
 strains , cell lines and media - escherichia coli dh5 
 ( invitrogen , usa ) was used for the general propagation of plasmids and e. coli 
 bl21 ( de3 ) was used to express the e2 protein .
bacterial 
 cells were grown under agitation at 37c in a low - salt luria - bertani medium containing 
 zeocin at a final concentration of 25 g / ml .
yeast cultures 
 were maintained in a yeast extract - peptone - dextrose ( yepd ) medium .
the media for growth 
 and induction were buffered complex glycerol medium ( bmgy ) and buffered complex methanol 
 medium ( bmmy ) , respectively , both at ph 4.0 huvecs ( atcc crl-2873 ) were 
 grown in rpmi-1640 medium ( sigma aldrich , usa ) containing 10% foetal bovine serum and a 
 mix of antibiotics and antifungals ( sigma aldrich ) .
the cultures were kept at 37c and 
 5% co2 and disassociated from the culture dish using trypsin . 

cloning , expression and purification of e2 protein in e. coli - hcv 
 cdna was obtained from viral rna extracted with the qiamp viral rna mini kit ( qiagen , 
 usa ) , according to the manufacturer s protocol , using pooled sera from individuals with 
 hcv genotype 1a provided by the laboratory of clinical immunology of the pharmaceutical 
 science school of araraquara , so paulo , brazil .
the hcv sequence was found by 
 comparison using the blastn local alignment program and its orf was entirely sequenced . 
 to express recombinant e2 protein , the soluble form of the protein without the 
 transmembrane domain
the mature orf was amplified with 
 the forward primer 5-ggccatgggggaaacccacgtcaccgg-3 and reverse 
 primer 5-gctcgaggctcggacctgtccctgtc-3 ( the underlined bases 
 indicate introduced restriction sites for ncoi and 
 xhoi , respectively ) ( rodrguez - rodrguez et al .
the pet42a plasmid was used to generate 
 the mature e2 protein orf flanked by glutathione s - transferase ( gst ) at the n - terminus 
 and a 6x his tag at the c - terminus .
the transformed e. coli bl21 were 
 induced for 3 h with isopropylthio--galactoside ( final concentration 0.4 mm ) at 37c 
 and 250 rpm when the optical density ( od ) at 600 nm reached 0.5 .
the cells were 
 pelleted , suspended in lysis buffer ( 10 mm tris - hcl , 50 mm nah2po4 
 and 100 mm nacl , ph 8.0 ) and subjected to sonication ( 5 pulses of 1 min each ) .
the 
 soluble phase was purified using glutathione sepharose 4 fast flow ( ge healthcare , usa ) . 

the binding buffer employed was 10 mm tris - base , 50 mm sodium phosphate and 100 mm 
 sodium chloride at ph 8.0 .
the gst - tagged protein was eluted with a two - fold resin 
 volume of elution buffer ( 10 mm reduced glutathione and 50 mm tris - hcl , ph 8.0 ) .
the 
 fractions containing the purified protein were dialysed against phosphate - buffered 
 saline ( pbs ) ( ph 8.0 ) , quantified using the pierce bca protein assay kit 
 ( thermo scientific , usa ) and stored at -20c .
cloning , expression and purification of recombinant protein in p. 
 pastoris - the e2 protein orf was cloned into ppicza and the mature orf was 
 amplified with the forward primer 
 5-aagaattcgaaacccacgtcaccgggggaa-3 and the reverse primer 
 5-aatctagattctcggacctgtccctgtcttcc-3 ( the underlined bases 
 indicate introduced ecori and xbai restriction sites , 
 respectively ) .
the cloning was performed to create a recombinant plasmid containing the 
 e2 protein orf flanked by the secretion signal peptide ( -factor ) at the n - terminus and 
 a 6x his tag at the c - terminus . before p. pastoris transformation ,
the 
 recombinant plasmid was linearised with pmei endonuclease and 
 introduced into the yeast by electroporation ( 1.5 kv , 25 f , 200  ) ( cregg 2007 ) ) .
transformants were cultivated in solid 
 yepd with 1 m sorbitol and 100 g / ml zeocin .
the yeast transformants were screened for 
 protein induction in 24-well plates ( boettner et al . 

( 2012 ) ) , differing only in the 
 use of bmgy and bmmy medium buffered with 100 mm mcilvaine s buffer , ph 4.0 .
the 
 supernatant was dialysed against pbs buffer ( ph 8.0 ) , concentrated using the labscale 
 tff system ( membrane pellicon xl50 , millipore , usa ) until 10-fold reduction and stored 
 at -20c .
cell viability - for the determination of cell viability , huvecs were 
 seeded at 5 x 10 cells / ml . the adherent cells were incubated for 24 h at 
 37c and 5% co2 with the recombinant proteins ( e2b and e2y ) at 250 , 125 , 
 62.5 , 31.25 , 15.63 and 7.81 g / ml or with 1.0 g / ml lipopolysaccharide ( lps ) , 10 ng / ml 
 tumour necrosis factor alpha ( tnf- ) , 10% sodium dodecyl sulfate ( sds ) ( positive 
 controls ) or rpmi medium and the culture supernatant of e. coli bl21 
 cells ( negative controls ) .
after incubation , the cells were incubated with 3-(4 , 
 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ( 1 mg / ml ) for 3 h. the resultant 
 formazan salt was dissolved in acidic 2-propanol and the od was measured ( 540/620 nm 
 filters ) .
the od of the untreated cells was taken as 100% cell viability ( mosmann 1983 ) ) .
cell death - huvecs were seeded at 5 x 10 cells / ml and 
 incubated for 24 h at 37c and 5% co2 with recombinant e2b and e2y using the 
 concentrations and controls as described in the cell viability section . 

the following controls were also added : cells without stimulation ( negative ) and annexin 
 and propidium iodide ( pi ) controls .
the evaluation of cell death was performed using the 
 annexin v - fitc annexin v apoptosis detection kit ( bd pharmingen , usa ) according to the 
 manufacturer s protocol .
the cells were analysed by flow cytometry ( using a facscanto 
 flow cytometer , bd biosciences and facsdiva software v.6.1.3 ) . in each run
n - acetylcysteine ( nac ) treatment - the effect of nac on cells exposed 
 to e2 recombinant proteins was studied with respect to apoptosis ( parp cleavage ) and no 
 and hydrogen peroxide ( h2o2 ) production .
huvecs were pre - incubated 
 with 5 mm nac for 1 h and treated with e2y , e2b and controls as described above . 

parp cleavage - huvecs at 5 x 10 cells / ml were 
 pre - incubated in the presence or absence of nac ( 5 mm ) for 1 h and incubated for 24 h at 
 37c and 5% co2 with recombinant e2 proteins ( e2b and e2y ) using the 
 concentrations and control stimuli described in the cell viability 
 section .
huvecs were lysed in 10 mm tris ( ph 7.4 ) , 1 mm edta , 0.5 mm egta , 150 mm nacl , 
 1% triton x-100 , 50 mm naf , 10 mm 
 na4p2o710h2o , 5 g / ml aprotinin , 5 g / ml 
 leupeptin and 1 mm pmsf . to evaluate cell apoptosis ,
20 g of lysate protein was 
 electrophoresed in 8% sds - polyacrylamide gels and transferred onto nitrocellulose 
 membranes ( hybond ecl ) .
the membranes were blocked with 5% skim milk in tris - buffered 
 saline ( tbs ) containing 0.1% tween-20 ( tbs - t ) and subsequently incubated with rabbit 
 parp antibody ( 1:2000 dilution , santa cruz biotechnology , inc , usa ) overnight at 4c to 
 detect full - length parp ( 116 kda ) and cleaved parp ( carboxyl - terminal catalytic 
 fragment , 89 kda ) . after washing with tbs - t for 1 h at room temperature ( rt )
, the 
 membranes were further incubated with a horseradish peroxidase - conjugated rabbit 
 polyclonal antibody ( 1:2000 dilution ; santa cruz biotechnology , inc ) for 2 h followed by 
 1 h washing ( with 3 - 5 wash buffer changes ) .
actin levels were used to control for protein levels and were detected with an 
 antibody against actin ( yang et al . 2004 ) ) . 
 no production - total no production was determined in the culture 
 supernatant of huvecs seeded at 5 x 10 cells / ml incubated with e2b and e2y 
 and controls as described in the cell viability section for 24 h at 
 37c and 5% co2 .
the samples were measured in a no analyser ( sievers nitric 
 oxide analyzer overview , model noa 280i , ge analytical instruments , usa ) , in which the 
 nitrites , nitrates and nitrosothiols present in the supernatant were converted into no 
 by a saturated solution of vanadium trichloride in 0.8 m hcl at 90c .
no was detected by 
 a chemiluminescent reaction in the gas phase between no and ozone ( archer 1993 , jaiswal et al . 

this 
 reaction is based on l - arginine hydrolysis by arginase in cell lysates ( corraliza et al .
briefly , huvecs were 
 cultured with recombinant proteins , lps , tnf- and culture medium or culture supernatant 
 of e. coli bl21 cells using the concentrations and stimuli described in 
 the cell viability section .
the cells were lysed using 100 l of 0.1% 
 triton x-100 for 30 min under agitation .
subsequently , 50 l of cell lysate was added to 
 50 l of 25 mm tris - hcl and 25 l of 100 mm mncl2 and the final solution was 
 incubated for 10 min at 56c for enzyme activation .
next , 50 l of 0.5 m l - arginine ( ph 
 9.7 ) was added and the test reaction was incubated at 37c for 60 min .
the reaction was 
 stopped by adding 400 l of stop solution ( 96% h2so4 , 85% 
 h3po4 and water , at a proportion of 1:3:7 v / v / v ) .
twenty - five 
 microlitres of 9% -isonitrosopropiophenone in 100% ethanol was added and the reaction 
 was incubated at 95c for 30 min . finally , the cells were incubated at rt for 10 min and 
 the absorbance was measured using a 540 nm filter .
the urea concentration was calculated 
 using a linear equation generated by known quantities of urea .
one unit of enzyme 
 activity was defined as the amount of enzyme capable of producing 1 mol of urea per 
 minute . 

h 
 2 
 o 
 2 
  production - huvecs at 5 x 10cells / ml were incubated for 
 2 h at 37c and 5% co2 with recombinant e2b and e2y using the concentrations 
 and stimuli described in the cell viability section .
approximately 600 
 ng / ml dihydrorhodamine 123 ( dhr ) ( sigma - aldrich ) was added and the cells were incubated 
 at 37c for 10 min .
the cells were washed with pbs ( ph 7.2 ) and centrifuged for 5 min at 
 300 g. the supernatant was discarded and the cells were resuspended in 
 150 l of pbs ( ph 7.2 ) .
the samples were read in the fl1 channel using a facscanto flow 
 cytometer ( bd biosciences ) and facsdiva software v.6.1.3 .
the experiment included a 
 control for spontaneous fluorescence ( cells only ) and a control for spontaneous 
 production of h2o2 ( dhr and cells without stimuli ) ( walrand et al .
il-8 , tnf- and vascular endothelial growth factor a ( vegf - a ) 
 production - huvecs at 5 x 10 cells / ml were incubated for 24 h 
 at 37c and 5% co2 with recombinant e2b and e2y using the concentrations and 
 controls described in the cell viability section . an additional control 
 using pma ( 0.50 m ) was used in the tnf- detection assay .
the negative control 
 consisted of 300 l of culture medium and 300 l of pbs ( ph 7.2 ; medium of the protein 
 dilution ) .
il-8 , tnf- and vegf - a production was measured by elisa using the kit 
 human vegf - a platinum elisa ( ebioscience inc , usa ) , according to the manufacturer s 
 instructions .
the results are expressed in pg / ml . 
 statistical analysis - the data were analysed by anova using a 5% level 
 of significance followed by multiple comparisons with the tukey test and graphic 
 representation of the data .
the recombinant e2 proteins were expressed in two different expression systems , the 
 e. coli rosetta strain ( e2b ) and the p. pastoris 
 km71h strain ( e2y ) ( fig .
1 ) . 
 the e2b protein exhibited a molecular weight of approximately 63.5 kda due to its 
 expression as a fusion protein with gst ( 26 kda ) and the 6x his tag ( 1 kda ) .
the e2y 
 protein exhibited a molecular weight of approximately 50.0 kda due to its expression as 
 a fusion protein with the 6x his tag ( 1 kda ) .
the n - glycosylation of the e2y protein was 
 confirmed by protein treatment with a peptide - n - glycosidase , pngase f ( new england 
 biolabs , usa ) , according to the manufacturer s protocol .
the proteins exhibited 
 different molecular weights ( e2b = 36.5 kda and e2y = 49 kda ) due to the types of 
 protein processing used in these two systems . 
 fig . 1 : produced envelope glycoprotein 2 ( e2 ) recombinant proteins ( sodium 
 dodecyl sulfate polyacrylamide gel electrophoresis 12% ) .
channel 1 : molecular 
 weight marker [ benchmarktm protein ladder ( 10 - 220 kda ) , invitrogen ] ; 2 : e2b 
 ( approximately 63.5 kda ) ; 3 : e2y ( approximately 50 kda ) . 
 
we observed that some concentrations of the recombinant e2 proteins ( e2y : 62.5 - 250 g / ml 
 and e2b : 125 - 250 g / ml ) were slightly cytotoxic to huvecs ( fig .
2 ) . at 250 g / ml , the decrease in viability was similar to that 
 obtained when the cells were incubated with lps or tnf-. the cellular events provoked 
 by the e2 proteins were evaluated using annexin v and pi assays , which indicated that 
 early apoptosis was the main cause of cell death ( fig . 

however , even at the highest concentration of e2 protein used in this 
 study , the proportion of apoptotic cells was always lower than 30% . 

2 : effect of envelope glycoprotein 2 ( e2 ) recombinant proteins on human 
 umbilical vein endothelial cells viability [ 3-(4 , 
 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay ] .
c- : negative control ; c+ : culture medium rpmi ; lps : 
 lipopolysaccharide ( 1.0 g / ml ) ; sup : culture supernatant escherichia 
 coli bl21 ; tnf- : tumour necrosis factor alpha ( 10 ng / ml ) ; * : p 
 <
0.05 compared to the negative control ; * * * : p < 0.001 compared to the 
 negative control . 
 

3 : cytotoxicity of envelope glycoprotein 2 ( e2 ) proteins on human umbilical 
 vein endothelial cells .
results presented as mean and standard deviation of 
 percentage obtained in the assay . in each run , 30,000 cells were analysed and 
 all experiments were performed in triplicate .
c- : negative control ; early 
 apoptosis : annexin v stained cells ; late apoptosis : cells double - positive for 
 annexin v and propidium iodide ( pi ) ; lps : lipopolysaccharide ( 1.0 g / ml ) ; 
 necrosis : cells stained with pi ; sup : culture supernatant escherichia 
 coli bl21 ;
tnf- : tumour necrosis factor alpha ( 10 ng / ml ) ; * * * : p 
 < 0.001 compared to the negative control . 
  to further explore the mechanism of e2 protein - induced apoptosis , we investigated the 
 degradation of parp , which is thought to be one of the targets of activated caspase-3 or 
 7 during apoptosis ( yang et al .
immunoblot analysis revealed that the recombinant e2 proteins induced the degradation of 
 endogenous 116 kda parp , as shown by the appearance of 89 kda fragments ( fig .
4a , b ) , 
 which were clearly detected in all samples treated with e2 protein or with the control 
 stimuli , tnf- ( fig .
these results indicate that parp 
 cleavage is associated with e2-induced apoptosis in huvecs . moreover , the pre - treatment 
 of the cells with the antioxidant nac protected against apoptosis by preventing parp 
 cleavage ( fig .
4 : parp cleavage in recombinant envelope glycoprotein 2 ( e2)-induced 
 apoptosis in human umbilical vein endothelial cells .
cells were pre - treated ( c , 
 d ) or no ( a , b ) with n - acetylcysteine ( nac ) for 1 h and 
 incubated with recombinant e2 proteins , e2y ( a , c ) and e2b ( b , d ) in different 
 concentrations .
line 2 : 7.81 
 g / ml ; 3 : 15.63 g / ml ; 4 : 31.25 g / ml ; 5 : 62.5 g / ml ; 6 : 125 g / ml ; 7 : 250 
 g / ml ; a - d1 : lysate of untreated cells ; e1 : cells without pre - treatment with 
 nac and treated with tumour necrosis factor alpha ( tnf- ) ; e2 : cells 
 pre - treated with nac and treated with tnf- ; e3 : cells without pre - treatment 
 with nac and treated with lipopolysaccharide ( lps ) ; e4 : cells pre - treated with 
 nac and treated with lps ; f1 - 4 : actin . 
 
statistical analysis of the results revealed 
 that there was a statistically significant difference ( p < 0.01 ) when compared with 
 the spontaneous control ( negative control ) or production stimulated by the bacterial 
 supernatant .
the e2 proteins were as effective as the classical stimuli , lps and tnf-. 
 one exception was the treatment with 7.81 g / ml of e2 protein , in which only a slight , 
 but significant elevation in no was observed relative to the more potent lps and tnf- 
 stimuli .
the higher production of no was not the result of increased or decreased 
 arginase activity relative to the negative control ( result not shown ) because no 
 significant differences were observed when compared with the negative control .
our data 
 also demonstrated that pre - treatment with nac significantly decreased ( p < 0.01 ) the 
 e2 protein - induced no production . 

fig . 5 : nitric oxide ( no ) production by envelope glycoprotein 2 ( e2)-stimulated 
 human umbilical vein endothelial cells .
c- : no spontaneous production , cells and culture medium ; lps : 
 lipopolysaccharide ( 1.0 g / ml ) ; nac : n - acetylcysteine ; sup : 
 culture supernatant escherichia coli bl21 ; tnf- : tumour 
 necrosis factor alpha ( 10 ng / ml ) ; * * * : p < 0.001 compared to the negative 
 control . 
 
the production of h2o2 was evaluated in huvecs after exposure to 
 recombinant proteins at different concentrations and control stimuli .
the relative 
 production of h2o2 , calculated as the mean fluorescence intensity , 
 is presented in fig .
the e2 proteins were able 
 to stimulate the production of h2o2 at all of the tested 
 concentrations .
again , the production was similar to that obtained by stimulation with 
 lps and was inferior to that of tnf-. of the two e2 proteins , e2y was more effective 
 than e2b at concentrations of 7.81 g / ml ( p < 0.05 ) .
the results of pre - treatment 
 with nac revealed a significant decrease ( p < 0.01 ) in the e2 protein - induced 
 h2o2 production . 

fig . 6 : hydrogen peroxide production by envelope glycoprotein 2 ( e2)-stimulated 
 human umbilical vein endothelial cells .
results presented as mean and standard 
 deviation of the mean fluorescence intensity ( mfi ) . the experiments were 
 performed in triplicate .
( 1.0 g / ml ) ; nac : 
 n - acetylcysteine ; sup : culture supernatant escherichia 
 coli bl21 ; tnf- : tumour necrosis factor alpha ( 10 ng / ml ) ; * * * : p 
 < 0.001 in relation to negative control . 
 
the e2 proteins were capable of inducing the production of il-8 compared with 
 non - stimulated cells .
7 . there was a statistically significant 
 difference ( p < 0.05 ) between all of the stimuli tested compared with the negative 
 control . unlike the il-8 results ,
the e2 proteins were not able to induce the production 
 of tnf- or lps by huvecs .
however , 0.50 m pma induced huvecs to produce 173.05 pg / ml 
 tnf-. 
 fig .
7 : interleukin-8 ( il-8 ) production by envelope glycoprotein 2 
 ( e2)-stimulated human umbilical vein endothelial cells .
c- : 
 cells and medium and phosphate - buffered saline ( ph 7.2 ) . lps : 
 lipopolysaccharide ( 1.0 g / ml ) ; sup : culture supernatant escherichia 
 coli bl21 ; tnf- : tumour necrosis factor alpha ( 10 ng / ml ) ; * * * : p 
 < 0.001 compared to negative control ; * : p < 0.05 compared to negative 
 control . 
  the detection of vegf - a production by huvecs in response to control stimuli and 
 recombinant proteins is presented in fig .
the 
 e2 proteins significantly induced ( p < 0.01 ) the production of vegf - a by huvecs . 

fig . 8 : vascular endothelial growth factor a ( vegf - a ) production by envelope 
 glycoprotein 2 ( e2)-stimulated human umbilical vein endothelial cells . results 
 presented as mean and standard deviation .
lps : lipopolysaccharide ( 1.0 g / ml ) ; tnf- : tumour necrosis factor 
 alpha ( 10 ng / ml ) ; sup : culture supernatant escherichia coli 
 bl21 ; c- : cells and medium and phosphate - buffered saline ( ph 7.2 ) ; * * : p < 
 0.01 compared to the negative control ; * * * : p < 0.001 compared to the 
 negative control . 
  the e2 protein - induced production of no
, h2o2 , il-8 and vegf by 
 huvecs strongly supports the cytotoxicity of these proteins .
there is evidence that endothelial cells are directly susceptible to infection by hcv 
 ( fletcher et al . 2012 ) ) and that the damage 
 caused by the infection leads to late complications , such as fibrosis , cirrhosis and 
 hepatocellular carcinoma .
these late complications are believed to be caused by numerous 
 inflammatory molecules in response to viral infection of the liver ( ming - ju et al .
consistent with this 
 hypothesis , we found that e2 proteins were able to induce apoptosis and several 
 inflammatory responses in huvecs .
the putative receptors for e2 proteins in this 
 cellular type have been described previously , including low - density lipoprotein receptor 
 ( agnello et al . 1999 ) ) , tetraspanin cd81 ( zhang et al .
2004 ) ) , scavenger receptor class b type 
 1 ( scarselli et al . 2002 ) ) , claudin-1 ( evans et al . 2007 ) ) , occludin ( ocln ) ( ploss et al . 2009 ) ) and transferrin receptor 1 
 ( tfr1 ) ( martin & uprichard 2013 ) ) .
no is an inorganic free radical molecule ( furchgott 
 & zawadzki 1980 ) ) that is highly diffusible and reactive ( bredt & snyder 1992 ) ) and is involved in various 
 physiological functions and pathological conditions when produced in excess ( kaufman 1999 , benali - furet et al .
1997 ) ) , which may play 
 an important role in the pathogenesis of cirrhosis associated with infection ( hassan et al . 2002 ) ) . here
, we have demonstrated for 
 the first time that no production by huvecs was induced by both recombinant e2 proteins . 

this no production may lead to later inflammation in the portal vein and subsequent 
 fibrosis and cirrhosis .
the increased no production could be the consequence of the increased expression of 
 arginase in huvecs .
hcv infection is associated with the development of hepatocellular 
 carcinoma ( okuda 2007 , tan et al .
2008 ) ) and can alter the expression of arginase , thereby 
 stimulating tumourigenesis and hepatocellular carcinoma ( cao et al .
. however , this pathway does not appear to be relevant to 
 endothelial cells because arginase expression was not altered by the e2 proteins .
the e2 proteins were also able to induce the production of h2o2 by 
 huvecs .
this is additional evidence of the role of e2 in the inflammatory response 
 mediated by hcv .
( 2011 ) ) and suggests the involvement of the e2 
 protein in h2o2 production and the development of inflammation in 
 the hepatic portal vein , with the increased expression of factors related to hepatic 
 fibrosis . 

( 2003 ) ) reported that the 
 hcv e2 protein was able to stimulate intracellular signalling pathways , leading to the 
 induction of secretion of pro - inflammatory cytokine il-8 .
il-8 is also observed in the serum of patients with 
 chronic hepatitis c ( polyak et al .
2001 , akbar et al . 2011 ) ) , demonstrating a correlation 
 between inflammation , il-8 serum levels and liver fibrosis ( kaplanski et al .
consistent with this hypothesis , recombinant e2 proteins stimulated the 
 production of il-8 in huvecs .
( 2005 ) ) . however , the e2 
 proteins were not able to induce the production of tnf- by huvecs . these results are 
 also consistent with the work of balasubramanian et al . 

( 2005 ) ) , who reported that hcv proteins can interact with the endothelium and 
 that e2 protein did not induce the production of cytokines such as monocyte chemotactic 
 protein-1 , tnf- and gamma interferon .
analysis of peripheral blood mononuclear cells and liver biopsy samples of individuals 
 chronically infected by the virus suggests that hcv infection may be able to induce 
 apoptosis , causing damage to the liver while helping the virus to evade the immune 
 system and facilitate viral dissemination ( hiramatsu et 
 al .
, we found that the e2 
 proteins were also able to induce apoptosis ( early and late ) as well as necrosis ( fewer 
 cells ) in huvecs .
the glycosylated protein expressed in 
 p. pastoris ( e2y ) was a more effective inducer of apoptosis as well 
 as necrosis relative to the non - glycosylated protein ( e2b ) , demonstrating the influence 
 of glycosylation on apoptosis .
moreover , the apoptosis induced by recombinant e2 protein was 
 effectively rescued in cells pre - treated with nac , suggesting that the generation of 
 reactive oxygen species is involved in e2-induced apoptosis in huvecs .
we also suggest 
 that the production of no , h2o2 , il-8 and vegf - a were not related 
 to cell death induced by high concentrations of the recombinant protein , but are 
 e2-specific effects .
vegf - a is a potent angiogenic factor that plays a key role in the development of 
 angiogenesis in various tumour types ( toi et al . 
 2000 ) ) , including hepatocellular carcinoma ( ng 
 et al . 2001 , poon et al . 2004 ) ) .
vegf - a 
 has a specific angiogenic effect on endothelial cells and can be stimulated by hcv 
 infection ( dvorak et al .
additionally , 
 vegf - a plays a role in the regulation of several cellular functions , including growth 
 ( nasimuzzaman et al . 2007 ) ) and apoptosis 
 ( hglinger et al . 2007 ) ) .
hepatic angiogenesis has been described in viral 
 hepatitis , autoimmune liver cirrhosis , primary biliary cirrhosis and hepatocellular 
 carcinoma ( garca - monzn et al .
hcv stimulates the synthesis and 
 secretion of vegf - a via virus - induced oxidative stress ( nasimuzzaman et al .
2007 ) ) . in our study , the 
 exposure of huvecs to both recombinant e2 proteins induced the production of vegf - a .
we 
 suggest that oxidative stress , as demonstrated by the production of no and 
 h2o2 in huvecs in response to e2 proteins , may represent the 
 stimulating factor of vegf - a production .
the literature reports that the hcv core 
 protein is able to stimulate the production of vegf - a , but there are no data regarding 
 the e2 protein ( hassan et al .
therefore , this is the first 
 demonstration that the e2 protein is also able to induce the production of vegf - a and , 
 consequently , angiogenesis .
hcv is a positive - stranded rna virus that is unable to integrate its genetic material 
 into the host cell genome .
the hcv genome does not contain oncogenes , suggesting that 
 hcv induces hepatocellular carcinoma indirectly by causing chronic inflammation , cell 
 death , proliferation and cirrhosis ( hassan et al . 

, we provide evidence that endothelial cells , such as huvecs , are 
 susceptible to e2 hcv envelope proteins . in conclusion , stimulation with e2 protein induced huvecs to produce inflammatory and 
 angiogenic factors . considering that endothelial inflammation is a determinant of 
 fibrosis progression and cirrhosis , we propose that these cellular effects might be 
 involved in the persistence and chronicity of hcv infection . these results
may 
 contribute to our understanding of the pathophysiology of hepatitis c and , consequently , 
 to the development of new therapeutic strategies against the interaction of hcv 
 structural proteins and the hepatic endothelium .
 the hepatitis c virus ( hcv ) encodes approximately 10 different structural and 
 non - structural proteins , including the envelope glycoprotein 2 ( e2 ) . 
 hcv proteins , 
 especially the envelope proteins , bind to cell receptors and can damage tissues . 
 
 endothelial inflammation is the most important determinant of fibrosis progression 
 and , consequently , cirrhosis . 
 the aim of this study was to evaluate and compare the 
 inflammatory response of endothelial cells to two recombinant forms of the hcv e2 
 protein produced in different expression systems ( escherichia coli 
 and pichia pastoris ) . 
 we observed the induction of cell 
 death and the production of nitric oxide , hydrogen peroxide , interleukin-8 and 
 vascular endothelial growth factor a in human umbilical vein endothelial cells 
 ( huvecs ) stimulated by the two recombinant e2 proteins . 
 the e2-induced apoptosis of 
 huvecs was confirmed using the molecular marker parp . 
 the apoptosis rescue observed 
 when the antioxidant n - acetylcysteine was used suggests that 
 reactive oxygen species are involved in e2-induced apoptosis . 
 we propose that these 
 proteins are involved in the chronic inflammation caused by hcv . 