse solution
(Becton Dickinson) was added to each 35 mm dish, and the cell/gel layer
was scraped into 50-ml conical tubes. An additional 2 ml of MatriSperse
solution was added to the dishes, which were then transferred to the
tubes, and the Matrigel dissolved at 4 °C for 1 h. Released
HUVEC were washed with phosphate-buffered saline three times and used
for isolation of mRNA with a MicroFast Track kit (Invitrogen, San Diego, CA).
Cloning by Suppression Subtractive Hybridization--
A
polymerase chain reaction-based cDNA subtraction methodology
employing reagents supplied in the polymerase chain reaction Select
cDNA Subtraction kit (CLONTECH, Palo Alto, CA),
was employed to identify genes preferentially expressed in tube-forming
endothelial cells (14). Tester DNA was derived from 2 µg of
tube-forming HUVEC poly(A) + RNA, and driver DNA was
derived from 2 µg of poly(A) + RNA from growth-arrested
HUVEC. Subtraction hybridization was performed according to the
manufacturer's instructions, and the products of secondary polymerase
chain reaction were evaluated by dideoxy sequencing. One cloned
cDNA fragment encoded a novel sequence and was used to screen a
HUVEC
gt10 cDNA library and a
gt11 5'-stretch mouse
11-day embryo cDNA library (CLONTECH) to
isolate clones encoding the entire open reading frame of the human and
mouse EDL transcripts.
Northern Blotting--
Poly(A)+ RNA was isolated
from a panel of human and mouse, adult and embryonic, tissues. The RNA
was preblotted to membranes for Northern blotting
(CLONTECH). For Northern blot analysis, 20 µg of
total RNA or 2 µg of poly(A)+ RNA was size-fractionated
on 1.3% agarose gels containing 2.2 M formaldehyde and
transferred to nylon membranes. These membranes were then hybridized
with 1 of 2 EDL probes, a 514-base pair human cDNA fragment or a
2272-base pair mouse cDNA fragment. The probes were labeled with
[32P]dCTP by random priming (Stratagene, La Jolla, CA)
and hybridized at 42 °C for 16-24 h in the presence of 48%
formamide and 10% dextran sulfate. After hybridization, the membranes
were washed at high stringency conditions: 65 °C in the presence of
2 × SSC (1×SSC = 0.15 M NaCl and 0.015 M sodium citrate) buffer and 0.5% SDS. Visualization was
achieved by exposure to Kodak Biomax MS film (Eastman Kodak Co.,
Rochester, NY).
In Situ Hybridization--
In situ hybridization slides were
generated from paraformaldehyde-fixed, paraffin-embedded mouse embryos
according to established methodology or were purchased from Novagen
(Madison, WI). A 611-base pair EcoRI mouse EDL cDNA
fragment encoding the carboxyl-terminal 52 amino acids and
3'-untranslated region was cloned into pBluescript KS(+). This fragment
was used for in vitro RNA probe transcription. Both
antisense and sense cRNA probes were labeled with
[35S]dUTP. Hybridization, washing, and probe detection
were performed as described by Hogan et al. (17). Once
developed, the sections were counterstained with nuclear fast red
(Zymed Laboratories Inc., South San Francisco, CA).
EDL Protein Expression and Functional Lipase Assays--
To
express a carboxyl-terminal c-Myc-tagged protein, we added an in-frame
DNA sequence to human EDL. The DNA sequence, which encodes a c-Myc
peptide tag (EQKLISEED), was added at the 3' end by polymerase chain
reaction. The following primers were utilized: 5'-GGCTCGAGCCACCATGAGCAACTCCGTTCCTCTGCTCTGT-3', and
5'-GGCTCGAGCTACAGATCTTCTTCAGAAATAAGTTTTTGTTCGGGAAGCTCCACAGTGGGACT-3'. This hybrid DNA fragment was then cloned into the eukaryotic
expression vector phbAPr-3-neo (kindly provided by Dr. L. Kedes,
University of Southern California, Los Angeles). Human EDL-c-Myc
expression constructs were subsequently transfected into COS7 cells
with LipofectAMINE (Life Technologies, Inc.). The cells were selected in the presence of 1000 µg/ml G418. Expression levels of human EDL
protein were determined by Western blotting using anti-c-Myc monoclonal
antibodies (Roche Molecular Biochemicals). Those clones that
synthesized c-Myc-tagged EDL proteins were selected for further study.
Negative control clones were randomly selected from a transfection with
the empty phbAPr-3-neo vector. The COS7 cells stably expressed EDL-c-Myc fusion protein (EDL-c-Myc/phbAPr-3-neo construct) or did not
express an exogenous protein (phbAPr-3-neo vector). To obtain the
c-Myc-tagged EDL protein, the COS7 cells were cultured in serum-free
DMEM with 2 units/ml heparin. After 36 h, the cells and
conditioned media were harvested. The medium was concentrated from 10 ml to 1 ml by Centricon 10 (Millipore, Bedford, MA), brought to 30%
final concentration glycerol, and stored at
80 °C. Cells were
harvested by scraping into lysis buffer (20 mM Tris-HCl, pH
7.4, 150 mM NaCl, 1% CHAPS, 10 mM EDTA, 10%
glycerol, 10 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride).
Triglyceride lipase activity were quantitated in triplicate using the
long chain triacylglycerol substrate [14C]triolein
activity assays (18). Phospholipase activity was measured using
dioleoylphosphatidylcholine liposomes, which were synthesized using a
modification of the triolein emulsion. Dioleoylphosphatidylcholine (20 mg/ml, Sigma) was used in place of egg yolk extract (18). Labeled
triolein was substituted with 1,2 di[1-14C]oleoyl-L-3-phosphatidylcholine
(Amersham Life Science) at an activity of 0.06 µCi/ml of substrate.
Substrate (200 µl) was added to 100 µl of medium from transfected
cells in a final volume of 330 µl (150 mM NaCl, 100 mM
Tris-HCl, pH 8.5, 2.5% bovine serum albumin, 2 units/ml heparin
(Elkins-Sinn, Cherry Hill, NJ) in the presence or absence of 10 µl of
human plasma as source of apoC-II) (19). The samples were incubated at
37 °C for 2 h followed by oleic acid extraction and
scintillation counting (20). The presence of functional catalytic
residues was established in an assay employing the water soluble
substrate p-nitrophenylbutyrate (21).
 |
RESULTS AND DISCUSSION |
Cloning and Sequence Analysis of EDL--
We performed subtractive
suppression hybridization to isolate genes preferentially expressed in
HUVEC undergoing tube formation on Matrigel, compared with
growth-arrested HUVEC in monolayers (14). Two cDNA fragments
isolated in this screen appeared to encode a novel lipoprotein
lipase-like gene and represented a gene that was documented by Northern
blot to be up-regulated in HUVEC in the early phase of tube formation
on Matrigel (Figs. 1 and
2). This novel lipase is also expressed
in cultured endothelial cells isolated from human coronary artery and
in murine cell lines derived from the primitive yolk sac that have
properties of endothelial progenitor cells (15, 16). Because there was
no prior evidence for a lipase produced within endothelial cells, this
new protein was named endothelial cell-derived
lipase (EDL). Through subsequent screening of a HUVEC
cDNA library and a mouse embryonic cDNA library, the entire
open reading frame of human and mouse EDL was determined.

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Fig. 1.
Primary sequence of human and murine
EDL. The deduced amino acid sequence of human EDL
(hEDL) and murine EDL (mEDL) proteins are aligned
and compared with human lipoprotein lipase (hLPL) and human
hepatic lipase (hHL). The mature EDL proteins have been
highly conserved in evolution, and there is significant sequence
similarity to the human LPL and HL sequences. Identical amino acids are
boxed, and conserved amino acid substitutions are
shaded. The signal peptide cleavage site of EDL and the
other lipases is indicated by a vertical arrow. Conserved
cysteines are indicated by dots, and the catalytic triad is
indicated by asterisks. Heparin binding clusters are
indicated by dashed boxes, and hydrophobic sequences are
indicated by dashed underlining.
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Fig. 2.
Northern blot analysis of EDL expression in
cultured cells. Murine and human cDNA fragments were employed
as 32P-labeled probes for hybridization to blots containing
total RNA or poly (A)+ RNA isolated from human and murine
cultured cells. For analysis of cell-specific expression of
EDL, total RNAs (20 µg) isolated from HUVEC, human
coronary artery endothelial cells (HCAEC), Namalwa (human
B-cell malignancy), MOLT4 (human T-cell malignancy), JEG3 (human
choriocarcinoma cells), HeLa (human epithelial cell tumor), 143B (human
osteosarcoma cells), A549 (human lung epithelial cell), MEL (mouse
erythroleukemia cell line), NIH3T3 (mouse embryonic fibroblast cell
line), C2C12 (mouse myoblast cell line), YSC (mouse yolk sac cells),
poly(A)+ RNA (2 µg) isolated from RAW 294.7 (mouse
monocyte/macrophage cell line), HepG2 (human hepatocyte cell line),
Hepa 1-6 (mouse hepatoma cell line) were used for Northern analysis.
For analysis of in vitro tube formation,
poly(A)+ RNA (2 µg) isolated from growth-arrested HUVEC
in monolayers or tube-forming HUVEC on Matrigel were used for Northern
analysis. A dominant band is observed at approximately 4.4 kilobases in
both the human and mouse samples. Bands of different size may represent
alternative spliced mRNAs from the EDL locus or transcripts from
different genes with significant nucleotide similarity. Message size
was determined by 28 S and 18 S ribosomal RNA. Cyph,
cyclophilin.
|
|
The open reading frames of both the human and mouse EDL sequences were
1500 nucleotides, encoding highly conserved proteins of 500 amino acids
(Fig. 1). A hydrophobicity analysis of the predicted amino acid
sequence revealed a hydrophobic leader peptide with a putative signal
cleavage site located 18 amino acids downstream of the translation
initiation site in both proteins (22). The mature human and mouse EDL
proteins thus consist of 482 amino acids. EDL shares sequence
similarity with the mammalian lipases, including 44% amino acid
identity to LPL and 41% amino acid identity to HL. Alignment with the
human LPL and HL amino acid sequences revealed conservation of the
catalytic residues serine (Ser-169), aspartic acid (Asp-193), and
histidine (His-274) as well as of the 10 cysteine residues involved in
disulfide bridge formation (23). Similarly, two stretches of
hydrophobic amino acids (163-172 and 272-281) that are adjacent to
the catalytic serine and histidine, respectively, and that presumably
are important for the interaction with lipid substrate are also
conserved. In addition, like LPL and HL, EDL possesses a lid consisting
of 19 amino acids. By analogy with the predicted three-dimensional
structures of LPL and HL, the EDL lid probably covers a catalytic
pocket and serves to confer substrate specificity for EDL (24, 25).
Because the EDL lid region has minimal sequence homology with the LPL
and HL lids, the EDL substrate may be different from those of other
mammalian lipases. Interestingly, the regions bordering the lid are
almost identical among EDL, LPL, and HL. In addition, alignment with the human LPL sequence indicates conservation of positively charged clusters involved in heparin binding (23). The corresponding clusters
in EDL include: cluster 1, Arg-327
Lys-329
Arg-330
Lys-333; cluster
2, Arg-312
Lys-313
Arg-315; cluster 3, Arg-188; and cluster 4, Lys-352
Arg-450
Lys-452-Lys-459). Finally, five potential
glycosylation sites are predicted by the presence of the universal
acceptor sequence Asn-Xaa-(Thr-Ser) at positions 80, 136, 393, 469, and 491. These glycosylation sites may modulate the heparin binding properties of EDL.
Expression Analysis--
The developmental-specific and
tissue-specific pattern of EDL expression was investigated by Northern
blotting and 35S-labeled cRNA in situ
hybridization. The tissue-specific expression pattern of EDL in the
adult is unique and complementary to that documented for LPL. LPL is
expressed in muscle tissues, adipose tissue, mammary gland, brain, and
macrophages, whereas EDL is expressed in placenta, liver, lung, testis,
thyroid, and corpus luteum of the ovary (6). This difference in
expression pattern suggests distinct and nonoverlapping functions for
these two lipases with similar primary sequence.
EDL expression in placenta was detected in both mouse and human by
Northern analysis and confirmed by in situ hybridization in
the mouse (Figs. 3 and
4). The signal in the 7-day embryonic mouse sample likely represents expression in the placenta, which was
included in the RNA extraction. Intense hybridization of the 35S-labeled cRNA in situ probe was detected in a
crescent shape in the placenta, consistent with labeling of trophoblast
cells. This very high level of EDL expression suggests that EDL may
play an important role in placental lipid metabolism. Fatty acids are required by the fetus, placenta, and fetal membranes for the synthesis of complex lipids such as phospholipids, triacylglycerols, and cholesterol esters. These lipids form cell membranes, are precursors for hormones, and may provide metabolic substrates. LPL hydrolyzes maternal very low density lipoprotein triglyceride to release free
fatty acid and diacylglycerol. The majority of these released fatty
acids are then transferred to the fetus by an unknown mechanism. There
is, however, evidence for placental phospholipid transfer involving
lipid breakdown and resynthesis (26). In addition, recent studies
suggest that cholesterol plays a crucial role in specific processes
during mammalian embryonic development, including modification of the
Hedgehog protein (27). By analogy with the hydrolytic action of HL,
which may facilitate transfer of lipoprotein-derived cholesterol to the
liver, the hydrolytic action of EDL may facilitate transfer of
lipoprotein-derived cholesterol to the feto-placental tissues (28).
Thus, EDL may facilitate the uptake of lipoprotein-derived lipids from
circulating maternal blood into the fetal membranes.

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Fig. 3.
Northern blot analysis of EDL expression in
tissue samples. Murine and human cDNA fragments were employed
as 32P-labeled probes for hybridization to blots containing
poly(A)+ RNA isolated from human and murine adult and
embryonic tissues. A dominant band is observed at approximately 4.4 kilobases in both the human and mouse samples. Bands of different size
may represent alternative spliced mRNAs from the EDL locus or
transcripts from different genes with significant nucleotide
similarity. Sizing is provided by an RNA ladder. Adr,
adrenal; Sm Intest, small intestine.
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Fig. 4.
In situ hybridization analysis of
EDL expression. 35S-Labeled cRNA sense and antisense
probes were hybridized to sections of murine embryos and adult tissues,
with only the antisense probes showing a signal above background.
Sections were photographed with brightfield (A,
C, E, G) and darkfield (B,
D, F, H) illumination. A
and B, murine embryo at 7.5 days of development shows an
intense signal over the placental tissue but no signal in the
developing embryo. C and D, low power view of the
lung reveals a punctate staining patter over the alveoli. E
and F, high power view of the lung, consistent with
expression by type II alveolar cells or macrophages. G and
H, mouse ovary shows an intense signal over corpus luteum.
Photographs: A, B, G, and H
at 4×; C and D at 10×, and E and
F at 20×.
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EDL mRNA was detected by Northern blot at high levels in mouse lung
tissue and low levels in human lung tissue. The punctate hybridization
pattern detected by in situ hybridization was not consistent
with alveolar type I epithelial cell or endothelial cell EDL expression
but was highly suggestive of EDL expression by macrophages or alveolar
type II epithelial cells (Fig. 4). Recent experiments have documented
EDL expression in differentiated RAW 264.7 cells, and EDL expression in
the spleen could represent macrophage expression (Figs. 2 and 3).
However, Northern blotting experiments with total RNA isolated from
primary mouse peritoneal macrophages have failed to detect EDL
transcripts, leaving unresolved the question of EDL expression in
macrophages in vivo. A role for EDL in alveolar type II
epithelial cells remains an intriguing possibility. These cells
synthesize surfactant, which maintains alveolar patency (29).
Disaturated phosphatidylcholine, which is synthesized from long chain
fatty acids, is the major lipid component of surfactant (30).
Surfactant synthesis is critically dependent on the availability of
free fatty acids. One source of these lipids may be very low density
lipoprotein-transported triglycerides, which are hydrolyzed locally by
lipases to free fatty acids (31). We propose that EDL may hydrolyze
lipoprotein-derived phospholipids and thereby play an integral role in
providing fatty acid or lysophospholipid substrates for surfactant
phospholipid synthesis by alveolar type II epithelial cells.
EDL expression was detected by Northern blot in testis and by in
situ hybridization in the corpus luteum of the ovary (Figs. 3 and
4). Expression in these tissues, in conjunction with the placenta,
raises the possibility that EDL might also play a role in the uptake of
lipoprotein-derived cholesterol into steroidogenic tissues to
synthesize steroid hormones. The main phospholipase function of HL (see
below) converts phospholipid-rich high density lipoprotein 2 to high
density lipoprotein 3. Hydrolysis of high density
lipoprotein 2 phospholipids may facilitate transfer of core
cholesterol esters to cells that synthesize steroid hormones (32). EDL
expression was absent in human samples from another steroid hormone
synthesizing tissue, the adrenal (Fig. 3). However, a weak
hybridization signal was detected in the mouse adrenal (data not
shown), suggesting that further study will be required to clarify
whether EDL is expressed in the adrenal. Surprisingly, there was a high
level of EDL expression identified in the thyroid. This endocrine organ
does not require lipid substrates for synthesis of thyroglobulin or
thyroid hormones. However, hydrolysis of lipoprotein-derived phospholipids by EDL may provide fatty acids for its energy needs.
In the liver, expression of EDL was detected by Northern blot in both
the adult mouse and human. EDL expression was detected in HepG2 cells
and Hepa 1-6 cells by Northern blotting, suggesting that EDL is
expressed at least in the hepatocyte of the liver (Fig. 2). In
situ studies in the adult mouse did not localize EDL expression to
a single cell type in the liver and could represent widespread
expression in this tissue (data not shown). Interestingly, a small
transcript was identified in the human embryonic liver and must
represent either an alternative spliced transcript from the EDL locus
or a transcript from a different homologous gene (Fig. 3). The role of
EDL in the liver, an organ which plays a central role in lipid
metabolism, warrants detailed study and careful comparison of the
activities of EDL and HL.
EDL message was detected in one of two mouse spleen samples, where it
may be expressed by macrophages. Further insights into the functional
role of EDL in this organ as well as in other tissues such as the
thyroid, heart, brain, and kidney will require in situ
hybridization experiments to localize expression to the cellular level.
In vitro studies with cultured cells provide compelling
evidence that endothelial cells can express EDL in a regulated fashion. Northern analysis of RNA isolated from HUVEC monolayers and HUVEC cultured on Matrigel verified that EDL mRNA was significantly increased in endothelial cells undergoing tube formation (Fig. 2).
Northern blotting revealed EDL was highly expressed in human coronary
artery endothelial cells (Fig. 2). In addition, an intense signal was
detected in yolk sac cells, which express a number of endothelial cell
markers and are felt to represent an in vitro model of
primitive endothelial cells (33). Expression by these cells supports a
potential role for EDL in embryonic vascular formation. EDL expression
was detected at moderate levels in vascular organs in the mid-gestation
human embryo and at low levels in the mouse embryo, consistent with a
vascular expression pattern (Fig. 3). Embryonic endothelial cell
expression of EDL was thus evaluated in a limited number of mouse
embryo sections. Unfortunately, these preliminary in situ
studies have not revealed embryonic endothelial cell expression (data
not shown). Whether EDL is expressed during vascular formation in the
embryo or in the adult in conditions of physiological or
pathophysiological angiogenesis will require further study.
Lipase Activity--
To characterize the functional activities of
EDL, and in particular, to determine its substrate specificity, we
expressed recombinant protein in eukaryotic cells. An expression
construct encoding a c-Myc-tagged human EDL fusion protein,
EDL-c-Myc/phbAPr-3-neo, was transfected into COS7 cells. By Western
blotting of cellular extracts and concentrated culture medium, it was
determined that greater than 95% of the EDL-c-Myc-tagged protein was
secreted into the supernatant (data not shown). The presence of
functional catalytic residues in EDL-c-Myc was investigated using the
water-soluble substrate p-nitrophenylbutyrate. The presence
of lipid-hydrolyzing triglyceride lipase activity was investigated
using the liposoluble substrate triolein. Because EDL demonstrates
highest sequence similarity with LPL, and LPL activity is enhanced in
the presence of apo-CII, assays were also conducted in the presence of
apoC-II. EDL hydrolyzed p-nitrophenylbutyrate (EDL-c-Myc,
11.79 ± 0.17 µmol p-nitrophenol/ml/h; vector alone,
2.76 ± 0.63 µmol p-nitrophenol/ml/h), demonstrating
a functional catalytic triad. However, EDL did not hydrolyze triolein
either in the presence or absence of apo-CII (both < 0.1 ± 0.01 µmol of free fatty acids/ml/h). These results suggest that EDL
does not recognize long chain triglycerides as substrates. To
investigate the phospholipase activities of EDL, we used
phosphatidylcholine labeled in the sn-1 position, because both HL and LPL exhibit phospholipase A1 activity. The EDL-c-Myc protein did show phopholipase activity, which was approximately twice
that of supernatant from vector alone-transfected cells (307 ± 25 versus 156 ± 34 nmol of free fatty acids/ml/h,
p < 0.00005, Table I).
Interestingly, this phospholipase A1 activity was partially inhibited
by apo-CII. Thus, this novel protein demonstrates functional as well as
structural characteristics of a lipase.
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Table I
Phospholipase activity of human endothelial cell lipase
Phospholipase activity was measured in the media of transfected COS7
cells.
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Summary and Implications--
Data presented here showing EDL
expression in a number of cultured endothelial cell lines and in a
single macrophage cell line suggest that EDL can be expressed in the
vessel wall. Such expression taken together with its structural and
functional similarities with HL and LPL, molecules known to influence
the atherosclerotic process, would suggest a role in vascular
pathophysiology. Specifically, EDL may modulate the atherosclerotic
process by facilitating cholesterol exchange between lipoproteins and
the vessel wall. In addition, as a local regulator of lipid metabolism,
EDL may support de novo angiogenesis by supplying fatty
acids for energy required for endothelial cell proliferation and migration.
 |
FOOTNOTES |
*
This work was supported in part by Grant KO8 HL 03865 01
(to H. L. D.) and a sponsored research agreement with Progenitor, Inc., Menlo Park, CA (to T. Q.).The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF118767 and AF118768.

To whom correspondence should be addressed: Div. of Cardiology,
Stanford University Medical School, Falk Bldg., 300 Pasteur Dr.,
Stanford, CA 94305. Tel.: 650-723-5013; Fax: 650-725-2178; E-mail:
tomq1{at}leland.stanford.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
HUVEC, human
umbilical vein endothelial cells;
apo, apolipoprotein;
EDL, endothelial
cell-derived lipase, HL, hepatic lipase;
LPL, lipoprotein lipase;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic
acid.
 |
REFERENCES |
-
Folkman, J.
(1995)
Nat. Med.
1,
27-31[Medline]
[Order article via Infotrieve]
-
Risau, W.
(1997)
Nature
386,
671-674[CrossRef][Medline]
[Order article via Infotrieve]
-
Yancopoulos, G. D.,
Klagsbrun, M.,
and Folkman, J.
(1998)
Cell
93,
661-664[Medline]
[Order article via Infotrieve]
-
Grant, D. S.,
Tashiro, K.,
Segui-Real, B.,
Yamada, Y.,
Martin, G. R.,
and Kleinman, H. K.
(1989)
Cell
58,
933-943[Medline]
[Order article via Infotrieve]
-
Kubota, Y.,
Kleinman, H. K.,
Martin, G. R.,
and Lawley, T. J.
(1988)
J. Cell Biol.
107,
1589-1598[Abstract]
-
Braun, J. E.,
and Severson, D. L.
(1992)
Biochem. J.
287,
337-347[Medline]
[Order article via Infotrieve]
-
Cryer, A.
(1987)
in
Lipoprotein Lipase (Borensztajn, J., ed), Evener Publisher, Chicago
-
Hide, W. A.,
Chan, L.,
and Li, W. H.
(1992)
J. Lipid Res.
33,
167-178[Abstract]
-
Goldberg, I. J.
(1996)
J. Lipid Res.
37,
693-707[Abstract]
-
Olivecrona, G.,
and Olivecrona, T.
(1995)
Curr. Opin. Lipidol.
6,
291-305[Medline]
[Order article via Infotrieve]
-
Appelbaum-Bowden, D.
(1995)
Curr. Opin. Lipidol.
6,
130-135[Medline]
[Order article via Infotrieve]
-
Santamarina-Fojo, S.,
Haudenschild, C.,
and Amar, M.
(1998)
Curr. Opin. Lipidol.
9,
211-219[CrossRef][Medline]
[Order article via Infotrieve]
-
O'Brien, K. D.,
Deeb, S. S.,
Ferguson, M.,
McDonald, T. O.,
Allen, M. D.,
Alpers, C. E.,
and Chait, A.
(1994)
Am. J. Pathol.
144,
538-548[Abstract]
-
Diatchenko, L.,
Lau, Y. F.,
Campbell, A. P.,
Chenchik, A.,
Moqadam, F.,
Huang, B.,
Lukyanov, S.,
Lukyanov, K.,
Gurskaya, N.,
Sverdlov, E. D.,
and Siebert, P. D.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
6025-6030[Abstract/Free Full Text]
-
Wei, Y.,
Quertermous, T.,
and Wagner, T. E.
(1995)
Stem Cells (Dayton)
13,
541-547[Abstract]
-
Corn, B. J.,
Reed, M. A.,
Dishong, S. L.,
Li, Y.,
and Wagner, T. E.
(1991)
Clin. Biotechnol.
3,
15-19
-
Hogan, B. L. M.,
Beddington, R.,
Costantini, F.,
and Lasy, E.
(1994)
Manipulating the Mouse Embryo, 2nd Ed., pp. 325-384, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
-
Iverius, P. H.,
and Brunzell, J. D.
(1985)
Am. J. Physiol.
249,
E107-E114[Abstract/Free Full Text]
-
Dichek, H. L.,
Parrott, C.,
Ronan, R.,
Brunzell, J. D.,
Brewer, H. B., Jr.,
and Santamarina-Fojo, S.
(1993)
J. Lipid Res.
34,
1393-1403[Abstract]
-
Belfrage, P.,
and Vaughan, M.
(1969)
J. Lipid Res.
10,
341-344[Abstract/Free Full Text]
-
Quinn, D. M.,
Shirai, K.,
Jackson, R. L.,
and Harmony, J. A.
(1982)
Biochemistry
21,
6872-6879[Medline]
[Order article via Infotrieve]
-
von Heijne, G.
(1987)
Sequence Analysis in Molecular Biology: Treasure Trove or Trivial Pursuit, p. 112, Academic Press, San Diego, CA
-
van Tilbeurgh, H.,
Roussel, A.,
Lalouel, J. M.,
and Cambillau, C.
(1994)
J. Biol. Chem.
269,
4626-4633[Abstract/Free Full Text]
-
Winkler, F. K.,
D'Arcy, A.,
and Hunziker, W.
(1990)
Nature
343,
771-774[CrossRef][Medline]
[Order article via Infotrieve]
-
Dugi, K. A.,
Dichek, H. L.,
and Santamarina-Fojo, S.
(1995)
J. Biol. Chem.
270,
25396-25401[Abstract/Free Full Text]
-
Biezenski, J. J.
(1969)
Am. J. Obstet. Gynecol.
104,
1177-1189[Medline]
[Order article via Infotrieve]
-
Farese, R. V., Jr.,
and Herz, J.
(1998)
Trends Genet.
14,
115-120[CrossRef][Medline]
[Order article via Infotrieve]
-
Rothblat, G. H.,
Mahlberg, F. H.,
Johnson, W. J.,
and Phillips, M. C.
(1992)
J. Lipid Res.
33,
1091-1097[Abstract]
-
Rooney, S. A.
(1985)
Am. Rev. Respir. Dis.
131,
439-460[Medline]
[Order article via Infotrieve]
-
Batenburg, J. J.
(1992)
Am. J. Physiol.
262,
L367-L385[Abstract/Free Full Text]
-
Mallampalli, R. K.,
Salome, R. G.,
Bowen, S. L.,
and Chappell, D. A.
(1997)
J. Clin. Invest.
99,
2020-2029[Abstract/Free Full Text]
-
Wang, N.,
Weng, W.,
Breslow, J. L.,
and Tall, A. R.
(1996)
J. Biol. Chem.
271,
21001-21004[Abstract/Free Full Text]
-
Hidai, C.,
Zupancic, T.,
Penta, K.,
Mikhail, A.,
Kawana, M.,
Quertermous, E. E.,
Aoka, Y.,
Fukagawa, M.,
Matsui, Y.,
Platika, D.,
Auerbach, R.,
Hogan, B. L. M.,
Snodgrass, R.,
and Quertermous, T.
(1998)
Genes Dev.
12,
21-33[Abstract/Free Full Text]
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