kits for cd34 and cd31 cells were , respectively , from stemcell technologies ( grenoble , france ) and dynal - biotech ( invitrogen , cergy - pontoise , france ) .
culture media , including endothelial cell basal medium ( ecbm ) , were from promocell ( heidelberg , germany ) .
antibodies for flow cytometry were from bd biosciences ( le pont - de - claix , france ) or caltag ( invitrogen , cergy - pontoise , france ) .
antibodies for immunofluorescence were from dakocytomation ( denmark , cd31 ) , epitomics ( cd34 ) , zymed ( invitrogen , france , icam1 ) , santa - cruz ( cliniscience , france , gpihbp1 ) , upstate ( millipore , france , phospho - histone--h2ax ) , and molecular probes ( invitrogen ; secondary antibodies coupled to alexafluor-488 or alexafluor-546 ) .
paired biopsies from abdominal sat ( periumbilical ) or vat ( greater omental ) were obtained from subjects undergoing bariatric surgery for obesity .
subjects had been weight stable for at least 3 months before surgery , and they included 29 women and 1 man ( mean bmi  sem = 43.35  1.05
kg / m , range 34.559.1 ; mean age  sem = 40.4  1.93 years , range 2661 years ) .
five subjects were hypertensive , 6 had hypercholesterolemia , and 3 had type 2 diabetes .
the mean time since obesity  sem had been diagnosed was 18.2  1.4 years .
abdominal sat were obtained from 51 nonobese healthy women undergoing plastic surgery for cosmetic purposes ( mean bmi  sem = 23.11  0.221 , range 19.527.2 ; mean age  sem = 43.57  1.753 , range 2474 ) .
fat collection protocols were approved by the institutional research board of inserm and the toulouse university hospital ethics committee . at ( 210 g ) was processed immediately after removal .
adipocytes and stromal - vascular fraction ( svf ) were obtained by collagenase digestion as previously described ( 10 ) . after digestion and filtration of the adipocyte suspension
( 250-m mesh nylon sieves ) , adipocytes were washed three times with krebs - ringer bicarbonate buffer supplemented with 10 mmol / l hepes ( krbh ) and 0.1% fatty acid - free bsa ( ph = 7.4 ) .
mature adipocytes were suspended in ecbm supplemented with 0.1% bsa ( 1/10 , vol / vol ) .
three different calibrated fields were examined to measure adipocyte diameters using nis software ( nikon , champigny - sur - marne , france ) .
svf cells were counted and analyzed by flow cytometry to determine the number of ecs .
[ pbs]/0.5% bsa , 2 mmol / l edta ) were incubated ( for 20 min , at 4c ) with fitc - cd31 and percp - cd34 antibodies or the appropriate isotype controls .
after washing , the labeled cells were analyzed by flow cytometry using a fluorescence - activated cell sorter calibur flow cytometer and diva software ( bd - biosciences ) .
when the quantity of svf cells was sufficient ( 11 subjects , paired sat and vat ) , native cd34/cd31 cells ( ec ) were isolated by immunoselection / depletion as previously described ( 10 ) .
mature adipocytes and ec were either lysed and stored at 20c for mrna extraction or further processed . sat and vat mature adipocytes were placed in fibrin gels ( 1.5 mg fibrinogen / ml ecbm supplemented with 25 units / ml thrombin ; 1/3 , vol / vol ) and cultured in ecbm/0.1% bsa supplemented with 100 units / ml penicillin and 100 g / ml streptomycin .
after 24 h , the adipocyte - conditioned media were collected and frozen at 80c . sat mature adipocytes were cultured in ecbm supplemented with 0.1% bsa , 100 units / ml penicillin , and 100 g / ml streptomycin in clinicell 25-culture cassettes ( 1/3 , vol / vol ) in normoxia or hypoxia chambers ( 1% o2 ; sanyo , avon , france ) .
after 24 h , mature adipocytes were lysed and stored at 20c for rna extraction .
tissue immunofluorescent staining of human sat and vat cut into small pieces ( 0.51 mm ) was performed .
after fixation in paraformaldehyde 4% for 1 h and subsequent washing in pbs , at pieces were incubated for 30 min in pbs/2% bsa in the presence of 0.1% triton . for -h2ax staining , at pieces
were permeabilized for 5 min in 95% ethanol and 5% acetic acid , and then incubated for 30 min in tris - buffered saline ( tbs)/3% bsa . at pieces
were incubated for 1 h with primary antibody ( cd31 [ 1/50 ] , cd34 [ 1/200 ] , icam1 [ 1/50 ] , gpihbp1 [ 1/40 ] , -h2ax [ 1/200 ] ) .
after several washings ( pbs/0.2% tween or tbs ) , at pieces were incubated for 30 min in pbs/2% bsa/0.1% triton or tbs/3% bsa and then incubated with the corresponding fluorescently - labeled secondary antibodies ( alexafluor-488 or alexafluor-546 , 1/200 ) .
after washing , at pieces were incubated for 10 min with 10 g / ml hoescht 33342 to stain nuclei .
they were placed between two mounting slides and examined with a fluorescence ( nikon ) or confocal ( zeiss 510 ) microscope .
quantification of phospho--h2ax positive nuclei in human sat and vat was performed on three calibrated fields using nis software ( nikon , champigny - sur - marne , france ) and normalized to the total number of nuclei ( hoescht 33342 staining ) .
total rna was extracted from ec and mature adipocytes and real - time pcr analyses were performed as previously described ( 10 ) .
primers ( taqman gene expression assays from applied biosystems , lifetechnologies , france ) are listed in the supplemental table in the online appendix available at http://diabetes.diabetesjournals.org .
senescence - associated--galactosidase ( sa--gal ) activity was determined in human sat - ec isolated from nonobese healthy women ( mean bmi  sem = 25.34  1.033 , range 22.028.5 ; mean age  sem = 48.5  3.074 , range 3557 ) and treated or not with adipocyte - conditioned media from sat and vat ( pretreated or not for 1 h with 1 g / ml vegfa neutralizing antibody [ r&d systems , lille , france ] , or with vegfa [ 10 ng / ml ; peprotech , levallois - perret , france ] ) .
cells were fixed ( 5 min , room temperature ) in 0.5% glutaraldehyde , washed with pbs , and incubated at 37c for 16 h with fresh sa--gal staining solution ( 1 mg / ml of 5-bromo-4-chloro-3-indolyl galactopyranoside in dimethylformamide ) supplemented with 0.12 mmol
/ l potassium ferricyanide , and 1 mmol / l mgcl2 ( ph 6 ) .
cells positive for sa--gal activity were counted by phase contrast microscopy through scanning the whole well and normalized to the total number of cells determined in four different fields with hoescht 33342 nuclear staining using nis software .
comparisons between the two groups ( paired biopsies from sat and vat ) were analyzed by paired student t tests .
comparisons among groups were made by one - way anova followed by a dunnett post hoc test .
kits for cd34 and cd31 cells were , respectively , from stemcell technologies ( grenoble , france ) and dynal - biotech ( invitrogen , cergy - pontoise , france ) .
culture media , including endothelial cell basal medium ( ecbm ) , were from promocell ( heidelberg , germany ) .
antibodies for flow cytometry were from bd biosciences ( le pont - de - claix , france ) or caltag ( invitrogen , cergy - pontoise , france ) .
antibodies for immunofluorescence were from dakocytomation ( denmark , cd31 ) , epitomics ( cd34 ) , zymed ( invitrogen , france , icam1 ) , santa - cruz ( cliniscience , france , gpihbp1 ) , upstate ( millipore , france , phospho - histone--h2ax ) , and molecular probes ( invitrogen ; secondary antibodies coupled to alexafluor-488 or alexafluor-546 ) .
paired biopsies from abdominal sat ( periumbilical ) or vat ( greater omental ) were obtained from subjects undergoing bariatric surgery for obesity .
subjects had been weight stable for at least 3 months before surgery , and they included 29 women and 1 man ( mean bmi  sem = 43.35  1.05
kg / m , range 34.559.1 ; mean age  sem = 40.4  1.93 years , range 2661 years ) .
five subjects were hypertensive , 6 had hypercholesterolemia , and 3 had type 2 diabetes . the mean time since
abdominal sat were obtained from 51 nonobese healthy women undergoing plastic surgery for cosmetic purposes ( mean bmi  sem = 23.11  0.221 , range 19.527.2 ; mean age  sem = 43.57  1.753 , range 2474 ) .
fat collection protocols were approved by the institutional research board of inserm and the toulouse university hospital ethics committee . at ( 210 g ) was processed immediately after removal .
adipocytes and stromal - vascular fraction ( svf ) were obtained by collagenase digestion as previously described ( 10 ) . after digestion and filtration of the adipocyte suspension
( 250-m mesh nylon sieves ) , adipocytes were washed three times with krebs - ringer bicarbonate buffer supplemented with 10 mmol / l hepes ( krbh ) and 0.1% fatty acid - free bsa ( ph = 7.4 ) .
mature adipocytes were suspended in ecbm supplemented with 0.1% bsa ( 1/10 , vol / vol ) .
three different calibrated fields were examined to measure adipocyte diameters using nis software ( nikon , champigny - sur - marne , france ) .
svf cells were counted and analyzed by flow cytometry to determine the number of ecs .
at least 100,000 cells ( in 100-l phosphate buffer saline [ pbs]/0.5% bsa , 2 mmol / l edta ) were incubated ( for 20 min , at 4c ) with fitc - cd31 and percp - cd34 antibodies or the appropriate isotype controls .
after washing , the labeled cells were analyzed by flow cytometry using a fluorescence - activated cell sorter calibur flow cytometer and diva software ( bd - biosciences ) .
when the quantity of svf cells was sufficient ( 11 subjects , paired sat and vat ) , native cd34/cd31 cells ( ec ) were isolated by immunoselection / depletion as previously described ( 10 ) .
mature adipocytes and ec were either lysed and stored at 20c for mrna extraction or further processed .
sat and vat mature adipocytes were placed in fibrin gels ( 1.5 mg fibrinogen / ml ecbm supplemented with 25 units / ml thrombin ; 1/3 , vol / vol ) and cultured in ecbm/0.1% bsa supplemented with 100 units / ml penicillin and 100 g / ml streptomycin .
after 24 h , the adipocyte - conditioned media were collected and frozen at 80c . sat mature adipocytes were cultured in ecbm supplemented with 0.1% bsa , 100 units / ml penicillin , and 100 g / ml streptomycin in clinicell 25-culture cassettes ( 1/3 , vol / vol ) in normoxia or hypoxia chambers ( 1% o2 ; sanyo , avon , france ) .
after 24 h , mature adipocytes were lysed and stored at 20c for rna extraction .
tissue immunofluorescent staining of human sat and vat cut into small pieces ( 0.51 mm ) was performed .
after fixation in paraformaldehyde 4% for 1 h and subsequent washing in pbs , at pieces were incubated for 30 min in pbs/2% bsa in the presence of 0.1% triton . for -h2ax staining , at pieces
were permeabilized for 5 min in 95% ethanol and 5% acetic acid , and then incubated for 30 min in tris - buffered saline ( tbs)/3% bsa . at pieces
were incubated for 1 h with primary antibody ( cd31 [ 1/50 ] , cd34 [ 1/200 ] , icam1 [ 1/50 ] , gpihbp1 [ 1/40 ] , -h2ax [ 1/200 ] ) .
after several washings ( pbs/0.2% tween or tbs ) , at pieces were incubated for 30 min in pbs/2% bsa/0.1% triton or tbs/3% bsa and then incubated with the corresponding fluorescently - labeled secondary antibodies ( alexafluor-488 or alexafluor-546 , 1/200 ) .
after washing , at pieces were incubated for 10 min with 10 g / ml hoescht 33342 to stain nuclei .
they were placed between two mounting slides and examined with a fluorescence ( nikon ) or confocal ( zeiss 510 ) microscope .
quantification of phospho--h2ax positive nuclei in human sat and vat was performed on three calibrated fields using nis software ( nikon , champigny - sur - marne , france ) and normalized to the total number of nuclei ( hoescht 33342 staining ) .
total rna was extracted from ec and mature adipocytes and real - time pcr analyses were performed as previously described ( 10 ) .
primers ( taqman gene expression assays from applied biosystems , lifetechnologies , france ) are listed in the supplemental table in the online appendix available at http://diabetes.diabetesjournals.org .
senescence - associated--galactosidase ( sa--gal ) activity was determined in human sat - ec isolated from nonobese healthy women ( mean bmi  sem = 25.34  1.033 , range 22.028.5 ; mean age  sem = 48.5  3.074 , range 3557 ) and treated or not with adipocyte - conditioned media from sat and vat ( pretreated or not for 1 h with 1 g / ml vegfa neutralizing antibody [ r&d systems , lille , france ] , or with vegfa [ 10 ng / ml ; peprotech , levallois - perret , france ] ) .
cells were fixed ( 5 min , room temperature ) in 0.5% glutaraldehyde , washed with pbs , and incubated at 37c for 16 h with fresh sa--gal staining solution ( 1 mg / ml of 5-bromo-4-chloro-3-indolyl galactopyranoside in dimethylformamide ) supplemented with 0.12 mmol
/ l potassium ferrocyanide , 0.12 mmol / l potassium ferricyanide , and 1 mmol / l mgcl2 ( ph 6 ) . the enzymatic reaction was stopped with water .
cells positive for sa--gal activity were counted by phase contrast microscopy through scanning the whole well and normalized to the total number of cells determined in four different fields with hoescht 33342 nuclear staining using nis software .
comparisons between the two groups ( paired biopsies from sat and vat ) were analyzed by paired student t tests .
comparisons among groups were made by one - way anova followed by a dunnett post hoc test .
human mature adipocytes were isolated from paired biopsies of sat and vat from obese subjects .
adipocytes were classified according to their diameter ( i.e. , small , with a diameter less than 60 m ; and large , with a diameter more than 100 m ) and the expression of genes was analyzed by real - time pcr .
the proportion of large adipocytes was higher in sat than vat ( table 1 ) .
expression of hypoxia - related genes , such as hypoxia - inducible factor ( hif)-1 , and certain hif1-responsive genes ( vascular endothelial growth factor a [ vegfa ] and glut1 , was higher in vat than sat adipocytes ( fig .
in contrast , other genes regulated by hypoxic conditions and induced by hif-1 , including leptin and the fasting - induced adipose factor ( fiaf ) , exhibited the inverse profile ( fig .
1a ) . to further define the impact of hypoxia and adipocyte size on adipocyte gene expression , correlations among transcript levels and percentages of large adipocytes were calculated .
adipocyte transcript levels of vegfa and glut1 were positively correlated with hif1- mrna , irrespective of fat depot origin ( * p = 0.03 ; spearman r = 0.2425 , n = 60 [ sat and vat ] ; and * * p < 0.0001 ; spearman r = 0.4744 , n = 60 [ sat and vat ] , respectively ) .
however , leptin and fiaf mrnas were positively correlated with the percentage of large adipocytes ( * p = 0.012 ; spearman r = 0.3302 , n = 60 [ sat and vat ] ; and * p = 0.014 ; spearman r = 0.3231 , n = 60 [ sat and vat ] , respectively ) . no correlation was found between the percentage of large adipocytes and transcript levels of hif-1 , glut1 , or vegfa .
finally , the specific impact of low oxygen tension on adipocyte vegfa and glut1 expression was confirmed by real - time pcr analysis of mature sat adipocytes maintained in culture for 24 h under normoxic or hypoxic ( 1% o2 ) conditions .
vegfa and glut1 transcript levels were increased under hypoxic culture conditions , whereas leptin and fiaf were not altered substantially ( fig .
thus , human vat adipocytes exhibit hypoxia - related characteristics , with increased hif-1 , vegfa , and glut1 expression , independently of fat cell size .
human sat and vat adipocyte sizes data are percentages  se of small ( < 60 m ) and large adipocytes ( 100 m ) in paired samples of sat and vat from n = 30 subjects ; * * p < 0.01 ; paired t test between sat and vat ; ns , not significant .
a : comparison of mature adipocyte gene expression in sat and vat ( n = 30 ) .
hypoxia inducible factor 1 or 2 ,  subunit ( hif-1 , hif-2 ) , glut1 , vascular endothelial growth factor a ( vegfa ) , and fasting - induced adipose factor ( fiaf ) .
open bars : genes upregulated , and solid bars : genes downregulated in vat compared with sat .
results are expressed as fold differences between vat and sat as means  sem . * p < 0.05 , * * p < 0.01 ; paired t tests between sat and vat .
b : sat adipocyte gene expression under normoxic ( nx ) and hypoxic conditions ( 1% o2 ) ( n = 6 ) .
open bars : gene expression under normoxic conditions , and solid bars : gene expression under hypoxic conditions .
results are expressed as means  sem * * p < 0.01 ; paired t tests between normoxic and hypoxic conditions . to test whether a less extensive vascular network in vat contributes to higher hypoxia - related gene expression in vat than sat in obese subjects , sat and vat were analyzed using three - dimensional confocal immunofluorescence microscopy for the ec markers cd34 and cd31 .
the apparent density of the vascular network , determined from colocalized cd34 and cd31 positive signals , was greater in vat than sat ( fig .
the number of cd34/cd31 cells , normalized for tissue weight , was significantly higher in vat than sat ( fig . 2c ) .
to note , the ec number in sat of lean ( n = 42 ) subjects was similar to that of obese subjects ( 20,378  2,554 vs. 25,476  3,211
cells / g at , respectively ; p = 0.1 ) , indicating that growth of sat is associated with concomitant expansion of its capillary network . vascular network and ec number in human sat and vat .
a and b : representative photomicrographs of three - dimensional confocal immunofluorescence analyses of human ( a ) sat and ( b ) vat using antibodies directed against cd34 ( green ) and cd31 ( red ) ( n = 5 ) . original magnification 10 .
c : flow cytometry analyses were performed on freshly harvested svf using fluorescently - labeled antibodies directed against cd34 and cd31 ( n = 30 ) .
results are means  sem of the number of cd34/cd31 cells per gram of at * p < 0.05 ; paired t tests between the two at depots . ( a high - quality digital representation of this figure is available in the online issue . ) to determine whether hypoxia - related gene expression in vat adipocytes is related to endothelial dysfunction , native human cd34/cd31 cells were isolated by immunoselection / depletion from sat and vat from obese subjects in biopsies that were of sufficient size .
genes involved in inflammation ( chemokines and adhesion molecules ) , angiogenesis , metabolism , and cellular senescence were analyzed by real - time pcr .
many of the genes encoding proinflammatory and angiogenic factor receptors were upregulated in vat compared with sat - ec , including cc motif ligand 20 ( ccl20 ) , chemokine cxc motif ligand 8 ( cxcl8 ) , intercellular adhesion molecule-1 ( icam1 ) , vascular endothelial growth factor receptor 2 ( vegfr2 ) , leptin receptor ( leptin r ) , and neuropilin 1 and neuropilin 2 ( nrp1 and nrp2 ; fig . 3a and b , respectively ) .
expression of genes involved in metabolism , such as endothelial lipase ( el ) , lpl , ppar , and gpihbp1 , tended to be higher in sat than vat - ec , although differences for el and lpl did not reach statistical significance ( fig .
immunohistochemical analyses confirmed the increased expression of icam1 and decreased expression of gpihbp1 in ec from vat compared with sat in obese subjects ( fig .
, both proteins were expressed at similar levels in sat from lean and obese subjects ( fig .
moreover , real - time pcr analyses performed on isolated sat - ec from lean subjects ( n = 6 ) did not differ substantially in ccl20 , cxcl8 , icam1 , leptin r , nrp2 , and el compared with obese sat - ec ( data not shown ) . finally ,
expression of the deacetylase , sirt1 , was decreased , whereas insulin - like growth factor binding protein 3 ( igfbp3 ) , a gene upregulated in senescent ec , was increased in vat - ec from obese subjects compared with sat - ec ( fig .
no differences in sirt1 and igfbp3 expression were detected in sat - ec from lean and obese subjects ( data not shown ) . to test whether cellular senescence is more extensive in vat than sat - ec
, immunofluorescence analyses were performed using an antibody directed against -h2ax , a marker of senescent nuclei .
5c and d , respectively ) , but at a higher density in vat - ec ( 3.3-fold increase in vat vs. sat , p = 0.0004 , * * n = 10 ) .
expression of inflammatory , angiogenic , and metabolic genes in human sat and vat ec .
cc motif ligand 20 ( ccl20 ) , cc motif ligand 2 ( ccl2 ) , chemokine cxc motif ligand 8 ( cxcl8 ) , platelet endothelial cell adhesion molecule-1 ( pecam1 ) , intercellular adhesion molecule-1 ( icam1 ) , vascular endothelial growth factor receptor 1 and vascular endothelial growth factor receptor 2 ( vegfr1 and vegfr2 ) , leptin receptor ( leptin r ) , neuropilin 1 and neuropilin 2 ( nrp1 and nrp2 ) , endothelial lipase ( el ) , lipoprotein lipase ( lpl ) , peroxisome proliferator - activated receptor gamma ( ppar ) , and gpi - anchored hdl - binding protein 1 ( gpihbp1 ) .
open bars : genes upregulated , and solid bars : genes downregulated in vat compared with sat .
results are expressed as fold differences between vat ec and sat ec as means  sem ( n = 7 to 11 ) * p < 0.05 , * * p < 0.01 ; paired t tests between sat ec and vat ec .
representative photomicrographs of immunofluorescence analyses of human sat from lean subjects ( n = 4 , 5 ) ( a and d ) , obese subjects ( n = 6 , 9 ) ( b and e ) and vat from obese subjects ( n = 6 , 9 ) ( c , f ) using antibodies directed against icam1 ( green ) and cd34 ( red ) ( a  c ) gpihbp1 ( green ) ( d  f ) with nuclear staining with hoescht 33342 ( blue ) .
( a high - quality digital representation of this figure is available in the online issue . ) markers of senescence in native ec in sat and vat .
b : expression of ( a ) sirtuin 1 ( sirt1 , n = 9 ) and ( b ) insulin - like growth factor binding protein 3 ( igfbp3 , n = 11 ) in sat ec and vat ec .
results are expressed as means  sem ; * p < 0.05 , * * p < 0.01 ; paired t tests between sat ec and vat ec .
d : representative photomicrographs of immunohistofluorescence analyses of human ( c ) sat and ( d ) vat using antibody directed against phospho--h2ax ( red ) and nuclear staining ( blue ) with hoescht 33342 ( n = 10 ) .
( a high - quality digital representation of this figure is available in the online issue . ) to define effects of the sat and vat microenvironments on ec , native sat - ec from nonobese women were treated with sat and vat adipocyte - conditioned media .
adipocyte - conditioned media clearly increased the number of sa--gal positive ec compared with control basal medium ( fig . 6b and a , respectively ) .
moreover , native sat - ec treated with vat conditioned medium developed a significantly higher percentage of senescent cells than the same native sat - ec cells treated with sat - derived conditioned medium ( fig .
ec proliferation was significantly increased by both sat and vat adipocyte - conditioned media , but to a greater extent in vat than sat - ec ( fig .
finally , to analyze the potential factor(s ) involved in such a senescence - promoting effect of adipocyte - conditioned media , sat - ec were treated with vegfa alone or with sat- and vat - derived conditioned media in the presence of neutralizing vegfa antibody .
although vegfa increased the number of sa--gal positive ec , the presence of a neutralizing vegfa antibody significantly reduced the effects of sat- and vat - conditioned media ( fig .
a and b : representative photomicrographs of native sat - ec cultured in the presence ( b ) or absence ( a ) of adipocyte - conditioned media ( n = 6 ) .
results are expressed as means  sem as percentages of control in the presence of vegfa ( n = 3 ) ( dotted ) or adipocyte - conditioned media from sat ( open ) or vat ( solid ) treated ( hatched ) or not with vegfa neutralizing antibody ( n = 3 ) , * p < 0.05 , * * p < 0.01 vs. control ; $ p < 0.05 , $ $ p < 0.01 , adipocyte - conditioned media alone versus containing vegfa neutralizing ab .
hatched bar : control , open bar : sat , and solid bar : vat .
results are expressed as means  sem as a percentage of control * p < 0.05 , * * p < 0.01 , n = 3 .
( a high - quality digital representation of this figure is available in the online issue . )
human mature adipocytes were isolated from paired biopsies of sat and vat from obese subjects .
adipocytes were classified according to their diameter ( i.e. , small , with a diameter less than 60 m ; and large , with a diameter more than 100 m ) and the expression of genes was analyzed by real - time pcr .
the proportion of large adipocytes was higher in sat than vat ( table 1 ) .
expression of hypoxia - related genes , such as hypoxia - inducible factor ( hif)-1 , and certain hif1-responsive genes ( vascular endothelial growth factor a [ vegfa ] and glut1 , was higher in vat than sat adipocytes ( fig .
in contrast , other genes regulated by hypoxic conditions and induced by hif-1 , including leptin and the fasting - induced adipose factor ( fiaf ) , exhibited the inverse profile ( fig .
1a ) . to further define the impact of hypoxia and adipocyte size on adipocyte gene expression , correlations among transcript levels and percentages of large adipocytes were calculated .
adipocyte transcript levels of vegfa and glut1 were positively correlated with hif1- mrna , irrespective of fat depot origin ( * p = 0.03 ; spearman r = 0.2425 , n = 60 [ sat and vat ] ; and * * p < 0.0001 ; spearman r = 0.4744 , n = 60 [ sat and vat ] , respectively ) .
however , leptin and fiaf mrnas were positively correlated with the percentage of large adipocytes ( * p = 0.012 ; spearman r = 0.3302 , n = 60 [ sat and vat ] ; and * p = 0.014 ; spearman r = 0.3231 , n = 60 [ sat and vat ] , respectively ) . no correlation was found between the percentage of large adipocytes and transcript levels of hif-1 , glut1 , or vegfa .
finally , the specific impact of low oxygen tension on adipocyte vegfa and glut1 expression was confirmed by real - time pcr analysis of mature sat adipocytes maintained in culture for 24 h under normoxic or hypoxic ( 1% o2 ) conditions .
vegfa and glut1 transcript levels were increased under hypoxic culture conditions , whereas leptin and fiaf were not altered substantially ( fig .
thus , human vat adipocytes exhibit hypoxia - related characteristics , with increased hif-1 , vegfa , and glut1 expression , independently of fat cell size .
human sat and vat adipocyte sizes data are percentages  se of small ( < 60 m ) and large adipocytes ( 100 m ) in paired samples of sat and vat from n = 30 subjects ; * * p < 0.01 ; paired t test between sat and vat ; ns , not significant .
a : comparison of mature adipocyte gene expression in sat and vat ( n = 30 ) .
hypoxia inducible factor 1 or 2 ,  subunit ( hif-1 , hif-2 ) , glut1 , vascular endothelial growth factor a ( vegfa ) , and fasting - induced adipose factor ( fiaf ) .
open bars : genes upregulated , and solid bars : genes downregulated in vat compared with sat .
results are expressed as fold differences between vat and sat as means  sem . * p < 0.05 , * * p < 0.01 ; paired t tests between sat and vat .
b : sat adipocyte gene expression under normoxic ( nx ) and hypoxic conditions ( 1% o2 ) ( n = 6 ) .
open bars : gene expression under normoxic conditions , and solid bars : gene expression under hypoxic conditions .
results are expressed as means  sem * * p < 0.01 ; paired t tests between normoxic and hypoxic conditions .
to test whether a less extensive vascular network in vat contributes to higher hypoxia - related gene expression in vat than sat in obese subjects , sat and vat were analyzed using three - dimensional confocal immunofluorescence microscopy for the ec markers cd34 and cd31 .
the apparent density of the vascular network , determined from colocalized cd34 and cd31 positive signals , was greater in vat than sat ( fig .
the number of cd34/cd31 cells , normalized for tissue weight , was significantly higher in vat than sat ( fig . 2c ) .
to note , the ec number in sat of lean ( n = 42 ) subjects was similar to that of obese subjects ( 20,378  2,554 vs. 25,476  3,211 cells / g at , respectively ; p = 0.1 ) , indicating that growth of sat is associated with concomitant expansion of its capillary network .
a and b : representative photomicrographs of three - dimensional confocal immunofluorescence analyses of human ( a ) sat and ( b ) vat using antibodies directed against cd34 ( green ) and cd31 ( red ) ( n = 5 ) . original magnification 10 .
c : flow cytometry analyses were performed on freshly harvested svf using fluorescently - labeled antibodies directed against cd34 and cd31 ( n = 30 ) .
results are means  sem of the number of cd34/cd31 cells per gram of at * p < 0.05 ; paired t tests between the two at depots .
( a high - quality digital representation of this figure is available in the online issue . )
to determine whether hypoxia - related gene expression in vat adipocytes is related to endothelial dysfunction , native human cd34/cd31 cells were isolated by immunoselection / depletion from sat and vat from obese subjects in biopsies that were of sufficient size .
genes involved in inflammation ( chemokines and adhesion molecules ) , angiogenesis , metabolism , and cellular senescence were analyzed by real - time pcr .
many of the genes encoding proinflammatory and angiogenic factor receptors were upregulated in vat compared with sat - ec , including cc motif ligand 20 ( ccl20 ) , chemokine cxc motif ligand 8 ( cxcl8 ) , intercellular adhesion molecule-1 ( icam1 ) , vascular endothelial growth factor receptor 2 ( vegfr2 ) , leptin receptor ( leptin r ) , and neuropilin 1 and neuropilin 2 ( nrp1 and nrp2 ; fig . 3a and b , respectively ) .
expression of genes involved in metabolism , such as endothelial lipase ( el ) , lpl , ppar , and gpihbp1 , tended to be higher in sat than vat - ec , although differences for el and lpl did not reach statistical significance ( fig .
immunohistochemical analyses confirmed the increased expression of icam1 and decreased expression of gpihbp1 in ec from vat compared with sat in obese subjects ( fig .
, both proteins were expressed at similar levels in sat from lean and obese subjects ( fig .
moreover , real - time pcr analyses performed on isolated sat - ec from lean subjects ( n = 6 ) did not differ substantially in ccl20 , cxcl8 , icam1 , leptin r , nrp2 , and el compared with obese sat - ec ( data not shown ) . finally ,
expression of the deacetylase , sirt1 , was decreased , whereas insulin - like growth factor binding protein 3 ( igfbp3 ) , a gene upregulated in senescent ec , was increased in vat - ec from obese subjects compared with sat - ec ( fig .
no differences in sirt1 and igfbp3 expression were detected in sat - ec from lean and obese subjects ( data not shown ) . to test whether cellular senescence is more extensive in vat than sat - ec ,
immunofluorescence analyses were performed using an antibody directed against -h2ax , a marker of senescent nuclei .
5c and d , respectively ) , but at a higher density in vat - ec ( 3.3-fold increase in vat vs. sat , p = 0.0004 , * * n = 10 ) .
expression of inflammatory , angiogenic , and metabolic genes in human sat and vat ec .
cc motif ligand 20 ( ccl20 ) , cc motif ligand 2 ( ccl2 ) , chemokine cxc motif ligand 8 ( cxcl8 ) , platelet endothelial cell adhesion molecule-1 ( pecam1 ) , intercellular adhesion molecule-1 ( icam1 ) , vascular endothelial growth factor receptor 1 and vascular endothelial growth factor receptor 2 ( vegfr1 and vegfr2 ) , leptin receptor ( leptin r ) , neuropilin 1 and neuropilin 2 ( nrp1 and nrp2 ) , endothelial lipase ( el ) , lipoprotein lipase ( lpl ) , peroxisome proliferator - activated receptor gamma ( ppar ) , and gpi - anchored hdl - binding protein 1 ( gpihbp1 ) .
open bars : genes upregulated , and solid bars : genes downregulated in vat compared with sat .
results are expressed as fold differences between vat ec and sat ec as means  sem ( n = 7 to 11 ) * p < 0.05 , * * p < 0.01 ; paired t tests between sat ec and vat ec .
representative photomicrographs of immunofluorescence analyses of human sat from lean subjects ( n = 4 , 5 ) ( a and d ) , obese subjects ( n = 6 , 9 ) ( b and e ) and vat from obese subjects ( n = 6 , 9 ) ( c , f ) using antibodies directed against icam1 ( green ) and cd34 ( red ) ( a  c ) gpihbp1 ( green ) ( d  f ) with nuclear staining with hoescht 33342 ( blue ) . original magnification 40 .
( a high - quality digital representation of this figure is available in the online issue .
b : expression of ( a ) sirtuin 1 ( sirt1 , n = 9 ) and ( b ) insulin - like growth factor binding protein 3 ( igfbp3 ,
results are expressed as means  sem ; * p < 0.05 , * * p < 0.01 ; paired t tests between sat ec and vat ec .
d : representative photomicrographs of immunohistofluorescence analyses of human ( c ) sat and ( d ) vat using antibody directed against phospho--h2ax ( red ) and nuclear staining ( blue ) with hoescht 33342 ( n = 10 ) .
( a high - quality digital representation of this figure is available in the online issue . )
to define effects of the sat and vat microenvironments on ec , native sat - ec from nonobese women were treated with sat and vat adipocyte - conditioned media .
adipocyte - conditioned media clearly increased the number of sa--gal positive ec compared with control basal medium ( fig . 6b and a , respectively ) .
moreover , native sat - ec treated with vat conditioned medium developed a significantly higher percentage of senescent cells than the same native sat - ec cells treated with sat - derived conditioned medium ( fig .
ec proliferation was significantly increased by both sat and vat adipocyte - conditioned media , but to a greater extent in vat than sat - ec ( fig .
finally , to analyze the potential factor(s ) involved in such a senescence - promoting effect of adipocyte - conditioned media , sat - ec were treated with vegfa alone or with sat- and vat - derived conditioned media in the presence of neutralizing vegfa antibody .
although vegfa increased the number of sa--gal positive ec , the presence of a neutralizing vegfa antibody significantly reduced the effects of sat- and vat - conditioned media ( fig .
a and b : representative photomicrographs of native sat - ec cultured in the presence ( b ) or absence ( a ) of adipocyte - conditioned media ( n = 6 ) .
results are expressed as means  sem as percentages of control in the presence of vegfa ( n = 3 ) ( dotted ) or adipocyte - conditioned media from sat ( open ) or vat ( solid ) treated ( hatched ) or not with vegfa neutralizing antibody ( n = 3 ) , * p < 0.05 , * * p < 0.01 vs. control ; $ p < 0.05 , $ $ p < 0.01 , adipocyte - conditioned media alone versus containing vegfa neutralizing ab .
hatched bar : control , open bar : sat , and solid bar : vat .
results are expressed as means  sem as a percentage of control * p < 0.05 , * * p < 0.01 , n = 3 .
( a high - quality digital representation of this figure is available in the online issue . )
we found greater expression of hypoxia - related genes and smaller sizes of vat than sat adipocytes in obese subjects .
the increased hypoxia in vat is not likely to be a consequence of capillary rarefaction since vascular density , as well as ec number , were higher in vat than sat .
however , the vat - ec phenotype in obese subjects was markedly proangiogenic and inflammatory , with decreased expression of metabolism - related genes , including el , gpihbp1 , and ppar. this phenotype of vat - ec in obese subjects could be related to premature ec senescence , as suggested by expression of the senescence markers , igfbp3 and -h2ax , as well as decreased expression of sirt1 .
adipocytes from vat appear to have reduced capacity for lipogenesis ( 17 ) and greater capacity for lipolysis than sat cells ( 15 ) , with vat containing more proinflammatory immune cells than sat ( 12 ) . in the present study and
consistent with other reports ( 18,19 ) , marked hypertrophy of sat compared with vat adipocytes was observed in obese subjects .
increased expression of the hypoxia - related genes , hif-1 , vegfa , and glut1 was found in vat compared with sat adipocytes . despite previous studies reporting that leptin and fiaf are induced under hypoxic conditions ( 20,21 ) , we found they were lower in vat than sat .
irrespective of at location , both leptin and fiaf transcript levels correlated with adipocyte size , as noted by others for leptin ( 22 ) .
expression of vegfa and glut1 was tightly linked with that of hif-1. moreover , sat adipocytes maintained under low oxygen conditions had higher vegfa and glut1 expresssion than vat , whereas the expression of both leptin and fiaf was not affected substantially .
together , the present results indicate that hypoxia - related processes are more highly activated in vat than sat and are not related to the extent of adipocyte hypertrophy in obese subjects
. moreover , qualitative analyses by confocal microscopy and flow cytometry , using both the ec markers cd34 and cd31 simultaneously , reveal that capillary network density was higher in vat than in sat .
treatment of native sat - ec originating from normal nonobese women with conditioned media originating from vat adipocytes from obese subjects led to marked proliferation compared with conditioned media from sat adipocytes from the same subjects .
this suggests that the microenvironment of vat is more proangiogenic than sat and is consistent with the greater capillary density in vat than sat .
whether increased proangiogenic induction by vat adipocytes is related to hypoxia- and inflammation - related events remains to be determined .
our results indicate that vat - ec from obese subjects exhibited a markedly proinflammatory and angiogenic activated state , with increased expression of chemokines , adhesion molecules , and angiogenic factor receptors .
this phenotype could contribute to the greater abundance of proinflammatory immune cells in vat than sat ( 12 ) .
el and gpihbp1 are ec - specific metabolic genes , at least in murine models ( 23 ) .
together with ppar ( the main regulator of gpihbp1 ) , these genes were downregulated in vat compared with sat .
a recent study in murine models of diet - induced obesity highlighted the key role of ppar in the modulation of ec function and metabolic alterations associated with obesity ( 7 ) .
together our results show that although the capillary density of vat is higher than that of sat , vat - ec exhibit a phenotype characterized by marked activation of inflammatory and angiogenic pathways associated with altered metabolic function in obese subjects .
this proangiogenic , proinflammatory phenotype might be related to premature endothelial cellular senescence . indeed ,
senescent ec exhibit an activated state that may by induced either by extensive cell replication , leading to premature irreversible cell growth arrest , or by various stresses , including oxidative stress ( 24 ) .
decreased expression of sirt1 , together with increased expression of igfbp3 and -h2ax , have been noted in senescent , replicatively exhausted human ec ( 2528 ) , and we found indications of cellular senescence found in vat - ec from obese subjects .
moreover , since conditioned media originating from vat adipocytes increased the number of sa--gal positive ec , it is tempting to speculate that the chronic proangiogenic microenvironment of vat promotes premature ec senescence , leading to endothelial dysfunction .
interestingly , vegfa alone enhanced the number of sa--gal positive ec , although to a lesser extent than adipocyte - conditioned media .
moreover , neutralization of vegfa reduced the senescence - promoting effect of the sat and vat adipocyte - conditioned media , suggesting that vegfa might be an adipocyte - derived factor involved in premature adipose tissue ec senescence , consistent with its effects on ec senescence in other systems ( 29,30 ) .
cellular senescence in other cell types is associated with a senescent secretory phenotype , with increased production of proinflammatory cytokines , chemokines , and extracellular matrix - modifying proteins , as well as angiogenic factors ( 3135 ) .
it will be important to define the secreted protein profile of senescent vat - ec and sat - ec and to determine whether such a phenotype is involved in the accumulation of immuno - inflammatory cells .
consistent with this possibility , we previously showed that treatment of sat - ec with sat adipocyte - conditioned media led to increased diapedesis of blood - derived monocytes ( 10 ) .
the subjects in our study were obese . whether regional variation in fat tissue ec properties
are already present in lean subjects or arise as a consequence of obesity needs to be determined .
since cellular senescence is increased in obesity ( 36 ) and since obesity is associated with aortic endothelial cell senescence ( 37 ) , it is tempting to speculate that obesity could induce senescence in vat - ec to a greater extent than in sat - ec .
consistent with this speculation , our study shows that sat - ec from lean and obese subjects did not exhibit marked phenotypic differences or numbers relative to at weight . if comparative studies of vat - ec from lean and obese subjects validate our speculation , interventions that limit the accumulation of senescent ec or their proinflammatory state could improve the approaches to limit inflammation attributable to obesity and its complications .
 objectiveregional differences among adipose depots in capacities for fatty acid storage , susceptibility to hypoxia , and inflammation likely contribute to complications of obesity . 
 we defined the properties of endothelial cells ( ec ) isolated from subcutaneous adipose tissue ( sat ) and visceral adipose tissue ( vat ) biopsied in parallel from obese subjects.research design and methodsthe architecture and properties of the fat tissue capillary network were analyzed using immunohistochemistry and flow cytometry . 
 cd34+/cd31 + ec were isolated by immunoselection / depletion . 
 expression of chemokines , adhesion molecules , angiogenic factor receptors , as well as lipogenic and senescence - related genes were assayed by real - time pcr . 
 fat cell size and expression of hypoxia - dependent genes were determined in adipocytes from both fat depots.resultshypoxia-related genes were more highly expressed in vat than sat adipocytes . 
 vat adipocytes were smaller than sat adipocytes . 
 vascular density and ec abundance were higher in vat . 
 vat - ec exhibited a marked angiogenic and inflammatory state with decreased expression of metabolism - related genes , including endothelial lipase , gpihbp1 , and ppar gamma . 
 vat - ec had enhanced expression of the cellular senescence markers , igfbp3 and -h2ax , and decreased expression of sirt1 . 
 exposure to vat adipocytes caused more ec senescence - associated -galactosidase activity than sat adipocytes , an effect reduced in the presence of vascular endothelial growth factor a ( vegfa ) neutralizing antibodies.conclusionsvat-ec exhibit a more marked angiogenic and proinflammatory state than sat - ec . 
 this phenotype may be related to premature ec senescence . 
 vat - ec may contribute to hypoxia and inflammation in vat . 