blood was recovered from 14 individuals with type 2 diabetes who arrived in our patient clinic ( sex [ m / f ] , 8/6 ; a1c , 8  1.2% ; age , 55.8  9.46 years ; bmi , 28.1  3.22 kg / m ; creatinine , 1.06  0.20 mg / dl ; waist circumference , 98.8  8.02 cm ; total cholesterol , 198  30 mmol / l ; hdl cholesterol , 48  12.98 mmol / l ; ldl cholesterol , 115 
38.94 mmol / l ; triglycerides , 159.5  55.23 mg / dl ; fasting glucose , 125  18.03 mg / dl ; no retinopathy ; hypertension in three patients ; blood pressure , 142/89 mmhg ; cholesterol / apolipoprotein b , 1.3  0.3 ) .
a total of 12 blood donors were used as control subjects ( sex [ m / f ] , 6/6 ; age , 47.8  4.85 years ; bmi , 21.25  8.07 kg / m ; creatinine , 0.90  0.083 mg / dl ; total cholesterol , 164.03  9.68 mmol / l ; hdl cholesterol , 50  8.4 mmol / l ; ldl cholesterol , 85.26  16.72
mmol / l ; triglycerides , 131.14  25.03 mg / dl ; no retinopathy ; no hypertension ; blood pressure , 126/70 mmhg ; cholesterol / apolipoprotein b , 1.6  0.2 ) .
ethics approval was obtained from both simt ( servizio immunoematologia e medicina trasfusionale ) and the institutional review board of s. giovanni battista hospital , turin , italy .
testing sessions were as follows : uag ( 3.0 g  kg  h i.v .
as infusion for 12 h , from 0 to 12 h ) ; ag ( 1.0 g  kg  h i.v . as infusion for 12 h , from 0 to 12 h ) ; and isotonic saline ( infusion from 0 to 12 h ) .
an indwelling catheter was placed into an forearm vein for slow infusion of isotonic saline .
cells were isolated from blood samples taken at 0 , 6 , and 12 h. to isolate cacs , peripheral blood mononuclear cells retrieved from healthy subjects ( ncacs ) or from individuals with type 2 diabetes ( dcacs ) were plated on fibronectin - coated dishes as described by hill et al .
briefly , the cells were cultured for 4 days in egm-2 medium ( cambrex , walkersville , md ) . to isolate epcs ( nepcs from healthy subjects , depcs from individuals with type 2 diabetes ) ,
peripheral blood mononuclear cells were recovered and cultured onto collagen-1coated dishes for 21 days in egm-2 medium as described by yoder et al .
( 26 ) . in selected experiments , cacs or epcs recovered from saline - infused healthy subjects were cultured with 1.2 mg / ml age , h2o2 ( 100
mol / l ) , 5 mmol / l glucose , or 25 mmol / l glucose alone or in combination with 1 mol / l uag or 1 mol / l ag ; 1 mol / l uag or 1 mol / l ag was also used alone .
fluorescence - activated cell sorter analysis was used to characterize cac and epc surface markers ( anti - cd45 , anti - cd14 , anti - cd34 , anti - cd31 , anti 
tie-2 , anti - kdr , anti - vwf antibodies ; see supplementary methods , available in an online appendix at http://diabetes.diabetesjournals.org/cgi/content/full/db09-0858/dc1 ) .
the plasma glucose and insulin determination for each group of mice ( charles river lab , lecco , italy ) are reported : 16 8-week - old ob / ob mice ( blood glucose , 296  19.6 mg / dl ; insulin , 55  9 ng / ml ) ; 16 8-week - old c57bl/6j wild - type mice ( blood glucose , 92  7.2 mg / dl ; insulin , 10  0.5 ng / ml ) . animal procedures conformed to the guide for the care and use of laboratory animals ( 27 ) .
blood glucose was measured with a one touch ii glucose meter ( lifescan , mountain view , ca ) .
serum insulin was measured with a mouse insulin radioimmunoassay kit ( linco research , st .
detection of ros ; gtp - rac1 loading assay ; senescence assay ; western blot analysis ; silencing of endogenous p53 , akt , and p47 by small interfering rnas ( sirnas ) ; matrigel plug assay ; immunohistochemistry and immunofluorescence ; human and mouse mobilization assays ; enzyme - linked immunosorbent assay and radioimmunoassay ; isolation and culture of bone marrow  derived cells ; and evaluation of mmp9 activation , cytofluorimetry analysis , and in vitro migration assays were described in detail in the supplementary methods . in vitro and in vivo results are representative of at least three independent experiments , performed at least in triplicate .
densitometric analysis using a bio - rad gs 250 molecular imager was used to calculate the differences in the fold induction of protein activation or expression .
significance of differences between experimental and control values ( * , # ,  , and  indicate p < 0.05 , statistically significant ) was calculated using anova with newman - keuls multicomparison test .
blood was recovered from 14 individuals with type 2 diabetes who arrived in our patient clinic ( sex [ m / f ] , 8/6 ; a1c , 8  1.2% ; age , 55.8  9.46 years ; bmi , 28.1  3.22 kg / m ; creatinine , 1.06  0.20 mg / dl ; waist circumference , 98.8  8.02 cm ; total cholesterol , 198  30 mmol / l ; hdl cholesterol , 48  12.98 mmol / l ; ldl cholesterol , 115 
38.94 mmol / l ; triglycerides , 159.5  55.23 mg / dl ; fasting glucose , 125  18.03 mg / dl ; no retinopathy ; hypertension in three patients ; blood pressure , 142/89 mmhg ; cholesterol / apolipoprotein b , 1.3  0.3 ) .
a total of 12 blood donors were used as control subjects ( sex [ m / f ] , 6/6 ; age , 47.8  4.85 years ; bmi , 21.25  8.07 kg / m ; creatinine , 0.90  0.083 mg / dl ; total cholesterol , 164.03  9.68 mmol / l ; hdl cholesterol , 50  8.4 mmol / l ; ldl cholesterol , 85.26  16.72
mmol / l ; triglycerides , 131.14  25.03 mg / dl ; no retinopathy ; no hypertension ; blood pressure , 126/70 mmhg ; cholesterol / apolipoprotein b , 1.6  0.2 ) .
ethics approval was obtained from both simt ( servizio immunoematologia e medicina trasfusionale ) and the institutional review board of s. giovanni battista hospital , turin , italy .
testing sessions were as follows : uag ( 3.0 g  kg  h i.v .
as infusion for 12 h , from 0 to 12 h ) ; ag ( 1.0 g  kg  h i.v . as infusion for 12 h , from 0 to 12 h ) ; and isotonic saline ( infusion from 0 to 12 h ) .
an indwelling catheter was placed into an forearm vein for slow infusion of isotonic saline .
to isolate cacs , peripheral blood mononuclear cells retrieved from healthy subjects ( ncacs ) or from individuals with type 2 diabetes ( dcacs ) were plated on fibronectin - coated dishes as described by hill et al .
briefly , the cells were cultured for 4 days in egm-2 medium ( cambrex , walkersville , md ) . to isolate epcs ( nepcs from healthy subjects , depcs from individuals with type 2 diabetes ) ,
peripheral blood mononuclear cells were recovered and cultured onto collagen-1coated dishes for 21 days in egm-2 medium as described by yoder et al .
( 26 ) . in selected experiments , cacs or epcs recovered from saline - infused healthy subjects were cultured with 1.2 mg / ml age , h2o2 ( 100
mol / l ) , 5 mmol / l glucose , or 25 mmol / l glucose alone or in combination with 1 mol / l uag or 1 mol / l ag ; 1 mol / l uag or 1 mol / l ag was also used alone .
fluorescence - activated cell sorter analysis was used to characterize cac and epc surface markers ( anti - cd45 , anti - cd14 , anti - cd34 , anti - cd31 , anti  tie-2 , anti - kdr , anti - vwf antibodies ; see supplementary methods , available in an online appendix at http://diabetes.diabetesjournals.org/cgi/content/full/db09-0858/dc1 ) .
the plasma glucose and insulin determination for each group of mice ( charles river lab , lecco , italy ) are reported : 16 8-week - old ob / ob mice ( blood glucose , 296  19.6 mg / dl ; insulin , 55  9 ng / ml ) ; 16 8-week - old c57bl/6j wild - type mice ( blood glucose , 92  7.2 mg / dl ; insulin , 10  0.5 ng / ml ) . animal procedures conformed to the guide for the care and use of laboratory animals ( 27 ) .
blood glucose was measured with a one touch ii glucose meter ( lifescan , mountain view , ca ) .
serum insulin was measured with a mouse insulin radioimmunoassay kit ( linco research , st .
detection of ros ; gtp - rac1 loading assay ; senescence assay ; western blot analysis ; silencing of endogenous p53 , akt , and p47 by small interfering rnas ( sirnas ) ; matrigel plug assay ; immunohistochemistry and immunofluorescence ; human and mouse mobilization assays ; enzyme - linked immunosorbent assay and radioimmunoassay ; isolation and culture of bone marrow  derived cells ; and evaluation of mmp9 activation , cytofluorimetry analysis , and in vitro migration assays were described in detail in the supplementary methods .
in vitro and in vivo results are representative of at least three independent experiments , performed at least in triplicate .
densitometric analysis using a bio - rad gs 250 molecular imager was used to calculate the differences in the fold induction of protein activation or expression .
significance of differences between experimental and control values ( * , # ,  , and  indicate p < 0.05 , statistically significant ) was calculated using anova with newman - keuls multicomparison test .
several lines of evidence indicate that the number and function of epcs are impaired in diabetes ( 17,19 ) and that these events rely mainly on nox - mediated ros production ( 15 ) .
the effect of in vivo uag and ag administration in protecting both cacs and epcs from oxidative damage was first evaluated . toward this end , cells isolated from uag- or ag - treated individuals with type 2 diabetes and healthy subjects were characterized for cac and epc markers ( supplementary fig .
1 ) ( 18 ) and subjected to dichlorofluorescein diacetate fluorescence assay . the results reported in fig .
1a and b demonstrate that uag , but not ag , treatment ( 6 h ) drastically reduced ros production in individuals with type 2 diabetes .
moreover , the number of viable cells was significantly higher after uag treatment compared with before ( fig .
a protective effect of uag on ros production was also demonstrated in cells cultured with age or high glucose ( supplementary fig .
these findings , together with the observation that neither in vitro nor in vivo uag administration ( supplementary fig . 2c and d ) changed age receptor ( rage ) expression , suggest that effector(s ) downstream of rage is the target for protective effect of uag . because similar results were obtained after 12 h of treatment ( data not shown ) , data throughout the study relates to 6-h uag treatment .
a and b : dichlorofluorescein diacetate assay was performed on cacs or epcs , recovered from healthy subjects ( ncacs , nepcs ) or from diabetic patients ( dcacs , depcs ) treated with saline , uag , or ag ( white area : no treatment ; dark gray area : uag infused ; light gray area : ag infused ) .
c : cacs and epcs isolated from healthy donors ( n ) and from diabetic patients ( d ) treated as above were cultured as described in the research design and methods section .
trypan blue  excluded viable cells were counted ( * and  indicate p < 0.05 , healthy donors vs. diabetic groups ; # p < 0.05 , saline vs. uag infusion ) .
d : ros production , in response to apocynin alone or in combination with uag , was evaluated on dcacs and depcs isolated from saline - treated patients ( white area : no treatment ; dark gray area : apocynin ; light gray area : apocynin + uag ) .
e : ros production was evaluated on dcacs and depcs isolated from saline - infused patients and transfected with the scrambled sequence or with p47 sirna .
cells were treated or not with uag ( white area : scramble ; dark gray area : p47 sirna ; light gray area : p47 sirna + uag ) .
f : dcacs and depcs retrieved from saline- or uag - infused patients were analyzed for rac1 activation .
cell extracts were either pulled down with gst - pak or directly subjected to sds - page .
cacs and epcs were recovered after a 6-h infusion with saline , uag , or ag as indicated .
apocynin , known to affect the assembly of nox subunits ( 28 ) , was used to investigate the role of nox in regulating ros production .
cells recovered from saline - infused individuals with type 2 diabetes were subjected to apocynin treatment .
1d ) , suggesting that nox activity might be controlled by uag . as the assembly of p47 and p67 subunits
is required for nox enzymatic activity ( 29 ) , silencing of p47 in dcacs and depcs ( supplementary fig .
once more , this effect could not be further enhanced by the addition of uag ( fig .
3b and c ) was prevented in cells expressing a dominant - negative racn17 construct .
indeed , rac1 activation was detected in cells recovered from saline - treated individuals with type 2 diabetes , but not from uag - infused patients ( fig .
hence , the modulation of rac1 activity is a crucial step in the uag anti - ros protective effect .
rac1 membrane localization and function rely on isoprenylation , which has been correlated with amp - activated protein kinase ( ampk)-dependent hydroxymethylglutaryl coa reductase activity ( 30 ) . to rule out the possibility that inhibition of rac1 activity by uag depends on this pathway , ampk phosphorylation was evaluated .
neither short- nor long - term exposure to uag alone or in combination with age affected ampk activity ( supplementary fig .
4 ) . p53 , p21 , and prb are major regulators of cell senescence ( 31 ) .
the above results prompted us to evaluate whether the increase in ros production , generally considered as an upstream signal , translates into an accelerated onset of senescence and whether uag could rescue this effect . because both senescence and ros generation ( fig . 2a and b ) were prevented by silencing p53 ( supplementary fig .
5a ) , we investigated the in vivo effect of uag on p53 expression . accordingly , uag treatment was able to prevent p53 accumulation , p21 expression , and rb phosphorylation ( fig . 2c ) and to reduce the number of senescence - associated -galactosidase ( sa--gal)-positive dcacs and depcs ( fig .
a : senescence was evaluated on dcacs and depcs recovered from saline - infused patients transfected with psuper retro containing p53 sirna or the scrambled sequence as control and expressed as the percentage of sa--gal  positive cells ( * p < 0.05 , scramble vs. p53 sirna ) .
b : ros production was evaluated on dcacs and depcs recovered from saline - infused patients transfected with psuper retro containing p53 sirna or the scrambled sequence ( white area : scramble ; gray area : p53 sirna ) .
c : dcacs and depcs retrieved from saline- or uag - infused patients were lysed and analyzed for p53 , p21 , and prb expression by western blotting . oxidized ldl  treated endothelial cells were used as positive control ( + ) .
d : senescence was evaluated on dcacs and depcs recovered from saline- or uag - infused patients and expressed as the percentage of sa--gal  positive cells ( * p < 0.05 , saline vs. uag infusion ) . to assess
whether the protective effect of uag also resulted in an enhancement of dcac and depc vasculogenic capability , de novo vessel formation was analyzed in severe combined immunodeficient mice injected with cells recovered from uag - treated individuals with type 2 diabetes . at 15 days after injection ,
plugs were recovered and analyzed by immunohistochemistry . as shown in fig . 3a and b ,
the number of functional vessels formed by cells recovered from uag - treated patients was significantly increased with respect to those from saline - treated patients .
the origin of neovessels from host vasculogenic cells was excluded because the majority of vessels were lined by human hla class i  positive cells ( supplementary fig .
a : immunohistochemistry of representative functional vessels formed by labeled dcacs and depcs , recovered from saline - infused ( left ) or from uag - infused ( right ) diabetic patients .
b : quantification of newly formed vessels was expressed as percentage  sd of the vessel area to the total matrigel area ( * p < 0.05 , saline infused vs. uag infused ) .
( a high - quality digital representation of this figure is available in the online issue . ) defective epc and cac mobilization has been reported in diabetes ( 19,20 ) . to further investigate the potential therapeutic effect of uag , cacs and epcs
were recovered from 10 normal healthy subjects and 10 individuals with type 2 diabetes , characterized , and counted .
uag treatment led to an increase in the number of recovered cells in individuals with type 2 diabetes compared with that of healthy subjects , and no effect of uag treatment was detected in healthy subjects ( fig .
4a ) . in contrast , no differences between ag- or saline - treated healthy and type 2 diabetic individuals were observed ( fig .
a : percentage of cacs and epcs recovered after 6 h of saline , uag , or ag treatment : ( n ) healthy donors and ( d ) diabetic patients ( * p < 0.05 , diabetic patients vs. healthy donors , # p < 0.05 saline vs. uag infusion ) .
b : senescence was evaluated on cacs and epcs recovered as described in a and expressed as the percentage of sa--gal  positive cells ( * p < 0.05 , diabetic patients vs. healthy donors , # p < 0.05 saline vs. uag infusion ) .
c : percentage of epcs recovered from wild - type or ob / ob mice infused for 12 h with saline , uag , or ag ( * p < 0.05 wt uag - infused vs. ob / ob uag - infused mice ) .
d : senescence was evaluated on murine epcs treated as described in c and expressed as the percentage of sa--gal  positive cells ( * p < 0.05 , wt vs. ob / ob mice , # p < 0.05 saline vs. uag infusion ) .
all data are the mean  sd obtained by three individual investigators . circulating stromal derived factor-1 ( sdf-1 ) ( 33 ) and vascular endothelial growth factor ( vegf ) ( 34 )
we herein demonstrate that 6- or 12-h ( data not shown ) uag or ag infusion did not change their serum concentrations ( supplementary fig .
similarly , igf-1 ( 35 ) serum concentration was not affected by uag treatment ( supplementary fig .
7 ) . for validation and characterization of the molecular mechanisms regulating bone marrow mobilization , a mouse model of type 2 diabetes ( ob / ob mice ) was used .
after treatment with saline , uag , or ag , recovered cells were subjected to fluorescence - activated cell sorter analysis for surface markers to confirm epc identity ( data not shown ) .
uag treatment induced a strong increase of recovered epcs only in ob / ob mice ( fig .
4c ) . finally , the number of senescent cells was significantly lower in uag - treated ob / ob mice compared with untreated or ag - treated animals ( fig .
as an impairment of enos phosphorylation contributes to defective epc mobilization in the diabetic setting ( 36 ) , we investigated whether uag modulated enos activity and the activation of its regulatory protein , akt ( 37 ) , in bone marrow stromal cells .
the stromal origin of the enos - expressing cells was confirmed by the presence of mbkitl ( fig .
consistent with the pivotal role of a local activation of mmp9 in promoting progenitor cell mobilization ( 22 ) , gelatin zymography revealed that mmp9 gelatinolytic activity was induced by uag ( fig .
the role of mmp9 activation and skitl release in controlling this event was further confirmed by functional studies using anti - mmp9 and anti - kitl neutralizing antibodies ( fig .
5c ) . accordingly , in parallel with effects on mmp9 activation , expression of the mbkitl was decreased in ob / ob mice subjected to uag treatment ( fig .
although we can not rule out the possibility that a paracrine effect of uag occurs in vivo , herein we have shown that in vitro uag treatment for 40 min elicited akt and enos phosphorylation in stromal cells obtained from ob / ob - derived total bone marrow pools ( fig .
5d ) . in agreement with the results measuring epc mobilization , ag failed to induce akt and enos phosphorylation ( fig .
the finding that uag failed to induce enos phosphorylation after knocking down akt ( fig .
the above data were validated by the lack of effect of uag treatment in nos3 mice ( 23 ) ( supplementary fig .
a : adherent cells ( from total bone marrow populations ) obtained from saline- , uag- , or ag - treated wt or ob / ob mice were lysed and analyzed by western blotting using the indicated antibodies .
the blots are representative of two wt ( 12 ) or three ob / ob ( 35 ) samples .
b : representative zymogram of mmp9 gelatinolytic activity on serum recovered from saline- or uag - treated mice . as positive control ,
c : migration assays performed on bone marrow cells using sera recovered from saline- or uag - treated ob / ob mice .
mmp9 or skitl neutralizing antibodies were added where indicated ( * p < 0.05 , saline vs. uag , # p < 0.05 , uag vs. uag + anti - mmp9 and uag + anti - skitl ) .
d : adherent cells obtained from ob / ob total bone marrow pools were treated as indicated for 40 min and processed for western blotting using the indicated antibodies .
e : adherent cells from ob / ob total bone marrow pools were depleted ( sirna akt ) or not ( scrambled ) of akt , subjected to uag treatment , and analyzed by western blotting using the indicated antibodies . based on our collective dataset
, we further investigated whether the biological response of cacs to uag was mediated by specific binding sites localized to the plasma membrane . to this end , double immunofluorescence experiments , using the uag analog 488-uag ( 100 nmol / l ) to label putative binding sites and anti  phycoerythrin - cd45 antibody as a membrane marker , were carried out at 4c . as shown in fig .
6a , at 4c 488-uag  binding sites colocalized with cd45 , indicating a plasma membrane localization . in agreement with the functional data , unlabeled uag ( 1 mol / l )
displaced the fluorescent signal from the cell surface , whereas ag ( 1 mol / l ) did not ( fig .
6b , after 20-min stimulation , receptor clusters undergoing internalization were visualized as labeled cytoplasmic vesicles . moreover , increasing concentrations of unlabeled uag specifically displaced the fluorescent ligand from both the plasma membrane and endocytic vesicles ( fig . 6c and supplementary fig .
a  c : living cells were incubated in the absence ( ctrl ) or presence of 100 nmol / l 488-uag with or without the indicated ligands .
the putative receptor is stained in green ( 488-uag ) ; the plasma membrane in red ( phycoerythrin - cd45 ) ; and the nucleus in blue ( dapi ) . a : visualization of 488-uag  binding site distribution on cac plasma membrane .
cac incubation with ligands was carried out at 4c for 4 h. 488-uag was displaced by 1 mol / l uag but not by 1 mol / l ag ( 40 magnification ) .
b and c : visualization of 488-uag  binding distribution on cacs upon incubation with the indicated ligands for 20 min at 25c .
b : representative photomicrograph showing the 488-uag  labeled cell surface binding sites internalized into endocytotic vesicles ( 20 magnification ) . c : 488-uag binding and internalization were completed by increasing concentrations of the unlabeled uag .
( a high - quality digital representation of this figure is available in the online issue . )
several lines of evidence indicate that the number and function of epcs are impaired in diabetes ( 17,19 ) and that these events rely mainly on nox - mediated ros production ( 15 ) .
the effect of in vivo uag and ag administration in protecting both cacs and epcs from oxidative damage was first evaluated . toward this end , cells isolated from uag- or ag - treated individuals with type 2 diabetes and healthy subjects were characterized for cac and epc markers ( supplementary fig .
1 ) ( 18 ) and subjected to dichlorofluorescein diacetate fluorescence assay . the results reported in fig .
1a and b demonstrate that uag , but not ag , treatment ( 6 h ) drastically reduced ros production in individuals with type 2 diabetes .
moreover , the number of viable cells was significantly higher after uag treatment compared with before ( fig .
a protective effect of uag on ros production was also demonstrated in cells cultured with age or high glucose ( supplementary fig .
these findings , together with the observation that neither in vitro nor in vivo uag administration ( supplementary fig . 2c and d ) changed age receptor ( rage ) expression , suggest that effector(s ) downstream of rage is the target for protective effect of uag . because similar results were obtained after 12 h of treatment ( data not shown ) , data throughout the study relates to 6-h uag treatment .
a and b : dichlorofluorescein diacetate assay was performed on cacs or epcs , recovered from healthy subjects ( ncacs , nepcs ) or from diabetic patients ( dcacs , depcs ) treated with saline , uag , or ag ( white area : no treatment ; dark gray area : uag infused ; light gray area : ag infused ) .
c : cacs and epcs isolated from healthy donors ( n ) and from diabetic patients ( d ) treated as above were cultured as described in the research design and methods section .
trypan blue  excluded viable cells were counted ( * and  indicate p < 0.05 , healthy donors vs. diabetic groups ; # p < 0.05 , saline vs. uag infusion ) .
d : ros production , in response to apocynin alone or in combination with uag , was evaluated on dcacs and depcs isolated from saline - treated patients ( white area : no treatment ; dark gray area : apocynin ; light gray area : apocynin + uag ) .
e : ros production was evaluated on dcacs and depcs isolated from saline - infused patients and transfected with the scrambled sequence or with p47 sirna .
cells were treated or not with uag ( white area : scramble ; dark gray area : p47 sirna ; light gray area : p47 sirna + uag ) .
f : dcacs and depcs retrieved from saline- or uag - infused patients were analyzed for rac1 activation .
cell extracts were either pulled down with gst - pak or directly subjected to sds - page .
cacs and epcs were recovered after a 6-h infusion with saline , uag , or ag as indicated .
apocynin , known to affect the assembly of nox subunits ( 28 ) , was used to investigate the role of nox in regulating ros production .
cells recovered from saline - infused individuals with type 2 diabetes were subjected to apocynin treatment .
1d ) , suggesting that nox activity might be controlled by uag . as the assembly of p47 and p67 subunits
is required for nox enzymatic activity ( 29 ) , silencing of p47 in dcacs and depcs ( supplementary fig .
once more , this effect could not be further enhanced by the addition of uag ( fig .
3b and c ) was prevented in cells expressing a dominant - negative racn17 construct .
indeed , rac1 activation was detected in cells recovered from saline - treated individuals with type 2 diabetes , but not from uag - infused patients ( fig .
hence , the modulation of rac1 activity is a crucial step in the uag anti - ros protective effect .
rac1 membrane localization and function rely on isoprenylation , which has been correlated with amp - activated protein kinase ( ampk)-dependent hydroxymethylglutaryl coa reductase activity ( 30 ) . to rule out the possibility that inhibition of rac1 activity by uag depends on this pathway , ampk phosphorylation was evaluated .
neither short- nor long - term exposure to uag alone or in combination with age affected ampk activity ( supplementary fig .
p53 , p21 , and prb are major regulators of cell senescence ( 31 ) .
the above results prompted us to evaluate whether the increase in ros production , generally considered as an upstream signal , translates into an accelerated onset of senescence and whether uag could rescue this effect . because both senescence and ros generation ( fig . 2a and b ) were prevented by silencing p53 ( supplementary fig .
5a ) , we investigated the in vivo effect of uag on p53 expression . accordingly , uag treatment was able to prevent p53 accumulation , p21 expression , and rb phosphorylation ( fig . 2c ) and to reduce the number of senescence - associated -galactosidase ( sa--gal)-positive dcacs and depcs ( fig .
similar results were obtained in age - treated cells ( supplementary fig . 5b and c ) .
a : senescence was evaluated on dcacs and depcs recovered from saline - infused patients transfected with psuper retro containing p53 sirna or the scrambled sequence as control and expressed as the percentage of sa--gal  positive cells ( * p < 0.05 , scramble vs. p53 sirna ) .
b : ros production was evaluated on dcacs and depcs recovered from saline - infused patients transfected with psuper retro containing p53 sirna or the scrambled sequence ( white area : scramble ; gray area : p53 sirna ) .
c : dcacs and depcs retrieved from saline- or uag - infused patients were lysed and analyzed for p53 , p21 , and prb expression by western blotting . oxidized ldl  treated endothelial cells were used as positive control ( + ) .
d : senescence was evaluated on dcacs and depcs recovered from saline- or uag - infused patients and expressed as the percentage of sa--gal  positive cells ( * p < 0.05 , saline vs. uag infusion ) . to assess
whether the protective effect of uag also resulted in an enhancement of dcac and depc vasculogenic capability , de novo vessel formation was analyzed in severe combined immunodeficient mice injected with cells recovered from uag - treated individuals with type 2 diabetes . at 15 days after injection
3a and b , the number of functional vessels formed by cells recovered from uag - treated patients was significantly increased with respect to those from saline - treated patients .
the origin of neovessels from host vasculogenic cells was excluded because the majority of vessels were lined by human hla class i  positive cells ( supplementary fig .
6 ) ( 32 ) . thus , these data provide evidence that uag restores dcac and depc vasculogenic activity .
a : immunohistochemistry of representative functional vessels formed by labeled dcacs and depcs , recovered from saline - infused ( left ) or from uag - infused ( right ) diabetic patients .
b : quantification of newly formed vessels was expressed as percentage  sd of the vessel area to the total matrigel area ( * p < 0.05 , saline infused vs. uag infused ) .
( a high - quality digital representation of this figure is available in the online issue . )
defective epc and cac mobilization has been reported in diabetes ( 19,20 ) . to further investigate the potential therapeutic effect of uag , cacs and epcs were recovered from 10 normal healthy subjects and 10 individuals with type 2 diabetes , characterized , and counted .
uag treatment led to an increase in the number of recovered cells in individuals with type 2 diabetes compared with that of healthy subjects , and no effect of uag treatment was detected in healthy subjects ( fig .
in contrast , no differences between ag- or saline - treated healthy and type 2 diabetic individuals were observed ( fig .
a : percentage of cacs and epcs recovered after 6 h of saline , uag , or ag treatment : ( n ) healthy donors and ( d ) diabetic patients ( * p < 0.05 , diabetic patients vs. healthy donors , # p < 0.05 saline vs. uag infusion ) .
b : senescence was evaluated on cacs and epcs recovered as described in a and expressed as the percentage of sa--gal  positive cells ( * p < 0.05 , diabetic patients vs. healthy donors , # p < 0.05 saline vs. uag infusion ) .
c : percentage of epcs recovered from wild - type or ob / ob mice infused for 12 h with saline , uag , or ag ( * p < 0.05 wt uag - infused vs. ob / ob uag - infused mice ) .
d : senescence was evaluated on murine epcs treated as described in c and expressed as the percentage of sa--gal  positive cells ( * p < 0.05 , wt vs. ob / ob mice , # p < 0.05 saline vs. uag infusion ) .
circulating stromal derived factor-1 ( sdf-1 ) ( 33 ) and vascular endothelial growth factor ( vegf ) ( 34 ) strictly control progenitor cell mobilization under stress conditions .
we herein demonstrate that 6- or 12-h ( data not shown ) uag or ag infusion did not change their serum concentrations ( supplementary fig .
similarly , igf-1 ( 35 ) serum concentration was not affected by uag treatment ( supplementary fig .
for validation and characterization of the molecular mechanisms regulating bone marrow mobilization , a mouse model of type 2 diabetes ( ob / ob mice ) was used . after treatment with saline , uag , or ag , recovered cells
were subjected to fluorescence - activated cell sorter analysis for surface markers to confirm epc identity ( data not shown ) .
uag treatment induced a strong increase of recovered epcs only in ob / ob mice ( fig .
4c ) . finally , the number of senescent cells was significantly lower in uag - treated ob / ob mice compared with untreated or ag - treated animals ( fig .
as an impairment of enos phosphorylation contributes to defective epc mobilization in the diabetic setting ( 36 ) , we investigated whether uag modulated enos activity and the activation of its regulatory protein , akt ( 37 ) , in bone marrow stromal cells .
the stromal origin of the enos - expressing cells was confirmed by the presence of mbkitl ( fig .
consistent with the pivotal role of a local activation of mmp9 in promoting progenitor cell mobilization ( 22 ) , gelatin zymography revealed that mmp9 gelatinolytic activity was induced by uag ( fig .
the role of mmp9 activation and skitl release in controlling this event was further confirmed by functional studies using anti - mmp9 and anti - kitl neutralizing antibodies ( fig .
5c ) . accordingly , in parallel with effects on mmp9 activation , expression of the mbkitl was decreased in ob / ob mice subjected to uag treatment ( fig .
although we can not rule out the possibility that a paracrine effect of uag occurs in vivo , herein we have shown that in vitro uag treatment for 40 min elicited akt and enos phosphorylation in stromal cells obtained from ob / ob - derived total bone marrow pools ( fig .
5d ) . in agreement with the results measuring epc mobilization , ag failed to induce akt and enos phosphorylation ( fig .
the finding that uag failed to induce enos phosphorylation after knocking down akt ( fig .
the above data were validated by the lack of effect of uag treatment in nos3 mice ( 23 ) ( supplementary fig .
a : adherent cells ( from total bone marrow populations ) obtained from saline- , uag- , or ag - treated wt or ob / ob mice were lysed and analyzed by western blotting using the indicated antibodies .
the blots are representative of two wt ( 12 ) or three ob / ob ( 35 ) samples .
b : representative zymogram of mmp9 gelatinolytic activity on serum recovered from saline- or uag - treated mice .
c : migration assays performed on bone marrow cells using sera recovered from saline- or uag - treated ob / ob mice .
mmp9 or skitl neutralizing antibodies were added where indicated ( * p < 0.05 , saline vs. uag , # p < 0.05 , uag vs. uag + anti - mmp9 and uag + anti - skitl ) .
d : adherent cells obtained from ob / ob total bone marrow pools were treated as indicated for 40 min and processed for western blotting using the indicated antibodies .
e : adherent cells from ob / ob total bone marrow pools were depleted ( sirna akt ) or not ( scrambled ) of akt , subjected to uag treatment , and analyzed by western blotting using the indicated antibodies .
based on our collective dataset , we further investigated whether the biological response of cacs to uag was mediated by specific binding sites localized to the plasma membrane . to this end , double immunofluorescence experiments , using the uag analog 488-uag ( 100 nmol / l ) to label putative binding sites and anti  phycoerythrin - cd45 antibody as a membrane marker , were carried out at 4c . as shown in fig .
6a , at 4c 488-uag  binding sites colocalized with cd45 , indicating a plasma membrane localization . in agreement with the functional data , unlabeled uag ( 1 mol / l )
displaced the fluorescent signal from the cell surface , whereas ag ( 1 mol / l ) did not ( fig .
6b , after 20-min stimulation , receptor clusters undergoing internalization were visualized as labeled cytoplasmic vesicles .
moreover , increasing concentrations of unlabeled uag specifically displaced the fluorescent ligand from both the plasma membrane and endocytic vesicles ( fig . 6c and supplementary
a  c : living cells were incubated in the absence ( ctrl ) or presence of 100 nmol / l 488-uag with or without the indicated ligands .
the putative receptor is stained in green ( 488-uag ) ; the plasma membrane in red ( phycoerythrin - cd45 ) ; and the nucleus in blue ( dapi ) . a : visualization of 488-uag  binding site distribution on cac plasma membrane .
cac incubation with ligands was carried out at 4c for 4 h. 488-uag was displaced by 1 mol / l uag but not by 1 mol / l ag ( 40 magnification ) .
b and c : visualization of 488-uag  binding distribution on cacs upon incubation with the indicated ligands for 20 min at 25c .
b : representative photomicrograph showing the 488-uag  labeled cell surface binding sites internalized into endocytotic vesicles ( 20 magnification ) . c : 488-uag binding and internalization were completed by increasing concentrations of the unlabeled uag .
( a high - quality digital representation of this figure is available in the online issue . )
the present data first demonstrate that uag , unlike ag , reverts diabetes - induced epc damage by inhibiting activation of the nox regulatory protein rac1 ; as a consequence , uag protects diabetic epcs from senescence and improves their vasculogenic capability ; again , only uag rescues epc mobilization under diabetic conditions by restoring enos phosphorylation , and specific uag - binding sites mediate its effects .
uag is the most abundant circulating form of ghrelin ( 3 ) and plays a positive role on glucose metabolism .
in contrast , basic and clinical studies have proposed ag as a diabetogenic hormone ( 8,38 ) . indeed , clinical conditions of insulin resistance are associated with an alteration in the circulating ghrelin profile with relative ag excess with respect to uag ( 12 ) .
thus , it is tempting to speculate that an altered uag / ag ratio might contribute both to metabolic changes and to diabetes - associated complications . among diabetes - associated complications , abnormal vascular remodeling is believed to play a major role in accelerating vascular disease ( 39 ) .
historically , it has been assumed that new blood vessels originate from sprouting cells and co - opting of neighboring preexisting vessels . however , both physiological and pathologic angiogenesis are also supported by mobilization and recruitment of other cell types , including the bone marrow  derived cells , such as epcs ( 16,18,40,41 ) .
interestingly , alterations in the number and function of these cells correlate with the risk factor profile ( 19,20 ) .
overproduction of ross in these pathologic settings seems to contribute to impaired vascular regenerative processes ( 42 ) .
the plasma membrane nox is recognized as one of the major regulators of ros generation ( 15,29 ) .
activation of the enzyme can occur via many upstream signaling pathways converging on phosphorylation of p47 and activation of rac1 leading to the oxidase assembly ( 29 ) .
our study shows that uag can protect diabetic epcs from oxidative stress by affecting the nox regulatory protein rac1 .
the accelerated onset of senescence contributes to the impaired epc bioavailability in patients with diabetes ( 17 ) .
the tumor suppressor p53 is a transcription factor involved in dna damage mechanisms and is recognized as a negative regulator of cell proliferation in human atherosclerotic and restenosis lesions ( 43 ) .
moreover , the p53-mediated pathway contributes to epc senescence - like growth arrest in diabetes ( 44 ) .
accordingly , the present study shows that silencing p53 in epcs isolated from individuals with type 2 diabetes or cultured with age prevents both ros production and senescence .
the finding that both in vitro and in vivo exposure to uag negatively modulates p53 accumulation identifies the p53-mediated signal as the primary mechanism through which uag protects epcs from senescence .
we also found that , by preventing oxidative stress , uag improves de novo vessel formation .
the efficacy of cell therapy certainly depends on the number , functional capability , and successful retention of cells in the site of action .
thus , our data strongly suggest that naturally occurring uag induces improvement of epc function and survival that may translate into a more efficient response to vascular dysfunction .
senescence is also associated with impaired mobilization of bone marrow  derived cells ( 17 ) .
the molecular interactions between stem cells and bone marrow stromal cells , and the molecular mechanisms controlling their mobilization in the bone marrow microenvironment , are poorly understood ( 45 ) .
considerable interest has arisen about agents able to mobilize and augment progenitor cell delivery to sites of vascular injury to enhance revascularization . among these ,
sdf-1 and vegf are recognized as primary regulators of bone marrow cell mobilization during stress conditions ( 22,46 ) .
in addition , under physiological stresses , the activation of matrix proteases within the bone marrow microenvironment results in the release of skitl , which enables endothelial and hematopoietic progenitor cells to transit from the quiescent to the proliferative niche and facilitates their mobilization into the circulation ( 22 ) .
the delivery of progenitor cells to sites of neovascularization also relies on functional enos activity ( 23 ) .
indeed , in pathologic settings associated with blunted enos activity and reduced systemic no bioavailability , defective epc mobilization and impaired vascular regenerative processes occur ( 24,25 ) .
enos activation through akt has been reported for ag acting on the gq - coupled ghs - r1a in cultured endothelial cells ( 47 ) .
we herein demonstrate that uag can restore enos activity via akt - mediated phosphorylation in a pathologic setting characterized by impaired enos phosphorylation .
such an event was found crucial to epc bone marrow mobilization , because uag had no effect in enos knockout mice .
we also showed that enos phosphorylation , occurring in response to uag , is associated with mmp9 activation and possibly with the release of skitl , as suggested by the reduced expression of the mbkitl on stromal cells recovered from uag - treated mice and by functional studies .
although we can not exclude that uag may act in a paracrine manner by locally inducing the release of vegf from bone marrow stromal cells , we demonstrate that short - term treatment with uag , but not ag , of bone marrow cells in vitro leads to akt and enos phosphorylation and that , in vivo , these events are associated with mmp9 activation and epc mobilization .
furthermore , uag , unlike ag , strongly induced epc mobilization in individuals with type 2 diabetes , but not in nondiabetic subjects .
notably , no change in serum concentrations of primary mobilization factors was detected after uag or ag systemic administration .
in addition , our results shed light on the earliest molecular events leading to uag- but not ag - mediated physiological regulation of epc bioavailability .
indeed , we showed that epcs possess specific uag - binding sites , which are not recognized by ag .
in addition to ghs - r1a , which is the ag - specific receptor ( 3 ) , other ghrelin receptor subtypes exist , whose molecular identities have not yet been characterized , but that recognize both ag and uag ( 3 ) .
thus , our data provide the first evidence of the existence of uag - specific binding sites and of their relevance in human - derived epcs , which could represent a novel target for pharmacological modulation .
preclinical and clinical studies generally support the therapeutic potential of autologous epcs in the treatment of cardiovascular diseases , such as tissue ischemia and myocardial infarction ( 49 ) .
however , epc mobilization may also accelerate atherosclerotic plaque progression ( 50 ) and induce tumor ( 51 ) or retina ( 52 ) neovascularization in individuals with type 2 diabetes .
nonetheless , therapies with statins , the main epc mobilization mediators ( 53 ) , revealed no concerns in terms of neovascularization . we now have reason to believe that the significant epc mobilization potential of uag , and the lack of its effects on serum levels of sdf-1 and vegf , may be exploited for clinical treatment of diabetes- and atherosclerosis - induced vascular impairment . in the presence of cardiovascular risk factors such as diabetes , epc availability
is reduced , restricting the possibility of treating patients in need with directed / cell - based therapies . besides displaying a positive influence on -cell viability and glucose homeostasis ( 3 ) , uag mobilizes epcs , protects epcs from oxidative stress and from senescence , and increases de novo vessel formation ( see model in fig .
this suggests that uag - related peptides or uag receptor  specific agonists may be further developed into lead compounds from the perspective of a novel pharmacologic intervention to ameliorate both metabolic control and impaired vascular growth in individuals with type 2 diabetes where ag has failed .
the resident bone marrow pool of early and late epcs was mobilized into circulation after uag - mediated akt activation in stromal cells , which leads to enos phosphorylation and mmp9 activation . in turn ,
mmp9 activation switched on the release of skitl , a determinant for epc exit into the bloodstream .
in addition , uag treatment restores diabetic viability of epcs and increases their vasculogenic capability by preventing ros generation and its downstream signaling pathway ( p53 accumulation , p21 expression , rb phosphorylation ) .
this effect results from uag - mediated inhibition of rac1 activation , a prerequisite for nadph oxidase assembly .
 objectiveacylated ghrelin ( ag ) is a diabetogenic and orexigenic gastric polypeptide . 
 these properties are not shared by the most abundant circulating form , which is unacylated ( uag ) . an altered uag / ag profile together with an impairment of circulating endothelial progenitor cell ( epc ) bioavailability 
 were found in diabetes . 
 based on previous evidence for the beneficial cardiovascular effects of ag and uag , we investigated their potential to revert diabetes - associated defects.research design and methodshealthy human subjects , individuals with type 2 diabetes , and ob / ob mice were ag or uag infused . 
 epc mobilization in patients and mice was evaluated , and the underlying molecular mechanisms were investigated in bone marrow stromal cells . 
 recovered epcs were also evaluated for the activity of senescence regulatory pathways and for nadph oxidase activation by knocking down p47phox and rac1 . 
 finally , uag modulation of human epc vasculogenic potential was investigated in an in vivo mouse model.resultsneither ag nor uag had any effect in healthy subjects . however , systemic administration of uag , but not ag , prevented diabetes - induced epc damage by modulating the nadph oxidase regulatory protein rac1 and improved the vasculogenic potential both in individuals with type 2 diabetes and in ob / ob mice . 
 in addition , unlike ag , uag facilitated the recovery of bone marrow epc mobilization . 
 crucial to epc mobilization by uag was the rescue of endothelial no synthase ( enos ) phosphorylation by akt , as uag treatment was ineffective in enos knockout mice . 
 consistently , epcs expressed specific uag - binding sites , not recognized by ag.conclusionsthese data provide the rationale for clinical applications of uag in pathologic settings where ag fails . 