The small GTPase Rab25 has been functionally linked to tumour progression and aggressiveness in ovarian cancer and promotes invasion in three-dimensional environments. This type of migration has been shown to require the expression of the hypoxia-inducible factor 1 alpha (HIF-1α). In this report we demonstrate that Rab25 regulates HIF-1α protein expression in an oxygen independent manner in a panel of cancer cell lines. Regulation of HIF-1α protein expression by Rab25 did not require transcriptional upregulation, but was dependent on de novo protein synthesis through the Erbb2/ERK1/2 and p70S6K/mTOR pathways. Rab25 expression induced HIF-1 transcriptional activity, increased cisplatin resistance, and conferred intraperitoneal growth to the A2780 cell line in immunocompromised mice. Targeting HIF1 activity by silencing HIF-1β re-sensitised cells to cisplatin in vitro and reduced tumour formation of A2780-Rab25 expressing cells in vivo in a mouse ovarian peritoneal carcinomatosis model. Similar effects on cisplatin resistance in vitro and intraperitoneal tumourigenesis in vivo were obtained after HIF1b knockdown in the ovarian cancer cell line SKOV3, which expresses endogenous Rab25 and HIF-1α at atmospheric oxygen concentrations. Our results suggest that Rab25 tumourigenic potential and chemoresistance relies on HIF1 activity in aggressive and metastatic ovarian cancer. Targeting HIF-1 activity may potentially be effective either alone or in combination with standard chemotherapy for aggressive metastatic ovarian cancer.

BACKGROUND

CMET represents an emerging therapy target for monoclonal antibodies and tyrosine kinase inhibitors in non-small cell lung cancer (NSCLC).

METHODS

We investigated CMET gene amplification status by fluorescence in-situ hybridization (FISH) and CMET protein expression by immunohistochemistry in a large series of 209 NSCLC brain metastases (BM; 165 adenocarcinoma, 20 squamous cell carcinoma, 11 adenosquamous carcinomas, 11 large cell carcinomas and two large cell neuroendocrine carcinomas) and correlated our results to clinic-pathological parameters and molecular data from previous studies.

RESULTS

We found CMET gene amplification in 36/167 (21.6%) and CMET protein expression in 87/196 (44.4%) of evaluable BM. There was a strong correlation between the presence of CMET gene amplification and CMET protein expression (P < 0.001, chi-square test). Furthermore, presence of CMET amplification correlated positively with presence of ALK amplifications (P = 0.039, chi-square test) and high HIF1 alpha index (P = 0.013, Mann-Whitney U-test). Neither CMET expression nor CMET gene amplification status correlated with patient outcome parameters or known prognostic factors.

CONCLUSIONS

CMET overexpression and CMET amplification are commonly found in NSCLC BM and may represent a promising therapeutic target.

Theileria annulata infects predominantly macrophages, and to a lesser extent B cells, and causes a widespread disease of cattle called tropical theileriosis. Disease-causing infected macrophages are aggressively invasive, but this virulence trait can be attenuated by long-term culture. Attenuated macrophages are used as live vaccines against tropical theileriosis and via their characterization one gains insights into what host cell trait is altered concomitant with loss of virulence. We established that sporozoite infection of monocytes rapidly induces hif1-α transcription and that constitutive induction of HIF-1α in transformed leukocytes is parasite-dependent. In both infected macrophages and B cells induction of HIF-1α activates transcription of its target genes that drive host cells to perform Warburg-like glycolysis. We propose that Theileria-infected leukocytes maintain a HIF-1α-driven transcriptional programme typical of Warburg glycolysis in order to reduce as much as possible host cell H2 O2 type oxidative stress. However, in attenuated macrophages H2O2 production increases and HIF-1α levels consequently remained high, even though adhesion and aggressive invasiveness diminished. This indicates that Theileria infection generates a host leukocytes hypoxic response that if not properly controlled leads to loss of virulence.

Traumatic brain injury (TBI) elicits identifiable changes within the adult subventricular zone (SVZ). Previously, we demonstrated that EphB3/ephrinB3 interaction inhibits neural stem/progenitor cell (NSPC) proliferation and downregulating this pathway following TBI plays a pivotal role in the expansion of the SVZ neurogenic compartment. It remains unclear, however, what early initiating factors may precede these changes. Using hypoxyprobe-1 (HPb) to identify regions of low oxygen tension or hypoxia (<1%), we found HPb uptake throughout the cortex (CTX), corpus callosum (CC) and SVZ within the first 24 h following controlled cortical impact (CCI) injury. At this early time point, HPb co-localized with EphB3 in the SVZ. NSPC specific markers also co-localized with HPb staining throughout the lateral wall of the ventricle. To determine the cell autonomous effects of hypoxia on EphB3/ephrinB3 signaling in NSPCs, we used an in vitro model of hypoxia to mimic 1% oxygen in the presence and absence of soluble aggregated ephrinB3 (eB3). As expected, hypoxia stimulated the uptake of 5-bromo-2'-deoxyuridine (BrdU) and reduced cell death. Coincident with these proliferative changes, both Hif1-α and phospho (p)-AKT were increased while EphB3 expression was decreased. Stimulation of EphB3 attenuated hypoxia-induced proliferation and prevented phosphorylation of AKT. Hif1-α accumulation, on the other hand, was not affected by EphB3/ephrinB3 signaling. These findings indicate that this pathway limits the NSPC response to hypoxic stimuli. These studies also suggest that early transient changes in oxygen tension following localized cortical injury may initiate a growth-promoting response in the SVZ.

Hypoxia-inducible transcription factor 1 (HIF-1), consisting of HIF-1 alpha and HIF-1 beta subunits, regulates the expression of a variety of genes involved in diverse adaptive processes in response to hypoxia. While oxygen availability regulates HIF-1 alpha by proteolytic degradation, some growth factors regulate HIF-1 alpha by protein synthesis in part through mammalian target of rapamycin complex 1 (TORC1) pathway. We herein report the role of nutrient availability on the regulation of HIF-1. A reduced availability of glucose, not amino acids, results in a decrease of the expression of HIF1-dependent genes and HIF-1 alpha protein in response to hypoxia. HIF-1 alpha mRNA expression was not significantly suppressed and DMOG, an inhibitor for proteasomal degradation of HIF-1 alpha, did not induce HIF-1 alpha protein expression under hypoxia combined with glucose depletion. In comparison to the effect in the presence of glucose, glucose depletion under hypoxia induced a much stronger activation of the AMP-dependent kinase pathway and phosphorylation of eIF2 alpha, and nearly complete inhibition of the TORC1 pathway. These findings imply that the reduced availability of glucose under hypoxia downregulates HIF-1 in part through the inhibition of HIF-1 alpha mRNA translation, which is occasionally observed in pathophysiological situations such as ischemic diseases.

BACKGROUND

The role of glucose transporter 14 (GLUT-14/SLC2A14) in tumor biology is entirely unknown, and the significance of hypoxia inducible factor 1-alpha (HIF1-α) for gastric adenocarcinoma is controversial. The impact of GLUT-1/SLC2A1 has never been confirmed in a Caucasian cohort.

METHODS

Between 1996 and 2007, 124 patients underwent gastrectomy for gastric adenocarcinoma. Tumor sections were incubated with GLUT-1, GLUT-14, and HIF1-α antibodies. Expression was analyzed for correlations with histopathology, marker coexpression, and patient survival by uni- and multivariate analyses.

RESULTS

Expressions of GLUT-1, GLUT-14, and HIF1-α were detectable in 50, 77.4, and 27.1 %, respectively. Expression of GLUT-1 was associated with pT-category (p = 0.019), pN-category (p = 0.019), tubular (WHO, p = 0.008), and intestinal (Lauren classification; p = 0.002) histologic subtypes. Expression of GLUT-14 was correlated with pT category (p = 0.043), whereas HIF1-α did not show any correlation with histopathology or survival. The median survival period was 14 months (95 % confidence interval [CI] 9.2-18.8 months) for GLUT-1-positive patients and 55 months (95 % CI 25.8-84.2; p = 0.01) for GLUT-1-negative patients. An inferior prognosis also was seen for GLUT-14-positive cases compared with GLUT-14-negative cases (p = 0.004). Thus, worst survival was seen with both GLUT-1- and GLUT-14-positive expression followed by single-positive and then double-negative cases (p = 0.004). In multivariate analysis including International Union Against Cancer (UICC) stages, R category, Lauren classification, surgery alone versus neoadjuvant/perioperative chemotherapy, and marker expression as covariates, GLUT-1 (p = 0.011) and GLUT-14 (p = 0.025) kept their prognostic independence.

CONCLUSIONS

The study findings suggest that detection of GLUT-1 and GLUT-14 is of high prognostic value. It gives additional information to UICC stages and identifies patients with inferior prognosis. If confirmed in prospective studies, these markers need to be considered for future classification systems.

Low (2%) oxygen conditions during postcompaction culture of bovine blastocysts improve embryo quality and are associated with small increases in the expression of glucose transporter 1 (SLC2A1), anaphase promoting complex (ANAPC1), and myotrophin (MTPN), suggesting a role for oxygen in the regulation of embryo development, mediated through oxygen-sensitive gene expression. However, bovine embryos, to at least the blastocyst stage, lack detectable levels of the key regulator of oxygen-sensitive gene expression, hypoxia-inducible 1 alpha (HIF1A), while the less well-characterized HIF2 alpha protein is readily detectable. Here we report that other key HIF1 regulated genes are not significantly altered in their expression pattern in bovine blastocysts in response to reduced oxygen concentrations postcompaction-with the exception of lactate dehydrogenase A (LDHA), which was significantly increased following 2% oxygen culture. Antioxidant enzymes have been suggested as potential HIF2 target genes, but their expression was not altered following low-oxygen culture in the bovine blastocyst. The addition of desferrioxamine (an iron chelator and inducer of HIF-regulated gene expression) during postcompaction stages significantly increased SLC2A1, LDHA, inducible nitric oxide synthase (NOS2A), and MTPN gene expression in bovine blastocysts, although development to the blastocyst stage was not significantly affected. These results further suggest that expression of genes, known to be regulated by oxygen via HIF-1 in somatic cells, is not influenced by oxygen during preimplantation postcompaction bovine embryo development. Oxygen-regulated expression of LDHA and SLC2A1 in bovine blastocysts suggests that regulation of these genes may be mediated by HIF2. Furthermore, the effect of a reduced-oxygen environment on gene expression can be mimicked in vitro through the use of desferrioxamine. These results further support our data that the bovine blastocyst stage embryo is unique in its responsiveness to oxygen compared with somatic cells, in that the lack of HIF1-mediated gene expression reduces the overall response to low (physiological) oxygen environments, which appear to favor development.

BACKGROUND

Treatment strategies for Retinoblastoma (RB), the most common primary intraocular tumor in children, have evolved over the past few decades and chemoreduction is currently the most popular treatment strategy. Despite success, systemic chemotherapeutic treatment has relevant toxicity, especially in the pediatric population. Antiangiogenic therapy has thus been proposed as a valuable alternative for pediatric malignancies, in particolar RB. Indeed, it has been shown that vessel density correlates with both local invasive growth and presence of metastases in RB, suggesting that angiogenesis could play a pivotal role for both local and systemic invasive growth in RB. We present here two cases of sporadic, bilateral RB that did not benefit from the conservative treatment and we provide evidence that the VEGF-A pathway is significantly up-regulated in both RB cases along with an over expression of hERG1 K+ channels.

METHODS

Two patients showed a sporadic, bilateral RB, classified at Stage II of the Reese-Elsworth Classification. Neither of them got benefits from conservative treatment, and the two eyes were enucleated. In samples from both RB cases we studied the VEGF-A pathway: VEGF-A showed high levels in the vitreous, the vegf-a, flt-1, kdr, and hif1-α transcripts were over-expressed. Moreover, both the transcripts and proteins of the hERG1 K+ channels turned out to be up-regulated in the two RB cases compared to the non cancerous retinal tissue.

CONCLUSIONS

We provide evidence that the VEGF-A pathway is up-regulated in two particular aggressive cases of bilateral RB, which did not experience any benefit from conservative treatment, showing the overexpression of the vegf-a, flt-1, kdr and hif1-α transcripts and the high secretion of VEGF-A. Moreover we also show for the first time that the herg1 gene transcripts and protein are over expressed in RB, as occurs in several aggressive tumors. These results further stress the relevance of the VEGF-A pathway in RB and the correlation with hERG1, making aggressive and recurrent RB cases good candidates for antiangiogenesis therapies based on the targeting of VEGF-A.

We aimed to analyze genotypes of VEGF-A, VEGFR2, Flt4, PDGFRα, HIF-1α and ERCC1 and their correlation with thymic tumor risk and patient outcome. DNA of 57 consecutive patients (43 thymomas and 14 thymic carcinomas) who underwent total thymectomy at our Institution was extracted from paraffin-embedded tissue. We selected polymorphisms in the following genes:HIF1-α (rs2057482T>> C, rs1951795A>> C, rs2301113C>> A, rs10873142C>> T, rs11158358G>> C, rs12434438G>> A, rs11549465C>> T, rs11549467G>> A), VEGF-A (rs2010963G>> C, rs699947A>> C), VEGFR-2 (rs2305948C>> T, rs1870377T>> A), VEGFR-3 (rs307826T>> C, rs307821C>> A), PDGFR-α (rs35597368C>> T) and ERCC1 (rs11615A>> G). Gene polymorphisms were determined by Real-Time PCR using TaqMan assays. As compared to the general population, the allele frequency of PDGFR-α rs35597368T was significantly higher (95% vs. 87%, p = 0.036), while the frequency of alleles HIF1-α rs2057482C (78% vs. 90%), rs1951795C (69% vs. 87%), rs2301113A (70% vs. 83%), rs10873142T (70% vs. 87%), rs11158358C (75% vs. 88%), rs12434438A (67% vs. 84%) were significantly lower. VEGFR-3 rs307821C frequency was significantly higher in thymomas vs. thymic carcinomas (79% vs. 72%, p = 0.0371). The following factors were significantly correlated with a longer overall survival: VEGFR-3 rs307826C, VEGFR-2 rs1870377A, PDGFR-α rs35597368T/C, HIF1-α rs2301113C, rs2057482C/T, rs1951795C, rs11158358G/C and rs10873142T/C, ERCC1 rs11615A (p < 0.05). Our results suggest, for the first time, that PDGFR-α, HIF-1α and VEGFR-3 SNPs are associated with thymic cancer risk and survival.

OBJECTIVE

Anemia is considered a major factor that counteracts the efficacy of radiotherapy, presumably because of reduced oxygen availability that leads to tumor hypoxia. Nevertheless, anemia is not the only factor defining oxygen availability, because a poor and/or immature vascular network may prevent blood flow and tumor oxygenation. Furthermore, the ability of tumors to upregulate hypoxia-regulated molecular pathways may affect radiosensitivity by mechanisms independent of the traditional concept of "oxygen effect."

METHODS

In this study, we investigated whether the preoperative blood hemoglobin levels affect the activation status of hypoxia/angiogenic pathways (hypoxia inducible factors [HIF1 alpha and HIF2 alpha], carbonic anhydrase 9, differentiated embryo-chondrocyte protein, vascular endothelial growth factor, and microvessel density), in squamous cell head-and-neck cancer.

RESULTS

Hypoxia/angiogenesis pathways were equally activated in tumors, independent of the patient's hemoglobin levels. The expression of HIF alphas was associated with microvessel density (p = 0.01).

CONCLUSIONS

In the present study, we failed to show that a patient's anemia is a main contributor to the activation of hypoxia-regulated molecular pathways in squamous cell head-and-neck cancer. Impaired intratumoral blood flow or tumor-related gene/protein pathologic features may account for this finding. Targeting the hypoxia-regulated molecular cascade emerged as a complementary radiosensitization strategy for a large group of patients with hypoxic tumors, who are unlikely to benefit from conventional approaches aiming to improve intratumoral oxygen delivery through anemia correction.

BACKGROUND

The beneficial impact of mesenchymal stem cells (MSC) on both acute and chronic liver diseases has been confirmed, although the molecular mechanisms behind it remain elusive. We aim to identify factors secreted by undifferentiated and hepatocytic differentiated MSC in vitro in order to delineate liver repair pathways potentially targeted by MSC.

METHODS

Secreted factors were determined by protein arrays and related pathways identified by biomathematical analyses.

RESULTS

MSC from adipose tissue and bone marrow expressed a similar pattern of surface markers. After hepatocytic differentiation, CD54 (intercellular adhesion molecule 1, ICAM-1) increased and CD166 (activated leukocyte cell adhesion molecule, ALCAM) decreased. MSC secreted different factors before and after differentiation. These comprised cytokines involved in innate immunity and growth factors regulating liver regeneration. Pathway analysis revealed cytokine-cytokine receptor interactions, chemokine signalling pathways, the complement and coagulation cascades as well as the Januskinase-signal transducers and activators of transcription (JAK-STAT) and nucleotide-binding oligomerization domain-like receptor (NOD-like receptor) signalling pathways as relevant networks. Relationships to transforming growth factor β (TGF-β) and hypoxia-inducible factor 1-α (HIF1-α) signalling seemed also relevant.

CONCLUSIONS

MSC secreted proteins, which differed depending on cell source and degree of differentiation. The factors might address inflammatory and growth factor pathways as well as chemo-attraction and innate immunity. Since these are prone to dysregulation in most liver diseases, MSC release hepatotropic factors, potentially supporting liver regeneration.

BACKGROUND

Brain metastases (BM) of gastro-oesophageal cancer are exceedingly rare and only limited data exist on their pathobiology.

METHODS

We identified tissue samples of BM of gastro-oesophageal cancer and analyzed the expression of human epidermal growth factor receptor-2 (HER2), phosphorylated signal transducer and activator of transcription-3 (pSTAT3), epithelial growth factor receptor (EGFR), V600E point mutation of the v-raf murine sarcoma viral oncogene homolog-B1 (BRAF V600E), cluster of differentiation molecule-34 (CD34), hypoxia inducible factor-1α (HIF 1-α) and Ki-67 by immunohistochemical methods.

RESULTS

Our series comprised of twenty adenocarcinomas and one oesophageal squamous cell carcinoma. Three (14%), 7 (33%), 9 (43%), 18 (86%) and 0 BM specimens were scored positively for HER2, EGFR, pSTAT3, HIF1-α and BRAF V600E expression. The median Ki-67 index was 59%. The microvascular density was moderate-to-high and active intratumoral microvascular sprouting was evident in 20/21 (95%) of BMs. The HER2 and EGFR expression status were consistent between primary tumors and BM in all three assessable cases. HIF1-α and pSTAT3 expression were significantly higher in HER2-positive cases.

CONCLUSIONS

Therapeutic use of agents targeting HER2, pSTAT3, EGFR and angiogenesis may be feasible for selected BM of gastro-esophageal cancer. HER2 positivity does not seem to predispose to brain colonization in gastro-esophageal cancer.

Hypoxia-inducible factor-1 is a heterodimer made up of an oxygen-regulated HIF1alpha subunit and a constitutively expressed HIF1beta subunit. Among the 70 target genes of HIF-1 known so far, several are involved in angiogenesis, erythropoiesis, cell proliferation, cell viability, and glucose and iron metabolisms. Intratumoral hypoxia or genetic alterations can lead to HIF-1 alpha over-expression. HIF-1 over-expression has been associated with an increased patient mortality rate in many cancer types. Also, in vitro suppression of hif1alpha gene expression has been shown to be efficient in tumour growth repression. During the past five years, drugs able to indirectly inhibit HIF1 activity have been rationally or empirically developed. Some are currently evaluated in clinical trials, but further work has still to be undertaken to rationally identify new specific inhibitors of HIF1 and to test their efficacy as anticancer therapeutics. This review focuses on HIF1 regulation, HIF1 involvement in tumour promotion, the different HIF-1 inhibitors currently tested and their mechanisms of action.

Persistent HPV infection alone is not sufficient for cervical cancer development, which requires additional molecular alterations for tumor progression and metastasis ultimately leading to a lethal disease. In this study, we performed a comprehensive analysis of HER family receptor alterations in cervical adenocarcinoma. We detected overexpression of HER protein, mainly HER2, which was an independent prognostic marker for these patients. By using in vitro and in vivo approaches, we provided evidence that HER inhibitors, allitinib and lapatinib, were effective in reducing cervical cancer aggressiveness. Furthermore, combination of these drugs with glucose uptake blockers could overcome the putative HIF1-α-mediated resistance to HER-targeted therapies. Thus, we propose that the use of HER inhibitors in association with glycolysis blockers can be a potentially effective treatment option for HER-positive cervical cancer patients.

High-altitude hypoxia generates spermiogram impairment due to germinal epithelium, Leydig cells, sperm and seminal plasma alterations, but precise mechanisms involved are unknown. The objective of this work was to analyse the effect of normobaric hypoxia on the morphology of testicular interstitium and some associated molecular and hormonal factors. Twenty-four mice were exposed to normobaric hypoxia (8.1% inspired oxygen fraction) during 20 days. The effects on body weight, testicular weight, vascularisation, testosterone, HIF1-α and VEGF were analysed at different periods of exposure and compared to controls. Hypoxic mice had lower body weight than mice kept in normoxia. Testicular weight raised significantly the 1st day, but remained normal during the rest of experiment. Number of blood vessels per field and mean diameter of vessels were higher in hypoxic mice. Plasmatic and testicular testosterone raised during first 24 h of hypoxia, but decreased on the 5th day. Vascular/interstitial ratio (proportion of interstice occupied by blood vessels) duplicated at the end of the experiment. Most substantial early effects of hypoxia were testicular oedema, increase in number and diameter of blood vessels and elevation of plasmatic and testicular testosterone. Normobaric hypoxia generates similar effects to those induced by hypobaric hypoxia.

We have studied the prophylactic administration of nifedipine and its molecular mechanism involved in reducing the transvascular leakage and inflammation in rats under hypoxia. Rats exposed to an altitude of 7620m for 6h resulted into significant increase in transvascular leakage, oxidative stress with increased NFkB expression in lungs followed by significant increase in pro inflammatory cytokines (IL-1, TNF-α) with up regulation of cell adhesion molecules (ICAM-I, VCAM-I, E-selectin, and P-selectin) in the lungs over control. Prophylactic administration of nifedipine significantly reduced the transvascular leakage, oxidative stress, inhibited the up regulation of NFkB in lungs of rats compared to control. In addition, nifedipine significantly suppressed the levels of proinflammatory cytokines and cell adhesion molecules and stabilized the HIF1-α accumulation in the lungs of rats compared to control. These results indicate that, nifedipine has an inhibitory effect on initial leaking and showed reduction in progression of inflammation through down regulation of NFkB activity in lungs of rats under hypoxia.

Neuroblastoma (NB) is the most common extracranial solid cancer in young children and malignant NB cells have been shown to possess cancer stem cell (CSC) characteristics. Thus, the successful elimination of CSCs represents a strategy for developing an effective preventive and chemotherapeutic agent. CSCs are characterized by differentiation and tumorigenicity. β-Carotene (BC) has been associated with many anticancer mechanisms, although the efficacy of BC on CSCs remains unclear. In the present study, the effects of BC on tumor cell differentiation and tumorigenicity was investigated using a xenograft model. Mice were pretreated with BC for 21 days, then received a subcutaneous injection of SK-N-BE(2)C cells. Both tumor incidence and tumor growth were significantly inhibited for mice that received BC supplementation compared to the control group. Treatment with BC has also been shown to induce tumor cell differentiation by up-regulating differentiation markers, such as vimentin, peripherin, and neurofilament. Conversely, BC treatment has been shown to significantly suppress tumor stemness by down-regulating CSC markers such as Oct 3/4 and DLK1. BC treatment also significantly down-regulated HIF1-α expression and its downstream target, vascular endothelial growth factor (VEGF). Taken together, these results suggest that BC is a potential chemotherapeutic reagent for the treatment of NB, and mediates this effect by regulating the differentiation and stemness of CSCs, respectively.

It is well established that thiamine deficiency results in an excess of metabolic intermediates such as lactate and pyruvate, which is likely due to insufficient levels of cofactor for the function of thiamine-dependent enzymes. When in excess, both pyruvate and lactate can increase the stabilization of the hypoxia-inducible factor 1-alpha (HIF-1α) transcription factor, resulting in the trans-activation of HIF-1α regulated genes independent of low oxygen, termed pseudo-hypoxia. Therefore, the resulting dysfunction in cellular metabolism and accumulation of pyruvate and lactate during thiamine deficiency may facilitate a pseudo-hypoxic state. In order to investigate the possibility of a transcriptional relationship between hypoxia and thiamine deficiency, we measured alterations in metabolic intermediates, HIF-1α stabilization, and gene expression. We found an increase in intracellular pyruvate and extracellular lactate levels after thiamine deficiency exposure to the neuroblastoma cell line SK-N-BE. Similar to cells exposed to hypoxia, there was a corresponding increase in HIF-1α stabilization and activation of target gene expression during thiamine deficiency, including glucose transporter-1 (GLUT1), vascular endothelial growth factor (VEGF), and aldolase A. Both hypoxia and thiamine deficiency exposure resulted in an increase in the expression of the thiamine transporter SLC19A3. These results indicate thiamine deficiency induces HIF-1α-mediated gene expression similar to that observed in hypoxic stress, and may provide evidence for a central transcriptional response associated with the clinical manifestations of thiamine deficiency.

Although excess iron induces oxidative stress in the liver, it is unclear whether it directly activates the hepatic stellate cells (HSC).

We evaluated the effects of excess iron on fibrogenesis and transforming growth factor beta (TGF-β) signaling in murine HSC. Cells were treated with holotransferrin (0.005-5g/L) for 24 hours, with or without the iron chelator deferoxamine (10µM). Gene expressions (α-SMA, Col1-αHif1-α, Tfrc and Slc40a1) were analyzed by quantitative real time-polymerase chain reaction, whereas TfR1, ferroportin, ferritin, vimentin, collagen, TGF-β RII and phospho-Smad2 proteins were evaluated by immunofluorescence, Western blot and enzyme-linked immunosorbent assay.

HSC expressed the iron-uptake protein transferrin receptor 1 (TfR1) and the iron-export protein ferroportin. Holotransferrin upregulated TfR1 expression by 1.8-fold (P < 0.03) and ferritin accumulation (iron storage) by 2-fold (P < 0.01), and activated HSC with 2-fold elevations (P < 0.03) in α-SMA messenger RNA and collagen secretion, and a 1.6-fold increase (P < 0.01) in vimentin protein. Moreover, holotransferrin activated the TGF-β pathway with TGF-β messenger RNA elevated 1.6-fold (P = 0.05), and protein levels of TGF-β RII and phospho-Smad2 increased by 1.8-fold (P < 0.01) and 1.6-fold (P < 0.01), respectively. In contrast, iron chelation decreased ferritin levels by 30% (P < 0.03), inhibited collagen secretion by 60% (P < 0.01), repressed fibrogenic genes α-SMA (0.2-fold; P < 0.05) and TGF-β (0.4-fold; P < 0.01) and reduced levels of TGF-β RII and phospho-Smad2 proteins.

HSC express iron-transport proteins. Holotransferrin (iron) activates HSC fibrogenesis and the TGF-β pathway, whereas iron depletion by chelation reverses this, suggesting that this could be a useful adjunct therapy for patients with fibrosis. Further studies in primary human HSC and animal models are necessary to confirm this.

OBJECTIVE

Glioblastoma multiforme (GBM) is the most common form of primary brain tumors. Although mutations in isocitrate dehydrogenase-1 (IDH1) have been identified in a number of cancers, their role in tumor development has not been fully elucidated. In this study, we aimed to investigate the association between IDH1 mutations, tumor tissue HIF-1 alpha, and serum VEGF levels in patients with primary GBM for the first time.

METHODS

32 patients (mean age, years: 58±14.0) diagnosed with primary glioblastoma multiforme were screened for IDH1 mutations (R132H, R132S, R132C and R132L) by direct sequencing. Serum VEGF and tumor tissue HIF1-alpha levels were measured by enzyme-linked immunosorbent assay. Associations between categoric variables were determined using chi-square tests. Differences between two groups were compared with t test for continuous variables.

RESULTS

Six percent of patients were found to be heterozygous for R132H mutation. Tumor HIF1-alpha and serum VEGF levels were found to be significantly increased in IDH1-mutated tumor tissues (p<0.0001 and p=0.0454, respectively).

CONCLUSIONS

Our results suggest that mutated IDH1 may contribute to carcinogenesis via induction of HIF-1 alpha pathway in primary GBM.

OBJECTIVE

To investigate how levels of vascular endothelial growth factor (VEGF) and VEGF receptor (VEGFR) change during corpus luteum formation in reproductive aging.

METHODS

Adult and reproductive aging female rats were given pregnant mare serum gonadotropin (PMSG) and the ovaries and sera were collected at 0, 3, 5, and 7 days after human chorionic gonadotropin (hCG) injection.

METHODS

Laboratory study.

METHODS

Animal model.

METHODS

Induction of corpus luteum formation with PMSG-hCG.

METHODS

Western blot analysis for ovarian VEGF, VEGFR-1, VEGFR-2, and hypoxia-induced factor 1 (HIF1-alpha). Enzyme-linked immunosorbent assay (ELISA) measurement of serum VEGF levels.

RESULTS

For VEGF and VEGFR-1, there was no difference between the adult and the reproductive aging ovaries. For total VEGFR-2 levels, the adult ovaries had a decline during corpus luteum formation. In contrast, the levels of the two forms of phosphorylated VEGFR-2 declined in aging ovaries. There was a decline in HIF1-alpha during corpora lutea formation in the reproductive aging rats.

CONCLUSIONS

The reproductive aging corpora lutea have a temporal decline in phosphorylated VEGFR-2 levels but not in the total VEGFR-2 levels. The data suggest that the VEGFR response mechanism is different in the aging ovary.

CITED2, a cardiac transcription factor, plays an important role in cardiac development. CITED2 mutations lead to a constellation of cardiac defects, which include tetralogy of Fallot and outflow tract malformations. However, the mechanisms underlying these mutations are poorly understood. We investigated the function and mechanism of two missense mutations, G184S and S192G, responsible for tetralogy of Fallot and aortic stenosis, respectively. We found that CITED2 variants decreased its ability to mediate the expression of vascular endothelial growth factor (VEGF) and the expression of the paired-like homeodomain transcription factor 2-gamma (PITX2C), both of which are closely related to cardiac development. Luciferase reporter and mammalian two-hybrid assays showed that G184S and S192G in CITED2 restored the expression of VEGF, which was due to a reduction in its competitiveness with hypoxia inducible factor 1-alpha (HIF1-α) for binding to CBP/p300. In addition, we found that the G184S and S192G mutant decreased cooperation between CITED2 and transcription factor AP2-gamma (TFAP2C) in the transactivation of the PITX2C gene. These results provide important evidence that the mutation of CITED2 may play a role in the development of congenital heart disease (CHD) in humans.

Pulmonary fibrosis is often complicated by pulmonary hypertension (PH), and previous studies have shown a potential link between bone morphogenetic protein receptor II (BMPR2) and PH secondary to pulmonary fibrosis. We exposed transgenic mice expressing mutant BMPR2 and control mice to repetitive intraperitoneal injections of bleomycin for 4 weeks. The duration of transgene activation was too short for mutant BMPR2 mice to develop spontaneous PH. Mutant BMPR2 mice had increased right ventricular systolic pressure compared to control mice, without differences in pulmonary fibrosis. We found increased hypoxia-inducible factor (HIF)1-α stabilization in lungs of mutant-BMPR2-expressing mice compared to controls following bleomycin treatment. In addition, expression of the hypoxia response element protein connective tissue growth factor was increased in transgenic mice as well as in a human pulmonary microvascular endothelial cell line expressing mutant BMPR2. In mouse pulmonary vascular endothelial cells, mutant BMPR2 expression resulted in increased HIF1-α and reactive oxygen species production following exposure to hypoxia, both of which were attenuated with the antioxidant TEMPOL. These data suggest that expression of mutant BMPR2 worsens secondary PH through increased HIF activity in vascular endothelium. This pathway could be therapeutically targeted in patients with PH secondary to pulmonary fibrosis.

Transcriptional activity of hypoxia-induced factor 1 (HIF1) - a heterodimer of HIF1α and ARNT (HIF1β) - is essential for cellular adaptation to environmental stress and plays an important role in skin development, wound healing, tumorigenesis and barrier function. Using primary mouse and human epidermal keratinocytes at ambient or hypoxic (1% O(2)) conditions we studied effects of hypoxia upon HIF protein expression. Significant nuclear levels of ARNT and HIF1α along with high HIF1 activity in normoxic keratinocytes suggest an as yet uncharacterised oxygen-independent role for HIF pathway in the epidermis. Acute hypoxia results in an instant but transient increase of HIF1α protein accompanied by a gradual decrease in its mRNA, while ARNT expression remains unchanged. In prolonged (chronic) hypoxia both HIF1α and Arnt are downregulated along with decline of HIF1 activity. However, expression of classical HIF1 targets such as Selenbp1 and Vegfa remains high. Thus, keratinocytes respond to acute hypoxia with immediate block of HIF1α protein degradation and concomitant increase of HIF activity, while under chronic hypoxia pro-angiogenic signalling is maintained through HIF1-independent pathway(s). Decline of HIF1α during chronic exposure is controlled at both mRNA and protein levels, while Arnt is downregulated post-translationally. Distinct transcription levels of Hif1α and Hif3α splice variants under normoxia and their differential response to hypoxia suggest functional diversity of Hif-α isoforms and highlight the complexity of HIF machinery control in epidermal keratinocytes.