Lynne Verna

c-Jun triggers apoptosis in human vascular endothelial cells.

In endothelial cells (ECs), the transcription factor c-Jun is induced by a variety of stimuli that perturb EC function. To extend our understanding of the role of c-Jun in EC physiology, we have directed overexpression of c-Jun in human umbilical vein ECs by using a tetracycline-regulated adenoviral expression system. In this study, we report a novel observation using this system. Specific expression of c-Jun is a sufficient trigger for ECs to undergo apoptosis, as demonstrated by a set of combined assays including an ELISA specific for histone-associated DNA fragmentation, DNA laddering, and TdT-mediated dUTP nick end labeling (TUNEL). Tetracycline can effectively shut off c-Jun overexpression and prevent EC apoptosis. Cleavage of poly(ADP-ribose) polymerase was also detected in ECs overexpressing c-Jun. Moreover, inhibitors of cysteine proteases blocked the apoptosis, suggesting a caspase-associated mechanism involved in proapoptotic effects of c-Jun. To gain further insight into the role of c-Jun as a pathophysiological regulator of EC death, TAM67, a dominant-negative mutant of c-Jun, was overexpressed in human umbilical vein ECs to abrogate endogenous c-Jun/activator protein-1 activation. H(2)O(2)-triggered apoptosis was largely attenuated in ECs overexpressing TAM67. Together, these results suggest that c-Jun, as a proapoptotic molecule, may play a role in mediating the cell death program in vascular endothelium.

Activation of ICAM-1 promoter by lysophosphatidylcholine: possible involvement of protein tyrosine kinases.

Lysophosphatidylcholine (lyso-PC) selectively upregulates the mRNA level of intercellular adhesion molecule-1 (ICAM-1) but not that of vascular cell adhesion molecule-1 (VCAM-1) in cultured human umbilical vein endothelial cells. Transfection studies show that lyso-PC activates the ICAM-1 promoter but not the VCAM-1 promoter. Gel mobility shift assays document an increase in NF-kappa B binding in cells treated with lyso-PC. The increases of ICAM-1 mRNA and NF-kappa B binding were inhibited by the protein tyrosine kinase inhibitors, genistein and lavendustin A, but not by inhibitors for cyclic AMP-dependent protein kinases or protein kinase C. Our results suggest that lyso-PC induces ICAM-1 expression most likely by activating NF-kappa B, and that the effect appears to be protein tyrosine kinase-dependent.

Adenovirus-Mediated Overexpression of c-Jun and c-Fos Induces Intercellular Adhesion Molecule-1 and Monocyte Chemoattractant Protein-1 in Human Endothelial Cells

As distal targets and mediators of signal transduction pathways, activator protein-1 (AP-1), c-Jun, and c-Fos are among the primary regulators of genes involved in cell function, proliferation, and differentiation. By using adenovirus-mediated gene transfer, we show that overexpression of AP-1 proteins directly causes coinduction of gene expression of an adhesion molecule, intercellular adhesion molecule-1 (ICAM-1), and a chemokine, monocyte chemoattractant protein-1 (MCP-1), in human vascular endothelial cells (ECs). The AP-1-induced gene expression occurs through a mechanism independent of nuclear factor-kappaB. Because the induced expression of ICAM-1 and MCP-1 in ECs has been implicated in endothelial activation and a number of important vascular disorders, it is suggested that AP-1 activation may play an important role in the pathogeneses of inflammation, angiogenesis, and atherogenesis.

Lipoprotein Promotes Caveolin-1 and Ras Translocation to Caveolae Role of Cholesterol in Endothelial Signaling

To explore the role of LDL in caveolin-Ras regulation in human endothelial cells (ECs), we incubated confluent human umbilical vein endothelial cells (HUVECs) with LDL. This resulted in a high steady-state caveolin-1 (Cav-1) expression at both the mRNA and protein levels. LDL exposure appeared not to regulate the abundance of Cav-1. Immunofluorescence staining showed that Cav-1 protein migrated from the cytoplasm to the cell membrane after LDL exposure. Cav-1 protein and cholesterol partitioned mainly into the caveola fractions, and LDL increased both Cav-1 and cholesterol in these fractions. Ras protein in caveola fractions was also increased by LDL. Increased Ras was detected in Cav-1 immunoprecipitated samples, and conversely, increased Cav-1 was found in Ras-immunoprecipitated samples. We also demonstrated LDL-increased Ras activity in HUVECs by measuring the GTP/GTP+GDP ratio of Ras with [32P]orthophosphate labeling in the cells. Finally, we determined the binding of [3H]-labeled free cholesterol and recombinant H-Ras to Cav-1 fusion proteins in vitro. Both cholesterol and Ras bound to full-length GST–Cav-1, scaffolding domain (61–101), and C-terminal (135–178) Cav-1 fusion peptides. Addition of cholesterol enhanced Ras binding to the full-length and scaffolding domain of Cav-1 but not to the C-terminal Cav-1. These findings strongly suggest a role for Cav-1 in cholesterol trafficking and cholesterol-mediated intracellular signaling, which may mediate EC activation by LDL.

Formation of 8-oxodeoxyguanosine in brain DNA of rats exposed to acrylonitrile

Abstract Acrylonitrile (ACN) produces tumors in rats, particularly gliomas of the brain, but tests for genotoxicity have yielded mixed results and no ACN-DNA adducts have been identified in the brain. To examine the possibility that ACN-related brain tumors were not a consequence of binding of ACN to brain DNA, experiments were conducted to investigate possible epigenetic mechanisms. Male Sprague-Dawley rats were exposed to 0, 3, 30, and 300 ppm ACN in drinking water for 21 days, a range that includes doses associated with brain tumorigenesis. In the 30 and 300 ppm ACN groups, 8-oxodeoxyguanosine (8-oxodG) levels were two fold greater than in the controls. Measures of glutathione levels, glutathione peroxidase and catalase were not significantly changed, but cyst(e)ine was somewhat increased. No changes were found in brain cytochrome oxidase activity, which indicates a lack of metabolic hypoxia. Also, no effects on thiobarbituric acid reactive substances were found, indicating a lack of lipid peroxidation. In an additional experiment, male Sprague-Dawley rats were exposed to 0 or 100 ppm ACN in drinking water for 94 days; interim sacrifices were conducted at 3, 10, and 31 days. Levels of brain nuclear DNA 8-oxodG were significantly increased in ACN-exposed rats compared with controls. Another group of animals were given weekly i.v. injections of 5 mg/kg methylnitrosurea and no increases in 8-oxodG were found. These studies suggest the possibility that ACN-induced tumors may be produced by a mode of action involving 8-oxodG. The formation of 8-oxodG is not understood, but does not appear to involve lipid peroxidation or disruption of antioxidant defenses.

In Vivo Low-Density Lipoprotein Exposure Induces Intercellular Adhesion Molecule-1 and Vascular Cell Adhesion Molecule-1 Correlated With Activator Protein-1 Expression

Objective—We tested the hypothesis that direct native low-density lipoprotein (LDL) injection into LDL receptor–deficient (LDLR−/−) mice would induce the adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in their aortic endothelial cells, and that transcriptional regulation of this pathway involved activator protein-1 (AP-1) but not nuclear factor &kgr;B (NF-&kgr;B). Methods and Results—Using tail vein injection of LDL into LDLR−/− mice, we were able to maintain atherogenic LDL blood levels, which induced ICAM-1 and VCAM-1 expression in their aortic endothelial cells after 24 hours. We were able to visualize and quantify this expression using immunohistochemistry and confocal microscopy. Under conditions in which ICAM-1 and VCAM-1 were expressed, the regulatory AP-1 proteins c-Fos and c-Jun were also highly expressed in the endothelial cell cytoplasm and observed within the cell nucleus. The NF-&kgr;B protein P65, although expressed in the endothelial cell cytoplasm after LDL injection, was not observed within the cell nucleus. Conclusions—Elevated LDL blood levels, maintained in vivo, increased the expression of the adhesion molecules ICAM-1 and VCAM-1 in aortic endothelial cells. This effect appeared to correlate with AP-1 but not NF-&kgr;B.

Cholesterol enrichment upregulates intercellular adhesion molecule-1 in human vascular endothelial cells.

Hypercholesterolemia is a major risk factor for atherosclerosis, but the mechanism by which cholesterol activates the endothelium remains undocumented. The present investigation was undertaken to investigate the role of cholesterol, one of the bioactive moieties of the low-density lipoprotein (LDL) particle, in initiating of intracellular signaling in endothelial cells (ECs) and culminating in increased abundance of the intercellular adhesion molecule-1 (ICAM-1). Cholesterol was delivered to human umbilical vein ECs (HUVECs) via cholesterol-enriched liposomes. In HUVECs, the cellular cholesterol:phospholipid ratio increased after 1 h of exposure to cholesterol. The level of ICAM-1 increased in both mRNA and protein after 24 h of cholesterol exposure. ICAM-1 mRNA half-life was not affected by cholesterol exposure. Promoter studies showed greater than two-fold activation of the ICAM-1 gene expression after cholesterol exposure. Electrophoretic mobility shift assay showed that activator protein-1 (AP-1) activity substantially increased after 2 h of exposure to cholesterol. In contrast, cholesterol did not affect nuclear factor-kappaB (NF-kappaB) activity. Results of trans-reporting assay revealed 2.5-fold increased expression of the AP-1-dependent reporter gene after cholesterol exposure whereas NF-kappaB-dependent expression was not affected. The AP-1/Ets (-891 to -908) site, one of the three AP-1-like sites in the ICAM-1 promoter, was most responsive to cholesterol. These data demonstrate for the first time that cholesterol enrichment phenotypically modulates ECs by transcriptionally upregulating ICAM-1 expression.

Low-density lipoprotein activates Jun N-terminal kinase (JNK) in human endothelial cells.

We have reported previously that native low-density lipoprotein (LDL) activates c-Jun and transcription factor AP-1 in human umbilical vein endothelial cells (HUVEC). The aim of this study was to elucidate the upstream signaling mechanisms mediating LDL activation of c-Jun/AP-1. Using a c-Jun NH2-terminal kinase (JNK) activity assay, we have detected an increase in JNK activity in LDL-exposed HUVEC, which started at 15 min and reached maximum activity after 1-2 h. This JNK activity, increased by LDL, occurred in a dose-dependent fashion starting at a concentration of 80 mg/dl of LDL and reaching maximum activation at a concentration of 160-240 mg/dl. Following cotransfection, the increase of AP-1 driven luciferase activity by LDL was attenuated 54% by a kinase-deficient JNK1. Furthermore, a specific trans-reporting system was utilized to confirm c-Jun activation by upstream signal mechanisms. The results show c-Jun activity increased by 3-fold after LDL exposure when compared with respective controls. In contrast, LDL exposure did not affect the activation of extracellular signal regulated kinase 1 and 2 (ERK1/2), even though phorbol 12-myristate 13-acetate treatment remarkably increased the activity of these kinases. Thus, this study demonstrates, for the first time, that JNK mediates LDL-induced endothelial cell activation.

Low-density lipoprotein augments interleukin-1-induced vascular adhesion molecule expression in human endothelial cells

In this study, the effect of low density lipoproteins (LDL) on the ability of the vascular endothelium to respond to vascular cell adhesion molecule 1 (VCAM-1) activation by a cytokine was investigated. After a 4-day pre-exposure to 240 mg/dl of LDL, human umbilical vein endothelial cells (HUVECs) were hyperresponsive to minute amounts of interleukin 1 alpha (IL-1 alpha) as demonstrated by an augmentation of VCAM-1 gene expression. Furthermore, in response to LDL exposure, endothelial recruitment of monocytes induced by minute amounts of IL-1 alpha was increased. This enhancing effect was blocked by an anti-VCAM antibody. The increased response appears not to be due to changes in IL-1 binding affinity or induction of endogenous IL-1 alpha. Transient transfection of HUVECs with a reporter driven by the VCAM promoter showed that LDL increased cellular response to IL-1 alpha by 46%. LDL itself does not increase NF-kappa B binding in endothelial cells (ECs). However, after a 2-day LDL incubation, NF-kappa B binding could be induced by over 63% with a very low dose of IL-1 alpha. IL-1 alpha at this dose (which activates NF-kappa B, but not AP-1) also enhanced LDL-activated AP-1 binding. This cross-enhanced effect may be an important intracellular signaling mechanism for EC activation. The results from this study provide new clues to understanding the mechanisms governing combined risk factors for atherosclerosis.

Selective activation of endothelial cells by the antioxidant pyrrolidine dithiocarbamate: involvement of C-jun N-terminal kinase and AP-1 activation.

The antioxidant agent pyrrolidine dithiocarbamate (PDTC) has been shown to protect endothelial cells (EC) from pro-inflammatory-induced and pro-oxidant-induced NF-kappaB activation. It also perturbs EC by altering activator protein-1 (AP-1) status and inducing ICAM-1. Experiments were performed to investigate the upstream mechanism by which PDTC produces these effects. We have demonstrated that PDTC not only induced AP-1 binding and ICAM-1 expression by itself, but it also augmented AP-1 activation and ICAM-1 induction in low-dose IL-1alpha treated cells. To dissect the mechanism of these effects, we measured c-Jun and c-Fos expression, and the activity of c-Jun NH2-terminal kinase (JNK) and extracellular signal regulated kinase (ERK) in human umbilical vein endothelial cells (HUVEC). We detected an increase in JNK activity in PDTC-treated HUVEC. Following cotransfection with JNK[K-M], a kinase-deficient JNK1, the PDTC-increased AP-1-driven-luciferase activity was attenuated. Utilizing a specific trans-reporting system we confirmed c-Jun activation by upstream signaling mechanisms. The results show that c-Jun activity was increased 9-fold after PDTC treatment. In addition, PDTC promoted more transient activation in ERK-c-fos. In contrast, PDTC produced sustained JNK-c-Jun activation, which translated into long-lasting ICAM-1 production. These results suggest that an antioxidant may contribute to chronic vascular endothelial activation.