the endoplasmic reticulum ( er ) fulfills multiple cellular functions . once er functions are perturbed by various pathological conditions , unfolded or misfolded proteins accumulate in the er lumen , resulting in er stress characterized by increasing er molecular chaperones and diminishing global protein synthesis .
activation of the signaling network in response to er stress is known as unfolded protein response ( upr ) .
there are three distinct upr signaling pathways triggered in response to er stress , which are mediated by ( 1 ) rna - dependent protein kinase - like endoplasmic reticulum kinase ( perk ) , ( 2 ) activating transcription factor 6 ( atf6 ) , and ( 3 ) inositol - requiring enzyme 1 ( ire1 ) , respectively , [ 2 , 3 ] . under physiological conditions , perk , atf6 , and ire1 are associated with the abundant luminal chaperone bip ( also known as glucose - regulated protein 78 , grp78 ) .
when the er is overloaded with newly synthesized proteins or is stimulated by agents that cause unfolded proteins to accumulate , grp78/bip preferentially associates with the unfolded proteins , releasing perk , atf6 , and ire1 to activate downstream signaling molecules . in the perk - mediated pathway ,
activated perk phosphorylates eif-2. the phosphorylated eif-2 subsequently inhibits global protein synthesis , preventing further influx of nascent proteins into an already saturated er lumen . paradoxically
, eif-2 phosphorylation enhances the translation of activating transcription factor-4 ( atf4 ) and subsequently upregulates the expression of atf4-target genes , including several protein chaperones .
recent studies also demonstrated that eif-2 phosphorylation upregulates atf6 expression and enhances atf6 activity and therefore increases the expression of its target genes .   although upr signaling pathways are activated classically by unfolded er proteins , increasing evidence demonstrates that lipids / lipoproteins can also trigger upr .
for instance , a prior study reported that accumulation of free cholesterol in the er was able to activate upr signaling pathways in mouse peritoneal macrophages .
also , studies from our laboratory demonstrated that treatment of mouse macrophages with lipoproteins carrying apolipoprotein ( apo ) b48 but lacking apoe (
e / b48 ) induced foam cell formation , and enhanced phosphorylation of perk and eif-2 , increased expression of atf4 and several molecular chaperons and reduced global protein synthesis [ 7 , 8 ] .
furthermore , we observed that overexpression of dominant - negative mutants of perk or eif-2 , or treatment with the eif-2 inhibitor , 2-aminopurine ( 2-ap ) , suppressed foam cell formation induced by e / b48 lipoproteins [ 7 , 8 ] .
these findings suggest that activation of the perk - eif2 signaling pathway is an underlying mechanism by which e / b48 lipoproteins induce foam cell formation .
such a transformation of macrophages into a foam cells is an early step of atherogenesis .
individuals with defective isoforms of apoe , such as apoe4 , develop postprandial hypercholesterolemia and atherosclerosis .
similarly , apoe knockout ( apoe ) mice manifest an elevated plasma cholesterol and develop atherosclerosis in a manner that resembles the human disease [ 10 , 11 ] .
the hypercholesterolemia in apoe mice results mainly from the increased levels of e / b48 lipoproteins .
it is interesting to note that atherosclerotic lesions in the aorta sinus of apoe mice show upr , as reflected by increased perk phosphorylation in the lesions [ 12 , 13 ] .
data from the present report demonstrated that treatment of apoe mice with the eif-2 inhibitor 2-ap reduced atherosclerotic lesions in their aortas compared to lesions observed in control mice . taken together , these findings suggest that activation of eif-2 signaling pathway is an underlying mechanism for the development of atherosclerosis in apoe mice and that inhibition of this upr pathway might provide a therapy strategy for treatment of atherosclerosis .
  male apoe mice were obtained from jackson laboratory ( bar harbor , me ) .
these mice were generated using embryonic stem cells from the 129 strain of mice   and were crossbred to c57bl/6 for over 10 generations .
they were maintained under barrier conditions in a temperature - controlled environment and fed with a mouse chow containing approximately 5% fat and 19% protein by weight ( harlan teklad , madison , wi ) . at 6 weeks of age
one group of the mice was gavage - fed 2-ap at a dose of 200  mg / kg body weight ( bw ) in 200  l water once every other day , and another group of mice was fed with same volume of water as a control .
after 24 weeks of treatment , mice were fasted overnight and anesthetized with ketamine hydrochloride ( 80  mg / kg  bw ) and xylazine hydrochloride ( 16  mg / kg  bw ) .
approximately 0.5  ml of blood was collected from the posterior vena cava of each mouse .
thereafter , a 23-gauge needle was inserted into the left ventricle and 4% paraformaldehyde was delivered into the animal at pressure of 80  mm  hg . a small incision was made into the liver to allow efflux of blood and fixative .
after fixation , the heart and the proximal aorta were removed from the body and stored in 4% paraformaldehyde at 4c before sectioning .  
the distal aorta ( 2  mm from the heart to the iliac bifurcation ) was opened longitudinally using microscissors and pinned flat on a black wax surface in a dissecting pan under a dissecting microscope ( smz1000 , nikon instruments inc . ,
this en face preparation was fixed overnight and stained with oil - red - o .
the photo - image of the aorta was captured with a coolsnap digital camera ( nikon instruments ) mounted on the smz1000 dissecting microscope .
the atherosclerotic lesion area and the total aortic area were measured using a metamorph imaging system ( nikon instruments ) .  
the proximal aorta attached to the heart was used to prepare cross - sections , as described previously .
briefly , the heart was sectioned transversely immediately below and parallel to a plane formed by the line between atrial leaflets .
the portion of the heart with the attached aorta was embedded either in oct or in paraffin , and sectioned from the attached aorta towards the root of the aorta where the aorta valves were attached .
sections ( 58  m ) were cut from the site where the aorta valve cups appear at the aorta root .
melville , ny ) equipped with a cool snaps color digital camera and a metamorph computer image acquisition system .
the average area ( m ) and morphological features ( foam cell deposits , cholesterol clefts , lipid cores and fibrous - caps ) of the lesions in 16 sections were determined for each mouse .
immunostaining was performed with the use of the vectastain abc system ( vector laboratories , inc . ,
the endogenous peroxidase activity was blocked with 5% hydrogen peroxide for 10  min . after blocking with 5% normal goat or rabbit serum
, the sections were incubated with primary antibodies against grp78 or phosphorylated eif-2 ( abcam inc . , cambridge , ma ) .
after treatment with a secondary antibody produced from goats or rabbits , sections were stained with diaminobenzidine or nova red substrates and counterstained with hematoxylin .
the immunostaining of the aortic sections were viewed using a microscope equipped with an hq2 coolsnap high - resolution camera ( nikon ) and the megamorth computer image acquisition system .
the levels of plasma cholesterol and triglycerides were measured by spectrophotometric quantification using reagents obtained from sigma chemical co. ( st .
cholesterol - reaction reagent was incubated at 37c for 30  min , and the absorbance was read at 530  nm with a dynex microplate reader ( thermo labsystems , franklin , ma ) .
for measuring triglycerides , the mixture of plasma and triglyceride - reaction reagent was incubated at 37c for 10  min , and the absorbance was read at 530  nm .
plasma concentrations of cholesterol and triglycerides were determined based on the absorbance obtained by incubation of the cholesterol and triglyceride standards provided by sigma .    for determination of the cholesterol level in various lipoproteins ,
a 100  l plasma sample obtained from individual mice was fractionated using a fast performance liquid chromatography ( fplc ) ( kta fplc 900 , amersham biosciences , piscataway , nj ) in a buffer containing 0.15  m nacl , 0.01  m na2hpo4 , 0.1  mm edta , ph 7.5 , at a flow rate of 0.5  ml / min .
it had already been established that fractions 1417 contain very low - density lipoprotein ( vldl ) and chylomicrons , fractions 1825 contain ldl , and fractions 2640 contain high - density lipoprotein ( hdl ) [ 14 , 15 ] .
the cholesterol content in various lipoproteins was calculated from the concentration in the fplc fractions [ 14 , 15 ] .   the antibodies against phosphorylated eif-2 , grp78 and lysosomal acid lipase ( lal ) were obtained from abcam inc .
( cambridge , ma ) , while the antibodies against eif-2 and -actin were obtained from santa cruz biotechnology inc .
aortas from two mice were pooled , homogenized in 20  mm tris - cl and centrifuged at 14,000  rpm for 10  min at 4c .
supernatants containing 15  g protein were separated by sodium dodecyl sulfate - polyacrylamide gel electrophoresis on 10% gels and transferred to polyvinylidene difluoride membranes .
membranes were blocked with 5% fat - free milk in tbs - t ( 2.5  mm tris , 15  mm nacl , 0.01% tween 20 ; ph 7.6 ) and then consequently incubated with primary antibodies against indicated proteins and horseradish peroxidase - conjugated secondary antibodies , as previously described .
immunoreactive bands were visualized using ecl - plus chemiluminescence reagent ( ge healthcare healthcare - amersham ) and analyzed with a gs-700 imaging densitometer ( bio - rad , hercules , ca ) .  
the differences between 2-ap treatment and control groups were analyzed by student 's t - test .
the pharmacological properties of 2-ap , such as effective dose 50 ( ed50 ) and effective half - life ( t1/2 ) , have not been tested in vivo . the material safety data sheet produced by invivogen (
san diego , ca ) indicates that the lethal dose 50 ( ld50 ) of 2-ap via oral administration and intraperitoneal injection of rats is 723 and 270  mg / kg body weight ( bw ) , respectively .
a preliminary study in our laboratory demonstrated that gavage feeding of 2-ap to apoe mice at doses greater than 400  mg / kg  bw once every other day reduced food intake and resulted in death ( data not show ) . in the present study , we observed that feeding 2-ap at a dose of 200  mg / kg  bw once every other day did not reduce food intake and body weight , and did not increase mortality within 24 weeks as compared to untreated control apoe mice ( table 1 ) . specifically , the average of food intake of apoe mice at 2430 weeks of age with or without 2-ap treatment was 0.24  0.03 and 0.25  0.07  g  chow / g  bw / day , respectively .
the body weight of these mice at 30 weeks of age was about 27  g. no significant difference was observed between the 2-ap treated and the untreated control mice .
we previously reported that 2-ap inhibited e / b48 lipoprotein - induced transformation of macrophages into foam cells .
figures 1(a ) and 1(b ) show examples of cross - sections and en face preparations of aortas obtained from apoe mice treated with or without 2-ap .
data in figures 1(c ) and 1(d ) show that the mean size of atherosclerotic lesions in the aorta sinus and the surface area of the entire aorta of 2-ap - treated apoe mice were reduced by about 55% and 39% , respectively , compared to those in the untreated control apoe mice .
consistent with the smaller lesion size , the advanced lesions were reduced markedly in 2-ap - treated mice .
for instance , the number of lipid cores ( acellular areas ) in the aortic sinus in the 2-ap - treated and untreated control apoe mice were 1.2  0.6 and 0.4  0.1/section ( p < 0.05 ) , respectively . these results , together with our previous findings that 2-ap inhibits e / b48 lipoprotein - induced foam cell formation , suggest that 2-ap is able to inhibit atherogenesis in vitro and in vivo .
an increase in plasma cholesterol and triglycerides has been suggested to be a risk factor for atherosclerosis . to determine whether the reduction in atherosclerotic lesions induced by 2-ap treatment was due to a change in plasma cholesterol and triglycerides , we measured the levels of plasma cholesterol and triglycerides in apoe mice with or without 2-ap treatment . as the data in table 1 show that the average plasma concentrations of total cholesterol and triglycerides were 513 and 146 , respectively , in apoe mice without 2-ap treatment and that 2-ap treatment did not significantly alter these plasma lipid levels . to investigate
whether 2-ap feeding affected the distribution of cholesterol among the lipoproteins , we fractionated mouse plasma with an fplc system ( figure 2(a ) ) .
as the data in figure 2(b ) show , the plasma cholesterol levels in vldl , ldl , and hdl fractions were comparable in 2-ap - treated and untreated control mice .
these results suggest that the reduced atherosclerotic lesions in 2-ap treated mice were not due to altered levels of plasma lipids .
we previously reported that transformation of macrophages into foam cells by e / b48 lipoproteins was associated with er stress , as reflected by increased phosphorylation of perk and eif-2. further that 2-ap inhibited e / b48
the present report studied the effect of 2-ap on the expression level of total and phosphorylated eif-2 in the aorta of apoe mice .
data in figure 3(a ) show that the level of total eif-2 was comparable in aortas obtained from apoe mice treated with or without 2-ap treatment .
in contrast , the level of phosphorylated eif-2 in the aorta was about 64% lower in 2-ap - treated apoe mice than in untreated control mice ( figure 3(b ) ) . the data in figure 3(c )
demonstrate that the protein level of chaperon grp78 was reduced by ~53% in 2-ap - treated apoe mice as compared to the untreated control littermates .
we previously reported that intralysosomal accumulation of lipids / lipoproteins induced by e / b48 lipoproteins was associated with decreased lysosomal hydrolase lal , and that 2-ap treatment restored e / b48 lipoprotein - reduced expression of lal .
therefore , the present report compared the protein levels of lal in the aorta of apoe mice with or without 2-ap treatment .
data in figures 3(a ) and 3(c ) show that the protein level of aortic lal was ~2.6 fold higher in 2-ap - treated apoe mice than in their untreated littermates .  
having demonstrated the inhibitory activity of 2-ap on er stress in the aorta , we then compared the expression of grp78 and phosphorylated eif-2 in the lesional area in the aorta sinus .
as the representative immunostaining sections in figure 4 show , the atherosclerotic lesions , especially the areas close to the plaque surface , accumulated cells positively stained by antibodies against grp78 or phosphorylated eif-2. the number of positively stained cells in 2-ap - treated apoe mice was much less than those in untreated control mice . these results are consistent with the western blot data shown in figure 3(a ) .
these findings provide evidence that 2-ap inhibits er stress in the aorta of apoe mice .
the most important finding of this report is that oral feeding of the eif-2 phosphorylation inhibitor 2-ap reduced atherosclerotic lesions in apoe mice .
this finding suggests that induction of eif-2 phosphorylation is a causal mechanism for the development of atherosclerosis in apoe mice .
four eif-2 kinases have been identified in mammalian cells , which each catalyze eif-2 phosphorylation in response to various stressful cellular events [ 18 , 19 ] .
specifically , er stress activates perk , heme deprivation activates heme - regulated inhibitor ( hri ) , virus infection activates double - stranded rna - activated protein kinase ( pkr ) and amino acid deprivation activates general control nonderepressible-2 ( gcn2 ) .
previous studies from our laboratory demonstrated that treatment of mouse macrophages with e / b48 lipoproteins induced phosphorylation of perk but no other eif-2 kinases and that overexpression of a nonphosphorylatable perk mutant attenuated e / b48 lipoprotein - induced eif-2 phosphorylation and foam cell formation [ 7 , 8 ] .
these findings suggest that perk is responsible for the increased eif-2 phosphorylation observed in e / b48 lipoprotein - treated macrophages .
a high plasma level of e / b48 lipoproteins is a hallmark feature of apoe mice [ 10 , 20 ] .
data from the present report demonstrated that 2-ap treatment reduced eif-2 phosphorylation in the aorta apoe mice .
it is highly likely that plasma e / b48 lipoproteins infiltrate into the arterial walls of apoe mice , where they activate perk and sequentially increase eif-2 phosphorylation in vascular cells , including resident macrophages .  
indeed , phosphorylation of eif-2 induced by e / b48 lipoproteins has been associated with increased expression of calreticulin and grp 78 .
inhibition of eif-2 by a nonphosphorylatable eif-2 mutant or an 2-ap attenuated e / b48 lipoprotein - induced expression of these protein chaperons .
data from the present report showed that 2-ap treatment reduced the expression of grp78 .  
reduction in global protein synthesis is another hallmark consequence of eif-2 phosphorylation . under physiological conditions , inhibition of global protein synthesis and upregulation of protein chaperones reduces the accumulation of newly synthesized proteins in the er , thereby relieving er stress [ 21 , 22 ] .
however , constant phosphorylation of eif-2 and inhibition of global protein synthesis might overpower normal cellular functions , causing pathological conditions [ 21 , 22 ] .
for example , we previously observed that e / b48 lipoprotein - induced eif-2 phosphorylation was associated with reduced global protein synthesis and reduced expression of lysosomal hydrolases , such as lysosomal acid lipase ( lal ) .
in addition , we previously observed that inhibition of eif-2 phosphorylation by 2-ap , or a nonphosphorylatable eif-2 mutant , attenuated the reduced expression of lal and inhibited foam cell formation induced by e / b48 lipoproteins [ 7 , 8 ] .
lal is the sole hydrolase responsible for cleavage of cholesteryl esters delivered to lysosomes [ 24 , 25 ] . taken together
, our previous studies suggest that reduction in lysosomal hydrolases could be a fundamental mechanism by which e / b48 lipoproteins trigger intracellular lipid / lipoprotein accumulation and transform macrophages into foam cells .
correspondingly , the present report demonstrated that treatment of apoe mice with 2-ap significantly elevated the protein level of lal in the apoe mouse aorta .
it is possible that the e / b48 lipoproteins deposited in the arterial wall induce eif-2 phosphorylation , which downregulates lysosomal hydrolases , such as lal , in the resident macrophages , and therefore reduces the degradation of e / b48 lipoproteins leading to intralysosomal e / b48 lipoprotein accumulation and resulting in foam cell formation and atherosclerosis development .
this postulation is supported by the evidence showing that patients with a deficiency of lal manifest both an accumulation of lipids in cells and develop premature atherosclerosis .
further more , overexpression of lal reduces atherosclerotic lesions in mice deficient in low - density lipoprotein receptor .
this report demonstrated that er stress occurred in the aorta of apoe mice , as reflected by increased phosphorylation of perk and eif-2 and by increased expression of chaperon grp78 .
we also observed that inhibition of eif-2 phosphorylation by 2-ap reduced atherosclerotic lesions in the aorta of apoe mice .
these observations suggest that induction of er stress , that is , activation of the perk - eif2 signaling cascade , is a causal mechanism for development of atherosclerosis in apoe mice .
such insights will inform novel strategies for using er stress inhibitors such as 2-ap to prevent or treat atherosclerosis .
 we previously reported that the apolipoprotein ( apo ) b48-carrying lipoproteins obtained from apoe knockout ( apoe/ ) mice , so called e/b48 lipoproteins , transformed mouse macrophages into foam cells and enhanced the phosphorylation of eukaryotic translation initiation factor 2 ( eif-2 ) . furthermore , the eif-2 phosphorylation inhibitor , 2-aminopurine ( 2-ap ) , attenuated e/b48 lipoprotein - induced foam cell formation . 
 the present report studied the effect of 2-ap on atherosclerosis in apoe/ mice . 
 our results showed that the level of food intake , bodyweight , plasma cholesterol , and triglycerides was comparable in apoe/ mice treated with or without 2-ap . 
 however , the mean size of atherosclerotic lesions in the aorta sinus as well as the surface area of the entire aorta of 2-ap - treated apoe/ mice were reduced by about 55% and 39% , respectively , compared to samples from untreated control apoe/ mice . in addition 
 , the 2-ap - treated apoe/ mice showed a significant decrease in glucose - regulated protein 78 ( grp78 ) and phosphorylated eif-2 in their aortic samples as compared to levels in untreated control apoe/ mice . 
 these observations suggest that endoplasmic reticulum stress is a causal mechanism for the development of atherosclerosis in apoe/ mice and that therapeutic strategies can be developed for using eif-2 phosphorylation inhibitors , such as 2-ap , to prevent or treat atherosclerosis . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion 
5. Conclusion