Protein kinase C beta II upregulates intercellular adhesion molecule-1 via mitochondrial activation in cultured endothelial cells

Activation of protein kinase C (PKC) is closely linked with endothelial dysfunction. However, the effect of PKCβII on endothelial dysfunction has not been characterized in cultured endothelial cells. Here, using adenoviral PKCβII gene transfer and pharmacological inhibitors, the role of PKCβII on endothelial dysfucntion was investigated in cultured endothelial cells. Phorbol 12-myristate 13-acetate (PMA) increased reactive oxygen species (ROS), p66shc phosphorylation, intracellular adhesion molecule-1, and monocyte adhesion, which were inhibited by PKCβi (10 nM), a selective inhibitor of PKCβII. PMA increased the phosphorylation of CREB and manganese superoxide dismutase (MnSOD), which were also inhibited by PKCβi. Gene silencing of CREB inhibited PMA-induced MnSOD expression, suggesting that CREB plays a key role in MnSOD expression. Gene silencing of PKCβII inhibited PMA-induced mitochondrial ROS, MnSOD, and ICAM-1 expression. In contrast, overexpression of PKCβII using adenoviral PKCβII increased mitochondrial ROS, MnSOD, ICAM-1, and p66shc phosphorylation in cultured endothelial cells. Finally, PKCβII-induced ICAM-1 expression was inhibited by Mito-TEMPO, a mitochondrial ROS scavenger, suggesting the involvement of mitochondrial ROS in PKC-induced vascular inflammation. Taken together, the results suggest that PKCβII plays an important role in PMA-induced endothelial dysfunction, and that the inhibition of PKCβII-dependent p66shc signaling acts as a therapeutic target for vascular inflammatory diseases.

Dynamic Regulation of APE1/Ref-1 as a Therapeutic Target Protein / 전남의대학술지

Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is a multifunctional protein that plays a central role in the cellular response to DNA damage and redox regulation against oxidative stress. APE1/Ref-1 functions in the DNA base excision repair pathway, the redox regulation of several transcription factors, and the control of intracellular redox status through the inhibition of reactive oxygen species (ROS) production. APE1/Ref-1 is predominantly localized in the nucleus; however, its subcellular localization is dynamically regulated and it may be found in the mitochondria or elsewhere in the cytoplasm. Studies have identified a nuclear localization signal and a mitochondrial target sequence in APE1/Ref-1, as well as the involvement of the nuclear export system, as determinants of APE1/Ref-1 subcellular distribution. Recently, it was shown that APE1/Ref-1 is secreted in response to hyperacetylation at specific lysine residues. Additionally, post-translational modifications such as phosphorylation, S-nitrosation, and ubiquitination appear to play a role in fine-tuning the activities and subcellular localization of APE1/Ref-1. In this review, we will introduce the multifunctional role of APE1/Ref-1 and its potential usefulness as a therapeutic target in cancer and cardiovascular disease.

Altered Secretory Activity of APE1/Ref-1 D148E Variants Identified in Human Patients With Bladder Cancer / 대한배뇨장애요실금학회지

PURPOSE: Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is a multifunctional protein involved in DNA repair and redox modulation. Recently, serum and urinary APE1/Ref-1 levels were reported to be increased in patients with bladder cancer. Genetic variations of APE/Ref-1 are associated with the risk of cancer. However, the effect of APE1/Ref-1 variants on its secretory activity is yet unknown. METHODS: APE1/Ref-1 variants were evaluated by DNA sequencing analysis of reverse transcription polymerase chain reaction products in coding DNA sequences (CDS) of APE1/Ref-1 in bladder tissue samples from patients with bladder cancer (n=10). Secretory activity of APE1/Ref-1 variants was evaluated with immunoblot and enzyme-linked immunosorbent assay of the culture medium supernatants. RESULTS: Four different substitution mutants (D148E, I64V/D148E, W67R/D148E, and E86G/D148E) of APE1/Ref-1 were identified in bladder cancer specimens. However, deletion mutants of APE1/Ref-1 CDS were not found. The secretory activity of the APE1/Ref-1 variants (D148E, I64V/D148E, and E86G/D148E) was increased compared to that of wild type APE1/Ref-1. Furthermore, the secretory activity in basal or hyperacetylated conditions was much higher than that in APE1/Ref-1 D148E-transfected HEK293 cells. CONCLUSIONS: Taken together, our data suggest that the increased secretory activity of D148E might contribute to increased serum levels of APE1/Ref-1 in patients with bladder cancer.

Dynamic Regulation of APE1/Ref-1 as a Therapeutic Target Protein / 전남의대학술지

Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is a multifunctional protein that plays a central role in the cellular response to DNA damage and redox regulation against oxidative stress. APE1/Ref-1 functions in the DNA base excision repair pathway, the redox regulation of several transcription factors, and the control of intracellular redox status through the inhibition of reactive oxygen species (ROS) production. APE1/Ref-1 is predominantly localized in the nucleus; however, its subcellular localization is dynamically regulated and it may be found in the mitochondria or elsewhere in the cytoplasm. Studies have identified a nuclear localization signal and a mitochondrial target sequence in APE1/Ref-1, as well as the involvement of the nuclear export system, as determinants of APE1/Ref-1 subcellular distribution. Recently, it was shown that APE1/Ref-1 is secreted in response to hyperacetylation at specific lysine residues. Additionally, post-translational modifications such as phosphorylation, S-nitrosation, and ubiquitination appear to play a role in fine-tuning the activities and subcellular localization of APE1/Ref-1. In this review, we will introduce the multifunctional role of APE1/Ref-1 and its potential usefulness as a therapeutic target in cancer and cardiovascular disease.

Elevation of the Serum Apurinic/Apyrimidinic Endonuclease 1/Redox Factor-1 in Coronary Artery Disease

BACKGROUND AND OBJECTIVES: Apurinic/apyrimidinic endonuclease 1/redox effector factor-1 (APE1/Ref-1) is a multifunctional protein involved in the DNA base excision repair pathway, inflammation, angiogenesis, and survival pathways. We investigated serum APE1/Ref-1 in patients with coronary artery disease (CAD). SUBJECTS AND METHODS: Serum APE1/Ref-1 was measured with a sandwich enzyme-linked immunosorbent assay from 360 patients who received coronary angiograms. They were divided into two groups; a control (n=57) and a CAD group (n=303), the latter included angina (n=128) and myocardial infarction (MI, n=175). RESULTS: The levels of APE1/Ref-1 were higher in the CAD than the control (0.63+/-0.07 vs. 0.12+/-0.07 ng/100 microL, respectively; p<0.01). They were also higher in MI than angina (0.81+/-0.10 vs. 0.38+/-0.11 ng/100 microL, respectively; p<0.01) and different according to the thrombolysis in myocardial infarction (TIMI) flow (0.88+/-0.09 for TIMI flow 0, 1, 2 vs. 0.45+/-0.13 ng/100 microL for TIMI flow 3, p<0.01) in acute coronary syndrome. In correlation analysis, the levels of APE1/Ref-1 were positively correlated with Troponin I (r=0.222; p<0.0001) and N-terminal pro-B type natriuretic peptide (NT-proBNP, r=0.217; p<0.0001) but not high sensitivity to C-reactive protein. Also, they revealed a negative correlation with ejection fraction (EF, r=-0.221; p=0.002). However, there were no significant differences among the three groups, were divided by their levels of APE1/Ref-1, for major adverse cardiovascular events (death, recurrent MI, stroke, revascularization) (8.2 vs. 14.0 vs. 12.5%, p=ns). CONCLUSION: The levels of serum APE1/Ref-1 are elevated in CAD, and are higher in MI than in angina. They are correlated with Troponin I, NT-proBNP, and EF.

Trichostatin A Modulates Angiotensin II-induced Vasoconstriction and Blood Pressure Via Inhibition of p66shc Activation

Histone deacetylase (HDAC) has been recognized as a potentially useful therapeutic target for cardiovascular disorders. However, the effect of the HDAC inhibitor, trichostatin A (TSA), on vasoreactivity and hypertension remains unknown. We performed aortic coarctation at the inter-renal level in rats in order to create a hypertensive rat model. Hypertension induced by abdominal aortic coarctation was significantly suppressed by chronic treatment with TSA (0.5 mg/kg/day for 7 days). Nicotinamide adenine dinucleotide phosphate-driven reactive oxygen species production was also reduced in the aortas of TSA-treated aortic coarctation rats. The vasoconstriction induced by angiotensin II (Ang II, 100 nM) was inhibited by TSA in both endothelium-intact and endothelium-denuded rat aortas, suggesting that TSA has mainly acted in vascular smooth muscle cells (VSMCs). In cultured rat aortic VSMCs, Ang II increased p66shc phosphorylation, which was inhibited by the Ang II receptor type I (AT1R) inhibitor, valsartan (10 microM), but not by the AT2R inhibitor, PD123319. TSA (1~10 microM) inhibited Ang II-induced p66shc phosphorylation in VSMCs and in HEK293T cells expressing AT1R. Taken together, these results suggest that TSA treatment inhibited vasoconstriction and hypertension via inhibition of Ang II-induced phosphorylation of p66shc through AT1R.

APE1/Ref-1 as a Serological Biomarker for the Detection of Bladder Cancer / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is a multifunctional protein that shows elevated expression in a number of cancers. We attempted to determine whether serum APE1/Ref-1 is elevated in patients with bladder cancer. MATERIALS AND METHODS: Serum APE1/Ref-1 levels were determined using enzyme-linked immunosorbent assay in serum from patients with bladder cancer who had not received chemotherapy or radiotherapy (n=51) and non-tumor controls (n=55). The area under the receiver operating characteristic area under the curve was applied to determine the correlation between clinical factors and the serum levels of APE1/Ref-1. RESULTS: Serum levels of APE1/Ref-1 in bladder cancer patients were significantly elevated compared to those of the control group (3.548+/-0.333 ng/100 muL [n=51] for bladder cancer vs. 1.547+/-0.319 ng/100 muL [n=55] for the control group), with a sensitivity and specificity of 93% and 59%, respectively. Serum APE1/Ref-1 levels are associated with tumor stage, grade, muscle invasion, and recurrence. CONCLUSION: Serum APE1/Ref-1 might be useful as a potential serologic biomarker for bladder cancer.

A standardized bamboo leaf extract inhibits monocyte adhesion to endothelial cells by modulating vascular cell adhesion protein-1

Bamboo leaves (Phyllostachys pubescens Mazel ex J. Houz (Poacea)) have a long history of food and medical applications in Asia, including Japan and Korea. They have been used as a traditional medicine for centuries. We investigated the mechanism of anti-inflammatory activity of a bamboo leaf extract (BLE) on tumor necrosis factor-alpha (TNF-alpha)-induced monocyte adhesion in human umbilical vein endothelial cells (HUVECs). Exposure of HUVECs to BLE did not inhibit cell viability or cause morphological changes at concentrations ranging from 1 microg/ml to 1 mg/ml. Treatment with 0.1 mg/ml BLE caused 63% inhibition of monocyte adhesion in TNF-alpha-activated HUVECs, which was associated with 38.4% suppression of vascular cell adhesion molecule-1 expression. Furthermore, TNF-alpha-induced reactive oxygen species generation was decreased to 47.9% in BLE treated TNF-alpha-activated HUVECs. BLE (0.05 mg/ml) also caused about 50% inhibition of interleukin-6 secretion from lipopolysaccharide-stimulated monocyte. The results indicate that BLE may be clinically useful as an anti-inflammatory or anti-oxidant for human cardiovascular disease including atherosclerosis.

Redox Regulating Protein APE1/Ref-1 Expression is Increased in Abdominal Aortic Coarctation-induced Hypertension Rats

BACKGROUND: Aim of study is designed to investigate whether apurinic/apyrimidinic endonuclease-1/redox factor-1 (APE1/Ref-1) expression is changed in abdominal aortic coarctation models. METHODS: Male Sprague-Dawley rats were randomly assigned with abdominal aortic coarctation, repaired group, sham, and control groups. Endothelial function was assessed with endothelium-dependent relaxations. Detection of superoxide anion and lipid peroxidation was performed by lucigenin chemiluminescence and thiobarbituric acid-reactive substances assay. APE1/Ref-1 expression was measured with Western blot and immunohistochemistry. RESULTS: In anesthetized condition, the abdominal aortic coarctation rats showed hypertension as systolic/diastolic arterial pressure of 171/114 mm Hg, compared with 114/94 mm Hg of control. Endothelium-dependent relaxations were significantly impaired in the aortic coarctation which was recovered in 1 week after coarctation repair. Superoxide production and lipid peroxidation were elevated in aortic coarctation rats. In immunohistochemistry, APE1/Ref-1 expressions were increased at aorta and kidney in aortic coarctation rats. Increased APE1/Ref-1 expression in aorta was recovered by repair of coarctation. CONCLUSIONS: Taken together, it suggests that APE1/Ref-1 expression was increased in aortic coarctation-induced hypertensive rats, suggesting a biomarker for hypertension. Impaired endothelium dependent relaxation in the aortic coarctation can be modulated by repair of coarctation or the modulation of blood pressure.

Vasorelaxing Activity of Ulmus davidiana Ethanol Extracts in Rats: Activation of Endothelial Nitric Oxide Synthase

Ulmus davidiana var. japonica Rehder (Urticales: Ulmaceae) (UD) is a tree widespread in northeast Asia. It is traditionally used for anticancer and anti-inflammatory therapy. The present study investigated the effect of an ethanol extract of UD on vascular tension and its underlying mechanism in rats. The dried root bark of UD was ground and extracted with 80% ethanol. The prepared UD extract was used in further analysis. The effect of UD on the cell viability, vasoreactivity and hemodynamics were investigated using propidium iodide staining in cultured cells, isometric tension recording and blood pressure analysis, respectively. Low dose of UD (10~100microg/ml) did not affect endothelial cell viability, but high dose of UD reduced cell viability. UD induced vasorelaxation in the range of 0.1~10microg/ml with an ED50 value of 2microg/ml. UD-induced vasorelaxation was completely abolished by removal of the endothelium or by pre-treatment with L-NAME, an inhibitor of nitric oxide synthase. UD inhibited calcium influx induced by phenylephrine and high K+ and also completely abolished the effect of L-NAME. Intravenous injection of UD extracts (10~100 mg/kg) decreased arterial and ventricular pressure in a dose-dependent manner. Moreover, UD extracts reduced the ventricular contractility (+dP/dt) in anesthetized rats. However, UD-induced hypotensive actions were minimized in L-NAME-treated rats. Taken together, out results showed that UD induced vasorelaxation and has antihypertensive properties, which may be due the activation of nitric oxide synthase in endothelium.

Development of a Method for the Immunological Measurement of Aspartate Aminotransferase with Monoclonal Antibodies / 대한간학회지

BACKGROUND/AIMS: For laboratory diagnostics in liver diseases, many enzymes have been used for the assessment of hepatocellular function. Among them, two transaminases, alanine and aspartate aminotransferase, have been regarded as the most sensitive indicators of hepatocellular damage. However, the enhanced enzyme activities of the enzymes do not exactly indicate or represent the cause and progression of diseases in the patients with liver disease. To overcome such limitations, immunological methods have been suggested as one of the alternatives for the replacement or supplement of the conventional enzymatic analysis. METHODS: In the hope of developing a new assay system for measuring the AST concentration rather than its activity, we have developed a new assay using fluorescence labeled anti-AST monoclonal antibodies. Blood was obtained from a normal population of 234 patients and 43 liver disease patients. The linearity, limit of detection, and performance of the new assay system were tested and evaluated. The comparability of assay was examined with an ELISA and biochemical assays. RESULTS: The linearity fell in the range of 0-1 mg/L of AST (R=0.995), and the analytical detection limit was 12 microgram/L of AST. The mean recovery of the control was 102.4 % in a working range. The precision of the intra- and inter-assay in a range of 50-800 microgram/L was CVs < 7% and CVs < 6%, respectively. In the normal population, the mean AST concentration was 35.5 microgram/L. The mean AST concentration in patients with liver disease was 266.5 microgram/L. The new assay system correlated well with an ELISA and biochemical assay for quantification of AST concentration (R=0.92 and 0.88, respectively; N=43). CONCLUSIONS: We have developed a new immunological assay using generated monoclonal antibodies to human cytosolic AST and used them for the development of a fluorescent assay measuring the enzyme mass. Cytosolic AST mass in sera could be measured reproducibly by the immunological method. In conclusion, this study has provided us with a new type of tool for an accurate measurement of the enzyme amount in circulation.