USP1 targeting impedes GBM growth by inhibiting stem cell maintenance and radioresistance.

BACKGROUND: Clinical benefits from standard therapies against glioblastoma (GBM) are limited in part due to intrinsic radio- and chemoresistance of GBM and inefficient targeting of GBM stem-like cells (GSCs). Novel therapeutic approaches that overcome treatment resistance and diminish stem-like properties of GBM are needed. METHODS: We determined the expression levels of ubiquitination-specific proteases (USPs) by transcriptome analysis and found that USP1 is highly expressed in GBM. Using the patient GBM-derived primary tumor cells, we inhibited USP1 by shRNA-mediated knockdown or its specific inhibitor pimozide and evaluated the effects on stem cell marker expression, proliferation, and clonogenic growth of tumor cells. RESULTS: USP1 was highly expressed in gliomas relative to normal brain tissues and more preferentially in GSC enrichment marker (CD133 or CD15) positive cells. USP1 positively regulated the protein stability of the ID1 and CHEK1, critical regulators of DNA damage response and stem cell maintenance. Targeting USP1 by RNA interference or treatment with a chemical USP1 inhibitor attenuated clonogenic growth and survival of GSCs and enhanced radiosensitivity of GBM cells. Finally, USP1 inhibition alone or in combination with radiation significantly prolonged the survival of tumor-bearing mice. CONCLUSION: USP1-mediated protein stabilization promotes GSC maintenance and treatment resistance, thereby providing a rationale for USP1 inhibition as a potential therapeutic approach against GBM.

Expression of synaptopodin in endothelial cells exposed to laminar shear stress and its role in endothelial wound healing.

We examined the hypothesis that certain actin binding proteins might be upregulated by laminar shear stress (LSS) and could contribute to endothelial wound healing. Analysis of mRNA expression profiles of human umbilical vein endothelial cells under static and LSS-exposed conditions provided a list of LSS-induced actin binding proteins including synaptopodin (SYNPO) whose endothelial expression has not been previously reported. Additional studies demonstrated that SYNPO is a key mediator of endothelial wound healing because small interfering RNA-mediated suppression of SYNPO attenuated wound closure under LSS whereas overexpression of exogenous SYNPO enhanced endothelial wound closure in the absence of LSS. This study suggests that LSS-induced actin binding proteins including SYNPO may play a critical role in the endothelial wound healing stimulated by LSS.

Laminar shear stress induces the expression of aquaporin 1 in endothelial cells involved in wound healing.

Laminar shear stress (LSS) due to blood flow contributes to the maintenance of endothelial health by multiple mechanisms including promotion of wound healing. The present study examined the hypothesis that the induction of water channel aquaporin 1 (AQP1) expression by LSS might be functionally associated with endothelial wound healing. When human umbilical vein endothelial cells were exposed to LSS at 12 dyn cm(-2) for 24h, significant increases in AQP1 expression were observed at the mRNA and protein levels as compared with static control. In the in vitro scratch wound healing assay, LSS treatments before and after wound creation enhanced endothelial wound healing and this effect was significantly attenuated by selective suppression of AQP1 expression using small interfering RNA. Ectopic expression of AQP1 enhanced wound healing in the absence of LSS. This study demonstrated that LSS stimulates the endothelial expression of AQP1 that plays a role in wound healing.

Analysis of serum cytokine/chemokine profiles affected by aging and exercise in mice.

Aging could be the cause of inflammation involved in the progression of many degenerative diseases while physical exercise might reduce the inflammation. This study examined the effects of aging versus exercise on serum profiles of cytokines and chemokines in mice models. Male C57BL/6N mice with different ages (2 and 20 months old) were subjected to treadmill exercise for 4 weeks. The exercise did not affect the body mass gain of the young mice but significantly reduced that of the old mice. Of 50 cytokines/chemokines analyzed using a multiplexed bead-based sandwich immunoassay, Eotaxin, Interleukin-9 and Thymus and activation-related chemokine showed significantly higher serum levels in old mice compared with young mice (p<0.05). The treadmill exercise did not alter serum cytokines/chemokines levels significantly. This study suggests that the cytokines and chemokines, whose serum levels were changed age-dependently, would provide useful markers of the systemic low-level inflammation associated with aging and age-related diseases.

A regulatory role of Kruppel-like factor 4 in endothelial argininosuccinate synthetase 1 expression in response to laminar shear stress.

Endothelial argininosuccinate synthetase 1 (ASS1) regulates the provision of l-arginine to nitric oxide synthase 3 (NOS3). Previous studies demonstrated that endothelial ASS1 expression was induced by laminar shear stress (LSS) and that this enzyme plays a role in maintaining anti-inflammatory microenvironments through enhancing NO production. However, differently from the case of NOS3, the regulatory mechanism for the endothelial ASS1 expression in response to LSS is not well understood. This study addressed a specific issue whether endothelial ASS1 expression is regulated by Kruppel-like factors (KLFs) that are presumed to coordinate endothelial gene expressions in response to LSS. The cDNA microarray data indicated that LSS stimulated the expression of numerous KLFs in human umbilical vein endothelial cells. KLF4 showed the highest fold increase and LSS-dependent increases of KLF4 and most other KLFs were similar in young versus senescent endothelial cells. LSS-induced KLF4 expression was verified by RT-PCR and Western blotting. LSS-induced ASS1 expression and NO production were suppressed by a small interfering RNA for KLF4. The ectopic expression of KLF4 led to the increase of ASS1 expression and NO production. The present study demonstrated a key regulatory role of KLF4 in the endothelial ASS1 expression and NO production in response to LSS.

Endothelial argininosuccinate synthetase 1 regulates nitric oxide production and monocyte adhesion under static and laminar shear stress conditions.

Laminar shear stress (LSS) is known to increase endothelial nitric oxide (NO) production, which is essential for vascular health, through expression and activation of nitric oxide synthase 3 (NOS3). Recent studies demonstrated that LSS also increases the expression of argininosuccinate synthetase 1 (ASS1) that regulates the provision of L-arginine, the substrate of NOS3. It was thus hypothesized that ASS1 might contribute to vascular health by enhancing NO production in response to LSS. This hypothesis was pursued in the present study by modulating NOS3 and ASS1 levels in cultured endothelial cells. Exogenous expression of either NOS3 or ASS1 in human umbilical vein endothelial cells increased NO production and decreased monocyte adhesion stimulated by tumor necrosis factor-α (TNF-α). The latter effect of overexpressed ASS1 was reduced when human umbilical vein endothelial cells were co-treated with small interfering RNAs (siRNAs) for ASS1 or NOS3. SiRNAs of NOS3 and ASS1 attenuated the increase of NO production in human aortic endothelial cells stimulated by LSS (12 dynes·cm(-2)) for 24 h. LSS inhibited monocyte adhesion to human aortic endothelial cells stimulated by TNF-α, but this effect of LSS was abrogated by siRNAs of NOS3 and ASS1 that recovered the expression of vascular cell adhesion molecule-1. The current study suggests that the expression of ASS1 harmonized with that of NOS3 may be important for the optimized endothelial NO production and the prevention of the inflammatory monocyte adhesion to endothelial cells.

Detection of low levels of nitric oxide using an electrochemical sensor.

Nitric oxide produced from nitric oxide synthases mediates various physiological and pathological events in biological systems. However, quantitative assessment of nitric oxide from biological sources remains a difficult task. Here we describe a procedure for the quantification of low levels of nitric oxide using a nitric oxide - selective electrochemical sensor. Nitric oxide is oxidized to nitrite and/or nitrate and accumulated in the aqueous media. First, nitrate in biological fluids or culture media is converted to nitrite by an enzymatic method. Nitrite is then chemically converted to equimolar NO in an acidic iodide bath, where nitric oxide is detected by the sensor. Using this method, the present study demonstrates siRNA -mediated suppression of nitric oxide synthase 3 leading to a significant decline of basal nitric oxide production in human umbilical vein endothelial cells. Basal nitric oxide production from HUVECs is also shown to be inhibited by N (G)-nitro-L: -arginine methyl ester but not by N (G)-nitro-D: -arginine methyl ester (D-NAME) D-NAME . The analytical method presented here provides a sensitive and convenient tool for measuring basal and stimulated nitric oxide production from biological sources.

Identification of CD44 as a senescence-induced cell adhesion gene responsible for the enhanced monocyte recruitment to senescent endothelial cells.

The mechanism that is responsible for progression of atherosclerosis seen with increasing age remains controversial. This issue was addressed in the present study, by searching for genes that are uniquely expressed in senescent endothelial cells and functionally involved in inflammatory leukocyte adhesion recognized as a critical step in the initiation of atherogenesis. Senescent human umbilical vein endothelial cells (HUVECs) prepared by continuous subculturing in vitro showed higher binding affinity for monocytes (THP-1 cells, human acute monocytic leukemia cell line) compared with young cells. Gene expression profiles between young and senescent endothelial cells were compared by the cDNA microarray method, and CD44 was identified as one of the "senescence-induced cell adhesion genes" whose expression was upregulated in senescent cells and whose gene ontology annotation indicated their role in cell adhesion. The enhanced gene expression of CD44 in senescent endothelial cells was verified both at the mRNA and protein levels. Adhesion of monocytes to senescent endothelial cells was significantly reduced following pretreatment of endothelial cells with the CD44 antibody or small-interfering RNA, thus reinforcing the critical role of CD44 in the inflammatory event. Exogenous expression of CD44 in young HUVECs and in human aortic endothelial cells led to an increase in monocyte adhesion. CD44 expression levels in the rat aorta endothelium were found to increase in an age-dependent manner, as determined by immunohistochemistry and Western blotting. CD44 and other senescence-induced cell adhesion genes identified in this study may provide the novel targets for the prevention of inflammatory leukocyte adhesion leading to the development atherosclerosis.

Evidence for the association of peroxidases with the antioxidant effect of p-coumaric acid in endothelial cells exposed to high glucose plus arachidonic acid.

Although many plant-derived phenolic compounds display antioxidant effects in biological systems, their mechanism of action remains controversial. In this study, the mechanism by which p-coumaric acid (p-CA) performs its antioxidant action was investigated in bovine aortic endothelial cells under oxidative stress due to high levels of glucose (HG) and arachidonic acid (AA), a free fatty acid. p-CA prevented lipid peroxidation and cell death due to HG+AA without affecting the production of reactive oxygen species. The antioxidant effect of p-CA was not decreased by buthionine-(S,R)-sulfoximine, an inhibitor of cellular GSH synthesis. In contrast, pretreatment with p-CA caused the induction of peroxidases that decomposed t-butyl hydroperoxide in a p-CA-dependent manner. Furthermore, the antioxidant effect of p-CA was significantly mitigated by methimazole, which was shown to inhibit the catalytic activity of 'p-CA peroxidases' in vitro. Therefore, it is suggested that the induction of these previously unidentified 'p-CA peroxidases' is responsible for the antioxidant effect of p-CA. [BMB reports 2009; 42(9): 561-567].

Differential gene expression in young and senescent endothelial cells under static and laminar shear stress conditions.

Laminar shear stress (LSS) caused by blood flow is known to regulate endothelial function and to contribute to vascular health. By way of contrast, endothelial cell senescence seems to increase the incidence of vascular disorders. In an attempt to identify genes associated with vascular health/disease states, this study assessed the differential gene expression of young and senescent human umbilical vein endothelial cells (HUVECs) under static and LSS conditions. Replicative cell senescence was induced by continuous subculture in vitro, and LSS was provided using a cone-and-plate device. Young (p4) and senescent (p18) cells were subjected to LSS at 12 dyn.cm(-2) or maintained under static conditions for 24 h. Total mRNA was subjected to cDNA microarray analysis using the Affymetrix GeneChip. Welch t test at a significance level of p < 0.05 provided 961 "LSS-responsive" genes, whose expression was altered by LSS in both young and senescent cells, and 529 "senescence-responsive" genes differentially expressed in young vs senescent cells under both static and LSS conditions. The LSS-responsive and senescence-responsive gene groups included 74 genes held in common; these may prove useful for the study of cellular responses commonly affected by LSS and senescence. Among them, 20 genes whose expression was increased by LSS and simultaneously decreased by cellular senescence are suggested as potential vascular health markers in the sense that LSS is antiatherogenic, whereas senescence is proatherogenic. These genes included argininosuccinate synthetase 1, which was determined to be critical for both basal and LSS-induced NO production in young HUVECs. Furthermore, its diminished expression, and not that of nitric oxide synthase 3, was implicated in the insufficient NO production exhibited by senescent HUVECs under LSS conditions. The genes identified in this study are expected to facilitate improvements in our current level of understanding regarding endothelial physiology in association with age-associated vascular disease.

Laminar shear stress inhibits lipid peroxidation induced by high glucose plus arachidonic acid in endothelial cells.

Elevated blood glucose and free fatty acids induce oxidative stress associated with the incidence of cardiovascular disease. In contrast, laminar shear stress (LSS) plays a critical role in maintaining vascular health. The present study examined the mechanism for the antioxidant effect of LSS attenuating the oxidative stress induced by high glucose (HG) and arachidonic acid (AA) in human umbilical vein endothelial cells. HG and AA synergistically decreased cell viability and increased glutathione (GSH) oxidation and lipid peroxidation. The lipid peroxidation was markedly prevented by LSS as well as tetrahydrobiopterin (BH4) and GSH. LSS increased BH4 and GSH contents, and expression of GTP cyclohydrolase-1 and glutamylcysteine ligase (GCL) involved in their biosynthesis. Inhibition of GCL activity by DL-buthionine-(S,R)-sulfoximine and small-interfering RNA-mediated knockdown of GCL lessened the antioxidant effect of LSS. Therefore, it is suggested that LSS enhances antioxidant capacity of endothelial cells and thereby attenuates the oxidative stress caused by cardiovascular risk factors.