the prevalence of type 2 diabetes ( t2 dm ) is increasing globally with approximately 3.2 million patients in the uk alone , plus a further 630,000 patients undiagnosed . the leading cause of mortality in such patients is cardiovascular disease ( cvd ) , with t2 dm patients suffering a threefold increase in cvd mortality over those without diabetes . insulin resistance is a feature of prediabetes , a silent condition that is difficult to diagnose early due to compensatory hyperinsulinaemia that can maintain glycaemia and delay diagnosis of the disease . accordingly , over 30% of newly diagnosed t2 dm patients present with cardiovascular complications   the treatment of which places a significant burden and inevitable impact on healthcare costs . hyperglycaemia is the hallmark of diabetes and clinical trials have revealed that intensive glycaemic control can ameliorate the microvascular complications of t2 dm . however , macrovascular complications persist at least in the medium term , particularly in patients with active coronary heart disease [ 5 , 6 ] , suggesting that factors other than glycaemia may contribute to persistent vascular dysfunction . t2 dm patients frequently present with a variety of metabolic disturbances such as hyperinsulinaemia , inflammation ( elevated inflammatory cytokines such as tumor necrosis factor alpha ; tnf- ) , and disturbed lipid profile ( elevated free fatty acids , e.g. , palmitate ) that may have a cumulative effect and impart persistent pathological changes on ec that are not easy to reverse . the endothelium is a highly metabolic monolayer of cells that lines the luminal surface of all blood vessels and is a principal moderator of vascular health ( reviewed in ) . however , endothelial dysfunction is perceived to precede the development of overt cvd . in health , however , in prediabetic patients with insulin resistance this balance is disturbed and favours prothrombotic and vasoconstrictive effects . endothelial dysfunction predisposes patients to atherosclerosis and coronary artery disease ; thus revascularisation procedures are almost a requirement in diabetic patients [ 8 , 9 ] . the principal method of revascularisation is coronary artery bypass grafting ( cabg ) , frequently performed using the autologous long saphenous vein ( sv ) to bypass diseased coronary arteries . during sv harvesting , the endothelial layer is damaged ; therefore the ability of endothelial cells ( ec ) to proliferate and migrate is essential to restore homeostasis . migration of ec is also vital for angiogenesis and the formation of collateral blood vessels , which provide alternate pathways of blood flow in the damaged coronary circulation . whilst premature coronary artery disease is evident in t2 dm patients , these individuals also suffer inferior outcomes post - cabg [ 10 , 11 ] . one particular study showed that failure of sv grafts occurred in 9.7% of nondiabetic patients 1-year post - cabg , which increased to 14.4% in the diabetic group . it is likely that the impaired capacity of ec to proliferate and migrate is at least partly due to metabolic disturbances in these patients . the aims of this study were twofold ; firstly , to examine the in vitro phenotype and function of sv - ec from patients with or without t2 dm , and secondly to investigate the effects of candidate  diabetic  stimuli on nondiabetic ( nd ) endothelial function . samples of undistended sv were collected from patients undergoing coronary artery bypass grafting at the leeds general infirmary . local ethical committee approval and informed patient consent were obtained . sv - ec were isolated using worthington type ii collagenase digestion ( 1  mg / ml , lorne laboratories , berkshire , uk ) as we described previously . cells were maintained in m199 medium ( sigma - aldrich , dorset , uk ) supplemented with 20% foetal calf serum ( fcs ; labtech international , sussex , uk ) , 1% l - glutamine , 1% antibiotic , 20  mm hepes ( all life technologies , paisley , uk ) , 15  g / ml endothelial cell growth supplement , 1  m pyruvate ( both sigma - aldrich ) , and 5  u / ml heparin ( leo laboratories ltd . , hurley , uk ) in a humidified atmosphere of 5% co2 in air at 37c and passaged using trypsin / edta ( life technologies ) as necessary . all experiments were performed on early passage cells ( p37 ) from patients with ( t2dm - ec ) or without ( nd - ec ) diabetes . subconfluent sv - ec monolayers were imaged at 100 magnification and spread cell areas measured from 50 cells per patient population as previously described . in addition , cell circularity was measured using imagej software ( http://imagej.nih.gov/ij/ ) and expressed relative to a perfect circle with a value of 1.0 . sv - ec were seeded at a density of 2  10 cells per well in 12-well plates and proliferation assays performed essentially as previously described [ 15 , 16 ] . following overnight quiescence in minimal medium ( mm ; containing 1% fcs ) , cells were reexposed to medium containing 20% fcs ( designated  day 0  ) for up to 5 days , with medium replenished on day 3 . viable cells were counted on days 0 , 3 , and 5 using a haemocytometer and trypan blue ( sigma - aldrich ) . further experiments were performed using a range of fcs concentrations ( 520% ) and all cell counts made on day 5 . data were expressed as both a percentage increase in cell number ( where day 0 counts = 100% ) and area under curve ( auc ) . to examine sv - ec proliferation / death in real time , time - lapse ptychography   was used . cells were seeded at a density of 7.5  10 cells per well in glass - bottomed 6-well plates . after overnight quiescence in mm , cells were reexposed to medium containing 20% fcs for 3  d and maintained in a humidified environment of 5% co2 in air at 37c in an incubator surrounding a phase focus virtual lens ( pfvl21 ) microscope . a 20x objective ( na 0.4 ) and a 635  nm illumination were used to obtain a 0.2  mm region at the centre of the dish . images were acquired every ~30  min for 3  d and used to create a time - lapse video . finally , these cells were trypsinised and viability was measured using a beckman coulter vi - cell . migration was analysed using a linear scratch wound model essentially as we previously described [ 15 , 16 ] . briefly , confluent sv - ec monolayers were quiesced in mm for 24  h , wounded with a sterile 10  ml pipette tip to generate a cell free area , imaged , and treated with medium containing fcs ( 120% ) for 24  h after which the number of cells migrated into the wounded area was quantified . data are represented as percentage increase in migration ( versus 1% fcs media ( mm ) = 100% ) . cells were quiesced in mm for 16 hours before preparing whole cell lysates and immunoblotting as previously described . antibodies were used at the following concentrations : phospho - enos ( ser1177 , bd biosciences , 1  :  800 ) , phospho - akt ( ser473 , cell signaling technology , 1  :  500 ) , phospho - erk1/2 ( thr202 and tyr204 , cell signaling technology , 1  :  800 ) , total akt ( cell signaling technology , 1  :  500 ) , total erk ( cell signaling technology , 1  :  1000 ) , and glyceraldehyde 3-phosphate dehydrogenase ( gapdh ; abcam , 1  :  4000 ) . films were scanned using an epson perfection 4490 flatbed scanner and densitometry analysis performed using imagej software ( http://imagej.nih.gov/ij/ ) . lysate ( designated as 100% ) that was loaded on all membranes to allow comparison between experiments . in additional experiments , cells were stimulated with 100  nm insulin ( sigma - aldrich ) for 5  min and lysates were immunoblotted for p - akt and p - erk . 
 in vitro angiogenesis was investigated using matrigel ( vwr international , lutterworth , uk ) tube - forming assays . briefly , 1  10 sv - ec were seeded in mm in duplicate wells onto polymerised matrigel 24-well plates . plates were incubated at 37c in a humidified chamber with 5% co2 in air for up to 24  h. the number of intact tubes was counted in ten random 100 fields by two independent observers . in further experiments , sv - ec were pretreated with either akt inhibitor ly294002 ( 1  m ; merck - millipore , watford , uk ) , enos inhibitor ( l - n - nitroarginine methyl ester , l - name ; 500  m ; sigma - aldrich ) , high glucose ( 25  mm ; sigma - aldrich ) , tnf- ( 1  ng / ml ; life technologies ) , palmitate ( 100  m ; sigma - aldrich ) , or appropriate control vehicle for 24  h prior to angiogenesis assays . cells were incorporated into assays without replenishment of the stimulus / inhibitor and tube formation was quantified after 8  h. in selected experiments , time - lapse ptychography   was performed in order to visualise any differences in nd and t2 dm tube dynamics , for example , the rate of formation or breakdown . in those experiments , cells were plated on matrigel - coated glass - bottomed 35  mm dishes and maintained in a humidified environment in 5% co2 ( in air ) at 37c by an incubator surrounding the pfvl21 microscope . images were acquired as described previously ; briefly a 10x objective ( na 0.3 ) and a 635  nm illumination were used to obtain a 5.88  mm region at the centre of the dish . image acquisition began 2  h after seeding of the cells , taken every 10  min for a total period of 24  h and used to create a time - lapse video such that tube formation and degradation could be monitored . sv - ec were seeded in gelatin - coated chamber slides at a density of 1  10 cells per well , cultured for 72  h in complete growth medium then fixed in 4% paraformaldehyde . to determine effects of  diabetic  stimuli , cells were quiesced in mm and then incubated in medium containing 5% fcs , either alone or supplemented with high glucose ( 25  mm ) , tnf- ( 1  ng / ml ) , palmitate ( 100  m ) , or appropriate controls for 24  h before fixing . the f - actin cytoskeleton was visualised using fluorescent rhodamine phalloidin ( life technologies ) as previously described . all images were captured on a zeiss axio imager z1 epifluorescence microscope at 200 and 400 magnification . all data are expressed as mean  sem , with n representing the number of experiments on cells from different patients . data were analysed using standard or ratio ( log transformed ) two - way anova or mann - whitney t - test as appropriate , using graphpad software ( www.graphpad.com ) , with p < 0.05 considered statistically significant . experiments were performed on endothelial cells from a total of 44 patients . the mean age of nd and t2 dm patients was not significantly different : nd ( n = 27 , 85% male ) , 64.5  1.6 ( range 5080 ) years versus t2 dm ( n = 17 , 82% male ) , and 67.0  2.1 ( range 4878 ) years ; p = 0.35 . all t2 dm patients were receiving oral therapy ( metformin / sulfonylureas / gliptins ) , and 30% of these were also receiving insulin . routine cardiovascular medications ( statins , -blockers , angiotensin modulating agents , anticoagulation therapies , and diuretics ) were similar in both cohorts as we have reported previously . in contrast to the distinct morphological and proliferative changes we have previously observed in sv smooth muscle cells ( sv - smc ) from t2 dm patients [ 14 , 21 , 22 ] , there were no distinct differences in morphology between nd and t2dm - ec , with all cells exhibiting a typical ec cobblestone appearance and no consistent difference in f - actin organisation ( figure 1(a ) ) . whilst there was inherent variability in cell size between patients , this neither did  circularity  vary between the two populations ( figure 1(c ) ) . although cell number of t2 dm ec was ~70% of nd - ec cell number at day 5 , this did not reach statistical significance ( figure 1(d ) ) . area under curve analysis for each cell population , whilst suggesting a trend towards reduced proliferation in the t2 dm cells , did not reach statistical significance ( figure 1(e ) ) . a similar degree of t2dm - ec proliferative impairment was visible across a range of concentrations of fcs ( figure 1(f ) ) . ptychography   is a novel label - free methodology that generates high quality / contrast phase images of living cells with no invasive labelling protocols or requirement for high energy illumination . the ability to digitally focus images after acquisition prevents loss of data due to focal drift of the microscope . nd and t2dm - ec were monitored for 3 d and a quantitative parameter related to cellular dry mass was calculated at each time point as a measure of proliferation . growth profiles of the 3 cell populations for both nd and t2 dm cells over time are represented in figure 1(g ) ( left panel ) . video data ( suppl . files 1 and 2 in supplementary material available online at http://dx.doi.org/10.1155/2015/409432 ) show that any reduction in dry mass was due to cells moving out of the field of view and not to increased cell death . two populations ( one each of nd and t2dm - ec ) did not proliferate readily , highlighting the inherent interpatient variability as shown by direct cell counting ( figures 1(d)1(f ) ) . importantly , the viability of all cell populations was similar between nd and t2 dm patients ( 96.2% and 96.8% , resp . ; figure 1(h ) ) suggesting that the trend towards reduced proliferation in t2dm - ec was not a result of increased cell death . indeed , time - lapse imaging showed multiple proliferation events and a lack of cell death events ( figure 1(g ) , right panel and suppl . ec motility was assessed using a scratch wound model where sv - ec migration into a denuded area over 24  h ( figure 2(c ) ) was evaluated . fcs - induced migration in both nd and t2dm - ec was concentration dependent , although the response in t2dm - ec was blunted ( figure 2(a ) ) . accordingly , in 10% fcs , t2dm - ec migration was reduced by > 30% compared to nd - ec ( figures 2(b ) and 2(c ) ) . we therefore explored the concept that basal differences in two principal insulin - stimulated signalling pathways , akt and erk ( figure 3(f ) ) , may be evident between nd and t2 dm sv - ec . cells were cultured in mm and phosphorylation of akt , enos , and erk quantified using western blotting . basal phosphorylation states of both akt ( figure 3(a ) ) and enos ( figure 3(b ) ) were significantly lower in t2dm - ec ( 56% and 73% of nd - ec , resp . ) . in contrast , phosphorylation of erk was similar in each population ( figure 3(c ) ) . diminished akt phosphorylation in t2 dm cells was maintained in response to stimulation with insulin ( figure 3(d ) ) whilst levels of erk phosphorylation were similar between populations ( figure 3(e ) ) . patients with t2 dm have impaired ability to form collateral blood vessels ; it is likely that the reduced migratory capacity is a factor contributing to inferior angiogenesis . using in vitro tube - forming assays , time courses were performed , revealing that the temporal profile of tube formation was similar in nd - ec and t2dm - ec . however , whilst each sv - ec population exhibited a similar temporal profile of tube formation which was maximal after 8  h , the number of tubes was significantly fewer in the t2 dm group ( ~60% nd ) ( figures 4(a ) and 4(b ) ) . to explore the kinetics of tube formation , we performed time - lapse ptychography over a 24  h interval . the establishment of tube - like networks was evident in both nd - ec and t2dm - ec after 2  h , the earliest time point at which the technique could detect all cells within the same plane . in nd - ec , tubes gradually expanded over the 24  h and formed stable , robust walls comprised of multiple cell layers ( denoted by red bars in figure 4(c ) ; also see suppl . file 3 ) . in t2dm - ec tubes appeared to expand more rapidly than nd - ec ( suppl . file 4 ) but tube walls appeared fragile ( generally only one cell thick ) and there were frequent broken / incomplete tubes ( illustrated by asterisks in figure 4(c ) ) . t2dm - ec exhibited marked reduction in basal levels of akt and enos phosphorylation ( figure 3 ) . to determine whether inhibiting akt or enos activity modulated in vitro angiogenesis , we treated nd - ec with appropriate inhibitors ( ly294002 and l - name , resp . ) prior to tube - forming assays . exposure to 1  m ly294002 for 24  h resulted in a reduction in akt phosphorylation confirming the efficacy of the inhibitor in these experiments ( figure 4(d ) ) . akt inhibition significantly impaired tube forming by 36% ( figure 4(e ) ) ; however the effect of l - name was inconsistent between cells from different patients ( figure 4(e ) ) . nd - ec were cultured under conditions designed to mimic the in vivo environment of t2 dm and the impact on angiogenesis assays was evaluated . we also examined the f - actin cytoskeleton due to its important role in migration and intercellular communication . surprisingly , 25  mm glucose had no impact on tube formation or the cytoskeleton ( figures 5(a ) and 5(b ) ) . in contrast , both the proinflammatory cytokine tnf- and free fatty acid palmitate significantly impaired angiogenesis , by 27% and 43% , respectively ( figures 5(c ) and 5(e ) ) . there was reduced f - actin staining intensity and reorganisation from peripheral fibres in control cells to more centrally aligned stress fibres in the tnf--treated cells ( figure 5(d ) ) . in palmitate treated cells a similar reduction in staining intensity was observed and although peripheral localisation was maintained , fibres in the body of the cell appeared more disorganised ( figure 5(f ) ) . a key strength of this study was the use of the exact cells whose dysfunction underlies failure or complications following bypass grafting in t2 dm patients , namely , sv - ec . during harvesting and grafting for cabg the endothelium is inevitably denuded and hence ec regeneration through both proliferation and migration is pivotal to restore a functional monolayer . in contrast to sv - smc [ 14 , 21 , 22 ] , gross sv - ec morphology was indistinguishable between nd and t2 dm patients ; although this does not rule out the possibility of subcellular alterations . instead , we focused on differences in cellular function that would be viewed as key to cardiovascular health . importantly , vital functions were persistently impaired , namely , migration , tube forming , and , to a lesser degree , proliferation . previously described a significant inhibition of proliferation in t2dm - ec isolated from human sv . indeed , the difference between the nd and t2 dm cells in our study appears more distinct at later time points and extending the study beyond 5 days would potentially answer this question . importantly , the modest reduction in t2dm - ec proliferation was not a result of increased cell death . previous studies have demonstrated impaired migration in bovine   and human aortic ec   under conditions of hyperglycaemia , suggesting that the diabetic milieu may harm the migratory capacity of resident cells . studies in a murine model of t2 dm also reported impaired ec migration in the db / db mouse . in human studies , reduced migration of endothelial progenitor cells from t2 dm patients was impaired relative to nd patients . accordingly we observed a significant impairment in migration of native t2dm - ec relative to nd - ec . importantly this divergence was observed under normoglycaemic conditions ( 5.5  mm glucose ) , lending credence to the notion of a persistent ec phenotype imprinted in vivo and that does not reverse easily in culture . insulin signals through the insulin receptor via two main arms , triggering the akt and erk pathways   ( figure 3(f ) ) . it is perceived that in t2 dm patients the akt pathway is impaired with signalling preferentially stimulating the erk pathway . to our knowledge this is the first direct demonstration of divergent akt and erk signalling , both basally and following insulin stimulation , in human sv - ec , another feature maintained in culture . this is in agreement with evidence in animal models of diabetes   and human skeletal muscle . since activation of akt leads to enos phosphorylation ( figure 3(f ) ) , it is perhaps not surprising that enos phosphorylation was also diminished in t2dm - ec . previous studies have revealed reduced akt and enos phosphorylation in atrial biopsies from t2 dm patients   which concurs with the data presented here ; however phosphorylation of enos was reportedly elevated in venous ec from t2 dm patients ' forearms . this highlights the importance of evaluating the strengths and weaknesses of different experimental models and advocates the use of an appropriate cellular model ( i.e. , sv - ec to explore dysfunction with respect to graft failure ) . an alternative compensatory pathway of restoring blood flow in arteries diseased by atherosclerosis is the formation of collateral blood vessels through angiogenic mechanisms . these small , new vessels help to revascularise the myocardium and bypass the occlusion . given the increased prevalence of cvd in t2 dm patients this is an important mechanism ; however it is well recognised that such patients suffer from impaired coronary collateral formation   which in turn increases the need for coronary revascularisation . animal models of diabetes exhibit reduced angiogenic capacity in vivo [ 35 , 36 ] . in our study we used a well validated in vitro model to study sv - ec angiogenesis and believe this is the first demonstration of impaired angiogenic capacity in human t2dm - ec versus nd - ec . ptychography was used to monitor tube formation dynamics without damaging or altering the cells , for example , by using fluorescent labels that potentially interfere with cell behaviour . this revealed that the initial establishment of tubes was not different between nd and t2dm - ec ; however t2dm - ec tubes expanded rapidly once established and exhibited thin , fragile walls that started to degenerate after 8  h. combining this knowledge with the reduced total number of tubes observed at 8  h , it is likely that t2dm - ec tubes are less stable than nd - ec . ptychography may be a useful technique to distinguish between the absolute capacity to form and maintain robust tubes and the ability to monitor degeneration . moreover , the absence of a requirement for cell  labelling  of any kind , together with a diffuse , low - powered 635  nm illumination , provides confidence that the cell has not been modified by such markers . having observed reduced akt and enos phosphorylation in native t2dm - ec , we explored whether akt and/or enos inhibition impaired angiogenesis in ec of nondiabetic origin . whilst akt inhibition significantly reduced tube formation , the response to enos inhibition was inconsistent ; four out of six cell populations showing reduced tube formation to varying degrees . other downstream effectors of akt , for example , vascular endothelial growth factor , may additionally be necessary in order to elicit a significant and robust inhibition . hyperglycaemia reportedly impairs ec migration [ 26 , 27 ] and inhibits angiogenesis in human microvascular ec   and huvec . somewhat unexpectedly we observed no effect of consistent high glucose on angiogenesis in sv - ec , possibly reflecting differences in the sources of ec . microvascular and macrovascular ec are phenotypically distinct , and huvecs express a foetal gene pattern which is divergent from sv - ec . another possible explanation is that variability in glycaemic control is more deleterious to endothelial cells than persistent hyperglycaemia . for example , some previous studies reported that fluctuating or intermittent high glucose increased expression of adhesion molecules icam-1 , vcam-1 , and e - selectin , augmented apoptosis , and downregulated no production . no studies have investigated hyperglycaemia per se on human sv - ec angiogenesis but the failure of intensive glycaemic control to normalise macrovascular complications in t2 dm in the medium term ( accord , advance , and vadt clinical trials [ 5 , 6 ] ) suggests involvement of other factors . circulating levels of tnf- are elevated in t2 dm patients   but reports of its effects on angiogenesis are contradictory . duration of exposure to tnf- is reportedly key to its pro- or antiangiogenic effects , and dysfunctional angiogenesis has been attributed to attenuated enos activity   and modification of rho gtpase activity ; the latter potentially associated with disturbed f - actin dynamics as observed in the present study and previously noted by others . raised plasma levels of free fatty acids are associated with metabolic syndrome . palmitate is proapoptotic to ec [ 53 , 54 ] although its effect on angiogenesis is not clear . in our study , in vitro tube - forming was markedly impaired with comparatively low ( 100  m ) concentrations of palmitate and could be attributable to inhibition of enos phosphorylation . our data also indicate reduced staining intensity of f - actin and disorganisation of identifiable fibres in palmitate - treated cells . palmitate was shown to reduce f - actin content in l6 myotubes , reportedly via accumulation of membrane cholesterol ; it is therefore possible that this mechanism exists in sv - ec . multiple cytokines and chemokines relevant to diabetic complications are induced by hyperglycaemia , including interleukin-1 beta ( il-1 ) . indeed , hyperglycaemia - induced il-1 expression in retinal endothelial cells is believed to underlie diabetic retinopathy . interestingly , a very recent study demonstrated that monocytes exposed to palmitate secreted il-1 , which in turn , induced ec adhesion molecule expression , an effect abolished by il-1 receptor antagonism . il-1 clearly plays an important role , both directly and indirectly via effects on other pathways . the maintenance of a persistent aberrant t2dm - ec phenotype under culture conditions is perhaps indicative of the phenomenon of metabolic memory ; that is , the concept that prior exposure to hyperglycaemia and/or other metabolic insults in vivo can lead to maintained cellular effects in vitro . this is likely to involve epigenetic mechanisms such as micrornas , histone modifications and/or dna methylation ( reviewed in ) . indeed , transient exposure to hyperglycaemia in human and bovine ec imparted persistent changes in the nuclear factor kappa b ( nfb ) signalling pathway that were retained even when cells were returned to normoglycaemia [ 60 , 61 ] . micrornas ( mirs ) are short , noncoding rnas that negatively regulate target gene expression and have been proposed as biomarkers for cardiovascular disease and diabetes . for example , mir-21 , widely expressed in the vasculature , has been reported to inhibit proliferation , migration , and angiogenesis in huvec . it is entirely possible that similar mechanisms exist in human sv - ec and studies to investigate this hypothesis are currently in progress . to our knowledge , this study is the first to demonstrate divergent migration and tube forming capacity in sv - ec cultured from patients with and without t2 dm . there was a nonsignificant trend towards impaired proliferation . importantly we provide direct evidence for selective impairment of the akt , but not erk signalling pathway in these clinically relevant cells . this t2 dm phenotype persisted throughout culture and passaging , suggestive of epigenetic changes inflicted in vivo and not easily reversed . nd - ec exposed to diabetic stimuli adopted a t2dm - ec phenotype , suggesting the importance of factors other than hyperglycaemia in promoting endothelial dysfunction in t2 dm . in conclusion , the blunted functionality of sv - ec from t2 dm patients in our study , together with our demonstration that diabetic stimuli can impair aspects of nd - ec behavior , may translate into an inadequate or delayed ability to reendothelialise , significantly impacting the homeostatic capabilities of the endothelium and predisposing to graft failure .
type 2 diabetes ( t2 dm ) confers increased risk of endothelial dysfunction , coronary heart disease , and vulnerability to vein graft failure after bypass grafting , despite glycaemic control . 
 this study explored the concept that endothelial cells ( ec ) cultured from t2 dm and nondiabetic ( nd ) patients are phenotypically and functionally distinct . 
 cultured human saphenous vein- ( sv- ) ec were compared between t2 dm and nd patients in parallel . 
 proliferation , migration , and in vitro angiogenesis assays were performed ; western blotting was used to quantify phosphorylation of akt , erk , and enos . 
 the ability of diabetic stimuli ( hyperglycaemia , tnf- , and palmitate ) to modulate angiogenic potential of nd - ec was also explored . 
 t2dm - ec displayed reduced migration ( ~30% ) and angiogenesis ( ~40% ) compared with nd - ec and a modest , nonsignificant trend to reduced proliferation . 
 significant inhibition of akt and enos , but not erk phosphorylation , was observed in t2 dm cells . 
 hyperglycaemia did not modify nd - ec function , but tnf- and palmitate significantly reduced angiogenic capacity ( by 27% and 43% , resp . ) , effects mimicked by akt inhibition . 
 aberrancies of ec function may help to explain the increased risk of sv graft failure in t2 dm patients . 
 this study highlights the importance of other potentially contributing factors in addition to hyperglycaemia that may inflict injury and long - term dysfunction to the homeostatic capacity of the endothelium .