previous studies have implicated that endothelial dysfunction plays an important role in the onset and progression of atherosclerosis .
tes , a novel tumor suppressor gene , is located in a common fragile site on human chromosome 7q31.2 , designated as fra7 g .
it is predicted to encode a highly conserved protein of 421 amino acids named testin .
testin has been found to play an important role in focal adhesion and is present in the cytoplasm .
recently , it has been reported that the expression of tes was decreased in the left anterior descending aortic tissue from patients with coronary artery disease ( cad ) compared with non - cad subjects . in this report
, we aimed to determine the cellular origin of tes expression in aorta and examine whether its expression is affected in the aortic tissue during the pathogenesis of atherosclerosis .
thirty - two healthy purebred new zealand rabbits were used at the age of 34 months with body weights from 1.5 to 2.0 kg .
all experimental protocols were approved by the institutional animal care and use committee at tianjin medical university .
the rabbits were randomly divided into two groups as the following : ( 1 ) the normal diet ( nd ) group ( n = 12 ) were fed on 100 g / d of nd ; ( 2 ) high fat diet ( hfd ) group ( n = 20 ) were fed on the mixture of 100 g / d of 1% cholesterol , 5% lard , 10% egg yolk powder and 84% nd .
body weight and serum lipid levels were measured at 0 , 4 and 12 weeks after enrollment .
thoracic aortas were rapidly removed , cleaned of adventitia , cut into 5-mm ring segments .
the specimens were fixed in 4% formaldehyde and sectioned for routine hematoxylin and eosin staining . tissue structure and pathological features of atherosclerosis
after fixation , the tissue sections were rehydrated twice for 5 min in phosphate buffer saline ( pbs ) , blocked for 1 h in pbs with 10% normal goat serum followed by incubation in a humid chamber with 50 l of anti - testin primary antibody ( 1:100 dilution ; abcam , ab78499 , usa ) and anti - cd31 primary antibody ( 1:50 dilution ; abcam , ab9498 , usa ) in 10% serum overnight at 4c .
the slides were rinsed twice for 5 min in pbs before being incubated for 2 h in a humid chamber with 50 l of anti - rabbit immunoglobulin g conjugated with fluorescein isothiocyanate ( 1:100 dilution ; sigma , usa ) solution to visualize testin .
stained sections were rinsed twice for 5 min in pbs , stained for 10 min with 50 l of 1 mg / ml 4,6-diamidino-2-phenylindole ( dapi ) in 200 ml pbs , then rinsed again twice for 5 min in pbs , and mounted with an aqueous mounting medium before being examined under confocal microscope ( olympus , japan ) .
total rna was isolated from aortic tissue using the trizol solution ( invitrogen , usa ) .
total of 1 g rna was used for first - strand complementary dna synthesis using random primer ( invitrogen , usa ) and superscript ii transcriptase ( invitrogen , usa ) according to manufacturer 's instruction .
real - time polymerase chain reaction ( pcr ) was performed using iq sybr green supermix ( bio - rad , usa ) with cfx-96 real - time pcr detection system ( bio - rad , usa ) .
the primers used for tes and glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) as an internal control in the reaction were : 5-cctgtccagaaccaggcatt - 3/5 - ttctttcggtactgtgcccc-3 ( tes gene , 98 bps ) , and 5-tgagaatctgcccctcttcac - 3/5-cgttgctgtcgagactttattga-3 ( gapdh , 110 bps ) .
total protein extract was prepared from aortic tissue by homogenize tissue in radio immunoprecipitation assay lysis buffer ( sigma , r0278 , usa ) .
proteins were separated on nupage sds - page gel ( invitrogen , usa ) and transferred to a nitrocellulose transfer membrane ( whatman , uk ) according to the manuscript 's instruction .
the primary antibodies used for protein detection included anti - testin ( 1:500 dilution ; abcam , ab78499 , usa ) and gapdh ( 1:1000 dilution ; santa cruz , usa ) . membranes were then incubated with horseradish peroxidase - conjugated secondary antibodies ( abcam , usa ) .
proteins were visualized by enhanced chemiluminescence system ( western blot detection kit , ge healthcare ) .
developed signals were digitally recorded and quantified with melanie two - dimensional gel software analysis ( sib , switzerland ) .
statistical analysis was performed by one - way analysis of variance among groups . for comparison of data before and after dietary intervention
thirty - two healthy purebred new zealand rabbits were used at the age of 34 months with body weights from 1.5 to 2.0 kg .
all experimental protocols were approved by the institutional animal care and use committee at tianjin medical university .
the rabbits were randomly divided into two groups as the following : ( 1 ) the normal diet ( nd ) group ( n = 12 ) were fed on 100 g / d of nd ; ( 2 ) high fat diet ( hfd ) group ( n = 20 ) were fed on the mixture of 100 g / d of 1% cholesterol , 5% lard , 10% egg yolk powder and 84% nd .
body weight and serum lipid levels were measured at 0 , 4 and 12 weeks after enrollment .
thoracic aortas were rapidly removed , cleaned of adventitia , cut into 5-mm ring segments .
the specimens were fixed in 4% formaldehyde and sectioned for routine hematoxylin and eosin staining . tissue structure and pathological features of atherosclerosis
the detection of testin on tissue samples was inspected via immunohistochemical and immunofluorescence staining . after fixation
, the tissue sections were rehydrated twice for 5 min in phosphate buffer saline ( pbs ) , blocked for 1 h in pbs with 10% normal goat serum followed by incubation in a humid chamber with 50 l of anti - testin primary antibody ( 1:100 dilution ; abcam , ab78499 , usa ) and anti - cd31 primary antibody ( 1:50 dilution ; abcam , ab9498 , usa ) in 10% serum overnight at 4c .
the slides were rinsed twice for 5 min in pbs before being incubated for 2 h in a humid chamber with 50 l of anti - rabbit immunoglobulin g conjugated with fluorescein isothiocyanate ( 1:100 dilution ; sigma , usa ) solution to visualize testin .
stained sections were rinsed twice for 5 min in pbs , stained for 10 min with 50 l of 1 mg / ml 4,6-diamidino-2-phenylindole ( dapi ) in 200 ml pbs , then rinsed again twice for 5 min in pbs , and mounted with an aqueous mounting medium before being examined under confocal microscope ( olympus , japan ) .
total rna was isolated from aortic tissue using the trizol solution ( invitrogen , usa ) .
total of 1 g rna was used for first - strand complementary dna synthesis using random primer ( invitrogen , usa ) and superscript ii transcriptase ( invitrogen , usa ) according to manufacturer 's instruction .
real - time polymerase chain reaction ( pcr ) was performed using iq sybr green supermix ( bio - rad , usa ) with cfx-96 real - time pcr detection system ( bio - rad , usa ) .
the primers used for tes and glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) as an internal control in the reaction were : 5-cctgtccagaaccaggcatt - 3/5 - ttctttcggtactgtgcccc-3 ( tes gene , 98 bps ) , and 5-tgagaatctgcccctcttcac - 3/5-cgttgctgtcgagactttattga-3 ( gapdh , 110 bps ) .
total protein extract was prepared from aortic tissue by homogenize tissue in radio immunoprecipitation assay lysis buffer ( sigma , r0278 , usa ) .
proteins were separated on nupage sds - page gel ( invitrogen , usa ) and transferred to a nitrocellulose transfer membrane ( whatman , uk ) according to the manuscript 's instruction .
the primary antibodies used for protein detection included anti - testin ( 1:500 dilution ; abcam , ab78499 , usa ) and gapdh ( 1:1000 dilution ; santa cruz , usa ) .
membranes were then incubated with horseradish peroxidase - conjugated secondary antibodies ( abcam , usa ) .
proteins were visualized by enhanced chemiluminescence system ( western blot detection kit , ge healthcare ) .
developed signals were digitally recorded and quantified with melanie two - dimensional gel software analysis ( sib , switzerland ) .
statistical analysis was performed by one - way analysis of variance among groups . for comparison of data before and after dietary intervention
we examined the extent of atherosclerosis in rabbits fed with hfd and nd for 12 weeks . as expected ,
all of the rabbits in hfd group had a significantly higher level of serum lipids [ table 1 ] and presence of atherosclerotic plaque in the thoracic aorta compared with nd group [ figure 1 ] .
levels of serum lipids in two groups at different periods ( mmol / l , mean  sd ) * compared with nd group , p < 0.05 .
tg : triglyceride ; tc : total cholesterol ; ldl - c : low - density lipoprotein cholesterol ; hdl - c : high- density lipoprotein cholesterol ; sd : standard deviation ; nd : normal diet ; hfd : high - fat diet .
high incidence of atherosclerotic plaque in high - fat diet ( hfd ) group when compared with natural diet ( nd ) group ( hematoxylin and eosin staining ) .
representative sections from aortic tissues in hfd group ( b and c ) showed atherosclerotic plaque , whereas nd group ( a ) was normal . by immunostaining in aortic tissues with anti - testin antibody and confocal microscopy , we observed testin signal was predominantly detected in the endothelium as identified with cd31 staining .
a relatively weaker signal was also detected in the subendothelial areas [ figure 2a2c ] .
moreover , the majority of atherosclerotic plaque displayed testin immunoreactivity on segments bearing the plaque but also on those distal from the plaque [ figure 2d2 g ] .
significant differences in testin signal intensities were observed among different segments , suggesting that testin expression may correlate and contribute to vascular phenotypic heterogeneity and its possible role in atherosclerosis expression of testin protein in the normal and atherosclerotic aortic tissues .
( a  c ,  10 ) the signal for testin co - localized with the cd31 signal , indicating that testin was expressed in endothelial cells ( arrow ) ; ( d and e ,  20 ; f and g ,  10 ) immunohistochemical and immunofluorescence staining analysis of the normal aorta and plaque with an anti - testin antibody .
it showed that testin was expressed predominantly in the endothelium of normal aorta and plaque ( arrows ) .
we assessed the expression levels of the tes gene in the aortic tissues obtained from hfd and nd groups .
real - time pcr analysis demonstrated that tes mrna was markedly reduced by approximately 10-fold in the hfd group compared with the nd group ( p = 0.015 ) [ figure 3a ] . using western blot analysis , on the same experimental subjects showed testin expression was decreased at protein level in the hfd group in comparison with atherosclerotic - free nd group ( p = 0.01 ) [ figure 3b and 3c ] .
these results suggested that reduced tes / testin expression was associated with hfd induced atherosclerosis . decreased tes / testin expression in atherosclerotic aorta compared with nonatherosclerotic samples .
( a ) tes mrna expression from 12 nonatherosclerotic aorta compared with 20 atherosclerotic aorta arteries by real - time reverse transcription - polymerase chain reaction
. the mrna expression level of tes in atherosclerotic aorta was significantly lower than in nonatherosclerotic aorta arteries ; ( b ) testin protein expression by western blot analysis with 12 nonatherosclerotic ( n ) aorta and 20 atherosclerotic aorta arteries ( a ) showed that testin expression was lower in atherosclerotic aorta arteries .
glyceraldehyde-3-phosphate dehydrogenase was used as loading control ; ( c ) western blot images in ( b ) was scanned , quantified , and plotted , which showed testin expression was significantly decreased in atherosclerotic aorta .
we examined the extent of atherosclerosis in rabbits fed with hfd and nd for 12 weeks . as expected ,
all of the rabbits in hfd group had a significantly higher level of serum lipids [ table 1 ] and presence of atherosclerotic plaque in the thoracic aorta compared with nd group [ figure 1 ] .
levels of serum lipids in two groups at different periods ( mmol / l , mean  sd ) * compared with nd group , p < 0.05 .
tg : triglyceride ; tc : total cholesterol ; ldl - c : low - density lipoprotein cholesterol ; hdl - c : high- density lipoprotein cholesterol ; sd : standard deviation ; nd : normal diet ; hfd : high - fat diet .
high incidence of atherosclerotic plaque in high - fat diet ( hfd ) group when compared with natural diet ( nd ) group ( hematoxylin and eosin staining ) .
representative sections from aortic tissues in hfd group ( b and c ) showed atherosclerotic plaque , whereas nd group ( a ) was normal .
by immunostaining in aortic tissues with anti - testin antibody and confocal microscopy , we observed testin signal was predominantly detected in the endothelium as identified with cd31 staining .
a relatively weaker signal was also detected in the subendothelial areas [ figure 2a2c ] .
moreover , the majority of atherosclerotic plaque displayed testin immunoreactivity on segments bearing the plaque but also on those distal from the plaque [ figure 2d2 g ] .
significant differences in testin signal intensities were observed among different segments , suggesting that testin expression may correlate and contribute to vascular phenotypic heterogeneity and its possible role in atherosclerosis expression of testin protein in the normal and atherosclerotic aortic tissues .
( a  c ,  10 ) the signal for testin co - localized with the cd31 signal , indicating that testin was expressed in endothelial cells ( arrow ) ; ( d and e ,  20 ; f and g ,  10 ) immunohistochemical and immunofluorescence staining analysis of the normal aorta and plaque with an anti - testin antibody .
it showed that testin was expressed predominantly in the endothelium of normal aorta and plaque ( arrows ) .
we assessed the expression levels of the tes gene in the aortic tissues obtained from hfd and nd groups .
real - time pcr analysis demonstrated that tes mrna was markedly reduced by approximately 10-fold in the hfd group compared with the nd group ( p = 0.015 ) [ figure 3a ] . using western blot analysis , on the same experimental subjects showed testin expression was decreased at protein level in the hfd group in comparison with atherosclerotic - free nd group ( p = 0.01 ) [ figure 3b and 3c ] .
these results suggested that reduced tes / testin expression was associated with hfd induced atherosclerosis . decreased tes / testin expression in atherosclerotic aorta compared with nonatherosclerotic samples .
( a ) tes mrna expression from 12 nonatherosclerotic aorta compared with 20 atherosclerotic aorta arteries by real - time reverse transcription - polymerase chain reaction .
the mrna expression level of tes in atherosclerotic aorta was significantly lower than in nonatherosclerotic aorta arteries ; ( b ) testin protein expression by western blot analysis with 12 nonatherosclerotic ( n ) aorta and 20 atherosclerotic aorta arteries ( a ) showed that testin expression was lower in atherosclerotic aorta arteries .
glyceraldehyde-3-phosphate dehydrogenase was used as loading control ; ( c ) western blot images in ( b ) was scanned , quantified , and plotted , which showed testin expression was significantly decreased in atherosclerotic aorta .
in this study , we characterized the expression change of tes / testin in aortic tissues during hfd induced atherosclerosis using a well - established rabbit model .
our data showed that tes / testin levels were significantly decreased in the hfd treated aortic tissues at both mrna and protein levels . using confocal analysis
, we detected that testin was mainly expressed in cd31 luminal endothelium while a weaker expression was also detected in the subendothelium area . in this study , tes
/ testin was expressed in endothelium , and was down - regulated in atherosclerotic tissues , which suggested a possible role for testin in the pathogenesis of atherosclerosis ; however , the underlying mechanism is not clear and needs to be further investigated .
first , the cellular composition of the aortic tissues in atherosclerotic subjects may be altered from basal status .
in fact , the loss of endothelial cells ( ecs ) is a hallmark pathological in atherosclerotic tissue , which can lead to reduced expression of testin .
second , endothelium dysfunction due to pro - atherosclerotic and pro - inflammatory stimulation in the aortic tissue following hfd can lead to down - regulation of testin expression by genetic and epigenetic regulatory machinery .
indeed , it has been demonstrated that 5 cpg island methylation of the tes gene can silence its expression in gastric cancer , and a similar mechanism may have occurred in atherosclerotic endothelium .
third , tes gene expression can also be regulated by mirnas , which may be induced in the process of atherosclerosis .
finally , single nucleotide polymorphisms ( snps ) may contribute to regulating the expression of tes gene .
these possible mechanisms should be the subject of future investigations . as a component of focal adhesions , testin interacts with the cytoskeletal protein such as zyxin , talin , vasodilator - stimulated phosphoprotein , mena , extractable nucler antigen ( evl ) , alphall - spectrin , actin and actin - related proteins 7a .
however , the functional role of testin in the cardiovascular system is not fully understood .
reported that overexpression of tes decreased monocyte adhesion to ecs or the migration of monocytes across the ec layer , while knockdown increased these activities .
this result indicated that tes may play a potentially important role in the homing and local infiltration of pro - inflammatory cells at atherosclerotic plaques , thus contributes to the initiation and progression of the pathology .
in addition , zhu et al . have demonstrated that mir-29b decreased tes mrna expression , whereas matrix metalloproteinases-2 ( mmp-2 ) increased tes expression , respectively , and it is well - established that mmp-2 is associated with atherosclerosis by regulating vascularization and inflammatory response . moreover , magno et al .
identified testin can also interact with calcium - sensing receptor ( car ) , and influence cytoskeletal function via enhancing the car - mediated rho signaling pathway .
all these results suggested that testin may play a protective role in endothelium , and its loss of expression contributes to the initiation and progression of atherosclerosis through multiple mechanisms . in short
, we characterized the expression of tes gene in the aortic tissue of atherosclerotic rabbits .
we showed that tes / testin expression was significantly decreased in aortic tissues with atherosclerotic lesions compared with normal vessels .
we further demonstrated that testin was strongly expressed in cd31 luminal lining with a weaker expression in the subendothelium area .
future studies are needed to fully uncover the underlying mechanisms of tes in the pathogenesis of cardiovascular diseases and to explore the possibility of targeting tes as a potential therapy .
 background : the expression of tes , a novel tumor suppressor gene , is found to be down - regulated in the left anterior descending aorta of patients with coronary artery disease ( cad ) compared with non - cad subjects . 
 this study aimed to investigate the expression of tes during the development of atherosclerosis in rabbits.methods:thirty-two new zealand rabbits were randomly divided into a normal diet ( nd ) and high - fat diet ( hfd ) groups . 
 body weight and serum lipid levels were measured at 0 , 4 , and 12 weeks after diet treatment . 
 the degree of atherosclerosis in thoracic aortas was analyzed by histological examinations . 
 the expression of testin in the tissue samples was inspected via immunohistochemical and immunofluorescence confocal microscopy . 
 real time - polymerase chain reaction and western blot analysis were performed to evaluate the expression of tes / testin at mrna and protein levels in the aortic tissues.results:after 12 weeks postenrollment , rabbits in hfd group had a higher level of serum lipids and atherosclerotic plaque compared to nd group ( p < 0.05 ) . 
 testin expression was detected at high levels in the endothelium and a weak expression on the subendothelium area . 
 the expression of tes mrna was markedly reduced by 10-fold in the aortic tissues in the hfd group compared with the nd group ( p = 0.015 ) , and the protein level was also significantly decreased in the hfd group ( p < 0.05).conclusions : reduced tes / testin expression is associated with the development of atherosclerosis , implicating a potentially important role in the pathogenesis of atherosclerosis . 