Patent Abstract:
osteopontin for the prediction and treatment of cardiovascular diseases the present invention relates to the use of endothelial progenitor cells and osteopontin for the treatment of cardiovascular diseases or complications . the invention also relates to the use of epc osteopontin levels as a marker of the risk of the development of these cardiovascular complications . in particular , the invention provides compositions and methods based on osteopontin and the genes encoding osteopontin .

Detailed Description:
patients with poorly controlled type 1 diabetes mellitus ( as defined by hba 1 / c & gt ; 10 %), who are on insulin for more than one year , and not on any other medications were recruited from the diabetes day centre , university college hospital galway , ireland . ethical approval for this study was obtained from the university college hospital galway clinical research and ethical committee . patients with micro - or macrovascular complications were excluded from the study . microvascular complications were defined as the presence of microalbuminuria , diabetic retinopathy and neuropathy . macrovascular complications were defined as the presence of any previous history of acute coronary syndrome , peripheral vascular disease and cerebrovascular disease . after signing consent , peripheral blood samples were collected from patients with type 1 diabetes mellitus and healthy volunteers . epcs were cultured according to previously described techniques . briefly , mononuclear cells ( mncs ) were isolated by ficollpaque density centrifugation method . after purification with 3 washing steps , 10 × 10 6 or 2 × 10 6 mncs were plated on fibronectin coated , 6 - well plates or 4 - well glass slides , respectively . cells were cultured in endothelial cell basal medium - 2 ( clonetics ) supplemented with egm - 2 single aliquots ( clonetics ) consisting of 5 % fbs , vascular endothelial growth factors ( vegf ), fibroblast growth factor - 2 , epidermal growth factor , insulin - like growth factor - 1 , and ascorbic acid . epcs were confirmed by dual staining with dii - acetylated low - density lipoprotein and fitc - lectin . diabetes was induced in male new zealand white rabbit using intravenous injection of alloxan ( 150 mg / kg ). rabbits with plasma glucose of & gt ; 22 were included for the study . phlebotomy was performed via the marginal artery under anaesthesia . this study was approved by the national university ireland , galway ( nuig ) animal care and use committee . fibronectin ( 100 g / ml ) was coated onto 24 - well plates for 2 hours at 37 ° c . wells were blocked with 1 % bsa in pbs for 2 hours and epcs ( 1 × 10 5 ) were added to each well to attach for 1 hour . adherent cells were stained with 0 . 1 % crystal violet and rinsed with 10 % acetic acid to elute the stain from the cells . attached cells were quantified by analyzing the optical density of the media at a wavelength of 600 nm with a microtiter plate reader . a monolayer of human umbilical vein endothelial cells ( huvecs ) was prepared 48 hours before the assay by plating 2 × 10 5 cells ( passage 5 to 8 ) in each well of 4 - well glass slides . huvecs were pretreated for 12 hours with tnf - alpha ( bd biosciences ) ( 1 ng / ml ) or media . epcs were labeled with dii and 1 × 10 5 cells were added to each well and incubated for 3 hours at 37 ° c . nonattached cells were gently removed with pbs , and adherent epcs were fixed with 4 % paraformaldehyde and counted by a blinded observer . matrigel ( sigma ) was thawed and placed in 4 - well glass slides at room temperature for 30 minutes to allow solidification . dii - labeled epcs ( 2 × 10 4 ) were coplated with 4 × 10 4 human umbilical vein endothelial cells ( huvecs ) and incubated with and without 5 ug / ml opn ( sigma ) at 37 ° c . for 12 hours . tubule formation was defined as a structure exhibiting a length 4 times its width . the number tubules formed was assessed by a blinded counter . to determine if the effect of osteopontin is rgd dependent , different rgd / rad concentrations were incubated with osteopontin . total rna was isolated from day 4 epcs using rneasy mini kit ( qiagen ) as described by the manufacturer . the concentration of isolated total rna was analyzed using nanodrop counter . quantit dna high sensitivity kit was used to detect presence of any genomic dna in the total rna samples . microarray analysis were performed using genechip human genome u133 plus 2 . 0 affymetrix array . gene expression profiles were compared between epcs derived from patients with poorly controlled t1dm and healthy volunteers , with epcs derived from healthy volunteers as baseline , using mas5 . 1 software ( affymetrix ). fold changes were calculated by comparing transcripts between the two groups . k - mean clustering was used to identify the detected ( present or absent ) and changed ( increased or decreased ) calls . primers were designed using primerexpress software and ordered from sigma genosys ( table . 1 ). the expression study was performed using a 96 well plate on an abi prism 7000 sequence detection system ( applied biosystems ) with one step quantitect sybr green pcr kit ( qiagen ). the reactions were performed according to the manufacturer &# 39 ; s instructions with minor modifications . the pcr program was initiated using sample volume of 25 μls at 50 ° c . for 30 mins for reverse transcription step , 95 ° c . for 15 mins for activation of taq dna polymerase and followed by 40 cycles of 15 seconds at 95 ° c ., and 30 seconds at 60 ° c . the dissociation curves were generated immediately after the real - time pcr using a temperature range between 60 ° c . and 95 ° c . each samples were analyzed in triplicates . all the reactions were further subjected to electrophoresis on 2 % agarose gels stained with sybrgreen dye to confirm the presence of the expected pcr products . c57bl / 6 ( wt ) and opn − / opn − mice were purchased from charles river lab and jackson lab respectively . opn - ko and wt mice aged between 8 - 10 weeks of age were used . the mice were housed at the animal facility in regenerative medicine institute ( remedi ), ncbes , nuig . all procedures were approved by the minister of health and children under the cruelty to animals act , 1876 . unilateral hind limb ischemia was created in c57bl / 6 and opn − / opn − mice as previously described 32 . in brief , an incision was performed in the skin overlying the middle portion of the left hind limb . after ligation of the proximal end of the femoral artery , the distal portion of the saphenous artery was ligated and the artery as well as , all side branches were dissected free and excised . the skin was closed using an absorbable suture . of note , the animals were anesthetized with ketamine and xylazine and maintained with isoflurane . the hind - limb blood flow on both hind limbs and feet were measured using a laser doppler blood flow ( ldbf ) analyzer ( periscan pimii , perimed inc ) immediately before surgery , and on postoperative days 0 , 7 , 14 , and 28 . blood flow was displayed as changes in the laser frequency using different color pixels . after scanning , stored images were analyzed to quantify blood flow . to avoid data variations caused by ambient light and temperature , hind limb blood flow was expressed as the ratio of left ( ischemic ) to right ( non - ischemic ) ldbf . all samples were processed within one hour . live cells were stained with conjugated antibodies to sca - 1 , c - kit , and cd31 ( bd biosciences ). facs aria coulter was used to perform the facs analysis . the frequency of bone marrow cells positive for the above reagents was determined by a two - dimensional side scatter - fluorescence dot plot analysis of the samples stained with the different reagents , after appropriate gating to exclude granulocytes . initially , sca - 1 + bone marrow cells were gated and then the resulting population was examined for dual expression of c - kit . for further analysis , sca - 1 + cells were studied for the expression of cd31 using a phycoerythrinconjugated anti - mouse cd31 monoclonal antibody ( bd biosciences ), reflecting endothelial differentiation of progenitor cells . data were processed using the macintosh cell quest software program ( bd biosciences ). a single trained operator ( t . b . ), who was blind to the status of the animal , performed all flow cytometric analyses throughout the study . results are expressed as mean ± sem . comparison between groups was performed by anova . post hoc analysis and pair wise multiple comparisons were performed using the 2 - sided t test with scheffe adjustment . probability values & lt ; 0 . 05 were considered statistically significant . all analyses were performed with spss software ( spss ver . 14 . 0 inc ). male sprague - dawley rats ( 225 - 250 g ) were anesthetized , and hearts were rapidly excised and immediately cannulated to a langendorff perfusion apparatus using a protocol adapted from tsuchida et al . ( circulation research 1994 ). briefly , hearts were perfused with krebs - ringer buffer at a constant pressure of 60 mm hg . all perfused hearts were stabilized for 20 min on the langandorff apparatus prior to induction of various treatments . three hearts were used per treatment group ( n = 3 ). perfused hearts were continuously perfused for 1 h 15 min following stabilisation . to mimic ischemia / reperfusion injury non - preconditioned hearts were continuously perfused for 30 min prior to a 30 min exposure to ischemia ( stoppage of kreb &# 39 ; s buffer flow ) followed by 15 min of reperfusion ( resumption of kreb &# 39 ; s buffer flow ). following treatment hearts were immediately removed to trizol reagent and homogenized ( invitrogen ). following addition of 20 % chloroform samples were mixed by inversion and centrifuged at 12 , 000 × g for 15 min at 2 - 8 ° c . the rna was removed and added to an eppendorf tube containing 0 . 5 ml isopropanol and vortexed vigorously to precipitate the rna . after a 10 min incubation at room temperature , the rna was pelleted by centrifugation at 12 , 000 × g for 10 min and washed in 1 ml of 75 % ethanol . rna was pelleted by centrifugation at 7 , 500 × g for 5 min , the supernatant removed and the pellet allowed to air - dry at room temperature for 10 min . the pellet was subsequently resuspended in 50 μl of depc treated water . rna was quantified by spectroscopy , based on its absorbance at 260 nm ( uv absorbance range ). quantitative pcr was carried out with 2 μg rna and oligo ( dt ) 12 - 18 ( invitrogen ) using amv reverse transcriptase ( sigma ). primers to glyceraldehyde - 3 - phosphate dehydrogenase ( gapdh ), osteopontinwere designed to published mrna sequences from the national centre for biotechnology information ( ncbi ) using primer express software ( applied biosystems , foster city , calif .) and sequence specificity was confirmed by performing a blast ( ncbi ) search . primer sets were synthesized by mwg biotech ( ebersberg , germany ). cdna quantification standards , containing a known number of cdna copies of each gene , were prepared by purifying pcr products for each gene using the qiagen qiaquick gel extraction kit . these purified products were then quantified by spectroscopy and appropriate dilutions were made . amplification reactions were carried out in real - time , with separate reactions set up for each primer set , each containing 12 . 5 μl of 1 × sybr green i pcr master mix ( applied biosystems ), 12 . 5 nm of each primer and 2 . 5 μl template ( 1 in 50 dilution of cdna ) in a final volume of 25 μl . amplification reactions were performed in 96 - well optical reaction plates on the abi 7000 . a dissociation curve was generated for each primer set at the end of each run and pcr products were run on 2 % agarose gels to confirm the size of the product and the specificity of the primers . cdna copy numbers for all differentially regulated genes were generated from their respective standard curves and normalised to the housekeeping gene gapdh . a fold increase was calculated relative to the expression levels of the perfused sample . real - time rt - pcr was carried out for each of the conditions in triplicate and results were then analysed using a one - way anova followed by scheffe &# 39 ; s test using the statistical package spss for windows version 12 . 0 . 1 ( spss inc ., chicago , ill ., usa ). primary cultures of neonatal cardiomyocytes were isolated from 1 - 4 day old sprague dawley rats . briefly , rats were euthanized and hearts excised . after scalpel homogenization , overnight trypsin digestion at 4 ° c . and a collagenase treatment for 20 min at 37 ° c ., cardiomyocytes were enriched by percoll gradient centrifugation ( amersham ) and plated at a density of 1 × 10 5 / ml in dmem / f12 medium supplemented with 10 % newborn calf serum , 100 u / ml penicillin , 100 μg / ml streptomycin , 1 mm sodium pyruvate ( gibco - brl ), 5 % insulin transferrin selenite ( its ) liquid supplement media , 100 μm 5 - bromo - 2 - deoxyuridine on culture plates coated with 0 . 2 % gelatin . cells were cultured at 37 ° c . and 5 % co 2 . to mimic endogenous ischemia , cultures were exposed to hypoxic conditions ( o 2 / n 2 / co 2 , 0 . 5 : 94 . 5 : 5 ), using a hypoxia gas chamber ( russkin ) in the absence of glucose and serum , using glucose - free dmem ( gibco - brl ) supplemented with 10 mm 2 - deoxyglucose , 100 u / ml penicillin , 100 μg / ml streptomycin , 1 mm sodium pyruvate , 5 % its liquid supplement media . cells were lysed in whole cell lysis buffer ( 20 mm hepes , ph 7 . 5 , 350 mm nacl , 1 mm mgcl 2 , 0 . 5 mm edta , 0 . 5 mm egta , 1 % igepal - 630 , 0 . 5 mm dithiothreitol ( dtt ), 100 μm pmsf and 1 μg / ml pepstatin ). cellular proteins were separated by electrophoresis on 10 % sds - polyacrylamide gels and transferred onto nitrocellulose membranes . after blocking ( 5 % non - fat milk , 0 . 05 % tween - 20 in pbs ), blots were incubated with antibodies to ostiopontin and were visualised using horseradish peroxidise - conjugated secondary antibodies ( pierce ) were used at a 1 : 5 , 000 dilution . protein bands were detected with supersignal ultra chemiluminescent substrate ( pierce ) on x - ray film ( agfa ). four patients with type 1 diabetes mellitus and four age - and gender - matched healthy volunteers were recruited ( table 2 ). patients with t1dm have lower number of epcs as compared to healthy volunteers ( 244 +/− 20 vs 334 +/− 7 , p = 0 . 02 ) ( fig1 ). patients with t1dm have normal adhesion to collagen ( 1 . 00 +/− 0 . 11 vs 1 . 34 +/− 0 . 15 , p = 0 . 13 ) ( fig2 ) and fibronectin ( 1 . 65 +/− 0 . 44 vs 2 . 13 +/− 0 . 20 , p = 0 . 16 ) ( fig3 ). the effect of diabetes on epc adhesion to endothelial cells was next assessed in quiescent endothelial cells and after exposure to tnf - α . epcs derived from patients with poorly controlled t1dm demonstrated normal adhesion to quiescent endothelial cells ( 7 . 01 +/− 0 . 91 vs 7 . 79 +/− 0 . 68 , p = 0 . 54 ) but impaired adhesion to activated endothelial cells ( 11 . 05 +/− 0 . 01 vs 21 . 03 +/− 1 . 13 , p = 0 . 001 ) ( fig4 ). formation of tubules in vitro a measure of the ability of epc to participate in angiogenesis was next assessed . epcs derived from patients with t1dm had impaired ability to form tubules compared to controls ( 1 . 7 +/− 0 . 9 vs 9 . 8 +/− 1 . 8 , p = 0 . 01 ) ( fig5 ). this defect was also seen in an animal model of insulin deficient diabetes mellitus when epcs derived from alloxan - induced diabetic rabbits also showed an impaired ability to form tubules as compared to the epcs derived from non - diabetic control rabbits ( 9 . 6 +/− 1 . 77 vs 13 . 0 +/− 0 . 65 : p = 0 . 049 ) ( fig6 ). expression of osteopontin in epcs from patients with poorly controlled diabetes mellitus : using real time pcr , it was demonstrated that opn expression is reduced in epcs derived from patients with poorly controlled diabetes mellitus as compared to healthy volunteers . having demonstrated reduced expression of opn in epcs derived from patients with poorly controlled t1dm , it sought to determine whether exposure of epcs to opn could reverse this defect . to do this , the effect of opn supplementation on epc function in vitro was assessed . incubation with opn augmented the number of tubules formed by epcs derived from non - diabetic rabbits ( 13 . 0 +/− 0 . 65 vs 16 . 5 +/− 1 . 15 : p = 0 . 039 ; fig7 ). incubation with opn also augmented the number of tubules formed by epcs derived from diabetic rabbits ( 9 . 88 +/− 2 . 48 vs 16 . 56 +/− 2 . 21 ; p = 0 . 01 ) ( fig8 ). next , investigations were made to determine if the mechanism of opn action is rgd - dependent . epcs were co - incubated with opn and rgd or rad ( scrambled peptide ). co - incubation of epcs with opn and rgd , but not rad , was associated with impaired epc tubule formation . the results of this experiment show that the effect of opn on epc function is rgd - dependent ( fig9 ). microarray analysis demonstrated that osteopontin was downregulated in the epcs derived from the diabetic subjects . this was further validated using realtime pcr . the mean fold change were compared with the microarray results ( table 3 ). to study the role of opn in angiogenesis in vivo , the extent of angiogenesis in a murine model of unilateral hind limb ischemia was assessed . the blood flow was assessed in the wt and opn - ko mice before and after the procedure . in opn - ko mice , measurement of the ldbf ratio between the ischemic and the non - ischemic limb indicated that restoration of perfusion in the ischemic hind limb was significantly impaired . at day 7 , 14 and 28 after surgery , ldpf ratio was reduced in the opn - ko mice , 0 . 31 ± 0 . 07 versus 0 . 68 ± 0 . 11 ( p = 0 . 021 ), 0 . 32 ± 0 . 03 versus 0 . 54 ± 0 . 05 ( p = 0 . 006 ) and 0 . 45 ± 0 . 06 versus 1 . 09 ± 0 . 13 ( p = 0 . 002 ) for the wt mice respectively ( fig1 and 11 ). next , the role of epc recruitment in the pathogenesis of the impaired angiogenesis observed in opn knockout mouse was explored . for this , circulating epc numbers were measured before and after induction of hind limb ischemia in opn knockout and wild type animals . flow cytometry analysis of epc number was performed before and three days after the induction of hind limb ischemia . at day zero there were no differences in epc number between both groups . furthermore , epc numbers increased in the opn knockout mice 3 days after induction of hind limb ischemia ( 0 . 33 ± 0 . 05 on day 0 versus 0 . 55 ± 0 . 05 on day 3 ; p = 0 . 036 ). this result suggests that opn is not implicated in epc mobilization . in contrast , epc numbers did not increase 3 days after induction of hind limb ischemia in control mice ( fig1 and 13 ). osteopontin expression levels , as determined by realtime pcr , were decreased 5 . 14 fold in ischemic / reperfused rat heart in comparison with expression levels in the perfused sample . in rat neonatal cardiomyocytes primary cultures , subjected to conditions to simulate endogenous ischemia conditions , osteopontin protein expression levels were reduced , as determined by western blot analysis ( see fig1 . ), in comparison to cardiomyocytes cultured under normal conditions . osteopontin expression levels , measured on 2 , 8 , 12 and 24 hours , did not recover to pre - ischemia levels . in summary , opn mrna and protein levels drop in response to ischemia or ischemia reperfusion . epc number and function can be affected by various factors 15 . reduced epc number were demonstrated in patients with type 1 and type 2 dm 16 , 17 . however , microvascular complications were not excluded in these studies . it has recently been shown that diabetic retinopathy increases epc number 20 , 21 . for this reason , a homogeneous population without diabetic retinopathy and other complications were chosen . it was desired to observe the effect of hyperglycaemia without other confounding factors in human with t1 dm . the data showed that epcs derived from patients with uncomplicated type 1 diabetes mellitus have a reduced number . these cells showed normal adhesion to collagen and fibronectin . they also showed normal adhesion to quiescent endothelial cells but impaired adhesion to activated endothelial cells . epcs derived from patients with t1dm have impaired ability to form tubules . these data were consistent with previous studies 16 , 17 . the role of opn in epc dysfunction in diabetes mellitus has been examined . it has been demonstrated for the first time that opn expression was markedly reduced in epcs isolated from subjects with poorly controlled t1dm . this result is the opposite of the effect observed by loomans et al using microarray analysis 22 . the reason for the discrepancy is unclear but may be due the patient population studied . furthermore , it has been demonstrated that epc dysfunction was reversed when cells from diabetic animals were cultured in the presence of recombinant opn . the effect of opn on epc function was also seen in cells from non - diabetic animals in which increased tubule formation was observed . thus the invention could allow treatment of diseases with are associated with poor tubule formation , or problems with angiogenesis , which includes peripheral vascular disease , ulcer , ischaemic heart disease , and cerebrovascular disease , and subarachnoid haemmorrhage secondary to cerebral aneurysm and diabetic retinopathy . the effect of opn on epc function was reversed by rgd but not rad showing the opn effect is rgd - dependent . the role of opn in angiogenesis was next explored using an opn knockout mouse . it was demonstrated that the restoration of perfusion in the ischemic hind limb was significantly impaired in opn - ko mice . at day 7 after surgery , ldpf ratio in the opn - ko mice was approximately half that of the wt mice . this impairment in blood flow recovery persisted up to 28 days after the surgery , suggesting that the absence of opn impairs neovascularisation in the murine model of unilateral hind limb ischemia . this defect could be due to decreased mobilization or impairment of epc incorporation into new vessels at the site of ischemia . the results show increased circulating levels of epcs after hind limb ischemia in the opn knockout suggests that impaired mobilization is not the mechanism . this hypothesis is supported by data from ballard et al who have shown that the expression of opn did not differ from baseline up to seven days after the induction of mid - dorsal cutaneous wound in diabetic mice . the opn level subsequently increased at day 7 and remained constitutively higher for a further four days . the authors suggested that the low expression of opn in diabetic mice may be in part responsible for the delayed healing of wounds in diabetic mice 33 . therefore , reduced opn expression in diabetic epc may explain the propensity of diabetic subjects to macrovascular complications . osteopontin exists as a secreted cytokine or adhesion molecule constitutively expressed in healthy myocardium . its expression is increased in non - myocytes following myocardial infarction to protect cellular viability and aid adaptive remodelling . loss of osteopontin impairs compensatory fibrosis and hypertrophy leading to decreased cardiac performance . osteopontin expression is also markedly increased in cardiomyocytes by myocardial infarction and heart failure . the mechanism of osteopontin cardioprotection is largely unknown . osteopontin suppresses cytokinc - induced nitric oxide synthase expression , preventing nitric oxide production and contractile impairment . cellular signalling is mediated through cell surface integrin receptor binding . integrin receptors communicate changes in the extracellular matrix to the cytoskeleton . increased expression of osteopontin is accompanied by the increased expression of its cardiac - receptor β1 integrin during hypertrophy . anti - integrin antibody blocked angiotensin ii induced cardiac remodelling an effect also blocked by anti - osteopontin antibody , suggesting signalling of angiotensin h proceeds via osteopontin . in osteopontin - deficient cardiac fibroblasts oxidative stress induced necrosis unlike wild type cells where apoptosis was predominant . this necrotic death was reduced on endogenous re - expression of osteopontin . however , in vivo study of myocardial infracted mouse hearts , have shown osteopontin deficient hearts to have the same number of apoptotic myocytes as wild type hearts . epc dysfunction in diabetes mellitus is associated with reduced opn expression and can be reversed by opn supplementation , which may explain why diabetic subjects are more prone to vascular complications . furthermore the studies in the opn knockout animals confirm the crucial role of opn in angiogenesis . the results suggest that this effect may be related to lower opn expression in epcs . epc dysfunction in diabetes mellitus is due to reduced opn expression identifying a new therapeutic target for this disorder . the words “ comprises / comprising ” and the words “ having / including ” when used herein with reference to the present invention are used to specify the presence of stated features , integers , steps or components but does not preclude the presence or addition of one or more other features , integers , steps , components or groups thereof . it is appreciated that certain features of the invention , which are , for clarity , described in the context of separate embodiments , may also be provided in combination in a single embodiment . conversely , various features of the invention which are , for brevity , described in the context of a single embodiment , may also be provided separately or in any suitable sub - 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