A ROLE FOR ARCHAEAL ORGANISMS IN DEVELOPMENT OF ATHEROSCLEROTIC VULNERABLE PLAQUES AND MYXOID MATRICES

PURPOSE: Vulnerable plaques are characterized by a myxoid matrix, necrotic lipidic core, reactive oxygen species, and high levels of microorganisms. Aerobic microbes such as Chlamydophila pneumoniae and Mycoplasma pneumoniae usually do not survive in oxidative stress media. Archaea are anaerobic microbes with powerful anti-oxidative enzymes that allow detoxification of free radicals whose presence might favor the survival of aerobic microorganisms. We searched for archaeal organisms in vulnerable plaques, and possible associations with myxoid matrix, chlamydia, and mycoplasma bodies. METHODS: Twenty-nine tissue samples from 13 coronary artherectomies from large excentric ostial or bifurcational lesions were studied using optical and electron microscopy. Infectious agents compatible with archaea, chlamydia, and mycoplasma were semiquantified using electron micrographs and correlated with the amounts of fibromuscular tissue, myxoid matrix, and foam cells, as determined from semi-thin sections. Six of the cases were also submitted to polymerase chain reaction with archaeal primers. RESULTS: All 13 specimens showed archaeal-compatible structures and chlamydial and mycoplasmal bodies in at least 1 sample. There was a positive correlation between extent of the of myxoid matrix and archaeal bodies (r = 0.44, P = 0.02); between archaeal and mycoplasmal bodies (r = 0.41, P = 0.03), and between chlamydial bodies and foam cells (r = 0.42; P = 0.03). The PCR test was positive for archaeal DNA in 4 of the 6 fragments. DISCUSSION: DNA and forms suggestive of archaea are present in vulnerable plaques and may have a fundamental role in the proliferation of mycoplasma and chlamydia. This seems to be the first description of apparently pathogenic archaea in human internal organ lesions.


INTRODUCTION
Vulnerable plaques are rich in foam cells, 1 necrotic lipid tissue, oxidized LDL (oxLDL), 2 and inflammation. 3,4he presence of oxLDL has been related to the inflammation at the subendothelial space 5 where microaggregated LDL is internalized by macrophages and smooth muscle cells forming the foam cells. 6,7Native LDL is considered nonatherogenic, but the presence of metals and reactive oxygen species (ROS) modifies the LDL resulting in oxLDL, which is quickly internalized by macrophages 8,9 that become chemoattractive for monocytes and T lymphocytes, which stimulates smooth cell proliferation and induces production of autoantibodies and immune complexes that facilitate LDL internalization by macrophages.The recruitment of inflammatory cells may result in continued oxidation of LDL and maintenance of the atherosclerotic lesion process. 10nfectious agents have been frequently detected in coronary atheromas, mainly Chlamydophila pneumoniae (CP) [11][12][13] with controversial interpretations; [14][15][16] Helicobacter pylori, Escherichia coli, and cytomegalovirus have been occasionally associated with cardiovascular events. 17Co-infections have also been described, for example, with Mycoplasma pneumoniae (MP) and CP, which have been detected in coronary vulnerable plaques 18,19 and in calcified foci of stenotic aortic valves in elderly patients. 20,21Multiple co-infections by mycoplasma, chlamydia, and viruses have been described in patients with chronic diseases, 22 and a higher prevalence of seropositivity for MP was observed in coronary artery disease of patients seropositive for CP. 23Therefore, the question arises as to whether infectious agents might be related to the development of ROS and oxLDL.Chlamydophila pneumoniae has already been related to the LDL oxidation process. 24Although CP and MP have been found in large amounts in vulnerable atherosclerotic plaques, they have also been detected in lower quantities in nonatherosclerotic vessels, suggesting that a co-factor could be determining their proliferation. 25t is intriguing to ponder that since vulnerable plaque is a rich ROS media, how is it that chlamydia, mycoplasma, and other microorganisms may exhibit greater proliferation at those sites?Relatively low steady-state levels of intracellular superoxide or H 2 O 2 causes oxidative stress-defined as an imbalance between oxidants and antioxidants in favor of the oxidants, potentially leading to damage 26 with reduction in the growth of aerobic or anaerobic bacteria. 27uperoxide dismutases (SOD) play an essential role in the defense against oxygen toxicity and have been found in the cytosol of anaerobic microorganisms, including archaeal representatives. 28Archaea occupy the deepest and shortest branches of the universal phylogenetic tree. 29One of their most particular characteristics is the ability to oxidize hydrogen and reduce metals such as Fe and Mn. 30,31he presence of SOD in archaea is a significant finding, as they exhibit unique properties of stability to extreme conditions.
In the present work, we explored whether archaeal organisms may be participating in the pathogenesis of atherosclerotic plaque vulnerability and proliferation of other infectious agents such as mycoplasma and chlamydia.

METHODS
This work was performed in patients , most of them with symptoms of unstable angina, undergoing interventional pre-stenting directional coronary atherectomy in ostial or bifurcational excentric atherosclerotic plaques at the Heart Institute (InCor), Hospital das Clínicas, São Paulo, University Medical School, after approval by the Ethical and Scientific Committee.Two or 3 fragments were obtained from each of 13 patients (10 men and 3 women, mean age, 64 +/-12 years and 69 +/-12 years, respectively, with a total of 29 fragments being studied.The specimens were fixed in 2% glutaraldehyde, processed to be embedded in araldite, and submitted to semi-thin and thin sectioning, which were analyzed by optical and transmission electron microscopy, respectively.In 6 of the 13 cases, samples were frozen in -70 o C liquid nitrogen for polymerase chain reaction (PCR) studies.

Tissue preparation for optical and transmission electron microscopy
Tissue fragments less than 0.1 cm thickness were fixed in 2% glutaraldehyde for 2 hours, postfixed in 1% osmium tetroxide, dehydrated, and embedded in araldite resin.Semi-thin sections (0.5 um.thickness) were stained with toluidine blue, and ultrathin sections (70 nm thickness) were stained with uranyl acetate and lead citrate.

Statistical analysis
The Spearman correlation test was used to test the number of infectious agent bodies from electron microscopy versus the histological aspects of plaque vulnerability.The Student t test was used to identify a possible association between archaeal elements and myxoid matrix and other infectious agents, ie, mycoplasmal and chlamydial bodies.

RESULTS
Electron microscopy revealed numerous elements compatible with archaea, i.e., rounded elements showing dimensional variation, surrounded by an irregular folded monolipid membrane, which frequently presented invaginations forming large rounded and flattened vesicles into a homogeneous clear cytoplasmic material.The morphology of these elements varied according to the section axis.(Figure 1).
The archaeal elements were frequently seen in close contact with chlamydial elementary bodies, which have a typical form, i.e., round dark structures enclosed by a double membrane with an small expansion that gives the characteristic pear shape to C. pneumoniae; alternatively, they were frequently seen with mycoplasmal forms characterized by rounded elements with granulous cytoplasmic material enclosed by a unique membrane (Figure 2A).
The PCR test for archaeal DNA was positive in 4 of the 6 cases.Figure 2B shows the PCR results, the first sample being the positive control.Our samples showed a constant positive reaction at 400 pb, using the same 1.110F and 1.400R generic archaeal primers that show positivity in the control at 300 pb.We divided the analyzed fragments into 2 groups, MM-negative samples and MM-positive samples.The semiquantitative analysis showed a significantly higher amount of archaeal-suggestive bodies and lipidic necrotic core in the MM-positive group than in the MMnegative group, with no difference regarding mycoplasmal and chlamydial bodies (Table 1).Considering all fragments, positive correlations were observed between extent of MM regions and the amount of archaeal-suggestive bodies (r = 0.44, P = 0.02); archaeal and mycoplasmal bodies (r = 0.41, P = 0.03), and chlamydial bodies and foam cells (r = 0.42; P = 0.03).

DISCUSSION
Atherosclerosis has different characteristics among individuals and among areas in a same individual and is related to oxidative LDL. 32How infectious agents may participate in the pathogenesis of different atherosclerotic vulnerability is our focus in this work.Chlamydophila pneumoniae has been related to the LDL oxidation process, 23 and previously we have identified LDL particles in  the serum of atherosclerotic elderly patients with antigens of MP in association with C-reactive protein. 33Archaea is a highly diverse and abundant group of prokaryotes, including a number of extremophiles that thrive in hostile environments such as hot springs, salt lakes, submarine volcanic habitats, in addition to more physiological sites. 346][37] They have been described in luminal intestinal microbial ecosystems producing methane, having CO 2 as the main carbon substrate. 38Genomes of archaea have been described in persistent pathogens and it is suggested that they may confer metabolic capabilities as adaptational strategies for survival even in hostile host niches by breaching host barriers that exclude other organisms. 39Recently, genetic sequences of members of domain archaea were found in necrotic material from apical periodontitis, suggesting that archaea may play a role as a human pathogen. 40In the present study we detected archaea sugestive microorganisms in human atherosclerotic lesions.
We analyzed atheroma-vulnerable tissues obtained from interventional atherotomy procedures and identified 16S rRNA archaeal genomic sequences by PCR assay in 4 of the 6 cases analyzed.Usually, archaeal sequences exhibit 300 pb in PCR technique, as indicated by our positive control of a cultive from H. sulpholobus.However, our cases showed a constant positivity at 400 pb, with the same sequences of archaeal primers, suggesting a different size of archaeal DNA.
Archaeal morphology has not been very well described, and the rare ultrastructural descriptions were of archaea from artificial cultures. 41In our cases, transmission electron microscopy exhibited numerous structures compatible with archaea in 100% of the cases.Archaea have a unique phospholipid membrane that can be structurally modified, allowing adaptation to high temperatures and high salinity; it is quite differently composed compared to a bacterial membrane. 42So it is reasonable to suppose that changes in the morphology, lipid composition, and fluidity of archaeal membranes occur according to the environmental needs. 43The archaeal-compatible bodies present at the vulnerable plaques were characterized by roundness and contained large clear intracytoplasmic vacuoles that might have contained gases.Although the membrane-related processes in archaea are poorly understood, inverted vesicle membranes have been described. 44This is in agreement with our observations, since the intracytoplasmic vacuoles seemed to be derived from invagination of the external enveloping membrane.Close associations between archaea and chlamydial or mycoplasmal bodies were frequently found.Myxoid matrix (MM) is a collagen alteration usually present in vulnerable plaques and a fundamental lesion of many other diseases with still unknown etiologies, such as prolapse valvar, aortic dissection, and arterial aneurysms.A significant association of archaeal-like elements containing large clear intracytoplasmic vacuoles with MM regions suggests a cause-effect relationship.We speculate that archaea may favor the survival and proliferation of chlamydia and mycoplasma at the vulnerable atherosclerotic plaques since archaea possess superoxide dismutases that would protect them from intra-and extracellular oxidative stress. 28In addition to the significant correlation between archaeal organisms and quantity of MM regions in the plaque, archaea correlated with the number of mycoplasmal bodies.Metalloproteinases have been found in association with mycoplasmal infection, 45 are present in archaea 46 and they are increased in valvar and aortic MM lesions. 47oam cells, another characteristic of vulnerable plaque, correlated with numbers of chlamydial bodies.These findings favor our hypothesis that archaea may be an important cofactor for the pathogenesis of plaque vulnerability, inducing proliferation of other microorganisms, which may favor the development of myxoid matrix lesions.

CONCLUSION
This pioneering description of archaeal elements in atherosclerotic lesions indicates that archaea may be a pathogenic organism involved in the pathogenesis of plaque vulnerability by favoring development of ROS and survival of aerobic microorganisms such as mycoplasma and chlamydia.This finding opens new directions for the comprehension of infection in the pathogenesis of atherosclerosis and myxoid matrix degeneration.
CLINICS 2006;61(5):473-8 A role for archaeal organisms in development of atherosclerotic Higuchi ML et al. trol, we used a culture of Halobacterium salinarum (positive reaction at 300 pb), kindly provided by Prof. Maysa Beatriz Mandetta Clementino, from the Bacteria and Archaea Reference Section of Microbiology Department, National Institute of Quality Control in Health, Oswaldo Cruz Foundation -FIOCRUZ, Rio de Janeiro.

Figure 1 -
Figure 1 -Electron micrograph exhibiting rounded and flattened bodies suggestive of archaeal origin sectioned in different positions: 1A) 2 elements transversally sectioned enveloped by a monolayer lipid membrane that invaginates (arrows) forming large, clear intracytoplasmic vacuoles (empty arrows).1B) Close view of an archaeal-suggestive element sectioned at the level of folded external membrane entrances (arrows) 1C) Close view of an archaeal-suggestive element sectioned on the sagittal axis, revealing a flattened and folded aspect (arrow).10000x original magnification

Figure 2 -
Figure 2 -A) Electron micrograph of a vulnerable plaque exhibiting a Chlamydophila pneumoniae elementary body in close contact with an archaeal-suggestive element, which contains intracytoplasmic vacuoles.A mycoplasmal element is also present.2B) PCR test for archaeal DNA was positive in 4 of the 6 cases (arrow points to the 400 bp product; left channel is the positive control).