the cyanobacteria metabolized much of toxin including microcystins ( mcs ) , which were the most widely spread [ 1 , 2 ] . in 2010 , wuxi ( in jiangsu , china ) had an outbreak of a water crisis , because of a massive bloom of the cyanobacteria microcystis spp . which occurred in lake taihu , which was the third largest freshwater lake in china .
more than 80 variants of microcystin have been found until now , and microcystin - lr ( mc - lr ) was the mostly toxic .
knowledge of mc - lr toxicity is necessary , because of both potencies of its acute cytotoxicity and tumor - inducing activity in animals and humans .
natural exposure of animals and humans to mc - lr is mostly related to drinking contaminated water .
moreover , toxicity was also reported in kidney , lung , and other cells of the human tissues .
simultaneously , endothelial cells ( ecs ) may be the most subjected to mc - lr during recreational activity and also be an important additional risk . the mc - lr strong effects on both whole organs and single cells
however , the effects of mc - lr on the ec have not been examined .
vascular endothelial integrity was influenced by ecs proliferation and apoptosis , which maintained blood vessel function .
therefore , whether mc - lr injured vessel functions via effecting ecs proliferation and apoptosis may be worth studying .
nowadays , more and more evidences implied that mc - lr leads to damage in liver and kidney via oxidative stress .
the metabolism of mc - lr in animal model induced overexpression of reactive oxygen species ( ros ) , such as superoxide radical ( o ) and hydrogen peroxide ( h2o2 ) . in physiological conditions , ros production in tissues
could be efficiently scavenged by the antioxidant enzymes and glutathione- ( gsh- ) related enzymes , such as catalase ( cat ) and superoxide dismutase ( sod ) .
but overexpression of ros could change endothelial function by many mechanisms , like peroxidation of membrane lipids and activation of nf-b . moreover , the vascular disorders induced more adhesion molecules , such as vascular cell adhesion molecule 1 ( vcam-1 ) and intracellular adhesion molecule 1 ( icam-1 ) , were thought to play an important role in controlling the pathological process of ecs inflammation . however , the effect of mc - lr cytotoxic activity induced oxidative stress was not cleared in vascular ecs yet . in this study , we treated human umbilical vein endothelial cells ( huvecs ) with mc - lr to detect the precise effects on ecs proliferation , apoptosis , and tube formation
. the possible signaling mechanisms by which mc - lr exerts its effects were also characterized .
our studies demonstrated for the first time that mc - lr induces vascular inflammation in huvecs via activation of nf-b .
mc - lr could also induce the expression of cam-1 and vcam-1 , which were associated with the inflammatory process .
all of these activated pathways may contribute to the vascular inflammatory action of mc - lr .
our findings reported that upregulation of ros and activation of nf-b signaling is a critical mode of action of mc - lr for vascular inflammation .
microcystin - lr ( purity 95% ) was purchased from the institute of hydrobiology , chinese academy of sciences , and protected from light until use .
rpmi 1640 medium and fetal bovine serum ( fbs ) were obtained from gibco ( grand island , ny , usa ) .
the cell lysis buffer ( ripa ) and electrochemiluminescence ( ecl ) kit were purchased from beyotime ( beijing , china ) .
all antibodies , including primary and horseradish peroxidase - conjugated secondary antibodies , were purchased from cell signaling technology ( beverly , ma , usa ) .
fitc - conjugated phalloidin was purchased from sigma ( st . louis , mo , usa ) .
primary huvecs were obtained from allcells ( shanghai , china ) and cells in passages 35 were used in this study .
cells were grown in an rpmi 1640 medium containing 20% fetal bovine serum ( gibco , grand island , ny , usa ) , 60  g / ml of endothelial cell growth supplement ( bd , san diego , ca ) , and 100  u / ml of penicillin with 100  g / ml of streptomycin ( gibco , grand island , ny , usa ) .
the viability of huvecs upon treatment of mc - lr was measured by a colorimetric mtt assay .
briefly , huvecs ( 2  10 cells / well ) were seeded on 24-well plates and cultured in rpmi 1640 medium containing 20% fbs for 24  h. after starvation in serum - free media for 12  h , huvecs were treated with mc - lr ( 040  m ) induced cells or an isovolumetric solvent control ( 0.1% dmso ) for 24 or 48  h. the cell number was measured using a colorimetric assay based on the ability of mitochondria in viable cells to reduce mtt .
the cell number index was calculated as the absorbance of treated cells / control cells  100% .
then the cells were treated with mc - lr ( 040  m ) for 24  h. they were then harvested , washed , and resuspended in pbs .
apoptotic cells were determined with an annexin v - fitc apoptosis detection kit ( bd biosciences , usa ) according to the manufacturer 's protocol .
apoptosis data were determined by using flowjo ( flowjo , ashland , or , usa ) .
the chemotactic motility of huvecs was determined using a transwell migration assay ( bd biosciences ) with 6.5  mm diameter polycarbonate filters ( 8  mm pore size ) .
briefly , the rpmi 1640 medium containing 10  ng / ml vegf ( 500  l ) was placed into the bottom chambers .
huvecs ( 5  10 ) were incubated with mc - lr ( 040  m ) in rpmi 1640 medium containing 0.5% fbs for 3 hours at 37c before seeding into the upper chambers . after 8  h of incubation , nonmigrated cells in the upper chamber were removed .
the migrated cells were fixed with 4% paraformaldehyde and stained with 1% calcein - am .
briefly , each well of a 96-well plate was coated with 80  l bd matrigel and incubated at 37c for polymerization .
huvecs were trypsinized and suspended in rpmi 1640 medium containing 10  ng / ml vegf ( 500  l ) medium .
various concentrations of mc - lr ( 040  m ) were added to the cells for 1  h at 37c before seeding .
cells were then plated onto the matrigel layer at a density of 2  10 cells per well for 24  h. cells with tube networks were visualized an olympus microscope .
10 cells / well ) were loaded with dcf - da ( 20  m ) and mitosox dye , respectively .
after treatment with mc - lr ( 040  m ) or a solvent control for 24  h , cells were washed with pbs and then detached using trypsin .
all experiments were repeated at least four times to ensure reproducibility . for evaluating the contribution of nadh oxidase ( nox ) and mitochondria on ros production induced by mc - lr in vitro
, cells were pretreated for 24  h with 40  m mc - lr and for 1  h with 500  nm of diphenyleneiodonium chloride ( dpi ; sigma aldrich , usa ) , a nox inhibitor , or 500  nm of the mitochondria - targeting antioxidant mito - tempo ( sigma aldrich , usa ) . afterwards
, total ros produced by cells were measured as described above for the dcfda probe .
the huvecs were treated with mc - lr ( 40  m ) for 24  h. western blot analyses were performed as lysates from each sample ( 40  g ) were mixed with 6x sample buffer ( 0.35  m tris , 10% w / v sds , 30% v / v glycerol , 0.6  m dtt , and 0.012% w / v bromophenol blue , ph 6.8 ) and heated to 95c for 5  min .
proteins were separated by electrophoresis and transferred onto polyvinylidene difluoride ( pvdf ) membranes for pnf-b , vcam-1 , icam-1 , and tnf-. the membranes were blocked with 5% nonfat milk in tbs-0.1% tween 20 and sequentially incubated with primary antibodies and hrp - conjugated secondary antibodies , followed by enhanced chemiluminescence ( ecl ) detection .
data of specific protein levels are presented as relative multiples in relation to the control .
differences between treatments were assessed by one - way anova followed by a tukey test ( bartlett test p > 0.05 ) to compare mean of treatments with controls .
to investigate the role of mc - lr on huvecs proliferation , it was observed that mc - lr at a concentration ( 40  m ) can significantly decrease huvecs proliferation for 24  h ( figure 1 ) .
considering that the maximum effect appeared when the mc - lr concentration was 40  m ; 40  m mc - lr was chosen for the following experiments .
these data taken together suggested that mc - lr effectively reduced huvecs proliferation . to determine whether mc - lr has a direct effect on huvecs apoptosis
, the cells were incubated with mc - lr ( 040  m ) for 24  h. as demonstrated in the flow cytometry results , mc - lr effectively induced apoptosis in huvecs at a dose of 10  m ( figure 2 ) .
the percentage of cells apoptotic increased from 5.1  0.6% to 35.8  3.8% after being exposed to 40  m mc - lr for 24  h. the endothelial cell migration is an essential process in angiogenesis .
the effects of mc - lr on the chemotactic motility of huvecs were also detected with transwell migration assay ( figure 3(a ) ) . compared with basal medium ,
rpmi 1640 medium with vegf triggered cell migration , but this effect was dose - dependently inhibited by mc - lr .
the minimal effective action of mc - lr was shown at 10  m ( figure 3(b ) ) . in the boyden chamber assay , 40  m mc - lr indicated significant inhibition of endothelial cell motility , demonstrating that inhibition cell migration was an important aim of mc - lr effect ( figure 3(b ) ) .
although angiogenesis is a complex process , tube formation is one of the key steps . to investigate the potential effects of mc - lr ( 040 
the huvecs were stimulated with vegf , and then seeded on the matrigel , the tube formation was exhibited figure 4(a ) .
however , tube formation was inhibited by mc - lr in a dose - dependent way ( figure 4(b ) ) .
the mc - lr indicated a similar degree of potency for inhibition tube formation as shown in the huvecs proliferation and migration assays .
the dcfda ( nonfluorescent in a resting state but fluorescent upon activation by ros ) was used to determine the efficiency of mc - lr ( 040  m ) induced ros formation in huvecs . in our study ,
mc - lr induced ros formation concentration - dependently as compared to resting ( untreated ) cells ( figure 5(a ) ) .
the overexpression ros could influence cell - cycle progression , cell migration , and growth factor signaling in many of normal cell types . at first
, we detected the ros generation induced by mc - lr ( figure 5(a ) ) .
the intracellular ros accumulated in spite of the catalytic action of nox and/or the mitochondrial respiratory chain . by using mitosox probe , we found that mitochondrial ros was notably increased in huvecs treated with mc - lr ( 40  m ) compared with the untreated cells ( figure 5(b ) ) .
as shown in figure 5(b ) , mc - lr ( 40  m ) induced twice total ros formation compared to the control , whereas mito - sox exhibited 90% increase in total ros compared to the control . to investigate the involvement of nox in ros formation induced by mc - lr ( 40  m ) , the cells was exposed to dpi .
dpi at the dose used in our experiment ( 500  nm ) was found to block nox enzymes without affecting mitochondrial ros production . as shown in figures 5(c ) and 5(d ) , the dpi did not reduce the mitochondrial ros and total ros production .
these results indicated that mitochondria , not nox , are responsible for the ros formation induced by mc - lr .
the ros overexpression has been shown to activate various pathways including nf-b in cultured cells . in this study
, it is proposed that the increased ros production in mc - lr - induced huvecs may partially cause the activation of nf-b .
therefore , we measured whether mc - lr ( 040  m ) induced nf-b activation in huvecs .
western blotting data showed that there was an increased expression of nf-b protein in huvecs treated with mc - lr ( figure 6 ) . to investigate whether mc - lr induces expression of vcam-1 and icam-1 in huvecs , we cultured huvecs at mc - lr ( 40  m ) for 24  h. immunoblot analysis showed that stimulation of huvecs with mc - lr increased the production of vcam-1 and icam-1 ( figure 7 ) .
so we analyzed whether the mc - lr ( 40  m ) inducted the tnf- expression .
the results showed a significant induction of tnf- expression which occurred after 24  h exposure to 40  m mc - lr ( figure 7 ) .
earlier studies have confirmed the nephrotoxicity and hepatotoxicity of mc - lr . in this study , we evaluated the effect of mc - lr on multiple steps of endothelial cell function . in huvecs viability assay ,
the higher concentration of mc - lr ( 40  m ) consistently exhibited potent antiproliferative activities .
the huvecs apoptosis enhanced from 10  m mc - lr because of cytotoxicity , but the proliferation of huvecs did not change at the same concentration .
it was important in the case of short - term exposure to the mc - lr . in the past
researches , the cell proliferation inhibition led to impairing wound healing and angiogenesis ; the mc - lr maybe delayed the healing process but did not complete destruction . however , pretreated huvecs in the presence of 10  m mc - lr for 48 hours decreased cells proliferation ( data not shown ) .
however , more researches should be needed to confirm the effect of mc - lr inside the cells .
we observed that mc - lr significantly influenced the migration of huvecs at a range of concentrations ( 1040  m ) .
the lower concentrations mc - lr was observed drastically regulated the huvecs migration activity . the mc - lr inhibited endothelial cell migration and tube formation at concentrations of 1040  m .
however , the endothelial cell proliferation was not changed at the lower concentration ( 10  m ) . meanwhile ,
the percentage of apoptotic cells increased about 10% after mc - lr treated ( 10  m ) .
in addition , the percentage of migration cell decreased about 30% after mc - lr treated ( 10  m ) . to some degree
, the results suggested an important target of mc - lr was the molecules that participated in cells motility .
this suggested that mc - lr toxicity might account at least in part for decreasing motile activity of huvecs .
our study was undertaken to know the mechanisms by which mc - lr impairs effects on human vascular .
for the first time , we have shown that mc - lr induced vascular inflammation via increasing of ros , cams , and nf-b in primary cultured huvecs .
the endothelial dysfunction could obviously enhance the risk of all forms of cardiovascular complications including critical limb ischemia and foot ulcers .
however , no previous studies have evaluated how mc - lr directly changes endothelial cells function . in our study , it was demonstrated that mc - lr can directly decrease huvecs functions .
moreover , regulating endothelial cells proliferation and apoptosis could significantly affect vascular endothelial integrity and sustain endothelium homeostasis .
so the mc - lr decreased huvecs proliferation and induced apoptosis , which may suggest blood vessel function damage .
this study also examined whether mc - lr affects oxidative stress of huvecs , as several lines of evidence showed that mc - lr induced ros production .
all results revealed that mc - lr significantly increased the ros production ( figure 5 ) .
the ros production has been linked closely with inflammatory responses [ 11 , 12 ] . both of the ros production and inflammation were potential mediators of vascular diseases . in our results ,
mc - lr induced increment of the cellular ros production may indicate that the mc - lr potential induces vascular inflammation .
another interesting finding of our study was the case that nf-b might be an aim of mc - lr induced vascular inflammation .
thus , improvement of approaches targeting this pathway could present a novel therapeutic tool for the treatment of many diseases .
but when the cell was stimulated by cytokines or endotoxin , the phosphorylation of inhibitor b and the activation of nf-b happened .
this in turn induced the overexpression of cell adhesion molecules , chemokines and macrophage migration inhibitory factor , and matrix metalloproteinases-1 and -9 , which could regulate cell apoptosis and proliferation .
the results of this study demonstrated that mc - lr induced nf-b activation in huvecs . in our study
, we also detected the expression of icam-1 , vcam-1 , and tnf- , which implied a signal for the huvecs activation .
these cell adhesion molecules participated in cell adherence and inflammatory responses . increased expression of icam-1 , vcam-1 , and tnf- was observed in mc - lr treated huvecs , and further research indicated that the level of tnf- was more strongly regulated by high content mc - lr treated .
these data showed a possible depiction of increased vascular permeability in mc - lr treated huvecs .
thus , the icam-1 and vcam-1 expression proved useful evidences that the mc - lr induced vascular inflammation .
accumulating evidences indicated that the inflammatory cytokine tnf- plays a key role in the vascular function lesions and the following development of vascular disease .
epidemiological studies have shown that tnf- expression was extremely increased in the plasma and arteries of humans with vascular complications .
moreover , tnf- overexpression has been estimated as an injury - related signal in many researches involving inflammation .
furthermore , tnf- has been shown to induce the expression and release of many adhesion molecules and chemokines involved in the inflammatory response .
thus , all the data including tnf- overexpression showed more evidences that the mc - lr induces vascular inflammation .
in conclusion , the most major findings of our study proved that mc - lr enhances inflammatory response in huvecs for the first time . in this study , we observed that mc - lr induced vcam-1 and icam-1 expression in huvecs through nf-b pathway .
therefore , this study suggested that mc - lr could be very harmful in the vascular inflammatory process and its precise mechanism needs to be further studied .
 microcystins ( mcs ) produced by toxic cyanobacteria cause serious water pollution and public health hazard to humans and animals . 
 however , direct molecular mechanisms of mc - lr in vascular endothelial cells ( ecs ) have not been understood yet . in this study 
 , we investigated whether mc - lr induces vascular inflammatory process in cultured human umbilical vein endothelial cells ( huvecs ) . 
 our data demonstrated that mc - lr decreased huvecs proliferation and tube formation and enhanced apoptosis . 
 mc - lr also induced intracellular reactive oxygen species formation ( ros ) in huvecs . 
 the mc - lr directly stimulated phosphorylation of nf-b . 
 furthermore , mc - lr also increased cell adhesion molecules ( icam-1 and vcam-1 ) expression in huvecs . taken together , the present data suggested that mc - lr induced vascular inflammatory process , which may be closely related to the oxidative stress , nf-b activation , and cell adhesion molecules expression in huvecs . 
 our findings may highlight that mc - lr causes potential damage to blood vessels . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusion