Chinese Medical Journal 2008;121(24):2599-2603
Roles of cyclooxygenase-2 in microvascular endothelial cell proliferation induced by basic fibroblast growth factor

QIAN Rui-zhe,  YUE Fei,  ZHANG Guo-ping,  HOU Li-kun,  WANG Xin-hong,  JIN Hui-ming

QIAN Rui-zhe (Department of Physiology and Pathophysiology, Shanghai Medical College, Fudan University, Shanghai 200032, China)

YUE Fei (Department of Physiology and Pathophysiology, Shanghai Medical College, Fudan University, Shanghai 200032, China)

ZHANG Guo-ping (Department of Physiology and Pathophysiology, Shanghai Medical College, Fudan University, Shanghai 200032, China)

HOU Li-kun (Department of Physiology and Pathophysiology, Shanghai Medical College, Fudan University, Shanghai 200032, China)

WANG Xin-hong (Department of Physiology and Pathophysiology, Shanghai Medical College, Fudan University, Shanghai 200032, China)

JIN Hui-ming (Department of Physiology and Pathophysiology, Shanghai Medical College, Fudan University, Shanghai 200032, China)

Correspondence to:JIN Hui-ming,Department of Physiology and Pathophysiology, Shanghai Medical College, Fudan University, Shanghai 200032, China (Tel: 86-21-54237081. Fax:86-21-64179832. E-mail:hmjin@shmu. edu.cn)
Keywords
cerebral ischemia; cerebral microvascular endothelial cell; basic fibroblast growth factor; cyclooxygenase; prostaglandin E2; vascular endothelial growth factor
Abstract

Background  The level of basic fibroblast growth factor (bFGF) increases rapidly after cerebral ischemia. However, the molecular mechanisms for the effects of bFGF on cerebral microvascular endothelial cells (cMVECs) have not yet been fully elucidated. In this study, a murine cMVEC line, bEnd.3, was employed to study the effects of bFGF on cyclooxygenase (COX) expression and its downstream effects in cMVECs.
Methods  After treatment with bFGF, RT-PCR and Western blotting analyses were carried out to evaluate the changes in COX-2 mRNA and protein expression, respectively. MTT assays were performed to measure cell proliferation. The prostaglandin E2 (PGE2) and vascular endothelial growth factor (VEGF) concentrations in the culture medium were measured by enzyme-linked immunosorbent assay (ELISA).
Results  COX-2 mRNA and protein expressions in bEnd.3 cells were induced by bFGF in time- and dose-dependent manners. The bFGF-induced COX-2 upregulation led to enhanced PGE2 production by bEnd.3 cells, and this effect was abolished by the selective COX-2 inhibitor NS-398. bFGF also increased VEGF production by bEnd.3 cells, and this effect was blocked by NS-398 and the EP1/2 (PGE2 receptors) antagonist AH6809. Furthermore, exogenous PGE2 increased VEGF production in bEnd.3 cells, and AH6809 blocked this effect.
Conclusion  bFGF increases VEGF production in an autocrine manner by increasing COX-2-generated PGE2 in cMVECs and subsequently stimulates MVEC proliferation and angiogenesis.

Cyclooxygenase (COX) is the rate-limiting enzyme for PGH2 synthesis from arachidonic acid. COX has attracted the interest of numerous researchers owing to its involvement in various physiological and pathological situations. To date, three COX proteins, namely COX-1, COX-2 and COX-3, have been identified. COX-1 is constitutively expressed as a housekeeping gene in most tissues, and its major function is to synthesize prostaglandin (PG) precursors for homeostatic regulation. COX-2 is expressed in some tissues and can be rapidly induced by a variety of stimuli, such as inflammation, cytokines and hormones.1-3 Recently, COX-2 was suggested to be a bystander molecule in the angiogenic pathway.4,5

Basic fibroblast growth factor (bFGF) is known to be a potent angiogenesis-inducing factor and can increase VEGF production during angiogenesis. It has been reported that bFGF may induce COX-2 expression in various endothelial cells (ECs), but the mechanisms of its effects on COX-2 expression and its downstream changes in microvascular endothelial cells (MVECs) remain unclear.6,7 Although COX-2 has been suggested to be a bystander molecule in the angiogenic pathway, little is known about its roles in MVEC proliferation and angiogenesis. To clarify the role of COX-2 in the angiogenesis induced by bFGF, the mechanisms of the effects of bFGF on COX expression and its downstream changes in MVECs were investigated in the present study.

METHODS

Reagents
Recombinant human bFGF was purchased from PeproTech (Rocky Hill, USA). Bovine serum albumin (BSA), 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT), The prostaglandin E2 (PGE2) and NS-398 were purchased from Sigma Chemical Co. (St. Louis, USA). AH6809 (AH) was purchased from Cayman Chemical Company (Ann Arbor, MI, USA). All other chemicals were purchased from commercial sources and qualified for biological research.

Cell culture and serum-free medium treatment
The bEnd.3 cell line was obtained from ATCC (Manassas, USA). bEnd.3 cells were previously characterized by immunocytochemical staining of von Willebrand factor (positive), CD31 (positive) and CD34 (positive), and cDNA microarray analysis.8,9 The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) in a humidified atmosphere containing 5% CO2 at 37°C. All experiments were carried out using bEnd.3 cells between passages 5 and 10. To avoid the potential interference of endogenous growth factors present within FBS, a serum-free medium treatment was performed as follows. After bEnd.3 cells reached approximately 80% confluence, the culture medium was aspirated and the cells were rinsed twice with sterile phosphate-buffered saline (PBS). Thereafter, the cells were cultured in serum-free DMEM containing 0.1% BSA in their normal atmosphere for 12 hours before further treatments.

RT-PCR analysis of murine COX mRNA expression
A semiquantitative RT-PCR method was used. Total RNA was isolated using the TRIzol reagent (Life Technologies, Gaithersburg, MD) according to the manufacturer’s instructions. The isolated RNA was then reverse- transcribed using a RevertAidTM First Strand cDNA Synthesis Kit (Fermentas Inc., Hanover, MD) following the manufacturer’s protocol. Specific intron-spanning primers for murine glyceraldehyde-3-phosphate dehydrogenase (GAPDH), COX-1 and COX-2 were designed as follows: GAPDH sense (5’-ACAGCCGC- ATCTTCTTGTGCAGTG-3’) and antisense (5’-GGCCT- TGACTGTGCCGTTGAATTT-3’); COX-1 sense (5’-CT- CACAGTGCGGCCAA-3’) and antisense (5’-AGGATG- AGGCGAGTGAT-3’); COX-2 sense (5’-TTGAGGAGA- GCAGATGGGATT-3’) and antisense (5’-GCTTCGGG- AGCACAACAGAG-3’).

Western blot analysis of murine COX-2 protein expression
Cells were washed with ice-cold PBS. Total cell proteins were harvested and solubilized using a lysis buffer comprising 20 mmol/L Tris-HCl (pH 7.5), 100 mmol/L NaCl, 10 mmol/L EDTA, 1% Nonidet P-40, 1% deoxycholate, 0.1% SDS and an appropriate amount of a protease inhibitor cocktail (Sigma Chemical Co.,USA). Protein concentrations were determined using the bicinchoninic acid assay. Aliquots of the cell lysates (80 µg of protein) were subjected by 10% SDS-PAGE, and the separated proteins were transferred to a PVDF membrane. An anti-COX-2 polyclonal antibody and a horseradish peroxidase-conjugated secondary antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, USA). The membranes were developed using an enhanced chemiluminescence mixture (Amersham, Buckinghamshire, UK).

MTT assay
The MTT assay was performed as previously described. The absorbances of the mixtures were measured at 492 nm with a correction wavelength of 540 nm.

PGE2 ELISA
The PGE2 concentrations in treated cell culture media were measured using a PGE2 Immunoassay (R&D Systems, Minneapolis, USA). Cell culture media collection and the ELISA were performed according to the manufacturer’s protocol. The concentration of PGE2 was expressed as pg/105 cells.

VEGF ELISA
Culture media were collected from treated cells, and measured for their VEGF concentrations using a Quantikine Mouse VEGF Immunoassay (R&D Systems). The ELISA was performed according to the manufacturer’s protocol. The concentration of VEGF was expressed as pg/105 cells.

Statistical analysis
Each experiment was performed in duplicate and repeated at least three times. Values were expressed as the mean ± standard deviation (SD) and analyzed by one-way analysis of variance (ANOVA) with SPSS software version 13.0 (SPSS Inc., Chicago, IL). A P <0.05 was considered statistically significant.

RESULTS

COX-2 mRNA expression is induced by bFGF in time- and dose-dependent manners in bEnd.3 cells
Several growth factors, including bFGF, are potent inducers of COX-2 expression. Besides glia and neurons, cerebral MVECs (cMVECs) represent a very important cell type in the central nervous system. RT-PCR analyses were employed to examine the effects of bFGF on COX-2 mRNA expression in cMVECs. bEnd.3 cells cultured in serum-free medium typically expressed low levels of COX-2 mRNA. After treatment with bFGF (10 ng/ml), COX-2 mRNA expression in bEnd.3 cells rapidly increased within 1 hour. The maximum level of COX-2 mRNA was observed at 2 hours, and the level gradually decreased thereafter (Figure 1A). Following treatment with different concentrations of bFGF ((0–15) ng/ml) for 2 hours, the level of COX-2 mRNA expression in bEnd.3 increased in a dose-dependent manner (Figure 1B). In contrast, bFGF had no apparent effects on COX-1 mRNA expression in bEnd.3 cells.
 

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Figure 1. Effects of bFGF on COX mRNA expression in bEnd.3 cells. (A) Effects of bFGF (10 ng/ml) after treatment for the indicated times. COX-2, *P <0.05 vs time 0. (B) Effects of treatment with the indicated concentrations of bFGF after 2 hours. COX-2, #P <0.05 vs 0 ng/ml bFGF.

bFGF-induced expression of COX-2 protein in bEnd.3 cells
Western blot analyses were employed to verify that bFGF was able to induce COX-2 protein expression in bEnd.3 cells. COX-2 protein expression was obviously induced in cMVECs after treatment with bFGF for 4 hours. The COX-2 protein concentration reached its maximum level at 8 hours (Figure 2A). bFGF also induced COX-2 protein expression in a dose-dependent manner (Figure 2B).
 

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Figure 2. Effects of bFGF on COX-2 protein expression in bEnd.3 cells. (A) Effects of treatment with bFGF (10 ng/ml) for the indicated times. #P <0.05 vs time 0. (B) Effects of treatments with the indicated concentrations of bFGF for 8 hours. *P <0.05 vs 0 ng/ml bFGF.

COX-2 plays an important role in bFGF-induced proliferation of bEnd.3 cells
bFGF is a pleiotropic mitogen toward a broad spectrum of cells. COX-2 has been confirmed to be involved in bFGF-induced angiogenesis by promoting EC migration and tube-like structure formation.10,11 Proliferation of ECs, especially MVECs, is an essential process in angiogenesis.

The MTT assay was employed to examine whether bFGF has any effects on cMVEC proliferation and the role of COX-2 in such effects. We found that bFGF ((0–15) ng/ml) stimulated bEnd.3 cell proliferation. This effect was blocked by NS-398 (20 µmol/L), a selective COX-2 inhibitor that acts by binding to the active site on COX-2. In contrast, NS-398 had no apparent effects on the proliferation of bEnd.3 cells under normal culture conditions (Figure 3).


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Figure 3. Effects of a COX-2 inhibitor on bEnd.3 cell proliferation. Proliferation was evaluated by MTT assay. bEnd.3 cells were incubated with the indicated concentrations of bFGF with or without NS-398 (20 µmol/L) for 36 hours. *P <0.05 vs 0 ng/ml bFGF; #P<0.05 vs cells treated with the same concentration of bFGF.

bFGF increases PGE2 production in a COX-2-dependent manner in bEnd.3 cells
In ECs, COX-2 is preferentially coupled with PGE2 synthesis, which results in the production of PGE2 as the predominant PG after COX-2 induction.12 We used an immunoassay specific for PGE2 to confirm that PGE2 production was increased by bFGF in a COX-2- dependent manner in cMVECs. After treatment with bFGF for 6 hours, the PGE2 concentration in the cell culture medium was increased by approximately 2-fold. After 12 hours of treatment with bFGF, PGE2 reached its highest concentration (Figure 4A). Furthermore, bFGF increased PGE2 production in bEnd.3 cells in a dose- dependent manner (Figure 4B). NS-398 (20 µmol/L) abolished the bFGF-induced PGE2 production in bEnd.3 cells.
 

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Figure 4. Effects of a COX-2 inhibitor on bFGF-induced PGE2 production in bEnd.3 cells. The PGE2 concentrations were measured by ELISA. (A) Effects of treatment with bFGF (10 ng/ml) with or without NS-398 (20 µmol/L) for the indicated times. *P<0.05 vs bFGF at 0 hour; #P<0.05 vs bFGF at the same time point. (B) Effects of treatment with the indicated concentrations of bFGF with or without NS-398 (20 µmol/L) for 12 hours. *P<0.05 vs 0 ng/ml bFGF, #P<0.05 vs bFGF at the same concentration.

VEGF production is increased by bFGF via COX-2-generated PGE2 in bEnd.3 cells
PGE2 was reported to promote VEGF production in ECs within tumor tissues.13 An immunoassay specific for mouse VEGF was employed to confirm that the bFGF-induced PGE2 also increased VEGF production in cMVECs. After treatment with bFGF (10 ng/ml), the VEGF concentrations in bEnd.3 cells culture medium were measured every 8 hours in 24 hours. The concentrations were apparently increased at 16 hours, compared with 0 hour and 8 hours, reached the maximum level at 24 hours and then decreased at 48 hours. The bFGF-induced VEGF production was abolished by NS-398 (20 µmol/L) and AH (10 µmol/L), a selective antagonist of the PGE2 receptors EP1/2. Furthermore, exogenous PGE2 (10 µmol/L) increased VEGF production in bEnd.3 cells, and AH (10 µmol/L) abolished this effect (Figure 5). The concentrations of NS-398 and AH in the above experiments were determined using concentration-effect curves (data not shown).
 

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Figure 5. bFGF increases VEGF production via PGE2 in bEnd.3 cells. (A) Effects of treatment with bFGF (10 ng/ml) with or without NS-398 (10 µmol/L) or AH (10 µmol/L) for 24 hours. *P <0.05 vs. bFGF alone. (B) Effects of treatment with the indicated combinations of PGE2 (10 µmol/L) and AH (10 µmol/L). The values are the mean ± SD of three experiments. *P <0.05 vs control (PGE2-, AH-); #P <0.05 vs PGE2 alone.

DISCUSSION

In this study, we have demonstrated that bFGF induced COX-2 mRNA and protein expressions in MVECs in time- and dose-dependent manners. In contrast, COX-1 expression was not apparently changed after bFGF treatment. The bFGF-induced COX-2 expression resulted in enhanced PGE2 production by MVECs. This effect was blocked by the selective COX-2 inhibitor NS-398, indicating that bFGF increased PGE2 production in MVECs in a COX-2-dependent manner. bFGF also stimulated MVEC proliferation. The selective COX-2 inhibitor NS-398 almost completely suppressed this effect, but had no apparent effects on MVEC proliferation under normal culture conditions. Furthermore, because the expression of COX-2, but not COX-1, was affected by bFGF treatment, the pro-proliferation effect of bFGF on bEnd.3 cells may be attributable to COX-2. Therefore, bFGF appears to exert its effects in a COX-2-dependent manner. bFGF has been confirmed to stimulate macrovascular EC proliferation by activation of cyclins through signal transduction cascades.10,11 Our results appeared to cast some doubts on at least some steps of this pathway in MVECs because COX-2 was involved in the bFGF-stimulated MVEC proliferation. NS-398 almost completely suppressed the bFGF-stimulated cMVEC proliferation. On the other hand, the issue of whether bFGF has other targets besides COX-2 in MVECs remains unclear, and the mechanism for the involvement of COX in bFGF-stimulated MVEC proliferation requires further investigation.

Our experimental data demonstrated that endogenous PGE2 induced by bFGF subsequently induced VEGF production in MVECs. Similar to PGE2, VEGF has been confirmed to exert its biological effects in autocrine and paracrine manners.13 The present results further indicated that bFGF signaling may transfer to VEGF signaling in the angiogenesis signal transduction pathway induced by bFGF. Although bFGF is a non-specific cell mitogen, VEGF is an EC-specific mitogen and a potent proangiogenic factor that induces and promotes angiogenesis. In other words, the bFGF signal has been transferred from a non-specific mitogen to a specific mitogen for the proliferation of MVECs.

Although several articles have reported that COX-2 expression is induced in macrovascular ECs by bFGF, macrovascular ECs differ enormously from MVECs in terms of their functions and roles in physiological and pathological situations.14 Furthermore, MVECs isolated from different tissues bear different properties and show different responses to stimuli.15 Studies based on ECs from other tissues may not be totally applicable to cMVECs. Therefore, studies based on cMVECs may have specific significance for certain cerebral diseases.16

Taking all the present results together, bFGF may induce COX-2 expression and trigger a series of downstream effects resulting in MVEC proliferation. The results further substantiate the roles of COX-2 as a bystander molecule in the angiogenic pathway induced by bFGF.

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  1. Shanghai Municipal Natural Science Foundation,No. 07ZR14009;