Key words

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Anna Wiktorowska-Owczarek

The Effect of Valdecoxib on the Production

of Growth Factors Evoked by Hypoxia and Bacterial

Lipopolysaccharide in HMEC-1 Cells*

Wpływ waldekoksybu na wydzielanie czynników wzrostu wywołane

hipoksją i bakteryjnym lipopolisacharydem w komórkach HMEC-1

Department of Pharmacology, Chair of Pharmacology and Clinical Pharmacology, Medical University of Lodz, Poland

Abstract

Background. Endothelial cells produce prostaglandins (PGE2 and PGI2) and growth factors (VEGF and bFGF).

These substances regulate proliferation of cells, inflammatory processes and neovascularization under physiologi-cal and pathologiphysiologi-cal conditions.

Objectives. The aim of this study was to check whether valdecoxib – a selective COX-2 inhibitor – inhibits VEGF

and/or bFGF secretion in the presence of LPS or cobalt chloride in normal human microvascular endothelial cells (HMEC-1).

Material and Methods. HMEC-1 cells were treated with valdecoxib at a concentration of 10 and 100 µM in the

presence of 100 µg/mL LPS or 200 µM CoCl2. The effect of NSAIDs and LPS on VEGF and bFGF proteins was

analyzed by ELISA kit (R&D Systems). Cell viability was measured using the 3-[4.5-dimethylthiazol-2-yl]-2.5- -diphenyltetrazolium bromide (MTT) method.

Results. Valdecoxib inhibited LPS-induced proliferation of endothelial cells and bFGF secretion in a

dose-depen-dent manner. Valdecoxib stimulated VEGF formation via HMEC-1 under inflammatory conditions.

Conclusion. Ultimately, the anti-angiogenic effect of the selective COX-2 inhibitor was a result of inhibition of

HMEC-1 cell proliferation and bFGF generation under inflammatory conditions (Adv Clin Exp Med 2013, 22, 6, 795–800).

Key words: LPS, VEGF, bFGF, valdecoxib, endothelial cells.

Streszczenie

Wprowadzenie. Komórki śródbłonka wytwarzają prostaglandyny (PGE2 i PGI2) i czynniki wzrostu (VEGF i bFGF).

Te substancje regulują proliferację komórek, proces zapalny oraz neowaskularyzację w warunkach fizjologicznych i patologicznych.

Cel pracy. Sprawdzenie czy waldekoksyb – selektywny COX-2 inhibitor hamuje wydzielanie VEGF i/lub bFGF

w obecności LPS lub chlorku kobaltu w linii komórkowej śródbłonka HMEC-1.

Materiał i metody. Komórki HMEC-1 były inkubowane z waldekoksybem w stężeniach 10 i 100 µM w obecności

100 µg/ml LPS lub 200 µM CoCl2. Efekt NLPZ-tu i LPS na wydzielanie VEGF i bFGF był analizowany za pomocą

ELISA kitu (R & D system). Żywotność komórek była mierzona testem MTT.

Wyniki. Waldekoksyb zahamował proliferację komórek śródbłonka indukowaną LPS oraz wydzielanie bFGF w sposób

zależny od dawki. Jednocześnie waldekoksyb pobudzał tworzenie VEGF przez HMEC-1 w warunkach zapalenia.

Wnioski. Ostatecznie antyangiogenne działanie selektywnego inhibitora COX-2 było rezultatem zahamowania

pro-liferacji komórek HMEC-1 i tworzenia bFGF w warunkach zapalenia (Adv Clin Exp Med 2013, 22, 6, 795–800).

Słowa kluczowe: LPS, VEGF, bFGF, waldekoksyb, komórki śródbłonka.

Adv Clin Exp Med 2013, 22, 6, 795–800 ISSN 1899–5276

ORIGINAL PAPERS

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Growth factors such as VEGF and bFGF are responsible for angiogenesis. Vascular endothelial growth factor (VEGF) is one of the factors regulat-ed by hypoxia and the HIF complex [1–3]. Nume-rous cells such as tumor cells, neutrophils, fibro-blasts and some endothelial cells (e.g. HMEC-1 cells) can secrete the most potent pro-angiogenic factors [3]. This growth factor influences the en-dothelial cells via VEGFR-1 (also known as Flt-1 – fms like tyrosine kinase) and VEGFR-2 (Flk-1 – fetal liver kinase-1 in mouse/KDR – kinase do-main region in human) receptors and leads to per-meability enhancement, cellular proliferation and migration of cells [4–6]. Apart from VEGF, fibro-blast growth factors (FGFs) possess a pro-angio-genic activity by binding to high affinity tyrosine kinase FGFRs on the surface of target cells. FGFs are a large family with 22 distinct members among which two of them were the first to be isolated from the peptide family: FGF1 and FGF2 (also named as acidic FGF; aFGF and basic FGF; bFGF). FGFs are responsible for mitogenesis, differentia-tion, migration and cell survival of endothelial cells and other cells [7–10].

Hypoxia promotes endothelial cells via VEGF. Bacterial cell wall lipopolysaccharide (LPS, endotoxin) causes VEGF and bFGF secretion and is also responsible for generation of vasoactive me-diators such as prostaglandins: PGI2, PGE2 [11].

Prostaglandins are synthesized from arachidonic acid by cyclooxygenase (COX). This enzyme exists in at least two isoforms: the constitutive isoform, COX-1, and the inducible isoform, COX-2. COX-2 is activated in response to inflammatory mediators such as cytokines, as well as mitogens, growth fac-tors, oncogenes and carcinogens, and also is con-stitutively expressed in the brain, kidney vascular endothelium, ovaries and uterus [12]. Both en-zymes are inhibited by nonsteroidal anti-inflam-matory drugs (NSAIDs). Valdecoxib and celecox-ib and other substances belong to selective COX-2 inhibitors and they block PGI2 formation by

endo-thelial cells. COX inhibitors show anti-angiogenic effect that is the result of endothelial cell survival inhibition and induction of apoptosis [1, 4, 12].

Endothelial cells produce PGs (PGE2 and

PGI2) [13] and growth factors (VEGF and

bFGF) [14]. These substances regulate prolifera-tion of cells, inflammatory processes and neovas-cularization under physiological and pathological conditions. Therefore, the aim of this study was to check whether valdecoxib inhibits VEGF and bFGF in the presence of LPS and cobalt chloride in normal human microvascular endothelial cells (HMEC-1).

Material and Methods

Chemicals

The substances used were the following: MCDB 131 medium, fetal bovine serum, penicillin-strep-tomycin solution (5,000 units/mL penicillin and 5.000 µg/mL streptomycin sulphate in normal sa-line), phosphate buffered saline (PBS; pH 7.4) and trypsine-EDTA (0.25% trypsin, 1mM EDTA-4 Na) were purchased from Invitrogen (Carlsbad, CA, USA). The cobalt chloride, thiazolyl blue tetrazoli-um bromide (MTT), EGF htetrazoli-uman, lipopolysaccha-rides from Salmonella enteritidis (LPS) and hydro-cortisone were purchased from Sigma Chemical Co. (St. Louis, USA). Valdecoxib was purchased from Cayman Chemical Company (Michigan, USA).

Cell Culture

HMEC-1 (human microvascular endothe-lial cells) were purchased from ATCC with cata-log number ATCC-CRL-10636 (depositor Centers for Disease Control, Dr. Edwin W. Ades, Atlanta). The cells were used between passages 10–31 and cultured in 25 cm3_{flasks in MCDB 131 medium}

supplemented with 10% fetal bovine serum, 10 ng/ mL epidermal growth factor, 1 µg/mL hydrocorti-sone and penicillin-streptomycin solution, in a hu-midified atmosphere of 95% O2 and 5% CO2 at

37°C. Cells were harvested every 3rd day in a tryp-sin-EDTA (0.25% trypsin, 1 mM EDTA) solution. HMEC-1 cells were cultured basing on the method described in the literature [15, 16] and the author’s own modification.

MTT Conversion

HMEC-1 cells proliferation was measured us-ing the 3-(4,5-dimethylthazol-2-yl)-2,5-diphenyl -tetrazolium bromide (MTT) conversion method. Cells were seeded (50.000 cells/well) into 96-well plates. The cells were incubated for 24 h with LPS 100 µg/mL, CoCl2 200 µM, valdecoxib 10 or

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ELISA Assays

VEGF and bFGF concentrations in cell culture media were determined by commercially available ELISA kits according to the vendor’s protocols (R&D System, Abingdon, UK).

Data Analysis

All data was presented as means ± SD (stan-dard deviation). Statistical comparisons between the groups were performed using ANOVA, and

post-hoc comparisons were performed using the

Student-Newman-Keuls test. The normal distribu-tion of parameters was checked by means of the Shapiro-Wilks test. If the data was not normally distributed or the values of the variance (test F) were different, ANOVA with Kruscal-Wallis and Mann-Whitney’s U test were used. All parameters were considered significantly different if p < 0.05. The statistical data analysis was performed using Statgraphics 5.0 plus software.

Results

The Effect of Valdecoxib

on VEGF Secretion Evoked

by Cobalt Chloride or Bacterial

LPS in HMEC-1

Basal HMEC-1 cells released only small amounts of VEGF (25.6 pg/mL) as measured by ELISA, but cobalt chloride (CoCl2), a hypoxia

mimicking agent, at a concentration of 200 µM in 24-h incubation stimulated the secretion of VEGF by 505% (p < 0.05) (Fig. 1). 100 µg/mL LPS, which mimics inflammation, increased the level of VEGF by 106% (p < 0.05) in a statistically significant manner. Valdecoxib – a selective COX-2 inhibi-tor – at a concentration of 10 µM did not influ-ence the level of VEGF, but at 100 µM stimulated its generation (p < 0.05). Neither of the valdecox-ib concentrations changed VEGF secretion un-der hypoxia conditions. However, valdecoxib at a higher concentration intensified VEGF secre-tion in the presence of LPS in comparison with LPS by 130% (p < 0.05) in a statistically signifi-cant manner and by 108% in comparison with si-multaneous application of valdecoxib 10 µM and LPS (p < 0.05).

The Effect of Valdecoxib on

bFGF Secretion Evoked by

Bacterial LPS in HMEC-1

The control group of HMEC-1 released ap-proximately 435 pg/mL bFGF (Fig. 2). LPS stim-ulated the release of bFGF by 50% (p < 0.05) in a statistically significant manner. Valdecoxib at the concentration of 10 µM increased the genera-tion of bFGF by 13% (p < 0.05). The simultane-ous application of the selective COX-2 inhibitor and LPS induced a higher secretion of bFGF by 8% (p < 0.05) in comparison with LPS. The ob-served results were statistically significant. In the next set of experiments, the influence of valde-coxib at 100 µM on the generation of bFGF was studied. 100 µM valdecoxib decreased the level of bFGF by 11% (p < 0.05) in comparison with the control. It also inhibited the secretion of bFGF in-duced by LPS by 13.5% (p < 0.05) in son with LPS and by 20% (p < 0.05) in compari-son with simultaneous application of valdecoxib 10 µM and LPS.

VE

G

F

[p

g/

m

l]

0 20 40 60 80 100 120 140 160 180

Control CoCl2 200 µM

LPS 100 µg/ml Valdecoxib 10 µM

Valdecoxib 10 µM & CoCl2 200 µM

Valdecoxib 10 µM & LPS 100 µg/ml Valdecoxib 100 µM

Valdecoxib 100 µM & CoCl2 200 µM

Valdecoxib 100 µM & LPS 100 µg/ml

*

* * a b

* *

*

Fig. 1. Effects of valdecoxib (10 and 100 µM) on VEGF levels in HMEC-1 cells in the presence of CoCl2 (200 µM) or LPS (100 µg/mL). Bars represent the means (± SEM of 3–5 experiments).

* p < 0.05 vs. control; a – p < 0.05 vs. LPS (100 µg/mL); b – p < 0.05 vs. LPS (100 µg/mL) & valdecoxib (10 µM)

Ryc. 1. Wpływ waldekoksybu (10 and 100 µM) na

poziom VEGF w komórkach HMEC-1 w obecności CoCl2 (200 µM) lub LPS (100 µg/ml). Kolumny przed-stawiają średnie (± SEM z 3–5 doświadczeń).

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The Effect of Valdecoxib on

Cell Viability in the Presence of

Cobalt Chloride or Bacterial LPS

in HMEC-1

Cobalt chloride at 200 µM decreased HMEC-1 cell viability (Fig. 3) in a statistically significant manner (p < 0.05). In contrast to hypoxia, LPS at 100 µg/mL stimulated proliferation of HMEC-1 cells by 33% (p < 0.05). Valdecoxib at a concentration of 10 and 100 µM did not change cell viability under hypoxia conditions. However, valdecoxib at both of the concentrations inhibited the proliferative effect of

LPS by 14% (p < 0.05) and 35% (p < 0.05), respec-tively. These observed effects were statistically signifi-cant. The decrease in cell viability was statistically sig-nificant (p < 0.05) after the application of valdecoxib 100 µM with LPS (100 µg/mL) in comparison with valdecoxib 10 µM with LPS (100 µg/mL).

Discussion

The major aim of this work was to check the effect of valdecoxib (a selective COX-2 inhibitor; valdecoxib inhibits the generation of prostaglan-dins) on the secretion of growth factors (VEGF

bFGF [pg/ml

]

0 200 400 600 800

Control

LPS 100 µg/ml

Valdecoxib 10 µM

Valdecoxib 100 µM

Fig. 2. Effects of valdecoxib (10 and 100 µM) on bFGF levels in HMEC-1 cells in the presence of LPS (100 µg/mL). Bars represent the means (± SEM of 3–5 experi-ments). * p < 0.05 vs. control; a – p < 0.05 vs. LPS (100 µg/mL); b – p < 0.05 vs. LPS (100 µg/mL) & valdecoxib (10 µM)

Ryc. 2. Wpływ waldekoksybu (10 and

100 µM) na poziom bFGF w komórkach HMEC-1 w obecności LPS (100 µg/ml). Kolumny przedstawiają średnie (± SEM z 3–5 doświadczeń). *p < 0.05 w stosunku do grupy kontrolnej; a – p < 0.05 w stosunku do grupy LPS (100 µg/ml); b – p < 0.05 w sto-sunku do grupy LPS (100 µg/ml) & walde-koksyb (10 µM)

(%

o

f c

on

tro

l)

M

TT

c

on

ve

rs

ion

0 20 40 60 80 100 120 140

Control CoCl2 200 µM

LPS 100 µg/ml Valdecoxib 10 µM

CoCl2 200 µM + Valdecoxib 10 µM

Valdecoxib 10 µM & LPS 100 µg/ml Valdecoxib 100 µM

CoCl2 200 µM + Valdecoxib 100 µM

*

* a

a b

*

Fig. 3. Effects of valdecoxib (10 and 100 µM) on the cell viability of cultured HMEC-1 cells in the presence of CoCl2 (200 µM) or LPS (100 µg/mL) measured by MTT conversion assay. The results are presented as a percent-age in relation to the control value. Bars represent the means (± SEM of 4–15 experi-ments). *p < 0.05 vs. control; a – p < 0.05 vs. LPS (100 µg/mL); b – p < 0.05 vs. LPS (100 µg/mL) & valdecoxib (10 µM)

Ryc. 3. Wpływ waldekoksybu (10 and

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and bFGF), which are the most potent angioge-nic factors. In previous studies the expression of VEGF by human microvascular endothelial cells (HMEC-1 cells) under hypoxia conditions evoked by low oxygen partial pressures (1 and 3%) and co-balt chloride was observed [2, 14, 16]. In the pre-sent work, the selective COX-2 inhibitor, valde-coxib, did not change the production of VEGF by HMEC-1 cells under chemical hypoxia (Fig. 1). Hypoxia as well as cobalt chloride potently inhi bits HIF-1α degradation and activates transcription of VEGF via binding of HIF-1 to HRE (hypoxia re-sponse element) on VEGF genes [2]. Palayoor et al. [17] have shown that higher concentrations of NSAIDs, than those needed to inhibit prostaglan-din synthesis, can reduce VEGF formation by af-fecting HIF-1α. In the present study, valdecoxib at 10 and 100 µM not only did not block VEGF gene-ration in the absence or presence of LPS but also, when used at the higher concentration of 100 µM, it stimulated VEGF production in HMEC-1 cells.

In the next set of experiments, the influence of valdecoxib (at 100 µM) on the generation of LPS- -stimulated bFGF in HMEC-1 cells was studied (Fig. 2). In the previous study, the lack of a CoCl2

effect on bFGF production in HMEC-1 cells was determined [14], therefore in this study valdeco-xib was used only in the presence of LPS. In this study, veldecoxib increased bFGF generation in a statistically significant manner only when used at 10 µM in the presence of LPS (100 µg/mL). In contrast to valdecoxib used at 10 µM, at a higher concentration (100 µM) valdecoxib inhibited the secretion of bFGF in the presence and absence of LPS in tested cells. Akarasereenont et al. demon-strated that LPS, as a potent inflammatory medi-ator, is responsible for stimulation of COX-2 and prostaglandins production in bovine aortic endo-thelial cells (BAEC) [18]. Thus, application of the selective COX-2 inhibitor evoked reduction of the bFGF levels in HMEC-1 cells that may indicate the participation of prostaglandins in bFGF syn-thesis. The inhibitory effect on bFGF of the high-er concentration of valdecoxib in this exphigh-eriment may be associated with the anti-angiogenic effect of valdecoxib. bFGF secreted by endothelial cells (HMEC-1 cells) and under inflammatory condi-tions (induced by LPS) can also affect other, non-endothelial cells. It is also known as a chemotac-tic and mitogenic factor for smooth muscle cells (SMC) [19]. The bFGF-evoked increase of SMC migration and proliferation underlies the mecha-nism responsible for atherosclerotic plaque forma-tion [4]. In this study LPS-induced inflammaforma-tion

evoked bFGF and VEGF secretion, which could stimulate SMC. Moreover, the decrease of bFGF in the presence of valdecoxib at the higher concentra-tion (100 µM) could inhibit the migraconcentra-tion and pro-liferation of SMC induced by LPS.

Incubation of endothelial cells with cobalt chlo-ride at 200 µM decreased cell survival in a statistical-ly significant manner (p < 0.05) but valdecoxib did not influence the viability of HMEC-1 cells under chemical hypoxia (Fig. 3). Moreover, 100 µg/mL LPS stimu lated proliferation of cells after 24-h incubation, which was attenuated by the selective COX-2 inhib-itor. Application of valdecoxib reduced HMEC-1 cell survival in the presence of LPS (100 µg/mL) and the observed effect of the drug was dose-dependent. These results are in close agreement with those of Flis et al. [13] who demonstrated that sulindac sul-fide (a non-selective COX-2 inhibitor) and celecoxib (a selective COX-2 inhibitor) inhibited the survival of HMEC-1 cells and induced their apoptosis. A simi-lar effect was shown by Niederburger et al. [20] in human umbilical vein endothelial cells (macrovessel-derived endothelial cells).

In conclusion, the anti-angiogenic effect of the selective COX-2 inhibitor is the result of both the inhibition of HMEC-1 proliferation and bFGF ge-neration under inflammatory conditions. Prosta-glandins (products of COX-2) are responsible for proliferation of endothelial cells [21, 22]. LPS can activate COX-2 and PGs formation [18, 23] but NSAIDs inhibit COX-2 and therefore they can re-duce endothelial cell survival. The obtained results and previous studies provide evidence that NSAIDs, due to inhibition of COX, can have an anti-angio-genic effect. NSAIDs can also inhibit bFGF forma-tion and proliferaforma-tion of microvascular endothe-lial cells (HMEC-1 cells) thus the inhibitory effect of these drugs on migration and proliferation pro-cesses can also be observed in other, non-endothe-lial cells. However, whether the observed valdeco-xib action in HMEC-1 can be described by a similar mechanism is still an open question and needs fur-ther studies. Angiogenesis and cell migration have been found to occur in the pathogenesis of rheuma-toid arthritis [24]. NSAIDs, including valdecoxib, are used in the treatment of rheumatoid arthritis; therefore their anti-angiogenic action can have an additional therapeutic importance [25].

The author concluded that valdecoxib inhibi-ted the LPS-induced proliferation of endotheli-al cells in a dose-dependent manner. The anti-an-giogenic effect of valdecoxib was associated with inhibiting bFGF secretion. Valdecoxib stimulated VEGF formation under inflammatory conditions.

Acknowledgments. The author thanks Prof. J.Z. Nowak for his valuable comments during the research. I thank

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Address for correspondence:

Anna Wiktorowska-Owczarek Department of Pharmacology

Chair of Pharmacology and Clinical Pharmacology Medical University of Lodz

Żeligowskiego 7/9 90-752 Łódź Poland

E-mail: [email protected]

Conflict of interest: None declared