in vitro effects of il-17 on angiogenic properties of endothelial cells in relation to oxygen levels

Cell Biology International ISSN 1065-6995doi: 10.1002/cbin.10144

RESEARCH ARTICLE

In vitro effects of IL-17 on angiogenic properties of endothelial cells inrelation to oxygen levelsJelena Krsti�c1*, Aleksandra Jaukovi�c1, Slavko Mojsilovi�c1, Ivana Oki�c Ðord-evi�c1, Drenka Trivanovi�c1,Vesna Ili�c2, Juan F. Santiba~nez1 and Diana Bugarski1

1 Laboratory for Experimental Hematology and Stem Cells, Institute for Medical Research, University of Belgrade, Dr. Suboti�ca 4, P.O. Box 102, 11129Belgrade, Serbia2 Laboratory for Immunology, Institute for Medical Research, University of Belgrade, Belgrade, Serbia

Abstract

The aim of this study has been to elucidate how different oxygen levels impact the effects of Interleukin-17 (IL-17) on angiogenicproperties of endothelial cells. Two endothelial cell lines, mouse MS-1 and human EA.hy 926, were grown in 20% and 3% O2

and their angiogenic abilities analyzed after IL-17 treatment: proliferation, apoptosis, migration and tubulogenesis. Expressionof endothelial nitric oxide synthase (eNOS) and cyclooxygenase-2 (Cox-2) was also measured. Considering EA.hy 926 cell line,hypoxia alone reduced proliferation, survival andmigration, but not their ability to form tubules. When cultured at 20%O2, IL-17 stimulated proliferation, migration and tubulogenesis, whereas a hypoxic environment did not affect their migration andproliferation, but increased their survival and tubulogenic properties. Expression of eNOS and Cox-2 increased by both IL-17and hypoxia, as well as with their combination. With the MS-1 cell line hypoxia did not affect proliferation, survival, migrationand tubule formation. At 20% O2, IL-17 did not alter their proliferation,but inhibited migration and stimulated tubuleformation. At 3%O2, only the stimulating effect of IL-17 on tubulogenesis was evident. The constitutive expression of eNOSwasunaffected by oxygen concentrations or IL-17 supplementation, whereas both IL-17 and hypoxia upregulated Cox-2 expression.Thus the effects of IL-17 on the angiogenic properties of endothelial cells depend on both the cell line used and the oxygenconcentration.

Keywords: angiogenesis; endothelial cells; EA.hy 926; hypoxia; IL-17; MS-1

Introduction

Endothelial cells present a very quiescent cell type(Fong, 2009; Mu~noz-Chápuli et al., 2004). However, muchinformation from their environment can eventually leadendothelial cells to progress along all stages of neovasculo-genesis: cell proliferation, increased resistance to apoptosis,migration and, finally differentiation and formation of newvasculature (Mu~noz-Chápuli et al., 2004). The processesinvolved are main mechanisms of vascularisation duringembryonic development, regeneration and wound healing,but can also be involved in pathological processes, includingtumor growth andmetastasis. In addition to being affected bysecreted paracrine factors (Mu~noz-Chápuli et al., 2004),growth of endothelial cells depends on adequate tissueoxygenation, which varies between 0.5% and 14% depending

on the tissue (Ivanovic, 2009a). Since these values aresignificantly below the normal oxygen partial pressure inambient room air, they are considered hypoxia (Fong, 2009)or in situ normoxia, as suggested by Ivanovic (2009b).

Interleukin (IL) 17A is a founding member of anintensively investigated family of proinflammatory cytokinesmainly produced by Th17 cells (Gaffen, 2008). IL-17 hasmultiple biological activities on numerous cell types due tothe ubiquitous expression of its receptor (Gaffen, 2008;Ivanov and Lind�en, 2009). In addition to its proinflammatoryfunctions, IL-17 has been recognized as a potent inducer ofangiogenesis in autoimmune diseases and malignancies,acting both directly on endothelial cell migration andvasculature formation, and indirectly via induction of variousangiogenic growth factors and chemokines (Pickenset al., 2010; Takahashi et al., 2005; Liu et al., 2011; Moran

*Corresponding author: e-mail: [email protected]

1162 Cell Biol Int 37 (2013) 1162–1170 � 2013 International Federation for Cell Biology

et al., 2011; Numasaki et al., 2005; Pickens et al., 2010).However, the impact of the lower oxygen concentrations, thatis physiological hypoxia, on the effects of IL-17 onangiogenesis and endothelial cells remained unexplored,although it has already been debated that the effects ofcytokines can differ in relation to O2 concentration in themicroenvironment, and even that in vitro research com-monly conducted at 20% O2 in air could provide misleadingevidence of the cellular response to cytokines, not compara-ble with physiological conditions (Ivanovic, 2009a).

We have examined how different oxygen levels influencethe effects of IL-17 on angiogenic properties of endothelialcells. Two endothelial cell lines of different originwere used asmodel systems: MS-1, a mouse pancreatic islet endothelialcell line (Arbiser et al., 1997), and EA.hy 926, humanendothelial hybrid cell line, established by fusing humanumbilical vein endothelial cells with a human carcinoma cellline (Emeis and Edgell, 1988). Cells were grown at 20% and3%O2 and treated with IL-17. Their angiogenic abilities, thatis proliferation, survival, migration and tubulogenesis wereanalysed in relation to IL-17 and the O2 levels. Consideringthat nitric oxide (NO) and particularly endothelial NOsynthase (eNOS), have a key role in maintaining vascularintegrity and angiogenesis by modulating endothelial cellresponses (Mu~noz-Chápuli et al., 2004), the effect of IL-17and/or O2 concentration on the expression of eNOSwas also followed. In addition, the influence of thesetwo environmental factors on the expression of cyclo-oxygenase-2 (COX-2), an inducible form of the enzymeinvolved in angiogenesis (I~niguez et al., 2003), was alsoanalysed.

Materials and methods

Cell culture

EA.hy 926 and MS-1 cell lines were purchased fromAmerican Type Culture Collection (ATCC, Rockville, MD,USA). Cells were grown in growth medium (GM) consistingof Dulbecco's modified Eagle's medium (DMEM, Sigma-Aldrich, St. Louis, MO, USA), supplemented with 10% fetalbovine serum (FBS) and 100 units/mL penicillin/streptomy-cin, both from PAA Laboratories GmbH (Pasching, Austria)in humidified environment at 378C and 5% CO2 in air, whilethe oxygen concentrations used in experiments were 20% or3% in air.

Proliferation assay

Proliferation rate of both cell lines was analysed by MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-mide) test. Briefly, 5 � 103 cells/well were seeded in 96-wellplates in GM and incubated overnight to adhere to plastic.

Cells were cultured at 20 or 3% O2 for 24, 48 and 72 h in thepresence of 0, 50 and 100 ng/mL recombinant human ormouse IL-17 (R&D Systems, Minneapolis, MN, USA). Afterappropriate incubation time, MTT (Sigma-Aldrich) wasadded to cells at 0.5 mg/mL, and after 2 h of incubation theformazan crystals were dissolved in 0.1 N HCl in isopropa-nol, and absorbance read at 540 nm.

Apoptosis assay

For the assay of apoptosis, EA.hy 926 and MS-1 cells weresubjected to flow cytometry analysis after Annexin V (AnnV)-FITC and propidium iodide (PI) (both from BeckmanCoulter International SA, Nyon, Switzerland) staining.Briefly, 105 cells per well were seeded in six-well plates inGM and allowed to adhere to plastic overnight. The next day100 ng/mL IL-17 was added to the cells, and cultures wereincubated at either 20 or 3% O2. After 72 h incubation, cellswere harvested by 1 mM EDTA, washed in cold PBSsupplemented with 0.5% BSA, and aliquots of 2 � 105 cellswere labelled with Ann V-FITC and PI, as suggested by themanufacturer. Flow cytometry was performed using aCyFlow CL (Partec, Münster, Germany).

Scratch test

The motility of EA.hy 926 and MS-1 cells was analysed by invitro scratch assay. Cells were seeded in a 24-well plate in GMand incubated at 20% O2. After they had reached confluence,a scratch in the cell monolayer over the total diameter of eachwell was made using a sterile pipette tip, and culturescontinued to grow for 24 h in GM supplemented with 0, 50and 100 ng/mL IL-17, at 20 and 3% O2. Cells were fixed withice-cold methanol and stained with 0.1% crystal violet.Migration of the cells into the scratch area was documentedby light microscopy.

Endothelial cell tubule-formation assay

To analyse the tubule forming capability of endothelial cells,tubule-formation assay was used. First, a collagen gel wasprepared: 2 mL of rat-tail collagen (3.5 mg/mL) was mixedwith 2.12 mL of a solution containing: 1 mL 5-timesconcentrated DMEM without NaHCO3, 0.5 mL FBS,0.5 mL 0.26 M NaHCO3, 0.02 mL 1 M NaOH and 0.1 mLH2O. After gentle mixing, 0.88 mL of DMEM was added tothe gel. Then, 500 mL of collagen gel was added per well of24-well plates and allowed to polymerise for 1 h at 378C and5% CO2 and 20% O2. The EA.hy 926 and MS-1 cells weretrypsinised and seeded over the collagen gel at 4 � 104/100 mL of GM and incubated for 24 h at 20% O2 or 3% O2

(378C, humidified atmosphere, 5% CO2) in the absence orpresence of 100 ng/mL IL-17. Tubule formation wasdocumented by light microscopy.

J. Krsti�c et al. Effects of Il-17 in relation to oxygen levels

1163Cell Biol Int 37 (2013) 1162–1170 � 2013 International Federation for Cell Biology

Western blot assay

A specific antibody against eNOS was obtained from R&DSystems, the antibody against Cox-2 from Santa CruzBiotechnology (Santa Cruz, CA, USA), and the antibodyagainst alpha-Tubulin was purchased from Sigma-Aldrich.After cultivation and adequate treatment of both cell lines(see Results Section), total protein extracts were isolatedusing lysis buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl,1% NP-40) containing 2 mM EDTA, 50 mM NaF andprotease inhibitor cocktail (SERVA Electrophoresis GmbH,Heidelberg, Germany). Protein samples from cell lysates wereseparated by SDS-PAGE and electrotransferred onto anitrocellulose membrane. Membranes were blocked with3% bovine serum albumin (BSA) in 0.5% Tween-20 in TBSand then incubated with primary antibodies against eNOS,Cox-2 or alpha-tubulin. Secondary antibodies conjugatedwith HRP (Sigma-Aldrich) were used to detect the immunecomplexes by enhanced chemiluminescence. Labeled pro-teins were visualized with enhanced chemiluminescencereagent system from Applichem (Darmstadt, Germany).Densitometry analysis of the protein bands was performedusing ImageJ software.

Statistical analysis

Data are presented as means � SEM. The statistical signifi-cance was evaluated using the Student's t-test. Differenceswere considered to be significant when P < 0.05. Allexperiments were performed at least three times.

Results

Combined effects of hypoxia and IL-17 on endothelialcell proliferation and survival

To investigate the effect of low O2 concentration onendothelial cells proliferation and the potential role of IL-17 in regard to different O2 levels, EA.hy 926 and MS-1 cellswere cultivated at 3 and 20% O2 in the presence of 0, 50 and100 ng/mL IL-17 for 24, 48 and 72 h. MTTassay showed thatboth cell lines proliferated intensively during culture at 20%O2, but addition of IL-17 did not induce significant changes(Figure 1Aa and Ba). In the contrast, 3% O2 reducedproliferation of both cell lines at 72 h of incubation, with EA.hy 926 cells being more susceptible to the hypoxia(Figure 1Ab and Bb). IL-17 induced a dose-dependent

Figure 1 Combined effects of hypoxia and IL-17 on the endothelial cell proliferation. (A) a. Proliferation of EA.hy 926 cells determined byMTT test

after 24, 48 and 72 h incubation at 20%O2 treated with 0, 50 or 100 ng/mL recombinant human IL-17. b. Proliferation of EA.hy 926 cells determined by

MTT test after 24, 48 and 72 h incubation at 3%O2 treatedwith 0, 50 or 100 ng/mL recombinant human IL-17. Optical density values were normalized to

control (cells incubated for 24 h without IL-17). (B) a. Proliferation of MS-1 cells determined byMTT test after 24, 48 and 72 h incubation at 20%O2 with

0, 50 or 100 ng/mL recombinant mouse IL-17. b. Proliferation of MS-1 cells determined by MTT test after 24, 48 and 72 h incubation at 3% O2 treated

with 0, 50 or 100 ng/mL recombinant human IL-17. Optical density values were normalized to control (cells incubated for 24 h without IL-17). Significant

difference versus indicated control (cells cultured at 3% O2 for 72 h) by t-test: ��P < 0.01.

Effects of Il-17 in relation to oxygen levels J. Krsti�c et al.


increase in the proliferation of both cell lines after 72 h at 3%O2; however, the positive effect of IL-17 onMS-1 cells was notstatistically significant.

To confirm the IL-17 protective effect, we analysed theapoptotic profile of cells treated with IL-17 and cultivated72 h at hypoxic and normoxic conditions. As expected, bothcell lines cultured at 20% O2, either in the presence and

absence of IL-17, showed a similar profile, with a low numberof apoptotic (Ann V-positive) cells (Figure 2Aa and Ba). Asimilar apoptotic profile with low Ann V-positive cells wasobtained forMS-1 cells cultured at 3%O2, regardless of IL-17supplementation (Figure 2Bb). However, when EA.hy 926cells were incubated at 3% O2, the high number of Ann V-positive cells correlated with the significant decline detected

Figure 2 Combined effects of hypoxia and IL-17 on the endothelial cell survival. (A) a. Apoptotic profile of EA.hy 926 cells determined by Flow

cytometry after 72 h incubation at 20%O2 in the absence or presence of 100 ng/mL IL-17. b. Apoptotic profile of the EA.hy 926 cells determined by Flow

cytometry after 72 h incubation at 3% O2 in the absence or presence of 100 ng/mL IL-17. (B) a. Apoptotic profile of MS-1 cells determined by flow

cytometry after 72 h incubation at 20%O2. b. Apoptotic profile ofMS-1 cells determined by flow cytometry after 72 h incubation at 3%O2 in the absence

or presence of 100 ng/mL IL-17. Ann�/PI� cells are viable, Annþ/PI� cells are apoptotic. Significant difference versus indicated control (Annþ/PI� cells

cultured at 3% O2) by t-test:��P < 0.01. Representative flow cytometry histograms from three independent readings are presented below the graph for

each cell culture condition.



in the proliferation assay. Moreover, after IL-17 treatment,this apoptotic pattern was reversed, as the percentage ofapoptotic cells in the co-cultures with IL-17 was significantlylower in comparison to the control cells not treated with IL-17 (Figure 2Ab).

Combined effects of hypoxia and IL-17 on theendothelial cell migration

To examine the effects of IL-17 in endothelial cells exposed todifferent O2 levels, the migratory capability of the cells wasanalyzed using scratch test. Cells were allowed tomigrate intothe scratch for 24 h at 3 and 20% O2, in the presence andabsence of IL-17. The results obtained differed depending onthe cell line. For the EA.hy 926 cells (Figure 3), the addition ofIL-17 at 20% O2 resulted in a dose-dependent stimulation oftheir migratory capacity. On the other hand, the low O2

concentration itself reduced their migration; however, at 3%O2 no effect of IL-17 was observed. Concerning the MS-1 cellline, opposite effects were noticed, since IL-17 inhibited theirmigratory capacity at both O2 concentrations, with a slightly

higher inhibition observed at 20%O2 (Figure 3). The hypoxicenvironment itself did not affect MS-1 cells' migration.

Combined effects of hypoxia and IL-17 on endothelialcell tubule-formation

The angiogenic potential of EA.hy 926 and MS-1 cells wasevaluated by endothelial cell tubule-formation assay after 24 hcultivation on collagen. Low oxygen concentration did notaffect the tubule forming capability of both endothelial celllines tested. IL-17 exerted positive effect, that is stimulatedtubule formation equally at both O2 concentrations tested,and more elongated cells leading to tubule formation wereobserved after IL-17 treatment in both cell lines (Figure 4).

Combined effects of hypoxia and IL-17 on eNOS andCox-2 expression

eNOS and Cox-2 are important proteins involved in themolecular response of endothelial cells exposed to hypoxia.Therefore, we investigated whether IL-17 can modulate their

Figure 3 Combined effects of hypoxia and IL-17 on the endothelial cell migration. Migration of EA.hy 926 and MS-1 cells analyzed by scratch

assay: a scratch was made in the confluent cell monolayer, and cells cultured in the presence of 0, 50 and 100 ng/mL IL-17 at 20% O2 and 3% O2. The

migration of the cells into the scratch area was documented after 24 h using a light microscope (magnification 10�).

Figure 4 Combined effects of hypoxia and IL-17 on the endothelial cell tubule-formation. Tubule forming capacity of EA.hy 926 and MS-1 cells

was determined by growing cells over collagen gel. Cells were grown for 24 h at 20%O2 and 3%O2 in the absence or presence of IL-17 (100 ng/mL). The

tubulogenic capacity of the cells was documented after 24 h using a light microscope (magnification 40�).



expression depending on O2 levels during culture. Consider-ing EA.hy 926 cells, the low basal expression of eNOS protein(detected in cells grown at 20%O2, since this concentration iscommonly used for cultures) was increased by either thecultivation at 3% O2 or the addition of IL-17, as well as bytheir combined effect (Figure 5A). Cox-2 protein expressionshowed a similar pattern, with both IL-17 and hypoxiaincreasing its expression (Figure 5A). With MS-1 cells, therelatively high constitutive expression of eNOS protein wasunaffected by low oxygen concentration or IL-17 supple-mentation, whereas Cox-2 expression was upregulated byboth IL-17 and hypoxia (Figure 5B).

Discussion

Regarding IL-17, there is increasing evidence that IL-17 isimplicated in the induction of angiogenesis both in tumour

microenvironment and the pathogenesis of inflammatorydiseases (Ivanov and Lind�en, 2009; Moran et al., 2011; Liuet al., 2011; Pickens et al., 2010). Hypoxia alone or incombination with inflammatory cells, has also been shownto stimulate tumor angiogenesis, as well as to be characteristicfeature of the inflammatory environment (Bennewith andDedhar, 2011; Ng et al., 2010; Keith et al., 2011; Tartouret al., 2011). Although hypoxic conditions associated withinflammation promote the differentiation of Th17 cells(Dang et al., 2011; Kurebayashi et al., 2013), the combinedeffects of IL-17 and hypoxia, not only related to the process ofangiogenesis and the endothelial cell functions, are much lessrecognized and investigated. In our previous work, weshowed that the enhancing effect of IL-17 on the mainte-nance and expansion of hematopoietic cells, co-cultured withmesenchymal stem cells, was dependent upon oxygenconcentration, reaching its maximum at 3% O2 (Krsti�c

Figure 5 Combined effects of hypoxia and IL-17 on the eNOS and Cox-2 expression. (A) Expression of eNOS and Cox-2 in EA.hy 926 cells grown at

20%O2 and 3%O2 treated or not with IL-17 (100 ng/mL) for 24 h. (B) Expression of eNOS and Cox-2 in MS-1 cells grown at 20%O2 and 3%O2 treated

or not with IL-17 (100 ng/mL) for 24 h. Protein expression was determined byWestern blot assay; a-tubulin was used as a loading control. Densitometric

quantification of the Western blot bands is indicated at the top of images, expressed relative to cells incubated at 20% O2 without IL-17, to which an

arbitrary value of 1 was given. Significant difference by t-test: �P < 0.05 and ��P < 0.01 compared to the expression obtained with 0 ng/mL IL-17 at 20%

O2 and #P < 0.05 compared to the expression obtained with 0 ng/mL IL-17 at 3% O2.



et al., 2009a). IL-17A may also promote rheumatoid arthritis(RA) synoviocyte migration and invasion under hypoxia (Liet al., 2013).

We found that a hypoxic environment per se cansignificantly modify the angiogenic properties of endothelialcells, reducing their proliferation, survival and/or migration,although it should be pointed out that the effect of the lowO2

concentration differed between the two lines, with EA.hy 926cells being more susceptible. Different effects of hypoxia onendothelial cells have previously been reported, but more interms of how different oxygen levels induce differentresponses, since moderate hypoxia (e.g. 5% oxygen)promotes proliferation, survival, migration, and vascularnetwork formation via increased expression of angiogenicmolecules in endothelial cells, while more severe hypoxiaincreased apoptosis (Fong, 2009). As for the IL-17, the effectson the angiogenic properties of endothelial cells were alsodependent on the line used. However, the influence of thiscytokine was also highly dependent upon the oxygenconcentration of the cultures. Similar to our previous reporton hematopietic cells (Krsti�c et al., 2009a), IL-17 at 3% O2

had a protective effect on the proliferation and survival of EA.hy 926 cells, significantly decreasing the number of hypoxia-induced apoptotic cells. A proangiogenic role of IL-17 inhuman dermal endothelial cells has been found (Moranet al., 2011). Human lung microvascular endothelial cells(Pickens et al., 2010) is in part mediated by the induction ofcell migration. In agreement with this data, conducted atstandard 20% O2, is the promoting effect of IL-17 on the EA.hy 926 cell migration seen in our study. Although loweroxygen concentrations are referred to as physiologicalhypoxia for the endothelium, the effect of IL-17 at 3% O2

on cell migration was negligible in both cell lines.Nevertheless, while hypoxic conditions did not reduce theability of both cell lines to form tubule, IL-17 stimulated thetubulogenic properties of both cell lines cultivated at bothoxygen concentrations, thus proving its proangiogenicpotential.

NO is a well-known reactive molecule with diversephysiological functions and general ubiquity (Michel andFeron, 1997), while eNOS has been identified as a keymolecule for endothelial cell activation and proliferation.Although constitutively expressed NOS isoform, eNOS is alsosubject to expressional regulation under physiologicalconditions and in pathology (Michel and Feron, 1997),an alternative is that eNOS regulates the expression of thepro-inflammatory molecules, thereby playing a role ininflammation and angiogenesis (Ying and Hofseth, 2007).NO acts as an important mediator of the various IL-17-induced effects on different hematopoietic progenitors(Krsti�c et al., 2009b; Bugarski et al., 2004; Jovci�cet al., 2004; Krsti�c et al., 2010). Constitutive proteinexpression of eNOS was detected in both cell lines and

hypoxia itself upregulated the expression of eNOS in EA.hy926 cells, while not affecting its expression in the MS-1 cellline. Regarding IL-17, the stimulating effect on eNOSexpression in EA.hy 926 cells occurred at both 20 and 3%O2, which can be associated with the IL-17-mediatedprotective effect on the proliferation and survival of thesecells in hypoxic conditions. Namely, in cell culture modelseNOS was shown to play an essential role in endothelial cellproliferation as a central mediator of several endotheliumgrowth stimulators, as well as to exhibit anti-apoptotic effects(Ying and Hofseth, 2007).

Endothelial cells express two cyclooxygenase isoforms,Cox-1 (constitutive) and Cox-2 (inducible), which catalyzethe conversion of arachidonic acid to prostaglandin H2(Cook-Johnson et al., 2006). The pro-angiogenic role of Cox-2 is thought to be mediated through the induction of thesynthesis of prostanoids, which then stimulate the expressionof other pro-angiogenic factors (I~niguez et al., 2003). Ourresults demonstrate increased expression of Cox-2 when bothcell lines were cultured at 3% O2, similar to datademonstrating that hypoxia can augment Cox-2 expressionin various cell types, amongst which are human umbilicalvein endothelial cells (Cook-Johnson et al., 2006). Our resultsalso demonstrated that Cox-2 expression is upregulated byIL-17, being in line with previous findings showing that,although Cox-2 is usually undetectable under normalphysiological conditions, it may be expressed at high levelsfollowing stimulation with proinflammatory cytokines (Kimet al., 2011), such as IL-17 (Hirata et al., 2008).

Our data confirm that IL-17 plays an important role inmodulating key events involved in the process of angiogene-sis, such as endothelial cell proliferation, apoptosis, migrationand tubule formation. Based on the existing data, IL-17 hasbeen strongly implicated as a proangiogenic cytokine indifferent pathogenic conditions. It is known to increasetumour angiogenesis and growth (Numasaki et al., 2003,2005). Also, experiments conducted on mouse RA modelshow that IL-17 increases vascularity in mouse ankle jointsand enhances blood vessel formation in vivo (Pickenset al., 2010). Importantly, both tumour microenvironmentand synovial tissue in RA are characterised by low-oxygenlevels (Ng et al., 2010). Wounded tissues are also typicallyhypoxic and rich in infiltrated immune cells expressing highlevels of angiogenic factors (Fong, 2009), with IL-17 amongthem. Regarding all these issues, investigation of the ability ofIL-17 to modulate various endothelial cellular functionsinvolved in angiogenesis, especially in context of the lowoxygen concentrations, is of great importance.

In summary, the effects of IL-17 on the angiogenicproperties of endothelial cells are not only dependent on thecell line used, but on the oxygen concentration used incultures, supporting the concept that physiological levels ofO2, mistakenly called hypoxia, should be considered as an



important environmental factor that significantly influencescytokine activity.

Acknowledgement and Funding

This work was supported by Grant no. 175062 from theMinistry of Education, Science and Technological Develop-ment of the Republic of Serbia.

Conflict of interest

The authors declare no conflict of interest.

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Received 1 April 2013; accepted 5 June 2013.Final version published online 8 July 2013.



in vitro effects of il-17 on angiogenic properties of endothelial cells in relation to oxygen levels

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