Inhibition of DNA2 nuclease as a therapeutic strategy targeting replication stress in cancer cells.

Replication stress is a characteristic feature of cancer cells, which is resulted from sustained proliferative signaling induced by activation of oncogenes or loss of tumor suppressors. In cancer cells, oncogene-induced replication stress manifests as replication-associated lesions, predominantly double-strand DNA breaks (DSBs). An essential mechanism utilized by cells to repair replication-associated DSBs is homologous recombination (HR). In order to overcome replication stress and survive, cancer cells often require enhanced HR repair capacity. Therefore, the key link between HR repair and cellular tolerance to replication-associated DSBs provides us with a mechanistic rationale for exploiting synthetic lethality between HR repair inhibition and replication stress. DNA2 nuclease is an evolutionarily conserved essential enzyme in replication and HR repair. Here we demonstrate that DNA2 is overexpressed in pancreatic cancers, one of the deadliest and more aggressive forms of human cancers, where mutations in the KRAS are present in 90-95% of cases. In addition, depletion of DNA2 significantly reduces pancreatic cancer cell survival and xenograft tumor growth, suggesting the therapeutic potential of DNA2 inhibition. Finally, we develop a robust high-throughput biochemistry assay to screen for inhibitors of the DNA2 nuclease activity. The top inhibitors were shown to be efficacious against both yeast Dna2 and human DNA2. Treatment of cancer cells with DNA2 inhibitors recapitulates phenotypes observed upon DNA2 depletion, including decreased DNA double strand break end resection and attenuation of HR repair. Similar to genetic ablation of DNA2, chemical inhibition of DNA2 selectively attenuates the growth of various cancer cells with oncogene-induced replication stress. Taken together, our findings open a new avenue to develop a new class of anticancer drugs by targeting druggable nuclease DNA2. We propose DNA2 inhibition as new strategy in cancer therapy by targeting replication stress, a molecular property of cancer cells that is acquired as a result of oncogene activation instead of targeting currently undruggable oncoprotein itself such as KRAS.

Assessment of flares in lupus patients enrolled in a phase II/III study of rituximab (EXPLORER).

The EXPLORER study was designed to assess the response to rituximab versus placebo in patients with moderate to severe extrarenal systemic lupus erythematosus (SLE) receiving background immunosuppression. The definition of response required reduced clinical activity without subsequent flares over 52 weeks, and the study did not meet its efficacy endpoint. The current exploratory analysis assessed flare rates in patients who achieved initial low disease activity response (British Isles Lupus Assessment Group [BILAG] C or better in all organs) during the study. Exploratory reanalysis of data from the EXPLORER trial was conducted, considering alternative definitions for flare. No difference was found between rituximab and placebo in preventing or delaying moderate to severe flares. However, when severe (BILAG A) flares alone were examined, rituximab reduced the risk of a subsequent first A flare (hazard ratio = 0.61; p = 0.052) and lowered mean ± SD annualized A flare rates (0.86 ± 1.47 vs. 1.41 ± 2.14; p = 0.038). Eighty-four (49.7%) rituximab-treated patients achieved low disease activity without subsequent A flares versus 31 (35.2%) placebo-treated patients (p = 0.027). Prednisone rescue for A flares was similar in rituximab- (24%) and placebo-treated (14%) patients (p = 0.204). This post hoc analysis evaluates the hypothesis that assessment of BILAG A flares may distinguish potential treatment effects with greater sensitivity than assessment of BILAG B flares.

Baseline autoantibody profiles predict normalization of complement and anti-dsDNA autoantibody levels following rituximab treatment in systemic lupus erythematosus.

B cells are thought to play a major role in the pathogenesis of systemic lupus erythematosus (SLE). Rituximab (RTX), a chimeric anti-CD20 mAb, effectively depletes CD20( +) peripheral B cells. Recent results from EXPLORER, a placebo-controlled trial of RTX in addition to aggressive prednisone and immunosuppressive therapy, showed similar levels of clinical benefit in patients with active extra-renal SLE despite effective B cell depletion. We performed further data analyses to determine whether significant changes in disease activity biomarkers occurred in the absence of clinical benefit. We found that RTX-treated patients with baseline autoantibodies (autoAbs) had decreased anti-dsDNA and anti-cardiolipin autoAbs and increased complement levels. Patients with anti-dsDNA autoAb who lacked baseline RNA binding protein (RBP) autoAbs showed increased complement and decreased anti-dsDNA autoAb in response to RTX. Other biomarkers, such as baseline BAFF levels or IFN signature status did not predict enhanced effects of RTX therapy on complement or anti-dsDNA autoAb levels. Finally, platelet levels normalized in RTX-treated patients who entered the study with low baseline counts. Together, these findings demonstrate clear biologic activity of RTX in subsets of SLE patients, despite an overall lack of incremental clinical benefit with RTX in the EXPLORER trial.

Pharmacist intervention in activated protein C therapy for severe sepsis: influence on health and economic outcomes.

OBJECTIVE: To assess the health and cost outcomes of pharmacist intervention versus non-intervention in activated protein C (drotrecogin alpha) therapy for patients with severe sepsis. METHOD: This is a retrospective study. We reviewed the medical records of patients aged 18 years and older who were admitted to our hospital for severe sepsis from January 1, 2003 to December 31, 2007. Only patients who are prescribed activated protein C for the treatment of severe sepsis according to the reimbursement criteria can be reimbursed by the Taiwan Bureau of National Health Insurance (BNHI). Our hospital stipulated that the criteria check list must be evaluated by a clinical pharmacist and the prescribing physician as to whether the patient is eligible to receive activated protein C. To assess the influence of pharmacist intervention on outcomes, we divided eligible patients into two groups, pharmacist-intervention group (Group A; n = 19) and non-pharmacist intervention group (Group B; n = 19). Both groups received a 96-h intravenous infusion of activated protein C at 24 microg/kg/h. We defined evident severe sepsis as concurrent antibiotic plus ventilator and/or vasopressor use. We compared group characteristics, 28-day in-hospital mortality, length of stay and direct medical costs between the two groups. One-way ANOVA was used for analysis. RESULTS: 50% of patients in each group met the reimbursement criteria of the BNHI. Activated protein C therapy was initiated within 1.37 +/- 0.4 days and 7.21 +/- 7.8 days of admission to the ICU in Group A and Group B, respectively (p < 0.01). All of the patients in Group A (19/19) and 42.1% of the patients in Group B (8/19) received activated protein C within 12 - 48 h of admission to the Intensive care unit (ICU) (p < 0.01). 28-day mortality was lower for Group A than for Group B (26.7% and 43.8%, respectively). The length of stay in the ICU for patients in Group A was shorter than that in Group B (14.1 +/- 7.7 vs. 19.7 +/- 11.1, respectively; p < 0.079). Total direct medical costs for survivors in Group A were less than those in Group B (US$ 20,632.3 vs. US$ 24,785.8, respectively; p < 0.05). CONCLUSIONS: Pharmacist intervention in prescribing activated protein C for patients with severe sepsis might reduce direct medical costs and promote earlier initiation of therapy. The potential impact of pharmacist intervention on the timing of activated protein C therapy and the direct medical costs of treatment warrant further study.

Cloning and heterologous expression of a novel ligninolytic peroxidase gene from poroid brown-rot fungus Antrodia cinnamomea.

A novel ligninolytic peroxidase gene (ACLnP) was cloned and characterized from a poroid brown-rot fungus, Antrodia cinnamomea. The genomic DNA of the fungus harboured two copies of ACLnP, with a length of 2111 bp, interlaced with 12 introns, while the full-length cDNA was 1183 bp, with a 66 bp signal peptide and an ORF of 990 bp. The three-dimensional molecular structure model was comparable to that of the versatile peroxidase of Pleurotus eryngii. ACLnP was cloned into vector pQE31, successfully expressed in Escherichia coli strain M15 under the control of the T5 promoter and produced a non-glycosylated protein of about 38 kDa, pI 5.42. The native and recombinant ACLnP was capable of oxidizing the redox mediator veratryl alcohol, and also decolorized bromophenol blue and 2,6-dimethoxyphenol dyes, implicating a functional extracellular peroxidase activity. The significance of discovering a functional ACLnP gene in A. cinnamomea in terms of wood degradation and colonization capacity in its unique niche is discussed.

Fatal amiodarone-induced hepatotoxicity: a case report and literature review.

OBJECTIVE: To report a fatal case of amiodarone-induced acute hepatotoxicity after intravenous amiodarone administration and similar fatal cases review. CASE SUMMARY: A 72-year-old woman with a history of hypertension, prior cardiovascular disease, atrial fibrillation and diabetes mellitus was admitted to the hospital with acute pyelonephritis and transferred to the intensive care unit due to cerebral infarction. An antidiabetic drug, a low dose of aspirin and intravenous amiodarone therapy was started. After receiving a second dose of amiodarone (1,200 mg; injection rate 1 mg/min), the woman developed ascites, jaundice, high levels of serum transaminases, decreased prothrombin time, and finally became unconscious. Immediately after treatment was discontinued, her extremely high hepatic parameters returned to normal. According to the Naranjo probability scale, this adverse reaction was highly probable. DISCUSSION: The occurrence of acute liver damage after intravenous amiodarone is rare but harmful. It can be induced by polysorbate 80, a solubilizer, by immunomediated centrilobular necrosis, or by the presence of a functional PPAR-I+/- gene. CONCLUSION: Amiodarone is an effective antiarrhythmic agent for preventing and treating atrial and ventricular arrhythmias. The molecular mechanism causing acute hepatic damage after amiodarone treatment is not clear. Therefore, amiodarone must be administered with care, and liver function should be monitored closely in patients treated with this drug.

First Report of Formosan Michelia Seedling Root Rot Caused by Pythium splendens in Taiwan.

Formosan michelia (Michelia compressa (Maxim.) Sargent) is a valuable evergreen tree in Taiwan that is distributed from low to medium (200 to 1,800 m) altitudes. In many nurseries in Taiwan, Formosan michelia seedlings grow poorly or wilt. The etiology of the disease observed in April 2004 in a nursery in Jinshan was investigated. Diseased seedlings with chlorotic leaves and decayed feeder roots lost leaves, died back, and then wilted. The putative pathogen, Pythium splendens Braun, was isolated and identified on a morphological basis (1). P. splendens was isolated from the roots of diseased seedlings on 2% water agar with 100 ppm of ampicillin. Isolates increased daily on potato dextrose agar at 24°C by 27 to 30 mm and on malt extract agar (MEA) by 23 to 25 mm. No zoosporangia and zoospores were produced. The main hyphae were as much as 9 µm wide on MEA. Hyphal swellings were abundant, globose, smooth, terminal, and 33 to 42 µm in diameter, often with dark, densely granulated contents. Attempted matings of four P. splendens isolates in V8 medium failed. To prove pathogenicity, the four isolates were cultured in 300-ml flasks containing 150 ml of 2% malt extract medium at room temperature for 14 days. The mycelia were homogenized in sterile water at 4,500 rpm for 5 min. The suspension was adjusted to 5 × 106 hyphal swellings per ml. Roots of the 2-month-old seedlings were immersed in the suspension for 2 h, whereas the control seedlings were immersed in sterilized water. Five seedlings of each of three replicates were inoculated with one of the four isolates for a total of 60 seedlings. Controls were replicated in the same way. The inoculated plants were transplanted into plastic flowerpots containing sterilized peat and moss and kept in the greenhouse at 20 to 24°C. After 14 days, inoculated seedlings developed symptoms like those of the original plants. The putative pathogen was reisolated from the roots of inoculated plants. Cultures are maintained at the Forest Pathology Lab of the National Taiwan University. To our knowledge, this is the first report of proof of pathogenicity of P. splendens on Formosan michelia seedlings. Reference: (1) A. J. Van der Plaats-Niterink. Stud. Mycol. 21:151, 1981.

First Report of Sclerotium rolfsii on Island Ash Seedlings in Taiwan.

Island ash (Fraxinus formosana Hay.) is a large, semideciduous tree in Taiwan. It is used for forestation, a shade tree, and producing wood for furniture. During the summer of 2001, a sudden wilt of 1-year-old plants was observed in a nursery in northern Taiwan. Initial symptoms included stem necrosis at the soil line and yellowing and tan discoloration of the leaves. As stem necrosis progressed, infected plants wilted and died. Necrotic tissues were covered with white mycelium that differentiated into reddish brown, spherical (1 to 2 mm in diameter) sclerotia. Sclerotium rolfsii was consistently recovered from the surface of symptomatic stem sections that were disinfected for 1 min in 0.5% NaOCl and then plated on potato dextrose agar (PDA) amended with 100 ppm of ampicillin. Pathogenicity of two S. rolfsii isolates was confirmed on 1-year-old island ash seedlings grown in 12.7 cm- (5-in) plastic pots in a sterilized mixture of peat moss and vermiculite (3:1). Seedlings were inoculated with mycelia and sclerotia of the pathogen grown on PDA. Three plants each were inoculated with four disks (5 mm) of agar with mycelium and three were inoculated with 10 sclerotia that were placed on the soil surface around the base of each plant. Noninoculated plants served as controls. All plants were kept in a growth chamber at 25 to 35°C and >95% relative humidity. The test was repeated once. All inoculated plants developed symptoms within 14 days, while control plants remained symptomless. Sclerotia developed on infected tissues, and S. rolfsii was reisolated from symptomatic tissues. This disease has been observed on many species of plants (1), but to our knowledge, this is the first report of Southern blight of Island ash seedlings caused by S. rolfsii in Taiwan. Reference: (1) Y. P. Tsai ed. List of Plant Diseases in Taiwan. The Plant Protection Society of the Republic of China and The Phytopathological Society of the Republic of China, 1991.

First Report of Sclerotium rolfsii on Star-cluster (Pentas lanceolata) in Taiwan.

Star-cluster (Pentas lanceolata (Forssk.) Deflers) has recently become popular as a bedding plant in Taiwan. During the summer of 2000, a sudden wilt of 60-day-old plants was observed in a nursery in Tainan City (southern Taiwan). Initial symptoms included stem necrosis at the soil line and yellowing and tan discoloration of leaves. As stem necrosis progressed, infected plants wilted and died. Necrotic tissues were covered with white mycelium that differentiated into reddish brown, spherical (1 to 2 mm in diameter) sclerotia. Sclerotium rolfsii was consistently recovered from the surface of symptomatic stem sections that were disinfected for 1 min in 0.5% NaOCl and plated on potato dextrose agar amended with 100 ppm streptomycin sulfate. Pathogenicity of three isolates of S. rolfsii was confirmed by inoculating 90-day-old plants of P. lanceolata that were grown in pots. Three plants each were inoculated with a 5-mm plug of agar with mycelium or two sclerotia of the pathogen. Inoculum was placed on the soil surface against the stem of each plant. Three noninoculated plants served as controls. All plants were kept in a growth chamber at 20 to 30°C with relative humidity >85%. The pathogenicity test was repeated. Inoculated plants developed symptoms within 7 days, while control plants remained symptomless. Sclerotia developed on infected tissues and S. rolfsii was reisolated from symptomatic tissues. Although this disease has been observed on many species of plants (1), to our knowledge, this is the first report of southern blight of P. lanceolata caused by S. rolfsii in Taiwan. Reference: (1) Tsai, Y. P., ed. List of Plant Diseases in Taiwan. The Plant Protection Society of the Republic of China and The Phytopathological Society of the Republic of China. 1991.

Detection of cytomegalovirus infection in a patient with febrile ulceronecrotic Mucha-Habermann's disease.

BACKGROUND: Febrile ulceronecrotic Mucha-Habermann's disease (FUMHD) is a severe and very rare variant of pityriasis lichenoides et varilioformis acuta, which is characterized by large coalescing, and ulceronecrotic maculopapules or plaques. Morphological changes of the skin accompanied by persistent high fever and several constitutional symptoms have suggested virus infection in patients with FUMHD. However, the available information of viral origin is limited. In this study we investigated the relationship of cytomegalovirus (CMV), Epstein-Barr virus (EBV), human herpesvirus 8 (HHV8), type I human T-cell lymphotropic virus (HTLV-I), and parvovirus B19 (PVB19) with FUMHD in a Taiwanese patient. METHODS: The existence of CMV, EBV, HHV8, HTLV-I, and PVB19 was determined by polymerase chain reaction (PCR). The presence of CMV in the endothelial cells was characterized by in situ hybridization (ISH) and immunohistochemistry (IHC). RESULTS: Serologic immunoglobulin to CMV and IHC identification of CMV late gene in the biopsy specimen indicated that the patient was infected with CMV. Detection of CMV was confirmed by PCR and ISH. CONCLUSIONS: These results indicate that FUMHD is associated with dermal CMV manifestation. Nonetheless, the induction mechanism of FUMHD with CMV infection has yet to be determined.

Cyclic strain induces redox changes in endothelial cells.

Our previous studies have shown that cyclic strain to endothelial cells (ECs) increases reactive oxygen species (ROS) that act as second messengers. The potential impact of these enhanced ROS levels on ECs was examined by studying the antioxidant activities and heme oxygenase-1 (HO-1) expression in strained ECs. Cyclic strain to ECs increased lipid peroxidation and augmented oxidation of low-density lipoproteins. ECs subjected to strain increased their superoxide dismutase activities. Concomitantly, glutathione peroxidase activities increased in 3 to 6 hr and returned to basal level 24 hr after continuous cyclic strain treatment. A decrease of glutathione (GSH) was accompanied with an increase of oxidized glutathione (GSSH) level in ECs 3 to 6 hr after strain treatment. This was followed with a return of both GSH and GSSH to basal levels in 24 hr. Consistently, H2O2 treatment of ECs decreased the GSH/GSSG ratio. ECs pretreated with catalase abolished the strain-induced change in GSH/GSSG. Strain treatment, similar to H2O2 exposure, induced HO-1 expression in a time-dependent manner. This induction was inhibited after treating ECs with catalase or free radical scavenger. ECs treated with N-acetyl-cysteine abolished HO-1 gene induction. Our results suggest that cyclic strain-induced ROS cause a transient increase of glutathione peroxidase activity that results in a decrease of GSH level in ECs and that this decrease is crucial to HO-1 induction.

Nitric oxide regulates shear stress-induced early growth response-1. Expression via the extracellular signal-regulated kinase pathway in endothelial cells.

Endothelial cells (ECs) subjected to shear stress constantly release nitric oxide (NO). The effect of NO on shear stress-induced endothelial responses was examined. ECs subjected to shear stress induced a transient and shear force-dependent increase in early growth response-1 (Egr-1) mRNA levels. Treatment of ECs with an NO donor, S-nitroso-N-acetylpenicillamine (SNAP) or 3-morpholinosydnonimine (SIN-1), inhibited this shear stress-induced Egr-1 expression. Conversely, an NO synthase inhibitor to ECs, N(G)-monomethyl-L-arginine, augmented this Egr-1 expression. NO modulation of Egr-1 expression was demonstrated by functional analysis of Egr-1 promoter activity using a chimera containing the Egr-1 promoter region (-698 bp) and reporter gene luciferase. In contrast to the enhanced promoter activity after N(G)-monomethyl-L-arginine treatment, shear stress-induced Egr-1 promoter activity was attenuated after ECs were treated with an NO donor. ECs cotransfected with a dominant negative mutant of Ras (RasN17), Raf-1 (Raf301), or a catalytically inactive mutant of extracellular signal-regulated kinase (ERK)-2 (mERK) inhibited shear stress-induced Egr-1 promoter activity. NO modulation of the signaling pathway was shown by its inhibitory effect on shear stress-induced ERK1/ERK2 phosphorylation and activity. This inhibitory effect was further substantiated by the inhibition of NO on both the shear stress-induced transcriptional activity of Elk-1 (an ERK substrate) and the promoter activity of a reporter construct containing serum response element. NO-treated ECs resulted in a reduction of binding of nuclear proteins to the Egr-1 binding sequences in the platelet-derived growth factor-A promoter region. These results indicate that shear stress-induced Egr-1 expression is modulated by NO via the ERK signaling pathway in ECs. Our findings support the importance of NO as a negative regulator in endothelial responses to hemodynamic forces.

Modulation of Ras/Raf/extracellular signal-regulated kinase pathway by reactive oxygen species is involved in cyclic strain-induced early growth response-1 gene expression in endothelial cells.

Endothelial cells (ECs) exposed to cyclic strain induce gene expression. To elucidate the signaling mechanisms involved, we studied the effects of cyclic strain on ECs by using early growth response-1 (Egr-1) as a target gene. Cyclic strain induced a transient increase of Egr-1 mRNA levels that resulted in an increase of binding of nuclear proteins to the Egr-1 binding sequences in the platelet-derived growth factor-A promoter region. ECs subjected to strain enhanced Egr-1 transcription as revealed by promoter activities. Catalase pretreatment inhibited this induction. ECs, transfected with a dominant positive mutant of Ras (RasL61), increased Egr-1 promoter activities. In contrast, transfection with a dominant negative mutant of Ras (RasN17) attenuated this strain inducibility. ECs transfected with a dominant negative mutant of Raf-1 (Raf301) or the catalytically inactive mutant of extracellular signal-regulated kinase (ERK)-2 (mERK2) diminished strain-induced promoter activities. However, little effect on strain inducibility was observed in ECs transfected with a dominant negative mutant of Rac (RacN17) or a catalytically inactive mutant of JNK (JNK[K-R]). Consistently, strain-induced Egr-1 expression was inhibited after ECs were treated with a specific inhibitor (PD98059) to mitogen-activated protein kinase kinase. Moreover, strain to ECs induced mitogen-activated protein kinase/ERK activity. The activation of the ERK pathway was further substantiated by an increase of strain-induced transcriptional activity of Elk1, an ERK substrate. This strain-induced ERK activity was attenuated after ECs were treated with N-acetylcysteine or catalase. Consequently, this Egr-1 gene induction was abolished after ECs were treated with N-acetylcysteine or catalase. Deletion analyses of the promoter region (-698 bp) indicated that cyclic strain and H2O2 shared a common serum response element. Our data clearly indicate that cyclic strain-induced Egr-1 expression is mediated mainly via the Ras/Raf-1/ERK pathway and that strain-induced reactive oxygen species can modulate Egr-1 expression at least partially via this signaling pathway.

Indoor environmental risk factors and childhood asthma: a case-control study in a subtropical area.

This study examined the relation between indoor environmental factors and childhood asthma in a subtropical area. A hospital-based case-control study was performed in Kaohsiung, Taiwan, between July of 1995 and June of 1996. Eighty-six children seen in the out-patient clinic of our university hospital and who had a first-time diagnosis of asthma made by a pediatrician were the test group; 86 control subjects were selected from children attending the Childhood Orthopaedic Clinic in the same hospital and who had no previous diagnosis of asthma or asthma symptoms and no history of physician confirmed atopic diseases. The control subjects were matched with test case children on the basis of gender and age. Information was obtained from parents using a structured questionnaire. Of the many indoor environmental factors included in this study, only home dampness showed an association with asthma (adjusted odds ratio=1.77; 95% confidence intervals, 1.24-2.53). We conclude that dampness in the home is a new public health risk factor related to asthma in subtropical areas.

Increase of reactive oxygen species (ROS) in endothelial cells by shear flow and involvement of ROS in shear-induced c-fos expression.

Intracellular reactive oxygen species (ROS) may participate in cellular responses to various stimuli including hemodynamic forces and act as signal transduction messengers. Human umbilical vein endothelial cells (ECs) were subjected to laminar shear flow with shear stress of 15, 25, or 40 dynes/cm2 in a parallel plate flow chamber to demonstrate the potential role of ROS in shear-induced cellular response. The use of 2',7'-dichlorofluorescin diacetate (DCFH-DA) to measure ROS levels in ECs indicated that shear flow for 15 minutes resulted in a 0.5- to 1.5-fold increase in intracellular ROS. The levels remained elevated under shear flow conditions for 2 hours when compared to unsheared controls. The shear-induced elevation of ROS was blocked by either antioxidant N-acetyl-cysteine (NAC) or catalase. An iron chelator, deferoxamine mesylate, also significantly reduced the ROS elevation. A similar inhibitory effect was seen with a hydroxyl radical (.OH) scavenger, 1,3-dimethyl-2-thiourea (DMTU), suggesting that hydrogen peroxide (H202), .OH, and possibly other ROS molecules in ECs were modulated by shear flow. Concomitantly, a 1.3-fold increase of decomposition of exogenously added H2O2 was observed in extracts from ECs sheared for 60 minutes. This antioxidant activity, abolished by a catalase inhibitor (3-amino-1,2,4-triazole), was primarily due to the catalase. The effect of ROS on intracellular events was examined in c-fos gene expression which was previously shown to be shear inducible. Decreasing ROS levels by antioxidant (NAC or catalase) significantly reduced the induction of c-fos expression in sheared ECs. We demonstrate for the first time that shear force can modulate intracellular ROS levels and antioxidant activity in ECs. Furthermore, the ROS generation is involved in mediating shear-induced c-fos expression. Our study illustrates the importance of ROS in the response and adaptation of ECs to shear flow.

Cyclic strain-induced monocyte chemotactic protein-1 gene expression in endothelial cells involves reactive oxygen species activation of activator protein 1.

Endothelial cells (ECs) are constantly exposed to blood pressure-induced mechanical strain. We have previously demonstrated that cyclic strain can induce gene expression of monocyte chemotactic protein-1 (MCP-1). The molecular mechanisms of gene induction by strain, however, remain unclear. Recent evidence indicates that intracellular reactive oxygen species (ROS) can act as a second messenger for signal transduction and thus affect gene expression. The potential role of ROS in strain-induced MCP-1 expression was investigated. ECs under cyclic strain induced a sustained elevated production of intracellular superoxide. ECs under strain or pretreated with either H2O2 or xanthine oxidase/hypoxanthine induced MCP-1 expression. Strain- or oxidant-induced MCP-1 mRNA levels could be inhibited by treating ECs with catalase or antioxidant N-acetyl-cysteine (NAC). Functional analysis of MCP-1 promoter and site-specific mutations indicates that the proximal tissue plasminogen activator-responsive element (TRE) in the -60-bp promoter region is sufficient for strain or H2O2 inducibility. Electrophoretic mobility shift assays demonstrated an increase of nuclear proteins binding to TRE sequences from ECs subsequent to strain or H2O2 treatment. NAC or catalase pretreatment of ECs inhibited the strain- or H2O2-induced AP-1 binding. These results clearly indicate that cyclic strain inducibility of MCP-1 in ECs uses the interaction of AP-1 proteins with TRE sites via the elevation of intracellular ROS levels in strained ECs. These findings emphasize the importance of intracellular ROS in the modulation of hemodynamic force-induced gene expression in vascular ECs.

Reactive oxygen species are involved in shear stress-induced intercellular adhesion molecule-1 expression in endothelial cells.

Vascular endothelial cells (ECs) are constantly subjected to flow-induced shear stress. Although the effects of shear stress on ECs are well known, the intracellular signal mechanisms remain largely unclear. Reactive oxygen species (ROS) have recently been suggested to act as intracellular second messengers. The potential role of ROS in shear-induced gene expression was examined in the present study by subjecting ECs to a shear force using a parallel-plate flow chamber system. ECs under shear flow increased their intracellular ROS as indicated by superoxide production. This superoxide production was maintained at an elevated level as shear flow remained. Sheared ECs, similar to TNF(alpha)-, PMA-, or H2O2-treated cells, increased their intercellular adhesion molecule-1 (ICAM-1) mRNA levels in a time-dependent manner. Pretreatment of ECs with an antioxidant, N-acetyl-cysteine (NAC) or catalase, inhibited this shear-induced or oxidant-induced ICAM-1 expression. ROS that were involved in the shear-induced ICAM-1 gene expression were further substantiated by functional analysis using a chimera containing the ICAM-1 promoter region (-850 bp) and the reporter gene luciferase. Shear-induced promoter activities were attenuated by pretreating sheared ECs with NAC and catalase. Flow cytometric analysis and monocytic adhesion assay confirmed the inhibitory effect of NAC and catalase on the shear-induced ICAM-1 expression on ECs. These results clearly demonstrate that shear flow to ECs can induce intracellular ROS generation that may result in an increase of ICAM-1 mRNA levels via transcriptional events. Our findings thus support the importance of intracellular ROS in modulating hemodynamically induced endothelial responses.

Fluid shear stress induces a biphasic response of human monocyte chemotactic protein 1 gene expression in vascular endothelium.

The focal distribution of atherosclerotic lesions in the arterial tree is related to the local shear stress generated by blood flow, but the molecular basis of the atherogenic response of endothelial cells in these lesion-prone areas is still unclear. We report that shear stress mediates a biphasic response of monocyte chemotactic protein 1 (MCP-1) gene expression in vascular endothelial cells (EC). Northern blot analysis indicated that the level of MCP-1 mRNA in human umbilical vein EC (HUVEC) subjected to a shear stress of 16 dynes/cm2 (1 dyne = 10 microN) for 1.5 hr increased by 2- to 3-fold when compared with static cells. The MCP-1 gene expression decreased to the basal level at 4 hr and then declined further to become completely quiescent at 5 hr after the onset of shear. Once the gene expression was fully suppressed, it remained quiescent even after static incubation for 1.5 hr and would not respond to reshearing after this static incubation. However, if the postshearing incubation extended from 1.5 to 24 hr, the MCP-1 mRNA returned to the basal level and was then able to increase after the reapplication of shear stress. Nuclear run-on experiments showed that the shear-induced increased MCP-1 mRNA in HUVEC was regulated at the transcriptional level. By using cycloheximide, it was shown that de novo protein synthesis was not necessary for the induction of MCP-1 by shear stress. The biphasic response of MCP-1 gene expression was found in experiments in which the applied shear stress was 6, 16, or 32 dynes/cm2, and it was observed not only in HUVEC but also in HeLa cells, glioma cell lines, and skin fibroblasts. This in vitro study demonstrates that the response of MCP-1 gene to shear stress represents an immediate early gene activation and suggests that this gene is probably suppressed in EC that have been exposed to a constant shear stress.

Human monocyte colony-stimulating factor stimulates the gene expression of monocyte chemotactic protein-1 and increases the adhesion of monocytes to endothelial monolayers.

The stimulation of the human umbilical vein endothelial cell (HUVEC) with recombinant human monocyte-derived colony-stimulating factor (MCSF) increased the gene expression of monocyte chemotactic protein (MCP-1). Northern blot analysis indicated that 50 U/ml of MCSF is the optimal concentration for this effect. The elevation of MCP-1 mRNA started as early as 1 h after stimulation and was maintained for at least 8 h. An increased MCP-1 level in MCSF-treated HUVEC was also demonstrated at the protein level by immunocytochemical staining using a polyclonal MCP-1-specific antibody. HUVEC activated by 50 U/ml of MCSF for 5 h showed a stronger immunofluorescence staining than control cells. Micropipette separation of THP-1 monocytes from HUVEC showed that the activation of both THP-1 and endothelium by MCSF led to an increase in the separation force by more than three times (36.2 +/- 6.7 x 10(-4) vs. 9.6 +/- 3.6 x 10(-4) dyn). An increased adhesiveness was also observed after MCSF activation of peripheral blood monocytes and HUVEC (16.7 +/- 2.7 x 10(-4) vs. 5.2 +/- 0.9 x 10(-4) dyn). The increased adhesive force in both systems was blocked by the use of anti-MCP-1 (5.5 +/- 0.8 x 10(-4) and 6.8 +/- 1.1 x 10(-4) dyn). Similar results were obtained in experiments in which only HUVEC, but not monocytes, were activated by MCSF. This increased adhesion of untreated monocytes to MCSF-activated HUVEC was also blocked by the addition of anti-MCP-1. In contrast, experiments in which only THP-1 or peripheral blood monocytes, but not HUVEC, were treated with MCSF did not show a significant increase of adhesion between these cells. These results indicate that MCSF augments monocyte-endothelium interaction primarily by its action on the endothelial cell and that this function is probably mediated through an increased expression of MCP-1. The MCSF/MCP-1-dependent adhesive mechanism might be operative in the arterial wall in vivo to lead to the trapping of the infiltrated monocyte-macrophage in the subendothelial space during atherogenesis.