Heat shock protein 90 is involved in regulation of hypoxia-driven proliferation of embryonic neural stem/progenitor cells

Hypoxia may regulate the proliferation of diverse stem cells. Our previous study showed that hypoxia promoted the proliferation of embryonic neural stem/progenitor cells (NPCs) and that hypoxia inducible factor-1(HIF-1) was critical in this process. HIF-1 could be stabilized under hypoxic conditions, and heat shock protein 90 (HSP90) is an essential protein that controls the activity and stabilization of HIF-1α. In the present work, we investigate whether HSP90 is involved in proliferation of NPCs under hypoxia by regulating HIF-1α stabilization. Geldanamycin (GA), an HSP90 inhibitor, decreased the expression of HIF-1α in NPCs during hypoxia-driven proliferation and reduced the expression level of HIF-1α protein under hypoxia in a time-dependent manner. The proliferation of NPCs induced by hypoxia was inhibited after GA treatment for 24 h. Another HSP90 inhibitor, radicicol, had the same effect on NPCs as GA. Furthermore, the expression of erythropoietin (EPO) and vascular endothelial growth factor (VEGF) in NPCs under hypoxia was suppressed by GA. The above data indicated that HSP90 might be involved in regulation of hypoxia-driven proliferation. Both institutes have contributed equally to this work.     

Short-term hyperhomocysteinemia-induced oxidative stress activates retinal glial cells and increases vascular endothelial growth factor expression in rat retina

Hyperhomocysteinemia is associated with an increase in the incidence of vascular diseases, including retinal vascular diseases. We examined the effects of high plasma levels of homocysteine on retinal glial cells and vascular endothelial growth factor (VEGF) expression. Male Sprague-Dawley rats were fed either a 3.0 g/kg homocystine diet or a control diet for 2 week. The homocystine-diet group had higher plasma levels of homocysteine and thiobarbituric acid reactive substances (TBARSs) and lower plasma levels of folate, retinol, alpha-tocopherol, and retinal expression of CuZn superoxide dismutase (SOD) than the controls. The rats fed the homocystine-diet showed an increase in vimentin, glial fibrillary acidic protein (GFAP), and VEGF immunoreactivity in the retina as compared to the controls. The increase in vimentin immunoreactivity in the hyperhomocysteinemic rats was correlated with changes in GFAP immunoreactivity in astrocytes within the ganglion cell layer. We found for the first time that short-term hyperhomocysteinemia-induced oxidative stress activates retinal glial cells and increases VEGF expression in the retina.     

Expression of αB-Crystallin in the Peripapillary Glial Cells of the Developing Chick Retina

Peripapillary glial cells (PPGCs) are a peculiar macroglia in avian species, located in the central retina adjacent to the optic nerve head. PPGCs have a similar shape and orientation to Müller cells, which traverse the entire layer of the retina; however, there are differences in protein expression between the two cell types. In the present study, we first demonstrated that PPGCs expressed αB-crystallin, which is not expressed in Müller cells, during retinal development. αB-crystallin was first faintly expressed in PPGCs of the E5 retina, adjacent to the optic nerve head. Further, αB-crystallin was exclusively expressed in PPGCs up to E14. The shape of these cells was bipolar with vitread and ventricular processes. The vitread processes of αB-crystallin+ PPGCs became finer at E18. Double labeling analysis clearly demonstrated that only vimentin+ or GFAP+ astrocytes were located in the optic nerve head and were demarcated from the retina by αB-crystallin+ PPGCs. Furthermore, we determined that αB-crystallin+ PPGCs, with a number of processes, completely wrapped the optic nerve head and were densely located in the junction of the optic nerve head and the retina in a whole mount preparation and in vertical-sectioned retinae. The results of present study, together with reports that retinal astrocytes migrate from the optic nerve head, suggest that PPGCs prevent astrocytes from migrating into the retina in avian species.     

Effect of hypoxia-inducible factor-1alpha silencing on the sensitivity of human brain glioma cells to doxorubicin and etoposide

Multidrug resistance (MDR) is a significant problem underlying the poor prognosis associated with gliomas. Hypoxia-inducible factor-1α (HIF-1α) is thought to induce the genes expression involved in MDR. To evaluate the effect of silencing HIF-1α in human glioma T98G cells, cells were transfected with HIF-1α-small interference RNA (HIF-1α-siRNA) and cultured under hypoxic conditions. The effect of HIF-1α-siRNA on HIF-1α and multidrug resistance-associated protein 1 gene (MRP1) and protein levels was determined. Silencing rates of HIF-1α were 90%, 85%, and 88% at 24, 48, 72 h post-transfection, respectively. Corresponding rates of HIF-1α protein were 74.5%, 61.1% and 59.1%. MRP1 protein levels decreased by 7.6%, 36.8% and 45.2%. HIF-1α-siRNA transfected cells were significantly more sensitive to doxorubicin and etoposide compared to non-transfected cells. These findings suggest that the HIF-1α plays a role in mediating chemotherapeutic drug resistance in glioma cells. HIF-1α silencing may prove to be an effective therapeutic means of treating gliomas.     

Concerted inhibition of HIF-1α and -2α expression markedly suppresses angiogenesis in cultured RPE cells

HIF-1α is known to play an important role in the induction of VEGF by hypoxia in retinal pigment epithelial (RPE) cells. However, the involvement of the other isoform, HIF-2α, in RPE cells remains unclear. Thus, the purpose of present study was to clarify the role of HIF-2α during induction of angiogenic genes in hypoxic RPE cells. When human RPE cells (ARPE-19) were cultured under hypoxic conditions, HIF-1α and HIF-2α proteins increased. This induced an increase in mRNA for VEGF, causing secretion of VEGF protein into the medium. This conditioned medium induced tube formation in human vascular endothelial cells (HUVEC). The increased expression of mRNA for VEGF in hypoxic RPE cells was partially inhibited by HIF-1α siRNA, but not by HIF-2α siRNA. However, co-transfection of HIF-1α siRNA and HIF-2α siRNA augmented downregulation of VEGF mRNA and protein in hypoxic RPE cells and inhibited formation of tube-like structures in HUVEC. GeneChip and PCR array analyses revealed that not only VEGF, but also expression of other angiogenic genes were synergistically downregulated by co-transfection of hypoxic RPE cells with HIF-1α and HIF-2α siRNAs. These findings suggest an important compensatory role for the HIF-2α isoform in the regulation of angiogenic gene expression. Thus, suppression of angiogenic genes for HIF-1α and HIF-2α may be a possible therapeutic strategy against retinal angiogenesis in Age-related macular degeneration (ARMD).     

Hypoxic damage to the periventricular white matter in neonatal brain: role of vascular endothelial growth factor, nitric oxide and excitotoxicity

The present study examined factors that may be involved in the development of hypoxic periventricular white matter damage in the neonatal brain. Wistar rats (1-day old) were subjected to hypoxia and the periventricular white matter (corpus callosum) was examined for the mRNA and protein expression of hypoxia-inducible factor-1alpha (HIF-1alpha), endothelial, neuronal and inducible nitric oxide synthase (eNOS, nNOS and iNOS), vascular endothelial growth factor (VEGF) and N-methyl-D-aspartate receptor subunit 1 (NMDAR1) between 3 h and 14 days after hypoxic exposure by real-time RT-PCR, western blotting and immunohistochemistry. Up-regulated mRNA and protein expression of HIF-1alpha, VEGF, NMDAR1, eNOS, nNOS and iNOS in corpus callosum was observed in response to hypoxia. NMDAR1 and iNOS expression was found in the activated microglial cells, whereas VEGF was localized to astrocytes. An enzyme immunoassay showed that the VEGF concentration in corpus callosum was significantly higher up to 7 days after hypoxic exposure. NO levels, measured by colorimetric assay, were also significantly higher in hypoxic rats up to 14 days after hypoxic exposure as compared with the controls. A large number of axons undergoing degeneration were observed between 3 h and 7 days after the hypoxic exposure at electron-microscopic level. Our findings point towards the involvement of excitotoxicity, VEGF and NO in periventricular white matter damage in response to hypoxia.     

The increased expression of DEC1 gene is related to HIF-1α protein in gastric cancer cell lines

Overexpression of differentiated embryo chondrocyte 1 (DEC1) has been reported to contribute to the cellular differentiation, proliferation, and apoptosis of various cancers. Our previous studies have shown that DEC1 was highly expressed in gastric cancer (GCa) tissues. However, there is no report about the expression of DEC1 in GCa cell lines until now. In this study, We evaluated the mRNA and protein expression of DEC1 and hypoxia-inducible factor 1α (HIF-1α) under normoxic and hypoxic conditions in six GCa cell lines: BGC-823, MGC80-3, MKN1, AGS, FU97 and SGC-7901. An HIF-1α protein inhibitor was used to analyze the association of DEC1 and HIF-1α expression. Under normoxia, the mRNA expression of both HIF-1α and DEC1 was moderate, whereas the protein expression of DEC1 was higher than that of HIF-1α. Hypoxia induced the mRNA expression of DEC1 and the protein expression of HIF-1α and DEC1 in a time-dependent manner but had no effect on the mRNA expression of HIF-1α. Furthermore, inhibition of HIF-1α protein expression resulted in a significant decrease in both the mRNA and protein expression of DEC1. Taken together, DEC1 expression is correlated with HIF-1α protein in GCa cell line, blockage of HIF-1α protein led to reduced DEC1 expression. The efficacy of inhibiting HIF-1α and DEC1 expression should be tested in clinical trials as possible treatment for GCa.     

Hesperidin inhibits expression of hypoxia inducible factor-1 alpha and inflammatory cytokine production from mast cells

The citrus unshiu peel has been used traditionally as a medicine to improve bronchial and asthmatic conditions or cardiac and blood circulation in Korea, China, and Japan. Here, we report the effects of citrus unshiu peel water extract (CPWE) on the phorbol myristate acetate (PMA) + calcium ionophore A23187-induced hypoxia-inducible factor-1α (HIF-1α) activation and inflammatory cytokine production from the human mast cell line, HMC-1 cells. We compared CPWE with hesperidin, a common constituent of citrus unshiu. CPWE and hesperidin inhibited the PMA + A23187-induced HIF-1α expression and the subsequent production of vascular endothelial growth factor (VEGF). In addition, CPWE suppressed PMA + A23187-induced phosphorylation of the extracellular signal-regulated kinase (ERK). We also show that the increased cytokines interleukin (IL)-1β, IL-8, and tumor necrosis factor (TNF)-α level was significantly inhibited by treatment of CPWE or hesperidin. In the present study, we report that CPWE and hesperidin are inhibitors of HIF-1α and cytokines on the mast cell-mediated inflammatory responses.     

The Role of Hypoxia in the Differentiation of P19 Embryonal Carcinoma Cells into Dopaminergic Neurons

Nervous system development at early stage is in hypoxic environment. Very little is known about the role of hypoxia in neuronal development. P19 embryonal carcinoma (EC) cells are a widely used model for studying early neuronal development. In this study we investigated the roles of hypoxia in differentiation of dopaminergic neurons derived from P19 EC cells. Results demonstrate that hypoxia increases the percentage of differentiated neurons, especially neurons of dopaminergic phenotype. To investigate the potential mechanism involved in hypoxia promoted differentiation of dopaminergic neurons, we measured the expression of hypoxia-inducible factor 1α (HIF-1α), based on its characteristic response to hypoxia. The result shows that HIF-1α mRNA level in P19 EC cells increases after hypoxia treatment. It is known that HIF-1α regulates the expression of tyrosine hydroxylase (TH) gene through binding to its promoter. Therefore, we propose that the underlying mechanism for hypoxia promoted differentiation of dopaminergic neurons was mediated by HIF-1α up-regulation under hypoxia. Yue Wang—Co-first author. Special Issue in honor of Dr. Ji-Sheng Han.     

TFF3 mediated induction of VEGF via hypoxia in human gastric cancer SGC-7901 cells

Increasing evidence indicates that in gastric epithelial cells, induction of TFF3 by hypoxia is mediated by HIF-1. Since VEGF is one of the most important angiogenic factors on cancer progression, we have started to investigate the possible link among HIF-1α, VEGF, and TFF3 in gastric cancer cells. We induced the hypoxic condition in SGC-7901cells using hypoxia-mimetic agent of CoCI2. SGC7901 cells were transfected with pcPUR + U6 plasmid carrying RNAi targeted to human TFF3 and selected puromycin-resistant pools to establish the stable knockdown of TFF3 cells. Our results showed the induction of HIF-1a via hypoxia and consequences of increased expressions of the TFF3 and VEGF in gastric cancer SGC-7901 cells. Overexpression of TFF3 upregulated the mRNA expressions of VEGF and HIF-1a induced by hypoxia, and stable knockdown of TFF3 impaired the mRNA upregulations of VEGF and HIF-1a induced by hypoxia. Furthermore, knockdown of TFF3 reduced the VEGF protein secretion: as VEGF secretion was increased time dependent manner in response to the hypoxia induction in TFF3-WT cells; however, VEGF production was significantly decreased in TFF3-KD cells (621 ± 89 vs. 264 ± 73 at 6 h and 969 ± 97 vs. 508 ± 69 at 12 h, P < 0.05). Our data demonstrated the TFF3 mediated regulation of VEGF expression induced by hypoxia, and implicated that TFF3 might be applied as a potential anti-angiogenic target for treatment of gastric cancer.     

Regulatory role of vHL/HIF-1alpha in hypoxia-induced VEGF production in hepatic stellate cells

Activated hepatic stellate cells (HSCs) produce cyclooxygenase-2 (COX-2) protein to induce vascular endothelial growth factor (VEGF) production that participates in angiogenesis in injured liver. To reveal the unknown regulatory mechanism, we used hypoxic atmosphere mimicking injured-tissue microenvironment to induce VEGF expression in a rat hepatic stellate cell line (T6-HSCs). The present study showed that hypoxia up-regulated the protein levels of COX-2 and hypoxia-inducible factor-1-alpha (HIF-1alpha), but rapidly effected degradation of von Hippel-Lindau (vHL) protein. As a result, expression of VEGF in HSCs was markedly elevated; and pretreatment with COX-2 inhibitors (nimesulide or indomethacin) could significantly ameliorate the angiogenic event. Collectively, hypoxic HSCs increased accumulation of HIF-1alpha protein and induced VEGF expression in a time-dependent manner. Inhibition of COX-2 activities would prevent vHL protein from degradation and suppress HIF-1alpha up-regulation. Thus, vHL/HIF-1alpha has a regulatory role in COX-2-mediated VEGF production in hypoxic stellate cells in injured liver.