several types of studies have suggested that infectious agents may play a role in atherosclerosis and coronary heart disease ( chd ) .
atherosclerosis in chickens is exacerbated by infection with marek 's disease virus , a member of the herpes virus family 12 , and numerous studies have suggested an association of viral pathogens such as cmv and bacterial pathogens such as helicobacter and chlamydia with human atherosclerosis 345 .
most 678 though not all 910 studies have found that patients with chd are more likely to carry anti - chlamydia antibodies than healthy subjects , and several studies have demonstrated that chlamydia can be detected immunohistologically in > 70% of atherosclerotic lesions , whereas it is virtually undetectable in undiseased arteries 5678 . c. pneuomonia is a gram - negative bacteria that may produce persistent infection through intracellular growth in macrophages 11 , a cell type that plays a dominant and necessary role in atherosclerotic lesions 1213 .
these observations have led to the hypothesis that chlamydia may play a causal role in atherosclerosis and chd 1415161718 .
this hypothesis is made plausible by studies on the association of helicobacter pylori and duodenal ulcer 1920 .
this condition was originally thought to be caused by excess gastric acid and was treated by reducing or neutralizing acid .
it is now clear that ulcers are most commonly caused by infection with h. pylori , and the most effective treatment involves antibiotics . in the same way
, atherosclerosis is currently thought to be caused by excess plasma cholesterol and is treated by agents that reduce cholesterol levels .
if chlamydia or any other pathogen causes atherosclerosis , then a more effective treatment may be an antibiotic or vaccine . we have sought evidence for a causal role of infectious agents in atherosclerosis using the murine apolipoprotein ( apo ) e ( deficient ) model .
because of the deficiency of apo e , these animals have high levels of plasma cholesterol and develop atherosclerosis with a reproducible time course 212223 . in a first experiment
, we bred the apo e with a strain of mouse that is unable to respond to bacterial lps , but found no alteration in the rate or extent of atherogenesis . in a second study
we conclude that neither the response to infectious agents nor the presence of infectious agents themselves is absolutely necessary for atherogenesis .
mice with the lps gene crossed onto a c57bl background ( c57bl/10scn ) were the gift of dr .
these mice exhibited minimal increases in serum levels of tnf- and il-6 in response to intraperitoneal injection of 50 g of lps , whereas c57bl and apo e animals exhibited > 100-fold elevations in both ( not shown ) .
c57bl/10scn mice were crossed with apo e mice on a c57bl/6 background ( jackson laboratories ) .
apo e/lpsanimals were identified in the f2 progeny by the presence of plasma cholesterol levels > 500 mg / dl and the absence of plasma elevations of tnf- 90 min after injection of 50 g lps .
germ - free and control apo e mice were generated and maintained at taconic farms .
offspring were weaned at 4 wk of age onto a high - fat western - type diet containing 21.22% ( g/100 g ) fat , 17.01% protein , 48.48% carbohydrate , and 0.15% cholesterol ( td88137 ; harlan teklad ) and maintained on the diet for the remainder of the study . autoclaved food and water were provided ad libitum .
the institutional animal care and use committee of merck research laboratories approved the animal use for experimentation , and all animals were cared for in accordance with the guide for the care and use of laboratory animals ( 1996 , national research council ) . to measure aortic cholesterol and cholesteryl ester ,
mice were killed and gently perfused through the left ventricle with cold pbs with 5 mm edta .
all branches and any adipose tissue connected to the aorta were removed , and each aorta was carefully excised from the aortic root to the right renal artery .
the aortas were blotted dry , minced , and extracted with chloroform / methanol ( 2:1 ) according to the method used by folch et al . 24 .
total and free cholesterol in the aortic extracts were determined using an enzymatic fluorometric assay modified from previously described methods 2526 , and data are expressed on a per aorta basis .
plasma cholesterol and triglyceride levels were determined using standard enzymatic kits ( sigma chemical co. ) .
for histology , mouse hearts were perfused in situ with pbs and removed with 1 mm of proximal aorta attached .
the top half of the heart was embedded in o.c.t . embedding medium ( fisher scientific ) and prepared for cryosectioning .
6-m sections were collected of the aortic root area , defined as having aortic valve leaflets present , and mounted on 10-well masked slides .
additional sections were immunolabeled with mab l3t4 for cd4 or m1/70 for cd11b ( pharmingen ) using the horseradish peroxidase method with 3 - 3-diamino benzadine as substrate .
the time course of atherosclerogenesis in apo e mice was followed by measuring aortic cholesteryl ester ( fig .
1 ) , a parameter that tracks with histological progression of atherosclerosis ( 23 ; data not shown ) . to determine a role for gram - negative bacteria such as chlamydia , we first used lps mice 27 , animals with a congenital deficiency in responses to the principal inflammatory component of these bacteria , lps ( endotoxin )
. lps is known to stimulate expression of cytokines ( tnf , monocyte chemotactic protein 1 [ mcp-1 ] , and others ) , adhesion molecules ( vascular cell adhesion molecule 1 [ vcam-1 ] , intercellular adhesion molecule 1 [ icam-1 ] , 2 integrins ) , and enzymes ( inducible nitric oxide synthetase [ inos ] , matrix metalloproteinase 9 [ mmp9 ] ) observed in atherosclerotic lesions 28 and is a prime candidate to mediate possible proatherogenic effects of chlamydia .
lps mice fail to respond to lps because of a deficiency in toll - like receptor 4 ( tlr-4 [ 29 , 30 ] ) , a transmembrane protein with homology to the il-1 receptor .
the inability of these animals to respond to lps was verified by measurements of plasma tnf in response to lps challenge , and their deficiency in apo e was verified by measurements of plasma cholesterol ( described in materials and methods ) .
we observed that development of atherosclerotic lesions in mice fed a high - fat western - type diet was not altered by the deletion of tlr-4 ( fig .
further studies showed that plasma cholesterol was also not affected by the absence of tlr-4 ( data not shown ) .
this result suggests that responses to gram - negative bacterial products are not critical for murine atherogenesis .
to more thoroughly ascertain the role of infectious agents in atherogenesis , we generated a germ - free colony of apo e mice .
founder germ - free apo e pups were delivered by caesarian section and reared by germ - free foster mothers .
the colony resulting from these founders was maintained germ free : weekly tests showed that no bacteria could be grown from feces , bedding , or swabs of the isolator .
in addition , animals chosen at random showed no antibodies against any of several viral pathogens .
the absence of intestinal bacteria is known to cause enlargement of the caecum 31 , and dramatically enlarged caeca were observed in all of the animals from the colony .
the progress of hyperlipidemia and atherogenesis in germ - free animals was compared with that of a second set of animals taken from the germ - free colony and reared with ambient pathogens .
as expected , control apo e animals exhibited extremely high levels of plasma cholesterol and moderately high triglyceride at both 22 and 32 wk of age ( fig .
germ - free animals showed an essentially identical plasma lipid profile , and lipid changes are thus unlikely to obscure effects of gnotobiosis on atherogenesis . in both male and female animals
, we observed that the exclusion of infectious agents caused no consistent differences in the rate or extent of free cholesterol or cholesteryl ester accumulation in the aorta ( fig . 2c and fig .
d ) . we did observe slightly reduced aortic cholesterol ester in the germ - free animals that attained statistical significance at 22 wk of age . however , these small differences disappeared upon correction for reduced body weight observed in the germ - free animals at the 22-wk time point ( 28  0.6 g for germ - free males vs. 34  0.8 g for control males , and 22  .02 g for germ - free females vs. 24  0.6 g for controls ; p = 0.001 ) .
the values for aortic cholesteryl ester in germ - free mice were also similar to those seen in a control colony that had never been made germ free ( values in fig .
the similarity of atherogenesis in control and germ - free animals was confirmed by histologic examination of the aortic root in animals taken at the 22-wk time point .
well - developed lesions with necrotic cores , large foam cells , and fibrous caps were observed in both control and germ - free specimens ( fig .
b ) , and neither quantitative nor qualitative differences could be detected in observations of either male or female lesions . a difference in aortic size reflecting the smaller body size of the germ - free animals 31 was readily detected histologically .
t cells are found in human and murine atherosclerotic lesions 1223 , suggesting activity of the adaptive immune system .
we identified t cells by immunohistochemistry in both control and germ - free animals ( fig . 3c and fig .
d ) , indicating that pathogens are not required to drive the influx of t cells into atherosclerotic lesions .
the observations presented here indicate that infectious agents , whether bacterial , viral , or fungal , are not necessary for murine atherogenesis .
infectious agents do not appear necessary either for initiation or progression of atherosclerosis , nor does the presence of infectious agents alter the morphology or cellular composition of the atherosclerotic lesions .
finally , the presence of infectious agents does not appear to alter the pattern of distribution of lesions along the aorta ( not shown ) .
rather , it appears that the high - circulating cholesterol levels in the apo e animals are sufficient to initiate and drive atherogenesis .
we conclude that koch 's postulates can not be fulfilled for any infectious agent in murine atherosclerosis .
atherosclerosis in apo e mice closely resembles that in humans with respect to histology , progression , and dependence on circulating cholesterol 23 , and we may thus speculate that infectious agents may not be necessary for the development of human atherosclerosis .
we wish to emphasize that the observations reported here relate to atherogenesis , not to plaque rupture , thrombus formation , or myocardial infarction .
it is possible that infectious agents play an important role in one or more of these acute processes .
several current studies are seeking to define a role for bacterial infection in human chd by studying endpoints such as myocardial infarction in patients randomized to placebo or antibiotic therapy of varying duration .
the largest of these studies to report results thus far has failed to demonstrate a decline in cardiovascular events in the 6 mo after treatment with antibiotic 32 .
although our results suggest that pathogens do not serve as etiologic agents for atherosclerosis , they may still have a role in exacerbating the disease .
recent reports suggest that inoculation of atherosclerosis - prone mice with high doses of chlamydia may cause an approximately twofold increase in the size of atherosclerotic lesions 3334 .
it is important to note that stresses such as infection provoke a set of physiological responses that are uniformly proatherogenic 353637 .
these include insulin resistance and hyperglycemia , elevation of plasma triglycerides , elevated white blood cell counts , elevated acute phase reactants ( e.g. , c - reactive protein and fibrinogen ) , and depression of high density lipoprotein ( hdl ) cholesterol levels .
it is thus possible that infection may exacerbate atherosclerogenesis , and antibiotics may ameliorate atherogenesis not through the action of an etiologic agent , but through indirect effects on metabolism and known risk factors .
 recent work has revealed correlations between bacterial or viral infections and atherosclerotic disease . 
 one particular bacterium , chlamydia pneumoniae , has been observed at high frequency in human atherosclerotic lesions , prompting the hypothesis that infectious agents may be necessary for the initiation or progression of atherosclerosis . to determine if responses to gram - negative bacteria are necessary for atherogenesis , we first bred atherosclerosis - prone apolipoprotein ( apo ) e/ ( deficient ) mice with animals incapable of responding to bacterial lipopolysaccharide . 
 atherogenesis was unaffected in doubly deficient animals . 
 we further tested the role of infectious agents by creating a colony of germ - free apo e/ mice . 
 these animals are free of all microbial agents ( bacterial , viral , and fungal ) . 
 atherosclerosis in germ - free animals was not measurably different from that in animals raised with ambient levels of microbial challenge . 
 these studies show that infection is not necessary for murine atherosclerosis and that , unlike peptic ulcer , koch 's postulates can not be fulfilled for any infectious agent in atherosclerosis . 