Serum cystatin C levels are associated with coronary artery disease and its severity.

OBJECTIVES: Serum cystatin C has been established as a predictor of cardiovascular events. The aim of this study was to evaluate the role of cystatin C in determining the presence and the severity of patients with coronary artery disease (CAD). DESIGN AND METHODS: A total of 936 subjects without overt renal disease were included in this cross-sectional study. Among them were 714 patients with CAD and 222 without based on coronary angiography. Subjects were further divided into four groups according to cystatin C quartile. Serum cystatin C was measured using particle-enhanced immunoassay method. The study analyzed the relationship of cystatin C levels with the presence and severity of CAD, including the number of stenotic vessels involved and Gensini score. RESULTS: Serum cystatin C levels were significantly higher in patients with CAD than those without (P<0.001), and significantly increased as the involvement of coronary vessels increased (P<0.001). The prevalence of CAD and its severity assessed by Gensini score were also significantly greater in the highest quartile of cystatin C (P<0.001). Moreover, cystatin C levels were independently correlated with the presence of CAD in a multivariate logistic regression model (P=0.023) and were positively correlated with Gensini score by linear regression analysis (standardized ß=0.083, P=0.010). CONCLUSIONS: Elevated serum cystatin C levels were significantly associated with the presence and severity of CAD in patients with normal renal function. It is suggested that cystatin C might play a role in CAD diagnosis and serve as a marker of CAD severity.

Effects of miR-19b overexpression on proliferation, differentiation, apoptosis and Wnt/ß-catenin signaling pathway in P19 cell model of cardiac differentiation in vitro.

MicroRNA (miR)-19b is part of the miR-17-92 cluster associated with cardiac development. Here, we investigated the effects of overexpressing miR-19b on proliferation, differentiation, apoptosis, and regulation of the Wnt/ß-catenin signaling pathway in the multipotent murine P19 cell line that can be induced to undergo cardiogenesis. P19 cells were transfected with the miR-19b plasmid or empty vector, and miR-19b overexpression was verified by Quantitative Real-Time PCR (qPCR). The miR-19b or vector control stable cell lines were selected using Blasticidin S HCl, and their proliferation, cell cycle, and apoptosis levels were analyzed using the Cell Counting Kit-8 and flow cytometry. P19 cell differentiation markers, apoptosis-related genes (bax, bcl-2), and Wnt/ß-catenin signaling pathway-related genes were detected by qPCR, the corresponding proteins by Western blot. Expression of the Wnt pathway and differentiation marker proteins was also verified by immunofluorescence. Morphological changes associated with apoptosis were observed by electron microscopy and Hoechst staining. On the basis of these results, we demonstrated that miR-19b overexpression promoted proliferation and differentiation but inhibited apoptosis in P19 cells; Wnt and ß-catenin expressions were decreased, while that of GSK3ß was increased with miR-19b overexpression. Overexpression of miR-19b inhibited activation of the Wnt/ß-catenin signaling pathway in P19 cells, which may regulate cardiomyocyte differentiation. Our findings may bring new insights into the mechanisms underlying cardiac diseases and suggest that miR-19b is a potential new therapeutic target for cardiovascular diseases.

Effect of organic matter on the performance of granular anammox process.

The presence of organic matter (OM) is considered to affect anammox process adversely, while practically wastewaters containing ammonia are not free from OM. In this study, the performance of anammox granules in presence of OM was evaluated under different COD to N ratios. Low OM concentration did not affect ammonia and nitrite removal significantly but improved the total nitrogen removal via denitrifiers. High OM could suppress anammox activity, resulting in a lower ammonia removal. PCR tests revealed that there was a reduction in the number of anammox bacteria and denitrifiers quantity increased when 400mg COD/L influent was applied. A COD to N threshold ratio for anammox inhibition, defined when ammonia removal dropped to 80%, was 3.1, higher than that of flocculent sludge. This study revealed that the coexistence of denitrification and anammox was an effective strategy to treat wastewaters containing high levels of nitrogen and OM.

Differential expression of microRNAs in cardiac myocytes compared to undifferentiated P19 cells.

microRNA (miRNA) expression is tightly controlled in a tissue-specific and developmental stage-specific manner; some are highly and specifically expressed in cardiovascular tissues. miRNA expression profiling, using miRNA microarrays facilitates studying the biological function of miRNAs. We investigated changes in miRNA expression profiles during differentiation of P19 cells into cardiac myocytes in order to elucidate the mechanisms of heart development. The morphology of P19 cells during differentiation was observed using an inverted microscope. Western blot analysis was performed to detect cardiac troponin I (cTnI) expression. Total RNA was extracted from P19 cells for microarray and real-time quantitative reverse transcription-polymerase chain reaction (real-time qRT-PCR) analyses to determine the miRNA expression profile. The miRNA microarray revealed differential expression of 49 miRNAs, of which 26 were down-regulated and 23 were up-regulated in differentiated cardiac myocytes, compared to normal P19 cells. This was confirmed by real-time qRT-PCR. We also utilized target prediction analysis to identify gene targets. Some miRNAs may have important roles in cardiac development and congenital heart defects (CHDs). Further analysis of miRNA function to confirm their target genes during cardiac development will determine the potential for novel miRNA-based therapeutic strategies.

LYRM1, a gene that promotes proliferation and inhibits apoptosis during heart development.

Congenital heart disease (CHD) is the most common type of birth defect, but its underlying molecular mechanisms remain unidentified. Previous studies determined that Homo sapiens LYR motif containing 1 (LYRM1) is a novel nucleoprotein expressed at the highest level in adipose tissue and in high levels in heart tissue. The LYRM1 gene may play an important role in the development of the human heart. This study was designed to identify the biological characteristics of the LYRM1 gene in heart development. On the basis of expression-specific differentiation markers identified with quantitative real-time RT-PCR and the morphology of LYRM1-overexpressing cells during differentiation, ectopic expression was not found to significantly affect differentiation of P19 cells into cardiomyocytes. MTT assays and cell cycle analysis showed that LYRM1 dramatically increases the proliferation of P19 cells. Furthermore, data from annexin V-FITC binding and caspase-3 activity revealed that LYRM1 can inhibit the apoptosis of P19 cells. Our data suggest that LYRM1 might have the potential to modulate cell growth, apoptosis, and heart development.

GATA-4 promotes the differentiation of P19 cells into cardiac myocytes.

The aim of this study was to investigate the effects of GATA-4 on the differentiation of P19 cells into cardiomyocytes and to examine the relationship between GATA-4 and cardiomyocytes. We constructed vectors to overexpress and silence GATA-4. These vectors, as well as empty ones were transfected into P19 cells. Subsequently, reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis were performed. The morphology of P19 cells during differentiation was observed using an inverted microscope. Total RNA was extracted from P19 cells. We used real-time PCR to evaluate the expression levels of 6 genes: GATA-4, GATA-6, transthyretin (TTR), alpha-fetoprotein (AFP), Nkx2.5, and alpha-myosin heavy chain (alpha-MHC). The gene expression pattern of these 6 genes is graphically shown for each group. The GATA-4 mRNA level in cells overexpressing GATA-4 was notably higher than that in the controls, whereas the levels in the controls were notably higher than those in the GATA-4-silenced P19 cells. The cell lines overexpressing GATA-4 expressed higher levels of Nkx2.5 and alpha-MHC than the controls. However, the controls expressed higher levels of AFP, GATA-6 and TTR than the cells overexpressing GATA-4. The RNAi group expressed lower levels of TTR, Nkx2.5, and alpha-MHC than the controls, but there were no differences in the RNAi group and the controls with regard to the expression levels of AFP and GATA-6. The gene expression pattern in the cells overexpressing GATA-4 was biased toward the Nkx2.5 and alpha-MHC. On the other hand, the gene expression pattern in GATA-4-silenced cells and the controls was biased toward the TTR and AFP. The overexpression of GATA-4 enhances the differentiation of P19 cells into cardiac myocytes, whereas its down-regulation suppresses this trend.