Background: Expression of the constitutively activated mutant EGFR variant III (EGFRvIII), the most common mutation in glioblastoma multiforme (GBMs), has been clinically correlated with tumor proliferation, invasion, and angiogenesis. In this study, we examined the role of EGFRvIII on the tumor microenvironment, especially on angiogenesis.Methods: To study the role of EGFRvIII in tumor angiogenesis, we prepared LN229 glioblastoma transfected with enhanced green fluorescent protein (EGFP), wild-type EGFR, or EGFRvIII (LN229-WT or -vIII), and examined tumor growth and microvessel density in the tumors. Additionally, the potential angiogenic factors were identified by real-time PCR analysis, and the functions in LN229-vIII cells were examined.Results: LN229-vIII cells showed more aggressive tumor growth and higher vascularity as compared to LN229-WT cells in vivo, although there was no significant difference in the cell growth rates in vitro. We next investigated the expression of 60 angiogenesis-related factors to clarify the mechanisms underlying the difference in vascularity between tumor xenografts of LN229-vIII and LN229-WT. We found that the mRNA and protein expressions of angiopoietin-like 4 (Angptl4), a secreted protein involved in angiogenesis and metabolism regulation, were significantly induced by EGFRvIII overexpression, both in vitro and in vivo. Constitutive knockdown of Angptl4 in LN229-vIII using shRNA significantly decreased the microvessel density in the tumor xenografts and suppressed tumor growth. To clarify the regulatory mechanisms of Angptl4 by EGFRvIII, we analyzed the signaling pathways and transcription factors by pharmacological inhibition and RNA interference. U0126, an ERK signal inhibitor dramatically suppressed Angptl4 expression. The transcription factor c-Myc, which is regulated by ERK, was activated in the LN229-vIII cells and knockdown of c-Myc using siRNA also attenuated Angptl4 expression in the LN229-vIII cells. Furthermore, chromatin immunoprecipitation (ChIP) assay revealed increased recruitment of c-Myc to the promoter region of Angptl4 in the LN229-vIII cells.Conclusions: In summary, we demonstrated that EGFRvIII induces Angptl4 expression through the ERK/c-Myc pathway and promotes tumor angiogenesis in malignant gliomas.

—Endothelin-1 (ET-1) acts not only as a growth-promoting peptide but also as a potent survival factor against myocardial cell apoptosis. However, the signaling pathways leading to myocardial cell protection by ET-1 are poorly understood. Using a culture system of primary cardiac myocytes derived from neonatal rats, we show in the present study that ET-1 almost completely blocked the hydrogen peroxide–induced increase in the percentage of TdT-mediated dUTP-biotin nick-end labeling–positive myocytes. Apoptosis inhibition by ET-1 was confirmed by cytofluorometric analysis as well as by examination of the ladder formation, morphological features, and caspase-3 cleavage. We have found that ET-1 converts the nuclear factor of activated T lymphocytes (NFATc) in cardiac myocytes into high-mobility forms and translocates cytoplasmic NFATc to the nuclei. In addition, ET-1 stimulates the interaction between NFATc and the cardiac-restricted zinc-finger protein GATA4 in these cells. The immunosuppressants cyclosporin A and FK506, which antagonize calcineurin, negated the inhibitory effect of ET-1 on apoptosis. Calcineurin activation de novo was sufficient to inhibit hydrogen peroxide–induced apoptosis. ET-1 induced the expression of an antiapoptotic protein bcl-2 in cardiac myocytes in a cyclosporin A–dependent manner, but it did not alter the expression of bax. Cyclosporin A also attenuated the ET-1–stimulated transcription of the bcl-2 gene in these cells. These findings demonstrate that the calcineurin pathway is required for the inhibitory effect of ET-1 on oxidant stress–induced apoptosis in cardiac myocytes.

Chronic inflammation plays a significant role in lifestyle-related diseases, such as cardiovascular diseases and obesity/impaired glucose tolerance. Curcumin is a natural extract that possesses numerous physiological properties, as indicated by its anti-inflammatory action. The mechanisms underlying these effects include the inhibition of nuclear factor-kappaB and Toll-like receptor 4-dependent signalling pathways and the activation of a peroxisome proliferator-activated receptor-gamma pathway. However, the bioavailability of curcumin is very low in humans. To resolve this issue, several drug delivery systems have been developed and a number of clinical trials have reported beneficial effects of curcumin in the management of inflammation-related diseases. It is expected that evidence regarding the clinical application of curcumin in lifestyle-related diseases associated with chronic inflammation will accumulate over time.

Purpose: COPD is mainly caused by tobacco smoking and is associated with a high frequency of coronary artery disease. There is growing recognition that the inflammation in COPD is not only confined to the lungs but also involves the systemic circulation and can impact nonpulmonary organs, including blood vessels. α1-antitrypsin–low-density lipoprotein (AT-LDL) complex is an oxidatively modified LDL that accelerates atherosclerosis. Curcumin, one of the best-investigated natural products, is a powerful antioxidant. However, the effects of curcumin on AT-LDL remain unknown. We hypothesized that Theracurmin^®, a highly absorptive curcumin with improved bioavailability using a drug delivery system, ameliorates the inflammatory status in subjects with mild COPD. Patients and methods: This is a randomized, double-blind, parallel-group study. Subjects with stages I–II COPD according to the Japanese Respiratory Society criteria were randomly assigned to receive 90 mg Theracurmin^® or placebo twice a day for 24 weeks, and changes in inflammatory parameters were evaluated. Results: There were no differences between the Theracurmin^® and placebo groups in terms of age, male/female ratio, or body mass index in 39 evaluable subjects. The percent changes in blood pressure and hemoglobin A1c and LDL-cholesterol, triglyceride, or high-density lipoprotein-cholesterol levels after treatment were similar for the two groups. However, the percent change in the AT-LDL level was significantly (P=0.020) lower in the Theracurmin^® group compared with the placebo group. Conclusion: Theracurmin^® reduced levels of atherosclerotic AT-LDL, which may lead to the prevention of future cardiovascular events in mild COPD subjects.

Oxidative stress is enhanced by various mechanisms. Serum oxidized low-density lipoprotein (LDL) is a useful prognostic marker in diabetic patients with coronary artery disease. To examine the effects of Theracurmin®, a highly absorbable curcumin preparation, on glucose tolerance, adipocytokines, and oxidized LDL, we conducted a double-blind placebo-controlled parallel group randomized trial in patients with impaired glucose tolerance or non-insulin-dependent diabetes mellitus. We randomly divided the patients with impaired glucose tolerance or non-insulin-dependent diabetes mellitus and stable individuals into the placebo group and the Theracurmin® (180 mg daily for 6 months) group. Of the 33 patients analyzed, 18 (14 males and 4 females) were administered placebo and 15 (9 males and 6 females) were administered Theracurmin®. The patient characteristics did not differ between the two groups. The primary endpoint, HbA1c, did not differ significantly between the two groups. However, the level of α1-antitrypsin-low-density lipoprotein (AT-LDL), the oxidized LDL, significantly increased (p=0.024) in the placebo group from the beginning of the trial up to 6 months, although there was no such change in the Theracurmin® group. The percentage change in BMI from the beginning of the trial up to 6 months tended to be higher in the Theracurmin® group than in the placebo group. Patients in the Theracurmin® group tended to have a larger percentage change in adiponectin and LDL-C than those in the placebo group. Patients in the Theracurmin® group showed a smaller percentage change in AT-LDL than those in the placebo group. This study suggests that the highly absorbable curcumin could potentially inhibit a rise in oxidized LDL in patients with impaired glucose tolerance or non-insulin-dependent diabetes mellitus. This trial is registered with UMIN000007361.

Hypertensive heart disease and post-myocardial-infarction heart failure (HF) are leading causes of cardiovascular mortality in industrialized countries. To date, pharmacological agents that block cell surface receptors for neurohormonal factors have been used, but despite such conventional therapy, HF is increasing in incidence worldwide. During the development and deterioration process of HF, cardiomyocytes undergo maladaptive hypertrophy, which markedly influences their gene expression. Regulation of histone acetylation by histone acetyltransferase (eg, p300) and histone deacetylase plays an important role in this process. Increasing evidence suggests that the excessive acetylation of cardiomyocyte nuclei is a hallmark of maladaptive cardiomyocyte hypertrophy. Curcumin inhibits p300-mediated nuclear acetylation, suggesting its usefulness in HF treatment. Clinical application of this natural compound, which is inexpensive and safe, should be established in the near future. (Circ J 2010; 74: 1059 - 1066)