Am J Physiol Heart Circ Physiol 284: H215–H224, 2003.
First published September 26, 2002; 10.1152/ajpheart.01118.2001.
Epoxygenase-driven angiogenesis in human lung
microvascular endothelial cells
MEETHA MEDHORA,1 JOHN DANIELS,1 KAVITA MUNDEY,1 BEATE FISSLTHALER,3
RUDI BUSSE,3 ELIZABETH R. JACOBS,1,2 AND DAVID R. HARDER2
1
Division of Pulmonary and Critical Care, Department of Medicine and 2Department
of Physiology, Cardiovascular Center, Medical College of Wisconsin,
Milwaukee, Wisconsin 53226; and 3Klinikum der Johann Wolfgang Goethe Universtitat,
Institut fur Kardiovaskulare Physiologie, D-60590 Frankfurt am Main, Germany
Submitted 20 December 2001; accepted in final form 24 September 2002
cytochrome P-450 2C9; recombinant adenovirus
P-450 (CYP450) enzymes belong to one of
the largest and most complicated family of isozymes
with over 500 known members (47). Even though products of these proteins have been characterized in detail
by xenobiologists and steroid chemists, unraveling a
functional role for these enzymes at the molecular level
has posed a challenge to investigators in cardiovascular physiology. Pharmacological inhibitors for these
enzymes have clearly demonstrated that they play a
crucial role in maintaining vascular tone in humans
(16, 33, 34) as well as other species (25, 29, 38, 41) in
addition to mediating cell proliferation (4, 6, 7, 18, 22,
CYTOCHROME
Address for reprint requests and other correspondence: M. Medhora,
Cardiovascular Center, Medical College of Wisconsin, 8701 Watertown
Plank Rd., Milwaukee, WI 53226 (E-mail: [email protected]).
http://www.ajpheart.org
43) and resisting inflammation (37). Using a specific
inhibitor for CYP450 isoforms that metabolize arachidonic acid (AA) and an in vitro assay made up of rat
cerebral endothelial cells and astrocytes in coculture
(35, 53), we indirectly demonstrated that the epoxygenase enzymes may mediate angiogenesis. This result
has important implications for the treatment of a number of brain injuries and may be applicable to other
organs and tissue.
Molecular definition of CYP450 epoxygenases in endothelial and surrounding cells has made characterization of specific CYP450s possible in the vascular system. Our laboratory demonstrated that the rat
epoxygenase 2C11 mediates astrocyte-driven functional hyperemia in the brain by RT-PCR, DNA sequencing, and antisense oligonucleotides in conjunction with functional analysis (2, 17). Also, with the use
of a combination of RT-PCR and antisense oligonucleotides, the endothelium-derived hyperpolarizing factor
in porcine coronary arteries was identified as epoxyeicosatrienoic acids (EETs) synthesized by the enzyme
CYP2C34 (porcine)/2C8 (human) (10). Human umbilical vein endothelial cells (HUVEC) express a number
of CYP450 epoxygenases of which CYP2E1, CYP3A,
and CYP1A2 were not detected by Northern blotting
with radiolabeled probes but were found to be present
when examined by the sensitive RT-PCR method (8).
Other CYP450 families present in microvascular endothelial cells are 1A, 2C, and 2J (10, 20). Overexpression
of CYP2J2, another epoxygenase in human endothelial
cells, resulted in decreased cytokine-induced expression of vascular cell adhesion molecule-1 (37). Knockout mice with deleted CYP4A (an AA ⍀-hydroxylase)
demonstrated a hypertensive phenotype that was more
severe in males (21). However, molecular characterization of an enzyme that promotes angiogenesis remains
to be defined.
Many CYP450 isoforms are readily induced by pharmacological agents, including CYP2C8, which is consistently enhanced by ␤-naphthoflavone (10). In addition,
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.
0363-6135/03 $5.00 Copyright © 2003 the American Physiological Society
H215
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.4 on June 18, 2017
Medhora, Meetha, John Daniels, Kavita Mundey,
Beate Fisslthaler, Rudi Busse, Elizabeth R. Jacobs,
and David R. Harder. Epoxygenase-driven angiogenesis in
human lung microvascular endothelial cells. Am J Physiol
Heart Circ Physiol 284: H215–H224, 2003. First published
September 26, 2002; 10.1152/ajpheart.01118.2001.—Angiogenesis is one of the most recent physiological functions
attributed to products of cytochrome P-450 (CYP450)
enymes. To test this at a molecular level in human cells, we
used a cloned cDNA for the human endothelial enzyme
CYP450 2C9 (CYP2C9) to study growth as well as differentiation of human microvascular endothelial cells from the
lung (HMVEC-L). Using adenoviral vectors overexpressing
mRNA for CYP2C9, we show that the presence of CYP2C9
doubles thymidine incorporation and stimulates proliferation
of primary cultures of endothelial cells compared with Ad5GFP (control) in 24 h. In addition, there is a significant
increase of tube formation in Matrigel after infection of
HMVEC-L with Ad5-2C9 than with Ad5-GFP. More interestingly, Ad5-2C9 expressing the antisense product of CYP2C9
(2C9AS) inhibited tube formation compared with both Ad5GFP as well as the Ad5-2C9 constructs. Finally, we tested the
most abundant arachidonic acid metabolite of CYP2C9,
14,15-epoxyeicosatrienoic acid, which induced angiogenesis
in vivo when embedded in Matrigel plugs and implanted in
adult rats. These data support an important role for CYP2C9
in promoting angiogenesis.
H216
EPOXYGENASE-DRIVEN ANGIOGENESIS
METHODS
Growth and culture of HMVEC-L. The HMVEC-Ls were
purchased from Clonetics (Walkersville, MD) and maintained exactly as recommended by the manufacturer. Cells
were cultured with the EGM-2-MV bullet kit containing
growth factors that maintain the phenotype of the cells as
judged by acetylated LDL uptake, von Willebrand factor
staining, and surface platelet endothelial cell adhesion molecule (PECAM) expression. The cells are primary cultures
and will remain differentiated for up to 15 passages if maintained as described above. We did not use cells beyond passage 13 and substituted the basal media EBM (Clonetics) as
the serum-free medium (SFM) described in some experiments only in the final stages of the experiment. The cells
were grown in a tissue culture water-jacketed incubator
under 95% air-5% CO2 following Biosafety 2 levels for human
samples as well as recombinant adenovirus.
Construction of functionally expressing recombinant Ad52C9. The full-length coding sequence of 2C9 was restricted
with EcoRI, cloned into the EcoRI site of pAdTrack-CMV
(kindly provided by Bert Vogelstein, Ref. 19), and checked for
orientation by restriction mapping. This construct was recombined into the adenovirus genome by homologous recombination in Escherichia coli as described (19) at the Adenoviral Core Facility at the Medical College of Wisconsin, with
a few modifications. Briefly, the shuttle vectors carrying
CYP2C9 (sense orientation) was digested with PmeI and
dephosphorylated by incubation at 37°C for 30 min with 0.5
units of alkaline phosphatase (calf intestinal mucosa) obtained from Amersham Pharmacia Biotech (Piscataway, NJ).
The enzyme was inactivated by heating to 85°C for 15 min.
The linear DNA was purified by phenol-chloroform as described (19) and then gel purified using Quantum Prep
AJP-Heart Circ Physiol • VOL
Freeze’N Squeeze DNA Gel Extraction Column (Bio-Rad;
Hercules, CA). This fragment was transformed along with
pAdEasy-1 into E. coli BJ5183 and kanamycin (70 ␮g/ml)resistant colonies analyzed by restriction digestion with
PacI. Plasmids having inserts were transformed into E. coli
DH10B for large-scale isolation.
The recombinant plasmids were transfected into HEK 293
cells after linearization with PacI as described (19). Transfected cells were incubated for up to 2 wk and monitored for
GFP expression (for inserts in pAdTrack-CMV). The virus
was obtained from these cells and infected into fresh HEK
293 cells to increase the titer to be used and to check for
expression of 2C9 protein in infected cells.
Construction of Ad5-2C9 antisense (Ad5-2C9AS). The 2C9
cDNA (11) was subcloned in an adenovirus shuttle plasmid
pCA3 (obtained from Microbix Biosystems; Toronto, Canada)
by restriction to completion with EcoRI. The orientation of
recombinant shuttle plasmid pCA3-2C9AS (antisense) was
determined by restriction analysis. The shuttle plasmid with
the insert in antisense orientation was restricted with ClaI,
and the 2C9-containing fragment along with adjacent DNA
was purified from the gel. This fragment was ligated into the
ClaI-digested adenovirus helper dl 327 (12) to generate an
intact virus by ligation in vitro. Subsequent transfection in
the permissive host cell line HEK 293 generated plaques
containing recombinant virus that were tested for 2C9 sequence by PCR with 2C9-specific primers. The plaque was
purified by two rounds of subplaquing (13, 14, 27). This
resultant virus along with Ad5-2C9 (sense orientation) were
tested by Western and Northern blotting. Constructs that
expressed 2C9 sequence (detected by Northern blotting) were
also tested by Western blot analysis for CYP2C9 protein.
Only one orientation (sense) demonstrated increased 2C protein expression. One virus for each orientation of 2C cDNA
was selected for amplification to a large-scale preparation
with titer ⬎1010 plaque-forming units (pfu)/ml by replicating
the recombinant virus in HEK 293 cells and purifying the
virions through two cesium-chloride gradients (13, 14).
Growth of adenovirus. The Ad5-GFP, Ad5-2C9, and Ad52C9AS were grown, amplified (13, 14, 27), and purified at the
Adenoviral Core Facility at the Medical College of Wisconsin.
Each batch of virus was assayed for toxicity in a preliminary
experiment using a multiplicity of infection (MOI) of 50
viruses/cell (29).
Western blot. Endothelial cells were cultured in 35-mm
dishes to 80% confluency, washed, incubated with SFM ⫹
0.1% FBS for 6 h. Virions (MOI 1:50) were added, and after
24 h the cells were washed three times with PBS and the
proteins solubilized and extracted with 50 ␮l RIPA buffer [50
mM Tris (pH 8.0), 150 mM NaCl, 0.5% SDS, 1% Nonidet
P-40, 0.5% sodium deoxycholate, 1 mM EDTA, and 1⫻ protease inhibitor cocktail (Pharmingen; San Diego, CA)]. The
lysates were used to estimate their protein content with the
Bio-Rad DC Protein Assay Reagent. Equal amounts of protein (10 ␮g) from each sample were electrophoresed on a 7.5%
SDS polyacrylamide gel with running buffer as previously
described (2, 28). Microsomes from the rat liver (2.5 ␮g) were
included as a positive control for the antibody. The proteins
in the gel were transferred to nitrocellulose membranes as
described (2). The membrane was treated with a primary
antibody (1:5,000 dilution of goat anti-rat 2C13 antibody
from Gentest manufactured by Daichi Pure Chemicals) for
18 h and washed three times before incubating with a secondary antibody (1:2,500 dilution of anti-goat horseradish
peroxidase) for 45 min. The protein bands were developed
with chemiluminescence reagents, and the film was inspected for bands corresponding to the positive control (⬃55
284 • JANUARY 2003 •
www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.4 on June 18, 2017
2C8 and 2C9 are inhibited by the drug sulfaphenazole,
and these properties make 2C8 and 2C9 favored targets
for molecular analysis. Both isoforms have been cloned
and characterized (10, 11). Although epoxygenase isoforms such as members of the 1A family as well as 2J2
might be present in pulmonary endothelial cells, we
chose the epoxygenase 2C9, which is found in human
endothelial cells (9, 11) and has been described to metabolize AA to EETs (51), with the most abundant product
being 14,15-EET (51), as a candidate to test the angiogenic properties of epoxygenases. Because this EET profile of 2C9 matches that of the lung where 14,15-EET is
also the most abundant endogenous EET (52), we used it
in conjunction with primary cells in culture from human
lung microvascular endothelium (HMVEC-L) in a model
of in vitro tube formation. Pharmacological regulation of
the 2Cs will enhance the ability to follow up on results
obtained with these isoforms. To manipulate 2C9 levels
in HMVEC-L, we constructed a recombinant adenovirus
carrying the coding sequence of the enzyme in sense and
antisense orientations and used it along with a control
virus expressing the reporter green fluorescent protein
(GFP). We have previously been successful in using adenoviral vectors to deliver functional CYP450 epoxygenases to cells in culture as well as in whole animals (29,
31). This study supports a role for epoxygenase 2C9 in
mediating proliferation and tube formation of cultured
HMVEC-L in vitro. It also demonstrates that 14,15-EET,
a product of this enzyme, promotes angiogenesis in vivo.
EPOXYGENASE-DRIVEN ANGIOGENESIS
AJP-Heart Circ Physiol • VOL
each well was treated with 25 ␮l of assay solution (CellTiter
Aqueous One Solution from Promega; Madison, WI) for 2 h at
37°C. The absorbance in each well was measured at 490 nm
in a plate reader to determine the formazan produced by this
assay, which is proportional to the number of cells.
Morphogenesis assay (tube formation) in Matrigel. HMVEC-L cells were infected with Ad5-GFP, Ad5-2C9, and Ad52C9AS separately at MOI of 1:50 in SFM ⫹ 2% FBS for 24 h.
The cells were lifted and seeded at 4 ⫻ 104 cells/well into
four-well Lab-Tek II chamber slides (Nalge Nunc; Naperville,
IL) coated with Matrigel (Bectin Dickinson Labware; Bedford, MA). The coating of Matrigel was applied after diluting
the stock (1:1) with SFM on ice to a final protein concentration of ⬃5.5 mg/ml. Matrigel (250 ␮l) was applied per squared
centimeter of each well, and the matrix was allowed to gel for
30 min at 37°C before the addition of the cells, which were
pretreated for some experiments as mentioned, with vehicle
or epoxygenase inhibitors miconazole (10 ␮M) or N-methylsulfonyl-6-(2-propargyloxyphenyl) hexanamide (MS-PPOH,
20 ␮M, 45) for 30 min. The inhibitors remained in the
samples during the experiment. All the wells were examined
after an 18-h incubation period in the tissue culture incubator after which cells were carefully removed and mounted for
microscopy. Each well was then scanned under low power
(⫻10), and equal numbers (3–5/experiment) of fields with
maximal tube formation were focused and captured using an
Eclips 600 (Nikon) microscope with attached digital camera
and SPOT software. The images were printed at a constant
magnification (⫻300), and the length of the tubes were manually measured and summated to give the total length of
tubes formed per image. Tube formation was measured after
a total of three independent infections each containing multiple wells of the same viral construct.
Angiogenesis in vivo. The protocols (24, 40) were modified
as follows: Sprague-Dawley rats (6 wk old) were lightly
anesthetized with pentobarbital sodium (Nembutal, 50
mg/kg ip) and were injected subcutaneously with 0.7 ml
Matrigel premixed with vehicle (ethanol) or 14,15-EET (150
␮M). The injection was made rapidly with a 22-gauge needle
to ensure the entire content was delivered in one plug. Each
rat carried both a control and 14,15-EET-treated plug along
the dorsal midline. The rats were allowed to recover and 7
days later the animals were anesthetized (pentobarbital sodium, 60 mg/kg ip), and the Matrigel plugs were harvested
from under the skin. The plugs were homogenized in a
hypotonic lysis buffer (⬃250 ␮l of 0.1% Brij-35/plug) and
centrifuged for 5 min at 5,000 g. The supernatant was used in
duplicate to measure hemoglobin with Drabkin’s Reagent
(Sigma) along with a hemoglobin standard, as directed by the
manufacturer. Protein was assayed with Bio-Rad Protein
Assay Reagent and compared with a known standard assayed at the same time. A total of 10 plugs carrying vehicle
and control were assessed from 5 animals.
Cryosections (⬃10 ␮m) were made from the Matrigel plugs
infused with EETs (described above), which were frozen
rapidly on crushed dry ice. The sections were fixed in 4%
paraformaldehyde for 10 min on ice, rinsed three times with
PBS, and treated for 1 h at room temperature with PBS
containing 0.1% BSA. The slides were incubated for 12 h at
4°C in the same buffer containing anti-rat PECAM-1 (1:200
dilution, Ref. 42) in a humidified chamber. The antibody was
removed, and the slides were rinsed four times with PBS
followed by treatment with fluorescein isothiocyanate isomer-conjugated secondary antibody in PBS ⫹ 0.1% BSA for
1 h. The sections were rinsed five times with PBS and once
with filtered tap water before being mounted and viewed on
284 • JANUARY 2003 •
www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.4 on June 18, 2017
kDa molecular mass). The membrane was stripped, rinsed,
and redeveloped with anti-␤-actin monoclonal antibody
Ac-74 (A5316, Sigma; St. Louis, MO).
Northern analysis. HEK 293 cells were infected with Ad5GFP and Ad5-2C9AS for 48 h at a MOI of 10. Northern
blotting was carried out as described before (28, 32), and total
RNA was extracted from the cells using the TRIZol reagent
(GIBCO-BRL; Gaithersburg, MD) as specified by the manufacturer. Equal amounts of denatured RNA from each sample
(20 ␮g) were loaded on a 1% formaldehyde agarose gel as
described (28, 32) and electrophoresed in MOPS buffer (20
mM MOPS, pH 6.8, 5 mM sodium acetate, and 1 mM EDTA)
at 10 V/cm, visualized under UV light, documented in a
Vistra Fluorimager, and blotted onto Nytran Plus Membrane
(Schleicher and Schuell, Keene, NH) using a TurboBlotter
(Schleicher and Schuell). The blots were prehybridized at
42°C for 3 h with 5 ml of buffer A [formamide 50%, 5⫻ SSPE
(150 mM NaCl, 10 mM NaH2PO4, and 1 mM EDTA), 1⫻
Denhardt’s solution, 10% dextran sulfate, 0.1% SDS, and 100
␮g/ml denatured salmon sperm DNA] and probed overnight
at the same conditions with denatured labeled cDNA, which
was prepared as follows: the cloned EcoRI fragment of 2C9
cDNA was isolated after appropriate restriction and electrophoreses on a 1% agarose gel, and a single band of ⬃2 kb was
purified using Quantum Prep Freeze’N Squeeze DNA Gel
Extraction Column (Bio-Rad). This fragment was used as a
cDNA probe by labeling 25 ng of it with [32P]dCTP (3,000
Ci/mmol, NEN Life Science Products; Boston, MA) using
Ready-To-Go ␣-dCTP DNA-labeling beads (Amersham Pharmacia Biotech). The labeled product was separated from
unincorporated nucleotides by Microspin S-200 HR columns
(Amersham, Pharmacia Biotech) and over 106 counts/min
put in the prehybridization solution after the probe was
boiled for 5 min to denature it. After overnight hybridization,
the filters were washed three times at room temperature
with 2⫻ SSC (0.3 M NaCl and 0.03 M sodium citrate, pH
7.0)-0.1% SDS followed by two washes with 0.1⫻ SSC-0.5%
SDS at 65°C. They were exposed to X-ray film (Kodak XOmat AR) for autoradiography and developed in an automatic developer.
Thymidine incorporation. Primary cultures of HMVEC-Ls
were transferred into 24-well tissue culture clusters at a
density of 20,000 cells/well. After 24 h, the wells were infected with a control virus (Ad5-GFP) or Ad5-2C9 at a MOI of
50 in SFM ⫹ 2% FBS. After 18 h, [3H]thymidine (2 ␮Ci/well)
was added and allowed to incorporate for 4 h. The wells were
washed three times with PBS (Sigma) and treated with
ice-cold 15% trichloroacetic acid for 30 min at 0°C to precipitate the macromolecules. The trichloroacetic acid was removed, and the cells were washed twice with distilled water
and dried in a hood. Precipitates were solubilized with 0.5
ml/well of 1 N NaOH for 20 min at 37°C. The NaOH was
neutralized with an equal volume of HCl (1 M), and the
contents were quantitatively removed to scintillation tubes.
Scintillation fluid (2.5 ml/ml solution of Ecoscint from National Diagnostics; Atlanta, GA) was added to the samples,
which were counted for 5 min each. The means ⫾ SE were
computed using SigmaStat 2.0 software and analyzed statistically in a t-test. There was a significant difference (P ⬍
0.05) between cells expressing recombinant GFP alone versus those overexpressing GFP ⫹ 2C9.
Cell proliferation. The HMVEC-L cells were cultured in
24-well clusters (1 ⫻ 104 cells/well) until they were 80%
confluent (2 days). The medium was changed to SFM ⫹ 0.1%
FBS to starve the cells for 6 h before they were infected with
Ad5-GFP or Ad5-2C9 (5 ⫻ 105 pfu/well) (29). The cells were
growth arrested for 20 h in SFM ⫹ 0.1% FBS after which
H217
H218
EPOXYGENASE-DRIVEN ANGIOGENESIS
an Eclips 600 (Nikon) microscope with attachments for fluorescent imaging, digital photography, and SPOT software.
Statistical analysis. Pooled data obtained in each experiment were used to calculate the means ⫾ SE for control
(vehicle treated) or experimental groups (treated with adenovirus or pharmacological agents). The data were tested for
significance by a nonpaired Student’s t-test using Jandel
SigmaStat softwares. All experiments with P ⬍ 0.05 and
power of performed test above 0.8 were considered significant.
RESULTS
Fig. 1. A: Western blot for 2C proteins
derived from human microvascular endothelial cells from lungs (HMVECLs). Lane 1, uninfected cells (control);
lane 2, cells infected with Ad5-GFP;
lane 3, cell infected with Ad5-2C9; lane
4, cells infected with Ad5-2C9 antisense (Ad5-2C9AS); lane 5, male rat
liver microsomes (positive control);
lanes 6 and 7, overexposed images of
lanes 1 and 4, respectively. Note dark
band in cells infected with 2C9, which
is of comparable size to the 2C13 band
in rat liver microsomes. Last two lanes
depict overexposure of uninfected cells
(lane 6) and cells infected with Ad52C9AS (lane 7) to show expression of
endogenous 2C protein before and after expression of antisense 2C9. B:
Northern blot analysis demonstrating
expression of 2C9 mRNA sequence in
HEK 293 cells infected with Ad52C9AS. Note a strong band that is absent in Ad5-GFP-infected cells, which
does not get translated into protein as
seen in A, lanes 4 and 7.
AJP-Heart Circ Physiol • VOL
284 • JANUARY 2003 •
www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.4 on June 18, 2017
Expression of CYP2C9 by recombinant adenovirus,
Ad5-2C9, and Ad5-2C9AS. The cloned 2C9 cDNA sequence that has been tested for activity in a number of
vascular endothelial cells (9, 11) was used to generate
recombinant virus. Protein products expressed by the
recombinants were analyzed by Western blotting to
ensure that the sense construct expressed full-length
epoxygenase protein, whereas the antisense did not.
The results are shown in Fig. 1A. Uninfected cells
(HMVEC-L) and those infected with Ad5-GFP (control), Ad5-2C9, and Ad5-2C9AS were lysed, and equal
amounts of protein were examined with goat anti-rat
2C13 antibody, which also recognizes human 2C enzymes, including 2C8 and 2C9. Rat liver microsomes
were loaded as a positive control (Fig. 1A). A very
strong band that migrated with the positive control
was seen in the lane with proteins from the 2C9infected HMVEC-L cells, indicating a high level of
expression by Ad5-2C9. Much longer (15 times) exposures of the membrane revealed a band in uninfected
cells (Fig. 1A, lane 6) implicating endogenous expression of 2C proteins in HMVEC-L cells, which was
attenuated by overexpression of 2C9AS. To ensure that
the Ad5-2C9AS expressed the 2C9 antisense RNA, we
carried out a Northern blot with cultured human embryonic kidney cells that have been passaged extensively to downregulate endogenous epoxygenses. These
cells were infected with Ad5-GFP and Ad5-2C9AS, and
the total RNA from each was hybridized with 32Plabeled 2C9 cDNA probe. A strong band at the expected
position (with respect to 18S rRNA, Ref. 51) was seen
in the RNA of cells infected with Ad5-2C9AS (Fig. 1B),
which was absent in the control Ad5-GFP.
2C9-mediated increase in DNA synthesis in HMVEC-L. Epoxygenases have been reported to induce
mitogenesis and proliferation in a number of cells,
including rat cerebral microvascular endothelial cells
(35). Because endothelial cell growth is an important
requisite for angiogenesis, we were interested to document increased proliferation of HMVEC-L cells after
intracellular delivery of 2C9 cDNA. Incorporation of
[3H]thymidine was measured 18–24 h after introduction of recombinant 2C9 using the Ad5-2C9 construct
with Ad5-GFP as a control in HMVEC-L cells (Fig. 2B).
The infections routinely gave over 90% delivery of
transgenes as determined by fluorescence (Fig. 2A), in
which HMVEC-L cells were infected with Ad5-2C9 and
viewed under phase contrast (Fig. 2A,i) as well as
fluorescence (Fig. 2A,ii). As seen in Fig. 2, a majority of
the cells expressed the GFP marker on the vector
pAdEasy-Track (19) and were fluorescent. Figure 2B
demonstrates the results in which DNA synthesis was
stimulated significantly in the cells expressing 2C9.
2C9-mediated proliferation of HMVEC-L. To measure whether cell number was also increased by overexpression of 2C9, the cells were infected with Ad5GFP and Ad5-2C9 for 18 h before cell density was
assayed with the use of the proliferation assay (Cell-
EPOXYGENASE-DRIVEN ANGIOGENESIS
H219
Titer Aqeous One Solution from Promega). There was a
significant increase in cell proliferation (⬎25%) as
shown in Fig. 2C. Preliminary results (not shown) with
Ad5-2C9AS did not show much decrease of cell number
below the Ad5-GFP control, indicating that even in the
absence of expression of endogenous CYP2C proteins,
cell numbers were maintained.
Effect of CYP2C9 on HMVEC-L-tube formation in
vitro. One of the characteristics of microvascular cells
is to form tubes when embedded in matrices containing
extracellular proteins like fibrinogen, gelatin, or Matrigel. In our hands, HMVEC-Ls were potent participants in tube formation in Matrigel and began to align
and make cell-cell contact within an hour after being
seeded on a matrix at a density of 4 ⫻ 104 cells/cm2.
We, therefore, tested the effect of CYP2C9 on tube
formation by first infecting the cells with Ad5-expressing recombinant products, then plating them on Matrigel for up to 18 h, and summating the length of the
tubes formed in three to five defined fields of each well.
Our results (see Fig. 3A) show that Ad5-2C9 was able
AJP-Heart Circ Physiol • VOL
to increase the already robust tube formation of these
cells compared with Ad5-GFP-infected cells (Fig. 3A)
as measured in 30 fields. The antisense orientation,
however, decreased tube formation by one-half compared with GFP-expressing cells and by one-third compared with cells infected with Ad5-2C9. This result
indicated that endogenous epoxygenase activity was
important for tube formation, unlike the effect we
observed on maintenance of cell number (see previous
section) and prompted us to treat uninfected cells with
an epoxygenase inhibitor before tube formation. As
seen in Fig. 3B, the epoxygenase inhibitor miconazole
(10 ␮M) significantly decreased tube formation. We
repeated this assay with similar result using a more
specific epoxygenase inhibitor, MS-PPOH (45) (Fig.
3C). Results are summarized in Fig. 3D,i–iii, showing
all the cells participated in forming slender, contiguous
tubes after infection with Ad5-2C9 (see red arrow in
Fig. 3C,i), but in cells infected with Ad5-2C9AS (ii) or
treated with miconazole (iii), there were shorter tubes
surrounded by many cells that did not participate in
284 • JANUARY 2003 •
www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.4 on June 18, 2017
Fig. 2. A: phase-contrast (i) and fluorescent (ii) images of HMVEC-Ls after infection with Ad5-2C9. Note high
frequency of gene transfer shown by presence of green fluorescent cells, which were not visible in uninfected cells
or cells infected with Ad5 that does not code for GFP (not shown). B: graphical representation of increase in
[3H]thymidine incorporation (expressed as counts per minute, CPM) in HMVEC-Ls infected with Ad5-2C9 versus
Ad5-GFP. C: CYP2C9-mediated cell growth of HMVEC-L containing Ad5-GFP and Ad5-2C9. The optical density
(OD; represented on the y-axis) is proportionate to cell number, which was determined independently in a standard
curve for HMVEC-Ls by titering known amounts of cells and assaying with CellTiter Aqeous One Solution.
H220
EPOXYGENASE-DRIVEN ANGIOGENESIS
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.4 on June 18, 2017
Fig. 3. A: representation of relative
lengths of tubes formed by cells infected with recombinant adenovirus as
marked. Note inhibition of tube formation after infection with Ad5-2C9AS
compared with Ad5-GFP and Ad5-2C9.
B: ⬃2.5-fold decrease in tube formation
by uninfected HMVEC-Ls after treatment with the epoxygenase inhibitor
miconazole (10 ␮M). C: significant inhibition of tube formation in HMVEC-L
after treatment with epoxygenasespecific inhibitor N-methylsulfonyl-6-(2propargyloxyphenyl) hexanamide (MSPPOH, 20 ␮M). D: morphogenesis in
Matrigel is demonstrated by fields
viewed by phase microscopy showing
tubes formed after treatment with
Ad5-2C9 (i), Ad5-2C9AS (ii), and miconazole (iii). Note formation of long
tubes marked by red arrow in A compared with the picture of tubes and
nonparticipating cells (black arrows) in
ii and iii. Magnification: ⫻100.
the reaction (marked with black arrows). In fact, Fig.
3C,ii–iii, was captured from fields that show the best
tubes in the well, which are atypical and difficult to
find after infection with Ad5-2C9AS or treatment with
inhibitors. Although most known angiogenic factors
like VEGF and basic fibroblast growth factor are generally protein in nature, EETs is an example of a lipid
that induces morphogenesis of endothelial cells. Sphingosine-1-phosphate is another example and exogenous
application of 300 nM induced a five- to sixfold increase
in tube formation of HUVECs (26).
14,15-EET supports angiogenesis in vivo. In vitro
tube formation in Matrigel is not conclusive evidence of
angiogenesis, and angiogenic agents should be tested
AJP-Heart Circ Physiol • VOL
in vivo. We therefore embedded 14,15-EET (150 ␮M) in
Matrigel and injected this mixture along with a similar
plug-carrying vehicle. Both plugs were infused subcutaneously on the dorsal midline of a rat, and angiogenesis was measured a week later by retrieving the plugs
and assaying hemoglobin content in each. Paired comparisions of four experiments showed that plugs carrying 14,15-EETs contained 1.6 times more hemoglobin
than the vehicle-treated controls (Fig. 4A), despite the
known short half-life of EETs, confirming that EETs
are potent angiogens. Frozen sections of plugs were
stained with anti-PECAM antibody and examined microscopically to verify the presence of endothelial-related vascularization (Fig. 4B).
284 • JANUARY 2003 •
www.ajpheart.org
EPOXYGENASE-DRIVEN ANGIOGENESIS
DISCUSSION
Endothelial epoxygenases have earned increasing
recognition as vasoactive agents in resistance arterioles (4a, 12a, 36, 38, 44), including in human coronary vessels (33, 34). The most common isoforms in
microvascular endothelium, as detected by RT-PCR,
belong to the 2B, 2C, and 2J (10, 20) families, of which
2C8 has been identified as the EDHF. Other members
of the 2C subfamily that are classified on the basis of
sharing significant homology include 2C9, 2C10, and
2C19. The 2C gene family is recognized as one of the
most complex human cytochrome subfamilies, and its
members were among the first to be purified and characterized (50). The translated proteins of 2C9 and 2C10
differ by only two amino acids and may even represent
the same gene or allelic variant (51). Their catalytic
abilities are identical, and they are the most prevalent
2C isoforms in the kidney. Molecular cloning and subsequent characterization of the product of 2C10
showed 14,15-EET was the most abundant AA metobolite of this enzyme representing 52% of the total
EETs [the other products being 11,12-EET (30%), 8,9EET (17%) and 5,6-EET ⬍1%] (51). Interestingly, the
AJP-Heart Circ Physiol • VOL
endogenous 14,15-EET concentration is also the highest of all four EET isomers in the rabbit lung (52). We
therefore decided to use the cloned CYP2C9 enzyme
(11) to test our hypothesis of epoxygenase-mediated
angiogenesis in human microvascular endothelium derived from the lung.
In addition to EETs, cytochrome epoxygenases also
generate superoxide and related free radicals (3, 11),
which affect a number of cellular signaling cascades as
well as normal and pathological endothelial phenotypes. Thus a study of angiogenesis by application of
exogenous EETs alone or by using pharmacological
inhibitors with nonspecific actions would not be as
specific as using gene delivery of a single epoxygenase
coding sequence and its complimentary antisense. We
chose an adenoviral vector for gene delivery in preference to liposome-mediated transfer because primary
cultures of human endothelial cells are notoriously
resistant to transfection. The viral vectors in our hands
routinely give ⬎90% efficiency in gene transfer. Another advantage for use of viral vectors is that they can
be delivered to the lung for in vivo testing in future
studies. There are, however, potential disadvantages of
these vectors, the most obvious being the viral proteins
they carry in cis, which are not favorable to the host
cell. We always included Ad5-GFP as a control in our
experiments to negate this effect but realize this potential pitfall of viral vectors.
Our results showed a statistically significant stimulation in growth of HMVEC-L after 24 h of infection
with CYP2C9. It is possible that a larger effect may
have resulted if the viral genome was not delivered
along with the CYP2C9. For example, recombinant
human VEGF (2.5 ng/ml) has been reported to more
than triple the cell number in bovine adrenal cortexderived endothelial cells after 5–7 days (39). Although
growth of HMVEC-L by overexpressing 2C9 for 18 h
was much less, the cells continued to show growth after
18 h as measured by increase in thymidine incorporation. Epoxygenase products may not be as potent mitogens as other vascular growth factors like VEGF or
they may regulate multiple biochemical pathways after introduction in a viral vector, and the growth we
observed was a net result of the summation of these.
Although endothelial cell proliferation and tube formation are separate events, they are sequential in angiogenesis, and our aim was to verify whether CYP2C9
induced both events. The growth-promoting ability of
EETs depend on activation of a number of pathways,
including mitogen-activated protein kinases and protein kinase B (Akt) (5, 7). Variations in the mechanisms in different cell types may represent cross-talk
between EETs and other growth pathways. For example, epidermal growth factor increases EETs in proximal tubule cells which in turn modulates intracellular
Ca2⫹ concentration to trigger mitogenesis (4, 7). The
mechanisms of EET-induced proliferation of microvascular endothelial cells including possible interaction
with VEGF and/or its receptor remain to be explored.
Our next goal was to assess the role of CYP2C9 in
the differentiation of HMVEC-L. The results pointed to
284 • JANUARY 2003 •
www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.4 on June 18, 2017
Fig. 4. A: hemoglobin measured in vehicle-containing Matrigel plugs
versus those carrying 14,15-epoxyeicosatrienoic acid (EET) (150
␮M). B: immunohistochemical staining of a 10-␮m frozen section
from a Matrigel plug treated with EET and developed with antibody
against rat platelet endothelial cell adhesion molecule-1. Note invasion of the Matrigel by endothelial cells participating to form a
network. Magnification: approximately ⫻1,000.
H221
H222
EPOXYGENASE-DRIVEN ANGIOGENESIS
AJP-Heart Circ Physiol • VOL
vine aortic endothelial cells protected against hypoxiareoxygenation injury (49), increasing the list of epoxygenase-driven vascular homeostatic functions.
In conclusion, we have evidence showing angiogenic
properties of a human epoxygenase, CYP2C9. This is
the first report of a specific CYP450 isoform to induce
both growth and differentiation of microvascular endothelium and promote angiogenesis. Because epoxygenases are inducible (10), they can now be targeted experimentally to initiate angiogenesis in pathological
conditions. Also because of the multiple functions of
EETs, molecular characterization of different epoxygenases will help to choose feasible candidates for gene
therapy.
We acknowledge the technical expertise of Xinnan Niu for construction and testing of the recombinant adenovirus, Ryan P. McAndrew for assistance with the thymidine incorporation and angiogenesis in vivo, and Ying Gao and Dr. Daling Zhu for support and
assistance. We are indebted to Dr. Chenyang Zhang for valuable
direction with experiments for tube formation and angiogenesis in
vivo.
Financial support was from National Institutes of Health Grants
PO1 HL-59996 and RO1 NS-32321.
REFERENCES
1. Alkayed NJ, Goyagi T, Joh HD, Klaus J, Harder DR,
Traystman RJ, and Hurn PD. Neuroprotection and P450
2C11 upregulation after experimental transient ischemic attack.
Stroke 33: 1677–1684, 2002.
2. Alkayed NJ, Narayanan J, Gebremedhin D, Medhora M,
Roman RJ, and Harder DR. Molecular characterization of an
arachidonic acid epoxygenase in rat brain astrocytes. Stroke 27:
971–979, 1996.
3. Bondy SC and Naderi S. Contribution of hepatic cytochrome
P450 systems to the generation of reactive oxygen species. Biochem Pharmacol 48: 155–159, 1994.
4. Burns KD, Capdevila J, Wei S, Breyer MD, Homma T, and
Harris RC. Role of cytochrome P-450 epoxygenase metabolites
in EGF signaling in renal proximal tubule. Am J Physiol Cell
Physiol 269: C831–C840, 1995.
4a.Campbell WB, Gebremedhin D, Pratt PF, and Harder DR.
Identification of epoxyeicosatrienoic acids as endothelium-derived hyperpolarizing factors. Circ Res 78: 415–423, 1996.
5. Chen JK, Capdevila J, and Harris RC. Cytochrome P450
epoxygenase metabolism of arachidonic acid inhibits apoptosis.
Mol Cell Biol 21: 6322–6331, 2001.
6. Chen JK, Falck JR, Reddy KM, Capdevila J, and Harris
RC. Epoxyeicosatrienoic acids and their sulfonimide derivatives
stimulate tyrosine phosphorylation and induce mitogenesis in
renal epithelial cells. J Biol Chem 273: 29254–29261, 1998.
7. Chen JK, Wang DW, Falck JR, Capdevila J, and Harris
RC. Transfection of an active cytochrome P450 arachidonic acid
epoxygenase indicates that 14,15-epoxyeicosatrienoic acid functions as an intracellular second messenger in response to epidermal growth factor. J Biol Chem 274: 4764–4769, 1999.
8. Farin FM, Pohlman TH, and Omiecinski CJ. Expression of
cytochrome P450s and microsomal epoxide hydrolase in primary
cultures of human umbilical vein endothelial cells. Toxicol Appl
Pharmacol 124: 1–9, 1994.
9. Fisslthaler B, Hinsch N, Chataigneau T, Popp R, Kiss L,
Busse R, and Fleming I. Nifedipine increases cytochrome
P4502C expression and endothelium-derived hyperpolarizing
factor-mediated responses in coronary arteries. Hypertension 36:
270–275, 2000.
10. Fisslthaler B, Popp R, Kiss L, Potente M, Harder DR,
Fleming I, and Busse R. Cytochrome P450 2C is an EDHF
synthase in coronary arteries. Nature 401: 493–497, 1999.
284 • JANUARY 2003 •
www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.4 on June 18, 2017
a significant attenuation by Ad5-2C9AS, which expressed a full-length antisense RNA, with very close
homology to members of the 2C subfamily (2C8 and
2C9 have 85% homology at the nucleotide level). The
data point out the importance of low levels of endogenous 2C epoxygenases in tube formation. It was therefore not surprising that one AA metabolite of CYP2C9,
14,15-EET, enhanced angiogenenesis in vivo. The single dose of EET was sufficient to increase functional
vasculature in a Matrigel plug. We observed invasion
of the Matrigel by endothelial cells (staining positive
for PECAM-1) upon sectioning harvested plugs and
examining the sections under the microscope (Fig. 4B).
Our results show the hemoglobin content in the plugs,
which is a more measurable and accepted quantitation
of these data (26, 40), were increased by 14,15-EET.
The concentration of 14,15-EET we used in the Matrigel was three times higher than that previously reported in the lung (147 ng/g, see Ref. 52) because this
treatment was a bolus and because EETs are labile
compounds. This experiment is a simplistic approach
to replacing CYP2C9 with EET, because epoxygenases
catalyze multiple substrates to form numerous products. However, EETs can also be released independent
of de novo synthesis by epoxygenase. This was observed when EETs potentiated endothelial-dependent
relaxation by being released from preformed stores
(and not by action of epoxygenase, Ref. 46) that were
esterified into the membrane phospholipids. In fact,
the majority (⬎85%) of EETs synthesized by epoxygenases that are present endogenously in mammalian
tissues are incorporated in cellular glycerophospholipids (50) and released on stimulation with varying agonists.
Results demonstrating that overexpression of epoxygenase enhance growth and differentiation of endothelial cells have important implications in normal as well
as pathological microvascular physiology. Vasoactive
agents such as bradykinin stimulate release of EETs
from endothelial cells (23, 30), and this function may,
in addition to mediating vascular tone, also promote a
functional endothelium especially after damage in vascular disease. EETs have been shown to be neuroprotective after experimental transient ischemic attack,
and this could be via the growth and angiogenic effects
following ischemia. Similarly, EETs also mediate protection to ischemia-reperfusion injury in the heart (48).
In addition, CYP450 enzymes in the vasculature may
be regulated by constant ingestion of pharmacological
drugs or exposure to toxins making study of their
cardiovascular effects very significant.
Besides CYP2C other epoxygenases also contribute
to EET biosynthesis in vascular endothelium (reviewed
recently, Ref. 50), although the relative contributions
by CYP2C versus molecules like CYP2J remains unknown. Overexpression of CYP2J2 prevented leukocyte adhesion to the vascular wall by inhibiting NF-␬B
(37) and I␬B kinase. It also increased cAMP-mediated
tissue plasminogen activator promoter activity (33).
More recently, introduction of CYP2J2 in cultured bo-
EPOXYGENASE-DRIVEN ANGIOGENESIS
AJP-Heart Circ Physiol • VOL
29. Medhora M and Harder D. Functional role of epoxyeicosatrienoic acids and their production in astrocytes: approaches for
gene transfer and therapy. Int J Mol Med 2: 661–669, 1998.
30. Medhora M, Narayanan J, Harder D, and Maier KG. Identifying endothelium-derived hyperpolarizing factor: recent approaches to assay the role of epoxyeicosatrienoic acids. Jpn
J Pharmacol 86: 369–375, 2001.
31. Medhora MM, Okamoto H, Narayanan J, Zhang C, Niu X,
Taheri MR, Kraemer R, Capdevila J, and Harder D. Expression of recombinant cytochrome P450 epoxygenase in rat
brain. In: EDHF 2000, edited by Vanhoutte PM. London: Taylor
& Francis, 2001, chapt. 26, p. 228–238.
32. Medhora MM, Teitelbaum S, Chapel J, Alvarez J, Mimura
H, Ross FP, and Hruska KA. 1␣-25 hydroxyvitamin D3 upregulates expression of the osteoclast integrin ␣V␤3. J Biol
Chem 268: 1456–1461, 1993.
33. Miura H and Gutterman DD. Human coronary arteriolar
dilation to arachidonic acid depends on cytochrome P-450 monooxygenase and Ca2⫹-activated K⫹ channels. Circ Res 83: 501–
507, 1998.
34. Miura H, Wachtel RE, Liu Y, Loberiza FR Jr, Saito T,
Miura M, and Gutterman DD. Flow-induced dilation of human coronary arterioles: important role of Ca2⫹-activated K⫹
channels. Circulation 103: 1992–1998, 2001.
35. Munzenmaier DH and Harder DR. Cerebral microvascular
endothelial cell tube formation: role of astrocytic epoxyeicosatrienoic acid release. Am J Physiol Heart Circ Physiol 278:
H1163–H1167, 2000.
36. Nishikawa Y, Stepp DW, and Chillian WM. In vivo location
and mechanism of EDHF-mediated vasodilation in canine coronary microcirculation. Am J Physiol Heart Circ Physiol 277:
H1252–H1259, 1999.
37. Node K, Huo Y, Ruan X, Yang B, Spiecker M, Ley K, Zeldin
DC, and Liao JK. Anti-inflammatory properties of cytochrome
P450 epoxygenase-derived eicosanoids. Science 285: 1276–1279,
1999.
38. Oltman CL, Weintraub NL, VanRollins M, and Dellsperger
KC. Epoxyeicosatrienoic acids and dihydroxyeicosatrienoic acids
are potent vasodilators in the canine coronary microcirculation.
Circ Res 83: 932–939, 1998.
39. Park JE, Chen HH, Winer J, Houck KA, and Ferrara N.
Placenta growth factor. Potentiation of vascular endothelial
growth factor bioactivity, in vitro and in vivo, and high affinity
binding to Flt-1 but not to Flk-1/KDR. J Biol Chem 269: 25646–
25654, 1994.
40. Passaniti A, Taylor RM, Pili R, Guo Y, Long PV, Haney JA,
Pauly RR, Grant DS, and Martin GR. A simple, quantitative
method for assessing angiogenesis and antiangiogenic agents
using reconstituted basement membrane, heparin, and fibroblast growth factor. Lab Invest 67: 519–528, 1992.
41. Quilley J and McGiff JC. Is EDHF an epoxyeicosatrienoic
acid? Trends Pharmacol Sci 21: 121–124, 2000.
42. Sagawa K, Swaim W, Zhang J, Unsworth E, and Siraganian RP. Aggregation of the high affinity IgE receptor results in
the tyrosine phosphorylation of the surface adhesion protein
PECAM-1 (CD31). J Biol Chem 272: 13412–13418, 1997.
43. Sellmayer A, Uedelhoven WM, Weber PC, and Bonventre
JV. Endogenous non-cyclooxygenase metabolites of arachidonic
acid modulate growth and mRNA levels of immediate-early
response genes in rat mesangial cells. J Biol Chem 266: 3800–
3807, 1991.
44. Shimokawa H, Yasutake H, Fujii K, Owada MK, Nakaike
R, Fukumoto Y, Takayanagi T, Nagao T, Egashira K, Fujishima M, and Takeshita A. The importance of the hyperpolarizing mechanism increases as the vessel size decreases in
endothelium-dependent relaxations in rat mesenteric circulation. J Cardiovasc Pharmacol 28: 703–711, 1996.
45. Wang MH, Brand-Schieber E, Zand BA, Nguyen X, Falck
JR, Balu N, and Schwartzman ML. Cytochrome P450-derived
arachidonic acid metabolism in the rat kidney: characterization
of selective inhibitors. J Pharmacol Exp Ther 284: 966–973,
1998.
284 • JANUARY 2003 •
www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.4 on June 18, 2017
11. Fleming I, Michaelis UR, Bredenkötter D, Fisslthaler B,
Dehghani F, Brandes RP, and Busse R. Endothelium-derived hyperpolarizing factor synthase (cytochrome P450 2C9) is
a functionally significant source of reactive oxygen species in
coronary arteries. Circ Res 88: 44–51, 2001.
12. Foschi M, Chari S, Dunn MJ, and Sorokin A. Biphasic
activation of p21ras by endothelin-1 sequentially activates the
ERK cascade and phosphatidylinositol 3-kinase. EMBO J 16:
6439–6451, 1997.
12a.Gauthier KM, Deeter C, Krishna UM, Reddy YK, Bondlela
M, Falck JR, and Campbell WB. 14,15-epoxyeicsa-5(Z)-enoic
acid a selective epoxyeicosatrienoic acid antagonist that inhibits
endothelium-dependent hyperpolarization and relaxation in coronary arteries. Circ Res 90: 1028–1036, 2002.
13. Graham FL, Smiley J, Russel WC, and Nairn R. Characteristics of a human cell line transformed by DNA from human
adenovirus type 5. J Gen Virol 36: 59–74, 1977.
14. Graham FL and van der Eb AJ. A new technique for the assay
of infectivity of human adenovirus 5 DNA. Virology 52: 456–467,
1973.
15. Halcox JP, Narayanan S, Cramer-Joyce L, Mincemoyer R,
and Quyyumi AA. Characterization of endothelium-derived
hyperpolarizing factor in the human forearm microcirculation.
Am J Physiol Heart Circ Physiol 280: H2470–H2477, 2001.
16. Hamilton CA, Williams R, Pathi V, Berg G, McArthur K,
McPhaden AR, Reid JL, and Dominiczak AF. Pharmacological characterisation of endothelium-dependent relaxation in
human radial artery: comparison with internal thoracic artery.
Cardiovasc Res 42: 214–223, 1999.
17. Harder DR, Alkayed NJ, Lange AR, Gebremedhin D, and
Roman RJ. Functional hyperemia in the brain: hypothesis for
astrocyte-derived vasodilator metabolites. Stroke 29: 229–234,
1998.
18. Harris RC, Homma T, Jacobson HR, and Capdevila J.
Epoxyeicosatrienoic acids activate Na⫹/H⫹ exchange and are
mitogenic in cultured rat glomerular mesangial cells. J Cell
Physiol 144: 429–437, 1990.
19. He TC, Zhou S, da Costa LT, Yu J, Kinzler KW, and Vogelstein B. A simplified system for generating recombinant
adenoviruses. Proc Natl Acad Sci USA 95: 2509–2514, 1998.
20. Hoebel BG, Steyrer E, and Graier WF. Origin and function of
epoxyeicosatrienoic acids in vascular endothelial cells: more
than just endothelium-derived hyperpolarizing factor? Clin Exp
Pharmacol Physiol 25: 826–830, 1998.
21. Holla VR, Adas F, Imig JD, Zhao X, Price E Jr, Olsen N,
Kovacs WJ, Magnuson MA, Keeney DS, Breyer MD, Falck
JR, Waterman MR, and Capdevila JH. Alterations in the
regulation of androgen-sensitive CYP4a monooxygenases cause
hypertension. Proc Natl Acad Sci USA 98: 5211–5216, 2001.
22. Homma T, Zhang JY, Shimizu T, Prakash C, Blair IA, and
Harris RC. Cyclooxygenase-derived metabolites of 8,9-epoxyeicosatrienoic acid are potent mitogens for cultured rat glomerular
mesangial cells. Biochem Biophys Res Commun 191: 282–288,
1993.
23. Imig JD, Falck JR, Wei S, and Capdevila JH. Epoxygenase
metabolites contribute to nitric oxide-independent afferent arteriolar vasodilation in response to bradykinin. J Vasc Res 38:
247–255, 2001.
24. Ito Y, Iwamoto Y, Tanaka K, Okuyama K, and Sugioka Y.
A quantitative assay using basement membrane extracts to
study tumor angiogenesis in vivo. Int J Cancer 67: 148–152,
1996.
25. Jacobs ER and Zeldin DC. The lung HETEs (and EETs) up.
Am J Physiol Heart Circ Physiol 280: H1–H10, 2001.
26. Lee MJ, Thangada S, Claffey KP, Ancellin N, Liu CH, Kluk
M, Volpi M, Sha’afi RI, and Hla T. Vascular endothelial cell
adherens junction assembly and morphogenesis induced by
sphingosine-1-phosphate. Cell 99: 310–312, 1999.
27. McGrory WJ, Bautista DS, and Graham FL. A simple technique for the rescue of early region-I mutations into infectious
human adenovirus type 5. Virology 163: 614–617, 1988.
28. Medhora MM. Retinoic acid upregulates the ␤1-integrin in
vascular smooth muscle cells and alters adhesion to fibronectin.
Am J Physiol Heart Circ Physiol 279: H382–H387, 2000.
H223
H224
EPOXYGENASE-DRIVEN ANGIOGENESIS
46. Weintraub NL, Fang X, Kaduce TL, VanRollins M, Chatterjee P, and Spector AA. Potentiation of endothelium-dependent
relaxation by epoxyeicosatrienoic acids. Circ Res 81: 258–267, 1997.
47. Werck-Reichhart D and Feyereisen R. Cytochromes P450: a
success story. Genome Biol 1: 3003.1–3003.9, 2000.
48. Wu S, Chen W, Murphy E, Gabel S, Tomer KB, Foley J,
Steenbergen C, Falck JR, Moomaw CR, and Zeldin DC.
Molecular cloning, expression, and functional significance of a
cytochrome P450 highly expressed in rat heart myocytes. J Biol
Chem 272: 12551–12559, 1997.
49. Yang B, Graham L, Dikalov S, Mason RP, Falck JR, Liao JK,
and Zeldin DC. Overexpression of cytochrome P450 CYP2J2 protects against hypoxia-reoxygenation injury in cultured bovine aortic endothelial cells. Mol Pharmacol 60: 310–320, 2001.
50. Zeldin DC. Epoxygenase pathways of arachidonic acid metabolism. J Biol Chem 276, 36059–36062, 2001.
51. Zeldin DC, DuBois RN, Falck JR, and Capdevila JH. Molecular cloning, expression and characterization of an endogenous human cytochrome P450 arachidonic acid epoxygenase
isoform. Arch Biochem Biophys 322: 76–86, 1995.
52. Zeldin DC, Plitman JD, Kobayashi J, Miller RF, Snapper
JR, Falck JR, Szarek JL, Philpot RM, and Capdevila JH.
The rabbit pulmonary cytochrome P-450 arachidonic acid metabolic pathway: characterization and significance. J Clin Invest
95: 2150–2160, 1995.
53. Zhang CZ and Harder DR. Cerebral capillary endothelial cell
mitogenesis and morphogenesis induced by astrocytic-epoxyeicosatrienoic acid. Stroke. In press.
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.4 on June 18, 2017
AJP-Heart Circ Physiol • VOL
284 • JANUARY 2003 •
www.ajpheart.org