HIF-1alpha Expression Research Articles

Prognostic factors in patients with advanced renal carcinoma.

391 Background: A retrospective cohort of 135 patients with advanced RCC treated with biological agents and/or cytokines (CK) was analysed between July 1996 and February 2010. Methods: The expression of several biomarkers by immunohistochemistry and 2 analytical variables: thrombocytosis and neutrophilia were analysed and were correlated with prognosis. Results: 67 patients were treated only with biological agents and 68 with CK (23 received also biological agents). The univariate statistical analysis showed that the enhanced expression of HIF-1alpha correlated with a poor prognosis in patients treated with sunitinib (PFS was 5.4 vs. 13.4 months in those with low expression, p=0.001). The overexpression of ACIX was associated to a better prognosis in patients that received biological agents (PFS was 18.3 vs. 5.2 months in those with decreased expression, p<0.001; OS was 32.1 vs. 7.8 months, p<0.001), including sunitinib (PFS was 16.8 vs. 5.5 months, p<0.001), sorafenib (PFS was 8 vs 3.5 months, p<0.001)) and CK (PFS was 6.3 vs. 2.7 months, p=0.003; OS was 32.9 vs. 5.9 months, p=0.001). Positive PTEN was related to a good prognosis in patients treated with sunitinib (PFS was 15.1 vs. 6.5 months, p=0.003) and CK (PFS was 7.5 vs. 3.8 months, p=0.037, OS was 13.7 vs 7.9 months, p=0.039). The increased expression of p21 was related to a poor prognosis in patients that received biological agents (PFS was 5.9 vs. 16.8 months with high expression, p=0.024), including sunitinib (PFS was 6.2 vs 18.9 months, p<0.001), sorafenib (PFS was 4 vs 9 months, p=0.013) and CK (PFS was 3.9 vs. 7.5 months, p<0.001). Thrombocytosis was related to a poor prognosis in patients treated with CK (PFS was 2.6 vs. 5.1 months p=0.017; OS was 5.9 vs. 14.3 months p=0.010). Neutrophilia was related to a poor prognosis in patients that received CK (PFS was 2.6 vs. 5.7 months, p=0.019; OS was 5.9 vs. 12.8 months, p=0.035). In the multivariate analysis, the overexpression of ACIX was a favorable prognostic factor independent of PFS with a HR of 0.107 (p<0.001) and OS with a HR of 0.055 (p<0.001). Conclusions: Our experience has suggested the utility of de HIF-1alpha, ACIX, PTEN, p21, thrombocytosis and neutrophilia as prognostic factors in patients with advanced RCC. ACIX has shown to be an independent prognostic factor. No significant financial relationships to disclose.

Hierarchical clustering analysis of tissue microarray immunostaining data on renal cell carcinomas.

393 Background: The clinical use of molecular expression profiles could result in more accurate and objective diagnoses of cancers as well as prognoses of disease or response to the treatment. Unsupervised hierarchical clustering (UHC) analysis is a common method to profile the molecular expression of tissue microarray data. Methods: TM4: a free, open-source system for microarray data management and analysis was used in order to identify expression patterns of interest in our cohort of renal cell carcinomas (RCC) (n=80). We investigated 5 pathological predictors: (a) the histological type, (b) Fuhrman grade, (c) depth of infiltration, (d) metastasis in lymph node, (e) TNM stages, and 27 immunohistochemical molecular predictors involved in different pathways of tumor development and progression including: p53 and VHL tumor suppressor proteins; Bcl-2 and Survivin antiapoptotic proteins; Hif1-alpha and Notch proteins as a transcription factors; EGFR, PDGFR-α and VEGF proteins involved in tumor growth and proliferation; Glut proteins involved in tumor cell metabolism. Results: Unfavorable prognosis was significantly correlated with pathological predictors. Clear RCC histological subtype had a worse prognosis than the other ones studied to be accompanied by increased expression of Hif1-alpha and CAIX (p<0.001 in both cases) in patients with a number of metastatic nodes weighed (p<0.001). We found in Fuhrman Grade III samples a higher expression of VEGF (p=0.029). UHC analysis was done using average method with Spearman rank test to group the data with different predictor profiles. A preliminary analysis by hierarchical clustering of RCC produced three separate clusters groups. Conclusions: UHC based on a extended immunoprofile might be a useful, promising and powerful tool for further translational studies and should lead us to define a diagnostic and prognostic signature for RCC. Our laboratory is currently involved in this issue. No significant financial relationships to disclose.

Rb1 postconditioning attenuates liver warm ischemia–reperfusion injury through ROS-NO-HIF pathway

AimsGinsenoside Rb1 could prevent ischemic neuronal death and focal cerebral ischemia, but its roles to liver warm I/R injury remain to be defined. We determined if Rb1 would attenuate warm I/R injury in mice. Main methodsMice were divided into sham, I/R, Rb1+I/R (Rb1 postconditioning, 20mg/kg, i.p. after ischemia), sham+L-NAME, I/R+L-NAME, and Rb1+I/R+L-NAME groups using 60min of the liver median and left lateral lobes ischemia. Serum levels of alanine aminotransferase (ALT) were measured and morphology changes of livers were evaluated. Contents of nitric oxide (NO) and nitric oxide synthase (NOS), malondialdehye (MDA) and activity of superoxide dismutase (SOD) were measured. Expressions of Akt, p-Akt, iNOS, HIF-1alpha, tumor necrosis factor-a (TNF-α) and intercellular adhesion molecule-1 (ICAM-1) were also determined by western blot or immunohistochemistry. Key findingsRb1 postconditioning attenuated the dramatically functional and morphological injuries. The levels of ALT were significantly reduced in Rb1 group (p<0.05). Rb1 upregulated the concentrations of NO, iNOS in serum, iNOS, and activity of SOD in hepatic tissues (p<0.05), while it dramatically reduced the concentration of MDA (p<0.05). Protein expressions of p-Akt, iNOS and HIF-1alpha were markedly enhanced in Rb1 group. Protein and mRNA expressions of TNF-α and ICAM-1 were markedly suppressed by Rb1 (p<0.05). SignificanceWe found that Rb1 postconditioning could protect liver from I/R injury by upregulating the content of NO and NOS, and also HIF-1alpha protein expression. These protective effects could be abolished by L-NAME. These findings suggested Rb1 may have the therapeutic potential through ROS-NO-HIF pathway for management of liver warm I/R injury.

Prognostic significance and mechanisms of patterned matrix vasculogenic mimicry in hepatocellular carcinoma

Vasculogenic mimicry (VM), including tubular VM and patterned matrix VM, has been generally recognized as a new pattern of tumor neovascularization. Pilot studies of tubular VM showed that it was present in hepatocellular carcinoma (HCC) and associated with poor clinical prognosis. However, whether patterned matrix VM is clinically significant in HCC is unknown. To elucidate the effects of patterned matrix VM on prognosis of HCC and the mechanisms involved in VM formation, we examined 151 cases of surgically resected human HCC by immunohistochemistry and transmission electron microscopy and conducted hypoxic experiments on human HCC cell line MHCC97-H. We observed 31 of 151 (20.5%) cases exhibited evidence of patterned matrix VM. The expression of patterned matrix VM was associated with larger tumors (P = 0.042), vascular invasion (P = 0.016), high-grade HCC (P = 0.022), and late-stage HCC (P = 0.013). Kaplan-Meier survival analysis revealed that cases of the VM group had lower overall survival (OS) rate (P < 0.001) and disease-free survival (DFS) rate (P = 0.002) than that of the non-VM group. Univariate and multivariate analysis indicated that the presence of patterned matrix VM was independent adverse prognostic factor for both OS (P = 0.004) and DFS (P = 0.011). Expression of hypoxia-inducible factor 1 alpha (HIF-1alpha), matrix metalloproteinase (MMP)-2, and MMP-9 were higher in the VM group than in the non-VM group (P = 0.001, P = 0.030, P = 0.007, respectively). After VM formation was induced by hypoxia, up-regulated expression of HIF-1α, MMP-2, and MMP-9 was also detected in cells cultured under hypoxia condition. Our results indicate that patterned matrix VM exists in HCC, and it might serve as an unfavorable prognostic factor for HCC patients. It is possible that hypoxia via induction of expression of HIF-1alpha, MMP-2, and MMP-9 may enhance VM formation in HCC.

Clinical significance of the expression of HIF-1α and VEGF gene in endometrial carcinoma tissue

Objective To explore the relationship between the expression of HIF-1 alpha,VEGF gene and cliulcal stage of endometrial cancer. Methods 30 patients with the membrane carcinoma were detected VEGF, HIF-1α expression by immunohistochemical method. The expression of different pathological type and clinical stage were analyzed. Another 10 pieces achieved from endometriosis dysplasia organization were taken as control. Results The positive rate of HIF-1 alpha, VEGF in endometriosis dysplasia and endometrial carcinoma were statistically significant differences( x2=11.87,8. 71, all P ＜ 0. 05 );There was correlated relationship between ultrasonic grading of tumor blood and HIF-1 alpha, VEGF; There was correlated relationship between Lesions and HIF-1 alpha, VEGF.Conclusion The expressions of HIF-1 alpha, VEGF were positively correlated to the degree,metastasis and prognosis of malignant endometrial carcinoma. Key words: Endometrial carcinoma; HIF-1α ; VEGF

The Antioxidant Quercetin Inhibits Cellular ProliferationviaHIF-1-Dependent Induction of p21WAF

Flavonoids are dietary antioxidants that may play a role as adjunct nutritional supplements in cancer or during inflammatory disorders. Hypoxia and the transcription factor hypoxia-inducible factor-1alpha also appear to play a key role in many human cancers. In this study, we investigated the role of quercetin in the hypoxia-dependent HIF-1alpha induction. It was shown that quercetin induced HIF-1alpha expression and HIF-1 activity under normoxia and hypoxia in human HepG2 hepatoma cells. By using actinomycin D and cycloheximide, we showed that quercetin acted post-transcriptionally by prolonging the HIF-1alpha protein half-life. Thereby quercetin interfered with the proline hydroxylation-dependent HIF-1alpha protein destabilization in the N-terminal HIF-1alpha transactivation domain. Experiments with quercetin analogues revealed that a flavonol structure and the presence of hydroxyl groups at position 3' and 4' are a prerequisite for the HIF-1alpha stabilizing effects. Further, quercetin inhibited cell proliferation and induced expression of the cell cycle inhibitor p21WAF and knocking-down HIF-1alpha disrupted these effects. These results provide evidence that quercetin inhibits the cell cycle and that induction of the HIF-system contributes to these effects of quercetin.

Inhibition of vascular endothelial growth factor expression by Chinese medicine of Hedyotis diffusa Willd herbal compounds

The Hedyotis diffusa Willd herbal compounds (HDWHCs) are commonly used as Chinese medicine to treat cancer patients with established clinical therapeutic efficacy in China. However, the underlying mechanisms remain to be elucidated. In this study, we used freeze-dried powder of the water extracts of HDWHCs to investigate the potential mechanisms of HDWHCs in cancer treatment. HDWHCs treatment significantly inhibited vascular endothelial growth factor (VEGF) mRNA levels and VEGF transcriptional activation in cancer cells. HDWHCs also had a remarkable inhibitory effect on the expression of hypoxia-inducible factor 1alpha (HIF-1alpha). Forced expression of HIF-1α restored VEGF transcriptional activation inhibited by HDWHCs, indicating that HDWHCs suppressed VEGF expression through decreasing HIF-1alpha expression. Moreover, HDWHCs inhibited cyclooxygenase-2 (COX-2) expression, and overexpression of HIF-1alpha restored HDWHCs’ inhibitory effect on COX-2 at transcriptional level. These findings may provide better understanding of HDWHCs’ anti-cancer mechanism in cancer treatment.

Increased Expression of Hypoxia-Inducible Factor 1α in Coeliac Disease

Previously, it has been suggested that hypoxia-inducible factor (HIF) 1 signaling may play determinative role in the maintenance of the barrier function of the intestinal epithelium in inflammatory bowel disease. Our aim was to depict the alteration of HIF-1alpha and related genes in celiac disease (CD) where the importance of the barrier function is well known. Duodenal biopsy specimens were collected from 16 children with untreated CD, 9 children with treated CD and 10 controls. HIF-1alpha, trefoil factor 1 (TFF1), ecto-5-prime nucleotidase (CD73), and multi drug resistance gene 1 (MDR1) mRNA and HIF-1alpha protein expression were determined by real-time PCR and Western blot, respectively. Localization of HIF-1alpha was determined by immunofluorescent staining. We found increased HIF-1alpha and TFF1 mRNA and HIF-1alpha protein expression in the duodenal mucosa of children with untreated CD compared with controls or children with treated CD (p < 0.05). In untreated CD children, HIF-1alpha staining was present in cytoplasmic and nuclear region of the villous enterocytes. In treated CD mRNA expression of CD73 and MDR1 were increased compared with controls (p < 0.01 and 0.05, respectively). Our results of increased mucosal HIF-1alpha expression in CD children suggest the contribution of this signaling pathway in the pathomechanism of CD.

Impaired retinal vascular development in anencephalic human fetus

This study was to investigate the effect of the absence of ganglion cells on the development of human retinal vasculature. Anencephaly (AnC) and age-matched control eyes derived from each three spontaneously aborted fetus (ranging from 15 to 20 weeks gestation) were subjected to immunofluorescence staining for HIF-1alpha, Thy-1, glial fibrillary acidic protein (GFAP) and platelet/endothelial cell adhesion molecule (PECAM) and apoptosis assay. In developing mouse retina, Western blotting for hypoxia-inducible factor-1alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF) was performed. Under hypoxic condition (O(2) < 1%), cellular proliferation and VEGF mRNA expression in astrocytes were measured. Apoptotic cells in AnC retina were primarily localized in the ganglion cell layer (GCL), whereas apoptotic cells in normal retina were distributed in the retinoblastic layer. With increase of apoptotic cells in GCL of AnC retina, HIF-1alpha expression were severely distinguished in avascular retina and GFAP expression in junctional area between avascular and vascular retina was much reduced, accompanied by decrease of PECAM expression compared to normal retina. In developing mouse retina, HIF-1alpha and VEGF expression were high in hypoxic retina of early stage with incomplete vascular development and then progressively decreased with regression to arborous pattern of matured vascular networks. In hypoxic condition, a significant increase in cellular proliferation and VEGF mRNA expression was observed in astrocytes. Therefore, our results suggest that vascular attenuation in AnC retina could be closely related to the absence of ganglion cells as the metabolic demander to induce retinal vascular development.

RNAi screen for telomerase reverse transcriptase transcriptional regulators identifies HIF1alpha as critical for telomerase function in murine embryonic stem cells.

In various types of stem cells, including embryonic stem (ES) cells and hematopoietic stem cells, telomerase functions to ensure long-term self-renewal capacity via maintenance of telomere reserve. Expression of the catalytic component of telomerase, telomerase reverse transcriptase (Tert), which is essential for telomerase activity, is limiting in many types of cells and therefore plays an important role in establishing telomerase activity levels. However, the mechanisms regulating expression of Tert in cells, including stem cells, are presently poorly understood. In the present study, we sought to identify genes involved in the regulation of Tert expression in stem cells by performing a screen in murine ES (mES) cells using a shRNA expression library targeting murine transcriptional regulators. Of 18 candidate transcriptional regulators of Tert expression identified in this screen, only one candidate, hypoxia inducible factor 1 alpha (Hif1alpha), did not have a significant effect on mES cell morphology, survival, or growth rate. Direct shRNA-mediated knockdown of Hif1alpha expression confirmed that suppression of Hif1alpha levels was accompanied by a reduction in both Tert mRNA and telomerase activity levels. Furthermore, gradual telomere attrition was observed during extensive proliferation of Hif1alpha-targeted mES cells. Switching Hif1alpha-targeted mES cells to a hypoxic environment largely restored Hif1alpha levels, as well as Tert expression, telomerase activity levels, and telomere length. Together, these findings suggest a direct effect of Hif1alpha on telomerase regulation in mES cells, and imply that Hif1alpha may have a physiologically relevant role in maintenance of functional levels of telomerase in stem cells.

Hypoxia and hypoglycaemia in Ewing's sarcoma and osteosarcoma: regulation and phenotypic effects of Hypoxia-Inducible Factor

BackgroundHypoxia regulates gene expression via the transcription factor HIF (Hypoxia-Inducible Factor). Little is known regarding HIF expression and function in primary bone sarcomas. We describe HIF expression and phenotypic effects of hypoxia, hypoglycaemia and HIF in Ewing's sarcoma and osteosarcoma.MethodsHIF-1α and HIF-2α immunohistochemistry was performed on a Ewing's tumour tissue array. Ewing's sarcoma and osteosarcoma cell lines were assessed for HIF pathway induction by Western blot, luciferase assay and ELISA. Effects of hypoxia, hypoglycaemia and isoform-specific HIF siRNA were assessed on proliferation, apoptosis and migration.Results17/56 Ewing's tumours were HIF-1α-positive, 15 HIF-2α-positive and 10 positive for HIF-1α and HIF-2α. Expression of HIF-1α and cleaved caspase 3 localised to necrotic areas. Hypoxia induced HIF-1α and HIF-2α in Ewing's and osteosarcoma cell lines while hypoglycaemia specifically induced HIF-2α in Ewing's. Downstream transcription was HIF-1α-dependent in Ewing's sarcoma, but regulated by both isoforms in osteosarcoma. In both cell types hypoglycaemia reduced cellular proliferation by ≥ 45%, hypoxia increased apoptosis and HIF siRNA modulated hypoxic proliferation and migration.ConclusionsCo-localisation of HIF-1α and necrosis in Ewing's sarcoma suggests a role for hypoxia and/or hypoglycaemia in in vivo induction of HIF. In vitro data implicates hypoxia as the primary HIF stimulus in both Ewing's and osteosarcoma, driving effects on proliferation and apoptosis. These results provide a foundation from which to advance understanding of HIF function in the pathobiology of primary bone sarcomas.

Hypoxia Enhances the Angiogenic Potential of Human Dental Pulp Cells

Introduction Trauma can result in the severing of the dental pulp vessels, leading to hypoxia and ultimately to pulp necrosis. Improved understanding of mechanisms underlying the response of dental pulp cells to hypoxic conditions might lead to better therapeutic alternatives for patients with dental trauma. The purpose of this study was to evaluate the effect of hypoxia on the angiogenic response mediated by human dental pulp stem cells (DPSCs) and human dental pulp fibroblasts (HDPFs). Methods DPSCs and HDPFs were exposed to experimental hypoxic conditions. Hypoxia-inducible transcription factor-1alpha (HIF-1alpha) was evaluated by Western blot and immunocytochemistry, whereas vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) expression was evaluated by enzyme-linked immunosorbent assay. YC-1, an inhibitor of HIF-1alpha, was used to evaluate the functional effect of this transcriptional factor on hypoxia-induced VEGF expression. Conditioned medium from hypoxic and normoxic pulp cells was used to stimulate human dermal microvascular endothelial cells (HDMECs). HDMEC proliferation was measured by WST-1 assay, and angiogenic potential was evaluated by a capillary sprouting assay in 3-dimensional collagen matrices. Results Hypoxia enhanced HIF-1alpha and VEGF expression in DPSCs and HDPFs. In contrast, hypoxia did not induce bFGF expression in pulp cells. YC-1 partially inhibited hypoxia-induced HIF-1alpha and VEGF in these cells. The growth factor milieu of hypoxic HDPFs (but not hypoxic DPSCs) induced endothelial cell proliferation and sprouting as compared with medium from normoxic cells. Conclusions Collectively, these data demonstrate that hypoxia induces complex and cell type–specific pro-angiogenic responses and suggest that VEGF (but not bFGF) participates in the revascularization of hypoxic dental pulps.

Tocopherol induced angiogenesis in placental vascular network in late pregnant ewes

BackgroundTocopherols have biphasic, proangiogenic and antiangiogenic therapeutic effects. The objective of this clinical trial was to clarify tocopherol's placental angiogenic potential in late pregnant ewes following oral supplementation.MethodsEighteen pregnant ewes during late gestation were selected for this study. Ewes were given oral supplementation of 500 mg of alpha-tocopherol (aT; N = 6) or 1000 mg of gamma-tocopherol (gT; N = 7) or placebo (CON; N = 5) once daily from 107 to 137 days post breeding. Serum was obtained at weekly intervals and tissue samples were obtained at the end of supplementation to: 1) evaluate tocopherol concentrations in serum, uterus and placentome; 2) evaluate relative mRNA expressions of Vascular Endothelial Growth Factor (VEGF), Placental Growth Factor (PlGF), endothelial Nitric Oxide Synthase (eNOS) and Hypoxia Inducible Factors (HIF) in uterus, caruncle and cotyledon; 3) analyze the morphometry of the placental vascular network.ResultsSupplementation of aT or gT resulted in increased concentrations in serum, placentome and uterus compared to control (P < 0.05). In aT group, mRNA expressions of PlGF, eNOS and HIF-1α in cotyledon were greater than the CON group. In gT group, mRNA expressions of VEGF, eNOS, HIF-1 alpha and HIF-2 alpha in caruncle and uterus, and HIF-1α in cotyledon, were greater than the CON group. Morphometry analysis revealed increased angiogenesis in the supplemented groups.ConclusionDaily oral supplementation of aT or gT increased angiogenesis in the placental vascular network in pregnant ewes during late gestation. Increase in placental angiogenesis may provide nutrients required for the development and growth of fetus during late pregnancy.

Muscle‐specific expression of hypoxia‐inducible factor in human skeletal muscle

Skeletal muscle is well known to exhibit a high degree of plasticity depending on environmental changes, such as various oxygen concentrations. Studies of the oxygen-sensitive subunit alpha of hypoxia-inducible factor-1 (HIF-1) are difficult owing to the large variety of functionally diverse muscle fibres that possess unique patterns of protein and gene expression, producing different capillarization and energy metabolism systems. In this work, we analysed HIF-1alpha mRNA and protein expression related to the fibre-type composition in untrained human skeletal muscle by obtaining muscle biopsies from triceps brachii (characterized by a high proportion of type II fibres), from soleus (characterized by a high proportion of type I fibres) and from vastus lateralis (characterized by an equal proportion of type I and II fibres). The hypothesis was that type I muscle fibres would have lower HIF-1alpha mRNA and protein owing to their higher oxidative capacity. We have shown, in normoxic conditions, a higher HIF-1alpha protein expression in predominantly oxidative muscles than in predominantly glycolytic muscles. However, the HIF-1alpha mRNA expression pattern was not in agreement with the HIF-1alpha protein level. Interestingly, none of the HIF-1alpha target genes, like the most studied angiogenic factor involved in muscle angiogenesis, vascular endothelial growth factor (VEGF), exhibited a muscle fibre-specific-related mRNA expression at rest in normoxia. However, soleus presented a significantly higher VEGF protein content than vastus lateralis and triceps muscle. In conclusion, we have shown that there are muscle-specific differences in HIF-1alpha and VEGF expression within human skeletal muscle at rest in normoxic conditions. Recent results, when combined with the findings described here, support a key role for HIF-1alpha for maintaining muscle homeostasis in non-hypoxic conditions.

Increased Expression of HIF-1α, VEGF-A and Its Receptors, MMP-2, TIMP-1, and ADAMTS-1 at the Venous Stenosis of Arteriovenous Fistula in a Mouse Model with Renal Insufficiency

A mouse model of renal insufficiency with arteriovenous fistula (AVF) and venous stenosis was created. The authors tested the hypothesis that there is increased gene expression of hypoxia-inducible factor-1 alpha (HIF-1alpha); vascular endothelial growth factor-A (VEGF-A) and its receptors (VEGFR-1, -2); matrix metalloproteinase-2 (MMP-2), -9 (MMP-9); tissue inhibitor of metalloproteinase-1, -2 (TIMP-1, -2); and a disintegrin and metalloproteinase thrombospondin-1 (ADAMTS-1) at the venous stenosis. Nineteen male C57BL/6 mice underwent a left nephrectomy and a surgical occlusion of the right upper pole to induce renal function characterized in eight animals. Twenty eight days later, an AVF (n = 11) was created from the right carotid artery to ipsilateral jugular vein, and the mice were killed at day 7 (n = 4) and day 14 (n = 4). The outflow and control veins were removed for gene expression. Three mice were killed at day 28 for histologic analysis. The mean serum blood urea nitrogen level remained significantly elevated for 8 weeks when compared with baseline (P < .05). By day seven, there was a significant increase in the expression of HIF-1alpha, VEGF-A, VEGFR-1, VEGFR-2, MMP-2, TIMP-1, and ADAMTS-1 at the outflow vein, with HIF-1alpha and TIMP-1 levels significantly elevated at day 14 (P < .05). By day 28, the venous stenosis was characterized by a thickened vein wall and neointima. A mouse model of renal insufficiency with AVF was developed that had increased expression of HIF-1alpha, VEGF-A, VEGFR-1, VEGFR-2, MMP-2, TIMP-1, and ADAMTS-1 at the outflow vein with venous stenosis by day 28.

Early Growth Response-1 Induces and Enhances Vascular Endothelial Growth Factor-A Expression in Lung Cancer Cells

Vascular endothelial growth factor-A (VEGF-A) is crucial for angiogenesis, vascular permeability, and metastasis during tumor development. We demonstrate here that early growth response-1 (EGR-1), which is induced by the extracellular signal-regulated kinase (ERK) pathway activation, activates VEGF-A in lung cancer cells. Increased EGR-1 expression was found in adenocarcinoma cells carrying mutant K-RAS or EGFR genes. Hypoxic culture, siRNA experiment, luciferase assays, chromatin immunoprecipitation, electrophoretic mobility shift assays, and quantitative RT-PCR using EGR-1-inducible lung cancer cells demonstrated that EGR-1 binds to the proximal region of the VEGF-A promoter, activates VEGF-A expression, and enhances hypoxia inducible factor 1alpha (HIF-1alpha)-mediated VEGF-A expression. The EGR-1 modulator, NAB-2, was rapidly induced by increased levels of EGR-1. Pathology samples of human lung adenocarcinomas revealed correlations between EGR-1/HIF-1alpha and VEGF-A expressions and relative elevation of EGR-1 and VEGF-A expression in mutant K-RAS- or EGFR-carrying adenocarcinomas. Both EGR-1 and VEGF-A expression increased as tumors dedifferentiated, whereas HIF-1alpha expression did not. Although weak correlation was found between EGR-1 and NAB-2 expressions on the whole, NAB-2 expression decreased as tumors dedifferentiated, and inhibition of DNA methyltransferase/histone deacetylase increased NAB-2 expression in lung cancer cells despite no epigenetic alteration in the NAB-2 promoter. These findings suggest that EGR-1 plays important roles on VEGF-A expression in lung cancer cells, and epigenetic silencing of transactivator(s) associated with NAB-2 expression might also contribute to upregulate VEGF-A expression.

Placenta Growth Factor-induced Early Growth Response 1 (Egr-1) Regulates Hypoxia-inducible Factor-1α (HIF-1α) in Endothelial Cells

Leukotrienes, the lipid inflammatory products derived from arachidonic acid, are involved in the pathogenesis of respiratory and cardiovascular diseases and reactive airway disease in sickle cell disease. Placenta growth factor (PlGF), elaborated from erythroid cells, increased the mRNA expression of 5-lipoxygenase and 5-lipoxygenase-activating protein (FLAP) in human pulmonary microvascular endothelial cells. PlGF-induced both promoter activity and mRNA expression of hypoxia-inducible factor-1alpha (HIF-1alpha), which was abrogated by early growth response-1 (EGR-1) small interfering RNA. PlGF showed a temporal reciprocal relationship in the mRNA levels of EGR-1 and NAB2, the latter a repressor of Egr-1. Moreover, Nab2, but not mutant Nab2, significantly reduced promoter activity and mRNA expression of HIF-1alpha and also reduced expression of the HIF-1alpha target gene FLAP. Furthermore, overexpression of Egr-1 led to increased promoter activities for both HIF-1alpha and FLAP in the absence of PlGF. Additionally, the Egr-1-mediated induction of HIF-1alpha and FLAP promoters was reduced to basal levels by EGR-1 small interfering RNA. The binding of Egr-1 to HIF-1alpha promoter was corroborated by electrophoretic mobility shift assay and chromatin immunoprecipitation assay, which showed increased Egr-1 binding to the HIF-1alpha promoter in response to PlGF stimulation. These studies provide a novel mechanism for PlGF-mediated regulation of HIF-1alpha via Egr-1, which results in increased FLAP expression. This study provides a new therapeutic target, namely Egr-1, for attenuation of elevated leukotriene levels in patients with sickle cell disease and other inflammatory diseases.

Expression characteristics of hypoxia inducible factor-1a and its clinical values in hepatocellular carcinoma

To investigate the dynamic expression of hypoxia inducible factor-1alpha (HIF-1alpha) and its clinical values in hepatocellular carcinoma (HCC). The dynamic changes of liver pathology, HIF-1alpha transcription and expression were observed through the hepatoma model. The self-control specimens from 35 human HCC patients were collected and the expression, cellular distribution, and clinicopathological features of HIF-1alpha and its gene was analyzed by immunohistochemistry, western blotting and nested- PCR, respectively. Both levels of hepatic HIF-1alpha and HIF-1alpha mRNA expression increased during the HCC development course. The incidence of HIF-1alpha and the ratio of HIF-1alpha to beta-actin was 0% and 0.16+/-0.02 in the control rats, 77.8% and 0.29+/-0.04 in the denatured rats, 88.9% and 0.52+/-0.03 in the precancerous rats, and 100% and 0.84+/-0.02 in the cancerous rats respectively, with significant difference between the control group and any of the experimental groups (P = 0.000). The positive HIF-1alpha was brown and granule-like and mainly presented in cytoplasm and few in nucleus. The incidence of HIF-1alpha was 80% (28/35) in HCC and 100% (35/35) in its surrounding tissues. The clinical pathological features indicated HIF-1alpha expression associated with tumor size and differentiation degree the of HCC. No correlation was found between HIF-1alpha and tumor numbers or positive-HBsAg. HIF-1alpha expression is associated with occurrence and development of HCC, and is perhaps a target molecule for HCC therapy.

RNA interference of HDAC7 expression in hepatocellular carcinoma

To investigate the role of histone deacetylase 7 (HDAC7) in the occurrence and development of hepatocellular carcinoma. HepG2 cells and human microvascular endothelial cells (HMEC-1) were divided into 3 groups after transfection pSUPER-HDAC7 retroviral interference plasmid: a pSUPER(HDAC7RNAi+) group, a pSUPER(HDAC7RNAi-) group, and a pSUPER group as the control group. The expression of HDAC7, p21, cyclin E, matrix metalloproteinases 10 (MMP10), and hypoxiainducible factor-1alpha (HIF-1alpha) were detected by Western blot. The expression of HDAC7 in cell lines was determined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometry, the nude mice modle, and vascular endothelial cells 2-dimensional tubulogenesis in vitro and in vivo. Compared with the control group and the pSUPER(HDAC7RNAi-) group, the expression of HDAC7 was downregulated, the rate of cell growth inhibition and apoptosis in the pSUPER(HDAC7RNAi+) group increased more significantly; the expression of p21 and HIF-1alpha was increased significantly, while the expression of cyclin E and MMP10 in the pSUPER(HDAC7RNAi+) group was downregulated (P<0.05). The expression of HDAC7 protein plays an important role in the apoptosis and vascular tubulogenesis of hepatocellular carcinoma by the upregulation of p21 and HIF-1alpha and the downregulation of cyclin E and MMP10.

Treatment with HIF-1α Antagonist PX-478 Inhibits Progression and Spread of Orthotopic Human Small Cell Lung Cancer and Lung Adenocarcinoma in Mice

PX-478 is a potent small-molecule inhibitor of hypoxia-inducible factor 1alpha (HIF-1alpha). In prior preclinical studies, it had antitumor activity against various solid tumors in subcutaneous xenografts but had no measurable activity against a non-small cell lung cancer (NSCLC) xenograft. To determine the effectiveness of PX-478 against lung tumors, we investigated HIF-1alpha expression in several lung cancer cell lines, both in vitro and in vivo, and treated orthotopic mouse models of human lung cancer with PX-478. Cells from two human lung adenocarcinoma cell models (PC14-PE6 and NCI-H441) or two human small cell lung cancer (SCLC) models (NCI-H187 and NCI-N417) were injected into the left lungs of nude mice and were randomized 16 to 18 days after injection with daily oral treatment with PX-478 or vehicle for 5 days. In the PC14-PE6 NSCLC model, treatment with 20 mg/kg PX-478 significantly reduced the median primary lung tumor volume by 87% (p = 0.005) compared with the vehicle-treated group. PX-478 treatment also markedly reduced mediastinal metastasis and prolonged survival. Similar results were obtained in a second NSCLC model. In SCLC models, PX-478 was even more effective. In the NCI-H187 model, the median primary lung tumor volume was reduced by 99% (p = 0.0001). The median survival duration was increased by 132%. In the NCI-N417 model, the median primary lung tumor volume was reduced by 97% (p = 0.008). We demonstrated that the PX-478, HIF-1alpha inhibitor, had significant antitumor activity against two orthotopic models of lung adenocarcinomas and two models of SCLC. These results suggest the inclusion of lung cancer patients in phase I clinical trials of PX-478.