in industrialized countries , cardiovascular disease ( cvd ) remains the predominant cause of morbidity and mortality .
it is current scientific opinion that cvd occurs as a part of a series of events that form a continuum .
the first stage of this continuum corresponds to the presence of risks factors that predispose to tissue injury , such as hypertension , dyslipidemia , and diabetes .
these risk factors usually activate a complex range of pathogenic pathways leading to the development of atherosclerosis and ischemic heart disease .
failure to effectively manage any stage of this continuum results in ventricular hypertrophy , heart failure , end - stage heart disease , and cardiovascular death .
one of the molecular pathways that could be activated in response to these risk factors and be involved in cvd development is that of osteoprotegerin ( opg ) and tnf - related apoptosis - inducing ligand ( trail ) , as reviewed in .
opg and trail are both members of the tnf superfamily , and they are widely expressed in different tissues , including the heart and the vessels , in health and disease states [ 4 , 5 ] . although initially opg actions seemed to be limited to bone metabolism   and those of trail to host defense against tumors , recent studies have suggested that both molecules might actually be involved in cvd development and progression .
epidemiological studies have shown that there is an association between circulating opg , trail , and cvd .
opg was found to be directly correlated [ 5 , 8 , 9 ] , while trail was inversely correlated , with cardiovascular morbidity and mortality [ 10 , 11 ] .
likewise , opg was significantly associated with the level of c - reactive protein , which is an independent predictor of acute vascular events and adverse outcomes , while trail was negatively correlated with it .
recently , secchiero and colleagues have shown that patients with coronary artery disease have an increased opg / trail ratio , which further increases in patients developing heart failure .
consistent with this epidemiological data , experimental studies suggest that opg and trail have opposite actions , as opg has proinflammatory   and proatherogenic effects [ 14 , 15 ] , while trail seems to be anti - inflammatory [ 16 , 17 ] and antiatherogenic [ 1820 ] . based on these premises
, we hypothesized that dyslipidemia and diabetes , which are two well known risk factors for cvd , could modify the vascular and cardiac expression of opg and trail , leading to an increased opg / trail ratio at a tissue level .
the aim of this study was to explore the vascular and cardiac changes of opg and trail in experimental models of dyslipidemia and diabetes .
a total of 12 male c57bl/6j ( cnt ) mice and 24 male apolipoprotein e null ( apoe ) mice on a c57bl/6j background , aged 6 weeks , were studied for 14 weeks . at 6 weeks of age ,
apoe mice were further randomized to saline ( apoe ) or streptozotocin ( apoe + dm ) , which were delivered intraperitoneally in five consecutive daily doses .
the daily dose of streptozotocin ( stz ) ( sigma - aldrich ) was 55  mg / kg of body weight .
after one week , blood glucose was checked and only the mice with blood glucose levels greater than 15  mm were included in the apoe + dm group . during the study period , all the mice were fed with a standard diet .
the animals were kept in a temperature - controlled room ( 22  1c ) on a 12  h light/12  h dark cycle with free access to food and water and they were fed ad libitum for the length of the study . after 14 weeks , the animals were anesthetized with an intraperitoneal injection of thiobutabarbital sodium ( inactin , sigma - aldrich ) ( 60  mg / kg body weight ) and sacrificed by exsanguination via cardiac puncture .
all the animal experiments were approved by the animal ethics committee of the university of trieste ( i d 28.0.2008 ) and the italian ministry of health , conforming to the guide for the care and use of laboratory animals . at the end of the study , we measured body weight and systolic blood pressure ( sbp ) .
in particular , sbp was assessed by a computerized noninvasive tail cuff system in conscious mice .
glucose , total cholesterol , hdl cholesterol , and triglycerides were measured in the sera by autoanalyzer technique ( hitachi 917 , tokyo , japan ) .
circulating levels of opg and trail were measured in the sera by elisa ( r&d systems duoset # dy459 and # dy1121 ) . in half of the animals in each group ,
aortae were collected and placed in 10% neutral - buffered formalin for atherosclerosis quantification , before being processed for immunohistochemical analyses . in the other half ,
aortae were snap - frozen in liquid nitrogen and stored at 80c for rna extraction and gene expression analyses .
briefly , aortae were cleaned of excess fat and stained with sudan iv - herxheimer 's solution .
images were acquired with a dissecting microscope ( olympus bx-50 ) equipped with a high - resolution camera ( sony xc-77ce ) .
digitized images were then evaluated with an image analysis system ( image - pro plus 6.3 software , media cybernetics ) .
total plaque area was quantified as the percentage area of aorta stained red . as for the hearts
, they were divided and half was put in formalin and half was put in liquid nitrogen .
the part that was fixed in formalin was embedded in paraffin , cut in 4  m thick sections , and stained with hematoxylin and eosin ( h&e ) in order to evaluate cardiomyocyte hypertrophy .
cardiomyocyte hypertrophy was assessed by measuring the shortest diameter of 200 cells , which were selected when they showed the spindle - shaped transverse section including the elliptical nucleus .
the shortest transverse diameter was measured 3 times per cell , and the values were averaged , as reported by .
the other half that was snap - frozen in liquid nitrogen was used either for gene expression analyses or for immunohistochemical stainings . gene expression of opg and trail was determined by real - time quantitative rt - pcr ( reverse transcription - polymerase chain reaction ) in aortic and cardiac tissue . in order to isolate mrna from the aorta and the heart , tissue was homogenized and processed as previously reported .
then , mrna was treated with the dnase inactivation reagent ( ambion dna - free product # am-1906 ) , and 3  g of treated mrna was subsequently used to synthesize cdna with superscript first strand synthesis system for rt - pcr ( gibco brl ) .
the gene expression of opg and trail was analyzed by real - time quantitative rt - pcr using the taqman system ( life technologies ) for opg and the sybr green system ( life technologies ) for trail .
fluorescence for each cycle was quantitatively analyzed by an abi prism 7900ht sequence detection system ( applied biosystems ) .
gene expression of opg was normalized to 18s mrna , while that of trail was normalized to rps9 .
results were reported as ratio compared with the level of expression in untreated controls , which were given an arbitrary value of 1 .
the presence of opg and trail in the aortae was evaluated by immunostainings on 4  m thick paraffin sections . in particular , after antigen retrieval in citrate buffer ( ph6 ) endogenous peroxidase was quenched for 10 minutes using 3% h2o2 in pbs . to localize opg
, we used a biotinylated goat anti - opg antibody ( r&d systems , 1  :  10 dilution ) followed by a catalyzed signal amplification system ( dako ) . to localize trail
, we used a monoclonal mouse anti - trail antibody ( r&d systems , 1  :  50 dilution ) , applied overnight at 4c . biotinylated goat anti - mouse immunoglobulins ( vector laboratories , dilution 1  :  200 )
were then used as secondary antibody , followed by streptavidin - hrp ( dako ) .
the presence of opg and trail in the hearts was evaluated by immunostaining on 5  m thick frozen sections .
after neutralization of endogenous peroxidase , sections were incubated overnight with the goat anti - opg antibody ( r&d systems , 1  :  10 dilution ) and the monoclonal mouse anti - trail antibody ( r&d systems , 1  :  50 dilution ) .
biotinylated immunoglobulins , diluted 1  :  200 , were then applied as secondary antibodies , followed by streptavidin - hrp ( dako ) . in both tissues ,
after counterstaining with hematoxylin , all the sections were examined by light microscopy ( olympus bx50wi ) and digitized using a high - resolution camera ( q - imaging fast 1394 ) .
the proportional area of brown staining was measured by an image analysis system ( image - pro plus 6.3 software , media cybernetics ) in order to quantify opg and trail expression in aortae and hearts .
apoe mice had higher levels of total cholesterol and triglycerides as compared to the cnt mice . in line with the study protocol , the mice in the apoe + dm group were hyperglycemic . moreover , diabetes was associated with a significant reduction of body weight and a significant increase of total cholesterol as compared to nondiabetic apoe mice .
apoe mice had a significant increase in total plaque area , which increased further after the induction of diabetes ( figures 1(a ) and 1(b ) ) .
as for cardiac changes , dyslipidemia was associated with a significant increase in cardiomyocyte size ( figures 1(c ) and 1(d ) ) , indicating cardiomyocyte hypertrophy , with no further increase after the induction of diabetes . in the aorta , the induction of diabetes was associated with a significant increase in opg gene expression and in the opg / trail ratio , while dyslipidemia had no effect on opg / trail ratio as compared to controls ( figure 2(a ) ) . by contrast , in the heart , dyslipidemia was the major determinant of opg / trail tissue changes , not only by increasing opg but also by reducing trail gene expression .
so dyslipidemia increased significantly cardiac opg / trail ratio , with no further differences after the induction of diabetes ( figure 2(b ) ) .
immunostainings showed that diabetes significantly increased aortic opg expression as compared to the other groups , while trail was unchanged ( figure 3 ) . on the other hand , in the heart , it was dyslipidemia that significantly increased opg protein expression , with no further changes after the induction of diabetes .
as for cardiac trail , trail protein expression decreased in the apoe and apoe + dm groups ( data not shown ) . the levels of circulating
circulating opg was 1.91  0.21  ng / ml in the control group , 2.05  0.1  ng / ml in the apoe group , and 3.1  0.19 in the apoe + dm group , such that opg increased significantly in diabetic mice as compared to the other groups ( p < 0.05 , apoe + dm versus cnt and versus apoe group ) .
as for trail , we were unable to measure its circulating levels , as they were below the detection levels of the elisa we used .
it has already been argued that opg , which circulates at much higher levels than its ligands ( rankl and trail ) , may be a more stable overall measure of opg / trail activity than trail .
patients with coronary artery disease have an increased opg / trail ratio , which further increases in the group of patients who develop heart failure . dyslipidemia and diabetes
, we evaluated the effects of dyslipidemia , alone and in combination with diabetes , on aortic and cardiac opg and trail expression and found that while diabetes is the major determinant of opg / trail tissue changes in the vessels , dyslipidemia is it in the heart .
this data is in line with previous experimental works showing that diabetes increased opg expression and the opg / trail ratio in the aorta   and that the onset of diabetes was associated with an increase of circulating opg .
consistent with these observations , in our study , we found that aortic and circulating opg increased significantly only in the group of mice with diabetes .
taken together , these findings seem to support the hypothesis that the vessels might be the source of increased circulating levels of opg in patients with diabetes and/or cvd .
it has to be noted that the increase of circulating and tissue opg could be not only a risk marker but also a risk factor for atherosclerosis and cvd development .
firstly , animal models point to a role for opg in glucose metabolism regulation , as opg injections increased significantly glucose levels [ 12 , 26 ] , which represent one of the risk factors for cvd .
this effect could be mediated by trail blockade , as trail lowers glucose levels . by offsetting trail effects ,
opg could also promote body weight gain and dyslipidemia , as well as atherosclerotic plaque development [ 18 , 20 ] .
secondly , several studies have shown that opg has proinflammatory and profibrotic effects on the vasculature .
consistent with these actions , our group has shown that opg increases leukocyte adhesion to endothelial cells , that it increases tgf - beta - mediated fibrogenesis and proliferation in vascular smooth muscle cells , and that it increases atherosclerosis extension in diabetic apoe mice .
by contrast , dyslipidemia alone did not affect opg / trail ratio in the vasculature .
this data is consistent with a few in vitro studies showing that oxidized ldl did not change opg expression in human coronary artery smooth muscle cells   and lymphocytes .
nevertheless , in our study , we found that dyslipidemia significantly increased opg / trail ratio in the heart , while diabetes had no additional effect on it .
it has been shown that dyslipidemia is independently associated with left ventricle ( lv ) hypertrophy [ 5 , 30 , 31 ] .
this was confirmed by experimental studies on apoe mice , where dyslipidemia was associated with increased lv mass in the absence of hemodynamic stress .
consistent with previous reports , here we found that dyslipidemia was associated with cardiomyocyte hypertrophy .
it has been demonstrated that triacylglycerol overload and the increased availability of other lipid intermediates , such as ceramides , diacylglycerol , and oxidized phospholipids , can all activate inflammatory pathways and oxidative stress , leading to cellular apoptosis and myocardial scarring [ 33 , 34 ] .
it is possible that the increase in cardiac opg / trail ratio that was associated with dyslipidemia represents one of the mediators of lipid - induced cardiac remodeling . as for opg
, a recent study has demonstrated that opg delivery induced lv hypertrophy , while , in vitro , cardiomyocytes transfected with aav - opg increased dramatically the expression of hypertrophy - related proteins , such as anp , -mhc , and troponin i . in the cardiac fibroblast ,
by contrast , we showed that trail administration to mice with diabetic cardiomyopathy had cardioprotective effects , as it reduced cardiac fibrosis and apoptosis , which are generally contributing to cardiac remodeling . in conclusion , here we found that both dyslipidemia and diabetes affect opg / trail ratio in the cardiovascular system .
this could contribute to the changes in circulating opg / trail which are observed in patients with diabetes and cvd .
most importantly , these changes could mediate / contribute to atherosclerosis development and cardiac remodeling .
 
 background . dyslipidemia and 
 diabetes are two of the most well established risk factors for the development of cardiovascular disease ( cvd ) . both of them usually activate a complex range of pathogenic pathways leading to organ damage . here 
 we hypothesized that dyslipidemia and diabetes could affect osteoprotegerin ( opg ) and tnf - related apoptosis - inducing ligand ( trail ) expression in the vessels and the heart . 
 materials and methods . gene and protein expression of opg , trail , and opg / trail ratio were quantified in the aorta and the hearts of control mice , dyslipidemic mice , and diabetic mice . 
 results . 
 diabetes significantly increased opg and the opg / trail ratio expression in the aorta , while dyslipidemia was the major determinant of the changes observed in the heart , where it significantly increased opg and reduced trail expression , thus increasing cardiac opg / trail ratio . conclusions . 
 this work shows that both dyslipidemia and diabetes affect opg / trail ratio in the cardiovascular system . 
 this could contribute to the changes in circulating opg / trail which are observed in patients with diabetes and cvd . 
 most importantly , these changes could mediate / contribute to atherosclerosis development and cardiac remodeling . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion