Gene expression profiling of human atrial myocardium with atrial fibrillation by DNA microarray analysis.

BACKGROUND: Atrial fibrillation (AF) is the most frequently encountered arrhythmia in the clinical setting. However, a comprehensive investigation of the molecular mechanism of AF has not been performed. The aim of this study was to clarify transcriptional profiling of genes modulated in the atrium of AF patients using DNA microarray technology. METHODS: We obtained 17 fresh cardiac specimens, right atrial appendages, isolated from 10 patients with normal sinus rhythm and seven chronic AF patients who underwent cardiac surgery. Affymetrix GeneChip (Human Genome U95A) investigating 12,000 human genes was used for each specimen. Quantitative analysis of selected genes was performed by the real-time PCR method. RESULTS: The left atrial diameter in the AF group was greater than that in the sinus rhythm group. We could identify 33 AF-specific genes that were significantly activated (>1.5-fold), compared with the sinus rhythm group, including an ion channel, an antioxidant, an inflammation, three cell growth/cell cycle, three transcription such as nuclear factor-interleukin 6-beta, several cell signaling and several protein genes, and seven expressed sequence tags (ESTs). In contrast, we found 63 sinus rhythm-specific genes, including several cell signaling/communication such as sarcoplasmic reticulum Ca2+-ATPase 2, several cellular respiration and energy production and two antiproliferative or negative regulator of cell growth genes, and 22 ESTs. CONCLUSIONS: The present study demonstrated that about one hundred genes were modulated in the atria of AF patients. These findings suggest that these genes may play critical roles in the initiation or perpetuation of AF and the pathophysiology of atrial remodeling.

Transcriptional profile of genes induced in human atrial myocardium with pressure overload.

BACKGROUND: The molecular response of human myocardium to mechanical stimuli, particularly the difference between pressure or volume overload cardiac hypertrophy, remains incompletely defined. METHODS: We investigated the transcriptional profile of genes induced in human pressure- or volume-overloaded myocardium with DNA microarray technology. We used right atrial tissue from patients who underwent cardiac surgery. On the basis of pressure data and echocardiographic findings, the patients were divided into three groups: control group (n=3), pressure overload group (mean right atrial pressure of >7 mm Hg, n=3), and volume overload group (moderate or severe tricuspid regurgitation, n=3). Expression profiles of 2139 human genes were investigated with mRNA obtained from the samples. RESULTS: In the pressure overload group, expression of genes of cyclin-dependent kinase inhibitor 1A (CDKI1A, 11.7+/-3.1-fold vs. control), and mitogen-activated protein kinase phosphatase-1 (MKP-1, 26.2+/-2.1-fold) was significantly increased compared with those in control or volume overload group (P<0.05). The specificity of these gene expressions was confirmed by a quantitative "real-time" polymerase chain reaction (PCR) analysis. In addition, mechanical strain induced CDKI1A and MKP-1 protein expressions in neonatal rat cardiac myocytes in an amplitude-dependent manner. In contrast, transcripts of growth factors did not significantly increase. CONCLUSIONS: This study demonstrated that gene expressions of CDKI1A and MKP-1, but not growth factors, are induced in chronic pressure-overloaded myocardium. These findings suggest that suppressors of the cell cycle or cell proliferation may play a critical role in the pathophysiology of pressure overload.

Mutations of BRAF are associated with extensive hMLH1 promoter methylation in sporadic colorectal carcinomas.

Activating mutations of BRAF have been frequently observed in microsatellite unstable (MSI+) colorectal carcinomas (CRCs), in which mutations of BRAF and KRAS are mutually exclusive. Previously, we reported that hypermethylation of hMLH1 might play an important role in the tumorigenesis of right-sided sporadic CRCs with MSI showing less frequency of KRAS/TP53 alteration. Therefore, we have assumed that BRAF mutations might be highly associated with hMLH1 methylation status rather than MSI status. In this study, mutations of BRAF and KRAS and their relationship with MSI and hMLH1 methylation status were examined in 140 resected specimens of CRC. The methylation status was classified into 3 types: full methylation (FM), partial methylation (PM) and nonmethylation (NM). Only FM closely linked to reduced expression of hMLH1 protein. BRAF mutations were found in 16 cases (11%), all leading to the production of BRAF(V599E). As for MSI status, BRAF mutations were found in 43% of MSI+ and 4% of MSI- cases (p < 0.0001). Among the MSI+ individuals, BRAF mutations were more frequent in cases with hMLH1 deficiency (58%) than those with hMSH2 deficiency (0%; p=0.02). Moreover, they were found in 69% of FM, 4% of PM and 4% of NM, revealing a striking difference between FM and the other 2 groups (FM vs. PM or NM; p < 0.0001). These findings suggest that BRAF activation may participate in the carcinogenesis of sporadic CRCs with hMLH1 hypermethylation in the proximal colon, independently of KRAS activation.

Effects of olmesartan, an angiotensin II receptor blocker, on mechanically-modulated genes in cardiac myocytes.

BACKGROUND: Angiotensin II plays an important role in cardiac hypertrophy or remodeling. Angiotensin II receptor blockers (ARB) are clinically useful for the treatment of hypertension and heart failure. However, the molecular effects of ARB in the mechanically-stressed myocardium have not been completely defined. We investigated the effects of ARB on mechanically-modulated genes in cardiac myocytes. METHODS: We used powerful DNA microarray technology to study the effects of the ARB, CS-886 (olmesartan), on genes modulated in neonatal rat cardiac myocytes using mechanical stimuli. Mechanical deformation was applied to a thin and transparent membrane on which neonatal rat cardiac myocytes were cultured in the presence or absence of RNH-6270, an active metabolite of CS-886. Expression profiles of 8000 rat genes using the Affymetrix GeneChip (Rat Genome U34A) were investigated with mRNA obtained from the samples above. RESULTS: Nine genes induced under 4% mechanical strain were significantly suppressed by RNH-6270 in rat cardiac myocytes: monoamine oxidase B, neuromedine B receptor, olfactory receptor, synaptotagmin XI, retinol-binding protein, and 4 expressed sequence tags (ESTs). In contrast, 21 genes suppressed under mechanical strain were significantly restored by RNH-6270: major acute phase alpha 1-protein, Sp-1, Bcl-Xalpha, JAK2, 2 genes encoding detoxification, few genes for receptor, structure, metabolism or ion channel, and 10 ESTs. CONCLUSIONS: As some of these genes may be involved in promoting or modulating cardiac remodeling, these findings suggest that ARB may affect cardiovascular morbidity and mortality partially via these molecular alterations.

DNA microarray analysis of stage progression mechanism in myelodysplastic syndrome.

Myelodysplastic syndrome (MDS) is a clonal disorder of haematopoietic stem cells. Despite the high incidence of MDS in the elderly, effective treatment of individuals in its advanced stages is problematic. DNA microarray analysis is a potentially informative approach to the development of new treatments for MDS. However, a simple comparison of 'transcriptomes' of bone marrow mononuclear cells among individuals at distinct stages of MDS would result in the identification of genes whose expression differences only reflect differences in the proportion of MDS blasts within bone marrow. Such a 'population shift' effect has now been avoided by purification of haematopoietic stem-like cells that are positive for the cell surface marker AC133 from the bone marrow of healthy volunteers and 30 patients at various stages of MDS. Microarray analysis with the AC133+ cells from these individuals resulted in the identification of sets of genes with expression that was specific to either indolent or advanced stages of MDS. The former group of genes included that for PIASy, which catalyses protein modification with the ubiquitin-like molecule SUMO. Induction of PIASy expression in a mouse myeloid cell line induced apoptosis. A loss of PIASy expression may therefore contribute directly to the growth of MDS blasts and stage progression.

Proteomic analysis of hematopoietic stem cell-like fractions in leukemic disorders.

DNA microarray analysis has been applied to identify molecular markers of human hematological malignancies. However, the relatively low correlation between the abundance of a given mRNA and that of the encoded protein makes it important to characterize the protein profile directly, or 'proteome,' of malignant cells in addition to the 'transcriptome.' To identify proteins specifically expressed in leukemias, here we isolated AC133(+) hematopoietic stem cell-like fractions from the bone marrow of 13 individuals with various leukemic disorders, and compared their protein profiles by two-dimensional electrophoresis. A total of 11 differentially expressed protein spots corresponding to 10 independent proteins were detected, and peptide fingerprinting combined with mass spectrometry of these proteins revealed them to include NuMA (nuclear protein that associates with the mitotic apparatus), heat shock proteins, and redox regulators. The abundance of NuMA in the leukemic blasts was significantly related to the presence of complex karyotype anomalies. Conditional expression of NuMA in a mouse myeloid cell line resulted in the induction of aneuploidy, cell cycle arrest in G(2)-M phases, and apoptosis. These results demonstrate the potential of proteome analysis with background-matched cell fractions obtained from fresh clinical specimens to provide insight into the mechanism of human leukemogenesis.

DNA microarray analysis of in vivo progression mechanism of heart failure.

Dahl salt-sensitive rats are genetically hypersensitive to sodium intake. When fed a high sodium diet, they develop systemic hypertension, followed by cardiac hypertrophy and finally heart failure within a few months. Therefore, Dahl rats represent a good model with which to study how heart failure is developed in vivo. By using DNA microarray, we here monitored the transcriptome of >8000 genes in the left ventricular muscles of Dahl rats during the course of cardiovascular damage. Expression of the atrial natriuretic peptide gene was, for instance, induced in myocytes by sodium overload and further enhanced even at the heart failure stage. Interestingly, expression of the gene for the D-binding protein, an apoptotic-related transcriptional factor, became decreased upon the transition to heart failure. To our best knowledge, this is the first report to describe the transcriptome of cardiac myocytes during the disease progression of heart failure.

Screening of genes specifically activated in the pancreatic juice ductal cells from the patients with pancreatic ductal carcinoma.

Pancreatic ductal carcinoma (PDC) is one of the most intractable human malignancies. Surgical resection of PDC at curable stages is hampered by a lack of sensitive and reliable detection methods. Given that DNA microarray analysis allows the expression of thousands of genes to be monitored simultaneously, it offers a potentially suitable approach to the identification of molecular markers for the clinical diagnosis of PDC. However, a simple comparison between the transcriptomes of normal and cancerous pancreatic tissue is likely to yield misleading pseudopositive data that reflect mainly the different cellular compositions of the specimens. Indeed, a microarray comparison of normal and cancerous tissue identified the INSULIN gene as one of the genes whose expression was most specific to normal tissue. To eliminate such a "population-shift" effect, the pancreatic ductal epithelial cells were purified by MUC1-based affinity chromatography from pancreatic juice isolated from both healthy individuals and PDC patients. Analysis of these background-matched samples with DNA microarrays representing 3456 human genes resulted in the identification of candidate genes for PDC-specific markers, including those for AC133 and carcinoembryonic antigen-related cell adhesion molecule 7 (CEACAM7). Specific expression of these genes in the ductal cells of the patients with PDC was confirmed by quantitative real-time polymerase chain reaction analysis. Microarray analysis with purified pancreatic ductal cells has thus provided a basis for the development of a sensitive method for the detection of PDC that relies on pancreatic juice, which is routinely obtained in the clinical setting.

Woman with postpartum ventricular tachycardia and hypomagnesemia.

A 28-year-old Japanese woman who had received continuous intravenous infusion of magnesium sulfate from 24 weeks of pregnancy until delivery underwent cesarean section at 30 weeks and gave birth to twins. Serum magnesium sharply declined to a subnormal level of 1.5 mg/dL on postpartum day 4. The patient exhibited sinus bradycardia (48 b.p.m.) with intermittent supraventricular contraction on postpartum day 2, intermittent ventricular bigeminy on postpartum day 3, and frequent selfterminated polymorphic ventricular tachycardia on postpartum day 4. The electrocardiogram (ECG) disclosed that the patient had prolonged QTc of 0.45-0.67. Correction of serum magnesium improved ECG findings promptly, resulting in the disappearance of arrhythmias. Hypomagnesemia due to postpartum diuresis may have played a role causing ventricular tachyarrhythmia in this patient.

HMG-CoA reductase inhibitors reduce interleukin-6 synthesis in human vascular smooth muscle cells.

Interleukin-6 (IL-6) is a key molecule in chronic inflammation and has been implicated in the progression of atherosclerosis. HMG-CoA reductase inhibitors (statins) may reduce the cardiovascular risk and vulnerability of atherosclerotic plaque through nonlipid as well as lipid-lowering mechanisms, but their anti-inflammatory effects on the vascular tissue have not been fully elucidated. We investigated the effects of fluvastatin on IL-6 synthesis in human vascular smooth muscle cells (VSMCs). Addition of fluvastatin decreased IL-6 synthesis in VSMCs in a time (0-24 hours)- and dose (10(-8)-10(-5) mol/L)-dependent manner. Fluvastatin also decreased IL-6 mRNA expression in VSMCs. The effects of fluvastatin on IL-6 expression were completely reversed in the presence of mevalonate or geranylgeranyl-pyrophosphate, but not squalene. Inhibition of Rho by C3 exoenzyme or Rho kinase by Y-27632 significantly decreased IL-6 expression in VSMCs. In conclusion, fluvastatin decreases IL-6 synthesis in human VSMCs through inhibition of Rho pathway. These findings suggested that reduction of IL-6 expression by statins may partially explain their therapeutic effects in patients with coronary artery disease.

Identification of mechanically induced genes in human monocytic cells by DNA microarrays.

BACKGROUND: Hypertension is a risk factor for coronary heart disease. Macrophages are critically involved in both atherogenesis and plaque instability. Although macrophages may be subjected to excess mechanical stress in these diseases, the way in which biomechanical forces affect macrophage function remains incompletely defined. OBJECTIVE: To investigate the molecular response to mechanical force in macrophages. DESIGN AND METHODS: We used a DNA microarray with 1056 genes to describe the transcriptional profile of mechanically induced genes in human monocytic THP-1 cells. Mechanical deformation was applied to a thin and transparent membrane on which cells were cultured. After THP-1 cells were pre-incubated in the presence of phorbol 12-myristate 13-acetate (0.2 micromol/l) for 24 h, THP-1 cells attached to the membrane were subjected to biaxial mechanical strain. Interleukin-8 concentrations were determined using an enzyme-linked immunosorbent assay. RESULTS: In DNA microarray analysis, cyclic mechanical strain at 1 Hz induced only three genes more than 2.5-fold at 3 and 6 h in THP-1 cells: prostate apoptosis response-4 (3.0-fold at 3 h, 6.7-fold at 6 h), interleukin-8 (4.3-fold at 6 h) and the immediate-early response gene, IEX-1 (2.6-fold at 6 h). Real-time reverse transcriptase polymerase chain reaction analysis confirmed the amplitude-dependent induction of these three genes. In addition, mechanical strain increased interleukin-8 protein expression. CONCLUSION: The present study demonstrates that human monocytic cells respond to mechanical deformation with induction of immediate-early and inflammatory genes. These findings suggest that mechanical stress in vivo, such as that associated with hypertension, may play an important part in atherogenesis and instability of coronary artery plaques, through biomechanical effects on vascular macrophages.