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Blood, Vol. 95 No. 7 (April 1), 2000:
pp. 2304-2311
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Departments of Radiation Oncology and Pathology, Wayne
State University School of Medicine, and the Karmanos Cancer Institute,
Detroit, MI.
Angiogenesis, the formation of new capillaries from preexisting
blood vessels, is a multistep, highly orchestrated process involving
vessel sprouting, endothelial cell migration, proliferation, tube
differentiation, and survival. Eicosanoids, arachidonic acid (AA)-derived metabolites, have potent biologic activities on vascular endothelial cells. Endothelial cells can synthesize various
eicosanoids, including the 12-lipoxygenase (LOX) product
12(S)-hydroxyeicosatetraenoic acid (HETE). Here we
demonstrate that endogenous 12-LOX is involved in endothelial cell
angiogenic responses. First, the 12-LOX inhibitor, N-benzyl-N-hydroxy-5-phenylpentanamide (BHPP), reduced endothelial cell
proliferation stimulated either by basic fibroblast growth factor
(bFGF) or by vascular endothelial growth factor (VEGF). Second,
12-LOX inhibitors blocked VEGF-induced endothelial cell migration, and this blockage could be partially reversed by the addition of 12(S)-HETE. Third, pretreatment of an angiogenic
endothelial cell line, RV-ECT, with BHPP significantly inhibited the
formation of tubelike/cordlike structures within Matrigel. Fourth,
overexpression of 12-LOX in the CD4 endothelial cell line significantly
stimulated cell migration and tube differentiation. In agreement with
the critical role of 12-LOX in endothelial cell angiogenic
responses in vitro, the 12-LOX inhibitor BHPP significantly
reduced bFGF-induced angiogenesis in vivo using a Matrigel implantation
bioassay. These findings demonstrate that AA metabolism in endothelial
cells, especially the 12-LOX pathway, plays a critical role in angiogenesis.
(Blood. 2000;95:2304-2311)
The formation of new capillaries from preexisting
vessels, a process termed angiogenesis, is tightly regulated in
physiologic processes such as embryonic development, wound repair, and
hypertrophy of normal organs. In contrast, persistent unregulated
angiogenesis underscores many pathologic conditions, such as tumor
growth and metastasis, diabetic retinopathy, atherosclerosis, and
chronic inflammation. Angiogenesis is a complex process involving an
extensive interplay between cells, soluble factors, and extracellular
matrix molecules that culminate in the proliferation, migration, and tube differentiation of endothelial cells.1 A plethora of
angiogenesis regulators such as vascular endothelial growth factor
(VEGF) and basic fibroblast growth factor (bFGF) can elicit various
angiogenic responses from endothelial cells.2 An
understanding of endothelial cell metabolism and the signaling that
underlies angiogenesis is important because it provides potential
therapeutic targets to inhibit or enhance angiogenesis.
12-lipoxygenases (12-LOX) are a family of isozymes that belong to the
LOX superfamily. These enzymes catalyze the stereospecific oxygenation
of arachidonic acid (AA) to form 12(S)-hydroperoxyeicosatetraenoic acid
(HPETE) and 12(S)-hydroxyeicosatetraenoic acid (HETE). At least 3 types
of 12-LOX have been well characterized: platelet-type, leukocyte-type,
and epidermal 12-LOX.3 Platelet-type 12-LOX exclusively
uses AA released from glycerophospholipid pools to synthesize 12(S)-HPETE and 12(S)-HETE, whereas leukocyte-type 12-LOX
can also synthesize 15(S)-HETE and 12(S)-HETE. In addition to
leukocytes and platelets, the expression of 12-LOX isozymes has been
detected in various types of cells, such as smooth muscle cells,4 keratinocytes,5 endothelial
cells,4,6 and tumor cells. Elevated 12-LOX activity has
been implicated in hypertension,7 vaso-occlusion in sickle
cell disease,8 inflammation,9
thrombosis,10 and mouse skin tumor
development.5 In human prostate carcinoma, the level of
12-LOX expression has been correlated with tumor stage.11
Along this line, we recently demonstrated that the overexpression of
platelet-type 12-LOX in human prostate cancer PC3 cells stimulated
tumor growth by elaborating tumor angiogenesis.12
In endothelial cells, it has been shown that 12-LOX activity is
required for serum- and bFGF-stimulated endothelial cell
proliferation13,14 and for minimally modified low-density
lipoprotein-induced monocyte binding to endothelial
cells.15 It has been shown that 12(S)-HETE can directly
stimulate endothelial cell mitogenesis,13,16
migration,12 and surface expression of
Inhibitors
Cell culture
Detection of 12-LOX expression by reverse
transcription-polymerase chain reaction
Immunoblot analysis of 12-LOX expression Semiconfluent confluent (70% to 80%) HUVEC, HMVEC, RV-EC, RV-ECT, and CD4 endothelial cells were rinsed with ice-cold PBS, scraped into lysis buffer containing 20 mmol/L Tris-HCl, pH 7.5, 2 mmol/L EDTA, 0.5 mmol/L EGTA, 0.5 mmol/L phenylmethylsulfonyl fluoride, 0.5 mmol/L leupeptin, 0.15 mmol/L pepstatin A, 1 mmol/L dithiothreitol, and 1% NP-40. Protein concentration was measured using BCA protein assay kit (Pierce, Rockford, IL). Human platelet lysates (10-40 ng) or human epidermoid carcinoma A431 cell lysates (30 µg) were used for positive control. Cell lysates (80 µg) from each sample were loaded onto a minigel for electrophoresis separation. The proteins in the gel were then transferred onto a polyvinylidene difluoride membrane and processed for immunodetection using a rabbit polyclonal antibody to human platelet-type 12-LOX obtained from Oxford Biomedical Research (Oxford, MI). This antibody reacts strongly with platelet-type 12-LOX from various species, with slight cross-reactivity with 5-LOX and 15-LOX at higher concentrations. Horseradish peroxidase-conjugated goat antirabbit IgG antibodies and enhanced chemiluminescent reagent was purchased from Amersham (Arlington Heights, IL).Measurement of 12(S)-HETE levels by enzyme immunoassay To measure 12(S)-HETE synthesis in cell culture, RV-ECT cells (4 × 106 cells) were plated and grown to 80% to 90% confluence in DMEM supplemented with 10% FBS and then serum starved overnight in serum-free DMEM. Fresh serum-free DMEM with 1 µmol/L arachidonic acid was added 1 hour before VEGF treatment. BHPP was added to a final concentration of 10 µmol/L 30 minutes before VEGF treatment. Recombinant human VEGF was added in a final concentration of 10 ng/mL. After 30 minutes of treatment, cells were washed in PBS once and harvested using cell scrapers. After centrifugation, the cell pellets were resuspended in cell lysis buffer and sonicated. Lipids were extracted from cell lysates by adding ethanol to a final concentration of 15%. After centrifugation at 375g for 10 minutes at 4°C, the supernatants were acidified to pH 3.5 with 3% formic acid and applied to BAKERBOND spe Octadecyl (C18) columns (J.T. Baker, Phillipsburg, NJ). After washing with ddH2O, 15% ethanol, and petroleum ether, lipids were eluted with ethyl acetate and dried under N2 gas, and 12(S)-HETE levels were measured using an EIA kit according to the manufacturer's instructions (Assay Designs, Ann Arbor, MI). To measure 12(S)-HETE levels in Matrigel (Becton Dickinson, Bedford, MA) implants, resected Matrigel plugs were homogenized in cell lysis buffer and processed for lipid extraction and 12(S)-HETE measurement.Stable transfection of CD4 endothelial cells and characterization Semiconfluent CD4 endothelial cells were transfected with a pcDNA 3.1 expression construct containing human platelet-type 12-lipoxygenase cDNA, which was a gift from Dr Colin Funk (University of Pennsylvania). Empty vector was used as a control. Transfection was performed using lipofectin reagent (Life Technologies). Transfectants were selected using 300 µg/mL geneticin (G418) in DMEM with 10% FBS. The expression of 12-LOX in CD4 transfectants was characterized by reverse transcription (RT)-PCR and Northern blot analysis for mRNA expression and by immunoblot for 12-LOX protein expression.Endothelial cell proliferation assay HUVEC cells were used to study the effects of 12-LOX inhibitors on endothelial cell proliferation. Cells were harvested by trypsinization, resuspended in EGM-2, and plated in a 96-well plate at 2000 cells per well. After overnight incubation, the media were changed to fresh EBM-2 with 1% FBS and treated with recombinant human VEGF-A or bFGF (R&D Systems, Minneapolis, MN) plus various amounts of BHPP. The concentrations of BHPP used were 0, 1, 10, and 50 µmol/L unless otherwise indicated. The final concentration of recombinant human VEGF and bFGF was 10 ng/mL. After 2 days, the plates were processed to quantitate the number of cells using an MTS cell proliferation assay kit (Promega, Madison, WI). The absorbance at 490 nm (A490) indicated the relative number of cells.Endothelial cell migration assay RV-ECT endothelial cell migration assay was performed essentially as previously described.12 VEGF (10 ng/mL), bFGF (10 ng/mL), or various other treatments were placed in the lower chamber. For each treatment, at least 3 chambers were used unless otherwise indicated. The migration assay for CD4 12-LOX transfectants was conducted in a similar way except that the number of cells seeded was 1 × 105 per chamber. The migrated cells were counted by a person unaware of the treatment regimen (blinded approach).Endothelial cell tube/cord formation assay Cord-forming RV-ECT cells or CD4 transfectants (1 × 105) were plated on a 24-well plate precoated with a thin layer of Matrigel (Becton Dickinson). After overnight incubation, BHPP was added. After 24 hours of treatment, the media were removed and the confluent monolayer was overlaid with 0.5 mL diluted Matrigel (Becton Dickinson; final concentration, 5 mg/mL). After solidifying at 37°C, 0.5 mL DMEM-10% FBS medium was carefully added without disturbing the gel. The formation of a tubelike structure was monitored microscopically every 6 hours and recorded.Matrigel implantation assay for angiogenesis The Matrigel (Becton Dickinson) implantation assay was performed as described by Ito et al28 with the following modifications. Matrigel 0.4 mL premixed with bFGF (5 µg/ml) with and without BHPP (0.9 mg/ml) was injected subcutaneously into nude mice (4 mice/group). Mice were killed 5 days after injection and dissected to expose the implants for recording using an SP SZ-4060 stereomicroscope (Olympus America, Melville, NY). The amount of blood retained in the Matrigel was further assessed by measuring the hemoglobin levels using Drabkin's reagent (Sigma Diagnostics, St. Louis, MO).
12-Lipoxygenase expression and activity in endothelial cells Several studies have provided evidence that endothelial cells synthesize various lipoxygenase products such as 5(S)-HETE, 12(S)-HETE, and 15(S)-HETE.16 The expression of platelet-type 12-LOX in HUVEC cells was previously detected by RT-PCR.6 Using primers selective for platelet-type 12-LOX, the expression of 12-LOX mRNA was confirmed in HUVEC cells and also detected in microvascular endothelial cells, HMVEC (Figure 2A). In RV-ECT, an endothelial cell line derived from rat brain resistance microvessel, a faint band of PCR product was also present (Figure 2A). To further confirm the expression of platelet-type 12-LOX in RV-ECT cells, we designed 7 primers on the basis of the partial sequence obtained from rat Walker 256 cells.24 The expression of platelet-type 12-LOX was detected by nested PCR using 3 different combinations of these 7 primers (Figure 2B). Interestingly, in addition to platelet-type 12-LOX, RV-ECT cells also expressed another isoform of 12-LOX that presumably was leukocyte-type as detected by using primers designed on the basis of the rat leukocyte-type 12-LOX sequence29 (data not shown), suggesting that RV-ECT cells expressed both platelet- and leukocyte-type 12-LOX.
Involvement of endogenous 12-lipoxygenase in endothelial cell migration Endothelial cell migration is a requisite step in angiogenesis. It has been shown that the activation of phospholipase A2 is required for endothelial cell migration in response to bFGF.30 To study whether AA released by phospholipase A2 modulates endothelial cell migration, we selected the RV-ECT cell line, which can grow in DMEM-10% FBS without bFGF or VEGF supplementation,26 for cell migration assay. First we examined the migratory response of RV-ECT cells toward exogenous AA, bFGF, and VEGF. As shown in Figure 3A, AA increased endothelial cell migration by 70% to 80%, a level comparable to that of bFGF but less than VEGF. This observation is consistent with the report that VEGF is a stronger chemotactic factor than bFGF.31 Because mobilization of AA has been observed in endothelial cells on stimulation with angiogenic factors such as VEGF,32 bFGF,33 and angiogenin,34 we studied the effect of various inhibitors of arachidonic acid metabolism on endothelial cell migration stimulated by VEGF. As shown in Figure 3B, ETYA, a promiscuous inhibitor for AA metabolism, inhibited VEGF-stimulated RV-ECT migration. NDGA, a general LOX inhibitor, also significantly reduced VEGF-stimulated RV-ECT migration. In contrast, indomethacin, a general COX inhibitor, had no effect. The results suggest that the LOX pathway, but not the COX pathway, of AA metabolism is involved in RV-ECT migration.
Endothelial 12-lipoxygenase was involved in RV-ECT tube differentiation Certain endothelial cell lines, under proper culture conditions, have the capacity to form tubelike structures. Confluent RV-ECT cells can spontaneously form cordlike structures under normal culture conditions.26 Therefore, this cell line was chosen for the current study. When RV-ECT cells were cultured between 2 layers of Matrigel, the formation of cordlike structures was expedited, as manifested by the formation of numerous vacuoles within 4 to 5 hours and interconnected tubelike structures within 24 hours (Figures 4A, 4C). Pretreatment of RV-ECT cells with BHPP (10 µmol/L) significantly impaired their ability to form cordlike structures (Figures 4B, 4D), suggesting the potential involvement of 12-LOX in endothelial cell cordlike differentiation.
Endothelial cells that overexpress 12-LOX had increased motility and enhanced tubelike differentiation To further explore the role of 12-LOX in endothelial cell migration and tubelike differentiation, we transfected CD4 endothelial cells with a pcDNA-12-LOX expression construct. The CD4 endothelial cell line was selected for 12-LOX overexpression study because of its low level of 12-LOX expression (Figure 2C). Stable transfectants were selected using G418, and the transfectant pools were characterized for the expression of 12-LOX mRNA by RT-PCR (Figure 5A) and Northern blot analysis (Figure 5B). The expression of 12-LOX was also increased in 12-LOX-transfected CD4 cells at protein levels as revealed by Western blot (Figure 5C). The growth rate of 12-LOX-transfected CD4 endothelial cells was similar to the mock transfectants (data not shown), suggesting that although 12-LOX is involved in bFGF- or VEGF-stimulated endothelial cell growth, the overexpression of 12-LOX in CD4 cells is not sufficient to stimulate endothelial cell proliferation. However, the overexpression of 12-LOX was able to stimulate CD4 endothelial cell migration (Figure 5D). Further, the increased motility in 12-LOX-transfected CD4 cells was inhibited by BHPP, suggesting that it is the increased 12-LOX activity in endothelial cells that enhances cell motility (Figure 5D).
Inhibition of angiogenesis in vivo by 12-lipoxygenase inhibitor The involvement of the 12-LOX pathway of arachidonic acid metabolism in endothelial cell proliferation, migration, and tube differentiation led us to study whether the inhibition of 12-LOX activity can compromise angiogenesis in vivo. Because the induction of angiogenesis in vivo by bFGF requires the angiogenic activities of VEGF,35 we used bFGF premixed with Matrigel to induce angiogenesis. As shown in Figure 6A, bFGF induced massive angiogenesis around and within the implant (upper panel, left). When dissected out, the implanted Matrigel retained a large volume of blood within the gel (upper panel, right). Matrigel implants without bFGF had little or no angiogenic activities in vivo (bottom panel). Inclusion of the 12-LOX inhibitor BHPP in the implants significantly reduced the ability of bFGF to induce angiogenesis (Figure 6A, middle panel), suggesting that 12-LOX is involved in angiogenesis in vivo. Figure 6B shows the hemoglobin levels in the dissected Matrigel. As shown in the Figure, BHPP significantly reduced the hemoglobin levels in Matrigel (P < .05), suggesting a reduction of angiogenesis. The reduction of angiogenesis was closely correlated with the levels of 12(S)-HETE in the Matrigel implants as shown in Figure 6C. Taken together, the data suggest a critical role of 12-LOX activity in angiogenesis in vivo.
In this study we demonstrated that endothelial cells from different species (rat and human) and different organs (brain, umbilical cord, and foreskin) express platelet-type 12-LOX and elucidated its important role in endothelial cell responses to angiogenic stimuli. Inhibition of 12-LOX activity by BHPP, a platelet-type selective inhibitor, attenuated the endothelial cell mitogenic and the migratory responses to the angiogenic factors bFGF and VEGF and the tubelike differentiation on Matrigel. Forced expression of 12-LOX in the CD4 endothelial cells stimulated cell migration and promoted tube differentiation. Inhibition of 12-LOX activity by BHPP significantly reduced angiogenesis in vivo. Our findings suggest that eicosanoids from the arachidonic acid metabolism through the 12-LOX pathway, ie 12(S)-HETE, are involved in modulating angiogenesis.
We thank Homan Kian, Yilong Cai, Alex Zacharek, and Kenny Hanna for their technical support and Dr Gerhard Furstenberger of Deutsches Krebsforschungszentrum (Germany) for sharing with us the unpublished data concerning the Ki of BHPP for various recombinant lipoxygenase enzymes.
Submitted June 2, 1999; accepted December 15, 1999.
Supported by National Institutes of Health grant CA-29997, United States Army Prostate Cancer Research Program DAMD 17-98-1-8502, the Harper Development Fund, a CaPCURE Foundation Award, and a Cancer Research Foundation of America Fellowship Award.
Reprints: Kenneth V. Honn, Department of Radiation Oncology, Wayne State University, 431 Chemistry Building, Detroit, Michigan 48202; e-mail: k.v.honn{at}wayne.edu.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
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Top
Abstract
Introduction
v
3 integrin.
5-, 8-, 11-, and 14-eicosatetraynoic acid
(ETYA), nordihydroguaiaretic acid (NDGA), 5-LOX-activating protein
(FLAP) inhibitor MK886, and cyclooxygenase (COX) inhibitor
indomethacin
), no reverse transcriptase present in RT reaction mixtures
as controls for the possible contamination of DNA in RNA samples;
RT(+), reverse transcription present. (B) RT-PCR detection of
platelet-type 12-LOX expression in RV-ECT cells. The primer
combinations for 1st, 2nd, and 3rd are described in "Materials and
methods." The target sizes of the final PCR product from 1st, 2nd,
and 3rd primer combinations are 111 bp, 62 bp, and 88 bp, respectively.
(C) Immunoblot analysis of 12-LOX expression in endothelial cells. The
blot was probed with a rabbit polyclonal antibody to human
platelet-type 12-LOX. (D) Inhibition of VEGF-stimulated 12-LOX activity
by BHPP. Cell treatment and measurement of 12(S)-HETE are detailed in
"Materials and methods." Columns, average levels of 12(S)-HETE
per 1 × 106 cells (n = 3); bars, SE. a,
P < .01 when compared with the unstimulated control; b,
P < .05 when compared to the VEGF-stimulated cells. (E)
Involvement of endothelial 12-LOX in bFGF- or VEGF-stimulated cell
proliferation. HUVEC cells were plated in 96-well culture plates. Cell
proliferation was stimulated with 10 ng/mL bFGF (open triangle) or 10 ng/mL VEGF (filled circle) in EBM-2 with 2% FBS. Cells with no bFGF or
VEGF stimulation were used as controls (open circle). 48 hours after
treatment with graded levels of BHPP, cell numbers were measured by an
MTS method as described in "Materials and methods." Data point,
mean from quadruplicate determination; bars, SE from quadruplicate of
treatment.
