miR-1322 regulates ChREBP expression via binding a 3'-UTR variant (rs1051943).

The carbohydrate response element-binding protein (ChREBP), also referred to as MLXIPL, plays a crucial role in the regulation of glucose and lipid metabolism. Existing studies have shown an association between genetic variations of the ChREBP gene and lipid levels, such as triglycerides and high-density lipoprotein cholesterol. However, mechanistic studies of this association are limited. In this study, bioinformatic analysis revealed that the polymorphism rs1051943A occurs in the complementary binding sequence of miR-1322 in the ChREBP 3'-untranslated region (UTR). Studies of potential mechanisms showed that the A allele could facilitate miR-1322 binding, and luciferase activity significantly decreased when co-transfected with a ChREBP 3'-UTR luciferase reporter vector and miR-1322 mimics in HepG2 cells. Furthermore, miR-1322 significantly regulated the expression of ChREBP downstream genes and reduced the synthesis of lipids. The expression of miR-1322 was up-regulated by glucose and palmitic acid stimulation. Population studies showed that rs1051943-A allele was only found in the Han Chinese and Uighur ethnic groups, different from European populations (G allele frequency = 0.07). In summary, we provide evidence that the rs1051943 A allele creates a functional miR-1322 binding site in ChREBP 3'-UTR and post-transcriptionally down-regulates its expression, possibly associated with levels of plasma lipids and glucose.

The role of endothelial cell in cardiac hypertrophy: Focusing on angiogenesis and intercellular crosstalk.

Cardiac hypertrophy is characterized by cardiac structural remodeling, fibrosis, microvascular rarefaction, and chronic inflammation. The heart is structurally organized by different cell types, including cardiomyocytes, fibroblasts, endothelial cells, and immune cells. These cells highly interact with each other by a number of paracrine or autocrine factors. Cell-cell communication is indispensable for cardiac development, but also plays a vital role in regulating cardiac response to damage. Although cardiomyocytes and fibroblasts are deemed as key regulators of hypertrophic stimulation, other cells, including endothelial cells, also exert important effects on cardiac hypertrophy. More particularly, endothelial cells are the most abundant cells in the heart, which make up the basic structure of blood vessels and are widespread around other cells in the heart, implicating the great and inbuilt advantage of intercellular crosstalk. Cardiac microvascular plexuses are essential for transport of liquids, nutrients, molecules and cells within the heart. Meanwhile, endothelial cell-mediated paracrine signals have multiple positive or negative influences on cardiac hypertrophy. However, a comprehensive discussion of these influences and consequences is required. This review aims to summarize the basic function of endothelial cells in angiogenesis, with an emphasis on angiogenic molecules under hypertrophic conditions. The secondary objective of the research is to fully discuss the key molecules involved in the intercellular crosstalk and the endothelial cell-mediated protective or detrimental effects on other cardiac cells. This review provides a more comprehensive understanding of the overall role of endothelial cells in cardiac hypertrophy and guides the therapeutic approaches and drug development of cardiac hypertrophy.

Characteristics of Electrocardiogram Findings in Fulminant Myocarditis.

Fulminant myocarditis (FM) is an acute and severe form of myocarditis with rapid progression and poor clinical outcomes in the absence of acute or chronic coronary artery disease. Electrocardiogram (ECG) abnormalities can provide preliminary clues for diagnosis; however, there is a lack of systemic descriptions on ECG changes in FM populations. Thus, a retrospective analysis of 150 consecutive FM patients and 300 healthy controls was performed to determine the characteristic ECG findings in FM. All patients included had markedly abnormal ECG findings. Specifically, 83 (55.33%) patients had significantly lower voltage with remarkably decreased QRS amplitudes in all leads compared with healthy controls (p < 0.01), and 77 (51.33%) patients had a variety of arrhythmias with lethality ventricular tachycardia/ventricular fibrillation in 21 (14.00%) patients and third-degree atrioventricular block in 21 (14.00%) patients, whereas sinus tachycardia was only found in 43 (28.67%) patients with the median heart rate (HR; 88.00 bpm, IQR: 76.00-113.50) higher than that of controls (73.00 bpm, IQR: 68.00-80.00) (p = 0.000). Conduction and repolarization abnormalities were common in patients. A longer QTc interval (452.00 ms, IQR: 419.00-489.50) and QRS duration (94.00 ms, IQR: 84.00-119.00) were observed in patients compared to controls (QTc interval = 399.00 ms, IQR: 386.00-414.00; QRS duration = 90.00 ms, IQR: 86.00-98.00) (p < 0.05). Additionally, HR > 86.50 bpm, QTc > 431.50 ms, and RV5 + SV1 < 1.715 mV can be used to predict FM. Thus, marked and severe ECG abnormalities provide preliminary clues for the diagnosis of FM.

Sphingosine-1-Phosphate Protects Against the Development of Cardiac Remodeling via Sphingosine Kinase 2 and the S1PR2/ERK Pathway.

OBJECTIVE: Cardiac remodeling is a common pathological change in various cardiovascular diseases and can ultimately result in heart failure. Thus, there is an urgent need for more effective strategies to aid in cardiac protection. Our previous work found that sphingosine-1-phosphate (S1P) could ameliorate cardiac hypertrophy. In this study, we aimed to investigate whether S1P could prevent cardiac fibrosis and the associated mechanisms in cardiac remodeling. METHODS: Eight-week-old male C57BL/6 mice were randomly divided into a sham, transverse aortic constriction (TAC) or a TAC+S1P treatment group. RESULTS: We found that S1P treatment improved cardiac function in TAC mice and that the cardiac fibrosis ratio in the TAC+S1P group was significantly lower and was accompanied by a decrease in α-smooth muscle actin (α-SMA) and collagen type I (COL I) expression compared with the TAC group. We also found that one of the key S1P enzymes, sphingosine kinase 2 (SphK2), which was mainly distributed in cytoblasts, was downregulated in the cardiac remodeling case and recovered after S1P treatment in vivo and in vitro. In addition, our in vitro results showed that S1P treatment activated extracellular regulated protein kinases (ERK) phosphorylation mainly through the S1P receptor 2 (S1PR2) and spurred p-ERK transposition from the cytoplasm to cytoblast in H9c2 cells exposed to phenylephrine. CONCLUSION: These findings suggest that SphK2 and the S1PR2/ERK pathway may participate in the anti-remodeling effect of S1P on the heart. This work therefore uncovers a novel potential therapy for the prevention of cardiac remodeling.

The epoxyeicosatrienoic acid-stimulated phosphorylation of EGF-R involves the activation of metalloproteinases and the release of HB-EGF in cancer cells.

AIM: To test the hypothesis that the epoxyeicosatrienoic acid (EET)-induced transactivation of EGF-R depends on the activation of metalloproteinases and the subsequent release of HB-EGF in cancer cells. METHODS: Exogenous 14,15-EET were added to four human-derived cancer cell lines Tca-8113, A549, HepG2, and MDA-MB-231, or these same cell lines were transfected with a mutant CYP epoxygenase (CYP102 F87V, an active 14,15-epoxygenase). The effects of elevated EET levels on the phosphorylation of tyrosine residues in the EGF receptor and on ERK1/2 activation were then assessed. RESULTS: Both the addition of 14,15-EET and the transfection of cells with CYP102 F87V markedly increased the phosphorylation of the tyrosine residues of EGF-R and ERK1/2, an effect that was blocked by a selective EGF-R tyrosine kinase inhibitor (tyrphostin AG1478), a broad-spectrum metalloproteinase inhibitor (1,10-phenanthroline), and an inhibitor of HB-EGF release (CRM197) in Tca-8113 cells. In addition, AG1478, 1,10-phenanthroline, and CRM197 also inhibited the tyrosine phosphorylation of EGF-R and ERK1/2 that was induced by 14,15-EET in the A549, HepG2, and MDA-MB-231 cell lines. CONCLUSION: These results suggest that the EET-induced transactivation of EGF-R depends on activation of metalloproteinases and the subsequent release of HB-EGF in cancer cell lines.

Cytochrome p450 epoxygenase promotes human cancer metastasis.

Cytochrome P450 (CYP) epoxygenases convert arachidonic acid to four regioisomeric epoxyeicosatrienoic acids (EET), which exert diverse biological activities in a variety of systems. We previously reported that the CYP2J2 epoxygenase is overexpressed in human cancer tissues and cancer cell lines and that EETs enhance tumor growth, increase carcinoma cell proliferation, and prevent apoptosis of cancer cells. Herein, we report that CYP epoxygenase overexpression or EET treatment promotes tumor metastasis independent of effects on tumor growth. In four different human cancer cell lines in vitro, overexpression of CYP2J2 or CYP102 F87V with an associated increase in EET production or addition of synthetic EETs significantly induced Transwell migration (4.5- to 5.5-fold), invasion of cells (3- to 3.5-fold), cell adhesion to fibronectin, and colony formation in soft agar. In contrast, the epoxygenase inhibitor 17-ODYA or infection with the antisense recombinant adeno-associated viral vector (rAAV)-CYP2J2 vector inhibited cell migration, invasion, and adhesion with an associated reduction in EET production. CYP overexpression also enhanced metastatic potential in vivo in that rAAV-CYP2J2-infected MDA-MB-231 human breast carcinoma cells showed 60% more lung metastases in athymic BALB/c mice and enhanced angiogenesis in and around primary tumors compared with control cells. Lung metastasis was abolished by infection with the antisense rAAV-CYP2J2 vector. CYP epoxygenase overexpression or EET treatment up-regulated the prometastatic matrix metalloproteinases and CD44 and down-regulated the antimetastatic genes CD82 and nm-23. Together, these data suggest that CYP epoxygenase inhibition may represent a novel approach to prevent metastasis of human cancers.

Regulation of endothelial nitric-oxide synthase activity through phosphorylation in response to epoxyeicosatrienoic acids.

Endothelial nitric oxide synthase (eNOS) is a key enzyme in NO-mediated cardiovascular homeostasis and its activity is modulated by a variety of hormonal and mechanical stimuli via phosphorylation modification. Our previous study has demonstrated that epoxyeicosatrienoic acids (EETs), the cytochrome P450 (CYP)-dependent metabolites of arachidonic acid, could robustly up-regulate eNOS expression. However, the molecular mechanism underlying the effects of EETs on eNOS remains elusive. Particularly, whether and how EETs affect eNOS phosphorylation is unknown. In the present study, we investigated the effects of EETs on eNOS phosphorylation with cultured bovine aortic endothelial cells (BAECs). BAECs were either treated with exogenous EETs or infected with recombinant adeno-associated virus (rAAV) carrying CYP2C11-CYPOR, CYP102 F87V mutant and CYP2J2, respectively, to increase endogenous EETs. Both addition of EETs and CYP epoxygenase transfection markedly increased eNOS phosphorylation at its Ser1179 and Thr497 residues. Inhibition of phosphatidylinositol 3-kinase (PI3K) with LY294002 prevented EETs-induced increases of eNOS-Ser(P)1179 but had no effect on the phosphorylation status of Thr497. However, inhibitors of protein kinase B (Akt), mitogen-activated protein kinase (MAPK) and MAPK kinase could block phosphorylation of eNOS at both sites. Inhibition of these kinases also attenuated the up-regulation of eNOS expression by EETs. Finally, administration of viral CYP epoxygenases expression vectors into rats enhanced eNOS phosphorylation and function in vivo. Thus, in addition to up-regulating eNOS expression, EETs also augment eNOS function by enhancing eNOS phosphorylation. EETs-induced up-regulation of eNOS phosphorylation and expression appears to involve in both PI3K/Akt and MAPK pathways.

Cytochrome P450 2J2 promotes the neoplastic phenotype of carcinoma cells and is up-regulated in human tumors.

Cytochrome P450 (CYP) arachidonic acid epoxygenase 2J2 converts arachidonic acid to four regioisomeric epoxyeicosatrienoic acids, which exert diverse biological activities in cardiovascular system and endothelial cells. However, it is unknown whether this enzyme highly expresses and plays any role in cancer. In this study, we found that very strong and selective CYP2J2 expression was detected in human carcinoma tissues in 101 of 130 patients (77%) as well as eight human carcinoma cell lines but undetectable in adjacent normal tissues and nontumoric human cell lines by Western, reverse transcription-PCR, and immunohistochemical staining. In addition, forced overexpression of CYP2J2, and CYP BM3F87V or addition of epoxyeicosatrienoic acids (EET) in cultured carcinoma cell lines in vitro markedly accelerated proliferation by analyses of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, cell accounts, and cell cycle analysis, and protected carcinoma cells from apoptosis induced by tumor necrosis factor alpha (TNF-alpha) in cultures. In contrast, antisense 2J2 transfection or addition of epoxygenase inhibitors 17-ODYA inhibited proliferation and accelerated cell apoptosis induced by TNF-alpha. Examination of signaling pathways on the effects of CYP2J2 and EETs revealed activation of mitogen-activated protein kinases and PI3 kinase-AKT systems and elevation of epithelial growth factor receptor phosphorylation level. These results strongly suggest that CYP epoxygenase 2J2 plays a previously unknown role in promotion of the neoplastic cellular phenotype and in the pathogenesis of a variety of human cancers.

An APOC3 3'UTR variant associated with plasma triglycerides levels and coronary heart disease by creating a functional miR-4271 binding site.

Apolipoprotein C-III (APOC3) is a key regulator of plasma triglycerides levels. Increasing evidence has shown that loss-of-function mutations in APOC3 is associated with reduction in plasma triglycerides levels and will confer a benefit in patients at high risk for cardiovascular disease. However, these favorable mutations were extremely distribution discrepant among different ethnics. In this study, the APOC3 gene was resequenced and we identified a common variant which located in the microRNA-binding site in APOC3 and would affect its expression and the risk of coronary heart disease (CHD). The molecular mechanism was explored. We found that the T allele of rs4225 suppressed APOC3 translation by facilitating miR-4271 binding, but not the G allele. Subjects carrying the GG genotype had higher plasma APOC3 levels (p for trend = 0.03) than those with the TT genotype. Furthermore, the T allele was significantly associated with decreased triglyceride levels [Beta (SE): -0.024 (0.020), P = 0.03]. Finally, the case-control study suggested that the TT genotype resulted in a significant reduction in overall CHD risk [OR, 0.89 (95% confidence interval, 0.77-0.98), P = 0.009]. In conclusion, our results provide evidence that the rs4225 in the 3'-UTR of APOC3 might contribute to the risk of CHD by interfering with miR-4271 binding.

Expression of cyclooxygenase-2 in human esophageal squamous cell carcinomas.

AIM: To determine whether cyclooxygenase-2 (COX-2) was expressed in human esophageal squamous cell carcinoma. METHODS: Quantitative reverse transcription-polymerase chain reaction (RT-PCR), western blotting, immunohistoc-hemistry and immunofluorescence were used to assess the expression level of COX-2 in esophageal tissue. RESULTS: COX-2 mRNA levels were increased by >80-fold in esophageal squamous cell carcinoma when compared to adjacent noncancerous tissue. COX-2 protein was present in 21 of 30 cases of esophageal squamous cell carcinoma tissues, but was undetectable in noncancerous tissue. Immunohistochemistry was performed to directly show expression of COX-2 in tumor tissue. CONCLUSION: These results suggest that COX-2 may be an important factor for esophageal cancer and inhibition of COX-2 may be helpful for prevention and possibly treatment of this cancer.

Expression of cytochrome P450 arachidonic acid epoxygenase 2J2 in human tumor tissues and cell lines.

BACKGROUND AND OBJECTIVE: Cytochrome P450 arachidonic acid epoxygenase 2J2 (CYP2J2) is a new metabolic pathway of arachidonic acid. However, its biological effects, especially pathophysiologic significance in human beings, remain to be further recognized. This study was to determine the expression of CYP2J2 in human tumor tissues and cell lines. METHODS: The expression of CYP2J2 mRNA and protein in 130 specimens of human carcinoma and related adjacent normal tissues, four specimens of inflammatory pseudotumor tissues, eight human tumor cell lines and two normal cell lines (as control) was detected by reverse transcription-polymerase chain reaction (RT-PCR), western blot and immunohistochemistry. RESULTS: CYP2J2 was highly expressed in 101 (78%) carcinoma tissues, but was not detected in adjacent normal tissues and inflammatory pseudo-tumor tissues. Its mRNA level was obviously correlated to its protein level (r = 0.613, p < 0.01). Immunohistochemistry analysis showed the same results as RT-PCR and western blot. Furthermore, CYP2J2 was only expressed in cancer cells but not in interstitial and inflammatory cells. CYP2J2 was highly expressed in all carcinoma cell lines, but not in two normal cell lines. CONCLUSION: CYP2J2 is highly and selectively expressed in human tumor tissues and cell lines and may be a novel biomarker of human tumors.

Effects of cytochrome P450 arachidonic acid epoxygenases on the proliferation of tumor cells.

BACKGROUND AND OBJECTIVE: Cytochrome P450 (CYP) arachidonic acid epoxygenases promote cell proliferation and inhibit apoptosis in endothelial cells. This study was to investigate the effects of CYP epoxygenases on the proliferation of tumor cells and possible signaling pathways. METHODS: The effects of recombinant adeno-associated virus (rAAV) mediated cytochrome P450 2J2 (CYP2J2), cytochrome P450 F87V (CYPF87V) and anti-CYP2J2 on proliferation of Tca-8113, A549, Ncl-H446 and HepG2 cells were measured using MTT and flow cytometry. Expressions of phosphorylated epidermal growth factor receptor (EGFR), extracellular signal-regulated kinase (ERK)1/2 and Akt before and after transfection were detected by western blot. Tca-8113 cells infected with rAAV-CYP2J2, rAAV-CYPF87V, rAAV-antiCYP2J2 and rAAV-GFP were inoculated into nude mice, to observe the effect of CYP epoxygenases on the growth of xenografts. RESULTS: Infection of Tca-8113, A549, Ncl-H446 and HepG2 cells with rAAV-CYP2J2 and rAAVCYPF87V significantly increased the proliferation of tumor cells by 1.7-, 1.4-, 1.6- and 2.2-fold, and 2.0-, 1.5-, 1.8- and 2.0-fold respectively, as compared with control cells. On the contrary, infection with rAAV-antiCYP2J2 inhibited the proliferation of the four tumor cell lines. Moreover, CYP epoxgenases remarkably enhanced phosphorylation of EGFR, ERK1/2 and Akt, and upregulated total PI3K by 2-, 2.3-, 2.4- and 1.9-fold in the four cell lines, while rAAV-antiCYP2J2 exerted an inhibition effect. Infection of CYP450 epoxygenase genes markedly increased the cell percentage in S/G(2)/M phases by 210% as compared to control Tca-8113 cells. rAAV-CYP2J2 and rAAV-CYPF87V promoted tumor growth of Tca-8113 cell xenografts in nude mice in comparison to the control and rAAV-antiCYP2J2 groups. CONCLUSION: CYP epoxygenases efficiently promote the proliferation of tumor cells, which may be related with the activation of EGFR, ERK1/2 and PI3K/Akt signaling pathways.

[Promotive effects of epoxyeicosatrienoic acids (EETs) on proliferation of tumor cells].

BACKGROUND & OBJECTIVE: Epoxyeicosatrienoic acids (EETs) are generated from arachidomic acid by cytochrome P450(CYP). Previous studies revealed very strong and selective expression of CYP expoxygenase in human cancer tissues, but almost none in adjacent normal tissues. This study was to investigate the promotive effect of EETs on proliferation of tumor cells and the possible mechanisms. METHODS: Four tumor cell lines, Tca-8113, A549, Ncl-H446 and HepG2, were treated with different concentrations of EETs (8,9-EET, 11,12-EET and 14,15-EET) for 12, 24, 48 and 72 h, respectively. Cell proliferation was measured using the MTT assay. The effect of exogenous EETs on cell cycle of Tca-8113 cells was assessed by flow cytometry. Signal transduction inhibitors of PI3K (LY294002), MAPKK (PD98059), MAPK (apigenin) and PKC (H7) were used to block EETs-induced cell proliferation. Expressions of the total protein and phosphorylated ERK1/2 and Akt were determined by Western blot. RESULTS: EETs promoted proliferation of tumor cells compared with the control and vehicle group in a dose-and time-dependent manner (P<0.01). Incubation of tumor cells with EETs markedly increased the cell number at S/G2-M phase. The percentages of Tca-8113 cells at S and G2-M phases were (49.7+/-7.5%) vs. (17.2+/-9.7%) (P<0.01) and (21.0+/-5.3%) vs. (4.9+/-7.3%), respectively(P<0.01) with and without the treatment of 11,12-EET. EETs incubation significantly enhanced phosphorylation of MARK as well as PI3K/Akt in tumor cells. LY294002, PD98059, apigenine and H7 reduced the stimulative effect of EETs on cell proliferation. CONCLUSION: EETs possess the promotive effect on proliferation of tumor cells via activation of MAPK and PI3K/Akt signal pathways.