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Cyclooxygenase-2-selective Nonsteroidal Anti-Inflammatory Drugs Inhibit Hepatocyte Growth Factor/Scatter Factor-induced Angiogenesis

¹ Angiogenesis Laboratory, Department of Pharmacology,
² Glaxo Institute of Applied Pharmacology,
³ Department of Oncology, and
⁴ Multi-Imaging Center, University of Cambridge, Cambridge, United Kingdom; and
⁵ Biological Engineering Division, Massachusetts Institute of Technology, Cambridge, Massachusetts

	ABSTRACT

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Epidemiological studies have indicated a reduced risk of malignancies with the use of nonsteroidal anti-inflammatory drugs (NSAIDs), although the exact mechanisms are debated. NSAIDs inhibit angiogenesis, which is a key step for tumor growth. Hepatocyte growth factor/scatter factor (HGF/SF), a potent and independent angiogenic factor, has been implicated in tumorigenesis, but limited knowledge exists on the potential targets for inhibiting HGF/SF-induced pathological angiogenesis. The current study was designed to elucidate the possible role of cyclooxygenase (COX) downstream of HGF/SF during angiogenesis and to evaluate the potential for harnessing NSAIDs as a therapeutic strategy. Known NSAIDs were classified as COX-1 or COX-2 selective based on their activity in a platelet aggregation experiment. The inhibitors were administered into a polyether polyurethane scaffold implant in mice at the selected doses, and the total neovascularization after the administration of HGF/SF was quantified using a ¹³³Xenon clearance technique, vessel counts, and immunohistochemistry. Angiogenesis was also quantized into chemoinvasion, migration, proliferation, and tube formation events in vitro, and the effects of the NSAIDs were evaluated on HGF/SF-induced activity of human umbilical vein endothelial cells (HUVECs). HGF/SF accelerated the angiogenic process in the murine implant, and this activity was inhibited by COX-2-selective meloxicam and NS398. The COX-1 inhibitors ketoprofen and SC560 failed to inhibit the HGF/SF-induced angiogenic events in vitro and in vivo. A COX-2 blockade inhibited the HGF/SF-induced chemoinvasion and migration of human umbilical vein endothelial cells, without affecting the proliferative or tubulogenic responses. Western blots revealed the induction COX-2 expression after HGF/SF treatment, and the pharmacological inhibition of COX-2 executed a temporal inhibition of phosphorylation of the mitogen-activated protein kinases. The current study, for the first time, implicates COX-2 as a downstream signal during HGF/SF-induced angiogenesis, temporally impinging on the mitogen-activated protein kinase signaling. However, the mediation is restricted to only the early events of the angiogenic process, emphasizing the chemopreventive role for NSAIDs. Few therapeutic options currently exist for HGF/SF-induced pathological angiogenesis, and the vast knowledge on COX-2 inhibitors can be harnessed to design a newer therapeutic approach.

	INTRODUCTION

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Epidemiological studies have indicated that the use of NSAIDs⁶ is associated with a reduced risk of malignancies of the gastrointestinal tract (1) and the pancreas (2) . Furthermore, a recent meta-analysis has suggested a reduced risk of breast cancer with the use of NSAIDs (3) . NSAIDs act primarily through the inhibition of the COX enzyme, which is involved in arachidonic acid metabolism, and which exists as a constitutive COX-1 and an inducible COX-2 isoform (4) . Indeed, numerous studies have demonstrated the overexpression of COX-2, but not COX-1, in premalignant lesions and human malignancies with poor prognosis (5 , 6) , providing a rationale for the chemopreventive activity of NSAIDs. In a murine model of human familial adenomatous polyposis, the genetic inactivation of COX-2 was shown to reduce substantially the frequency and size of intestinal polyps (7) , whereas the selective inhibition of COX-2 was shown to reduce the polyp burden in patients (8) . Interestingly, a recent study reported the separation of COX-2 inhibition from the apoptosis-inducing activity of NSAIDs (9) , spurring further an intense debate focusing on many mechanisms for the anticancer activity of NSAIDs.

In a classical study, Tsujii et al. (10) demonstrated that COXs are implicated in tumor angiogenesis, suggesting a novel mechanism for the anticancer activity of NSAIDs. Angiogenesis, the formation of new blood vessels from an existing vascular bed, is a key step in tumor growth and metastasis (11) . Many molecules that induce tumor angiogenesis have been characterized, including VEGF, FGF, angiopoietins, HGF/SF, and others, acting through mediators such as VEGF, tyrosine kinases, nitric oxide, prostaglandins, and so forth (12) . The past decade has seen a concentration of efforts to develop inhibitors and antagonists to block pathological angiogenesis, primarily targeting VEGF (13) . However, in a recent study, we have demonstrated that HGF/SF, which is associated with an aggressive phenotype in multiple cancer pathologies and has been implicated in angiogenesis (14) , can independently and sufficiently induce angiogenesis even in the presence of a VEGF blockade (15) . This finding emphasized the need for newer strategies that target a broad spectrum, but site-specific, blockade.

HGF/SF was discovered independently both as a mitogen for hepatocytes and as a fibroblast-derived factor that induced scattering in polarized epithelial cells (16 , 17) . An elevated level of HGF/SF has been implicated in malignancies of the gastrointestinal tract (18) . Lamzus et al. (19) demonstrated that HGF/SF conferred a growth advantage to human breast cancer xenotransplants, linked with a higher microvessel density. Strategies to block HGF/SF-induced angiogenesis and tumor growth have included using a mixture of three antibodies or ribozymes (20 , 21) ; however, these technologies need to be evaluated further before clinical applications. Interestingly, in a recent study, HGF/SF was shown to trigger the activation of the COX-2 gene in gastric epithelial cells (22) . The same group also demonstrated that angiogenesis was susceptible to the inhibition of COX (23) . Therefore, the current study was designed to elucidate the interaction between HGF/SF and COX and evaluate the possibility of harnessing NSAIDs in the modulation of HGF/SF-induced angiogenesis.

	MATERIALS AND METHODS

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Preparation of HGF/SF.
Murine recombinant HGF/SF was used for the mouse experiments and was generated using the NSO mouse melanoma cell line transfected by electroporation with a mouse HGF/SF cDNA. For experiments involving human endothelial cells, the NSO cells were transfected with a human HGF/SF cDNA to generate recombinant human HGF/SF. The proteins were purified by elution through a heparin-Sepharose column and a Mono-S column.

Chemicals.
The 5-LOX inhibitor NDGA and the preferential COX-1 inhibitor ketoprofen were from Sigma Chemical Co. The selective COX-2 inhibitor NS398 [N-(2-(Cyclohexyloxy)-4-nitrophenyl)methanesulfonamide] was procured from RBI, whereas the selective COX-1 inhibitor SC560 [5-(4-Chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethylpyrazole] was obtained from Calbiochem. The 5-LOX-activating protein inhibitor L-655-238 was obtained from Alexis Corporation, whereas LY294002, a PI3K inhibitor, was purchased from Tocris. Meloxicam was a gift from Prof. S. K. Gupta (All India Institute of Medical Sciences, New Delhi, India). Hypnorm and Hypnovel were obtained from Janssen Pharmaceutica and Roche, respectively. Radioactive ¹³³Xe was purchased from DuPont Pharma. Biomatrix was obtained from TCS Biologicals.

Platelet Aggregation Experiments.
Platelet aggregation was performed according to the protocol of Born (24) , using a Payton Dual Channel Aggregation Module linked to a Kipp and Zonen chart recorder. Light transmission through platelet-poor plasma was fixed as 100%, whereas light transmission through platelet-rich plasma was set as 0%. Exclusion criteria included intake of NSAIDs or eicosa-pentaenoic acid-rich diet within 7 days before donating blood. Informed consent and approval from the donors was requisitioned before collection of 3 ml of venous blood. Platelet aggregation was induced using collagen (40 µg/ml^-1) after pretreatment with the various inhibitors of COXs. Data were represented as light transmission as percentages of untreated controls.

Murine Angiogenesis Model.
Male BALB/c mice (20 g; Tucks, United Kingdom) were anesthetized with 4% isoflurane and maintained on 2% isoflurane, using a mixture of oxygen (0.8 liters/min) and nitrous oxide (0.6 liters/min). Two bilateral s.c. air pockets were created in the dorsal subscapular region using a pair of curved blunt forceps. Two sterile polyether polyurethane scaffolds (160 mm³; Acer Associates, Bucks, United Kingdom) were inserted into the pockets, and the distant incision was closed with silk sutures (Mersilk, United Kingdom).

The treatment with vehicle or peptides/drugs was started 24 h (considered as day 0) after the implantation of the scaffolds and continued for 10 days. A Precision Glide 30-gauge needle (Sigma Chemical Co.) was used to deliver the injection, and the total volume administered into the scaffold was kept constant at 40 µl/scaffold. All of the in vivo procedures were approved and conformed to the United Kingdom Home Office guidelines.

Assay for Functional Status of Neovasculature.
On day 15, the animals were anesthetized using a combination of fentanyl citrate-fluanisone and midazolam (diluted 1:1:20 in saline). Vascularization was assessed as a function of the blood flow through the implants by direct injection of ¹³³Xe-containing saline into the scaffold and by monitoring its clearance over a 6-min period. Radioactivity was measured using a microprocessor scalar ratemeter (Nuclear Enterprise), linked to a collimated low energy X-ray/gamma ray NaI-crystal with an Al-entrance window, on an HG-type mount coupled to an NE 5289C preamplifier. The rate meter was connected to an Epson LX850 printer. The data were expressed as the percentage of ¹³³Xe cleared at every 40 s, calculated according to the formula:

Although the background radioactivity was negligible, it was subtracted from the counts in the calculations. The neovascularization in the scaffold was correlated with the total ¹³³Xe cleared in a 6-min period and also as a function of the T_1/2 clearance of ¹³³Xe from the scaffold. The T_1/2 clearance was calculated by nonlinear regression curve fitting using a one-phase exponential decay equation.

Macroscopic Vessel Count.
After the ¹³³Xe clearance measurements, the animals were sacrificed by cervical dislocation, and the dorsal skin flap was everted. The gross angiogenic response was photographed using a macrolens connected to a Nikon Single Lens Reflex (SLR) camera. Prints were developed on Kodak plates. Angiogenesis was quantified as the in-growth of vessels in the scaffold-granuloma tissue.

Immunostaining for Endothelial Cells.
Thin sections (12 µm) were probed overnight with the primary antibody against human von Willebrand factor (1:2000 dilution; DAKO). Antibody-binding sites were visualized using a goat antirabbit antibody conjugated to FITC (1:150 dilution; Vector Laboratories, United Kingdom). The fluorochrome was excited using a 488-nm laser line, and the emitted light was captured using 530/30-nm bandpass filter. The images were captured at a resolution of 512 x 512 pixels using a Leica TCS-NT confocal microscope. Controls were run alongside, by omitting the primary antibody.

Morphometry.
An axial strip sampling technique (25) was used to calculate the numerical density of blood vessels. Using a 96-point square lattice, the volume fraction (V_v) occupied by the fibrovascular growth and the number of vascular profiles within the field area were recorded. Length density per mm³ of sponge was then calculated as:

Numerical density of blood vessels, Q_A = number of vessels/unit area

Length density, L_V = 2 x Q_A

Length density per mm³ of sponge = L_V x V_V.

In Vitro Tube Formation Assay.
HUVECs, between passages 2 and 6, were plated on 6-well plates and grown to confluence. The cells were synchronized in 1% serum-containing media for 24 h, after which they were plated in 24-well plates (Costar), each well coated with 200 µl of Biomatrix (extracellular matrix extracted from Engelbreth-Holm-Swarm murine sarcoma, diluted 1:3 in PBS). The drugs and growth factors were added in the appropriate dilutions in the media, and the cells were allowed to incubate for 16 h. Wherever appropriate, cells were pretreated with the inhibitors, which were maintained for the entire duration of the experiments. At the end of 16 h, the cells were fixed in 10% formalin and visualized with a Nikon Diaphot inverted microscope using a x20 objective. This time point was selected to avoid any contribution of the cell proliferation component to tubulogenesis.

Endothelial cells form connected tube-like structures with branches when plated on appropriate matrices. The number of branches per view field was counted, and an average of 18 fields were recorded across the two perpendicular axes per well.

Mechanical Injury Regeneration Model.
A confluent monolayer of synchronized endothelial cells was scraped with a multichannel wounder, thereby causing 11 parallel lesions, 400 µm wide. Some of the coverslips were fixed immediately after wounding and served as time zero (T₀) controls. At 24 h after lesion, the experiments were stopped by washing the coverslips with ice-cold PBS and fixing in 4% formaldehyde. Recovery of the denuded area was quantified by use of a Leica Q500, semiautomated, computerized image analysis system. The image analysis system integrates the discrete shifts in the boundaries of the injury with time and ignores the stray migration of individual cells to avoid any bias. For each coverslip, four fields of view were selected at random. The area of lesion in each field of view was measured, and by use of the data for T₀, the lesion area was then converted to give percentage of regeneration relative to T₀ values.

Proliferation Assay.
The monolayer of endothelial cells were "wounded" as above and cultured in 1% FCS with vehicle or peptides/drugs for a period of 24 h. At the end of this period, they were washed in ice-cold PBS, trypsinized, and counted with a hemocytometer using the trypan blue exclusion method.

Chemoinvasion Assay.
Synchronized HUVECs were trypsinized and plated on the top chamber of a biomatrix-coated Transwell (pore size, 8 µM; Costar) at a density of 10⁴ cells/well. The drugs and growth factors were added in the appropriate chambers, and the cells were allowed to incubate for 16 h. The cells that had migrated to the lower side of the membrane were fixed in 10% formalin and stained with H&E. The cells on the top side of the membrane were wiped off using a cotton bud, and those on the lower surface were visualized at x20 objective using a Nikon Diaphot inverted microscope. The number of cells in a view field was counted, and an average of five random fields were recorded across the two perpendicular axes per well.

Western Blot Detection of the Expression of COX-2 and Phosphorylation of ERKs.
A confluent monolayer of cells was wounded as described previously, and the cellular proteins were solubilized by rapid mixing with sample buffer (three times) under reducing conditions. Equivalent amounts of protein per sample were resolved electrophoretically on 10% polyacrylamide gels and transferred onto a nitrocellulose (0.22 µm) membrane. Antiphospho-ERK1/2 antibody (1:800 dilution; New England Biolabs and Cell Signaling Technologies, respectively), and anti-ERK1/ERK2 total protein antibodies (1:500 dilution; Santa Cruz Biotechnology) were used to probe the membrane. For the detection of COX-2, the membranes were probed with a 1:200 dilution of a monoclonal antibody against COX-2 (Santa Cruz Biotechnology). Protein level normalization was achieved by probing the blots using a 1:1000 dilution of a polyclonal antibody against ß-actin. Where LY294002, the PI3K inhibitor was used, the cells were incubated with the drug for 1 h before wounding. The signal was amplified using a 1:2000 dilution of the appropriate horseradish peroxidase-conjugated secondary antibody (Bio-Rad), and the immunocomplexes were visualized using enhanced chemiluminescence detection (Amersham Life Science).

Statistics.
All experiments were repeated at least three times with replicates, and the data were expressed as mean SE. Data were tested using ANOVA, followed by Newman-Keuls or Bonferroni’s post hoc test, with the level of significance set at P < 0.05.

	RESULTS

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Selectivity of NSAIDs for COX-1 and COX-2 Isoenzymes.
To test for the concentration range at which the NSAIDs exhibit selectivity for COX isoforms, we studied their activity on the aggregation of human platelets. As shown in Fig. 1A , collagen induced complete aggregation of the platelets, which was completely inhibited by ketoprofen at a concentation of 10^-6 M, the concentration at which neither NS398 nor meloxicam had any inhibitory effects. Significant inhibition of aggregation was observed at 10^-5 M for meloxicam and 10^-4 M for NS398 (Fig. 1, A and B) , suggesting that the selectivity ratio for COX1:COX-2 decreases in the order of ketoprofen>meloxicam>NS398 (Fig. 1C) . Indeed, in an extensive study using Chinese hamster ovary cells transfected with COX-1 or COX-2, Riendeau et al. (26) demonstrated that meloxicam and NS398 were selective inhibitors of COX-2 with an IC₅₀ in the nanomolar range and a COX-2/COX-1 selectivity ratio of 300, although we found NS398 to be less potent than meloxicam in inhibiting COX-1. The same study also reported that ketoprofen failed to completely inhibit COX-2.

View larger version (38K): [in this window] [in a new window]   Fig. 1. Effect of ketoprofen, meloxicam, and NS398 on collagen-induced platelet aggregation. Platelet aggregation was performed according the protocol of Born (24) , using a Payton Dual Channel Aggregation Module linked to a Kipp and Zonen chart recorder. Light transmission through platelet-poor plasma was considered as 100%, whereas light transmission through a platelet-rich plasma was set as 0%. Platelet aggregation was induced using a 40 µg/ml-1 dose of collagen. Where the COX-inhibitors were used, the platelets were incubated in the presence of the inhibitors for 45 min before the addition of collagen. The tracings in A depict a typical aggregation response as quantified by translating the light-transmission into electrical deflections that were recorded on a chart. B, graph depicts the total inhibition of collagen-induced platelet aggregation by the COX inhibitors. C, The panel shows the selectivity of the inhibitors for the COX isoforms. Data are expressed as percentages of collagen-induced aggregation and are mean ± SE of n = 3–5. **, P < 0.01 versus collagen treatment.

View larger version (51K): [in this window] [in a new window]   Fig. 2. Temporal effect of HGF/SF on angiogenesis into a scaffold implanted in vivo. A sterile polyether-polyurethane scaffold was implanted s.c. on the dorsum of a mouse. Treatment with HGF/SF (30 ng/scaffold) or vehicle was started 24 h after implantation and continued for 10 days. Angiogenesis into the scaffold was quantified using immunolabeling, 133Xe clearance, and vessel count. A, the confocal immunofluorescence micrographs of scaffold sections labeled with an antibody against von Willebrand factor to delineate all endothelial cells. The blood vessels are seen as white overlaid on the phase contrast image of the scaffold section. The images were at an original magnification of x400; the final image size is 141 x 141 µm in area. Images were captured with a resolution of 512 x 512 pixels using a Leica TCS-NT confocal microscope. Antibody binding sites were visualized using a species-specific secondary antibody conjugated to FITC. The fluorochrome was excited using a 488-nm laser line, and the emitted light was captured using 530/30 nm bandpass filters. Controls undertaken by omitting the primary antibody were imaged using the same settings for laser power and gain and showed no specific fluorescence. Angiogenesis was quantified using 133Xe clearance from the scaffolds (B), vessel counts (C), and vessel density (D). E, graph shows the clearance of 133Xe from the skin on the experimental days, included as a control for environmental effects.Table 1 shows the T1/2 of clearance of the radioactive Xe from the scaffold. A shorter T1/2 denotes a more functional vasculature. *, P < 0.05; ***, P < 0.001 versus day 7 vehicle-treated control values; +, P < 0.01 versus concurrent vehicle-treated group. Data shown are mean ± SE (n = 6–10).

View larger version (67K): [in this window] [in a new window]   Fig. 3. Effect of NSAIDs on HGF/SF-induced angiogenesis in a murine scaffold-granuloma model. Sterile polyether polyurethane scaffolds were implanted s.c. on the dorsum of mice. Treatment was started 24 h after implantation and continued for 10 days. Angiogenesis into the scaffold was quantified using immunohistochemistry, 133Xe clearance, and vessel count. A, confocal immunofluorescence micrographs of HGF/SF- and HGF/SF + meloxicam-treated scaffold sections labeled with antibody against von Willebrand factor to delineate all endothelial cells. The images were at an original magnification of x400; the final image size is 141 x 141 µm in area. Images were captured with a resolution of 1024 x 1024 pixels using a Leica TCS-NT confocal microscope. The fluorochrome was excited using a 488-nm laser line, and the emitted light was captured using 530/30-nm bandpass filters. Controls undertaken by omitting the primary antibody were imaged using the same settings for laser power and gain and showed no specific fluorescence. Nuclear staining was done with DAPI. The effect of meloxicam on HGF/SF-induced angiogenesis was quantified using the total 133Xe cleared from the implant at the end of 6 min (B), and as the total vessel count (C), quantified as the number of vessels entering an implant. The graph shows the effect of NS398 on HGF/SF-induced neovascularization quantified as 133Xe clearance (D) and vessel counts (E). F, the effect of treatment with the NSAIDs on the body weight of the animals. Treatment with ketoprofen, a COX-1 inhibitor, failed to block HGF/SF-induced increase in the 133Xe clearance (G) and vessel counts (H). *, P < 0.05; ***, P < 0.001 versus vehicle-treated controls; #, P < 0.05 versus HGF/SF-treated group.Table 2 shows the T1/2 of clearance of 133Xe from the scaffolds in the different treatment groups. Data shown are mean ± SE (n = 3–8).

Ketoprofen, at a dose range where it selectively inhibited COX-1, did not inhibit the HGF/SF-induced neovascularization (Fig. 3, G and H) . Fig. 3 (inset, Table 2) shows the half-life (T_1/2) of clearance of ¹³³Xe from the implants, in which a shorter T_1/2 indicates a faster clearance resulting from enhanced blood flow. Ketoprofen failed to block the HGF/SF-induced increase in functional vasculature, which was inhibited by both meloxicam and NS398.

Effect of NSAIDs on HGF/SF-induced wound Regeneration and Cell Proliferation.
To elucidate the interaction between COX isoenzymes and HGF/SF, we used a multiple scratch model to mimic the pathophysiological situation and amplify biochemical signals. Contact-inhibited endothelial cells were injured using a multichannel wounder (27) , switching them from a physiological "off" state to a pathophysiological "on" state. As shown in Fig. 4 , the addition of HGF/SF (10^-9 M) induced a significant regeneration into the denuded area. Furthermore, HGF/SF induced a significant proliferation of endothelial cells (Fig. 4) . Both NS398 and meloxicam blocked the HGF/SF-induced regeneration in a concentration-dependent manner without affecting the basal regeneration or the HGF/SF-induced cell proliferation (Fig. 4, A–E) .

View larger version (59K): [in this window] [in a new window]   Fig. 4. Effect of COX-2-selective NSAIDs on the HGF/SF-induced repair process. A, photomicrographs depict the regeneration in different treatment groups 24 h after injury. B and C, the concentration-effect curves of NS398 on the basal and HGF/SF-induced wound regeneration and cell count, respectively. D and E, the concentration-effect curves of meloxicam on the basal and HGF/SF-induced wound regeneration and cell count, respectively. Where the NSAIDs were used, the cells were preincubated with the inhibitors for 1 h before the lesioning and addition of HGF/SF. COX blockade was maintained for the entire duration of the study. Data expressed are mean ± SE of at least six separate experiments with duplicate/quadruplicate wells in each. In the wound recovery experiment, data are shown as percentages of 0 h values. *, P < 0.05; **, P < 0.01; ***, P < 0.001 versus HGF/SF-treated controls; +, P < 0.001 versus vehicle-treated controls.

View larger version (60K): [in this window] [in a new window]   Fig. 5. Effect of COX-1-selective NSAIDs on the HGF/SF-induced repair process. A, photomicrographs depict the recovery in different treatment groups 24 h after injury. B and C, the concentration-effect curves of ketoprofen on the basal or HGF/SF-induced wound regeneration and cell count, respectively. D and E, the concentration-effect curves of SC560 on the basal or HGF/SF-induced wound regeneration and cell count, respectively. Where the NSAIDs were used, the cells were preincubated with the inhibitors for 1 h before the lesioning and addition of HGF/SF. COX blockade was maintained for the entire duration of the study. Data expressed are mean ± SE of at least three separate experiments with duplicate/quadruplicate wells in each. In the wound recovery experiment, data are shown as percentages of 0 h values. +, P < 0.001 versus vehicle-treated controls.

View larger version (92K): [in this window] [in a new window]   Fig. 6. Effect of COX-1 and COX-2 inhibition on HGF/SF-induced chemoinvasion. A, pictomicrographs of H&E images of cells migrated under defined conditions. B, the chemoinvasion of cells through Biomatrix under different conditions. Data shown are mean ± SE where n = 3. ***, P < 0.001 versus vehicle-treated control; #, P < 0.001 versus HGF/SF (lower chamber)-induced chemoinvasion.

View larger version (97K): [in this window] [in a new window]   Fig. 7. Effect of COX-inhibitors on HGF/SF-induced cord-like structure/tube formation by HUVECs. A, photomicrographs depict a random field of view and show the alignment of HUVECs under different treatment conditions. Where the selective COX inhibitors were used, they were added to the cell media 1 h before plating. B, the effect of the treatments as quantified by number of branch-points per field of view. The data represents mean ± SE from n = 3, with 18 fields of view captured per well, across the two axes. ***, P < 0.001 versus vehicle-treated group. V, vehicle; H, HGF/SF (1 nM); NS, NS398 (1 µM), a COX-2-selective inhibitor; M, meloxicam (10 µM), a preferential inhibitor of COX-2; K, ketoprofen (1 µM), a selective COX-1 inhibitor.

View larger version (27K): [in this window] [in a new window]   Fig. 8. Western blot to detect changes in the levels of COX-2 expression after treatment with HGF/SF and the effect of COX-inhibitors on the HGF/SF-induced changes in phosphorylation of ERK1 and ERK2 as a function of time. A confluent monolayer of HUVECs was injured using a multichannel wounder and incubated for fixed lengths of time in the presence of HGF/SF (1 nM). A, probing with an antibody against COX-2 demonstrates the HGF/SF-induced expression of COX-2, which is susceptible to the PI3K LY294002. The blot was normalized to ß-actin levels. B, meloxicam and NS398, both selective for COX-2, were added 1 h before injury. At fixed time points, the cells were lysed with 3x sample buffer, and equal amounts of proteins were resolved using a 10% SDS-PAGE gel. The phosphorylation changes of ERK1 and ERK2 were probed using a 1:800 diluted monoclonal phospho-specific antibody against ERK raised in rabbit. Equal loading of ERK1 and ERK2 protein was confirmed by reprobing the blots using a 1:500 dilution of a polyclonal antibody raised in goat, against ERK1 and ERK2. V, vehicle; H, HGF/SF (1 nM); NS, NS398 (10-6 M); M, meloxicam (10-6 M); LY, LY294002.

Effect of the Inhibition of the Lipoxygenase Pathway on HGF/SF-induced Angiogenesis.
The arachidonate metabolism can traverse down the LOX pathway and result in the systhesis of leukotrienes that have been implicated in angiogenesis (29) . To dissect out any role of LOXs in HGF/SF-induced angiogenesis, we used NDGA and L655238, which at the concentrations used inhibit LOX (30) and 5-LOX-activating protein (31) , respectively. As shown in supplementary Fig. 1 , neither NDGA nor L655238 exerted any effect on HGF/SF-induced regeneration or cell proliferation, although loss of cells was evident at high concentrations.

	DISCUSSION

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In the present study, we report that HGF/SF induces angiogenesis by signaling through COX-2, and targeting this isoenzyme using selective COX-2-selective NSAIDs could be harnessed as a therapeutic approach for the inhibition of HGF/SF-induced pathological angiogenesis. Although the NSAIDs were administered locally to precisely dissect the role of COX isoforms in HGF/SF-induced angiogenesis, preliminary results suggest that orally administered NS398, but not ketoprofen, retained the inhibitory effect (see supplementary Fig. 2 ). Furthermore, we demonstrated that there is a temporal relationship between COX-2 and HGF/SF-induced phosphorylation of MAPK. Using a deconstructional approach in which we replicated the key steps of angiogenesis in vitro, we demonstrated that HGF/SF could promote the tubulogenesis, chemoinvasion, proliferation, and chemokinesis of endothelial cells but that COX-2 is implicated only during the chemoinvasive and the chemokinesis steps.

HGF/SF has been implicated in tumorigenesis and associated angiogenesis (32) . Furthermore, it also promotes angiogenesis underlying diabetic retinopathy (33) , psoriasis (34) , and arthritis (35) , both in cohesion and independently of other angiogenic factors. Interestingly, however, research into the mechanisms of angiogenic action of HGF/SF and the development of effective therapeutics against it has been overshadowed by the importance being placed on VEGF.

An interesting approach for inhibiting the HGF/SF-induced angiogenesis would be to block the signal transduction cascade. A few studies had reported a link between HGF/SF and COX-2. For example, it was demonstrated that prostaglandins strongly induce HGF/SF expression in skin fibroblasts (36) . Jones et al. (22) demonstrated that HGF/SF could trigger the activation of the COX-2 gene, and HGF/SF was shown to enhance the activity of cytosolic phospholipase A2 and COX and increase the levels of prostaglandin E₂ in human gastric carcinoma (37) . Furthermore, COX-2 products were shown to accelerate the healing of ulcers by HGF/SF (38) . Indeed, the products of COX-mediated metabolism have been implicated in inducing angiogenesis (39) . However, despite the circumstantial evidence supporting the involvement of COX in HGF/SF signaling, there has been no study to elucidate the exact implications. In the current study, we evaluated the neovascular response induced by a daily dosing of HGF/SF into a polymer scaffold implant (25) . Compared with vehicle-treated controls, by day 15, the administration of HGF/SF induced a significant increase in the total ¹³³Xe cleared, vessel counts, and vessel density, with a decrease in the T_1/2 of ¹³³Xe clearance, allowing the pharmacological modulation with NSAIDs to dissect out the involvement of COX in HGF/SF-induced angiogenesis. Interestingly, this discrimination was lost by day 28, suggesting that HGF/SF did not alter the maximal angiogenesis but accelerated the process. Furthermore, at day 28, although the total ¹³³Xe clearance was similar to day 15 in the HGF/SF-treated group, the vessel counts were lower and the half-life was higher. This was consistent with the observations of Reed et al. (40) and could possibly be attributable to active vascular remodeling.

Although the involvement of COX in the angiogenic process is well documented, there exists some disagreement on the extent to which each isoform is involved. We used NS398 as a highly selective COX-2 inhibitor, meloxicam as a nonselective inhibitor with a preference for COX-2, and ketoprofen as a selective COX-1 inhibitor (41 , 42) . However, before using the NSAIDs, the dosage range at which each selectively blocks the COX-1 isoform were established using a platelet aggregation experiment, which involves only the COX-1 isoform (43) . At a concentration range at which NS398 and meloxicam had no inhibitory activity on platelet aggregation, both the drugs blocked HGF/SF-induced angiogenesis. In contrast, ketoprofen failed to block HGF/SF-induced angiogenesis, although it inhibited platelet aggregation, suggesting that COX-2 is the key isoform involved in mediating HGF/SF-induced angiogenesis. This is supported further by the overexpression of COX-2 after treatment with HGF/SF, and, indeed, a recent study reported that HGF/SF and prostacyclin, a COX-mediated metabolite, synergize for enhanced angiogenesis (44) . Other eicosanoids, such as thromboxane-A2, were shown to modulate COX-2-dependent endothelial cell migration (45) , and COX-2 inhibitors were demonstrated to block angiogenesis induced via a prostaglandin E₂-EP3 signaling (46) . An interesting point of note is that the pharmacological inhibition of COX-2 did not completely inhibit HGF/SF-induced angiogenesis, suggesting the involvement of additional pathways.

The angiogenic phenotype is a culmination of sequential temporal events. We, therefore, recruited in vitro assays using endothelial cells for a quantized study of the angiogenic process. Blockade of the monolayer regeneration after a pharmacological inhibition of COX-2 using NS398 or meloxicam and the inability of ketoprofen or SC560, a highly selective COX-1 inhibitor, supported the in vivo findings that COX-2 was required for HGF/SF-induced angiogenesis. Interestingly, even though the LOX products have been implicated in promoting angiogenesis (47) , this alternative pathway of arachidonic acid metabolism seems not to be involved with HGF/SF-induced angiogenesis in the current study. In a previous study, we had dissected out a chemokinetic and a proliferation component in this assay, using paclitaxel and actinomycin D (48) . This same study also revealed that endothelial cells start replicating after 18 h, and based on the finding, any experiment in which cell proliferation could skew results was terminated at 16 h. Interestingly, in the present study, although treatment with the COX-2 inhibitors blocked the total regeneration, HGF/SF-induced proliferation was not altered, suggesting that it was the migratory component that was being inhibited. The inhibition of HGF/SF-induced chemoinvasion by NS398 and the failure of SC560 to do so, further supported the implication of COX-2 in mediating the migratory phenotye in endothelial cells. This was in contrast with the reported implication of COX-2 in the VEGF pathway, in which COX-2 selective NSAIDs were found to inhibit cell proliferation (49) . Furthermore, VEGF-induced tube formation was also reportedly inhibited by NS398 (23 , 50) , whereas in the present study HGF/SF-induced tubulogenesis remained unaltered by either the COX-1- or COX-2-selective NSAIDs. This suggests that the temporal implication of COX-2 during angiogenesis is dependent on the cytokine and that HGF/SF-induced tubulogenesis proceeds via a COX-2-independent mechanism. Interestingly, Tsujii et al. (10) demonstrated that COX-2 could up-regulate the expression of COX-1, and this could be suppressed by a combination of neutralizing antibodies (anti-VEGF, anti FGF-2, and anti-platelet-derived growth factor or anti-transforming growth factor ß) that could not inhibit COX-2 expression. This observation, in light of the current findings, suggests that COX-2 induction could proceed through a VEGF- or FGF-independent pathway and that HGF/SF could be a likely candidate. This is supported further by our previous observation that HGF/SF could induce angiogenesis in the presence of a VEGF receptor blockade (15) .

Inhibition of integrins can block the migration of endothelial cells, and a recent observation by Dormond et al. (51) suggests that COX-2-selective NSAIDs can block _Vß₃ integrin-mediated angiogenesis (51) . Furthermore, in different cell types, HGF/SF has been reported to exert antiapoptotic effects through Bcl-2, the expression of which can be modulated by prostaglandins (52 , 53) . Similar mechanisms could be active here and need additional studies, but we explored the involvement of MAPK. We have demonstrated recently that HGF/SF induced "a rapid and a delayed" biphasic phosphorylation of the MAPK module in endothelial cells and the pharmacological inhibition of MEK could block HGF/SF-induced migration and proliferation. Furthermore, Jones et al. (23) demonstrated that NSAIDs could block a VEGF-induced rapid phosphorylation of MAPK and inhibit angiogenesis (23) . In the current study, both meloxicam and NS398 inhibited the delayed phase of MAPK phosphorylation without altering the rapid phase. This temporal distinction in activation may explain the differences in the phenotypic outcomes when endothelial cells are treated with different cytokines in the presence of NSAIDs. Furthermore, the escape of the early MAPK phosphorylation from NSAID inhibition could possibly account for pathways that contribute to residual angiogenesis in the presence of a COX-2 blockade. Intriguingly, we also demonstrated that the inhibition of PI3K could block the HGF/SF-induced delayed phase phosphorylation of MEK and block angiogenesis (54) . Furthermore, in a recent study, LY294002, a PI3K inhibitor, was shown to decrease the expression of COX-2 in keratinocytes (55) . This was consistent with the findings of this study in which treatment of an injured monolayer of HUVECs with HGF/SF resulted in the overexpression of COX-2, which was susceptible to the inhibition by the PI3K inhibitor LY294002. This raises the possibility of the existence of a HGF/SFPI3KCOX-2MAPK link during angiogenesis, but additional studies are warranted.

Although epidemiological data strongly support the chemopreventive effects of NSAIDs for gastrointestinal malignancies and benefits in other solid tumors, the precise mechanism is not yet clear. The current study for the first time demonstrates that COX-2 plays a significant role in the early stages of HGF/SF-induced angiogenesis, and stresses further the position of NSAIDs in chemopreventive therapeutics. This enthusiasm should be tempered by caution arising from suggestions that COX-2 inhibitors may be associated with adverse cardiovascular events as reported in the Vioxx Gastrointestinal Outcome Research trial (56) . Contrasting reports to this trial has evolved from the Celexocib Long-Term Arthritis Safety Study (57) . Nonetheless, the selective expression of COX-2 in pathological vasculature and the normal vasculature expressing COX-1 makes the former isoform an attractive therapeutic target (58) . Furthermore, because COX-2 and HGF/SF are coexpressed at high levels in multiple malignancies, it may be possible to generalize the paradigm developed for the use of NSAIDs in HGF/SF-induced angiogenesis to these conditions, especially given the extensive clinical data that exist on the COX-2-selective NSAIDs, which can be harnessed to design clinical trials.

	FOOTNOTES

 
Grant support: Cambridge Nehru Trust, Committee of Vice Chancellors and Principals (CVCP) United Kingdom, and Trinity College (all to S. S.).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Lynda A. Sellers, Babraham Institute, Babraham, Cambridge, United Kingdom.

Requests for reprints: Ram Sasisekharan/Shiladitya Sengupta, Biological Engineering Division, 16-561, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139. Phone: (617) 258-9494; Fax: (617) 258-9409; E-mail: rams{at}MIT.edu/shiladit@MIT.edu

⁶ The abbreviations used are: NSAID, nonsteroidal anti-inflammatory drug; COX, cyclooxygenase; VEGF, vascular endothelial growth factor; FGF, fibroblast growth factor; HGF/SF, hepatocyte growth factor/scatter factor; NDGA, nordihydroguaiaretic acid; PI3K, phosphatidylinositol 3'-kinase; ¹³³Xe, ¹³³Xenon; HUVEC, human umbilical vein endothelial cell; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; LOX, lipoxygenase.

Received 3/13/03. Revised 7/29/03. Accepted 9/11/03.

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