BACKGROUND
atherosclerosis is a chronic disease characterized by endothelial dysfunction and inflammation .
in addition to the significant monetary burden, atherosclerotic vascular disease undermines functional capacity, leads to a greater dependence on hospitalizations and long-term care, and is a risk factor for the development of myocardial infarction and heart failure  <cit> .
a greater understanding of mechanisms and mediators of vascular dysfunction and inflammatory processes in the aortic vasculature that manifest as atherosclerotic disease are needed in order to promote the development of novel prevention and treatment strategies.
clinical studies have shown that atherosclerotic plaque in the aortic arch and innominate arteries is commonly observed in patients at risk for stroke, myocardial infarction, atrial fibrillation, and peripheral artery disease and that plaque in the aorta is an indication of generalized atherosclerosis  <cit> .
while retention of lipoprotein into the sub-endothelial vascular layer is believed to be the initiating stimulus leading to the development of atherosclerosis, activation of multiple pathways related to vascular inflammation and dysfunction sustain the process by stimulating recruitment of leukocytes and immune cells into the sub-endothelial layer  <cit> .
differentiation of monocytes into tissue resident macrophages that engulf and oxidize lipids to become inflammatory foam cells is also a hallmark of atherosclerosis  <cit> .
one of the well-defined risk factors for the development of atherosclerosis is diet-induced obesity, which is on the rise in western societies  <cit> .
mounting evidence in humans supports an etiological role for the microbiota in inflammatory atherosclerosis.
recent studies have established that common chronic infections may account for up to 40% of newly developed atherosclerosis independent of genetic risk factors  <cit> .
the gram-negative bacteria, porphyromonas gingivalis  and chlamydia pneumoniae , have been associated with the development and acceleration of plaque burden in humans and these observations have been validated in animal models .
both of these pathogens have a high prevalence of infection in the general population.
p. gingivalis is an oral pathogen strongly associated with periodontal disease, one of the most common chronic diseases with a prevalence between 10-60% of adults  <cit> .
p. gingivalis promotes chronic systemic inflammation by disrupting host immune responses and altering the composition of microbial communities
.
c. pneumoniae is an obligate intracellular bacterium that infects the respiratory tract and is a major cause of pneumonia in humans.
an estimated 2- <dig> million cases of pneumonia each year in the united states are attributed to c. pneumoniae infection  <cit> .
approximately 50% of adults have evidence of past infections by age  <dig> and re-infection throughout life is common  <cit> .
although extensive research has shown endogenous mediators arising from excessive dietary lipids and the pathogens p. gingivalis and c. pneumoniae are pro-atherogenic  <cit> , the specific inflammatory/atherogenic pathways induced by these individual stimuli in plaque progression are not well defined.
in this study, we examined how exposure to two pathogens associated with atherosclerosis induces modulation of gene expression in aortic tissues using apoe-/- mice that spontaneously develop atherosclerosis in the absence of an additional pro-atherogenic stimulus.
apoe-/- mice are the most widely used mouse model for the development of atherosclerosis in the absence of additional stimuli and are characterized by increased total plasma cholesterol levels .
furthermore, we compared pathogen-induced gene signatures to apoe-/- mice fed a western diet.
comparison of gene expression profiles obtained from pathogen treated mice at the acute and chronic time points was also examined to define how these pathogens modulate gene expression during the natural progression of atherosclerosis in apoe-/- mice.
RESULTS
pca and qrt-pcr analysis
principal component analysis  performed using all genes across all samples showed that there was strong separation between chronic and acute p. gingivalis treatment samples  along the pc <dig> axis, indicating that there was strong time-dependent differential gene expression in this treatment group.
mean fold changes  obtained by taqman real time rt-pcr analysis for  <dig> genes were in good agreement with microarray results .
chronic treatment with p. gingivalis, c. pneumoniae,and wd induces distinct gene expression patterns in aortic tissue
genes with significant differential expression  in response to the three pro-atherogenic stimuli are tabulated in figure  <dig>  p. gingivalis treatment resulted in a 2- <dig> times greater number of unique differentially expressed genes compared to the other two treatments.
the number of differentially expressed genes common to the two pathogen-treated groups was also substantially larger than the number common to either pathogen-treated group and the wd group.figure  <dig>
chronic time point venn diagrams.
venn diagrams depicting the number of genes with significantly increased  or significantly decreased  expression vs. the chronic control group for each chronic treatment group.
the sets of differentially expressed genes result from differential expression analyses for each chronic treatment group vs. the chronic control group using the r environment for statistical computing  with a cut off of fdr q <  <dig>  as described in methods.
identification of pathways altered by chronic treatment with p. gingivalis, c. pneumoniae,or wd
to provide insight into the biological pathways and processes that are altered in response to each treatment, we used gene set enrichment analysis   to determine whether the members of gene sets involved in curated pathways and processes  were nonrandomly distributed across all genes interrogated by the array with respect to each treatment.
the gene sets that were coordinately upregulated  in response to p. gingivalis were related to proliferative signaling, matrix remodeling, execution of apoptosis, pdgf signaling, the secretin g-protein receptor family, and the lysosome .
the gene sets with the strongest coordinate downregulation  with p. gingivalis treatment were involved in mitochondrial function and glucose metabolism , suggesting that p. gingivalis treatment may lead to mitochondrial dysfunction and metabolic imbalance in the aorta.
c. pneumoniae treatment was associated with the coordinate upregulation of genes involved in redox signaling, lipid metabolism, carbohydrate and amino acid metabolism, the mitochondrion, and vitamin metabolic processes , and the downregulation of genes related to muscle contraction and differentiation and channel activity .
these findings suggest that c. pneumoniae treatment alters the redox state and metabolism of lipids that may promote the dedifferentiation of smooth muscle cells in aortic tissue.
the majority of gene sets that were coordinately upregulated in response to wd were those involved in defense and immune function; others included pathways related to macromolecular degradation and cell cycle regulation .
gene sets that were negatively enriched in response to wd included tight junction regulation, receptor signaling, muscle proteins and channel activity .
these findings suggest that dietary lipids and cholesterol are sensed by the innate and adaptive immune system in aortic tissue in a manner that promotes inflammation and loss of vascular barrier function.table  <dig>
gene set enrichment analysis
1
basal cell carcinoma
3
nucleachromosome part
5
regulation of gene expression epigenetic
6
kegg melanogenesis
8
dna replication
9
proteinaceous extracellular matrix
10
kegg hedgehog signaling pathway
12
reactome cell extracellular matrix interactions
14
reactome e2f mediated regulation of dna replication
15
reactome apoptotic execution phase
16
collagen
17
reactome inactivation of apc via direct inhibition of the ap complex
19
reactome smooth muscle contraction
20
reactome conversion from apc cdc <dig> to apc cd <dig> in late anaphase
21
reactome signaling by pdgf
22
kegg lysosome
23
microtubule cytoskeleton
24
cell division
25
reactome class b <dig> secretin family receptors
positive enrichment: chronic p. gingivalis-treated group vs. chronic control group.
the top  <dig> p. gingivalis gene sets whose member genes are predominantly upregulated with respect to the chronic control group.
column  <dig> indicates the rank of the gene set based on normalized enrichment score .
column  <dig> lists the gene set name  as provided by msigdb.
gene set names and ranks that are not in bold are gene sets that are redundant or partially redundant and included within larger bolded gene sets.
the nes ranged from  <dig>   to  <dig>  .
fdr q range:  <dig> - <dig> .
gene set enrichment analysis
2
reactome integration of energy metabolism
4
kegg oxidative phosphorylation
5
kegg parkinsons disease
7
mitochondrial part
8
mitochondrian
9
kegg citrate cycle tca cycle
12
kegg alzheimers disease
13
organelle inner membrane
14
kegg huntingtons disease
15
reactome pyruvate metabolism and tca cycle
19
kegg cardiac muscle contraction
20
reactome diabetes pathways
21
reactome glucose metabolism
22
energy derivation by oxidation of organic compounds
24
regulation of heart contraction
25
kegg ppar signaling pathway
negative enrichment: chronic p. gingivalis-treated group vs. chronic control group.
the top  <dig> p. gingivalis gene sets whose member genes are predominantly downregulated with respect to the chronic control group.
column  <dig> indicates the rank of the gene set based on normalized enrichment score .
column  <dig> lists the gene set name  as provided by msigdb.
gene set names and ranks that are not in bold are gene sets that are redundant or partially redundant and included within larger bolded gene sets.
the nes ranged from - <dig>   to - <dig>  .
fdr q <  <dig> × 10- <dig>
gene set enrichment analysis
1
microbody
2
peroxisome
3
kegg peroxisome
4
kegg ppar signaling pathway
5
reactome metabolism of lipids and lipoproteins
8
kegg valine leucine and isoleucine degradation
7
9
kegg fatty acid metabolism
10
reactome metabolism of vitamins and cofactors
11
kegg pyruvate metabolism
13
kegg glycerolipid metabolism
15
lipid catabolic process
16
mitochondrion
18
reactome branched chain amino acid catabolism
19
cofactor metabolic process
21
kegg biosynthesis of unsaturated fatty acids
25
vitamin metabolic process
positive enrichment: chronic c. pneumoniae-treated group vs. chronic control group.
the top  <dig> c. pneumoniae gene sets whose member genes are predominantly upregulated with respect to the chronic control group.
column  <dig> indicates the rank of the gene set based on normalized enrichment score .
column  <dig> lists the gene set name  as provided by msigdb.
gene set names and ranks that are not in bold are gene sets that are redundant or partially redundant and included within larger bolded gene sets.
the nes ranged from  <dig>   to  <dig>  .
fdr q range: 0- <dig> .
gene set enrichment analysis
1
kegg hypertrophic cardiomyopathy
2
kegg dilated cardiomyopathy
3
reactome muscle contraction
5
muscle development
6
actin cytoskeleton
8
structural molecule activity
9
regulation of multicellular organismal process
10
kegg cardiac muscle contraction
11
cytoskeletal protein binding
13
kegg arrhythmogenic right ventricular cardiomyopathy arvc
14
contractile fiber
15
contractile fiber part
16
heart development
20
gated channel activity
22
cation channel activity
24
cytoskeletal part
negative enrichment: chronic c. pneumoniae-treated group vs. chronic control group.
the top  <dig> c. pneumoniae gene sets whose member genes are predominantly downregulated with respect to the chronic control group.
column  <dig> indicates the rank of the gene set based on normalized enrichment score .
column  <dig> lists the gene set name  as provided by msigdb.
gene set names and ranks that are not in bold are gene sets that are redundant or partially redundant and included within larger bolded gene sets.
the nes ranged from - <dig>   to - <dig>  .
fdr q <  <dig> × 10- <dig>
gene set enrichment analysis
1
kegg lysosome
2
reactome signaling in immune system
3
kegg natural killer cell mediated cytotoxicity
4
kegg systemic lupus erythematosus
6
kegg b cell receptor signaling pathway
7
leishmania infection
8
kegg toll like receptor signaling pathway
9
immune system process
11
reactome toll receptor cascades
13
reactome s phase
14
kegg fc gamma r mediated phagocytosis
16
reactome host interactions of hiv factors
17
reactome m g <dig> transition
18
kegg t cell receptor signaling pathway
19
defense response
20
kegg nod like receptor signaling pathway
21
kegg hematopoietic cell lineage
22
reactome g <dig> s transition
23
kegg chemokine signaling pathway
25
reactome cell cycle checkpoints
positive enrichment: western diet group vs. chronic control group.
the top  <dig> western diet gene sets whose member genes are predominantly upregulated with respect to the chronic control group.
column  <dig> indicates the rank of the gene set based on normalized enrichment score .
column  <dig> lists the gene set name  as provided by msigdb.
gene set names and ranks that are not in bold are gene sets that are redundant or partially redundant and included within larger bolded gene sets.
the nes ranged from  <dig>   to  <dig>  .
fdr q <  <dig> × 10- <dig>
gene set enrichment analysis
1
g protein coupled receptor activity
2
apical junction complex
3
apicolateral plasma membrane
4
neurotransmitter binding
9
kegg neuroactive ligand receptor interaction
11
reactome amine ligand binding receptors
12
intercellular junction
13
structural constituent of muscle
15
calcium channel activity
16
feeding behavior
18
voltage gated channel activity
19
kegg basal cell carcinoma
21
contractile fiber
22
anion transmembrane transporter activity
23
reactome tight junction interactions
25
digestion
negative enrichment: western diet group vs. chronic control group.
the top  <dig> western diet gene sets whose member genes are predominantly downregulated with respect to the chronic control group.
column  <dig> indicates the rank of the gene set based on normalized enrichment score .
column  <dig> lists the gene set name  as provided by msigdb.
gene set names and ranks that are not in bold are gene sets that are redundant or partially redundant and included within larger bolded gene sets.
the nes ranged from - <dig>   to - <dig>  .
fdr q range:  <dig> –  <dig> .
functional classification of clusters of genes differentially expressed with respect to chronic atherogenic stimuli
the  <dig> genes with the greatest significance by one-way anova across the four chronic treatment groups  were assigned to several groups using hierarchical clustering .
at the arbitrary cutoff of  <dig> genes, the chronic time point anova fdr q value was <  <dig>  and the p value was <  <dig>  × 10- <dig>  the david functional classification tool was used to extract biological meaning from each of the clusters .
the three pro-atherogenic stimuli produced strikingly different patterns of differential gene expression.
genes involved in immunity and inflammation  were coordinately upregulated in response to wd, but were largely unchanged in response to either pathogen.
by contrast, genes involved in lipid synthesis and ppar signaling  were markedly increased in response to c. pneumoniae treatment but were unchanged in the other groups.
finally, two smaller clusters suggest that the hedgehog pathway is suppressed in wd mice  and that treatment with either pathogen decreased the expression of contractile proteins ; however, these results were based on a small number of genes and should be interpreted with caution .figure  <dig>
chronic time point cluster analysis.
the top  <dig> differentially expressed genes at the chronic time point with  <dig> distinct clusters.
a. heat map shows relative expression among all groups.
clusters are color-coded by row sidebars: red , chartreuse , mint green , blue , and magenta ; and dendrogram is left of the color-coded sidebars.
each row corresponds to a gene  and each column to a sample.
the colors are scaled by row; red and blue indicate  <dig> standard deviations above or below the mean , respectively.
at the arbitrary cutoff of  <dig> genes, the chronic time point one-way anova fdr q value was <  <dig>  and the p value was <  <dig>  × 10- <dig>  b. david analysis of clusters  <dig> and  <dig>  gene enrichment is indicated by p values .
c. box and whisker plots of the mean expression  for clusters  <dig> and  <dig> reflect patterns seen on heat map.
*** = p <  <dig>  chronic treatment group vs. chronic control group by mann-whitney test.
time-dependent changes in aortic gene expression in mice treated with p. gingivalisor c. pneumoniae
to examine the acute response in aortic tissue following pathogen exposure, rna samples were obtained one day after the last treatment with p. gingivalis or c. pneumoniae .
genes with nominally significant differential expression  between the chronic and acute time points in untreated or p. gingivalis- or c. pneumoniae-treated apoe-/- mice are tabulated in figure  <dig>  differential expression in all three groups was greater than expected by chance, and as in the comparison between groups at the chronic time point alone , p. gingivalis treatment produced the largest amount of differential gene expression, and the genes regulated by the two pathogens overlapped substantially.
for the top  <dig> differentially expressed genes observed at the acute to chronic time points, aortic tissue from apoe-/- mice fed a normal chow diet was characterized by a balance of genes with increased and decreased expression; p. gingivalis treatment was skewed towards decreased expression .
because time-dependent differential expression was less robust in the c. pneumoniae-treated group, these changes may reflect inhibition of natural changes that occur with time in the aortic tissue from apoe-/- mice fed a normal chow diet.figure  <dig>
acute to chronic time point venn diagrams.
venn diagrams depicting the number of genes with significantly increased  or significantly decreased  expression vs. the acute group for each chronic group.
the sets of differentially expressed genes result from differential expression analyses for each acute group vs. its corresponding chronic group using the r environment for statistical computing  with a cut off of p <  <dig>  as described in methods.
functional classification of genes with varying kinetics of response to p. gingivalisor c. pneumoniaetreatment
using two-factor anova, we then analyzed the capacity of either pathogen to alter normal time-dependent changes in gene expression that occurred in apoe-/- mice.
a clustering heat map of the  <dig> genes with the strongest interaction effect between time and treatment is shown in figure  <dig>  at the arbitrary cutoff of  <dig> genes, the anova interaction  fdr q value was <  <dig>  and the p value was <  <dig>  × 10- <dig>  the expression of genes encoding myofibril, cytoskeletal, and ion binding/transport proteins  greatly increased over time in untreated apoe-/- mice.
however, treatment with either pathogen prevented or reversed this effect: the expression of these genes was increased and subsequently downregulated in the acute or chronic p. gingivalis-treated groups, respectively, and was unchanged in either c. pneumoniae treatment group.
conversely, the expression of a number of zinc-finger transcription factors  was downregulated over time in untreated mice, but was decreased and then subsequently upregulated in the acute or chronic c. pneumoniae-treated groups, respectively, and was unchanged in either p. gingivalis treatment group.
a third noteworthy pattern was defined by a group of genes with functions in fatty acid metabolism and ppar signaling whose expression was moderately downregulated over time in untreated animals ; this downregulation was greatly amplified in mice treated with p. gingivalis but was completely abrogated in mice treated with c. pneumoniae .figure  <dig>
acute to chronic time point cluster analysis.
the top  <dig> differentially expressed genes for acute and chronic time points: the effect of group, time, and group x time interactions determined by two-way anova are grouped into  <dig> distinct clusters.
a. heat map depicts relative expression among all groups and time points.
clusters are color-coded by row sidebars: red , chartreuse , mint green , blue , and magenta ; and dendrogram is left of the color-coded sidebars.
each row corresponds to a gene  and each column to a sample.
the colors are scaled by row; red and blue indicate  <dig> standard deviations above or below the mean , respectively.
at the arbitrary cutoff of  <dig> genes, the two-way anova interaction  fdr q value was <  <dig>  and the p value was <  <dig>  × 10- <dig>  b. david analysis of clusters  <dig> and  <dig>  gene enrichment is indicated by p values .
c. box and whisker plots of the mean expression  for clusters  <dig> and  <dig> reflect patterns seen on heat map.
***p <  <dig>  vs. acute control; ###p <  <dig>  vs. chronic control; +++p <  <dig>  vs. acute treatment; ++p <  <dig>  vs. acute treatment by mann-whitney test.
functional classification of clusters of genes differentially expressed with respect to acute atherogenic stimuli
as before, the  <dig> genes with the greatest significance by one-way anova across the three acute treatment groups  were assigned to several groups using hierarchical clustering .
acute treatment with p. gingivalis produced little differential gene expression, whereas acute treatment with c. pneumoniae resulted in large changes in gene expression.
genes whose expression increased with c. pneumoniae treatment  represented g-protein coupled signaling, viral myocarditis, antigen processing and presentation, and membrane genes.
by contrast, nearly half of the genes whose expression decreased with c. pneumoniae treatment  encoded phosphoproteins, with the remainder encoding proteins involved in alternative splicing, the endoplasmic reticulum, and glycoproteins and secreted proteins.
the remaining clusters were not remarkable due to lack of annotation to a david pathway or of marginal significance .
the three remaining clusters were not noteworthy .
analysis of genes associated with unstable plaque
a recent study identified  <dig> genes whose expression in the aortic tissue of apoe-/- mice was associated with unstable plaque  <cit> .
we observed increased expression of several of these genes in aortic tissue from untreated apoe-/- mice fed a normal chow diet, which represents the natural progression of atherosclerosis ; and, in apoe-/- wd mice, we observed an even greater increase in expression of some of these genes.
p. gingivalis treatment blunted the increase in expression of some genes and induced the expression of others.
treatment with c. pneumoniae prevented the increase in all of the unstable plaque genes with the exception of mmp <dig>  which was increased over time in the c. pneumoniae-treated group but decreased over time in untreated apoe-/- mice.figure  <dig>
genes associated with unstable plaque.
heat map showing expression of genes associated with unstable plaque identified in chen, et al.
<cit> .
discussion
in this study, we applied microarray analysis to define gene signatures in aortic tissues obtained from mice fed a western diet  or in mice treated with the oral pathogen p. gingivalis , or with the pulmonary pathogen c. pneumoniae .
examination of gene expression profiles at the chronic time point enabled us to identify treatment-specific pro-atherogenic pathways rather than simply the genes expressed in established plaque.
our results in untreated apoe-/- mice are consistent with a recent microarray study by papadodima et al.
<cit>  that documented changes in gene expression in aortic tissue of apoe-/- mice at three time points during the natural progression of atherosclerosis in the absence of additional pro-atherogenic stimuli.
we identified common core sets of genes that were either increased or decreased in response to each of these pro-atherogenic stimuli.
contemporary analysis tools including gsea and david analysis were then applied.
gsea approach is more powerful than single gene analysis, which merely highlights the top up- or downregulated genes and may overlook effects on specific pathways  <cit> .
pathway analysis showed a markedly greater number of gene sets that were different among the three pro-atherogenic stimuli, suggesting that underlying vascular inflammation and dysfunction leading to atherosclerosis differ mechanistically and in the functional pathways leading to atherosclerosis progression depending upon stimulus.
these findings are significant in that this is the first study that has performed direct, side-by-side, comparison of genome-wide aortic gene expression changes in three well-recognized models of atherosclerosis development.
treatment with wd, p. gingivalis or c. pneumoniae has pro-atherogenic effects that include alterations in macrophage function, cholesterol homeostasis, and endothelial dysfunction  <cit> .
signaling through innate immune toll-like receptors  expressed on immune cells and the endothelium may be a common link among the three stimuli; however, notable differences in tlr usage as well as features unique to each of the two pathogens may account for the differences in signaling pathways activated and repressed.
tlr <dig>  tlr <dig>  and tlr <dig> rna and protein are expressed at high levels in endothelial cells and macrophages in human atherosclerotic plaque biopsies and the lipid-rich atherosclerotic lesions in the aortic root of mice  <cit> .
a role for tlrs in high-fat-diet-induced atherosclerosis in animal models has been shown in studies using apoe-/- tlr2-/- and apoe-/- tlr4-/- mice.
interestingly specificity in tlr <dig> and tlr <dig> signaling by p. gingivalis and c. pneumoniae has been proposed to define specific inflammatory pathways unique to each organism .
p. gingivalis expresses heterogeneous lps lipid a structures that weakly activate tlr <dig> but can also act as a tlr <dig> antagonist  <cit> , resulting in alterations in signaling through tlr <dig> that promote low-grade, chronic inflammation at distant sites including the aorta  <cit> .
c. pneumoniae induces its proinflammatory signaling primarily through tlr <dig> but also expresses lps and signals through tlr <dig>  <cit> .
the unique feature of c. pneumoniae is that it gains entry into monocytes as elementary bodies , which are infectious but metabolically inert.
after entering macrophages or monocytes, ebs can rapidly differentiate to a replicative form known as reticulate bodies , and start bacterial replication  <cit> .
rbs use the host’s metabolic metabolism and may find a favorable environment within a lipid-laden plaque to complete its replication.
infected monocytes may circulate to distant sites to promote vascular inflammation  <cit> .
c. pneumoniae may increase adherence of macrophages to endothelial cells through expression of its virulence factor, hsp  <dig>  which has been shown to promote monocyte attachment to endothelium, and to promote extravasation to sub-endothelial layer, where it oxidizes ldl and promotes foam cell formation  <cit> , a hallmark of atherosclerosis.
most published studies involving animal models of atherosclerosis have used high dietary lipids and cholesterol , and these studies have formed the basis for consensus around the mechanisms underlying the development of atherosclerosis.
as a result, the use of statin drugs, which target cholesterol and lipid handling, are the most widely used class of drugs in the field of cardiovascular medicine.
a recent study in circulation  <cit>  showed that despite the improvement of risk with optimum statin therapy in patients with cardiovascular disease, many patients demonstrate atheroma progression and additional cardiovascular events, suggesting that additional mechanisms are at play, highlighting the need to identify novel therapeutic strategies that combat the additional cardiovascular risk.
recent research implicates vascular inflammation and endothelial dysfunction as a factor involved in the initiation, progression, and instability of atherosclerotic plaques and elevations of serum inflammatory biomarkers consistently associated with the risk of experiencing a cardiovascular event, providing further evidence for systemic inflammation involvement in atherosclerotic cardiovascular disease  <cit> .
given the prevalence of diet-induced obesity and infection with p. gingivalis and c. pneumoniae in the general population and the likelihood of co-morbidity of obesity with chronic or recurring infection with these common pathogens, these findings suggest that the development of atherosclerosis in humans is likely more complex and multifactorial than previously appreciated.
atherosclerotic plaques undergo both progressive and regressive changes, which affect their size and stability; regression in lesion area can increase plaque stability.
plasma cholesterol lowering has been associated with regression, and a recent study identified ppar-gamma as a “master regulator” of regression in early lesions  <cit> .
our gsea analysis showed that c. pneumoniae treatment upregulated the ppar pathway while p. gingivalis treatment downregulated this pathway and thus may indicate a role for c. pneumoniae in regression and stability of early lesions, while p. gingivalis may inhibit plaque regression.
p. gingivalis decreased pathways involved in mitochondrial function, suggesting that p. gingivalis promotes mitochondrial dysfunction, which is associated with cardiovascular risk, vascular dysfunction and plaque development  <cit> .
western diet group had significant reduction in tight junction pathways and expression of genes encoding claudins, proteins involved in maintenance of cell-cell junctions.
this finding is consistent with a recent study that showed that western diet promotes atherosclerosis in part via loss of tight junction control  <cit> .
a surprising finding from the present study was that immune and inflammatory pathways were markedly upregulated in response to western diet.
it has recently come to light that certain lipids in western style diets are either endogenous ligands of tlrs or assist with pattern recognition of tlrs  <cit> .
consistent with this, prior studies have shown that tlr <dig>  tlr <dig>  and myd <dig> knockout mice on an apoe-/- background had decreased atherosclerotic aortic plaque in response to a western diet  <cit> .
a recent microarray study by hyvarinen et al., compared gene expression in adipose tissue following chronic infection with c. pneumoniae and/or a periodontal pathogen, a. actinomycetemcomitans  and revealed significant enrichment in inflammation associated pathways by aa or a combination of aa and c. pneumoniae but not by c. pneumoniae alone as compared to a control group  <cit> .
of interest was the increased expression of a number of genes associated with vulnerable plaque previously identified by chen et al.
<cit>  in aortic tissues obtained from mice at the chronic phase compared to tissues obtained at the acute phase.
these genes were bmper, hdc, ifitm <dig>  s100a <dig>  upp <dig>  adamts <dig>  fpr <dig>  clec4n, hmox <dig> and irg <dig>  our findings demonstrate that expression of these genes increases as part of the natural progression of atherosclerosis in apoe-/- mice in the absence of an additional pro-atherogenic stimulus.
importantly, the increased expression of these genes was blunted at the chronic time point in mice treated with c. pneumoniae.
expression of kit was increased in the c. pneumoniae -treated group compared to the chronic control group.
in addition, p. gingivalis treatment blunted the increase in expression of bmper, hdc, ifitm <dig>  s100a <dig>  upp <dig> and hmox <dig>  expression of egln <dig> was decreased in the p. gingivalis-treated group compared to the chronic control group.
expression of clec4n and hmox <dig> was increased in the western diet group compared to the chronic control group.
the identification of differences in the effects of each of the pro-atherogenic stimuli on expression of important vulnerable plaque genes may result in differences in the nature and composition of the atherosclerotic plaque, which will be explored in future studies.
limitations of the study
several limitations of the study should be noted.
we could not simultaneously perform a side-by-side quantification of the degree of the aortic plaque and the pathological features assessed by histology induced by the three atherogenic stimuli.
we also could not determine the contribution of possible alterations in gut microbiota induced by each of the pro-atherogenic treatments or following antiobiotics pre-treatment in the p. gingivalis group.
we could not validate our findings with regard to establishing that the pathways we identified are mechanistically linked to each pro-atherogenic stimulus at the protein level.
we also acknowledge the limitations of using mouse models to draw conclusions about human diseases, which are more complex and multifactorial.
future studies will also investigate the effects of comorbidity with  <dig> or more pro-atherogenic stimuli, which resembles scenarios that occur clinically.
CONCLUSIONS
the present study suggests that p. gingivalis treatment may promote atherosclerosis by stimulating mitochondrial dysfunction and inhibiting the egress of endogenous lipids from the vessel wall, whereas c. pneumoniae may promote atherosclerosis by enhancing lipid uptake and metabolism into the vessel wall, and wd may promote atherosclerosis by activation of inflammatory and immune pathways that enhance leukocyte adhesion and endothelial dysfunction.

 atherosclerosis is a chronic disease characterized by endothelial dysfunction and inflammation. in addition to the significant monetary burden, atherosclerotic vascular disease undermines functional capacity, leads to a greater dependence on hospitalizations and long-term care, and is a risk factor for the development of myocardial infarction and heart failure. a greater understanding of mechanisms and mediators of vascular dysfunction and inflammatory processes in the aortic vasculature that manifest as atherosclerotic disease are needed in order to promote the development of novel prevention and treatment strategies. clinical studies have shown that atherosclerotic plaque in the aortic arch and innominate arteries is commonly observed in patients at risk for stroke, myocardial infarction, atrial fibrillation, and peripheral artery disease and that plaque in the aorta is an indication of generalized atherosclerosis. while retention of lipoprotein into the sub-endothelial vascular layer is believed to be the initiating stimulus leading to the development of atherosclerosis, activation of multiple pathways related to vascular inflammation and dysfunction sustain the process by stimulating recruitment of leukocytes and immune cells into the sub-endothelial layer. differentiation of monocytes into tissue resident macrophages that engulf and oxidize lipids to become inflammatory foam cells is also a hallmark of atherosclerosis. one of the well-defined risk factors for the development of atherosclerosis is diet-induced obesity, which is on the rise in western societies. mounting evidence in humans supports an etiological role for the microbiota in inflammatory atherosclerosis. recent studies have established that common chronic infections may account for up to 40% of newly developed atherosclerosis independent of genetic risk factors. the gram-negative bacteria, porphyromonas gingivalis and chlamydia pneumoniae, have been associated with the development and acceleration of plaque burden in humans and these observations have been validated in animal models. both of these pathogens have a high prevalence of infection in the general population. p. gingivalis is an oral pathogen strongly associated with periodontal disease, one of the most common chronic diseases with a prevalence between 10-60% of adults. p. gingivalis promotes chronic systemic inflammation by disrupting host immune responses and altering the composition of microbial communities. c. pneumoniae is an obligate intracellular bacterium that infects the respiratory tract and is a major cause of pneumonia in humans. an estimated 2- million cases of pneumonia each year in the united states are attributed to c. pneumoniae infection. approximately 50% of adults have evidence of past infections by age and re-infection throughout life is common. although extensive research has shown endogenous mediators arising from excessive dietary lipids and the pathogens p. gingivalis and c. pneumoniae are pro-atherogenic, the specific inflammatory/atherogenic pathways induced by these individual stimuli in plaque progression are not well defined. in this study, we examined how exposure to two pathogens associated with atherosclerosis induces modulation of gene expression in aortic tissues using apoe-/- mice that spontaneously develop atherosclerosis in the absence of an additional pro-atherogenic stimulus. apoe-/- mice are the most widely used mouse model for the development of atherosclerosis in the absence of additional stimuli and are characterized by increased total plasma cholesterol levels. furthermore, we compared pathogen-induced gene signatures to apoe-/- mice fed a western diet. comparison of gene expression profiles obtained from pathogen treated mice at the acute and chronic time points was also examined to define how these pathogens modulate gene expression during the natural progression of atherosclerosis in apoe-/- mice.  pca and qrt-pcr analysis principal component analysis performed using all genes across all samples showed that there was strong separation between chronic and acute p. gingivalis treatment samples along the pc axis, indicating that there was strong time-dependent differential gene expression in this treatment group. mean fold changes obtained by taqman real time rt-pcr analysis for genes were in good agreement with microarray results. chronic treatment with p. gingivalis, c. pneumoniae,and wd induces distinct gene expression patterns in aortic tissue genes with significant differential expression in response to the three pro-atherogenic stimuli are tabulated in figure  p. gingivalis treatment resulted in a 2- times greater number of unique differentially expressed genes compared to the other two treatments. the number of differentially expressed genes common to the two pathogen-treated groups was also substantially larger than the number common to either pathogen-treated group and the wd group.figure chronic time point venn diagrams. venn diagrams depicting the number of genes with significantly increased or significantly decreased expression vs. the chronic control group for each chronic treatment group. the sets of differentially expressed genes result from differential expression analyses for each chronic treatment group vs. the chronic control group using the r environment for statistical computing with a cut off of fdr q <  as described in methods. identification of pathways altered by chronic treatment with p. gingivalis, c. pneumoniae,or wd to provide insight into the biological pathways and processes that are altered in response to each treatment, we used gene set enrichment analysis to determine whether the members of gene sets involved in curated pathways and processes were nonrandomly distributed across all genes interrogated by the array with respect to each treatment. the gene sets that were coordinately upregulated in response to p. gingivalis were related to proliferative signaling, matrix remodeling, execution of apoptosis, pdgf signaling, the secretin g-protein receptor family, and the lysosome. the gene sets with the strongest coordinate downregulation with p. gingivalis treatment were involved in mitochondrial function and glucose metabolism, suggesting that p. gingivalis treatment may lead to mitochondrial dysfunction and metabolic imbalance in the aorta. c. pneumoniae treatment was associated with the coordinate upregulation of genes involved in redox signaling, lipid metabolism, carbohydrate and amino acid metabolism, the mitochondrion, and vitamin metabolic processes, and the downregulation of genes related to muscle contraction and differentiation and channel activity. these findings suggest that c. pneumoniae treatment alters the redox state and metabolism of lipids that may promote the dedifferentiation of smooth muscle cells in aortic tissue. the majority of gene sets that were coordinately upregulated in response to wd were those involved in defense and immune function; others included pathways related to macromolecular degradation and cell cycle regulation. gene sets that were negatively enriched in response to wd included tight junction regulation, receptor signaling, muscle proteins and channel activity. these findings suggest that dietary lipids and cholesterol are sensed by the innate and adaptive immune system in aortic tissue in a manner that promotes inflammation and loss of vascular barrier function.table gene set enrichment analysis 1 basal cell carcinoma 3 nucleachromosome part 5 regulation of gene expression epigenetic 6 kegg melanogenesis 8 dna replication 9 proteinaceous extracellular matrix 10 kegg hedgehog signaling pathway 12 reactome cell extracellular matrix interactions 14 reactome e2f mediated regulation of dna replication 15 reactome apoptotic execution phase 16 collagen 17 reactome inactivation of apc via direct inhibition of the ap complex 19 reactome smooth muscle contraction 20 reactome conversion from apc cdc to apc cd in late anaphase 21 reactome signaling by pdgf 22 kegg lysosome 23 microtubule cytoskeleton 24 cell division 25 reactome class b secretin family receptors positive enrichment: chronic p. gingivalis-treated group vs. chronic control group. the top p. gingivalis gene sets whose member genes are predominantly upregulated with respect to the chronic control group. column indicates the rank of the gene set based on normalized enrichment score. column lists the gene set name as provided by msigdb. gene set names and ranks that are not in bold are gene sets that are redundant or partially redundant and included within larger bolded gene sets. the nes ranged from to. fdr q range: -. gene set enrichment analysis 2 reactome integration of energy metabolism 4 kegg oxidative phosphorylation 5 kegg parkinsons disease 7 mitochondrial part 8 mitochondrian 9 kegg citrate cycle tca cycle 12 kegg alzheimers disease 13 organelle inner membrane 14 kegg huntingtons disease 15 reactome pyruvate metabolism and tca cycle 19 kegg cardiac muscle contraction 20 reactome diabetes pathways 21 reactome glucose metabolism 22 energy derivation by oxidation of organic compounds 24 regulation of heart contraction 25 kegg ppar signaling pathway negative enrichment: chronic p. gingivalis-treated group vs. chronic control group. the top p. gingivalis gene sets whose member genes are predominantly downregulated with respect to the chronic control group. column indicates the rank of the gene set based on normalized enrichment score. column lists the gene set name as provided by msigdb. gene set names and ranks that are not in bold are gene sets that are redundant or partially redundant and included within larger bolded gene sets. the nes ranged from - to -. fdr q <  × 10- gene set enrichment analysis 1 microbody 2 peroxisome 3 kegg peroxisome 4 kegg ppar signaling pathway 5 reactome metabolism of lipids and lipoproteins 8 kegg valine leucine and isoleucine degradation 7 9 kegg fatty acid metabolism 10 reactome metabolism of vitamins and cofactors 11 kegg pyruvate metabolism 13 kegg glycerolipid metabolism 15 lipid catabolic process 16 mitochondrion 18 reactome branched chain amino acid catabolism 19 cofactor metabolic process 21 kegg biosynthesis of unsaturated fatty acids 25 vitamin metabolic process positive enrichment: chronic c. pneumoniae-treated group vs. chronic control group. the top c. pneumoniae gene sets whose member genes are predominantly upregulated with respect to the chronic control group. column indicates the rank of the gene set based on normalized enrichment score. column lists the gene set name as provided by msigdb. gene set names and ranks that are not in bold are gene sets that are redundant or partially redundant and included within larger bolded gene sets. the nes ranged from to. fdr q range: 0-. gene set enrichment analysis 1 kegg hypertrophic cardiomyopathy 2 kegg dilated cardiomyopathy 3 reactome muscle contraction 5 muscle development 6 actin cytoskeleton 8 structural molecule activity 9 regulation of multicellular organismal process 10 kegg cardiac muscle contraction 11 cytoskeletal protein binding 13 kegg arrhythmogenic right ventricular cardiomyopathy arvc 14 contractile fiber 15 contractile fiber part 16 heart development 20 gated channel activity 22 cation channel activity 24 cytoskeletal part negative enrichment: chronic c. pneumoniae-treated group vs. chronic control group. the top c. pneumoniae gene sets whose member genes are predominantly downregulated with respect to the chronic control group. column indicates the rank of the gene set based on normalized enrichment score. column lists the gene set name as provided by msigdb. gene set names and ranks that are not in bold are gene sets that are redundant or partially redundant and included within larger bolded gene sets. the nes ranged from - to -. fdr q <  × 10- gene set enrichment analysis 1 kegg lysosome 2 reactome signaling in immune system 3 kegg natural killer cell mediated cytotoxicity 4 kegg systemic lupus erythematosus 6 kegg b cell receptor signaling pathway 7 leishmania infection 8 kegg toll like receptor signaling pathway 9 immune system process 11 reactome toll receptor cascades 13 reactome s phase 14 kegg fc gamma r mediated phagocytosis 16 reactome host interactions of hiv factors 17 reactome m g transition 18 kegg t cell receptor signaling pathway 19 defense response 20 kegg nod like receptor signaling pathway 21 kegg hematopoietic cell lineage 22 reactome g s transition 23 kegg chemokine signaling pathway 25 reactome cell cycle checkpoints positive enrichment: western diet group vs. chronic control group. the top western diet gene sets whose member genes are predominantly upregulated with respect to the chronic control group. column indicates the rank of the gene set based on normalized enrichment score. column lists the gene set name as provided by msigdb. gene set names and ranks that are not in bold are gene sets that are redundant or partially redundant and included within larger bolded gene sets. the nes ranged from to. fdr q <  × 10- gene set enrichment analysis 1 g protein coupled receptor activity 2 apical junction complex 3 apicolateral plasma membrane 4 neurotransmitter binding 9 kegg neuroactive ligand receptor interaction 11 reactome amine ligand binding receptors 12 intercellular junction 13 structural constituent of muscle 15 calcium channel activity 16 feeding behavior 18 voltage gated channel activity 19 kegg basal cell carcinoma 21 contractile fiber 22 anion transmembrane transporter activity 23 reactome tight junction interactions 25 digestion negative enrichment: western diet group vs. chronic control group. the top western diet gene sets whose member genes are predominantly downregulated with respect to the chronic control group. column indicates the rank of the gene set based on normalized enrichment score. column lists the gene set name as provided by msigdb. gene set names and ranks that are not in bold are gene sets that are redundant or partially redundant and included within larger bolded gene sets. the nes ranged from - to -. fdr q range: –. functional classification of clusters of genes differentially expressed with respect to chronic atherogenic stimuli the genes with the greatest significance by one-way anova across the four chronic treatment groups were assigned to several groups using hierarchical clustering. at the arbitrary cutoff of genes, the chronic time point anova fdr q value was <  and the p value was <  × 10- the david functional classification tool was used to extract biological meaning from each of the clusters. the three pro-atherogenic stimuli produced strikingly different patterns of differential gene expression. genes involved in immunity and inflammation were coordinately upregulated in response to wd, but were largely unchanged in response to either pathogen. by contrast, genes involved in lipid synthesis and ppar signaling were markedly increased in response to c. pneumoniae treatment but were unchanged in the other groups. finally, two smaller clusters suggest that the hedgehog pathway is suppressed in wd mice and that treatment with either pathogen decreased the expression of contractile proteins; however, these results were based on a small number of genes and should be interpreted with caution.figure chronic time point cluster analysis. the top differentially expressed genes at the chronic time point with distinct clusters. a. heat map shows relative expression among all groups. clusters are color-coded by row sidebars: red, chartreuse, mint green, blue, and magenta; and dendrogram is left of the color-coded sidebars. each row corresponds to a gene and each column to a sample. the colors are scaled by row; red and blue indicate standard deviations above or below the mean, respectively. at the arbitrary cutoff of genes, the chronic time point one-way anova fdr q value was <  and the p value was <  × 10- b. david analysis of clusters and gene enrichment is indicated by p values. c. box and whisker plots of the mean expression for clusters and reflect patterns seen on heat map. *** = p <  chronic treatment group vs. chronic control group by mann-whitney test. time-dependent changes in aortic gene expression in mice treated with p. gingivalisor c. pneumoniae to examine the acute response in aortic tissue following pathogen exposure, rna samples were obtained one day after the last treatment with p. gingivalis or c. pneumoniae. genes with nominally significant differential expression between the chronic and acute time points in untreated or p. gingivalis- or c. pneumoniae-treated apoe-/- mice are tabulated in figure  differential expression in all three groups was greater than expected by chance, and as in the comparison between groups at the chronic time point alone, p. gingivalis treatment produced the largest amount of differential gene expression, and the genes regulated by the two pathogens overlapped substantially. for the top differentially expressed genes observed at the acute to chronic time points, aortic tissue from apoe-/- mice fed a normal chow diet was characterized by a balance of genes with increased and decreased expression; p. gingivalis treatment was skewed towards decreased expression. because time-dependent differential expression was less robust in the c. pneumoniae-treated group, these changes may reflect inhibition of natural changes that occur with time in the aortic tissue from apoe-/- mice fed a normal chow diet.figure acute to chronic time point venn diagrams. venn diagrams depicting the number of genes with significantly increased or significantly decreased expression vs. the acute group for each chronic group. the sets of differentially expressed genes result from differential expression analyses for each acute group vs. its corresponding chronic group using the r environment for statistical computing with a cut off of p <  as described in methods. functional classification of genes with varying kinetics of response to p. gingivalisor c. pneumoniaetreatment using two-factor anova, we then analyzed the capacity of either pathogen to alter normal time-dependent changes in gene expression that occurred in apoe-/- mice. a clustering heat map of the genes with the strongest interaction effect between time and treatment is shown in figure  at the arbitrary cutoff of genes, the anova interaction fdr q value was <  and the p value was <  × 10- the expression of genes encoding myofibril, cytoskeletal, and ion binding/transport proteins greatly increased over time in untreated apoe-/- mice. however, treatment with either pathogen prevented or reversed this effect: the expression of these genes was increased and subsequently downregulated in the acute or chronic p. gingivalis-treated groups, respectively, and was unchanged in either c. pneumoniae treatment group. conversely, the expression of a number of zinc-finger transcription factors was downregulated over time in untreated mice, but was decreased and then subsequently upregulated in the acute or chronic c. pneumoniae-treated groups, respectively, and was unchanged in either p. gingivalis treatment group. a third noteworthy pattern was defined by a group of genes with functions in fatty acid metabolism and ppar signaling whose expression was moderately downregulated over time in untreated animals; this downregulation was greatly amplified in mice treated with p. gingivalis but was completely abrogated in mice treated with c. pneumoniae.figure acute to chronic time point cluster analysis. the top differentially expressed genes for acute and chronic time points: the effect of group, time, and group x time interactions determined by two-way anova are grouped into distinct clusters. a. heat map depicts relative expression among all groups and time points. clusters are color-coded by row sidebars: red, chartreuse, mint green, blue, and magenta; and dendrogram is left of the color-coded sidebars. each row corresponds to a gene and each column to a sample. the colors are scaled by row; red and blue indicate standard deviations above or below the mean, respectively. at the arbitrary cutoff of genes, the two-way anova interaction fdr q value was <  and the p value was <  × 10- b. david analysis of clusters and gene enrichment is indicated by p values. c. box and whisker plots of the mean expression for clusters and reflect patterns seen on heat map. ***p <  vs. acute control; ###p <  vs. chronic control; +++p <  vs. acute treatment; ++p <  vs. acute treatment by mann-whitney test. functional classification of clusters of genes differentially expressed with respect to acute atherogenic stimuli as before, the genes with the greatest significance by one-way anova across the three acute treatment groups were assigned to several groups using hierarchical clustering. acute treatment with p. gingivalis produced little differential gene expression, whereas acute treatment with c. pneumoniae resulted in large changes in gene expression. genes whose expression increased with c. pneumoniae treatment represented g-protein coupled signaling, viral myocarditis, antigen processing and presentation, and membrane genes. by contrast, nearly half of the genes whose expression decreased with c. pneumoniae treatment encoded phosphoproteins, with the remainder encoding proteins involved in alternative splicing, the endoplasmic reticulum, and glycoproteins and secreted proteins. the remaining clusters were not remarkable due to lack of annotation to a david pathway or of marginal significance. the three remaining clusters were not noteworthy. analysis of genes associated with unstable plaque a recent study identified genes whose expression in the aortic tissue of apoe-/- mice was associated with unstable plaque. we observed increased expression of several of these genes in aortic tissue from untreated apoe-/- mice fed a normal chow diet, which represents the natural progression of atherosclerosis; and, in apoe-/- wd mice, we observed an even greater increase in expression of some of these genes. p. gingivalis treatment blunted the increase in expression of some genes and induced the expression of others. treatment with c. pneumoniae prevented the increase in all of the unstable plaque genes with the exception of mmp which was increased over time in the c. pneumoniae-treated group but decreased over time in untreated apoe-/- mice.figure genes associated with unstable plaque. heat map showing expression of genes associated with unstable plaque identified in chen, et al.. discussion in this study, we applied microarray analysis to define gene signatures in aortic tissues obtained from mice fed a western diet or in mice treated with the oral pathogen p. gingivalis, or with the pulmonary pathogen c. pneumoniae. examination of gene expression profiles at the chronic time point enabled us to identify treatment-specific pro-atherogenic pathways rather than simply the genes expressed in established plaque. our results in untreated apoe-/- mice are consistent with a recent microarray study by papadodima et al. that documented changes in gene expression in aortic tissue of apoe-/- mice at three time points during the natural progression of atherosclerosis in the absence of additional pro-atherogenic stimuli. we identified common core sets of genes that were either increased or decreased in response to each of these pro-atherogenic stimuli. contemporary analysis tools including gsea and david analysis were then applied. gsea approach is more powerful than single gene analysis, which merely highlights the top up- or downregulated genes and may overlook effects on specific pathways. pathway analysis showed a markedly greater number of gene sets that were different among the three pro-atherogenic stimuli, suggesting that underlying vascular inflammation and dysfunction leading to atherosclerosis differ mechanistically and in the functional pathways leading to atherosclerosis progression depending upon stimulus. these findings are significant in that this is the first study that has performed direct, side-by-side, comparison of genome-wide aortic gene expression changes in three well-recognized models of atherosclerosis development. treatment with wd, p. gingivalis or c. pneumoniae has pro-atherogenic effects that include alterations in macrophage function, cholesterol homeostasis, and endothelial dysfunction. signaling through innate immune toll-like receptors expressed on immune cells and the endothelium may be a common link among the three stimuli; however, notable differences in tlr usage as well as features unique to each of the two pathogens may account for the differences in signaling pathways activated and repressed. tlr tlr and tlr rna and protein are expressed at high levels in endothelial cells and macrophages in human atherosclerotic plaque biopsies and the lipid-rich atherosclerotic lesions in the aortic root of mice. a role for tlrs in high-fat-diet-induced atherosclerosis in animal models has been shown in studies using apoe-/- tlr2-/- and apoe-/- tlr4-/- mice. interestingly specificity in tlr and tlr signaling by p. gingivalis and c. pneumoniae has been proposed to define specific inflammatory pathways unique to each organism. p. gingivalis expresses heterogeneous lps lipid a structures that weakly activate tlr but can also act as a tlr antagonist, resulting in alterations in signaling through tlr that promote low-grade, chronic inflammation at distant sites including the aorta. c. pneumoniae induces its proinflammatory signaling primarily through tlr but also expresses lps and signals through tlr. the unique feature of c. pneumoniae is that it gains entry into monocytes as elementary bodies, which are infectious but metabolically inert. after entering macrophages or monocytes, ebs can rapidly differentiate to a replicative form known as reticulate bodies, and start bacterial replication. rbs use the host’s metabolic metabolism and may find a favorable environment within a lipid-laden plaque to complete its replication. infected monocytes may circulate to distant sites to promote vascular inflammation. c. pneumoniae may increase adherence of macrophages to endothelial cells through expression of its virulence factor, hsp which has been shown to promote monocyte attachment to endothelium, and to promote extravasation to sub-endothelial layer, where it oxidizes ldl and promotes foam cell formation, a hallmark of atherosclerosis. most published studies involving animal models of atherosclerosis have used high dietary lipids and cholesterol, and these studies have formed the basis for consensus around the mechanisms underlying the development of atherosclerosis. as a result, the use of statin drugs, which target cholesterol and lipid handling, are the most widely used class of drugs in the field of cardiovascular medicine. a recent study in circulation showed that despite the improvement of risk with optimum statin therapy in patients with cardiovascular disease, many patients demonstrate atheroma progression and additional cardiovascular events, suggesting that additional mechanisms are at play, highlighting the need to identify novel therapeutic strategies that combat the additional cardiovascular risk. recent research implicates vascular inflammation and endothelial dysfunction as a factor involved in the initiation, progression, and instability of atherosclerotic plaques and elevations of serum inflammatory biomarkers consistently associated with the risk of experiencing a cardiovascular event, providing further evidence for systemic inflammation involvement in atherosclerotic cardiovascular disease. given the prevalence of diet-induced obesity and infection with p. gingivalis and c. pneumoniae in the general population and the likelihood of co-morbidity of obesity with chronic or recurring infection with these common pathogens, these findings suggest that the development of atherosclerosis in humans is likely more complex and multifactorial than previously appreciated. atherosclerotic plaques undergo both progressive and regressive changes, which affect their size and stability; regression in lesion area can increase plaque stability. plasma cholesterol lowering has been associated with regression, and a recent study identified ppar-gamma as a “master regulator” of regression in early lesions. our gsea analysis showed that c. pneumoniae treatment upregulated the ppar pathway while p. gingivalis treatment downregulated this pathway and thus may indicate a role for c. pneumoniae in regression and stability of early lesions, while p. gingivalis may inhibit plaque regression. p. gingivalis decreased pathways involved in mitochondrial function, suggesting that p. gingivalis promotes mitochondrial dysfunction, which is associated with cardiovascular risk, vascular dysfunction and plaque development. western diet group had significant reduction in tight junction pathways and expression of genes encoding claudins, proteins involved in maintenance of cell-cell junctions. this finding is consistent with a recent study that showed that western diet promotes atherosclerosis in part via loss of tight junction control. a surprising finding from the present study was that immune and inflammatory pathways were markedly upregulated in response to western diet. it has recently come to light that certain lipids in western style diets are either endogenous ligands of tlrs or assist with pattern recognition of tlrs. consistent with this, prior studies have shown that tlr tlr and myd knockout mice on an apoe-/- background had decreased atherosclerotic aortic plaque in response to a western diet. a recent microarray study by hyvarinen et al., compared gene expression in adipose tissue following chronic infection with c. pneumoniae and/or a periodontal pathogen, a. actinomycetemcomitans and revealed significant enrichment in inflammation associated pathways by aa or a combination of aa and c. pneumoniae but not by c. pneumoniae alone as compared to a control group. of interest was the increased expression of a number of genes associated with vulnerable plaque previously identified by chen et al. in aortic tissues obtained from mice at the chronic phase compared to tissues obtained at the acute phase. these genes were bmper, hdc, ifitm s100a upp adamts fpr clec4n, hmox and irg our findings demonstrate that expression of these genes increases as part of the natural progression of atherosclerosis in apoe-/- mice in the absence of an additional pro-atherogenic stimulus. importantly, the increased expression of these genes was blunted at the chronic time point in mice treated with c. pneumoniae. expression of kit was increased in the c. pneumoniae -treated group compared to the chronic control group. in addition, p. gingivalis treatment blunted the increase in expression of bmper, hdc, ifitm s100a upp and hmox expression of egln was decreased in the p. gingivalis-treated group compared to the chronic control group. expression of clec4n and hmox was increased in the western diet group compared to the chronic control group. the identification of differences in the effects of each of the pro-atherogenic stimuli on expression of important vulnerable plaque genes may result in differences in the nature and composition of the atherosclerotic plaque, which will be explored in future studies. limitations of the study several limitations of the study should be noted. we could not simultaneously perform a side-by-side quantification of the degree of the aortic plaque and the pathological features assessed by histology induced by the three atherogenic stimuli. we also could not determine the contribution of possible alterations in gut microbiota induced by each of the pro-atherogenic treatments or following antiobiotics pre-treatment in the p. gingivalis group. we could not validate our findings with regard to establishing that the pathways we identified are mechanistically linked to each pro-atherogenic stimulus at the protein level. we also acknowledge the limitations of using mouse models to draw conclusions about human diseases, which are more complex and multifactorial. future studies will also investigate the effects of comorbidity with or more pro-atherogenic stimuli, which resembles scenarios that occur clinically.  the present study suggests that p. gingivalis treatment may promote atherosclerosis by stimulating mitochondrial dysfunction and inhibiting the egress of endogenous lipids from the vessel wall, whereas c. pneumoniae may promote atherosclerosis by enhancing lipid uptake and metabolism into the vessel wall, and wd may promote atherosclerosis by activation of inflammatory and immune pathways that enhance leukocyte adhesion and endothelial dysfunction. 
for the top differentially expressed genes unique to each group, we observed a high percentage that exhibited decreased expression in p. gingivalis-treated mice in contrast to a high percentage that exhibited increased expression in wd mice. gene set enrichment analysis revealed distinct stimuli-associated phenotypes, including decreased expression of mitochondrion, glucose metabolism, and ppar pathways in response to p. gingivalis but increased expression of mitochondrion, lipid metabolism, carbohydrate and amino acid metabolism, and ppar pathways in response to c. pneumoniae; wd was associated with increased expression of immune and inflammatory pathways. our results suggest that pathogen exposure results in dysregulated cellular responses that may impact plaque progression and regression pathways. similar to endogenous mediators arising from excessive dietary lipids, these gram-negative pathogens are pro-atherogenic in animal models, although the specific inflammatory/atherogenic pathways induced by these stimuli are not well defined. in this study, we identified gene expression profiles that characterize p. gingivalis, c. pneumoniae, and western diet at acute and chronic time points in aortas of apolipoprotein e mice. at the chronic time point, we observed that p. gingivalis was associated with a high number of unique differentially expressed genes compared to c. pneumoniae or wd. david analysis of gene clusters identified by two-way anova at acute and chronic time points revealed a set of core genes that exhibited altered expression during the natural progression of atherosclerosis in apoe-/- mice; these changes were enhanced in p. gingivalis-treated mice but attenuated in c. pneumoniae-treated mice. porphyromonas gingivalis and chlamydia pneumoniae are associated with inflammatory atherosclerosis in humans. apoe-/- miceporphyromonas gingivalischlamydia pneumoniaewestern dietgene expression profilinggseaatherosclerosisvascular inflammationvulnerable plaquepparissue-copyright-statement© the author 2014 notable differences in the expression of genes associated with unstable plaques were also observed among the three pro-atherogenic stimuli. c. pneumoniae treatment resulted in approximately equal numbers of genes that exhibited increased and decreased expression. despite the common outcome of p. gingivalis, c. pneumoniae, and wd on the induction of vascular inflammation and atherosclerosis, distinct gene signatures and pathways unique to each pro-atherogenic stimulus were identified.
 atherosclerosis is a progressive disease characterized by inflammation and accumulation of lipids in vascular tissue. porphyromonas gingivalis and chlamydia pneumoniae are associated with inflammatory atherosclerosis in humans. similar to endogenous mediators arising from excessive dietary lipids, these gram-negative pathogens are pro-atherogenic in animal models, although the specific inflammatory/atherogenic pathways induced by these stimuli are not well defined. in this study, we identified gene expression profiles that characterize p. gingivalis, c. pneumoniae, and western diet at acute and chronic time points in aortas of apolipoprotein e mice.  at the chronic time point, we observed that p. gingivalis was associated with a high number of unique differentially expressed genes compared to c. pneumoniae or wd. for the top differentially expressed genes unique to each group, we observed a high percentage that exhibited decreased expression in p. gingivalis-treated mice in contrast to a high percentage that exhibited increased expression in wd mice. c. pneumoniae treatment resulted in approximately equal numbers of genes that exhibited increased and decreased expression. gene set enrichment analysis revealed distinct stimuli-associated phenotypes, including decreased expression of mitochondrion, glucose metabolism, and ppar pathways in response to p. gingivalis but increased expression of mitochondrion, lipid metabolism, carbohydrate and amino acid metabolism, and ppar pathways in response to c. pneumoniae; wd was associated with increased expression of immune and inflammatory pathways. david analysis of gene clusters identified by two-way anova at acute and chronic time points revealed a set of core genes that exhibited altered expression during the natural progression of atherosclerosis in apoe-/- mice; these changes were enhanced in p. gingivalis-treated mice but attenuated in c. pneumoniae-treated mice. notable differences in the expression of genes associated with unstable plaques were also observed among the three pro-atherogenic stimuli.  despite the common outcome of p. gingivalis, c. pneumoniae, and wd on the induction of vascular inflammation and atherosclerosis, distinct gene signatures and pathways unique to each pro-atherogenic stimulus were identified. our results suggest that pathogen exposure results in dysregulated cellular responses that may impact plaque progression and regression pathways. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords apoe-/- miceporphyromonas gingivalischlamydia pneumoniaewestern dietgene expression profilinggseaatherosclerosisvascular inflammationvulnerable plaquepparissue-copyright-statement© the author 2014 