FOXO1 impairs whereas statin protects endothelial function in diabetes through reciprocal regulation of Kruppel-like factor 2.
Krüppel-like factor 2 (KLF2) is implicated as a key molecule maintaining endothelial function. This study was designed to evaluate the reciprocal regulation of KLF2 by the forkhead transcription factor FOXO1, and the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor atorvastatin, in hyperglycaemic conditions.



Exposure of human umbilical vein endothelial cells to 30 mM glucose activated FOXO1 and suppressed KLF2. These effects were reversed by FOXO1 small interfering RNA. Adenoviral transfection of constitutively active FOXO1 suppressed KLF2 expression. Interestingly, atorvastatin inhibited FOXO1 by increasing phosphorylation and also by inhibiting nuclear localization and replenished KLF2 in high-glucose conditions. This effect of atorvastatin was attenuated by mevalonate. Chromatin immunoprecipitation analysis demonstrated that glucose increased whereas atorvastatin decreased FOXO1 binding to the promoter region of the KLF2 gene. In the vessels of Otsuka Long-Evans Tokushima Fatty rats, animal models of type 2 diabetes, FOXO1 was activated and KLF2 was suppressed, and this was reversed by atorvastatin treatment. The arteries from Otsuka Long-Evans Tokushima Fatty rats showed impairment of endothelium-dependent vasodilatation, and both atorvastatin and KLF2 gene therapies restored it.



Suppression of KLF2 by FOXO1 may be a plausible mechanism of diabetic endothelial dysfunction. High-glucose-induced, FOXO1-mediated KLF2 suppression was reversed by atorvastatin, suggesting that intensive statin treatment could be a therapeutic option in diabetic vascular dysfunction.

title: Acute oliguric renal failure associated with angiotensin II receptor antagonists.

title: Forkhead factor, FOXO3a, induces apoptosis of endothelial cells through activation of matrix metalloproteinases.
abstract: The forkhead factor, FOXO3a, is known to induce apoptosis in endothelial cells (ECs). However, its effects on extracellular matrices (ECM), which are important in EC survival, remained unknown. Here, we evaluated the role of FOXO3a on EC-ECM interaction.



Constitutively active FOXO3a was transduced to human umbilical vein endothelial cells by adenoviral vector (Ad-TM-FOXO3a). Ad-TM-FOXO3a transfection led to dehiscence of ECs from fibronectin-coated plates, resulting in anoikis, which was significantly reversed by matrix metalloproteinase (MMP) inhibitor, GM6001. FOXO3a increased the expression of MMP-3 (stromelysin-1) but decreased the expression of tissue inhibitors of metalloproteinases-1 (TIMP-1), which was associated with increased MMP enzymatic activity in zymography. Pathophysiologic conditions such as serum starvation or heat shock also induced activation of endogenous FOXO3a, leading to activation of MMP-3 and apoptosis, which was reversed by GM6001. Delivery of Ad-TM-FOXO3a to the intraluminal surface in vivo led to EC denudation, disrupted vascular integrity, and impaired endothelium-dependent vasorelaxation.



Activation of MMPs and possible ECM disruption represent novel mechanisms of FOXO3a-mediated apoptosis in ECs.

title: FOXO3a turns the tumor necrosis factor receptor signaling towards apoptosis through reciprocal regulation of c-Jun N-terminal kinase and NF-kappaB.
abstract: We evaluated the full range effects of FOXO3a in endothelial cells (ECs) by microarray analysis and investigated the role of FOXO3a regulating TNF receptor signaling pathway.



Human umbilical vein endothelial cells (HUVECs) were transfected with adenoviral vectors expressing constitutively active FOXO3a (Ad-TM-FOXO3a). Ad-TM-FOXO3a transfection caused remarkable apoptosis, which were accompanied with upregulation of genes related with TNF receptor signaling, such as TNF-alpha, TANK (TRAF-associated NF-kappaB activator), and TTRAP (TRAF and TNF receptor-associated protein). Furthermore, kappaB-Ras1 (IkappaB-interacting Ras-like protein-1) which is known to block IkappaB degradation was found increased, and intranuclear translocation of NF-kappaB was inhibited. GADD45beta and XIAP, negative regulators of c-Jun N-terminal kinase (JNK), were suppressed and JNK activity was increased. Attenuation of TNF signaling pathway either by blocking antibody for TNF receptor or by blocking JNK with DMAP (6-dimethylaminopurine) or Ad-TAM67 (dominant negative c-Jun) cotransfection, significantly reduced FOXO3a-induced apoptosis. Finally, treatment of vasculature with heat shock, an activator of endogenous FOXO3a, resulted in EC apoptosis, which was completely rescued by Ad-TAM67.



FOXO3a promotes apoptosis of ECs, through activation of JNK and suppression of NF-kappaB. These data identify a novel role of FOXO3a to turn TNF receptor signaling to a proapoptotic JNK-dependent pathway.

title: Aging and arterial stiffness.
abstract: Arterial walls stiffen with age. The most consistent and well-reported changes are luminal enlargement with wall thickening and a reduction of elastic properties at the level of large elastic arteries. Longstanding arterial pulsation in the central artery causes elastin fiber fatigue and fracture. Increased vascular calcification and endothelial dysfunction are also characteristic of arterial aging. These changes lead to increased pulse wave velocity, especially along central elastic arteries, and increases in systolic blood pressure and pulse pressure. Vascular aging is accelerated by coexisting cardiovascular risk factors, such as hypertension, metabolic syndrome and diabetes. Vascular aging is an independent risk factor for cardiovascular disease, from atherosclerosis to target organ damage, including coronary artery disease, stroke and heart failure. Various strategies, especially controlling hypertension, show benefit in preventing, delaying or attenuating vascular aging.

title: Phase IV, 8-week, multicenter, randomized, active treatment-controlled, parallel group, efficacy, and tolerability study of high-dose candesartan cilexetil combined with hydrochlorothiazide in Korean adults with stage II hypertension.
abstract: The objective of this study was to evaluate the efficacy and tolerability of high-dose (32 mg) candesartan in Asians.



In Korean adult patients with stage II hypertension, we evaluated the efficacy and tolerability of candesartan 16 mg/hydrochlorothiazide (HCT) 12.5 mg and candesartan 32 mg/HCT 12.5 mg compared with candesartan 16-mg and 32-mg monotherapy, respectively. This Phase IV, 8-week, multicenter, randomized, active treatment-controlled, parallel group, efficacy, and tolerability study, named CAESAR (Candesartan Effect in Second Stage Arterial Hypertension), enrolled 253 patients with stage II hypertension. Treatment started with either candesartan 16 mg or candesartan 16 mg/HCT 12.5 mg. After 4 weeks, the candesartan dose was forced titrated to 32 mg in both groups. The primary and secondary objectives were to compare the blood pressure (BP) changes after 4 weeks and 8 weeks between candesartan-HCT combination therapy and candesartan monotherapy. The proportion of patients achieving target BP (systolic blood pressure [SBP] <140 mm Hg, but <130 mm Hg for patients with diabetes mellitus or chronic kidney disease; diastolic blood pressure (DBP) <90 mm Hg, but <80 mm Hg for those with diabetes mellitus or chronic kidney disease) after 4 and 8 weeks of therapy was also evaluated. Adverse events were investigated both by spontaneous report by the patient and by the investigators' evaluations in each visit. Laboratory tests were performed at the end of the study to evaluate drug tolerability.



Study patients were all Asians, mostly male (65.7%), with a mean (SD) age of 49.4 (10.6) years and a mean body weight of 68.9 (12.1) kg; there were no between-group variances in demographic profiles except that the mean age in the candesartan-HCT group (51.0 [10.2] years) was about 3.2 years higher than that in the candesartan monotherapy group (47.8 [10.7] years; P = 0.02) despite random allocation. A total of 80.4% of the study patients had not been treated before, whereas 19.6% were previously treated and enrolled after 2 weeks of washout period. Baseline sitting systolic/diastolic BPs (SBP/DBP) were 160.7 (13.0)/104.6 (9.5) mm Hg. After 4 weeks, patients treated with candesartan 16 mg/HCT 12.5 mg showed significant decreases in SBP/DBP of 28.7 (17.5)/17.8 (10.2) mm Hg, and those in the candesartan 16 mg monotherapy group showed decreases of 20.5 (14.5)/14.1 (10.1) mm Hg (P < 0.01 between treatments for both SBP and DBP). After forced titration of candesartan from 16 mg to 32 mg at week 8, there was an additional reduction in SBP/DBP of 4.1 (12.7)/3.8 (8.3) mm Hg in the candesartan-HCT combination therapy group and 4.0 (11.6)/2.0 (8.5) mm Hg in the candesartan monotherapy group (P < 0.001 for SBP and DBP in both groups compared with values at week 4). A greater proportion of patients (70.6%) attained the target BP in the candesartan-HCT combination therapy group than in the candesartan monotherapy group (53.2%, P = 0.014). A total of 32 mg of candesartan was well tolerated both in combination therapy with HCT and in monotherapy. Dizziness was the most common adverse event in both groups (5 and 2 patients, respectively).



The candesartan-HCT combination was associated with a statistically significant lowering of BP compared with candesartan monotherapy.

title: Insulin-like growth factor binding protein-3 inhibits the growth of non-small cell lung cancer.
abstract: Insulin-like growth factors (IGFs) have mitogenic and antiapoptotic properties and have been implicated in the development of lung cancer. The effects of IGFs are modulated by insulin-like growth factor binding proteins (IGFBPs). This study explored the effects of IGFBP-3 on non-small cell lung cancer (NSCLC) cells after infection with an adenovirus constitutively expressing IGFBP-3 under the control of the cytomegalovirus promoter (Ad5CMV-BP3). We found that IGFs, especially IGF-I, stimulated the growth of NSCLC cells, and Ad5CMV-BP3 suppressed this IGF-I-induced NSCLC cell growth. We also found that the clonogenicity of H1299 cells in soft agar was markedly reduced by Ad5CMV-BP3. Furthermore, direct injection of Ad5CMV-BP3 into H1299 NSCLC xenografts s.c. established in athymic nude mice induced massive destruction of the tumors. Ad5CMV-BP3 did not induce detectable cytotoxicity on normal human bronchial epithelial cells, suggesting therapeutic efficacy of this virus. Ad5CMV-BP3 infection was accompanied by apoptotic cell death in vitro as detected by flow cytometry, DNA fragmentation analysis, and Western blot analysis on the expression of Bcl-2 and on the cleavage of poly(ADP-ribose) polymerase, a substrate of caspase 3. Immunofluorescence confocal microscopy was also used to show the apoptotic effect of Ad5CMV-BP3 in H1299 tumors established in nude mice. These findings indicated that IGFBP-3 was a potent inducer of apoptosis in NSCLC cells in vitro and in vivo. To delineate the underlying mechanism, we examined the effect of IGFBP-3 on Akt/protein kinase B and glycogen synthase kinase-3beta, downstream mediators of the phosphatidylinositol 3-kinase pathway, and on mitogen-activated protein kinase (MAPK), all three of which are activated by IGF-mediated signaling pathways and have important roles in cell survival. IGFBP-3 overexpression inhibited the phosphorylation of Akt and glycogen synthase kinase-3beta and the activity of MAPK. Furthermore, IGF-I rescued the NSCLC cells from serum depletion-induced apoptosis, and this rescue was blocked in Ad5CMV-BP-3-infected H1299 NSCLC cells. Transient transfection with activated Akt or constitutively active MAPK kinase-1, an upstream activator of MAPK, partially blocked IGFBP-3-induced apoptosis of NSCLC cells. These findings suggested that the growth-regulatory effect of IGFBP-3 on NSCLC cells was attributable in part to the inhibition of the IGF-induced survival pathway. These data demonstrate the importance of IGFBP-3 in the regulation of NSCLC cell proliferation, clonogenicity, and tumor growth, suggesting that IGFBP-3 is a target for the treatment of lung cancer and that Ad5CMV-BP3 is a potential therapeutic agent.

title: Effects of insulin-like growth factor binding protein-3 and farnesyltransferase inhibitor SCH66336 on Akt expression and apoptosis in non-small-cell lung cancer cells.
abstract: Overexpression of insulin-like growth factor binding protein-3 (IGFBP-3) induces apoptosis in non-small-cell lung cancer (NSCLC) cells in vitro and in vivo. However, Ras-mediated signaling pathways could develop resistance to apoptotic activities of IGFBP-3 in NSCLC cells. We thus evaluated the therapeutic potential of the combination of IGFBP-3 and SCH66336, a farnesyltransferase inhibitor that blocks Ras activation, in NSCLC cell lines.



The effects of the combination of adenoviral IGFBP-3 (Ad-IGFBP3) and SCH66336 on proliferation and apoptosis of NSCLC cell lines (H1299, H596, A549, H460, H358, H322, and H226B) were assessed in vitro and in vivo by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, a flow cytometry-based terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling assay, western blot analyses, and an NSCLC xenograft tumor model. The specific effects of Ad-IGFBP 3 and SCH66336 on mitogen-activated protein kinase and Akt were assessed by using adenoviral vectors that express constitutively active MEK1 or constitutively active Akt. Synergy was assessed by median effect analysis.



The combination of Ad-IGFBP3 and SCH66336 had synergistic antiproliferative effects in five cell lines (H1299, H596, A549, H460, and H322). Antiproliferative effects were accompanied by increased apoptosis in H460 cells in vitro. Overexpression of a constitutively active Akt but not a constitutively active MEK-1 rescued H460 cells from apoptosis induced by single or combined treatment of Ad-IGFBP3 and SCH66336. In H1299 tumor xenografts, Ad-IGFBP3 and SCH66336 was associated with decreased tumor volume, increased apoptosis, and decreased Akt levels.



The combination of Ad-IGFBP3 and SCH66336 decreased Akt expression and increased apoptosis in NSCLC cells in vitro and in vivo. Simultaneous treatment with IGFBP-3 and SCH66336 may have the potential to be an effective therapeutic strategy in NSCLC.

title: Differential distribution of human mitochondrial DNA in somatic tissues and hairs.
abstract: To investigate mitochondrial DNA (mtDNA) distribution within tissues during life, we observed length heteroplasmy in a polycytosine tract of the mitochondrial HV2 region by size-based separation of PCR products, using a mutagenic primer which was designed to avoid stutter production. Blood, brain, heart, liver, skeletal muscle and hair shaft samples were collected during autopsies of 25 individuals. Here, we demonstrate differences in the level of mtDNA length heteroplasmy both within and between individuals and tissues. We also show that mtDNA is distributed randomly in varying proportions in various somatic tissues during growth, resulting in an imbalance in the composition of mtDNA pools among tissues. This mtDNA distribution appears not to be strictly random, and can be explained by the random somatic segregation of nucleoids. On the other hand, significant qualitative/quantitative mtDNA peak pattern variations in hair shafts are thought to be a result of the different developmental origins of hairs. Each hair shaft may have a restricted or clonal set of mtDNA molecules derived from a discrete group of stem cells.

title: Amicrobial Pustulosis-Like Rash in a Patient with Crohn’s Disease under Anti-TNF-Alpha Blocker
abstract: Amicrobial pustulosis of the folds (APF) is a recently described entity characterized by relapsing pustular lesions predominantly involving the cutaneous flexures and scalp. This disease typically occurs in association with systemic lupus erythematosus and a variety of other autoimmune diseases. We here describe an APF-like pustular eruption predominantly affecting the scalp, face and trunk, occurring during long-term infliximab treatment for Crohn’s disease. Immunohistochemical staining of skin biopsy specimens for myxovirus resistance protein A, a marker for type 1 interferon-inducible proteins, showed increased staining in the epidermis and dermal mononuclear inflammatory infiltrate. Our observation further extends the spectrum of cutaneous adverse reactions potentially related to anti-tumor necrosis factor-α, the clinical context in which APF can occur as well as its clinical presentations.

title: Triquinanes from linear alkylidene carbenes via trimethylenemethane diyls.
abstract: The intramolecular [2+3] cycloaddition reaction of the trimethylenemethane diyl generated from the intramolecular cyclopropanation reaction of alkylidene carbene produced linearly fused triquinanes regio- and stereoselectively. The current tandem cycloaddition reaction was applied to a 14-step total synthesis of hirsutene from methallyl alcohol.