Identification of Periopathogens in Atheromatous Plaques Obtained from Carotid and Coronary Arteries.

Increasing attention has been paid to the possible link between periodontal disease and atherosclerosis over the past decade. The aim of this study is to investigate the presence of five periopathogens: Porphyromonas gingivalis (P.g.), Aggregatibacter actinomycetemcomitans (A.a.), Tannerella forsythia (T.f.), Treponema denticola (T.d.), and Prevotella intermedia (P.i.) in atheromatous plaques obtained from the carotid and coronary arteries in patients who underwent coronary artery bypass graft surgery and carotid endarterectomy. Group I (carotid arteries) consisted of 30 patients (mean age: 54.5 ± 14.8), and group II (coronary arteries) consisted of 28 patients (mean age: 63 ± 12.1). Clinical periodontal examinations consisted of plaque index, gingival index, sulcus bleeding index, and periodontal probing depth and were performed on the day of vascular surgery. The presence of periopathogens in periodontal pockets and atherosclerotic vessels was detected using polymerase chain reaction. In both subgingival plaque and atherosclerotic plaque of carotid arteries, P.g., A.a., T.f., T.d., and P.i. were detected in 26.7%, 6.7%, 66.7%, 10.0%, and 20.0%, respectively, while for coronary arteries, P.g. was detected in 39.3%, A.a. in 25%, T.f. in 46.4%, T.d. in 7.1%, and P.i. in 35.7%. The presence of five periopathogens in carotid and coronary atherosclerotic vessels showed correlation in regard to the degree of periodontal inflammation. The present study suggests the relationship between periodontal pathogenic bacteria and atherogenesis. Further studies are necessary in relation to the prevention or treatment of periodontal disease that would result in reduced mortality and morbidity associated with atherosclerosis.

Coronary Vasculitis Induced in Mice by Cell Wall Mannoprotein Fractions of Clinically Isolated Candida Species.

Kawasaki disease (KD) is an inflammatory disease that was identified by Professor Tomisaku Kawasaki in 1961. Candida albicans-derived substances (CADS) such as the hot water extract of C. albicans and Candida water-soluble fractions (CAWS) induce coronary vasculitis similar to KD in mice. An increasing proportion of deep-seated candidiasis cases are caused by non-albicans Candida and are often resistant to antifungal drugs. We herein investigated whether the mannoprotein fractions (MN fractions) of clinically isolated Candida species induce vasculitis in mice. We prepared MN fractions from 26 strains of Candida species by conventional hot water extraction and compared vasculitis in DBA/2 mice. The results obtained revealed that the induction of vasculitis and resulting heart failure were significantly dependent on the species; namely, death rates on day 200 were as follows: Candida krusei (100%), Candida albicans (84%), Candida dubliniensis (47%), Candida parapsilosis (44%), Candida glabrata (32%), Candida guilliermondii (20%), and Candida tropicalis (20%). Even for C. albicans, some strains did not induce vasculitis. The present results suggest that MN-induced vasculitis is strongly dependent on the species and strains of Candida, and also that the MN fractions of some non-albicans Candida induce similar toxicity to those of C. albicans.

Proteus mirabilis Targets Atherosclerosis Plaques in Human Coronary Arteries via DC-SIGN (CD209).

Bacterial DNAs are constantly detected in atherosclerotic plaques (APs), suggesting that a combination of chronic infection and inflammation may have roles in AP formation. A series of studies suggested that certain Gram-negative bacteria were able to interact with dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin [DC-SIGN; cluster of differentiation (CD) 209] or langerin (CD207), thereby resulting in deposition of CD209s at infection sites. We wondered if Proteus mirabilis (a member of Proteobacteria family) could interact with APs through CD209/CD207. In this study, we first demonstrated that CD209/CD207 were also receptors for P. mirabilis that mediated adherence and phagocytosis by macrophages. P. mirabilis interacted with fresh and CD209s/CD207-expressing APs cut from human coronary arteries, rather than in healthy and smooth arteries. These interactions were inhibited by addition of a ligand-mimic oligosaccharide and the coverage of the ligand, as well as by anti-CD209 antibody. Finally, the hearts from an atherosclerotic mouse model contained higher numbers of P. mirabilis than that of control mice during infection-challenging. We therefore concluded that the P. mirabilis interacts with APs in human coronary arteries via CD209s/CD207. It may be possible to slow down the progress of atherosclerosis by blocking the interactions between CD209s/CD207 and certain atherosclerosis-involved bacteria with ligand-mimic oligosaccharides.

Repeated Porphyromonas gingivalis W83 exposure leads to release pro-inflammatory cytokynes and angiotensin II in coronary artery endothelial cells.

The role of Porphyromonas gingivalis (P. gingivalis) or its virulence factors, including lipopolysaccharide (LPS) not only has been related with periodontitis but also with endothelial dysfunction, a key mechanism involved in the genesis of atherosclerosis and hypertension that involving systemic inflammatory markers as angiotensin II (Ang II) and cytokines. This study compares the effect of repeated and unique exposures of P. gingivalis W83 LPS and live bacteria on the production and expression of inflammatory mediators and vasoconstrictor molecules with Ang II. Human coronary artery endothelial cells (HCAEC) were stimulated with purified LPS of P. gingivalis (1.0, 3.5 or 7.0 µg/mL) or serial dilutions of live bacteria (MOI 1: 100 - 1:0,1) at a single or repeated exposure for a time of 24 h. mRNA expression levels of AGTR1, AGTR2, IL-8, IL-1ß and MCP-1 were determined by RT-qPCR, and IL-6, MCP-1, IL-8, IL-1ß and GM-CSF levels were measured by flow cytometry, ELISA determined Ang II levels. Live bacteria in a single dose increased mRNA levels of AGTR1, and repeated doses increased mRNA levels of IL-8 and IL-1ß (p < 0.05). Repeated exposure of live-P. gingivalis induced significant production IL-6, MCP-1 and GM-CSF (p < 0.05). Moreover, these MCP-1, IL-6 and GM-CSF levels were greater than in cells treated with single exposure (p < 0.05), The expression of AGTR1 and production of Ang II induced by live-P. gingivalis W83 showed a vasomotor effect of whole bacteria in HCAEC more than LPS. In conclusion, the findings of this study suggest that repeated exposure of P. gingivalis in HCAEC induces the activation of proinflammatory and vasoconstrictor molecules that lead to endothelial dysfunction being a key mechanism of the onset and progression of arterial hypertension and atherosclerosis.

Bacterial footprints in aspirate of infarct-related artery in ST-elevation myocardial infarction patients underwent primary percutaneous coronary intervention.

BACKGROUND: Bacterial infections can trigger acute coronary syndromes. This study aimed to examine bacterial footprints in the aspirate of infarct-related artery. PATIENTS AND METHODS: We studied 140 patients with ST-elevation myocardial infarction who underwent a primary coronary intervention using thrombus aspiration catheters. The aspirate was sent for bacteriological and pathological examinations and immunoassay for pneumolysin toxin. RESULTS: Bacterial culture showed different bacteria in 14 samples. Leukocyte infiltrate was detected in all pathologically examined samples. Pneumolysin toxin was detected in only two samples. Patients with bacteria had similar baseline data as those without, except for the median age [46 (44-50) vs. 55 (47-62) years, P = 0.001, respectively], and white blood cells (WBCs) (16670 vs. 7550 cells/µl, P < 0.0001, respectively). In hospital-major clinical events (death, stroke, reinfarction, lethal arrhythmia, and heart failure) were not significantly different between the 2 groups with and without bacteria [4 (28.6%) vs. 20 (18.6%) events, respectively, odds ratio (OR) 1.8 (95% CL: 06-6.3), P = 0.5]. Patients with bacteria, heavy infiltration, and pneumolysin had insignificant higher events compared with those without [10/35 (28.6%) vs. 16/105 (15.2%) events, OR 2.2 (95% CL: 0.92-5.43), P = 0.13]. However, the difference was not significant. By multivariate analysis, bacteria, leukocyte infiltration, and pneumolysin were not predictors for in-hospital clinical events. Higher WBCs and younger age were significant predictors of bacterial footprints (P < 0.0001 and P = 0.04, respectively). CONCLUSION: Bacterial footprints existed in the aspirate of infarct-related artery of ST-elevation myocardial infarction patients. Predictors were higher WBCs and younger age. Bacterial markers were not predictors for in-hospital clinical events. The presence of bacterial footprints supports the infectious hypothesis of atherosclerosis.

An unusual case of mycobacterium tuberculous coronary arteritis and thrombosis resulting in acute myocardial infarction.

Tuberculosis (TB) is a prevalent infection worldwide and an endemic infection in Singapore. The most common presentation is that of pulmonary tuberculosis. Extra pulmonary tuberculosis usually involves the lymph nodes, pleura, central nervous system, or abdominal cavity. Involvement of the heart is rare (0.14-2% of TB cases), and when it is involved, it tends to be in the pericardium or myocardium. Here, a rare case of sudden death due to tuberculous coronary arteritis with tuberculous coronary thrombosis resulting in acute myocardial infarction is presented.

The changes of gut microbiota after acute myocardial infarction in rats.

Recent studies suggested that gut microbiota was involved in the development of coronary artery disease. However, the changes of gut microbiota following acute myocardial infarction (AMI) remain unknown. In this study, a total of 66 male Wistar rats were randomly divided into control, AMI and SHAM groups. The controls (n = 6) were sacrificed after anesthesia. The AMI model was built by ligation of left anterior descending coronary artery. The rats of AMI and SHAM groups were sacrificed at 12 h, 1 d, 3 d, 7 d and 14 d post-operation respectively. Gut microbiota was analyzed by 16S rDNA high throughput sequencing. The gut barrier injuries were evaluated through histopathology, transmission electron microscope and immunohistochemical staining. The richness of gut microbiota was significantly higher in AMI group than SHAM group at 7 d after AMI (P<0.05). Principal coordinate analysis with unweighted UniFrac distances revealed microbial differences between AMI and SHAM groups at 7 d. The gut barrier impairment was also the most significant at 7 d post-AMI. We further identified the differences of microorganisms between AMI and SHAM group at 7 d. The abundance of Synergistetes phylum, Spirochaetes phylum, Lachnospiraceae family, Syntrophomonadaceae family and Tissierella Soehngenia genus was higher in AMI group compared with SHAM group at 7 d post-operation (q<0.05). Our study showed the changes of gut microbiota at day 7 post AMI which was paralleled with intestinal barrier impairment. We also identified the microbial organisms that contribute most.

Evidence for polymicrobial communities in explanted vascular filters and atheroma debris.

RATIONALE: Microbial communities have been implicated in a variety of disease processes and have been intermittently observed in arterial disease; however, no comprehensive unbiased community analysis has been performed. We hypothesize that complex microbial communities may be involved in chronic vascular diseases as well and may be effectively characterized by molecular assays. OBJECTIVE: The main objective is to survey vascular debris, atheroma, and vascular filters for polymicrobial communities consisting of prokaryotic and eukaryotic microbes, specifically eukaryotic microbes. METHODS AND RESULTS: We examined vascular aspirates of atheromatous debris or embolic protection filters in addition to matched peripheral blood samples, from fifteen patients, as well as three cadaveric coronary arteries from two separate patients, for microbial communities. General fluorescence microscopy by Höechst and ethidium bromide DNA stains, prokaryotic and eukaryotic community analysis by Next Generation DNA Sequencing (NGS), and a eukaryotic microbial 9 probe multiplexed quantitative PCR were used to detect and characterize the presence of putative polymicrobial communities. No prokaryotes were detected in peripheral blood; however, in 4 of 9 sequenced filters and in 2 of 7 sequenced atheroma debris samples, prokaryotic populations were identified. By DNA sequencing, eukaryotic microbes were detected in 4 of 15 blood samples, 5 of the 9 sequenced filters, and 3 of the 7 atheroma debris samples. The quantitative multiplex PCR detected sequences consistent with eukaryotic microbes in all 9 analyzed filter samples as well as 5 of the 7 atheroma debris samples. Microscopy reveals putative polymicrobial communities within filters and atheroma debris. The main contributing prokaryotic species in atheroma debris suggest a diverse and novel composition. Additionally, Funneliformis mosseae, an arbuscular mycorrhizal fungus in the Glomeraceae family, was detected in the coronary hard plaque from two patients. Well studied biofilm forming bacteria were not detectable in circulating peripheral blood and were not universally present in atheroma or filters. Analyses of the sequenced eukaryotes are consistent with a diverse of array poorly studied environmental eukaryotes. In summary, out of 15 patients, 6 exhibited molecular evidence of prokaryotes and 14 had molecular evidence of eukaryotic and/or polymicrobial communities in vivo, while 2 post-mortem coronary plaque samples displayed evidence of fungi. CONCLUSION: Prokaryotes are not consistently observed in atheroma debris or filter samples; however, detection of protozoa and fungi in these samples suggests that they may play a role in arterial vascular disease or atheroma formation.

Heterologous expression of Streptococcus mutans Cnm in Lactococcus lactis promotes intracellular invasion, adhesion to human cardiac tissues and virulence.

In S. mutans, the expression of the surface glycoprotein Cnm mediates binding to extracellular matrix proteins, endothelial cell invasion and virulence in the Galleria mellonella invertebrate model. To further characterize Cnm as a virulence factor, the cnm gene from S. mutans strain OMZ175 was expressed in the non-pathogenic Lactococcus lactis NZ9800 using a nisin-inducible system. Despite the absence of the machinery necessary for Cnm glycosylation, Western blot and immunofluorescence microscopy analyses demonstrated that Cnm was effectively expressed and translocated to the cell wall of L. lactis. Similar to S. mutans, expression of Cnm in L. lactis enabled robust binding to collagen and laminin, invasion of human coronary artery endothelial cells and increased virulence in G. mellonella. Using an ex vivo human heart tissue colonization model, we showed that Cnm-positive strains of either S. mutans or L. lactis outcompete their Cnm-negative counterparts for tissue colonization. Finally, Cnm expression facilitated L. lactis adhesion and colonization in a rabbit model of infective endocarditis. Collectively, our results provide unequivocal evidence that binding to extracellular matrices mediated by Cnm is an important virulence attribute of S. mutans and confirm the usefulness of the L. lactis heterologous system for further characterization of bacterial virulence factors.

Streptococcus agalactiae infective endocarditis complicated by large vegetations at aortic valve cusps along with intracoronary extension: An autopsy case report.

Streptococcus agalactiae infective endocarditis is a rare condition with high mortality owing to complications of large vegetations and systemic emboli. A 49-year-old man was found dead in his house. He had a history of hepatic cirrhosis and had been diagnosed with type 2 diabetes 2years previously. He had presented with a high fever 10days before his death. An autopsy revealed 50mL of purulent pericardial effusion, and S. agalactiae was detected from the culture of this pericardial effusion. Two slender rope-like vegetations were present at the right aortic valve cusp and noncoronary aortic valve cusp. The vegetation at the right aortic valve cusp extended into the right coronary artery. The right coronary artery was broadly occluded by white rod-like material. The mitral valves were also affected, and the posterior papillary muscle was ruptured. Myocardial infarction was not observed. Systemic microscopic Gram-positive bacterial masses were observed in several organs. The death was attributed to acute myocardial ischemia caused by occlusive intracoronary extension of the vegetation at the proximal right coronary artery.

Candida glabrata binds to glycosylated and lectinic receptors on the coronary endothelial luminal membrane and inhibits flow sense and cardiac responses to agonists.

Candida glabrata (CG) is an opportunistic fungal pathogen that initiates infection by binding to host cells via specific lectin-like adhesin proteins. We have previously shown the importance of lectin-oligosaccharide binding in cardiac responses to flow and agonists. Because of the lectinic-oligosaccharide nature of CG binding, we tested the ability of CG to alter the agonist- and flow-induced changes in cardiac function in isolated perfused guinea pig hearts. Both transmission and scanning electron microscopy showed strong attachment of CG to the coronary endothelium, even after extensive washing. CG shifted the coronary flow vs. auricular-ventricular (AV) delay relationship upward, indicating that greater flow was required to achieve the same AV delay. This effect was completely reversed with mannose, partially reversed with galactose and N-acetylgalactosamine, but hyaluronan had no effect. Western blot analysis was used to determine binding of CG to isolated coronary endothelial luminal membrane (CELM) receptors, and the results indicate that flow-sensitive CELM receptors, ANG II type I, α-adrenergic 1A receptor, endothelin-2, and VCAM-1 bind to CG. In addition, CG inhibited agonist-induced effects of bradykinin, angiotensin, and phenylephrine on AV delay, coronary perfusion pressure, and left ventricular pressure. Mannose reversed the inhibitory effects of CG on the agonist responses. These results suggest that CG directly binds to flow-sensitive CELM receptors via lectinic-oligosaccharide interactions with mannose and disrupts the lectin-oligosaccharide binding necessary for flow-induced cardiac responses.

Presence of Periodontopathic Bacteria DNA in Atheromatous Plaques from Coronary and Carotid Arteries.

OBJECTIVES: Interest in periodontitis as a potential risk factor for atherosclerosis and its complications resulted from the fact that the global prevalence of periodontal diseases is significant and periodontitis may induce a chronic inflammatory response. Many studies have analyzed the potential impact of the Porphyromonas gingivalis, major pathogen of periodontitis, on general health. The purpose of this study was to find the presence of the Porphyromonas gingivalis DNA in the atherosclerotic plaques of coronary and carotid arteries and in the periodontal pockets in patients with chronic periodontitis, who underwent surgery because of vascular diseases. METHODS AND RESULTS: The study population consisted of 91 patients with coronary artery disease or scheduled for carotid endarterectomy. The presence of Porphyromonas gingivalis DNA in atheromatous plaques and in subgingival samples was determined by PCR. Bacterial DNA was found in 21 of 91 (23%) samples taken from vessels and in 47 of 63 (74.6%) samples from periodontal pockets. CONCLUSIONS: Porphyromonas gingivalis DNA is frequently found in atheromatous plaques of patients with periodontitis. That is why more research should be conducted to prove if this periopathogen may have an impact on endothelium of patients at risk of atherosclerosis.

Chlamydia Pneumoniae and Immunoinflammatory Reactions in an Unstable Atherosclerotic Plaque in Humans.

Comparative study of the walls of the aorta, coronary artery, and a. basilaris detected for the first time intra- and extracellular depositions of Chlamydia pneumoniae in unstable atherosclerotic plaques. No chlamydia were detected in the intima of normal sites of the vascular wall and just negligible levels thereof in stable atherosclerotic plaques. An unstable plaque with intra- and extracellular colonies was characterized by infiltration of the cap and intima adjacent to the atheromatous core with mononuclear cells, primarily T cells. These data suggested that Chlamydia pneumoniae could play an important role in the development of immunoinflammatory processes in the vascular wall and promote destabilization and progressive development of atherosclerotic plaques in humans.

Genomic factors related to tissue tropism in Chlamydia pneumoniae infection.

BACKGROUND: Chlamydia pneumoniae (Cpn) are obligate intracellular bacteria that cause acute infections of the upper and lower respiratory tract and have been implicated in chronic inflammatory diseases. Although of significant clinical relevance, complete genome sequences of only four clinical Cpn strains have been obtained. All of them were isolated from the respiratory tract and shared more than 99% sequence identity. Here we investigate genetic differences on the whole-genome level that are related to Cpn tissue tropism and pathogenicity. RESULTS: We have sequenced the genomes of 18 clinical isolates from different anatomical sites (e.g. lung, blood, coronary arteries) of diseased patients, and one animal isolate. In total 1,363 SNP loci and 184 InDels have been identified in the genomes of all clinical Cpn isolates. These are distributed throughout the whole chlamydial genome and enriched in highly variable regions. The genomes show clear evidence of recombination in at least one potential region but no phage insertions. The tyrP gene was always encoded as single copy in all vascular isolates. Phylogenetic reconstruction revealed distinct evolutionary lineages containing primarily non-respiratory Cpn isolates. In one of these, clinical isolates from coronary arteries and blood monocytes were closely grouped together. They could be distinguished from all other isolates by characteristic nsSNPs in genes involved in RB to EB transition, inclusion membrane formation, bacterial stress response and metabolism. CONCLUSIONS: This study substantially expands the genomic data of Cpn and elucidates its evolutionary history. The translation of the observed Cpn genetic differences into biological functions and the prediction of novel pathogen-oriented diagnostic strategies have to be further explored.

Vasculitis and anaphylactoid shock induced in mice by cell wall extract of the fungus Candida metapsilosis.

To investigate whether cell wall mannan from Candida metapsilosis induces vasculitis similar to that in Kawasaki syndrome and anaphylactoid shock in mice, we examined the pathogenic effects of C. metapsilosis cell wall extracts. Our results show that intraperitoneal injection of cell wall extracts induced severe coronary arteritis, and intravenous injection induced acute anaphylactoid shock similar to extracts from Candida albicans (C. albicans). Structural analysis of cell wall mannan from C. metapsilosis using NMR spectroscopy showed it to contain only a-mannan, indicating that a-mannan might be contributing to Candida pathogenicity by inducing coronary arteritis and acute shock.

Differential inflammasome activation by Porphyromonas gingivalis and cholesterol crystals in human macrophages and coronary artery endothelial cells.

OBJECTIVE: Observational evidence suggests association between periodontitis and atherosclerotic vascular disease (ASVD), however the cause-effect remains unclear. In this study, we investigated the mechanistic link of the two diseases by measuring production of interleukin (IL)-1ß, a potent inflammatory cytokine, induced via inflammasome activation by a key periodontal pathogen--Porphyromonas gingivalis LPS and cholesterol crystals (CC). METHODS: An in vitro model of primary human monocyte-derived macrophages (M1 and M2 macrophages) and coronary artery endothelial cells (HCAEC) was employed as a source of inflammasome product-IL-1ß. Both cell types are essential in initial inflammatory process of ASVD. As inflammasome activation requires 2 signals, P. gingivalis LPS was used as a signal1 and CC as a signal2. RESULTS: We found markedly release of IL-1ß from P. gingivalis LPS-primed M1 and M2 macrophages treated with CC. Unlike macrophages, HCAEC showed no release of IL-1ß in response to P. gingivalis LPS priming and subsequent treatment with either CC or extracellular danger molecule adenosine-5'-triphosphate (signal2). However, HCAEC, which were primed with pro-inflammatory cytokine TNF-α (signal1) and treated with adenosine-5'-triphosphate, consistently secreted minimal IL-1ß. The amount of IL-1ß released from activated HCAEC was much lower than that from M1 or M2 macrophages. CONCLUSIONS: P. gingivalis LPS and CC induced a differential activation of the inflammasome between human macrophages and HCAEC. The mechanistic role of periodontal infection in inflammasome activation as a cause of ASVD requires further investigation.

Perturbation of human coronary artery endothelial cell redox state and NADPH generation by methylglyoxal.

Diabetes is associated with elevated plasma glucose, increased reactive aldehyde formation, oxidative damage, and glycation/glycoxidation of biomolecules. Cellular detoxification of, or protection against, such modifications commonly requires NADPH-dependent reducing equivalents (e.g. GSH). We hypothesised that reactive aldehydes may modulate cellular redox status via the inhibition of NADPH-generating enzymes, resulting in decreased thiol and NADPH levels. Primary human coronary artery endothelial cells (HCAEC) were incubated with high glucose (25 mM, 24 h, 37°C), or methylglyoxal (MGO), glyoxal, or glycolaldehyde (100-500 µM, 1 h, 37°C), before quantification of intracellular thiols and NADPH-generating enzyme activities. Exposure to MGO, but not the other species examined, significantly (P<0.05) decreased total thiols (∼35%), further experiments with MGO showed significant losses of GSH (∼40%) and NADPH (∼10%); these changes did not result in an immediate loss of cell viability. Significantly decreased (∼10%) NADPH-producing enzyme activity was observed for HCAEC when glucose-6-phosphate or 2-deoxyglucose-6-phosphate were used as substrates. Cell lysate experiments showed significant MGO-dose dependent inhibition of glucose-6-phosphate-dependent enzymes and isocitrate dehydrogenase, but not malic enzyme. Analysis of intact cell or lysate proteins showed that arginine-derived hydroimidazolones were the predominant advanced glycation end-product (AGE) formed; lower levels of N(ε)-(carboxyethyl)lysine (CEL) and N(ε)-(carboxymethyl)lysine (CML) were also detected. These data support a novel mechanism by which MGO exposure results in changes in redox status in human coronary artery endothelial cells, via inhibition of NADPH-generating enzymes, with resultant changes in reduced protein thiol and GSH levels. These changes may contribute to the endothelial cell dysfunction observed in diabetes-associated atherosclerosis.