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Background. A disintegrin and metalloproteinase-8 (ADAM8) is a potential surrogate of inflammation which has recently been associated with myocardial infarction. We evaluated in a rat cardiac transplantation model whether ischemia-reperfusion injury alone (IRI) or with early regional myocardial infarction (MI) would suffice to induce inflammatory myocardial remodeling and ADAM8 expression. Material and methods. Isogenic heterotopic cardiac transplantation after cardiac arrest was performed to 48 Fischer 344 rats to induce ischemia-reperfusion injury (IRI), of which 27 rats also underwent ligation of the left anterior coronary artery (LAD) of the heart to yield MI. Histology was performed at 0.5, 24 and 48 h after transplantation. ADAM8 was evaluated by qRT-PCR after graft harvesting. Results. After 0.5 and 48 h respectively, edematous intramyocardial artery nuclei and periadventitial inflammation were more prominent in MI after transplantation, as compared with IRI alone and Controls (57.0 vs 40.0 and 5.0; 1.9 vs 1.1 and 0.9, point score units, p < 0.05, respectively). The expression of ADAM-8 was increased in MI as compared with Controls (1.9 vs 1.0, 1.9 fold increase) at 48 h. In grafts with MI, ADAM8 was localized using immunohistochemistry to the vicinity of the area corresponding to the developing infarction as well as in intramyocardial arteries remote to the infarction area. Conclusions. Remote histopathological changes of ischemic cardiac grafts are associated with increased expression of ADAM8 thus emphasizing a global myocardial impact of MI. [ABSTRACT FROM AUTHOR]

Background and aims. Carbonic anhydrases (CA) play a central role in osteoclast function and bone remodeling by catalyzing the formation of bicarbonate and proton from carbon dioxide. According to previous histochemical studies, advanced atherosclerotic plaques share similarities with bone. However, whether CAs are expressed in plaques is not known. Methods and results. Whole genome expression array of arterial samples ( n = 24) confirmed that several genes indicating osteoblastogenesis and osteoclastogenesis were up-regulated in plaques when compared to control vessel samples from internal thoracic arteries ( n = 6), including CA2 and CA12, expression of which was also verified with quantitative reverse transcription polymerase chain reaction (RT-PCR). In atherosclerotic plaques there was 11.6-fold ( P < 0.0001) and 11.4-fold ( P < 0.0001) up-regulation of CA2 and CA12, compared to controls, respectively. According to quantitative PCR, CA2 expression was elevated in carotid (12.3-fold, P < 0.0001), femoral (13.2-fold, P < 0.01), and aortic plaques (7.5-fold, P < 0.0001). CA12 expression was elevated in carotid (11.6-fold, P < 0.0001), femoral (11.5-fold, P < 0.01), and aortic plaques (9.7-fold, P < 0.0001). CAII, CAXII, and CD68 and tartrate-resistant acid phosphatase (TRAP), a marker of osteoclast-like cells, were found to be co-localized in multinucleated giant cells in the atherosclerotic plaques using immunohistochemistry and double-staining immunofluorescence analysis. Conclusions. The present findings provide evidence for the involvement of CAs in advanced atherosclerosis in osteoclast-like cells of monocyte-macrophage lineage. [ABSTRACT FROM AUTHOR]

Human embryonic stem cell (hESC) differentiation in embryoid bodies (EBs) provides a valuable tool to study the interplay of different germ layers and their influence on cell differentiation. The gene expression of the developing EBs has been shown in many studies, but the protein expression and the spatial composition of different germ layers in human EBs have not been systematically studied. The aim of the present work was to study the temporal and spatial organisation of germ layers based on the expression of mesoderm (Brachyury T), endoderm (AFP) and ectoderm (SOX1) markers during the early stages of differentiation in eight hESC lines. Tissue multi-array technology was applied to study the protein expression of a large number of EBs. According to our results, EB formation and the organisation of germ layers occurred in a similar manner in all the lines. During 12 days of differentiation, all the germ layer markers were present, but no obvious distinct trajectories were formed. However, older EBs were highly organised in structure. Pluripotency marker OCT3/4 expression persisted unexpectedly long in the differentiating EBs. Cavity formation was observed in the immunocytological sections, and caspase-3 expression was high, suggesting a role of apoptosis in hESC differentiation and/or EB formation. The expression of Brachyury T was notably low in all the lines, also those with the best cardiac differentiation capacity, while the expression of SOX1 was higher in some lines, suggesting that the neural differentiation propensity may be detectable already in the early stages of EB differentiation. [ABSTRACT FROM AUTHOR]

Objective. We aimed to characterize the expression of indoleamine 2,3-dioxygenase (IDO) or IDO-induced tryptophan degradation-dependent pathways, which may lead to suppression of T cells and possible protection against atherosclerosis. Methods and results. Expression of IDO and IDO-related pathway components was analyzed in advanced human atherosclerotic plaques ( n = 24) and in non-atherosclerotic arteries ( n = 6). Up-regulation of IDO and genes related to the IDO pathway was found to be pronounced in atherosclerotic plaques. Immunohistochemistry demonstrated IDO protein in the atheromatous core and co-distribution with monocyte-macrophages (CD68-positive cells). In gene-set enrichment analysis, the IDO pathway revealed a significant (false discovery rate (FDR) = 0.07) regulatory T cell, fork-head box protein 3 (FoxP3)-initiated CD28-cytotoxic T lymphocyte-associated antigen 4 (CTLA-4)-inducible T cell co-stimulator (ICOS)-driven pathway leading to activation of IDO expression in antigen-presenting cells (APCs). Expression of these IDO pathway genes varied between 2.1- and 16.8-fold as compared to control tissues ( P < 0.05 for all). Conclusions. IDO and the IDO-related pathway are important mediators of the immunoinflammatory responses in advanced atherosclerosis offering new viable therapeutic targets for the development of antiatherogenic immunosuppressive therapies. [ABSTRACT FROM AUTHOR]

Objective. Previously, we scanned all 23,000 human genes for differential expression between normal and atherosclerotic tissues and found the involvement of ADAM8. Methods. We investigated the expression of ADAM8 mRNA and protein level in human atherosclerotic tissues and non-atherosclerotic internal thoracic arteries as well as the association of ADAM8 2662 T/G single nucleotide polymorphism (SNP) with the extent of coronary atherosclerosis and with the risk of fatal myocardial infarction. Results. ADAM8 mRNA was up-regulated in carotid, aortic, and femoral atherosclerotic plaques (n=24) when compared with non-atherosclerotic arteries. ADAM8 protein expression was increased in advanced atherosclerotic plaques as compared to control vessels wherein it was localized to macrophages and smooth muscle cells The G allele carriers of the ADAM8 2662 T/G SNP had significantly larger areas of fibrotic, calcified, and complicated plaques in coronary arteries (P=0.027, P=0.011, and P=0.011, respectively) and significantly higher occurrence of myocardial infarction (MI) (P=0.004) and fatal pre-hospital MI (P=0.003) than did the TT homozygotes. Conclusion. ADAM8 is a promising candidate to be involved in atherosclerosis, and its 2662 T/G allelic variant significantly associates with advanced atherosclerotic lesion areas and MI. [ABSTRACT FROM AUTHOR]

Aim. The differentiation efficiencies of human embryonic stem cell (hESC) lines differ from each other. To assess this in more detail we studied the cardiac differentiation of eight hESC lines derived in the same laboratory. Results. Substantial variation in growth and in the ability to form beating areas was seen between the different hESC lines; line HS346 gave the best efficiency (9.4%), while HS293 did not differentiate into beating colonies at all. Nine germ layer and differentiation markers were quantified during early differentiation in four hESC lines. The expression levels of Brachyury T, MESP1 and NKX2.5 were highest in the most efficient cardiac line (HS346). A systematic characterization of the beating cells revealed proper cardiac marker expression, electrophysiological activity, and pharmacological response. Conclusions. The hESC lines derived in the same laboratory varied considerably in their potential to differentiate into beating cardiomyocytes. None of the expression markers could clearly predict cardiac differentiation potential, although the expression of early cardiomyogenic genes was upregulated in the best cardiac line. The proper cardiomyocyte characteristics and pharmacological response indicate that these cells could be used as a model for human cardiomyocytes in pharmacological and toxicological analyses when investigating new heart medications or cardiac side-effects. [ABSTRACT FROM AUTHOR]

Background and aims. The expression of disintegrin and metalloprotease ADAM-9, ADAM-15, and ADAM-17 has been associated with cell-cell, cell-platelet, and cell-matrix interactions and inflammation. They are possibly implicated in the pathophysiology of atherosclerosis. Methods and results. Whole-genome expression array and quantitative real-time polymerase chain reaction (PCR) analysis confirmed that ADAM-9, ADAM-15, and ADAM-17 are upregulated in advanced human atherosclerotic lesions in samples from carotid, aortic, and femoral territories compared to samples from internal thoracic artery (ITA) free of atherosclerotic plaques. Western analysis indicated that the majority of these ADAMs were in the catalytically active form. ADAM-9, ADAM-15, and ADAM-17-expressing cells were shown to co-localize with CD68-positive cells of monocytic origin in the atherosclerotic plaques using immunohistochemistry and double-staining immunofluorescence analysis. Co-localization was demonstrated in all vascular territories. In the carotid territory, cells expressing the ADAMs co-distributed also with smooth muscle cells and, in femoral territory, with CD31-positive endothelial cells, indicating that the ADAM expression pattern depends on vascular bed territory. Conclusions. Present findings provide strong evidence for the involvement of catalytically active ADAM-9, ADAM-15, and ADAM-17 in advanced atherosclerosis, most notably associated with cells of monocytic origin. [ABSTRACT FROM AUTHOR]

Nuclear receptors are master regulators of metazoan gene expression with crucial roles during development and in adult physiology. Fushi tarazu factor 1 (FTZ-F1) subfamily members are ancient orphan receptors with homologues from Drosophila to human that regulate diverse gene expression programs important for developmental processes, reproduction and cholesterol homeostasis in an apparently ligand-independent manner. Thus, developmental and tissue-specific cofactors may be particularly important in modulating the transcriptional activities of FTZ-F1 receptors. In Drosophila, the homeodomain protein Fushi tarazu acts as a cofactor for FTZ-F1 (NR5A3), leading to the hypothesis that a similar type of homeodomain cofactor-nuclear receptor relationship might exist in vertebrates. In this study, we have identified and characterized the homeodomain protein Prox1 as a co-repressor for liver receptor homologue 1 (LRH1/NR5A2), a master regulator of cholesterol homeostasis in mammals. Our study suggests that interactions between LRH1 and Prox1 may fulfil roles both during development of the enterohepatic system and in adult physiology of the liver. [ABSTRACT FROM AUTHOR]

The effect of preconditioning (PC) on the changes of nerve growth factor-induced gene A (NGFI-A) expression induced by severe hypobaric hypoxia was studied by in situ hybridization. A PC consisted of three trials of mild hypobaric hypoxia (360 Torr, 2 h) spaced at 24 h. The last trial was followed by severe hypoxia (SH; 180 Torr, 3 h) 24 h later. The PC hypoxia prevented the NGFI-A messenger RNA (mRNA) increase in the cortex, neostriatum, piriform cortex, amygdala and hippocampus detected 3 h after SH. The preconditioned SH caused a peak in NGFI-A mRNA expression at the 24 h time-point and thus abolished the dramatic decrease of the mRNA in vulnerable areas seen by 24 and 72 h after SH. The findings suggest a role of brain NGFI-A in the protective effect of hypoxic/ischemic PC. [Copyright &y& Elsevier]

Abstract: Background: Proprotein convertase subtilisin/kexin (PCSK) enzymes cleave proproteins into mature end products. Previously, MBTPS1 and PCSK9 have been shown to regulate cholesterol metabolism and LDL receptor recycling, whereas FURIN and PCSK5 have been suggested to inactivate lipases and regulate inflammation in atherosclerosis. Here, we systematically analyzed the expression of PCSKs and their targets in advanced atherosclerotic plaques. Methods and results: Microarray and quantitative real-time PCR experiments showed that FURIN (42.86 median fold, p =2.1e−8), but no other PCSK, is universally overexpressed in the plaques of different vascular regions. The mRNA expression screen of PCSK target proteins in plaques identified many known factors, but it also identified the significant upregulation of the previously overlooked furin-processed B cell activating cytokines APRIL (TNFSF13, 2.52 median fold, p =3.0e−5) and BAFF (TNFSF13B, 2.97 median fold, p =7.6e−6). The dysregulation of FURIN did not associate with its htSNPs or the previously reported regulatory SNP (−229, rs4932178) in the promoter. Immunohistochemistry experiments showed the upregulation of FURIN in the plaque lymphocytes and macrophages where it was co-expressed with BAFF/TNFSF13B and APRIL/TNFSF13. Conclusions: Our data unequivocally show that FURIN is the primary PCSK that is dysregulated in the immune cells of advanced human atherosclerotic plaques, which implies a role for this enzyme in plaque pathology. Therefore, drugs that inhibit FURIN in arteries may modulate the course of this disease. [Copyright &y& Elsevier]