Patent Application: US-93743009-A

Abstract:
recent studies have demonstrated that lymphocyte - derived microparticles impair endothelial cell function . the present invention concerns the use of lmps in the regulation of angiogenesis or diseases such as cancer or retinopathy of prematurity . having long been considered as cellular debris , microparticles constitute reliable markers of vascular damage . released into biological fluids , microparticles are involved in the modulation of key functions including immunity , inflammation , vascular remodeling and angiogenesis . the present data demonstrates that lmps have considerable impact on angiogenesis in vitro and in vivo . in view of this , lmps may be important contributors to the pathogenesis of diseases that are accompanied by impaired angiogenesis and could thus influence vascular function ( microvascular angiogenesis and vasopermeability of ischemic tissue , alerting the body for special attention and the need for emergency repair procedures .

Description:
actinomycin d , 3 -( 4 , 5 - dimethyl thiazol - 2yl )- 2 , 5 - diphenyl tetrazolium bromide ( mtt ), lucigenin ( n , n - dimethyl - 9 , 9 ′ biacridinium dinitrate ) ( sigma aldrich ); β - actin ( novus biologicals ); flk - 1 ( vegfr2 ) rabbit polyclonal antibody and horseradish peroxidase linked anti - rabbit igg , antibodies against gp91 phox , p22 phox ( fl - 195 ), p47 phox , erk1 / 2 , phospho - erk1 / 2 , tsp - 1 , and rabbit polyclonal cd36 antibody ( santa cruz biotechnology ; santa cruz , calif . ); u83836e and u74389g ( biomol , pa , usa ); [ 3 h ]- thymidine ( amersham , mississauga , ontario , canada ); hrvegf and apocynin ( calbiochem , la jolla , calif ., usa ); mitomycin c ( fluka biochemika ); annexin - v - cy5 ( bd pharmagen , sandiego , calif . ); vybrant apoptosis assay kit , propidium iodide ( pi ) and fluorescent microbeads , 1 μm ( molecular probes , eugene , oreg ., u . s . a ); nadph ( roche diagnostics , laval , qc canada ); diphenyliodonium ( dpi ) ( calbiochem , la . jolla , calif ., usa ). cem t cells were purchased from atcc ( manassas , usa ) and cultured with x - vivo medium ( cambrex , walkersville , md .). human umbilical vein endothelial cells ( huvec ) were purchased from cambrex ( walkersville , md .) and cultured as recommended . the immortalized human microvascular endothelial cell line - 1 ( hmec - 1 ) was kindly supplied by dr candal fj ( centers for disease control and prevention , atlanta , ga .). hmec - 1 were grown in endothelial basal medium ( cambrex , walkersville , md .) supplemented with 10 % fetal bovine serum ( gibco , gaithersburg , md ., usa ), 100 μg / ml streptomycin , 100 u / ml penicillin , 10 ng / ml epidermal growth factor ( bd , oakville , ontario , canada ) and 1 μg / ml hydrocortisone ( sigma ). mouse lewis lung carcinoma cells ( llc ) ( llc1 , crl - 1642 ) and human breast cancer cell ( m4a4 ) were purchased from atcc ( manassas , usa ) and cultured in dulbecco &# 39 ; s modified eagle &# 39 ; s medium ( dmem ) ( gibco brl , long island , n . y .) supplemented with 10 % fbs , penicillin at 100 units / ml and streptomycin at 100 μg / ml . lmps were generated as described . ( 16 ) briefly , cem t cells were treated with 0 . 5 μg / ml of actinomycin d for 24 hours , a supernatant was obtained by centrifugation at 750 g for 15 min , then 1500 g for 5 min to remove cells and large debris . mps from the supernatant were washed after three centrifugation steps ( 50 min at 12 , 000 g ) and recovered in saline . washing medium from last supernatant was used as control . lmps were characterized with annexin v staining by facs analysis , and gated using 1 . 0 μm beads in which 97 % of mps (≦ 1 μm ) were annexin - v - cy5 positive . the concentrations of lmps were determined by the biorad protein assay . using the same protocol , lmps were also generated from hyperoxia ( 95 % o 2 , 36 h ) or hypoxia ( 5 % o 2 , 36 h ) exposed cem t cells . six week old male c57bl / 6 mice purchased from charles river ( st - constant , quebec , canada ) were used according to a protocol approved by the sainte - justine research center animal care committee . aortic ring angiogenesis assay the aortic ring assay was performed as described previously . 7 in brief , 1 mm thoracic aortas were embedded in 3 - dimensional growth factor reduced matrigel ( bd biosciences ) and cultured in egm - 2 medium at 37 ° c . the culture medium was changed on day 3 and the aortic rings were treated with saline or 30 μg / ml of lmps until day 7 . aortic rings were photographed on days 5 and 7 using a nikon eclipse te300 inverted microscope . the angiogenic response was determined by measuring the area of neovessel formation using image pro plus software . angiogenesis was investigated in vivo using a murine model of corneal neovascularization ( cnv ) as described previously . 7 briefly , each mouse was anesthetized with isoflurane ( abbott , canada ), and topical proparacaine ( alcon , canada ) and 2 μl of 0 . 15m naoh were applied to the central cornea . the corneal and limbal epithelium were removed by scraping with a scalpel . gentamicin sulfate ophthalmic solution ( sabex inc ., quebec , canada ) was instilled immediately following epithelial denudation . buprenorphine ( 0 . 05 mg / kg ; schering - plough ltd ) was administered post - operatively for analgesia . twenty - four hours after corneal injury , mice were randomly divided into two groups that received either saline or 50 μg / ml lmps . treatments were administered topically three times daily for 7 days , after which corneas were harvested , flatmounted and immunostained with fitc - conjugated anti - cd31 . images were captured with a nikon digital camera dxm 1200 using nikon act 1 version 2 . 62 software . the cnv was quantified in a masked fashion using adobe photoshop 7 . 0 image analysis software . the total corneal surface area was outlined using the innermost vessel of the limbal arcade as the border and the ratio [( neovascularized area / total cornea area )× 100 ] was used to provide a measure of the percentage vascularized cornea . cells at approximately 60 % confluence were incubated for 24 hours with vehicle or the indicated concentrations of lmps . cell viability was estimated by mitochondrial - dependent reduction of mtt . essentially , mtt ( 0 . 5 mg / ml in pbs [ ph 7 . 4 ]) was added to the culture medium and incubated at 37 ° c . for 3 hours , media was aspirated , formazan product solubilized with acidified isopropanol , and the optical density was read at 545 nm with reference wavelength at 690 nm . 4 × 10 4 huvec were plated and serum starved for 24 hours . after synchronization , cells were cultured in complete medium with vehicle or 10 μg / ml lmps for an additional 24 hours . thereafter , 1 μci / ml [ 3 h ]- thymidine was added to each well , and incubated for 24 hours . [ 3 h ]- thymidine dna incorporation was assayed by scintillation counting . huvec were treated with or without 10 μg / ml lmps for 8 , 18 and 24 hours , then treated with reagents from the vybrant apoptosis assay kit ( molecular probes , invitrogen ) followed by flow cytometry analysis according to the manufacturer &# 39 ; s protocol . the rate of apoptosis or necrosis was expressed as the percentage of apoptotic cells relative to the total number of cells per condition . induction of reactive oxygen species ( ros ) was measured using the fluoroprobe dcfda ( molecular probes ). endothelial cells were cultured in 24 - well plates and treated with lmps and / or apocynin at indicated concentrations for three hours , or angiotensin ( ang ii ; 100 nm ) for 45 minutes as a positive control . cells were stained with dcfda ( 10 μm ) for another 30 minutes . after staining , the extracellular dye was washed twice with 10 . 0 mm hepes buffer ( ph 7 . 4 ), and the fluorescence was measured at an excitation wavelength of 485 nm and an emission wavelength of 535 nm , using a multi - well fluorescent plate reader ( wallac 1420 victor multilabel counter ). nadph oxidase activity was measured by the lucigenin - enhanced chemiluminescence method . briefly , huvec were treated with 10 μg / ml lmps for different time periods , washed in ice - cold pbs , harvested , and homogenized via sonication ( 1 second ) ( brandson sonifier 150 , usa ) in lysis buffer ( 20 mm kh 2 po4 , ph 7 . 0 , 1 mm egta , 10 mm complete protease inhibitor cocktail ). homogenates were centrifuged at 800 × g at 4 ° c . for 10 min to remove the unbroken cells and debris , and aliquots were used immediately . to initiate the assay , 100 - μl aliquots of the homogenates were added to 900 μl of 50 mm phosphate buffer , ph 7 . 0 , containing 1 mm egta , 150 mm sucrose , 5 μm lucigenin , and 100 μm nadph . photon emission in terms of relative light units was measured in a luminometer every 2 mins for 30 min . there was no measurable activity in the absence of nadph . superoxide anion production was expressed as relative chemiluminescence ( light ) units ( rlu )/ μg protein . protein content was determined by the bio - rad protein assay . cells were plated at a density of 1 × 10 6 cells per 100 - mm plate and incubated with 7 . 5 , or 10 and 15 μg / ml lmps for 24 hours . soluble proteins were extracted using cell lysis buffer ( 10 mm tris - hcl , 1 . 5 mm mgcl 2 , 1 mm dtt , 1 μm pepstatin , 0 . 75 mm edta , 1 % ( v / v ) sds , 10 mm protease inhibitor cocktail ( roche , ph 7 . 5 ). following centrifugation , the supernatant was collected and total protein concentration was determined ( bio - rad assay ). 25 μg of protein was fractionated by sds - page . the resolved proteins were transferred onto a pvdf membrane on a semi - dry electrophoretic transfer cell ( bio - rad ) for western blot analysis . membranes were blocked , and then incubated overnight at 4 ° c . with an anti - vegfr2 polyclonal antibody ( 1 : 500 dilution ), anti - gp91 phox ( 1 : 100 ), anti - p22 phox antibody ( 1 : 200 ), anti - p47 phox ( 1 : 200 ), phospho - erk1 / 2 antibody ( 1 : 200 ), erk1 / 2 ( 1 : 200 ), tsp - 1 ( 1 : 400 ), and anti - cd36 polyclonal antibody ( 1 : 400 ). after washing , membranes were incubated with a horseradish peroxidase linked anti - rabbit igg ( 1 : 5000 ) for 1 h at room temperature . β - actin was used as a loading control ( 1 : 10000 ). proteins were visualized using the ecl western blotting detection system ( perkin elmer ). two cell migration assays were used to facilitate analysis . cell migration was first determined using a coverslip border migration assay . briefly , 0 . 5 × 10 6 huvec were seeded onto 12 mm - coverslips in a 24 - well plate . cells were serum starved for 4 hours and proliferation was inhibited by adding 10 μg / ml mitomycin c for 30 minutes . next , coverslips were carefully removed , washed with fresh media , and transferred into a 12 - well plate containing 10 ng / ml vegf in the presence or absence of 10 μg / ml lmps . images were captured between 48 and 72 hours using an axiovert 200m inverted microscope ( zeiss ). at 72 hours , the coverslips were removed and the proportion of migrated cells was quantified by mtt assay . the boyden chamber migration assay was also used . a 96 - well chemotaxis chamber with five micron polycarbonate filter was purchased from corning incoporated , ny . the filter was placed over a bottom chamber containing 10 ng / ml of hrvegf . 10 , 000 huvec were seeded to each well in the upper chamber . for testing the effects of lmps and apocynin on the cell migration , huvec were incubated with lmps and / or apocynin in the upper chambers . the assembled chemotaxis chamber was incubated for 24 hours at 37 ° c . with 5 % co 2 to allow cells to migrate through the filter . non - migrated cells on the upper surface of the filter were removed by scraping with a wiper tool ( neuro probe , inc ., gaithersburg , md .) and a cotton swab , and the filter was stained with coomassie blue . the total number of migrated cells per well were counted ; the assays were performed in quadruplicate . newborn rat pups were anesthetized with isoflurane and injected intravitreally on postnatal days 2 and 4 with 5 μl of vehicle ( saline ) in the right eye or 50 μg / ml lmp in the left eyes [ final intraocular concentrations are based on estimated eye volume 8 . pups were killed at postnatal day 6 , and retinal flatmounts obtained . after tritc - conjugated lectin staining , vascularized area was analyzed using image - pro plus software . a well - established rat model of proliferative retinopathy ( rop ) will be applied . in order to induce retinal vaso - obliteration , sprague - dawley rats and their nursing mothers were exposed to a variable oxygen environment consisting of alternating cycles of 50 % oxygen ( 24 h ) and 10 % oxygen ( 24 h ) from p1 to p14 ( postnatal day 1 to day 14 ). animals were subsequently returned to room air for 4 days generating retinal ischemia . the above conditions were achieved using an oxycycler ( model a820cv , biospherix ltd ., redfield , n . y ., usa ). a control group of animals were kept in normoxic conditions and analysed simultaneously . retinal tissue and blood were collected from control ( normoxic ) and rop - exposed rat pups at p14 and p18 . mp were isolated based on canault et al 9 . retinal tissue was isolated , minced in 0 . 2 μm filtered dulbecco &# 39 ; s modified eagles medium , and centrifuged at 400 g ( 15 min ) followed by 12500 g ( 5 min ). the supernatant ( retinal homogenate ) was used for flow cytometry . mps were also isolated from whole blood drawn from the retroorbital sinus as described by combes et al 10 . microparticles were extracted by 2 sequential centrifugations for 15 minutes at 1 , 500 g , followed by 1 minute at 13 , 000 g . supernatant containing microparticles was used for flow cytometry after staining with annexin v . levels of lymphocytic microparticles were determined by double labelling with annexin and cd4 . a group of newborn rat pups subjected to the rop model and a group of control pups in normoxic condition were anesthetized with isoflurane and injected intravitreally on postnatal days p1 , p3 , p6 , p9 , p12 , p15 with vehicle ( saline ) in the right eye or lmp ( 100 μg / ml ) in the left eye . pups were sacrificed on postnatal day 20 , and retinal flatmounts obtained . after tritc - conjugated lectin staining , vascularized area and vascular density were analyzed using image - pro plus software . assessment of vegf - induced vasopermeability was determined in male sprague - dawley rats , weighing ˜ 300 g 11 . after induction of generalized anesthesia , the vitreous of one eye was injected with 50 ng recombinant murine vegf 164 ( r & amp ; d systems inc ., minneapolis , minn .) in 5 μl pbs buffer . the contralateral eye , received an equal volume of pbs , or solution of vegf containing 6 μg lmp . approximately 24 hours later , evans blue was injected through a tail vein at a dosage of 45 mg / kg . after the dye had circulated for 90 minutes , the chest cavity was opened , and rats were perfused via the left ventricle with normal saline . immediately after perfusion , both eyes were enucleated and the retinas were then carefully dissected . after measurement of the retinal wet weight , evans blue dye was extracted by incubating each retina in formamide for 16 hours at 72 ° c ., and the concentration measured by spectrophotometry ( uv - 1600pc ; shimadzu , kyoto , japan ). the concentration of dye in the extracts was calculated from a standard curve of evans blue in formamide . rat cortical neuronal ( rcn ) were isolated from sprague - dawley rat cortices with the described method . briefly , cortices from adult sprague - dawley rat embryos were cleaned from their meninges and blood vessels in krebs - ringer &# 39 ; s bicarbonate buffer containing 0 . 3 % bovine serum albumin ( bsa , gibco brl ). cortices were then minced and dissociated in the same buffer with 1 , 800 u / ml trypsin ( sigma ) at 37 ° c . for 20 min . following the addition of 200 u / ml dnase i ( sigma ) and 3 , 600 u / ml soybean trypsin inhibitor ( sigma ) to the suspension , cells were triturated through a 5 ml pipet . after the tissue was allowed to settle for 5 - 10 min , the supernatant was collected , and the remaining tissue pellet was retriturated . the combined supernatants were then centrifuged through a 4 % bsa layer and the cell pellet was resuspended in neuronal seeding medium , which consisted of neurobasal medium ( gibco ), supplemented with 1 . 1 % 100 × antibiotic - antimycotic solution ( biofluids ), 25 um naglutamate , 0 . 5 mm l - glutamine , and 2 % b27 supplement ( gibco ). cells were seeded at a density of 1 . 5 × 10 5 cells onto each well of 24 - well tissue culture plates ( corning ) pre - coated with poly - d - lysine ( 70 - 150 kd , sigma ). on the following day , cells were treated with different concentrations of lmps for 24 hours and cell viability was analyzed by mtt assay . six week old female c57bl / 6 mice purchased from charles river ( st - constant , quebec , canada ) were used according to a protocol approved by the sainte - justine research center animal care committee . mice were housed in a room maintained at 25 ± 1 ° c . with 55 % relative humidity and given food and water ad libitum . one week later , mice were anesthetized and each was inoculated with 0 . 5 × 10 6 llc cells by subcutaneous ( s . c .) injection in 50 μl pbs on the right flank . seven days after llc inoculation , mice were randomly grouped and each mouse was given an intravenous ( i . v .) or intratumoral injection of lmps at 2 . 5 mg / kg in 50 μl of pbs every two days for consecutive 4 injections . meanwhile control mice were administered 50 μl of pbs . mice were observed at least 4 times weekly for signs of toxicity ( lethargy , bloating , and ruffling of fur ) during and after each treatment . all mice were killed 15 days after llc inoculation and locally growing tumors were separated from skin and muscles and weighed . the nu / nu nude mice ( charles river laboratories international , inc .) were used for xenografting at the age of 5 - 7 weeks . the mice were housed in sterile enclosures under specific virus and antigen - free conditions . they are fed ad libitum . the weight and general appearance of the animals were recorded every other day throughout the experiments . the human neuroblastoma cells ( sh - sy5y , 20 × 10 6 cells in 0 . 1 ml of pbs ) were implanted s . c . in the right flank using a 23 g needle . tumour volume measurements were started when the tumour become palpable (˜ 100 mm 3 ). lmps treatment began on day 12 once per day for 16 days . for weighing , measurement of tumour volume , and drug injection , the animals will be anesthetized with isofluoran . all experiments were repeated at least three times and values are presented as means ± sem . data were analyzed by one - way anova followed by post - hoc bonferroni tests for comparison among means . statistical significance was set at p & lt ; 0 . 05 . the first objective was to determine whether lmps affect vessel development . for this purpose , the aortic ring angiogenesis assay was used as well as a pathophysiologically relevant cnv model that is largely driven by vegf . incubation of aortic rings with saline or 30 μg / ml lmps for 48 and 96 hours significantly reduced neovessel formation by 50 % ( 2 . 2 ± 0 . 2 mm 2 vs . 1 . 1 ± 0 . 1 mm 2 ; p & lt ; 0 . 05 ) and 58 % ( 7 . 7 ± 0 . 3 mm 2 vs . 3 . 2 ± 0 . 5 mm 2 ; p & lt ; 0 . 001 ) ( fig1 a , b ) respectively . having established that lmps inhibit ex vivo angiogenesis , the significance of this in vivo was determined by treating mice subjected to cnv with saline or 50 μg / ml lmps three times daily for 7 days . compared with saline treatment , lmps caused a 23 % reduction in cnv ( 80 . 0 ± 3 . 6 % vs . 61 . 6 ± 2 . 3 %; p & lt ; 0 . 001 ; fig1 c , d ). cell survival and proliferation are critical steps during angiogenesis . to determine the effect of lmps on vascular cell survival , huvec and hmec - 1 were exposed to different concentrations of lmps and their viability was assessed by mtt assay . lmps significantly diminished cell viability in both cell types in a concentration dependent manner ( fig2 a - b ). in order to determine whether the effect of lmps on cell proliferation is stimulus - dependent , lmps were generated from hyperoxia or hypoxia exposure . lmps produced under both hyperoxic and hypoxic conditions potently suppressed huvec proliferation ( 45 . 8 ± 1 . 4 and 50 . 8 ± 2 . 3 , respectively ; p & lt ; 0 . 001 vs . control ) to a comparable degree as actinomycin d - derived lmps ( 49 . 0 ± 0 . 8 ; p & lt ; 0 . 001 vs . control ). this indicates that the effects of lmps are not stimulus - dependent . the observed reduction in cell survival could be caused by decreased cell proliferation or increased apoptosis or necrosis . [ 3 h ]- thymidine dna incorporation was applied and lmps ( 10 μg / ml ) reduced huvec proliferation by 60 % ( p & lt ; 0 . 001 ) ( fig2 c ). to ascertain whether lmps were inducing apoptosis or necrosis , both lmps treated and control huvec were double labelled with fitc - conjugated annexin - v and pi ; however , induction of apoptosis or necrosis was not observed under all test conditions ( p & gt ; 0 . 05 ) ( fig2 d ). previous studies have shown that emps increase superoxide production and lead to impairment of angiogenic pattern 12 . moreover , nox , a major source of superoxide free radicals , was highly expressed by endothelial cells . it was therefore postulated that lmps were exerting their anti - angiogenic properties via oxidative stress mechanisms . to address this hypothesis , utilization was made of two well known lipid peroxidation inhibitors , namely u83836e and u74389g , were tested for their ability to attenuate the anti - proliferative effects of lmps . u83836e and u74389g at 5 and 10 μm concentrations , respectively , led to a partial but statistically significant increase in cell proliferation compared to lmps treatment alone ( p & lt ; 0 . 05 ) ( fig3 a ). additionally , pre - treatment of huvec with two specific nox inhibitors , apocynin ( 1 . 5 mm ) and diphenyleneiodonium ( dpi ; 5 μm ), significantly abrogated the lmp - induced anti - proliferative effects (* p & lt ; 0 . 05 and *** p & lt ; 0 . 001 respectively ; fig3 b - c ). having demonstrated the important role of oxidative stress in lmp - mediated activities , the effects of lmps on ros generation were investigated next . the latter was determined by measurement of intracellular ros levels using dcf fluorescence following a 3 hour pretreatment with 10 μg / ml lmps . as shown in fig4 a , compared to control , lmps significantly increased ros production as indicated by a rise in the dcf signal ( p & lt ; 0 . 05 ). moreover , lmp - induced ros generation was significantly attenuated by pretreatment with apocynin ( 1 . 5 mm ; p & lt ; 0 . 05 ). because the superoxide - generating nadph oxidase has been described to largely contribute to ros formation in endothelial cells , the effect of lmps on superoxide generation from this enzyme was studied . superoxide anion production was measured in lmp - treated huvecs as lucigenin - enhanced chemiluminescence using nadph as the substrate . as indicated in fig4 b , lmps increased the rate of superoxide formation after 1 hour incubation and reached a peak after 8 hours ( p & lt ; 0 . 001 ). lmps induce protein levels of p22 phox , p47 phox , gp91 phox , and the cd36 scavenger receptor owing to the ability of lmps to induce nox activity , their effect on the expression of p22 phox , p47 phox and gp91 phox , which are critical subunits of nadph oxidase was determined . indeed , lmps strongly upregulated p22 phox , p47 phox and gp91 phox protein expression in a concentration - dependent fashion ( p & lt ; 0 . 05 ) ( fig5 a - f ). although lmps demonstrated very low level expression of p22 phox , p47 phox and gp91 phox were undetected . the cd36 scavenger receptor and its endogenous ligand tsp - 1 are potent inhibitors of in vitro and in vivo angiogenesis 7 , whose expression are potentiated in pro - oxidative environments as well as by nadph oxidase activation . in this context , human microvascular endothelial cells treated with 10 and 15 μg / ml lmps dose - dependently augmented cd36 protein levels by 1 . 9 and 2 . 3 fold , respectively ( fig5 g , h ), whereas expression of tsp - 1 was not significantly changed . moreover , tsp - 1 was not detected in lmps per se , which is in agreement with the published results from the proteomic analysis of lmps . because cell migration plays a pivotal role in angiogenesis , the effect of lmps on vegf - induced cell migration was considered . huvecs were plated onto coverslips and exposed to 10 ng / ml vegf with or without lmps . cell migration was substantially decreased by 58 % after 72 hours of lmps treatment ( p & lt ; 0 . 001 ; fig6 a , b ). cell migration was also evaluated using the modified boyden chamber assay . lmps strongly inhibited vegf - induced cell migration by 40 % ( p & lt ; 0 . 001 ; fig6 c ) and apocynin ( 1 . 5 mm ) was able to partially rescue lmps mediated anti - migratory effects ( p & lt ; 0 . 01 vs . lmp ; fig6 c ). having observed that lmps induced cd36 expression , it was surmised that lmps were further suppressing angiogenesis by antagonizing the vegf signaling pathway . this hypothesis is corroborated by evidence that activation of cd36 leads to suppression of vegf - induced vegfr2 phosphorylation . accordingly , huvec proliferation was assessed following pre - incubation with 1 . 5 μg / ml anti - vegfr2 antibody in the presence or absence of lmps ( 10 μg / ml ). as expected , the anti - vegfr2 antibody alone strongly decreased cell proliferation ( p & lt ; 0 . 01 ); however , co - treatment with the anti - vegfr2 antibody and lmps did not result in a synergistic reduction of cell proliferation (# p & gt ; 0 . 05 , compared to ab - vegfr2 group fig7 a ). consistent with this data , western blot analysis of huvec treated with 7 . 5 and 15 μg / ml lmps , caused a dose - dependent downregulation of vegfr2 protein expression by 50 % and 65 %, respectively , vs . control (** p & lt ; 0 . 01 ; fig7 b , c ). phospho - erk1 / 2 levels were also significantly inhibited by 35 % (* p & lt ; 0 . 05 ; fig7 d , e ) microparticles ( mps ) are known to contribute to the pathogenesis of cardiovascular diseases , including inflammation and vascular dysfunction . another important action of mps in the vascular system is their ability to modulate angiogenesis 13 . nevertheless , despite the escalation in mps research , very little is known regarding the role of t lymphocyte - derived microparticles ( lmps ) in regulating angiogenesis . the present experimental findings demonstrate that lmps inhibit angiogenesis in vivo and in vitro by suppressing vascular cell survival , proliferation and migration . significantly , the data demonstrate that lmps induce ros production via nox activation while antioxidants and nox inhibitors attenuate the anti - angiogenic effects of lmps . furthermore , through cd36 induction and vegfr2 and phospho - erk1 / 2 down regulation , evidence is provided to the effect that lmps interfere with the vegf signalling pathway . taken together , these results strongly support a role for lmps in regulating angiogenesis during pathological conditions . mps are released from the plasma membrane during cell activation by apoptosis , shear stress , or agonists . the present studies , mps were obtained by apoptosis from t lymphocytes treated with actinomycin d . moreover , the characteristics of mps appear to depend on the mechanism of stimulation and the activation status of the cell from which they originate 6 , 12 . this is clearly highlighted by the reported effects of mps on angiogenesis . for example , although it is shown that lmps possess anti - angiogenic properties , others have shown that mps from endothelial cells inhibit , whereas platelet - derived mps promote angiogenesis 6 , 12 . the anti - angiogenic effects of lmps seem to occur as a result of decreased cell proliferation rather than increased cell apoptosis or necrosis ( fig2 ). this is in agreement with observations by andriantsitohaina &# 39 ; s group who showed that pathophysiological levels of lmps failed to induce endothelial cell apoptosis 14 . it has been documented that oxidative stress is one of the central mechanisms responsible for endothelial cell dysfunction . the major sources of ros in endothelial cells are endothelial nitric oxide synthase ( enos ) and nox . in line with this , there is a general consensus that nitric oxide ( no ) inhibits both vascular smooth muscle and endothelial cell proliferation . in this study , nitrite levels were unchanged by lmp treatment and enos blockers did not prevent the anti - proliferative effects of lmps ( data not shown ). conversely , lmps increased ros levels and nox activity ( fig4 ) as well as upregulated expression of the gp 91 phox , p22 phox and p47 phox nox subunits ( fig5 ). consistent with this , inhibition of nox partly abrogated the inhibitory effects of lmps on both cell proliferation and migration ( fig6 c ). collectively , these results support that the nox , and not the enos - no signaling pathway , is involved in generating ros that mediate the angiostatic effects of lmps . one of the detrimental consequences of oxidative stress is peroxidation of membrane lipids . lipid peroxidation induces site - specific changes in the organization of the phospholipid bilayer thus leading to cellular dysfunction . the lipid peroxidation inhibitors , u74389g and u83836e , are lipophilic steroid compounds that intercalate into biological membranes , thus enhancing their stability in the event of oxidative stress . in this study , both compounds partially attenuated the anti - proliferative effects of lmps ( fig3 a ), thus suggesting that lmps &# 39 ; angiostatic activities also involve increased lipid peroxidation . several studies have demonstrated that oxidative stress stimulates cd36 expression and that antioxidants attenuate its expression and function . 15 it was therefore intriguing to observe that lmps upregulated cd36 expression , which is consistent with the pro - oxidant actions of lmps ( fig3 , 4 , 5 ). however , it is presumed that the lmp - mediated upregulation of cd36 is tsp - 1 independent since lmps had no significant effect on tsp - 1 expression . moreover , because cd36 is a well established anti - angiogenic receptor , it is tempting to speculate that the generation of ros by lmps occurs upstream of the induction of cd36 with subsequent suppression of the vegf / vegfr2 signaling pathway , as has been proposed here and by others 7 . it is well known that vegf plays a pivotal role in developmental and pathological angiogenesis . vegf stimulates angiogenesis through vegfr2 ( kdr / flk - 1 ), which is expressed mainly on endothelial cells . 16 in the present study , several lines of evidence supported the hypothesis that lmps antagonized the vegf / vegfr2 pathway . first , lmps were shown to potently inhibit vegf - induced inflammatory corneal neovascularization ( fig1 c , d ). secondly , vegf - induced endothelial cell migration was dramatically reduced by lmps ( fig6 ). thirdly , inhibition of vegfr2 activity had no synergistic effect on the anti - proliferative effects of lmps , suggesting that both vegfr2 and lmps signal via the same pathway ( fig7 a ). finally , it was shown that lmps significantly downregulated vegfr2 and phosphorylated erk1 / 2 expression ( the main downstream effector of the vegf signaling pathway ) ( fig7 ), while increasing cd36 protein levels ( fig5 g , h ), a known negative regulator of this pathway . in conclusion , the studies described herein provide evidence for the first time that mps from t cells inhibit angiogenesis in vivo and in vitro . it was demonstrated that lmps impair vascular cell survival , proliferation , and migration . the present data also suggests that lmps regulate angiogenesis by acting through the nad ( p ) h oxidase and vegfr2 pathways . given the pivotal role of the vegf / vegfr2 signaling pathway in angiogenesis , understanding the mechanisms of how lmps interrupt vegfr2 signaling could provide attractive therapeutic strategies aimed at reducing the deleterious effects of mps on the vascular system . retinopathy of prematurity ( rop ) is a potentially blinding eye disorder that primarily affects premature infants weighing about 2¾ pounds ( 1250 grams ) or less that are born before 31 weeks of gestation . the smaller a baby is at birth , the more likely that baby is to develop rop . this disorder , which usually develops in both eyes , is one of the most common causes of visual loss in childhood and can lead to lifelong vision impairment and blindness . today , with advances in neonatal care , smaller and more premature infants are being saved . these infants are at a much higher risk for rop . not all babies who are premature develop rop . there are approximately 3 . 9 million infants born in the u . s . each year ; of those , about 28 , 000 weigh 2¾ pounds or less . about 14 , 000 - 16 , 000 of these infants are affected by some degree of rop . the disease improves and leaves no permanent damage in milder cases of rop . about 90 percent of all infants with rop are in the milder category and do not need treatment . however , infants with more severe disease can develop impaired vision or even blindness . about 1 , 100 - 1 , 500 infants annually develop rop that is severe enough to require medical treatment . about 400 - 600 infants each year in the us become legally blind from rop . rop occurs when abnormal blood vessels grow and spread throughout the retina , the tissue that lines the back of the eye . these abnormal blood vessels are fragile and can leak , scarring the retina and pulling it out of position . this causes a retinal detachment . retinal detachment is the main cause of visual impairment and blindness in rop . lmps can be considered a hallmark of stress - injured or dying lymphocytic cells and may be recognized in the future as a marker of lymphocytic dysfunction . alterations in the retinal blood barrier by disorganizing cell - cell junctions , promote shedding of lmps . however , mps can no longer be described as passive bystanders awaiting removal by professional phagocytes , as was the previous misconception . it has been reported that in vitro lmps , at concentrations that can be reached in circulating blood under immunological dysfunction ( e . g ., hiv ), impair endothelium dependent relaxation in conductance and small resistance arteries in response to agonists and shear stress , respectively . also , lmps can affect vascular contraction by acting directly on smooth muscle cells . lmps are highly generated in the retinal circulation and exist in the retinal tissue during oxygen - induced retinopathy conditions ( fig8 ). lmps exert their antiangiogenic effects primarily through vegfr - 2 and by suppression of its downstream signaling effectors and cascades including akt and the pkc - erk1 / 2 pathway ( fig1 ). data indicate that hyperoxia - induced lmps generated from an in vivo model of retinopathy significantly reduced developmental retinal angiogenesis ( fig1 ). in the rat model of angiogenesis retinopathy , a significant reduction in retinal neovascularization was observed following lmps administration ( fig1 ). because ischemia - induced vegf is a primary stimulus in this animal model , the present results point towards an anti - proliferative and anti - migration role for lmps in hrec ( fig9 and 10 ). such a suppression of retinal neovascularization in vivo corroborates the hypothesis for lmp - mediated vegf inhibition . in the leakage animal model , lmps significantly reduced vegf - induced retinal vascular leakage ( fig1 ). this suggest that lmps interfere with vegfr - 2 - mediated pkc activation . because pkc - β , one of the pkc isozymes , has been postulated as a therapeutic target for vegf - dependent events in diabetic retinopathy , this result suggest a potent inhibitory action of lmps in both vegf and vegf - dependent pkc signaling in the retina . the above findings provide a novel therapeutic avenue for the use of lmps in treating vegf - based ocular neovascular and vasopermeability conditions including diabetic retinopathy . fig1 - 35 demonstrate the effects of lmps on cancer cell survival and growth . the results of these figures may be briefly summarized by the following : 1 ) lmps inhibit the growth of several tumor cell lines in vitro . 2 ) lmps have no effect on terminal differentiated neuron cell death . 3 ) lmps modulate gene expression in neuroblastoma cells ( n2a ), downregulate vegfa and several oncogenes , and upregulate tumor supressors . the following examples will deal more particularly with three different cancer types : lung carcinoma ( example 2 ), neuroblastoma ( example 3 ) and breast cancer ( example 4 ). lung cancer is the most common cancer worldwide , and is also the leading cause of cancer death in both men and women in the north american . angiogenesis , the growth of new vessels from preexisting vessels , is a fundamental step in tumor growth and progression . vascular endothelial growth factor ( vegf ) is a key angiogenic factor implicated in tumor blood vessel formation and permeability . overexpression of vegf has been observed in a variety of cancers and has been associated with a worse relapse - free and overall survival . mammalian cells obtain the cholesterol necessary for the synthesis of membranes , and rely predominantly on the uptake of lipoprotein derived cholesterol via low density lipoprotein receptor ( ldlr ) for their cholesterol needs . the ldlr family is a group of receptors that mediate endocytosis leading to lysosomal degradation of an enormous repertoire of ligands . it has been reported that some malignant cell lines have higher ldlr activity than the corresponding normal cells . ldl uptake has been shown to be higher in lung tumour tissue than in the corresponding normal tissue . it is likely that tumour cells need a very high level of cholesterol during their growth phase . this cholesterol might be used for membrane synthesis and could be related to cell growth . lmps can function as subcellular vectors to deliver proteins and lipids into target cells and alter the phenotypic properties of the target cells as well as induce a variety of functional changes . base on the strong antiangiogenic property , we assume that lmps play an important role in the modulation of lung carcinoma tumor growth . fig1 - 23 provide evidence that lmps are effective in reducing lewis lung carcinoma ( llc ) cell viability . specifically , the results show the following : lmps generated from three different t lymphocyte sources reduce llc viability , but not cem t cells per se ( fig1 ). lmps reduce llc viability and proliferation rate in a dose - dependent manner ( fig1 ). lmps induce llc apoptosis in a dose - dependent manner ( fig1 ). lmps down - regulate vegf - a expression in llc cells and tumors ( fig1 ). lmps reduce microvessel density in llc tumors ( fig2 ). lmps suppress llc tumor development and progression in vivo ( fig2 and 22 ). lmps down - regulate ldlr expression in llc . ( fig2 ). based on this data , it is surmised that the lmps - relatd reduction of vegf / vegfr and ldlr may result from the ldlr mediates endocytosis . the regulatory proteins , lipids and cholesterol were uptaken by tumor cells which highly expressed ldlr and showed high ldlr activities . proteins , lipids and cholesterol activate certain signalling pathways and consequently may down - regulate vegf - a and ldlr expression to interfere with the tumor cell growth . neuroblastoma ( nb ) is the most common solid cancer of early childhood , and accounts for 15 % of all cancer deaths in children . despite significant advances in the past three decades , high risk nb has remained an enigmatic challenge to clinical and basic scientists . anaplastic lymphoma kinase ( alk ), a receptor tyrosine kinase , was recently identified as a familial nb predisposition gene . somatic point mutations in the alk gene have been found in sporadic cases of nb . these mutations lead to alk kinase activation , cell transformation and tumorigenicity in vivo 17 . like most human cancers , vigorous neovascularisation is one of the prominent characteristics of high - risk neuroblastic tumours . tumour angiogenesis offers a uniquely attractive therapeutic target . vascular endothelial growth factor ( vegf ), one of the most important angiogenic factors , is not only specific for the vasculature , but also plays a role in tumour cell survival and motility . agents blocking vegf signalling pathways have shown promising results in clinical trials against various tumours . microparticles ( mps ) are a heterogeneous population of membrane - coated vesicles which can be released from virtually all cell types during activation or apoptosis 1 . these particles serve as mediators of intercellular cross - talk and induce a variety of cellular responses . our previous studies have demonstrated that lymphocyte - derived microparticles ( lmps ) effectively inhibit endothelial cell proliferation , and potently suppress microvascularization in vitro and in an in vivo disease model of neovascularization through interfering with the vegf signaling pathway 18 . nb is a frequent pediatric tumor with a poor outcome in spite of aggressive treatment , and new therapeutic strategies are urgently needed . the current project introduces the challenging new concept that the biological message carried by lmps could target both the tumour vasculature and the tumour cells themselves . we anticipate that our findings will provide new insights into the anti - cancer effect of lmps in advanced nb and help for the development of more attractive therapeutic options . fig2 - 32 reveal the effect of lmps on neuroblastoma ( nb ) cell viability . the results may be summarized as follows : lmps have no effect on confluent primary cultured adult rat cortical neurons ( fig2 ). lmps reduce high proliferating ( rgc - 5 ) viability in a dose - dependent manner ; whereas they have no effect on the growth of terminal differentiated rgc - 5 ( fig2 a , b ). lmps reduce the viability of three different neuroblastoma ( nb ) cells ( fig2 ). lmps inhibit nb cell proliferation and induce human nb cells death ( fig2 ). lmps strongly down - regulate vegf expression in human nb cells ( fig3 ). lmps inhibit nb growth through down regulating alk expression ( fig3 ). lmps suppress human nb tumor growth in xenograft mice ( fig3 ). breast cancer is the most commonly diagnosed cancer and the 2nd leading cause of cancer death , in women in canada . in 2008 , an estimated 22 , 600 women will be diagnosed with breast cancer and 5 , 400 will die of it . one in 9 women is expected to develop breast cancer during her lifetime . hormone - insensitive breast cancers represent approximately 30 % of all breast cancers . they are unresponsive to antiestrogens , more likely to be poorly differentiated , of higher histological grade and are associated with a higher recurrence rate and decreased overall survival . there remains a significant unmet medical need to improve cancer - related clinical outcomes in hormone - insensitive breast cancer patients . http :// caonline . amcancersoc . orq / cqi / content / full / 54 / 3 / 150 — r115 - 7 # r115 - 7http :// caonline . amcancersoc . orq / cgi / content / full / 54 / 3 / 150 — r117 - 7 # r117 - 7http :// caonline . amcancersoc . orq / cgi / content / full / 54 / 3 / 150 — r118 - 7 # r118 - 7http :// caonline . amcancersoc . orq / cgi / content / full / 54 / 3 / 150 — r119 - 7 # r119 - 7the non - stopping proliferation and vigorous neovascularisation are two prominent characters of cancer . anti - angiogenic agents are now being used successfully to treat several types of cancer and they predominantly act through inhibiting the vascular endothelial growth factor ( vegf ) pathway . however , vegf or vegf receptor ( vegfr ) inhibitors can have toxic effects on normal tissues , and tumours can develop resistance to these agents . there is a need to find other angiogenic factors that could be targeted to either circumvent resistance or reduce toxicity . lymphocytes - derived microparticles ( lmp ) are submicron vesicles shed from plasma membranes in response to cell activation , injury , and / or apoptosis . the measurement of the phospholipid content ( mainly phosphatidylserine ) of microparticles and the detection of proteins specific for these cells has allowed their quantification and characterization . our laboratory has demonstrated that lmps generated by apoptosis exert potent anti - angiogenic and anti - cancer effects . in vitro , lmps inhibit the proliferation of several tumor cell lines and , can significantly reduce tumor size in vivo . in summary , given the pivotal role of the uncontrolled cell growth and angiogenesis in metastatic breast cancer , understanding the mechanisms of how lmps interrupt angiogenesis and breast cancer cells proliferation could provide attractive therapeutic strategies aimed at anti - metastatic breast cancer effects of lmps . fig3 - 35 provide evidence that lmps reduce breast cancer cell viability . in addition to the specific illustration of this in fig3 , fig3 shows that lmps derived from three different t lymphocyte sources reduce mcf - 7 cell viability and dose - dependently induce breast cancer cells death . morever , as revealed by the findings in fig3 , lmps abrogate vegf expression in human breast cancer cells ( m4a4 ). although the present invention has been described hereinabove by way of preferred embodiments thereof , it can be modified without departing from the spirit , scope and nature of the subject invention , as defined in the appended claims . 1 . hugel b , martinez m c , kunzelmann c , freyssinet j m . membrane microparticles : two sides of the coin . physiology ( bethesda , md . february 2005 ; 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