Although mortality rates from coronary heart disease in the western countries have declined in the last few decades, morbidity caused by this disease is increasing and a substantial number of ...patients still suffer acute coronary syndrome (ACS) and sudden cardiac death. Acute coronary syndrome occurs as a result of myocardial ischaemia and its manifestations include acute myocardial infarction and unstable angina. Culprit plaque morphology in these patients varies from thrombosis with or without coronary occlusion to sudden narrowing of the lumen from intraplaque haemorrhage. The coronary artery plaque morphologies primarily responsible for thrombosis are plaque rupture, and plaque erosion, with plaque rupture being the most common cause of acute myocardial infarction, especially in men. Autopsy data demonstrate that women <50 years of age more frequently have erosion, whereas in older women, the frequency of rupture increases with each decade. Ruptured plaques are associated with positive (expansive) remodelling and characterized by a large necrotic core and a thin fibrous cap that is disrupted and infiltrated by foamy macrophages. Plaque erosion lesions are often negatively remodelled with the plaque itself being rich in smooth muscle cells and proteoglycans with minimal to absence of inflammation. Plaque haemorrhage may expand the plaque rapidly, leading to the development of unstable angina. Plaque haemorrhage may occur from plaque rupture (fissure) or from neovascularization (angiogenesis). Atherosclerosis is now recognized as an inflammatory disease with macrophages and T-lymphocytes playing a dominant role. Recently at least two subtypes of macrophages have been identified. M1 is a pro-inflammatory macrophage while M2 seems to play a role in dampening inflammation and promoting tissue repair. A third type of macrophage, termed by us as haemoglobin associated macrophage or M(Hb) which is observed at site of haemorrhage also can be demonstrated in human atherosclerosis. In order to further our understanding of the specific biological events which trigger plaque instability and as well as to monitor the effects of novel anti-atherosclerotic therapies newer imaging modalities in vivo are needed.
Objectives The goal of this study was to identify histomorphologic characteristics of atherosclerotic plaques and to determine the amenability of some of these components to be used as markers for ...invasive and noninvasive imaging. Background Rupture of the atherosclerotic plaques is responsible for the majority of acute coronary events, and the culprit lesions demonstrate distinct histopathologic features. It has been tacitly believed that plaque rupture (PR) is associated with angiographically minimally occlusive lesions. Methods We obtained 295 coronary atherosclerotic plaques, including stable (fibroatheroma FA; n = 105), vulnerable (thin-cap fibroatheroma TCFA; n = 88), and disrupted plaques (plaque rupture PR; n = 102) from the hearts of 181 men and 32 women who had died suddenly. The hierarchical importance of fibrous cap thickness, percent luminal stenosis, macrophage area, necrotic core area, and calcified plaque area was evaluated by using recursive partitioning analysis. Because clinical assessment of fibrous cap thickness is not possible by noninvasive imaging, it was excluded from the second set of partitioning analysis. Results Thickness of the fibrous cap emerged as the best discriminator of plaque type; the cap thickness measured <55 μm in ruptured plaques, and all FA were associated with >84-μm cap thickness. Although the majority of TCFA were found in the 54- to 84-μm thickness group, those with <54-μm thickness were more likely to show <74% luminal stenosis (area under the curve: FA, 1.0; TCFA, 0.89; PR, 0.90). After exclusion of cap thickness, analysis of the plaque characteristics revealed macrophage infiltration and necrotic core to be the 2 best discriminators of plaque types (area under the curve: FA, 0.82; TCFA, 0.58; PR, 0.72). More than 75% cross-section area stenosis was seen in 70% of PR and 40% of TCFA; only 5% PR and 10% TCFA were <50% narrowed. Conclusions This postmortem study defines histomorphologic characteristics of vulnerable plaques, which may help develop imaging strategies for identification of such plaques in patients at a high risk of sustaining acute coronary events.
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N6-Methyladenosine (m6A) modification is the most prevalent RNA modification in mammals. We have recently demonstrated that inhibition of m6A modification by 3-deazaadenosine results ...in an increase in the expression of the cytochrome P450 (CYP) isoforms CYP1A2, CYP2B6, and CYP2C8 in human liver-derived cells. In the present study, we aimed to clarify the mechanism of m6A-mediated regulation of CYP2B6 expression. RNA immunoprecipitation using an anti-m6A antibody revealed that CYP2B6 mRNA in human liver and hepatocarcinoma-derived HepaRG cells was m6A-modified around the stop codon. In contrast to the treatment with 3-deazaadenosine, double knockdown of methyltransferase like (METTL) 3 and METTL14 (METTL3/14) resulted in a decrease in the levels of CYP2B6 mRNA in Huh-7 and HepaRG cells and a decrease in bupropion hydroxylase activity, a marker activity of CYP2B6, in HepaRG cells. The stability of CYP2B6 mRNA was not influenced by siMETTL3/14. Reporter assays using the plasmids containing the last exon or 5′-flanking region of CYP2B6 indicated that reporter activities were not influenced by knockdown of METTL3/14. The expression levels of the constitutive androstane receptor, pregnane X receptor, and retinoid X receptor, which are the nuclear receptors regulating the transcription of CYP2B6, were not influenced by siMETTL3/14. The chromatin immunoprecipitation and formaldehyde-assisted enrichment of regulatory elements assays revealed that H3K9me2, a repressive histone marker, was enriched in the vicinity of the upstream region of CYP2B6, and knockdown of METTL3/14 induced the condensation of the chromatin structure in this region. In conclusion, we demonstrated that METTL3/14 upregulated CYP2B6 expression by altering the chromatin status.
Microorganisms that activate plant immune responses have attracted considerable attention as potential biocontrol agents in agriculture because they could reduce agrochemical use. However, ...conventional methods to screen for such microorganisms using whole plants and pathogens are generally laborious and time consuming. Here, we describe a general strategy using cultured plant cells to identify microorganisms that activate plant defense responses based on plant-microbe interactions. Microbial cells were incubated with tobacco BY-2 cells, followed by treatment with cryptogein, a proteinaceous elicitor of tobacco immune responses secreted by an oomycete. Cryptogein-induced production of reactive oxygen species (ROS) in BY-2 cells served as a marker to evaluate the potential of microorganisms to activate plant defense responses. Twenty-nine bacterial strains isolated from the interior of Brassica rapa var. perviridis plants were screened, and 8 strains that enhanced cryptogein-induced ROS production in BY-2 cells were selected. Following application of these strains to the root tip of Arabidopsis seedlings, two strains, Delftia sp. BR1R-2 and Arthrobacter sp. BR2S-6, were found to induce whole-plant resistance to bacterial pathogens (Pseudomonas syringae pv. tomato DC3000 and Pectobacterium carotovora subsp. carotovora NBRC 14082). Pathogen-induced expression of plant defense-related genes (PR-1, PR-5, and PDF1.2) was enhanced by the pretreatment with strain BR1R-2. This cell-cell interaction-based platform is readily applicable to large-scale screening for microorganisms that enhance plant defense responses under various environmental conditions.
Today's concept of vulnerable plaque has evolved primarily from the early pioneering work uncovering the pivotal role of plaque rupture and coronary thrombosis as the major cause of acute myocardial ...infarction and sudden cardiac death. Since the first historical description of plaque rupture in 1844, several key studies by leading researchers and clinicians have lead to the current accepted views on lesion instability. Important to the complex paradigm of plaque destabilization and thrombosis are many discoveries beginning with the earliest descriptions of advanced plaques, reminiscent of abscesses encapsulated by fibrous tissue capable of rupture. It was not until the late 1980s that studies of remodeling provided keen insight into the growth of advanced plaques, beyond the simple accumulation of lipid. The emphasis in the next decade, however, was on a focused shift toward the mechanisms of lesion vulnerability based on the contribution of tissue proteolysis by matrix metalloproteinases as an essential factor responsible for thinning and rupture of the fibrous cap. In an attempt to unify the understanding of what constitutes a vulnerable plaque, morphological studies, mostly from autopsy, suggest the importance of necrotic core size, inflammation, and fibrous cap thickness. Definitive proof of the vulnerable plaque, however, remains elusive because animal or human data supporting a cause-and-effect relationship are lacking. Although emerging imagining technologies involving optical coherence tomography, high-resolution MRI, molecular biomarkers, and other techniques have far surpassed the limits of the early days of angiography, advancing the field will require establishing relevant translational animal models that produce vulnerable plaques at risk for rupture and further testing of these modalities in large prospective clinical trials.
Dihydrofolate reductase (DHFR) plays a key role in folate metabolism and is a target molecule of methotrexate. An increase in the cellular expression level of DHFR is one of the mechanisms of tumor ...resistance to methotrexate. The present study investigated the possibility that adenosine-to-inosine RNA editing, which causes nucleotide conversion by adenosine deaminase acting on RNA (ADAR) enzymes, might modulate DHFR expression. In human breast adenocarcinoma-derived MCF-7 cells, 26 RNA editing sites were identified in the 3′-UTR of DHFR. Knockdown of ADAR1 decreased the RNA editing levels of DHFR and resulted in a decrease in the DHFR mRNA and protein levels, indicating that ADAR1 up-regulates DHFR expression. Using a computational analysis, miR-25-3p and miR-125a-3p were predicted to bind to the non-edited 3′-UTR of DHFR but not to the edited sequence. The decrease in DHFR expression by the knockdown of ADAR1 was restored by transfection of antisense oligonucleotides for these miRNAs, suggesting that RNA editing mediated up-regulation of DHFR requires the function of these miRNAs. Interestingly, we observed that the knockdown of ADAR1 decreased cell viability and increased the sensitivity of MCF-7 cells to methotrexate. ADAR1 expression levels and the RNA editing levels in the 3′-UTR of DHFR in breast cancer tissues were higher than those in adjacent normal tissues. Collectively, the present study demonstrated that ADAR1 positively regulates the expression of DHFR by editing the miR-25-3p and miR-125a-3p binding sites in the 3′-UTR of DHFR, enhancing cellular proliferation and resistance to methotrexate.
Clinical trials have demonstrated that the second-generation cobalt-chromium everolimus-eluting stent (CoCr-EES) is superior to the first-generation paclitaxel-eluting stent (PES) and is noninferior ...or superior to the sirolimus-eluting stent (SES) in terms of safety and efficacy. It remains unclear whether vascular responses to CoCr-EES are different from those to SES and PES because the pathology of CoCr-EES has not been described in humans.
A total of 204 lesions (SES=73; PES=85; CoCr-EES=46) from 149 autopsy cases with duration of implantation >30 days and ≤3 years were pathologically analyzed, and comparison of vascular responses was corrected for duration of implantation. The observed frequency of late and very late stent thrombosis was less in CoCr-EES (4%) versus SES (21%; P=0.029) and PES (26%; P=0.008). Neointimal thickness was comparable among the groups, whereas the percentage of uncovered struts was strikingly lower in CoCr-EES (median=2.6%) versus SES (18.0%; P<0.0005) and PES (18.7%; P<0.0005). CoCr-EES showed a lower inflammation score (with no hypersensitivity) and less fibrin deposition versus SES and PES. The observed frequency of neoatherosclerosis, however, did not differ significantly among the groups (CoCr-EES=29%; SES=35%; PES=19%). CoCr-EES had the least frequency of stent fracture (CoCr-EES=13%; SES=40%; PES=19%; P=0.007 for CoCr-EES versus SES), whereas fracture-related restenosis or thrombosis was comparable among the groups (CoCr-EES=6.5%; SES=5.5%; PES=1.2%).
CoCr-EES demonstrated greater strut coverage with less inflammation, less fibrin deposition, and less late and very late stent thrombosis compared with SES and PES in human autopsy analysis. Nevertheless, the observed frequencies of neoatherosclerosis and fracture-related adverse pathological events were comparable in these devices, indicating that careful long-term follow-up remains important even after CoCr-EES placement.
Atherosclerotic plaque rupture with luminal thrombosis is the most common mechanism responsible for the majority of acute coronary syndromes and sudden coronary death. The precursor lesion of plaque ...rupture is thought to be a thin cap fibroatheroma (TCFA) or “vulnerable plaque”. TCFA is characterised by a necrotic core with an overlying thin fibrous cap (≤65 μm) that is infiltrated by macrophages and T-lymphocytes. Intraplaque haemorrhage is a major contributor to the enlargement of the necrotic core. Haemorrhage is thought to occur from leaky vasa vasorum that invades the intima from the adventitia as the intima enlarges. The early atherosclerotic plaque progression from pathologic intimal thickening (PIT) to a fibroatheroma is thought to be the result of macrophage infiltration. PIT is characterised by the presence of lipid pools which consist of proteoglycan with lipid insudation. The conversion of the lipid pool to a necrotic core is poorly understood but is thought to occur as a result of macrophage infiltration which releases matrix metalloproteinase (MMPs) along with macrophage apoptosis that leads to the formation of a acellular necrotic core. The fibroatheroma has a thick fibrous cap that begins to thin over time through macrophage MMP release and apoptotic death of smooth muscle cells converting the fibroatheroma into a TCFA. Other causes of thrombosis include plaque erosion which is less frequent than plaque rupture but is a common cause of thrombosis in young individuals especially women <50 years of age. The underlying lesion morphology in plaque erosion consists of PIT or a thick cap fibroatheroma. Calcified nodule is the least frequent cause of thrombosis, which occurs in older individuals with heavily calcified and tortious arteries.
RNA editing is a post-transcriptional process that alters the nucleotide sequence of RNA transcripts to generate transcriptome diversity. Among the various types of RNA editing, adenosine-to-inosine ...(A-to-I) RNA editing is the most frequent type of RNA editing in mammals. Adenosine deaminases acting on RNA (ADAR) enzymes, ADAR1 and ADAR2, convert adenosines in double-stranded RNA structures into inosines by hydrolytic deamination. Inosine forms a base pair with cytidine as if it were guanosine; therefore, the conversion may affect the amino acid sequence, splicing, microRNA targeting, and miRNA maturation. It became apparent that disrupted RNA editing or abnormal ADAR expression is associated with several diseases including cancer, neurological disorders, metabolic diseases, viral infections, and autoimmune disorders. The biological significance of RNA editing in pharmacokinetics/pharmacodynamics (PK/PD)-related genes is starting to be demonstrated. The authors conducted pioneering studies to reveal that RNA editing modulates drug metabolism potencies in the human liver, as well as the response of cancer cells to chemotherapy agents. Awareness of the importance of RNA editing in drug therapy is growing. This review summarizes the current knowledge on the RNA editing that affects the expression and function of drug response-related genes. Continuing studies on the RNA editing that regulates pharmacokinetics/pharmacodynamics would provide new beneficial information for personalized medicine.
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