adipose tissue dysfunction relies on many and heterogeneous factors , including impaired secretory function , insulin resistance , and lipolysis , which are believed to be caused by the activation of inflammatory mechanisms . in turn
, inflammation is activated as a consequence of excessive lipid uptake , adipocyte hypertrophy , and hypoxia .
besides the limited ability of oxygen to diffuse between hypertrophic adipocytes , hypoxia also results from microvascular dysfunction and decreased compensatory angiogenesis [ 14 ] .
these mechanisms are thought to contribute to the development and progression of type 2 diabetes .
recently , our group demonstrated that microvascular dysfunction and concomitant hypoxia may be caused by methylglyoxal - induced glycation , what may constitute a new factor for adipose tissue dysfunction during type 2 diabetes progression .
thus , new strategies improving microvascular function of adipose tissue may strongly contribute to prevent these mechanisms .
methylglyoxal ( mg ) is a highly reactive dicarbonyl compound , endogenously formed during lipid peroxidation and glycolysis .
irreversible formation of advanced glycation end - products ( age ) from methylglyoxal is one of the pathways responsible for diabetes - associated complications , not only due to increased aminoacid crosslinks and protein misfolding , but also due to the rage activation ( age receptor ) [ 2 , 3 ] .
we showed that increased methylglyoxal - induced ages accumulation in adipose tissue of wistar rats leads to hypoadiponectinemia , increased plasma free fatty acids , fibrosis , decreased irrigation , hypoxia , higher levels of apoptotic markers ( ratio bcl-2/bax and caspase 3 ) , and macrophage recruitment . as well , other authors showed that mg - induced age accumulation leads to microvascular dysfunction in other models [ 58 ] .
pyridoxamine is a vitamin b6 derivative , which belongs to the b vitamins family , a group of vitamins known as coenzymes of several metabolic cellular pathways .
pyridoxamine is known to be an inhibitor of the maillard reaction and a blocker of age formation from amadori products , mainly because it acts as a dual scavenger of free radical and carbonyl species .
pyridoxamine was observed to decrease age accumulation , in part due to increased glyoxalase - i expression and decreased oxidative stress and rage levels in several models of diabetic complications [ 6 , 912 ] .
this work was designed to assess the usefulness of age inhibition in the adipose microvasculature , after methylglyoxal - induced glycation .
unless otherwise stated all reagents were purchased to merck darmstadt ( germany ) , sigma - aldrich ( eua ) or pancreac qumica sa ( spain ) .
antibodies used were directed to actin ( mab1501r , millipore , usa ) , cel ( kh025 , transgenic inc . ,
japan ) , rage ( ab3611 , abcam , uk ) , tgf- ( mab240 , r&d systems inc . ) , bax , bcl-2 ( sc 6236 and sc7382 , santa cruz biotechnology , usa ) , and vwf ( a0082 , dako , usa ) . in this work we studied 6-month - old wistar rats , from our breeding colonies at the faculty of medicine , university of coimbra .
a group of wistar rats was treated with methylglyoxal in the daily water ( sigma , usa ) during 8 weeks ( 75  mg / kg / day , diluted in the water ) .
after this period , these rats were divided in two subgroups : one was treated with pyridoxamine ( pyr ) ( sigma , usa ) during the following 4 weeks ( 100  mg / kg / day diluted in the daily water ) [ 13 , 14 ] , whereas the other did not have any kind of treatment ( mg ) .
the control group ( c ) did not have any treatment during the whole period of the experiment ( n = 6 per group ) .
mg administration was suspended at the moment of pyridoxamine treatment initiation , in order to avoid ex vivo interactions with pyridoxamine .
animals were kept ( 2 per cage ) under standard ventilation , temperature ( 2224c ) , humidity ( 5060% ) , and light ( 12 hours light/12 hours darkness ) with free access to water and food ( standard diet ao4 , panlab , barcelona ) . in treated animals ,
the experimental protocol was approved by the local institutional animal care and use committee , and all the procedures were performed by licensed users ( felasa ) . at the end of the treatment ,
glycemia , fasting ( 16 hours ) and 2 hours after intraperitoneal glucose administration ( 1.8  g / kg ) , was measured in the tail vein through the glucose oxidase method , using a glucometer and reactive test stripes ( elite - bayer sa , portugal ) .
blood samples were obtained through cardiac puncture from anesthetized animals ( ketamine chloride ( 75  mg / kg , i.m . , parke - davis , ann arbor , usa ) and chlorpromazine chloride ( 2.65 
serum and plasma were collected using tubes bd vacutainer and bd vacutainer k3e , with 5.4  mg edta ( uk ) , respectively .
blood was centrifuged at 3500  g , 4c , 10 minutes and plasma and serum were aliquoted and stored at 80c .
after blood collection , animals were sacrificed and epididymal adipose tissue was harvested , washed in 0.9% nacl , and immediately stored in 10% formalin or frozen at 80c .
serum levels of cholesterol ( total and hdl ) and triglycerides were determined using commercial kits ( olympus - diagnstica , portugal , produtos de diagnstico sa , portugal ) .
serum concentration of adiponectin was determined by commercially available elisa kits ( adiponectin immunoassay kit krp0041 , invitrogen ) .
plasma levels of free fatty acids were assessed spectrophotometrically using the half - micro test ( 11383175001 roche diagnostic , germany ) .
tissue sections of 300  mg were homogenized in a buffer containing 25  mm tris , 150  mm nacl , 1% triton x-100 , 1  mm edta , 1  mm egta , 10  mm pmsf , and 40  l / g tissue of proteases inhibitor cocktail ( sigma , usa ) , ph = 7.7 and centrifuged at 14000  g , 20 minutes , 4c .
supernatants were collected , centrifuged again , and protein concentration was determined using the bca method ( pierce , usa ) .
samples ( 50  g ) were separated by sds - page in 10% acrylamide gels and transferred to pvdf membranes .
membranes were blocked with tbst solution ( 25  mm tris - hcl , 150  mm nacl , 0.1% tween , ph = 7.6 ) supplemented with 5% bsa .
membranes were then incubated overnight at 4c with the respective primary antibodies ( cel , rage , tgf- , bcl-2 and bax , diluted 1  :  1000 in tbst solution supplemented with 1% bsa ) and during 2 hours at room temperature with the secondary antibodies ( anti - rabbit / mouse 1  :  10000 ; ge healthcare , uk ) .
membranes were incubated with ecf and revealed using the typhoon system ( ge healthcare life sciences , uk ) .
membrane analysis was performed using the software image quant ( molecular dynamics , usa ) .  
tissue sections ( 4  m ) were stained with periodic acid schiff ( pas ; carbohydrates and glycoconjugates ) and masson trichrome ( collagen / connective tissue ) , two slices per animal , three animals per group .
immune staining was performed after paraffin removal , hydration , and blocking , following the recommendation of the manufacturer ( dab150 immunoperoxidase secondary detection system jh1743622 , millipore , usa ) .
sections were incubated overnight at 4c with the primary antibody ( tgf- and vwf , diluted 1  :  100 in pbs ) and during 1 hour at room temperature with the secondary antibodies ( alexa fluor 488 goat anti - rabbit igg and alexa fluor 568 goat anti - mouse igg , invitrogen , usa ) .
finally , sections were mounted with mounting medium ( dako , usa ) and analyzed in a fluorescent microscope ( leica dmire2 , leica microsystems , germany ) .
quantification ten images were captured randomly ( images containing large vessels were excluded ) from two slices per animal / three animals per group .
mg administration during 8 weeks ( mg group ) , as well as the treatment with pyridoxamine during 4 weeks ( pyr group ) , did not result in significant alterations of body weight , glycemia , hba1c , triglycerides , and total cholesterol , compared to the control ( c ) group ( table 1 ) .
however , mg group showed a significant decrease of hdl cholesterol levels , compared to the c group ( p < 0.05 ) , which was reverted by pyridoxamine treatment ( pyr group ) ( p < 0.05 ) ( table 1 ) . regarding serum adiponectin levels ,
, mg administration led to increased circulating free fatty acids levels , when compared to c group ( p < 0.05 ) , suggesting adipocyte dysfunction . in turn , the treatment with pyridoxamine normalized plasma free fatty acids levels ( p < 0.01 ) ( figure 1(b ) ) .
mg treatment during 8 weeks resulted in increased n - epsilon-(carboxyethyl)lysine ( cel ) accumulation in adipose tissue , compared to the c group ( p < 0.01 ) ( figure 2(a ) ) , but did not cause significant alterations of rage expression ( figure 2(b ) ) .
pyridoxamine administration significantly reverted cel accumulation in adipose tissue ( p < 0.01 ) , leading to values similar to control rats ( figure 2(a ) ) .
pas staining represents the accumulation of carbohydrates and nonspecific glycated materials . in accordance with cel levels , a substantial increase of pas material was observed in the adipose tissue of mg - treated rats , when compared to the c group .
this accumulation was reverted by pyridoxamine treatment , leading to a phenotype similar to control rats ( figure 3(a ) ) .  
the administration of mg during 8 weeks resulted in more fibrotic components in the adipose tissue .
once again , the treatment with pyridoxamine resulted in decreased fibrotic material , resulting in a phenotype similar to control rats ( figure 3(b ) ) . as well
, the transforming growth factor- ( tgf- ) precursor was observed to be decreased after pyridoxamine treatment , when compared to the mg group ( p < 0.05 ) ( figure 4(a ) ) .
no significant differences were observed in the cleaved form of the tgf- ( figure 4(b ) ) .
the histological staining of tgf- showed accumulation in the capillaries , as suggested by its colocalization with the von willebrand factor ( figure 4(c ) ) .
the ratio between bcl-2 and bax can be used as an indicator of the predisposition for cellular death by apoptosis .
the decrease of this ratio in the mg group , when compared to the c group ( p < 0.01 ) , indicates that mg - induced glycation leads to a cellular death predisposition .
these effects were completely reverted by the treatment with pyridoxamine ( p < 0.01 ) ( figure 5(a ) ) .  
quantification of vwf fluorescent staining was used as a marker of adipose tissue microvasculature , as it is a marker of endothelial cells and is present in the circulation .
mg administration resulted in a significant decrease of adipose tissue vwf levels , when compared to control rats ( p < 0.05 ) .
after pyridoxamine treatment a significant difference in relation to the c group was no longer observed , despite that no significant differences were observed between mg and pyr groups ( figure 5(b ) ) .
this study investigates the scavenger properties and beneficial actions of pyridoxamine in methylglyoxal - induced microvascular damages in the adipose tissue .
the significant alterations of hdl cholesterol , free fatty acids , cel , pas staining , tgf- precursor , masson trichrome staining , bcl-2/bax ratio , and partially vwf were reverted by pyridoxamine . in accordance with previous reports from our laboratory
, mg administration during 8 weeks did not have significant alterations in body weight , glycemia , and hba1c [ 1 , 7 ] .
once no alterations were found in the glucose metabolism , the lesions observed after mg treatment are only due to direct mg effects . however , mg administration increased systemic free fatty acids levels , suggesting adipose tissue dysfunction , due to a higher rate of lipolysis .
these results are also in accordance with our previous observations after mg treatment during 14 weeks .
as demonstrated before in zucker diabetic rats ( zdf ) [ 13 , 15 ] , pyridoxamine effects led to lower systemic levels of free fatty acids .
our previous and present results show that mg treatment leads to decreased serum adiponectin levels , but these effects may be acquired during the time , as they are significant only after 14 weeks of mg administration [ 1 , 7 ] .
mg treatment during 8 weeks may have a lower impact on adiponectin levels , or the effects may have been lost because the rats were kept with water during 4 weeks after mg treatment .
previously , correlation between cel levels and mg administration was observed in the aorta of spontaneously hypertensive rats   and in retina of glo1 transgenic rats .
as expected , mg administration during 8 weeks led to increased cel levels and accumulation of pas - positive material in adipose tissue , in accordance to previous reports [ 1 , 17 ] .
however , 8 weeks of mg treatment were not sufficient to induce major alterations in rage levels in adipose tissue , what is in accordance with previous data from our laboratory ( unpublished data ) .
however , that kind of effect was observed in aorta after 14 weeks of mg administration .
previous in vitro studies from onorato and colleagues showed that pyridoxamine inhibits ages formation as well as subsequent protein modifications , features commonly observed in type 2 diabetes .
several authors described that pyridoxamine inhibits age formation through the reaction with and scavenging of dicarbonyl intermediates [ 11 , 19 ] .
the role of pyridoxamine in decreasing age formation in in vitro systems   and in different animal models , including stz - induced diabetic rats and zdf rats , was also observed . in accordance , in our study the treatment with pyridoxamine during 4 weeks after mg administration caused a significant decrease of cel levels and pas - positive components in adipose tissue .
in vivo studies using wistar rats showed an increase of tgf- in glomerular , tubular , interstitial , and endothelial cells of the kidney . in vitro ,
collagen glycation caused by mg stimulates the differentiation of human cardiac fibroblasts to myofibroblast , in a tgf--dependent manner .
as well , we reported that mg administration during 14 weeks also led to increased tgf- expression , as well as increased masson trichrome staining . in the present study ,
we show that even after 8 weeks of mg treatment a higher accumulation of masson trichrome components occurred . in vitro studies with pyridoxamine
revealed that it protects from mg - induced integrin binding and cell adhesion , a major event of vessel thickening . here
, we show that the treatment with pyridoxamine decreased the expression of tgf- precursor and inhibited the accumulation of masson trichrome - positive material , suggesting a causal role between increased glycation markers and profibrotic responses .
colocalization of tgf- and vwf suggests tgf- expression to occur mostly in blood vessels regions .
this demonstrates the presence of fibrosis in the microvasculature of adipose tissue , what may be an important factor for local microvascular lesions .
the ratio between bcl-2 and bax decreased after mg treatment , what is in accordance with several reports from our and other laboratories [ 1 , 21 , 22 ] .
our group demonstrated that decreased bcl-2/bax ratio is associated with increased endothelial cell death and higher vessel dysfunction in the retina .
furthermore , we demonstrated that this event is also related with decreased blood supply to adipose tissue , what may lead to hypoxia , a major contributor to adipocyte dysfunction .
pyridoxamine was observed to prevent cell death by apoptosis in the human lens epithelial cell line hle - b3 and in the cataractous lenses of zdf rats , through the inhibition of argpyrimidine formation [ 5 , 23 ] . in b6 db
/ db mice , a model of type 2 diabetes , pyridoxamine decreased the progression of established diabetic nephropathy . as mentioned , we previously reported that mg administration during 14 weeks causes a decrease of blood supply to the adipose tissue , mainly through increased endothelial cell death and impaired angiogenesis . in this study , we show that these effects were similar after 8 weeks , although more modest . despite pyridoxamine was effective in the reduction of most of the lesion markers , it was not able to significantly raise adipose vwf levels in relation to mg - treated rats .
however , vwf levels were no longer decreased in relation to control rats , suggesting a partial effect .
we show that reducing adipose tissue glycation and fibrosis using pyridoxamine may be a useful strategy to improve adipose tissue microvascular lesions .
 
 background and aims . adipose tissue dysfunction results from many factors , including glycation - induced microvascular damages . 
 we tested the usefulness of inhibiting methylglyoxal - induced glycation to adipose tissue microvasculature in this work , using the antioxidant and dicarbonyl scavenger drug pyridoxamine . 
 methods . 
 a group of wistar rats was treated daily with methylglyoxal ( mg , 75  mg / kg / day , 8 weeks ) . 
 half of this group was treated with pyridoxamine in the following 4 weeks ( pyr ) ( 100  mg / kg / day ) and the other half did not have any further treatment ( mg ) . 
 a group of wistar rats without mg treatment was used as control ( c ) . 
 results . 
 mg group showed decreased hdl cholesterol and increased plasma free fatty acids levels , what was reverted by pyridoxamine . 
 mg also caused an increase of tissue cel levels ( glycation marker ) , as well as increased staining of pas and masson trichrome - positive components . 
 pyridoxamine led to cel and tgf- levels similar to those observed in control rats and inhibited the accumulation of pas and masson trichrome - positive components . 
 mg caused a decrease of bcl-2/bax ratio ( marker of apoptosis ) and vwf staining ( microvascular marker ) , what was partially reverted by the treatment with pyridoxamine . 
 conclusions . preventing methylglyoxal - induced accumulation of glycated and fibrotic materials using pyridoxamine 
 improves the microvascular lesions of the adipose tissue . 
 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusions