Low oxygen tension enhances hepatitis C virus replication.

Low oxygen tension exerts a significant effect on the replication of several DNA and RNA viruses in cultured cells. In vitro propagation of hepatitis C virus (HCV) has thus far been studied under atmospheric oxygen levels despite the fact that the liver tissue microenvironment is hypoxic. In this study, we investigated the efficiency of HCV production in actively dividing or differentiating human hepatoma cells cultured under low or atmospheric oxygen tensions. By using both HCV replicons and infection-based assays, low oxygen was found to enhance HCV RNA replication whereas virus entry and RNA translation were not affected. Hypoxia signaling pathway-focused DNA microarray and real-time quantitative reverse transcription-PCR (qRT-PCR) analyses revealed an upregulation of genes related to hypoxic stress, glycolytic metabolism, cell growth, and proliferation when cells were kept under low (3% [vol/vol]) oxygen tension, likely reflecting cell adaptation to anaerobic conditions. Interestingly, hypoxia-mediated enhancement of HCV replication correlated directly with the increase in anaerobic glycolysis and creatine kinase B (CKB) activity that leads to elevated ATP production. Surprisingly, activation of hypoxia-inducible factor alpha (HIF-α) was not involved in the elevation of HCV replication. Instead, a number of oncogenes known to be associated with glycolysis were upregulated and evidence that these oncogenes contribute to hypoxia-mediated enhancement of HCV replication was obtained. Finally, in liver biopsy specimens of HCV-infected patients, the levels of hypoxia and anaerobic metabolism markers correlated with HCV RNA levels. These results provide new insights into the impact of oxygen tension on the intricate HCV-host cell interaction.

Mannose-binding lectin is present in human semen and modulates cellular adhesion of Neisseria gonorrhoeae in vitro.

Mannose-binding lectin (MBL) is an innate immune molecule present in blood and some mucosal tissues, which can influence microbial attachment and inflammatory responses of host cells during infection. In this study MBL was found to be present at a low concentration in semen samples in the range 1.2-24.9 ng/ml. Co-incubation of bacteria with semen resulted in the binding of MBL to the bacterial surface. Neisseria gonorrhoeae is a common cause of genitourinary infection. MBL bound to N. gonorrhoeae with strain-to-strain variation in the intensity of binding and nature of the bacterial receptor. Pretreatment with MBL concentrations similar to those found in human serum modulated the adhesion of N. gonorrhoeae strain FA1090 but not strain MS11 to epithelial cells. This effect was dose-dependent. This work demonstrates that MBL is present in human semen and modifies cellular responses to N. gonorrhoeae in a concentration-dependent manner.

Phase II trial of irinotecan/gemcitabine as second-line therapy for relapsed and refractory small-cell lung cancer: Cancer and Leukemia Group B Study 39902.

BACKGROUND: This phase II study evaluated the efficacy and safety of the irinotecan/gemcitabine combination in patients with relapsed/refractory small-cell lung cancer (SCLC). PATIENTS AND METHODS: Patients with measurable tumor who had received one previous chemotherapy or chemotherapy/radiation regimen were eligible. Gemcitabine 1000 mg/m(2) was administered i.v. over 30 min followed immediately by irinotecan 100 mg/m(2) i.v. over 90 min, both on days 1 and 8 every 21 days. Patients were stratified based on response to initial treatment [i.e. primary sensitive disease with progression >or=3 months (group A), or refractory disease (group B)]. RESULTS: Seventy-three patients were enrolled but one never received treatment and one ineligible patient did not have SCLC. Median patient ages of the remaining patients were 61 and 63 years in groups A (n = 35) and B (n = 36), respectively, with performance status of 0 or 1 in 85% of 71 patients. Primary grade 3/4 toxic effects in groups A versus B were neutropenia (36% versus 43%), thrombocytopenia (36% versus 26%), nausea (12% versus 11%), vomiting (0 versus 11%), diarrhea (12% versus 9%), and pulmonary (12% versus 12%). Two patients had fatal events including pneumonitis (n = 1) and acute respiratory distress syndrome (n = 1). Responses occurred in 11 group A [two complete responses and nine partial responses (PRs)] and four group B (all PRs) patients, for response rates of 31% [95% confidence interval (CI) 17%, 49%) and 11% (95% CI 3%, 26%), respectively. Median survival and progression-free survival times were 7.1 (95% CI 6, 10.5) versus 3.5 (95% CI 3.1, 5.7) months, and 3.1 (95% CI 1.6, 5.3) versus 1.6 (95% CI 1.4, 2.8) months for group A versus B. CONCLUSION: The irinotecan/gemcitabine combination is active and well tolerated as second-line therapy in SCLC patients. Additional studies are warranted as second-line therapy in patients who progressed 90 days or more after first-line therapy. However, the observed efficacy results in refractory SCLC patients indicate that this regimen should not be further explored in this population.

Haplotype-specific gene expression profiles in a telomeric major histocompatibility complex gene cluster and susceptibility to autoimmune diseases.

The telomeric class III region of the major histocompatibility complex is gene dense, but apart from the three tumour necrosis factor (TNF) superfamily members (TNF, lymphotoxin alpha and lymphotoxin beta) little is known of the expression and function of the majority of the genes. Recent genetic studies in autoimmune diseases, particularly rheumatoid arthritis (RA), have suggested a human leukocyte antigen (HLA)-DR-independent disease effect in this region. To gain further insights into these associations, we used lipopolysaccharide-stimulated human macrophages to examine inducible mRNA expression and genotype-phenotype relationships for genes in this region. Following stimulation in addition to the expected induction of TNF mRNA, a 14-fold increase of ATP6V1G2 at 18 h (P<0.001) was seen, whereas B-associated transcript (BAT)2 (P<0.001) and leucocyte-specific transcript (LST)1 (P<0.001) were both downregulated. By genotyping single-nucleotide polymorphisms spanning a 70 kb interval centred on the TNF locus, we constructed haplotypes and determined associated expression profiles for 10 genes in the cluster using quantitative real-time polymerase chain reaction. Overexpression of BAT1 mRNA was associated with carriers of a haplotype containing the LST1 marker transmitted to RA cases in a family study and also DRB1(*)15 associated with susceptibility to nephritis in systemic lupus erythematosus. The implications of our findings for the understanding of genetic associations with disease susceptibility in this region are discussed.

Role of macrophage-expressed adipocyte fatty acid binding protein in the development of accelerated atherosclerosis in hypercholesterolemic mice.

Atherosclerosis is an inflammatory disease process associated with elevated levels of plasma cholesterol, especially low-density lipoproteins. The latter become trapped within the arterial wall and are oxidized and taken up by macrophages to form foam cells. This process is an initiating event for atherosclerosis. Fatty acid binding proteins (FABP) are involved in fatty acid metabolism and cellular lipid transport, and adipocyte FABP (aP2) is also expressed in macrophages. We recently generated mice lacking both apolipoprotein (Apo)E and aP2 (ApoE-/-aP2-/-) and found that these mice, compared with ApoE-/- mice, developed markedly smaller atherosclerotic lesions that contained fewer macrophages. Here we investigated the mechanism(s) responsible for this prevention of atherosclerotic lesion formation. Bone marrow transplantations were performed in ApoE-/- mice, receiving cells from either ApoE-/- or ApoE-/-aP2-/- mice. The lack of aP2 in donor marrow cells led to the development of smaller (5.5-fold) atherosclerotic lesions in the recipient mice. No differences were found in plasma cholesterol, glucose, or insulin levels between recipients of bone marrow cells from ApoE-/- or ApoE-/-aP2-/- mice. However, the expression of chemoattractant and inflammatory cytokines was decreased in macrophages from ApoE-/-aP2-/- mice compared with ApoE-/- mice, which may contribute to the decrease in atherosclerotic lesion formation. Taken together, we demonstrate the importance of macrophage aP2 in the development of atherosclerotic lesions.

Impaired abdominal wall development and deficient wound healing in mice lacking aortic carboxypeptidase-like protein.

Aortic carboxypeptidase-like protein (ACLP) is a member of a diverse group of proteins that contain a domain with similarity to that of the Dictyostelium discoideum protein discoidin I. The discoidin domain has been identified in mammalian milk fat globule membrane proteins, blood coagulation factors, and receptor tyrosine kinases, where it may facilitate cell aggregation, adhesion, or cell-cell recognition. Here we show that ACLP is a secreted protein that associates with the extracellular matrix (ECM). During mouse embryogenesis, ACLP is abundantly expressed in the ECM of collagen-rich tissues, including the vasculature, dermis, and the developing skeleton. We deleted the ACLP gene in mice by homologous recombination. The majority of ACLP(-/-) mice die perinatally due to gastroschisis, a severe disruption of the anterior abdominal wall and herniation of the abdominal organs. ACLP(-/-) mice that survived to adulthood developed nonhealing skin wounds. Following injury by a dermal punch biopsy, ACLP(-/-) mice exhibited deficient wound healing compared with controls. In addition, dermal fibroblasts isolated from ACLP(-/-) 18.5-day-postconception embryos exhibited a reduced proliferative capacity compared with wild-type cells. These results indicate that ACLP is an ECM protein that is essential for embryonic development and dermal wound healing processes.

Exacerbation of chronic renovascular hypertension and acute renal failure in heme oxygenase-1-deficient mice.

Heme oxygenase (HO) is a cytoprotective enzyme that degrades heme (a potent oxidant) to generate carbon monoxide (a vasodilatory gas that has anti-inflammatory properties), bilirubin (an antioxidant derived from biliverdin), and iron (sequestered by ferritin). Because of properties of HO and its products, we hypothesized that HO would be important for the regulation of blood pressure and ischemic injury. We studied chronic renovascular hypertension in mice deficient in the inducible isoform of HO (HO-1) using a one kidney-one clip (1K1C) model of disease. Systolic blood pressure was not different between wild-type (HO-1(+/+)), heterozygous (HO-1(+/-)), and homozygous null (HO-1(-/-)) mice at baseline. After 1K1C surgery, HO-1(+/+) mice developed hypertension (140+/-2 mm Hg) and cardiac hypertrophy (cardiac weight index of 5.0+/-0.2 mg/g) compared with sham-operated HO-1(+/+) mice (108+/-5 mm Hg and 4.1+/-0.1 mg/g, respectively). However, 1K1C produced more severe hypertension (164+/-2 mm Hg) and cardiac hypertrophy (6.9+/-0.6 mg/g) in HO-1(-/-) mice. HO-1(-/-) mice also experienced a high rate of death (56%) within 72 hours after 1K1C surgery compared with HO-1(+/+) (25%) and HO-1(+/-) (28%) mice. Assessment of renal function showed a significantly higher plasma creatinine in HO-1(-/-) mice compared with HO-1(+/-) mice. Histological analysis of kidneys from 1K1C HO-1(-/-) mice revealed extensive ischemic injury at the corticomedullary junction, whereas kidneys from sham HO-1(-/-) and 1K1C HO-1(+/-) mice appeared normal. Taken together, these data suggest that chronic deficiency of HO-1 does not alter basal blood pressure; however, in the 1K1C model an absence of HO-1 leads to more severe renovascular hypertension and cardiac hypertrophy. Moreover, renal artery clipping leads to an acute increase in ischemic damage and death in the absence of HO-1.

Akt participation in the Wnt signaling pathway through Dishevelled.

Inactivation of glycogen synthase kinase 3beta (GSK3beta) and the resulting stabilization of free beta-catenin are critical steps in the activation of Wnt target genes. While Akt regulates GSK3alpha/beta in the phosphatidylinositide 3-OH kinase signaling pathway, its role in Wnt signaling is unknown. Here we report that expression of Wnt or Dishevelled (Dvl) increased Akt activity. Activated Akt bound to the Axin-GSK3beta complex in the presence of Dvl, phosphorylated GSK3beta and increased free beta-catenin levels. Furthermore, in Wnt-overexpressing PC12 cells, dominant-negative Akt decreased free beta-catenin and derepressed nerve growth factor-induced differentiation. Therefore, Akt acts in association with Dvl as an important regulator of the Wnt signaling pathway.

Using radon risk to motivate smoking reduction: evaluation of written materials and brief telephone counselling.

OBJECTIVE: Radon and cigarette smoking have synergistic effects on lung cancer, even when radon concentrations are relatively low. Working through an electric utility company, we sought to reach smoking households with low radon concentrations and motivate smoking cessation or prohibiting smoking in the home. DESIGN: Eligible homes (n = 714) were randomised to receive: (1) the Environmental Protection Agency's (EPA's) "A citizen's guide to radon"; (2) a specially developed pamphlet; or (3) that pamphlet plus brief telephone counselling. PROCEDURE: Utility company "bill stuffers" offered free radon test kits to smoking households. All households received radon test results with an explanatory cover letter. Both the specially developed pamphlet and the telephone counselling emphasised that smoking cessation or prohibiting smoking in the home were the optimal risk reduction strategies. Households were followed up at 3 and 12 months after receiving materials. RESULTS: The specially developed pamphlet and the EPA guide yielded similar outcomes. There was a non-significant trend for telephone counselling to produce greater sustained quitting than the specially developed pamphlet, and phone counselling led to significantly more new household smoking bans. CONCLUSIONS: Working through a public utility company is an efficient way to reach smoking households, and brief telephone counselling is a promising method for promoting household smoking bans and cessation in homes alerted to the risk posed by the combination of radon and smoking.

Universal healthcare: a bold proposal.

One problem with the American health-care system today is not that it's too expensive but that it's too wasteful. The amount spent on health-care in the United States is sufficient to take care of the medical needs of every citizen. The reason that it does not is that the money is far too often is misspent. America already is spending 14% of the gross domestic product and yet we have health-care chaos and 44 million uninsured. Another problem is the lack of trust between payors, insurance carriers, regulators, employers, employees, providers, and patients. Although the financing aspect of health-care is tremendously important, it is even more important to design a plan to spend the money wisely, align the incentives of insurers, payors, patients, and providers, and restore trust between parties. Otherwise we shall continue to be in chaos regardless of how much money we spend on health-care. In this article, we outline our proposal for an ideal comprehensive national health-care plan that will guarantee that we spend our health-care dollars wisely. Our plan would cover all Americans, including the 44 million uninsured, and it would do so at less than the cost of the current system. Because insurance companies are not equipped to oversee medical practice, our plan would place the day-to-day management of the health-care system in the hands of physicians and local physician-run, physician-owned "provider groups." The physicians in these provider groups would be charged with two primary responsibilities: 1) clinically, they would be responsible for providing total quality cradle-to-grave health-care for every patient in their group and 2) economically, they would be responsible for the budget and to spend it wisely. Physicians will be compensated fee-for-service plus an incentive for efficiency, patient satisfaction, and outcome in a broad sense. Physicians would enjoy wide latitude in clinical decision-making without being second-guessed by distant third parties. Our plan places the fiscal responsibility on physicians while at the same time establishing a system of checks and balances to ensure that patients are protected and well cared for. Unlike outwardly similar plans, under this proposal the physicians are owners of the provider groups and the incentives between payors, insurers, providers, and patients are better aligned. It will eliminate the debate about giving patients the right to sue health plans and employers. It would empower large legally organized physician groups to negotiate with insurers. Our plan is a model for spending money wisely. We believe it would benefit, and therefore be embraced, by all parties--physicians, other healthcare providers, employers, insurance companies, the government, and above all the American public.

Role of gut-enriched Krüppel-like factor in colonic cell growth and differentiation.

Cancer cells differ from normal cells in many aspects, including hyperproliferation and loss of differentiation. Recent research has focused on the role of transcription factors in regulating abnormal cell growth. Gut-enriched Krüppel-like factor (GKLF) is a newly identified eukaryotic zinc finger protein expressed extensively in the gastrointestinal tract. In the current study, we demonstrated that GKLF mRNA levels were significantly decreased in the dysplastic epithelium of the colon, including adenomatous polyp and cancer. GKLF immunostains in the normal colon were higher at the surface epithelium and gradually decreased toward the crypt, but this gradient was not present in the adenomatous and cancerous mucosa. Constitutive overexpression of GKLF DNA in a human colonic adenocarcinoma cell line (HT-29) decreased [(3)H]thymidine incorporation, whereas suppression of GKLF gene increased DNA synthesis, indicating that downregulation of the GKLF gene might contribute to cellular hyperproliferation. Cyclin D1 (CD1) protein level and CD1-associated kinase activity were decreased in HT-29 cell overexpressed GKLF cDNA, and CD1 promoter activity was profoundly suppressed by GKLF. When HT-29 cells were cultured in the presence of sodium butyrate, GKLF mRNA levels increased as cells acquired more differentiated phenotypes. These results suggest that GKLF plays an important role in regulating cell growth and differentiation in the colonic epithelium and that downregulation of GKLF expression may cause colonic cells to become hyperproliferative. Furthermore, GKLF appears to be a transcriptional repressor of the CD1 gene.

Conscientiousness, perceived risk, and risk-reduction behaviors: a preliminary study.

Participants (N = 343) from an Oregon community completed surveys at baseline, 3 months, and 12 months to assess personality, the perceived health risk of radon in combination with smoking, and changes in smoking behavior. Conscientiousness predicted instituting a more restrictive household smoking rule (p < .01), and perceived risk predicted reduction in cigarettes smoked per day for men (p < .001). Perceived risk predicted a reduction in the proportion of cigarettes smoked in the home for those who had high (p < .05) but not low or moderate levels of Conscientiousness, a dimension in one personality model. The results demonstrate the importance of Conscientiousness in the prediction of health behavior, particularly behavior that affects others as well as oneself.

Hematopoietic response to lineage-non-specific (rrIL-3) and lineage-specific (rhG-CSF, rhEpo, rhTpo) cytokine administration in SIV-infected rhesus macaques is related to stage of infection.

The present study reports the hematopoietic response to the exogenous administration of recombinant rhesus interleukin-3 (rrIL-3) or a combination of recombinant human granulocyte colony-stimulating factor (rhG-CSF)/erythropoietin (Epo)/thrombopoietin (Tpo) at two different stages of SIV infection: Early-stage (n = 6, CD4 + > 1000/microl and mild splenomegaly) and late-stage (n = 6, CD4 + < 500/microl, progressive hepatosplenomegaly and/or weight loss). SIV-infected animals exhibited significantly impaired bone marrow (BM) and peripheral blood (PB) responses to both rrIL-3 and rhG-CSF/Epo/Tpo administration, as compared to historic controls. In addition, compared to early-stage SIV-infected animals, late-stage SIV-infected macaques demonstrated a more marked dysfunction, as assessed by PB and BM CD34 + content and clonogenic progenitors (colony-forming unit). Neither rrIL-3 nor rhG-CSF/Epo/Tpo administration during either early-stage or late-stage SIV infection increased the viral load, as assessed by bDNA assay. These data suggest that hematopoietic reserve and the response to various cytokines is decreased even in early-stage SIV infection, with the hematopoietic dysfunction progressing in parallel to SIV infection.

Radon and cigarette smoking: perceptions of this synergistic health risk.

Past approaches to the measurement of the perceived risk of combined hazards have failed to demonstrate awareness of synergy (S. E. Hampson et al., 1998; D. Hermand, E. Mullet, & B. Coutelle, 1995; D. Hermand, E. Mullet, & S. Lavieville, 1997). Respondents (N = 650) were provided with information about the synergistic risk of lung cancer from the combination of smoking and radon, and their risk perceptions were assessed on two occasions. At Time 1, using Likert-type scales, there was no evidence of synergistic risk perception. At Time 2, using a scale based on the appraisal of relative risk with anchors allowing for the expression of synergy, the combined hazard of radon and smoking was rated as significantly more of a health risk than the single hazards. The findings are discussed in terms of methodological issues in assessing synergistic risk.

In vitro and in vivo responses to interleukin 12 are maintained until the late SIV infection stage but lost during AIDS.

The in vitro proliferative responses of macaque peripheral blood mononuclear cells (PBMCs) to IL-12 appeared similar before and early after SIV infection, whereas macaque PBMCs sampled during symptomatic stages of SIV infection showed markedly decreased responses. IL-12 was administered to SIVmac239-infected rhesus macaques either during the asymptomatic or the AIDS stage of infection in efforts to evaluate the effect of this cytokine on immune responses, viral loads, and hematopoietic functions in vivo. IFN-gamma secretion levels induced during the asymptomatic or early symptomatic phase were similar to preinfection induced levels, whereas in later AIDS stages this response was lost. The constitutive levels of other measured cytokines were not affected by IL-12 administration in vivo. The frequency and activity of circulating NK cells were markedly enhanced at early stages but not at symptomatic stages of SIV infection. pCTL frequencies were enhanced at early symptomatic stages but not at late AIDS stages. Despite its immunomodulatory effect, IL-12 did not seem to exacerbate or inhibit the replication of SIV in vivo, or the frequency of circulating infected lymphocytes. IL-12 administration was associated with a significant yet subclinical and transient decrease in hematocrit and hemoglobin levels without evidence of hemolysis, hemodilution, or reduction in the frequency of colony-forming unit potential of bone marrow CD34+ cells. This phenomenon may be explained by a functional inhibition of differentiation rather than an altered generation of bone marrow precursors. Thus, these results suggest that IL-12 may benefit HIV-1-infected patients only as long as their immune system retains its capability to respond to cytokine stimulation.

Transforming growth factor-beta 1 inhibits cytokine-mediated induction of human metalloelastase in macrophages.

Matrix metalloproteinases (MMP) have been identified in vulnerable areas of atherosclerotic plaques and may contribute to plaque instability through extracellular matrix degradation. Human metalloelastase (MMP-12) is a macrophage-specific MMP with broad substrate specificity and is capable of degrading proteins found in the extracellular matrix of atheromas. Despite its potential importance, little is known about the regulation of MMP-12 expression in the context of atherosclerosis. In this study, we report that in human peripheral blood-derived macrophages, MMP-12 mRNA was markedly up-regulated by several pro-atherosclerotic cytokines and growth factors including interleukin-1beta, tumor necrosis factor-alpha, macrophage colony-stimulating factor, vascular endothelial growth factor, and platelet-derived growth factor-BB. In contrast, the pleiotropic anti-inflammatory growth factor transforming growth factor-beta1 (TGF-beta1) inhibited cytokine-mediated induction of MMP-12 mRNA, protein, and enzymatic activity. Analyses of MMP-12 promoter through transient transfections and electrophoretic mobility shift assays indicated that both its induction by cytokines and its inhibition by TGF-beta1 depended on signaling through an AP-1 site at -81 base pairs. Moreover, the inhibitory effect of TGF-beta1 on MMP-12 was dependent on Smad3. Taken together, MMP-12 is induced by several factors implicated in atherosclerosis. The inhibition of MMP-12 expression by TGF-beta1 suggests that TGF-beta1, acting via Smad3, may promote plaque stability.

Recombinant human CD40 ligand inhibits simian immunodeficiency virus replication: a role for interleukin- 16.

CD40 ligand (CD40L), expressed on activated T cells, binds its receptor, CD40, on dendritic cells, B cells, and monocytes/ macrophages. Human immunodeficiency virus (HIV)-infected individuals exhibit normal B-cell CD40 expression but diminished expression of CD40L on CD4 + T cells. Thus, we studied recombinant human CD40L (huCD40L) in an in vitro rhesus macaque model of acquired immunodeficiency syndrome (AIDS). huCD40L induced peripheral blood mononuclear cell (PBMC) proliferation independent of mitogenic cytokines and led to a 70% reduction in p27 production by simian immunodeficiency virus (SIV) mac239 infected PBMCs (P < 0.05). Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis showed reduced expression of SIV gag and increased expression of interleukin (IL)-16 mRNA. Supernatants from huCD40L-stimulated PBMC and control cultures contained similar amounts of IL-16, suggesting an intracellular antiviral effect by IL-16. Phytohemagglutinin (PHA)-stimulated PBMCs similarly cultured with huCD40L showed only slight increases in chemokine production (P > 0.05). These results suggest that huCD40L inhibits replication (antigen and mRNA production) of SIVmac239. This response involves huCD40L induction of IL16 mRNA expression and appears to be independent of beta-chemokines.

Generation of a dominant-negative mutant of endothelial PAS domain protein 1 by deletion of a potent C-terminal transactivation domain.

Endothelial PAS domain protein 1 (EPAS1) is a basic helix-loop-helix/PAS domain transcription factor that is preferentially expressed in vascular endothelial cells. EPAS1 shares high homology with hypoxia-inducible factor-1alpha (HIF-1alpha) and, like HIF-1alpha, has been shown to bind to the HIF-1-binding site and to activate its downstream genes such as vascular endothelial growth factor (VEGF) and erythropoietin. In this report, we show that EPAS1 increased VEGF gene expression through the HIF-1-binding site. This transactivation was enhanced further by cotransfection of an aryl hydrocarbon receptor nuclear translocator expression plasmid. Deletion analysis of EPAS1 revealed a potent activation domain (amino acids 486-639) essential for EPAS1 to transactivate the VEGF promoter. We confirmed the ability of this domain to activate transcription using a Gal4 fusion protein system. Because a truncated EPAS1 protein lacking the transactivation domain at amino acids 486-639 eliminated induction of the VEGF promoter by wild-type EPAS1, the truncated protein functions as a dominant-negative mutant. Most important, infection of the cells with an adenoviral construct expressing this mutant inhibited the induction of VEGF mRNA under conditions that mimic hypoxia. Our results suggest that EPAS1 is an important regulator of VEGF gene expression. Since VEGF plays a crucial role in angiogenesis, the ability of dominant-negative EPAS1 to inhibit VEGF promoter activity raises the possibility of a novel approach to inhibiting pathological angiogenesis.