HIF-1alpha Gene Research Articles

Evolution and Regulation of the Downstream Gene of Hypoxia-Inducible Factor-1α in Naked Carp (Gymnocypris przewalskii) from Lake Qinghai, China

The naked carp (Gymnocypris przewalskii) is a native teleost of Lake Qinghai (altitude, 3.2 km) on the Qinghai-Tibet Plateau in China. Hypoxia-inducible factor (HIF)-1alpha of Gymnocypris przewalskii was cloned and a phylogenetic tree for vertebrate HIF-1alpha was constructed. By analysis of maximum likelihood models of codon substitutions for HIF-1alpha, three positive sites in the branch lineages of crucian carp, eelpout, and flounder, and a higher proportion of neutral sites in naked carp, antarctic eelpout, rainbow trout, and grayling, were detected among all teleosts. It seems that low habitat temperatures relax the purifying selection of HIF-1alpha in these stenothermal coldwater fish, and both cold and hypoxic lake water contributed to the evolution of the HIF-1alpha gene in the naked carp. Furthermore, Glut 1 mRNA, a gene downstream from HIF-1alpha, has a time-course- and tissue-specific dependent response to hypoxic challenge.

A Molecular Approach to Apoptosis in the Human Heart During Brain Death

Brain death induces changes in tissues and organs destined for transplant at the cell, molecular, and endocrine level including cell death through apoptosis. This study was designed to examine apoptotic damage in cardiac tissue obtained from brain dead donors. Fifty tissue specimens from the left ventricles of individual donors were processed to evaluate changes in the expression levels of five genes involved in apoptosis (BAX, BCL2, CASPASE 3, CYTOCHROME C, and FAS) using the real time-polymerase chain reaction technique. Expression levels were quantified by the relative standard method and results normalized to the levels recorded for the endogenous control peptidylprolyl isomerase A. The HIF1alpha gene was also determined to check for the possibility of hypoxic damage. Control ventricular tissue specimens were obtained from patients undergoing mitral valve replacement. Using a mixed linear model it was determined that the sample type (donor vs. control patient) significantly affected (P<0.0001) expression levels of the genes examined reflected by their Ct values. Three of the genes (BAX, CASPASE 3, and FAS) showed significantly higher (Student's t test, P<0.05) expression levels (4.89-, 7.85-, and 12.14-fold endogenous control values, respectively) in donors compared with control patients (2.31-, 2.64-, and 3.57-fold endogenous control values, respectively) indicating the activation of apoptosis during brain death. Our findings suggest the possibility of using antiapoptosis agents to prevent cardiac injury and improve posttransplant behavior.

Inhibition of HIF-1α by the anticancer drug TAS106 enhances X-ray-induced apoptosis in vitro and in vivo

In a previous study, we showed that a novel anticancer drug, 1-(3-C-ethynyl-β-D-ribo-pentofuranosyl)cytosine (TAS106, ECyd) increased the antitumour efficacy of X-irradiation. However, its effects on hypoxic cells in tumours remain unclarified. Here, we show that TAS106 enhances the induction of apoptosis in X-irradiated human gastric adenocarcinoma MKN45 and MKN28 cells under hypoxia in vitro. At the same time, the accumulation of HIF-1α observed under hypoxia was shown to be decreased to the level of normoxia in the presence of 0.1 μM TAS106. To study the function of HIF-1α protein in apoptosis of hypoxic cells, we employed an HIF-1α reductive approach using its specific antisense oligodeoxynucleotide. The reduction of HIF-1α gene expression dramatically enhanced X-ray-induced apoptosis in hypoxic cells. In in vivo experiments in which MKN45 cells were transplanted into severe combined immunodeficient (SCID) mice, TAS106 (0.5 mg kg−1) suppressed HIF-1α expression and subsequently reduced the area of the hypoxic region in the tumour and enhanced the induction of apoptosis in the hypoxic region when combined with 2 Gy of X-irradiation. These results suggest the possibility that TAS106 acts as a potent radiosensitiser through the inhibition of HIF-1α expression and can be a useful agent against radiotherapy-resistant hypoxic cells in solid tumours.

Expression and role of factor inhibiting hypoxia-inducible factor-1 in pulmonary arteries of rat with hypoxia-induced hypertension

Hypoxia-inducible factor-1alpha subunit (HIF-1alpha) plays a pivotal role during the development of hypoxia-induced pulmonary hypertension (HPH) by transactivating it' target genes. As an oxygen-sensitive attenuator, factor inhibiting HIF-1 (FIH) hydroxylates a conserved asparagine residue within the C-terminal transactivation domain of HIF-1alpha under normoxia and moderate hypoxia. FIH protein is downregulated in response to hypoxia, but its dynamic expression and role during the development of HPH remains unclear. In this study, an HPH rat model was established. The mean pulmonary arterial pressure increased significantly after 7 d of hypoxia. The pulmonary artery remodeling index became evident after 7 d of hypoxia, while the right ventricular hypertrophy index became significant after 14 d of hypoxia. The messenger RNA (mRNA) and protein expression of HIF-1alpha and vascular endothelial growth factor (VEGF), a well-characterized target gene of HIF-1alpha, were markedly upregulated after exposure to hypoxia in pulmonary arteries. FIH protein in lung tissues declined after 7 d of hypoxia and continued to decline through the duration of hypoxia. FIH mRNA had few changes after exposure to hypoxia compared with after exposure to normoxia. In hypoxic rats, FIH protein showed significant negative correlation with VEGF mRNA and VEGF protein. FIH protein was negatively correlated with mean pulmonary arterial pressure, pulmonary artery remodeling index and right ventricular hypertrophy index. Taken together, our results suggest that, in the pulmonary arteries of rat exposed to moderate hypoxia, a time-dependent decrease in FIH protein may contribute to the development of rat HPH by enhancing the transactivation of HIF-1alpha target genes such as VEGF.

Expression and role of factor inhibiting hypoxia-inducible factor-1 in pulmonary arteries of rat with hypoxia-induced hypertension

Hypoxia-inducible factor-1alpha subunit (HIF-1alpha) plays a pivotal role during the development of hypoxia-induced pulmonary hypertension (HPH) by transactivating it' target genes. As an oxygen-sensitive attenuator, factor inhibiting HIF-1 (FIH) hydroxylates a conserved asparagine residue within the C-terminal transactivation domain of HIF-1alpha under normoxia and moderate hypoxia. FIH protein is downregulated in response to hypoxia, but its dynamic expression and role during the development of HPH remains unclear. In this study, an HPH rat model was established. The mean pulmonary arterial pressure increased significantly after 7 d of hypoxia. The pulmonary artery remodeling index became evident after 7 d of hypoxia, while the right ventricular hypertrophy index became significant after 14 d of hypoxia. The messenger RNA (mRNA) and protein expression of HIF-1alpha and vascular endothelial growth factor (VEGF), a well-characterized target gene of HIF-1alpha, were markedly upregulated after exposure to hypoxia in pulmonary arteries. FIH protein in lung tissues declined after 7 d of hypoxia and continued to decline through the duration of hypoxia. FIH mRNA had few changes after exposure to hypoxia compared with after exposure to normoxia. In hypoxic rats, FIH protein showed significant negative correlation with VEGF mRNA and VEGF protein. FIH protein was negatively correlated with mean pulmonary arterial pressure, pulmonary artery remodeling index and right ventricular hypertrophy index. Taken together, our results suggest that, in the pulmonary arteries of rat exposed to moderate hypoxia, a time-dependent decrease in FIH protein may contribute to the development of rat HPH by enhancing the transactivation of HIF-1alpha target genes such as VEGF.

Role of Hypoxia-inducible factor-1α and Survivin in colorectal carcinoma progression

Hypoxia-inducible factor-1 alpha (HIF-1 alpha) is the main active subunit of HIF-1 that promoted tumor cells survival and critical steps in tumor progression and aggressiveness. The authors aimed to investigate the role of HIF-1 alpha and Survivin in colorectal cancer (CRC) progression. Plasmid expressing small interfering RNA (siRNA) against HIF-1 alpha was constructed and transfected into LS174T cells with Lipofectamine. The LS174T cells were incubated for 24 h under hypoxic condition. The inhibitory effects of siRNA on HIF-1 alpha gene was determined by semiquantitative reverse transcriptase polymerase chain reaction and Western blot. Expression of HIF-1 alpha and Survivin was investigated by immunohistochemistry in colorectal adenocarcinomas tissue microarrays. HIF-1 alpha and Survivin expressions were markedly downregulated after the siRNA expression vector against HIF-1 alpha was transfected into the LS174T cells. Of the eight adenoma lesions, one case (12.25%) and four cases (50%) were positive for HIF-1 alpha and Survivin, respectively. Of the 69 cases of CRCs, 46 cases (66.7%) and 39 cases (56.5%) were positive for HIF-1 alpha and Survivin, respectively. The positive rate of HIF-1 alpha protein in CRCs was significantly higher than that in colorectal adenoma lesions (P < 0.05). HIF-1 alpha protein expression was significantly higher in patients with stage III than in patients with stage I-II CRCs (P < 0.01). In addition, overexpression of HIF-1 alpha in higher stages of CRCs was found to correlate positively with Survivin levels (P < 0.001). Our data demonstrate that HIF-1 alpha and Survivin are mostly expressed in invasive CRCs. Inhibition of HIF-1 alpha may lead to exploration of its potential as a diagnostic tool and possibly a target for gene therapy for colorectal carcinoma.

Small hairpin loop RNA targeting HIF-1α down-regulates VEGF and up-regulates PEDF in human retinal pigment epithelial cells under hypoxic condition

The aim of this study was to explore the effect of small hairpin loop RNA (shRNA) silencing hypoxia-induced factor 1alpha (HIF-1alpha) gene on the expression of vascular endothelial growth factor (VEGF) and pigment epithelium derived factor (PEDF) in human retinal pigment epithelium (RPE) cells under hypoxic condition. Two target sites of HIF-1alpha mRNA were chosen and two kinds of shRNA were designed and synthesized against the target sites. Then the two kinds of shRNA were transfected into human RPE cells in vitro, respectively. These cells were cultured under hypoxic condition that was simulated by using 150 mumol/L CoCl(2). The mRNA expressions of HIF-1alpha, VEGF and PEDF were tested by semi-quantitative reverse transcription PCR (RT-PCR). The protein levels of HIF-1alpha, VEGF and PEDF were analyzed by Western blotting. After the two kinds of HIF-1alpha-specific shRNA were transfected into RPE cells respectively, the expression of HIF-1alpha mRNA and the levels of HIF-1alpha protein were decreased significantly in RPE cells under hypoxic condition. The expression of VEGF mRNA and the levels of protein significantly were also decreased. However, the levels of PEDF protein was significantly increased, but the expression of PEDF mRNA showed no significant changes. In conclusion, HIF-1alpha-specific shRNA can effectively silence the HIF-1alpha gene, and consequently down-regulate VEGF and up-regulate PEDF expression against hypoxia. These results reveal that HIF-1 is associated with posttranslational mechanism for down-regulating PEDF under hypoxia and provide an explanation for hypoxia-provoked increases in VEGF/PEDF ratios. These results also suggest that HIF-1 is one of the key cytokines to retinal neovascularization.

Hyperbaric Oxygen Attenuates Apoptosis and Decreases Inflammation in an Ischemic Wound Model

The molecular mechanisms whereby hyperbaric oxygen (HBO) improves ischemic wound healing remain elusive. In this study, a rat model of wound ischemia was used to test the hypothesis that HBO enhances wound healing by modulating hypoxia-inducible factor-1alpha (HIF-1alpha) signaling. Male Sprague-Dawley rats underwent creation of a previously validated ischemic flap. Three groups underwent daily treatment: HBO (90 minutes, 2.4 atm); systemic administration of the free radical scavenger, N-acetylcysteine (NAC 150 mg kg(-1) intraperitoneal); control (neither HBO nor NAC). HBO treatment improved healing of the ischemic wounds. Analysis of ischemic wound tissue extracts demonstrated significantly reduced expression of HIF-1alpha, p53, and BNip3. Additionally, HBO increased expression of Bcl-2 while decreasing cleaved caspase-3. DNA fragmentation was abolished and the number of TUNEL-positive cells was reduced compared to the other groups. Vascular endothelial growth factor, cyclooxygenase-2, and neutrophil infiltration were reduced in ischemic wounds treated with HBO. These results indicate that HBO improves ischemic wound healing by downregulation of HIF-1alpha and subsequent target gene expression with attenuation of cell apoptosis and reduction of inflammation.

Protective effect of puerarin on diabetic retinopathy in rats

Puerarin is a major active ingredient extracted from the traditional Chinese medicine Ge-gen. The purpose of this study is to investigate the protective effect of puerarin on diabetic retinopathy (DR) and its mechanisms in rats. Seventy-two male Wistar rats were selected and divided at random into three main groups: control group, streptozotocin (STZ) group and puerarin + STZ group. Retinal histopathological observation and electron microscopic examination were performed; retinal vascular endothelial growth factor (VEGF) and hypoxia inducible factor 1 (HIF-1alpha) gene expressions were examined by Reverse transcription-polymerase chain reaction (RT-PCR) analysis. Results showed that the DR induced by STZ was significantly reduced by the treatment of puerarin as judged by the reduction of morphological changes of inner nuclear layer and outer nuclear layer at any time-point. Puerarin regulates expressions of VEGF and HIF-1alpha stimulated by STZ. It was concluded that puerarin exerts significant protective effects against DR in rats, likely regulating angiogenesis factors expressions, and thus may be an effective and promising medicine for treatment of DR.

Inhibition of proliferation, migration and tube formation of choroidal microvascular endothelial cells by targeting HIF-1α with short hairpin RNA-expressing plasmid DNA in human RPE cells in a coculture system

Retinal pigment epithelial (RPE) cells and choroidal microvascular endothelial cells (CECs) are the main cells involved in choroidal neovascularization (CNV), and hypoxia plays an important role in CNV formation via hypoxia inducible factor 1 (HIF-1). Our aim was to evaluate the role of HIF-1 in human RPE cells with regard to proliferation, migration and tube formation of CECs under hypoxia. RPE cells were cultured under chemical hypoxia induced by 200 muM CoCl(2), and RNA interference (RNAi) technique was used to knock down HIF-1alpha gene in RPE cells. mRNA and protein expression of HIF-1alpha and VEGF in RPE cells were investigated by real-time RT-PCR and Western blot. Three kinds of coculture models were used to observe the effects of RPE cells transfected by short hairpin RNA (shRNA)-expressing plasmid DNA (pDNA) (pshHIF-1alpha) on the proliferation, migration and tube formation of CECs respectively. Transfection of shRNA-expressing pDNA targeting HIF-1alpha to RPE cells resulted in the knock down of HIF-1alpha gene and reduction of the corresponding mRNA and protein of HIF-1alpha and VEGF under hypoxia. Consequently, the proliferation, migration and tube formation of CECs were significantly inhibited by the knocked-down RPE cells compared with the control in the coculture system. The proliferation rates of CECs decreased by 40.2%, 36.6% and 36.8% on days 3, 4 and 5 respectively. Migration reduced by 49.6% at 5 h, and tube formation decreased by 40.4% at 48 h. RNAi of HIF-1alpha in RPE cells can inhibit angiogenesis in vitro and provide a possible strategy for treatment of choroidal neovascularization diseases by targeting HIF-1alpha.

The transcription factor Egr1 regulates the HIF‐1α gene during hypoxia

Using oligonucleotide expression microarrays we have examined the modulation of gene expression in the DU145 prostate cancer cell line. Our findings confirm that the Egr1 transcription factor is rapidly and transiently upregulated by hypoxia. Furthermore, we have demonstrated that HIF-1alpha mRNA is also transiently upregulated, as is its target gene VEGF. To elucidate the mechanism of the transcriptional upregulation of the HIF-1alpha gene, we have shown that Egr1 is able to directly bind to the HIF-1alpha promoter using chromatin immunoprecipitation. We also provide evidence that the binding of Egr1 is necessary for the trans-activation of the HIF-1alpha promoter. These studies highlight the importance for the Egr1 transcription factor in the hypoxic response in cultured prostate cancer cell lines, and indicate that the response of Egr1 is upstream of HIF-1 in these cells. These studies are the first demonstration that the HIF-1alpha transcription factor is targeted directly by Egr1 in hypoxia.

Age‐dependent impairment of HIF‐1α expression in diabetic mice: Correction with electroporation‐facilitated gene therapy increases wound healing, angiogenesis, and circulating angiogenic cells

Wound healing is impaired in elderly patients with diabetes mellitus. We hypothesized that age-dependent impairment of cutaneous wound healing in db/db diabetic mice: (a) would correlate with reduced expression of the transcription factor hypoxia-inducible factor 1alpha (HIF-1alpha) as well as its downstream target genes; and (b) could be overcome by HIF-1alpha replacement therapy. Wound closure, angiogenesis, and mRNA expression in excisional skin wounds were analyzed and circulating angiogenic cells (CACs) were quantified in db/db mice that were untreated or received electroporation-facilitated HIF-1alpha gene therapy. HIF-1alpha mRNA levels in wound tissue were significantly reduced in older (4-6 months) as compared to younger (1.5-2 months) db/db mice. Expression of mRNAs encoding the angiogenic cytokines vascular endothelial growth factor (VEGF), angiopoietin 1 (ANGPT1), ANGPT2, platelet-derived growth factor B (PDGF-B), and placental growth factor (PLGF) was also impaired in wounds of older db/db mice. Intradermal injection of plasmid gWIZ-CA5, which encodes a constitutively active form of HIF-1alpha, followed by electroporation, induced increased levels of HIF-1alpha mRNA at the injection site on day 3 and increased levels of VEGF, PLGF, PDGF-B, and ANGPT2 mRNA on day 7. CACs in peripheral blood increased 10-fold in mice treated with gWIZ-CA5. Wound closure was significantly accelerated in db/db mice treated with gWIZ-CA5 as compared to mice treated with empty vector. Thus, HIF-1alpha gene therapy corrects the age-dependent impairment of HIF-1alpha expression, angiogenic cytokine expression, and CACs that contribute to the age-dependent impairment of wound healing in db/db mice.

C1772T polymorphism of hypoxia-inducible factor-1 alpha gene in Han ethnic population from Northern China

To analyze the distribution of single nucleotide polymorphism C1772T (SNP/C1772T) in hypoxia-inducible factor -1 alpha (HIF-1 alpha) gene in Han ethnic population from Northern China. Allele frequencies in a sample of 206 healthy Chinese Hans were determined by polymerase chain reaction followed by restriction fragment length polymorphism. The genotype frequencies of C1772T observed in Chinese Hans were 93 % and 7% for CC and CT respectively; and allele frequencies were 96% and 4 %for C and T respectively. There was significant difference in distribution of allele frequencies between Chinese Hans and Caucasian. The single nucleotide polymorphism of C1772T in HIF-1 alpha gene has difference in different ethnic population.

Preconditioning with cobalt chloride or desferrioxamine protects oligodendrocyte cell line (MO3.13) from tumor necrosis factor‐α‐mediated cell death

Hypoxia-inducible factor-1alpha (HIF-1alpha) is a transcription factor induced under hypoxic conditions. HIF-1alpha promotes the expression of genes encoding proteins that increase the cellular supply of oxygen and promote survival in periods of cellular stress and availability of cellular energy. We examined the effect of desferrioxamine (DFO) and cobalt chloride (CoCl(2)), two agents known to increase the stability of HIF-1alpha, and its effect on the survivability of an oligodendroglial cell line, MO3.13, when cultured with tumor necrosis factor-alpha (TNFalpha). Our studies showed that, unlike a murine microglial cell line (BV-2), MO3.13 cells do not induce HIF-1alpha in the presence of TNFalpha. MO3.13 cells do stabilize HIF-1alpha in the presence of DFO or CoCl(2). When MO3.13 cell were preconditioned with either DFO or CoCl(2), addition of TNFalpha further increased protein levels of HIF-1alpha. The mechanisms that underlie the increase in protein levels of HIF -1alpha seen, following addition of TNFalpha in preconditioned cells is due to an increase in transcription of the HIF-1alpha gene. Increased cellular levels of HIF-1alpha is associated with improved survival of MO3.13 cells, when cultured with TNFalpha after a period of preconditioning by DFO or CoCl(2). These studies suggest that compoundsthat increase HIF-1alpha can function as neuroprotective agents in inflammatory disorders of the CNS.

Differential expressions and roles of hypoxia-inducible factor-1alpha, -2alpha and -3alpha in the rat carotid body during chronic and intermittent hypoxia.

The HIF-1alpha expression in the carotid body (CB) is central to the transcriptional regulation of the CB structural and functional changes in chronic hypoxia (CH). The CB plays pathogenic roles in cardiovascular morbidity in patients with sleep-disordered breathing; yet, the expression and role of HIF-alpha subtypes in intermittent hypoxia (IH), resembling recurrent episodic apnea, are unclear. We hypothesized a divergent role of HIF-alpha subtypes, regulated by differential expression in the CB response to IH. A time-course analysis of the CB volume, and expression profiles of the HIF-1alpha, -2alpha, -3alpha and HIF-regulated gene products, including vascular endothelial growth factor (VEGF), endothelin-1 (ET-1), and tyrosine hydroxylase (TH), showed a significant difference in the lack of increase in the rat CB volume, HIF-1alpha and VEGF expression during IH, despite an increase in the mRNA level of HIF-1alpha and the prominent increase of volume and expression in the CH group. In contrast, there were increased CB expressions of HIF-2alpha and -3alpha, and also ET-1 and TH in both IH and CH groups. Results demonstrated a significant role played by HIF-2alpha and -3alpha in the CB response to IH, which could be complementary to the expression and role of HIF-1alpha under hypoxic conditions. This differential regulation of the HIF-alpha subtypes and pathways could account for the morphological and neurochemical discrepancy in the CB responses to IH and CH.

Activation of fractalkine/CX3CR1 by vascular endothelial cells induces angiogenesis through VEGF-A/KDR and reverses hindlimb ischaemia

The present study investigated the detailed mechanism by which fractalkine (Fkn), a CX3C chemokine, induces angiogenesis and its functional implication in alleviating ischaemia in vivo. Fkn induced new vessel formation on the excised rat aorta and chick chorioallantoic membrane (CAM) through CX3CR1 activation. Immunoblotting analysis, promoter assay and electrophoretic mobility shift assay showed that Fkn upregulated hypoxia-inducible factor-1 alpha (HIF-1alpha) by cultured human aortic endothelial cells (ECs), which in turn induced mRNA and protein levels of vascular endothelial growth factor (VEGF)-A through a p42/44 mitogen-activated protein kinase pathway. In vivo Fkn-induced angiogenesis on CAM was completely blocked by functional inhibition of VEGF receptor 2 kinase insert domain-containing receptor (KDR) and Rho GTPase. C57/BL6 mice with CX3CR1(-/-) bone marrow-derived cells developed angiogenesis in the implanted Fkn-mixed Matrigel plug, suggesting CX3CR1 activation in vascular ECs is sufficient for Fkn-induced angiogenesis in vivo. The condition of rat hindlimb ischaemia, which rapidly stimulated mRNA expression of both Fkn and VEGF-A, was significantly alleviated by the injection of whole-length Fkn protein. Fkn-induced activation of CX3CR1 by ECs leads to in vivo angiogenesis through two sequential steps: the induction of HIF-1alpha and VEGF-A gene expression by CX3CR1 activation and the subsequent VEGF-A/KDR-induced angiogenesis. The potent induction of angiogenesis by Fkn can be used as a therapeutic strategy for alleviating peripheral ischaemia.

Single Nucleotide Polymorphisms in the Hypoxia-inducible Factor-1α (HIF-1α) Gene in Human Sporadic Breast Cancer

DNA sequence variations in hypoxia-inducible factor-1alpha (HIF-1alpha) gene, which have been demonstrated to be correlated with tumor angiogenesis, may yield changes both in the production outcomes and in the activities of the gene. In this study, we investigated the relationship between three single nucleotide polymorphisms (SNPs) [C1772T and G1790A in exon 12 and C111A in exon 2 of the HIF-1alpha gene] in the HIF-1alpha gene coding regions and development of sporadic breast cancer in the Turkish population. These three polymorphisms result in an amino acid change from proline 582 to serine, from alanine 588 to threonine and from serine 28 to tyrosine, respectively. Genomic DNA was isolated from 102 sporadic breast cancer patients and 102 healthy female controls. All three HIF-1alpha gene regions were amplified by PCR, and genotypes were determined by RFLP and DNA sequencing. There were no significant differences between patients and controls in terms of the distribution of C1772T and G1790A polymorphisms of HIF-1 gene (p >0.05). As for HIF-1alpha C111A polymorphism, we did not find CA and AA variants of the gene in either controls or patients. Multivariable logistic regression analysis was performed between CC and CT + TT genotypes of C1772T polymorphism. No significant differences were found between these two genotypes in terms of clinicopathological characteristics of the patients including age at enrollment, age at menarche and first delivery, number of full-term pregnancies, body mass index, use of oral contraceptives and postmenopausal hormones, family history of breast and ovarian cancers, menopausal status, histopathological features, oophorectomy, smoking habits, and alcohol consumption (p >0.05). Our results suggest that none of the polymorphisms studied in the HIF-1alpha gene influence susceptibility to sporadic breast cancer. The present study is the first case-control study that investigates the association of HIF-1alpha polymorphisms with sporadic breast cancer in the Turkish population.

The Effect of HIF-1α siRNA on Growth and Chemosensitivity of Mia-paca Cell Line

PurposeHypoxia-inducible factor-1α (HIF-1α) primarily mediates the hypoxic response. HIF-1α induction by various stimuli contributes to cell proliferation and survival. To investigate the effect of HIF-1α, we used small interfering RNA (siRNA), and expected that cell apoptosis and sensitivity to chemotherapeutic drug increase, when we blocked the HIF-1α gene. Thus we performed in vitro and in vivo experiment to clarify the effect of hypoxia-inducible factor-1α on tumor growth.Materials and MethodsWe made control and HIF-1α siRNA using vector plasmid and then transfected Mia-paca cell lines with these RNAs. After selection with geneticin, two new cell lines were made, confirmed via immunoblotting. After treating with gemcitabine, each cell line was assayed to confirm the effect of HIF-1α siRNA using the cell proliferation assay and capase-3 assay. And then in vivo study was performed using female athymic nude mice. After subcutaneously injecting each new cell lines, intraperitoneal gemicitabine chemotherapy was performed for 3 weeks. During that period, we analyzed the difference of tumor growth rate.ResultsThe tumor growth of HIF-1α siRNA-transfected group was slower than that of the control group both in vitro and in vivo experiment.ConclusionThe suppression of HIF-1α results in decrease of cell proliferation and increase of chemosensitivity of pancreatic cancer cell line. Therefore, targeting the HIF-1α may be useful treatment modality for some pancreatic cancers.

Angiogenesis in Day-30 Bovine Pregnancies Derived from Nuclear Transfer

Impaired placental angiogenesis during early pregnancy may result in placental defects that adversely affect development of nuclear-transfer (NT) embryos later in pregnancy. These experiments were designed to quantify and compare development of placental microvasculature and expression of genes associated with angiogenesis, including members of the VEGF and angiopoietin (Ang) families, in maternal and embryonic placental tissues of day 30 bovine concepti derived from NT or in vitro fertilization (IVF) followed by in vivo development to the blastocyst stage in the sheep oviduct. Microvascular volume density (MVD) within the caruncular tissues, as determined using Periodic Acid-Schiff's staining as well as immunohistochemical staining for von Willebrand's factor, was not different between NT- and IVF- derived pregnancies. Expression of genes implicated in angiogenic mechanisms, including VEGF-A and -C, placental growth factor (PlGF), VEGF receptors (Flt-1, Flk-1, and Flt-4), angiopoietin-1 (Ang1), Ang2, Tie1, Tie2, and hypoxia-inducible factor (HIF), were determined. In chorio-allantoic membranes, levels of PlGF transcripts were significantly lower in NT- than IVF-derived tissues (p<0.05), whereas HIF-1alpha transcription in chorio-allantoic membranes of cloned concepti was higher at p<0.10. Caruncular expression of HIF-1alpha and Ang1 also was increased in NT-derived pregnancies at p <or= 0.10. Immunohistochemical staining of caruncular tissues for VEGF-A and the Flt-1 receptor revealed few differences in protein expression between NT- and IVF-derived pregnancies. These results indicate that expression of most angiogenic factors at day 30 of gestation is not altered as a result of the NT procedure; however, given reports of impaired placental vascular development in NT-derived bovine embryos, perturbations in angiogenesis may occur subsequently during early placental development and throughout gestation. Elevated expression of the HIF-1alpha gene in maternal and chorio-allantoic tissues of cloned concepti may suggest a generalized hypoxic condition in early placental tissues of NT-derived concepti, which could adversely affect subsequent development of the placenta.

Induction of renoprotective gene expression by hypoxia-inducible transcription factor-1α ameliorates renal damage

Hypoxia can cause not only acute kidney injury but also chronic kidney diseases. Hypoxia-inducible transcription factors activated by hypoxia play an important role in the cellular and tissular adaptation to low oxygen conditions. Hypoxia-inducible transcription factor-1 is composed of alpha and beta subunits. Hypoxia-inducible transcription factor-1alpha (HIF-1alpha) is known as a key regulator in protective role of renal damage by induction of downstream protective genes such as erythropoietin, heme oxygenase-1, vascular endothelial growth factor and glucose transporter-1. These proteins coded by protective genes are sufficient to induce renoprotection. So we hypothesize that recombinant adeno-associated viral HIF-1alpha gene will offer potential as a novel means to treat renal disease and can ameliorate renal damage efficiently by the injection of renal vein.