diabetic retinopathy ( dr ) is a devastating complication of diabetes , manifesting primarily as vascular structural and functional changes in the retina , eventually leading to vision loss . dr is the most common cause of blindness in north america in the 2574-years age group . glucose - induced increased production of vasoactive factors are characteristics of all chronic diabetic complications including dr . vascular endothelial growth factor ( vegf ) vegf is an important mediator of increased vascular permeability in early dr and a major contributor of retinal neovascularization in proliferative dr [ 14 ] . in human , vegf mrna expression is increased in the neovascular membranes from diabetic patients obtained by vitrectomy compared to those removed from the nondiabetic individuals . augmented vegf protein production was further observed in human retina in diabetes using immunohistochemistry [ 2 , 3 ] . we and others have demonstrated increased vegf expression in the retina of streptozotocin - induced diabetic rat model causing increased microvascular permeability [ 5 , 6 ] . in a murine model of ischemic retinopathy , inhibition of vegf furthermore exposure of endothelial cells ( ecs ) to high glucose causes increased vegf expression [ 8 , 9 ] . vegf mrna and protein expression are stimulated by long - term high glucose treatment in bovine microvascular retinal ecs . previous studies in our lab have demonstrated that vegf interacts with other vasoactive factors such as endothelin-1 ( et-1 ) in mediating glucose - induced increased permeability in the ecs [ 5 , 8 ] . these data indicate that vegf plays an important role in causing increased vascular permeability and angiogenesis in dr [ 2 , 3 ] . several glucose induced signaling mechanisms such as protein kinase c ( pkc ) activation , nonenzymatic glycation and mitogen - activated protein kinase ( mapk ) activation are instrumental in causing glucose induced alteration of vasoactive factors in diabetes . erk5 , also known as big mapk1 ( bmk1 ) , was identified as a member of the mapk family in 1995 [ 12 , 13 ] . mapk / erk kinase 5 ( mek5 ) is the specific mapk kinase for erk5 . studies on erk5 knockout mice have shown that the erk5 pathway is critical for endothelial function and for maintaining blood vessel integrity . in addition , erk5 signaling mediates stress response in the ecs [ 14 , 16 ] . more recently , studies have shown that erk5 signaling controls migration and morphology of the ecs . it has been demonstrated that in the nondiabetic conditions , erk5 have a regulatory role on vegf expression [ 1820 ] . in mouse , mutant erk5 gene ( homozygous knockout ) increases expression of vegf mrna and protein [ 19 , 20 ] . investigations have shown that overexpression of krupple - like factor 2 ( klf2 ) , a target gene of erk5 , inhibited vegf receptor promoter activation . in addition , microarray studies have revealed that klf2 overexpression decreases mrna expression of human vegf receptor 2 [ 23 , 24 ] . hence it is potentially possible that erk5 through klf2 may also have a regulatory role on the production of vegf in dr . however such possibilities have not been investigated . here , we examined the mechanisms of glucose - induced erk5-mediated regulation of vegf in the ecs and in the retina of diabetic rats . a dermal - derived human microvascular endothelial cell ( hmvec ) was obtained from lonza , inc . hmvecs were grown in endothelial cell basal medium 2 ( ebm-2 , lonza , walkersville , md ) containing human epidermal growth factor ( hegf ) , 1 ; hydrocortisone , 0.4 ; gentamycin , 1 ; fetal bovine serum ( fbs ) , 10 ; vascular endothelial growth factor ( vegf ) , 1 ; human fibroblast growth factor - basic ( hfgf - b ) , 4 ; long r3 insulin - like growth factor ( r3-igf-1 ) , 1 ; ascorbic acid , 1. in ebm2 , the glucose concentration was 5 cells were grown in 25  cm tissue culture flasks and maintained in a humidified atmosphere containing 5% co2 at 37c . cells at 80% confluence were growth arrested by incubation in serum - free medium overnight prior to incubation with high glucose ( 25  mmol / l d - glucose ) or osmotic control ( l - glucose ) of the same concentration . for gain of function study , constitutively active human recombinant mek5 ( camek5 , 20moi ) adenovirus ( cell biolabs , san diego , ca ) was used to activate erk5 . cells were seeded in 6-well plate , cultured overnight and infected with adenovirus for 48  hrs . a nonspecific gfp adenovirus with the same multiplicity of infection ( 20moi ) was used as a negative control . for loss of function study , erk5 sirna ( sierk5 ) was used to knock down erk5 expression in endothelial cells . endothelial cells were transfected with erk5 sirnas ( on - targetplus sirna , 100  nmol / l ; dharmacon inc . lafayette , co ) for 48 hrs using sirna transfection reagent ( dharmafect 4 ; dharmacon inc . lafayette , co ) with the same concentration of erk5 sirna was used as a negative control ( control sirna ) . trizol reagent ( invitrogen , burlington , on , canada ) was used to isolate rna as previously described in . rna was extracted with chloroform followed by centrifugation to separate the sample into aqueous and organic phases . rna was recovered from the aqueous phase by isopropyl alcohol precipitation and suspended in diethylpyrocarbonate - treated water . total rna ( 24  g ) was used for cdna synthesis with high capacity cdna reverse transcription kit ( applied biosystem , foster city , ca ) . real - time rt - pcr was performed using the lightcycler ( roche diagnostics canada , laval , pq , canada ) as previously described in . for a final reaction volume of 20  l , the following reagents were added : 10  l sybr advantage qpcr premix ( clontech , mountain view , ca ) , 1  l of each forward and reverse 10  m primers ( table 1 ) , 7  l h2o , and 1  l cdna template . messenger rna ( mrna ) levels were quantified using the standard curve method . the data were normalized to 18s ribosomal rna or -actin rna to account for differences in reverse transcription efficiencies and the amount of template in the reaction mixtures . total proteins from endothelial cells were isolated by homogenizing cells in lysis buffer ( contains 25  mmol / l trishcl , ph 7.5 , 150  mmol / l nacl , 5  mmol / l mgcl2 , 1% np-40 , 1  mmol / l dtt and 5% glycerol ) and protease inhibitor ( complete mini tablet , roche ) and phosphatase inhibitor cocktail 1 and 2 ( sigma - aldrich , saint louis , mo ) . protein concentrations were determined by bicinchoninic acid ( bca ) protein assay kit ( pierce , rockford , il ) . 30  g of protein was resolved by 10% sodium dodecyl sulfate - polyacrylamide gel electrophoresis and transferred to a pvdf membrane ( bio - rad , hercules , ca ) . the membrane was then incubated with the rabbit antiphospho - erk5 antibody ( 1  :  1000 ; cell signaling technology , ma ) . horseradish peroxidase - conjugated antirabbit antibody ( 1  :  10000 ; upstate biotechnology , charlottesville , va ) was used for detection . the signals from the western blots were visualized with an ecl plus chemiluminescence detection kit ( amersham pharmacia biotechnology , buckinghamshire , uk ) . blots were stripped with reblot plus strong antibody stripping solution ( millipore corporation , billerica , ma ) and reprobed with erk5 antibody ( 1  :  1000 ; cell signaling technology , ma ) . blots were then stripped again and reprobed with -actin antibody ( 1  :  1000 , santa cruz biotechnology , ca ) as a control for protein loading . blots were quantified by densitometry using mocha software ( spss , chicago , il ) and the data expressed as a ratio of phosphor - erk5 to -actin . the angiogenic responses to high glucose ( 25  mmol / l ) and erk5 sirna transfection were assessed using an in vitro matrigel analysis . following appropriate treatment , hmvecs were seeded in 96-well culture plates precoated with ecmatrix ( in vitro angiogenesis assay kit , milipore , billerica , ma ) at 1  10 cells / well . images were captured with infinity capture software version 3.5.1 at 10 magnification after 6  hrs incubation . to quantify the image of tube formation , branch points were counted in several random microscopic fields ( 35 ) per sample and the values averaged . male sprague - dawley rats ( charles river ) weighing between 200 and 250  g were used . diabetes was induced by a single intravenous injection of streptozotocin ( 65  mg / kg , in citrate buffer , ph 5.6 ) . age - and sex - matched rats were used as controls and given equal volume of citrate buffer . the animals were monitored for glucosuria and ketonuria ( uriscan gluketo ; yeong dong , seoul , south korea ) . all diabetic rats were implanted with slow release insulin implants to prevent ketosis ( approximately 2  u / day ) ( linshin , scarborough , on , canada ) . we have previously demonstrated that they develop diabetes induced tissue damage in the retina and kidney at this time . the retinas of the right eye were gently peeled off , snap - frozen in liquid nitrogen , and stored at 70c . the left retinas were fixed in 4% paraformaldehyde ( pfa ) , as described before in . all animals were cared for according to the association for research in vision and ophthalmology 's guiding principles in the care and use of animals . all experiments were approved by the university of western ontario council on animal care committee . formalin fixed retinal tissues were embedded in paraffin , sectioned at 4  m thickness , and placed on positively charged slides for phosphor - erk5 ( perk5 ) immunohistochemical staining . briefly , the sections were incubated with rabbit anti - perk5 antibody ( invitrogen , carlsbad , ca , usa ) at 1  :  200 dilutions overnight at 4c , followed by incubation with labeled polymer - hrp anti - rabbit antibody ( dako north america , carpinteria , ca , usa ) for 30 minutes at room temperature . visualization was conducted using diaminobenzidine ( dab , dako north america , carpinteria , ca , usa ) as hrp substrate . images were recorded by an olympus bx51 microscope ( olympus canada inc , on , canada ) with northern eclipse software ( empix inc , on , canada ) . statistical significance of difference between groups was tested using student 's t - test or if there were more than two groups , using one way analysis of variance ( anova ) followed by posthoc analysis . we initially established whether in the ecs , glucose causes any alteration of erk5 signaling . no change in the mrna expression of erk5 and its downstream molecules mef2c , klf2 , or vegf were seen after exposure of the cells to 25  mmol / l glucose ( hg ) for 1 hour compared to 5  mmol / l glucose ( lg ) ( figures 1(c)1(f ) ) . l - glucose was used as an osmotic control ( oc ) . following 24  hrs of glucose exposure , erk5 signaling was significantly activated as evidenced by increased phosphorylation ( figures 1(a ) and 1(b ) ) and mrna expression of erk5 ( figure 1(c ) ) and augmented mrna expression of mef2c ( figure 1(d ) ) and klf2 ( figure 1(e ) ) , which are downstream mediators of erk5 signaling . interestingly , at this time point there were no increases of vegf mrna expression ( figure 1(f ) ) . however , with increased duration of high - glucose treatment ( 48  hrs ) , erk5 phosphoryation and mrna expression ( figures 1(a)1(c ) ) as well as mef2c and klf2 mrna expression ( figures 1(d ) and 1(e ) ) were decreased , while vegf mrna expression was increased ( figure 1(f ) ) . similar results were also observed after 72  hrs of hg treatment ( data not shown ) . since expression of total erk5 was also changed after glucose treatment , western blot of perk5 was normalized to -actin . to further delineate the mechanistic role and significance of glucose - induced erk5 activation , especially with its regulatory effects on vegf expression , gain and our initial investigation indicated that vegf mrna expression was inversely related with erk5 activation following high - glucose treatment . to further explore the interaction of erk5 signaling and vegf expression , loss of function study was performed in the hmvecs using erk5 sirna . erk5 's downstream substrates , mef2c and klf2 , were also significantly reduced ( figures 2(b ) and 2(c ) ) . as expected , vegf mrna expression was increased following erk5 sirna transfection measured by real - time pcr ( figure 2(d ) ) . to further study the effect of erk5 knockdown on glucose induced vegf upregulation , hmvecs were transfected with erk5 sirna and then treated with high glucose for short period ( 24  hrs ) . results showed that erk5 sirna transfection abolished high - glucose - induced increase of erk5 , mef2c , klf2 mrna ( figures 2(a)2(c ) ) , whereas promoted upregulation of vegf mrna ( figure 2(d ) ) . we further expanded the investigations to examine functional significance of glucose - induced erk5 mediated vegf expression . as glucose - induced vegf upregulation plays an important role in neovascularization in proliferative dr , we examined whether alteration of erk5 and subsequent change in vegf has any effects on endothelial tube formation using and an in vitro angiogenesis assay . high - glucose treatment ( 48  hrs ) stimulated branching and tube formation in endothelial cells transfected with control sirna ( figures 3(a ) and 3(b ) ) , which was similar as our finding in endothelial cells without transfection ( data not shown ) . glucose induced tube formation paralleled increased vegf mrna expression measured by real - time pcr ( figure 1(e ) ) . endothelial cells transfected with erk5 sirna rapidly formed capillary - like tube structures ( figure 3(c ) ) . high - glucose treatment further augmented the number and size of tube - like structure formation ( figure 3(d ) ) . such increase in tube formation was associated with pronounced vegf mrna expression ( figure 2(d ) ) . we then investigated whether erk5 upregulation can protect endothelial cells in pathological conditions mediated by glucose . as glucose - induced tissue damage is mediated by vasoactive factors such as vegf , we proceeded to examine whether glucose induced upregulation of vegf mrna can be prevented by constitutively active mek5 ( camek5 ) . mek5 is a specific mapk kinase for erk5 [ 13 , 30 , 31 ] . camek5 not only activated erk5 phosphorylation , but also augmented erk5 transcription ( figures 4(a ) and 4(b ) ) . western blot confirmed increased perk5 after camek5 infection in hmvecs ( figure 4(a ) ) . real - time pcr showed that erk5 mrna level after camek5 infection was significantly higher than that of gfp control and untreated control ( figure 4(b ) ) . erk5 activation caused upregulation of klf2 mrna expression ( figure 4(c ) ) and downregulation of vegf mrna expression . ( figure 4(d ) ) .   to study the effect of camek5 on glucose - induced vegf expression , hmvecs was infected with camek5 and then treated with high glucose for 24  hrs . vegf mrna was slightly decreased by camek5-induced activation of erk5 in lg groups , while constitutive activation of erk5 by camek5 infection led to a significant decrease of vegf after hg treatment ( figure 4(e ) ) . from the perspective of dr , it is important to examine whether the alterations demonstrated in the endothelial cells are indeed important in a clinically relevant model of diabetic retinal microangiopathy . hence , we investigated retinas from a well - established model of diabetic retinal microangiopathy . phospho - erk5 immunohistochemical staining was performed in the retinal tissues of stz - induced diabetic rats after 4 month of followup . diabetic animals showed increased blood glucose levels , reduced body weight gain , glucosuria , and occasional ketonuria ( data not shown ) . positive perk5 staining was localized in the ganglion cell layer and in the inner nuclear layer . microscopic examination further revealed that such positivity was in the microvasculature as well as in other cells . the number of positive cells and the intensity of staining were reduced in the retina of diabetic animals compared with that of controls ( figures 5(a ) and 5(b ) ) . we also examined the mrna expression of erk5 , klf2 , and vegf in retinal tissues of 4-month diabetic and control rats . real - time pcr showed that erk5 mrna expression in diabetic group was too low to be detected ( data not shown ) . in parallel , mrna expression of klf2 , a downstream molecule of erk5 signaling , in diabetic group was markedly lower than that of control group ( figure 5(c ) ) . in keeping with our previous data from our lab and others , real - time pcr analyses demonstrated a significant increase of vegf mrna expression in the retina of diabetic rats ( figure 5(d ) ) . here we have demonstrated a novel mechanism of glucose mediated vegf gene upregulation in diabetes . we have shown that in the ecs glucose causes transient activation of erk5 followed by deactivation . we have confirmed such negative regulation of erk5 on vegf using erk5 silencing and overexpression . furthermore , we have found similar alteration of erk5 and vegf in the retina of diabetic animals . a substantial body of evidence indicates that vegf is a major angiogenic factor involved in dr [ 32 , 33 ] . in addition , knockout animal study showed that jnk regulated vegf expression at the transcriptional level in hypoxia induced retinal vegf production . a recent publication revealed that wnt signaling is activated in dr and upregulates vegf expression . on the other hand erk5 , as demonstrated in this paper , is the only protective signaling that is activated by high glucose . in this study , we examined the effect of erk5 on high - glucose - induced vegf expression and demonstrated a novel pathway that potentially contributes to vegf expression and subsequent angiogenesis in dr . erk5 is different from other mapks because of its unique c - terminal , which contains transcriptional activation domain . expression of this transcription domain is sufficient to drive mef2 and regulate mef2-dependent gene expression . the importance of transcriptional activation erk5 have also been previously demonstrated by its effects on inhibition of erk5 sumoylation and prevention of diabetes - mediated left ventricular dysfunction . studies have shown that the activated kinase activity of erk5 undergoes autophosphorylation on its most c - terminal region , which is required for the c - terminal - half to enhance the erk5 activity . in this study we have demonstrated both attenuated erk5 transcription and phosphorylation in high - glucose treated endothelial cells . our data indicate that both mechanisms may be operating in glucose - induced vegf upregulation . it has been previously reported that mef2-klf2 counteracted vegf - mediated inflammatory responses in endothelial cells . in addition , klf2 , a downstream molecule of erk5 , is a transcriptional regulator of angiogenesis , and overexpression of klf2 counteracts vegf - mediated angiogenesis due to a potent inhibition of vegfr2 expression and promoter activity . our study found that reduced erk5 , mef2c , and klf2 expression were along with increased vegf level after 48  hrs glucose exposure ( figure 1 ) . on the other hand , after 24  hrs , although erk5 was activated , we failed to observe vegf upregulation . possible explanation may include other regulatory factors and cell specific factors , which need further characterization . nevertheless , erk5 sirna transfection significantly reduced mef2c and klf2 mrna expression ( figure 4 ) , suggesting that erk5 negatively regulates vegf through mef2c and klf2 upon high glucose treatment . under hypoxic conditions , erk5 inhibits vegf via hypoxia inducible factor 1 ( hif1 ) in endothelial cells . in addition , it has been shown that klf2 inhibits hif1a and hypoxia - mediated angiogenesis . high glucose induced a state of pseudo - hypoxia in diabetic complications [ 43 , 44 ] . it is therefore possible that depletion of erk5/klf2 signaling may promote high glucose - induced angiogenesis via hif1a . in keeping with our finding it has been demonstrated that erk5 activation is induced transiently by high glucose in endothelial cells , which ultimately decreased after long - term treatment . as following long - term glucose exposure in ecs and in the retina of chronically diabetic animals , similar pattern of erk5 and vegf were seen , it is possible that in long - term diabetes inhibitory effects of erk5 is lost , leading to vegf upregulation . in this study this is in keeping with studies in endothelial cells from other sources [ 9 , 46 ] . various sources of cells and culture conditions may in part be responsible for such discrepancy . in addition simultaneously other mechanisms may also be responsible for glucose induced vegf upregulation . both pkc and erk1/2 activation we observed that after 24  hrs glucose treatment , although there was significant activation of erk5 , there were no significant downregulation of vegf in this system . this suggests that other mechanisms mentioned above may also modulate glucose - induced vegf , further investigation is required to delineate the relationship of erk5 and other signaling pathways . exact mechanism of glucose induced erk5 alteration is still not known , however it is possible that glucose - induced oxidative stress is a key player in erk5 change . growth factors such as epidermal growth factor ( egf ) and nerve growth factor ( ngf ) activate erk5 [ 48 , 49 ] . hence several additional mechanisms may potentially regulate glucose induced erk5 alteration . we , however , understand some of the limitations of this study as the in vitro experiments were performed in the microvascular cells of nonretinal origin . we tried to address some of these problems with simultaneous experiments at levels of complexities , that is , retinal tissues . however , additional future studies are needed in various cell types to further characterize these changes . in summary , this is the first study to show that erk5 may potentially regulate vegf upon high - glucose treatment in the ecs and in the retina of diabetic rats . although this study was done in the context of diabetic retinopathy , it is possible that such process is of importance in other diabetic complications involving vegf signaling . erk5 may also provide an attractive target for drug development in dr and other diabetic vascular complications .
diabetic retinopathy is one of the most common causes of blindness in north america . 
 several signaling mechanisms are activated secondary to hyperglycemia in diabetes , leading to activation of vasoactive factors . 
 we investigated a novel pathway , namely extracellular signal regulated kinase 5 ( erk5 ) mediated signaling , in modulating glucose - induced vascular endothelial growth factor ( vegf ) expression . 
 human microvascular endothelial cells ( hmvec ) were exposed to glucose . in parallel , retinal tissues from streptozotocin - induced diabetic rats 
 were examined after 4 months of follow - up . in hmvecs , glucose caused initial activation followed by deactivation of erk5 and its downstream mediators myocyte enhancing factor 2c ( mef2c ) and kruppel - like factor 2 ( klf2 ) mrna expression . 
 erk5 inactivation further led to augmented vegf mrna expression . 
 furthermore , sirna mediated erk5 gene knockdown suppressed mef2c and klf2 expression and increased vegf expression and angiogenesis . on the other hand , constitutively active mek5 , an activator of erk5 , increased erk5 activation and erk5 and klf2 mrna expression and attenuated basal- and glucose - induced vegf mrna expression . 
 in the retina of diabetic rats , depletion of erk5 , klf2 and upregulation of vegf mrna were demonstrated . 
 these results indicated that erk5 depletion contributes to glucose induced increased vegf production and angiogenesis . 
 hence , erk5 may be a putative therapeutic target to modulate vegf expression in diabetic retinopathy .