State-of-the-art technology for cardiovascular research

Heart and vascular diseases are responsible for tens of millions of deaths annually, underscoring an urgent need to improve the existing clinical practice in order to benefit patients. Advancement of basic science and technology enables understanding of disease etiology and pathogenesis at a deeper level of complexity. This mini-review article provides a summary of recent methods of cell and molecular biology applicable for current cardiovascular research. Widespread application of these approaches in cardiovascular disease research will be a key factor in the prolonged longevity and life expectancy of the general population.Heart and vascular diseases are responsible for tens of millions of deaths annually, underscoring an urgent need to improve the existing clinical practice in order to benefit patients. Advancement of basic science and technology enables understanding of disease etiology and pathogenesis at a deeper level of complexity. This mini-review article provides a summary of recent methods of cell and molecular biology applicable for current cardiovascular research. Widespread application of these approaches in cardiovascular disease research will be a key factor in the prolonged longevity and life expectancy of the general population

ASSOCIATION OF TREM-1 GENE POLYMORPHISMS WITH INFECTIVE ENDOCARDITIS

Infective endocarditis (IE) is a septic inflammation of endocardium, which generally involves the lining of the heart chambers and heart valves. The development of IE depends in many respects on how properly and efficiently the immune system responds to the occurrence of an infection. Innate immunity, which carries out the response to a transient bacteremia, is genetically determined in a large extent. Pattern recognition receptors, which identify pathogenand danger-associated molecular patterns, are the main effectors of innate immune response; one of these receptors is triggering receptor expressed on myeloid cells-1 (TREM-1). We hypothesized that inherited variation in TREM-1 gene may affect individual susceptibility to IE. The distribution of genotypes and alleles of rs1817537, rs3804277, rs6910730, rs7768162, rs2234246, rs4711668, rs9471535, and rs2234237 gene polymorphisms was investigated in 110 Caucasian (Russian) subjects with IE and 300 age-, sex-, and ethnicity-matched healthy blood donors. Odds ratios with 95% confidence intervals were calculated. We found that rs1817537 polymorphism was associated with decreased IE risk (OR = 0.60; 95%CI = 0.37–0.99; р = 0.046, dominant model); however, this was not significant after an adjustment for multiple comparisons. Therefore, we observed no statistically significant association between the investigated polymorphisms within TREM-1 gene and IE. Further in-depth investigations in this field are necessary to shed the light on the impact of inherited variation within innate immune response genes on the development of IE

LYSOSOME-DEPENDENT CELL DEATH DEFINES SPECIFIC ENDOTHELIAL TOXICITY OF CALCIUM PHOSPHATE BIONS

Aim of the study was to identify the mechanism of specific endothelial toxicity related to calcium phosphate bions (CPB). Material and methods. CPB and magnesium phosphate bions (MPB) were artificially synthesised through supersaturation of culture medium with respective salts and then added to human endothelial cells (EA.hy 926) and murine endothelial cells (2H-11) to study: 1) spatiotemporal aspects of bion internalisation by means of transmission electron microscopy and confocal microscopy; 2) whether blocking of H+-ATPase by lysosomal inhibitor bafilomycin A1 affects endothelial toxicity of bions; 3) expression of caspase-3 and its substrate poly(ADP-ribose) polymerase (PARP-1). Results. CPB were internalized by endothelial cells as early as 1 h upon their addition and were localized in lysosomes; after 4 h, we detected release of calcium ions (Ca2+) from lysosomes to cytosol accompanied by multifold increase in cleaved caspase 3 and its substrate PARP-1. Bafilomycin A1 rescued endothelial cells from death induced by slightly soluble CPB regardless of exposure time and dose; however, freely soluble MPB did not evince endothelial toxicity regardless of bafilomycin A1 addition. Conclusion. Upon internalization by endothelial cells, CPB cause their death due to dissolution in lysosomes and subsequent release of calcium ions into the cytosol, ultimately leading to cleavage of executioner caspases. MPB lack endothelial toxicity because their dissolution does not lead to release of calcium ions. Therefore, specific endothelial toxicity of CPB is defined by lysosome-dependent cell death

Colorectal cancer liver metastatic growth depends on PAD4-driven citrullination of the extracellular matrix

Citrullination of proteins, a post-translational conversion of arginine residues to citrulline, is recognized in rheumatoid arthritis, but largely undocumented in cancer. Here we show that citrullination of the extracellular matrix by cancer cell derived peptidylarginine deiminase 4 (PAD4) is essential for the growth of liver metastases from colorectal cancer (CRC). Using proteomics, we demonstrate that liver metastases exhibit higher levels of citrullination and PAD4 than unaffected liver, primary CRC or adjacent colonic mucosa. Functional significance for citrullination in metastatic growth is evident in murine models where inhibition of citrullination substantially reduces liver metastatic burden. Additionally, citrullination of a key matrix component collagen type I promotes greater adhesion and decreased migration of CRC cells along with increased expression of characteristic epithelial markers, suggesting a role for citrullination in promoting mesenchymal-to-epithelial transition and liver metastasis. Overall, our study reveals the potential for PAD4-dependant citrullination to drive the progression of CRC liver metastasis

Proteomic profiling of metastatic matrisome reveals citrullination as a marker of colorectal liver metastasis

Colorectal cancer is one of the most frequently occurring malignancies and a major cause of cancer death. Distant metastases in this disease most commonly develop in the liver and are often untreatable. Here, we use proteomics to characterise, qualitatively and quantitatively, extracellular matrix (ECM) from colorectal cancer liver metastases. We show that citrullination of the ECM by cancer cell derived peptidyl arginine deiminase 4 (PAD4) is important for the growth of liver metastases. Citrullination of proteins, a posttranslational conversion of arginine residues to citrulline, is well recognised in rheumatoid arthritis, but largely undocumented in cancer. PAD4, a key enzyme responsible for catalysing citrullination, is produced by metastatic colorectal cancer cells and found at higher levels in human liver metastases than in normal liver. Functional significance for citrullination in metastatic growth was evident in murine models where inhibition of citrullination, either globally by pharmacologic inhibition of PADs or specifically in colorectal cancer cells by PAD4 knockdown reduced liver metastatic burden by 3- to 4-fold (P &lt; 0.05). Additionally, citrullination of key extracellular matrix (ECM) component collagen type I in vitro led to greater adhesion and 25-30% decreased migration of colorectal cancer cells (P &lt; 0.05) along with increased expression of characteristic epithelial markers, indicating a role for citrullination in promoting mesenchymal-to-epithelial transition (MET). Overall, our study revealed PAD4- dependent citrullination of the ECM altering mesenchymal-epithelial plasticity in colorectal cancer cells and the progression of liver metastasis. These data indicate that inhibition of citrullination could be exploited to potentially prevent the development of liver metastases in colorectal cancer.</p

Criteria for standartization of probiotic components in functional food products

The increasing volume of consumption of probiotics and functional food products requires determination of standardized criteria for cultures and their exometabolites used in functional products manufacturing. The study was aimed at developing criteria for the estimation and standardization of exometabolites and the colony-forming ability of probiotic strains for functional food production. The work included such microbiological and physicochemical methods as GC-MS, GPC, UV, and FT-IR-spectroscopy. Based on the results of the study, the comparative analysis of the microbiological properties of probiotic Bifidobacterium strains was provided, the fatty acid composition of the cell wall was described, and the physical and chemical study of the exopolymers produced by them was carried out. According to the data of FT-IR- spectroscopy, the characteristic features of the components of the cell wall of Bifidobacterium strains were established. Bifidobacteria form the unique composition of organophosphorus structures of lipoteichoic acids, which determines the adhesive ability of strains. The authors studied the molecular weight distribution of the samples of exometabolites isolated from the nutrient medium after the cultivation of bifidobacteria, under conditions of gel-permeation chromatography. The spectral (UV, FT-IR) characteristics of the produced metabolites and their chromatographic fractions were compared. The fatty acids of the Bifidobacterium cell membrane were analyzed using the GC/MS method. The fatty acids were extracted from bacterial cells with different hydrophobicity with a mixture of chloroform and hexane. It has been established that the hydrophobicity is determined by different contents of unsaturated and branched fatty acids in the bacterial membrane. Hydrophobic bifidobacteria are the only that contain the isopentadecane (isoC15:0) and methyl-tetradecanoic (13Me-C14:0) acids. With the mean hydrophobicity, a high content of the isopalmitic (isoC16:0) and stearic (C18:0) acids was established. Low-hydrophobic strains are characterized by a low content of monounsaturated fatty acids

A core matrisome gene signature predicts cancer outcome

Background: Accumulating evidence implicates the tumour stroma as an important determinant of cancer progression but the protein constituents relevant for this effect are unknown. Here we utilised a bioinformatics approach to identify an extracellular matrix (ECM) gene signature overexpressed in multiple cancer types and strongly predictive of adverse outcome. Methods: Gene expression levels in cancers were determined using Oncomine. Geneset enrichment analysis was performed using the Broad Institute desktop application. Survival analysis was performed using KM plotter. Survival data were generated from publically available genesets. Results: We analysed ECM genes significantly upregulated across a large cohort of patients with ovarian, lung, gastric and colon cancers and defined a signature of nine commonly upregulated genes. Each of these nine genes was considerably overexpressed in all the cancers studied, and cumulatively, their expression was associated with poor prognosis across all data sets. Further, the gene signature expression was associated with enrichment of genes governing processes linked to poor prognosis, such as EMT, angiogenesis, hypoxia, and inflammation. Conclusions: Here we identify a nine-gene ECM signature, which strongly predicts outcome across multiple cancer types and can be used for prognostication after validation in prospective cancer cohorts.</p

Tumor-infiltrating monocytes/macrophages promote tumor invasion and migration by upregulating S100A8 and S100A9 expression in cancer cells

Myeloid cells promote development of distant metastases, but little is known about the molecular mechanisms underlying this process. Here we have begun to uncover the effects of myeloid cells on cancer cells in a mouse model of liver metastasis. Monocytes/macrophages, but not granulocytes, isolated from experimental liver metastases stimulated migration and invasion of MC38 colon and Lewis lung carcinoma cells. In response to conditioned media from tumor-infiltrating monocytes/macrophages, cancer cells upregulated S100a8 and S100a9 mRNA expression through an ERK-dependent mechanism. Suppression of S100A8 and S100A9 in cancer cells using shRNA significantly diminished migration and invasion in culture. Downregulation of S100A8 and S100A9 had no effect on subcutaneous tumor growth. However, colony size was greatly reduced in liver metastases with decreased invasion into adjacent tissue. In tissue culture and in liver colonies derived from cancer cells with knockdown of S100A8 and S100A9, MMP2 and MMP9 expression were decreased, consistent with the reduction in migration and invasion. Our findings demonstrate that monocytes/macrophages in the metastatic liver microenvironment induce S100A8 and S100A9 in cancer cells, and that these proteins are essential for tumor cell migration and invasion. S100A8 and S100A9, however, are not responsible for stimulation of proliferation. This study implicates S100A8 and S100A9 as important mediators of tumor cell aggressiveness, and highlights the therapeutic potential of S100A8 and S100A9 for interference of metastasis

Whole-Transcriptome Sequencing: A Powerful Tool for Vascular Tissue Engineering and Endothelial Mechanobiology

Among applicable high-throughput techniques in cardiovascular biology, whole-transcriptome sequencing is of particular use. By utilizing RNA that is isolated from virtually all cells and tissues, the entire transcriptome can be evaluated. In comparison with other high-throughput approaches, RNA sequencing is characterized by a relatively low-cost and large data output, which permits a comprehensive analysis of spatiotemporal variation in the gene expression profile. Both shear stress and cyclic strain exert hemodynamic force upon the arterial endothelium and are considered to be crucial determinants of endothelial physiology. Laminar blood flow results in a high shear stress that promotes atheroresistant endothelial phenotype, while a turbulent, oscillatory flow yields a pathologically low shear stress that disturbs endothelial homeostasis, making respective arterial segments prone to atherosclerosis. Severe atherosclerosis significantly impairs blood supply to the organs and frequently requires bypass surgery or an arterial replacement surgery that requires tissue-engineered vascular grafts. To provide insight into patterns of gene expression in endothelial cells in native or bioartificial arteries under different biomechanical conditions, this article discusses applications of whole-transcriptome sequencing in endothelial mechanobiology and vascular tissue engineering

Macrophage migration inhibitory factor : a key cytokine and therapeutic target in colon cancer

Macrophage migration inhibitory factor (MIF) was one of the first cytokines to be discovered, over 40 years ago. Since that time a burgeoning interest has developed in the role that MIF plays in both the regulation of normal physiology and the response to pathology. MIF is a pleotropic cytokine that functions to promote inflammation, drive cellular proliferation, inhibit apoptosis and regulate the migration and activation state of immune cells. These functions are particularly relevant for the development of cancer and it is notable that various solid tumours over express MIF. This includes tumours of the gastrointestinal tract and MIF appears to play a particularly prominent role in the development and progression of colonic adenocarcinoma. Here we review the role that MIF plays in colonic carcinogenesis through the promotion of colonic inflammation, as well as the progression of primary and metastatic colon cancer. The recent development of various antagonists and antibodies that inhibit MIF activity indicates that we may soon be able to classify MIF as a therapeutic target in colon cancer patients.11 page(s