Unveiling the role of hypoxia-inducible factor 2alpha in osteoporosis:Implications for bone health

BACKGROUND Osteoporosis(OP)has become a major public health problem worldwide.Most OP treatments are based on the inhibition of bone resorption,and it is necessary to identify additional treatments aimed at enhancing osteogenesis.In the bone marrow(BM)niche,bone mesenchymal stem cells(BMSCs)are exposed to a hypoxic environment.Recently,a few studies have demonstrated that hypoxiainducible factor 2alpha(HIF-2α)is involved in BMSC osteogenic differentiation,but the molecular mechanism involved has not been determined.AIM To investigate the effect of HIF-2αon the osteogenic and adipogenic differentiation of BMSCs and the hematopoietic function of hematopoietic stem cells(HSCs)in the BM niche on the progression of OP.METHODS Mice with BMSC-specific HIF-2αknockout(Prx1-Cre;Hif-2αfl/fl mice)were used for in vivo experiments.Bone quantification was performed on mice of two genotypes with three interventions:Bilateral ovariectomy,semilethal irradiation,and dexamethasone treatment.Moreover,the hematopoietic function of HSCs in the BM niche was compared between the two mouse genotypes.In vitro,the HIF-2αagonist roxadustat and the HIF-2αinhibitor PT2399 were used to investigate the function of HIF-2αin BMSC osteogenic and adipogenic differentiation.Finally,we investigated the effect of HIF-2αon BMSCs via treatment with the mechanistic target of rapamycin(mTOR)agonist MHY1485 and the mTOR inhibitor rapamycin.RESULTS The quantitative index determined by microcomputed tomography indicated that the femoral bone density of Prx1-Cre;Hif-2αfl/fl mice was lower than that of Hif-2αfl/fl mice under the three intervention conditions.In vitro,Hif-2αfl/fl mouse BMSCs were cultured and treated with the HIF-2αagonist roxadustat,and after 7 d of BMSC adipogenic differentiation,the oil red O staining intensity and mRNA expression levels of adipogenesis-related genes in BMSCs treated with roxadustat were decreased;in addition,after 14 d of osteogenic differentiation,BMSCs treated with roxadustat exhibited increased expression of osteogenesis-related genes.The opposite effects were shown for mouse BMSCs treated with the HIF-2αinhibitor PT2399.The mTOR inhibitor rapamycin was used to confirm that HIF-2αregulated BMSC osteogenic and adipogenic differentiation by inhibiting the mTOR pathway.Consequently,there was no significant difference in the hematopoietic function of HSCs between Prx1-Cre;Hif-2αfl/fl and Hif-2αfl/fl mice.CONCLUSION Our study showed that inhibition of HIF-2αdecreases bone mass by inhibiting the osteogenic differentiation and increasing the adipogenic differentiation of BMSCs through inhibition of mTOR signaling in the BM niche.

Wortmannin influences hypoxia-inducible factor-1 alpha expression and glycolysis in esophageal carcinoma cells

AIM: To investigate the influence of phosphatidylinositol-3-kinase protein kinase B(PI3K/AKT)-HIF-1α signaling pathway on glycolysis in esophageal carcinoma cells under hypoxia. METHODS: Esophageal carcinoma cell lines Eca109 and TE13 were cultured under hypoxia environment, and the protein, m RNA and activity levels of hypoxia inducible factor-1 alpha(HIF-1α), glucose transporter 1, hexokinase-Ⅱ, phosphofructokinase 2 and lactate dehydrogenase-A were determined. Supernatant lactic acid concentrations were also detected. The PI3K/AKT signaling pathway was then inhibited with wortmannin, and the effects of hypoxia on the expression or activities of HIF-1α, associated glycolytic enzymes and lactic acid concentrations were observed. Esophageal carcinoma cells were then transfected with interference plasmid with HIF-1α-targeting si RNA to assess impact of the high expression of HIF-1α on glycolysis.RESULTS: HIF-1α is highly expressed in the esophageal carcinoma cell lines tested, and with decreasing levels of oxygen, the expression of HIF-1α and the associated glycolytic enzymes and the extracellular lactic acid concentration were enhanced in the esophageal carcinoma cell lines Eca109 and TE13. In both normoxia and hypoxic conditions, the level of glycolytic enzymesand the secretion of lactic acid were both reduced by wortmannin. The expression and activities of glycolytic enzymes and the lactic acid concentration in cells were reduced by inhibiting HIF-1α, especially the decreasing level of glycolysis was significant under hypoxic conditions.CONCLUSION: The PI3K/AKT pathway and HIF-1α are both involved in the process of glycolysis in esophageal cancer cells.

Is the hypoxia-inducible factor-1 alpha mRNA expression activated by ethanol-induced injury, the mechanism underlying alcoholic liver disease?

BACKGROUND: Excessive alcohol consumption can result in multiple organ injury, of which alcoholic liver disease (ALD) is the most common. With economic development and improvement of living standards, the incidence of diseases caused by alcohol abuse has been increasing in China, although its pathogenesis remains obscure. The aim of this study was to investigate the role of hypoxia in chronic ALD. METHODS: Twenty-eight male Sprague-Dawley rats were randomized into a control group (n=12) with a normal history and an experimental group (n=16) fed with 10 ml/ kg of 56% (vol/vol) ethanol once per day by gastric lavage for 24 weeks. At 24 weeks, blood samples were collected and then the rats were killed. Liver samples were frozen at -80 ℃ and used for RT-PCR; other liver samples were obtained for immunohistochemical staining. RESULTS: When the period of alcohol consumption increased, the positive rate of expression of hypoxia- inducible factor-1 alpha (HIF-1α) mRNA was more significantly elevated in the liver of the alcohol group than in the control group (P≤0.05). The HIF-1α protein located in the cytoplasm was seldom expressed in the control group, but significantly in the alcohol group (P≤0.01). CONCLUSION: HIF-1α mRNA expression was activated by ethanol-induced injury in this study, suggesting that hypoxia is involved in the underlying mechanism of ALD.

Clinicopathological and Prognostic Significance of Hypoxia-inducible Factor-1 alpha in Lung Cancer: a Systematic Review with Meta-analysis

Hypoxia-inducible factor-1 alpha（HIF-1α） plays a vital role in the initiation, evaluation and prognosis in lung cancer. The prognostic value of HIF-1α reported in diverse study remains disputable. Accordingly, a meta-analysis was implemented to further understand the prognostic role of HIF-1α in lung cancer. The relationship between HIF-1α and the clinicopathological characteristics and prognosis of lung cancer were investigated by a meta-analysis. Pub Med and Embase were searched from their inception to January 2015 for observational studies. Fixed-effects or random-effects meta-analyses were used to calculate odds ratios and 95% confidence intervals of different comparisons. A total of 20 studies met the criteria. The results showed that HIF-1α expression in lung cancer tissues was significantly higher than that in normal lung tissues. Expression of HIF-1α in patients with squamous cell carcinoma was significantly higher than that of patients with adenocarcinomas. Similarly, non-small cell lung cancer（NSCLC） patients had higher HIF-1α expression than small cell lung cancer（SCLC） patients. Moreover, lymph node metastasized tissues had higher HIF-1α expression than non-lymph node metastasized tissues. A high level HIF-1α expression was well correlated with the expression of vascular endothelial growth factor and epidermal growth factor receptor in the NSCLC. Notably, NSCLC or SCLC patients with positive HIF-1α expression in tumor tissues had lower overall survival rate than patients with negative HIF-1α expression. It was suggested that HIF-1α expression may be a prognostic biomarker and a potential therapeutic target for lung cancer.

Phosphatase and tensin homology deleted in chromosome 10,hypoxia-inducible factor-1 alpha gene expression in colorectal adenoma and adenocarcinoma and their relation to vascular endothelial growth factor protein expression

To examine phosphatase and tensin homology deleted in chromosome 10 (PTEN),hypoxia-inducible factor-1 alpha (HIF-1 alpha) gene expressions and their relation to vascular endothelial growth factor(VEGF) protein expression in the patients with human colorectal adenomas and adenocarcinomas.Methods The expression of PTEN,HIF-1 alpha gene was detected by using in situ hybridization,and the VEGF expression levels by immunohistochemistry in colorectal adenomas and primary colorectal adenocarcinoma.Results Strong expression of HIF-1 alpha was detectable in the majority of colorectal dadenocarcinoma,particularly surrounding areas of necrosis in adenocarcinoma.PTEN,HIF-1 alpha mRNA and VEGF protein were positive in 51.6%,67.7% and 59.7% respectively in 62 cases of adenocarcinomas,and 77.8%,44.4% and 33.3% respectively in 18 cases of adenomas.The positive rate of VEGF was higher in the patients with colorectal adenocarcinomas than that in those with adenomas,whereas that of PTEN mRNA was contrary.HIF-1 mRNA expression was correlated significantly with lymph node metastasis,liver metastasis,Duke’s stage and recurrence.During colorectal tumor progression,the expression of HIF-1 alpha mRNA was positively correlated with the VEGF protein expression (χ2= 4.751 ,P<0.05),but negatively with the PTEN mRNA expression(χ2=21.84,P<0.01).Conclusion The absence or low expression of PTEN and the increased levels of HIF-1α and VEGF may paly an important role in carcinogenesis and progression of colorectal carcinoma.These results suggest that VEGF upregulated by HIF-1 alpha gene may be involved in angiogenesis of colorectal adenocarcinoma.4 refs,1 tab.

Inhibition of Expression of Hypoxia-inducible Factor-1α mRNA by Nitric Oxide in Hypoxic Pulmonary Hypertension Rats

In order to study the effect of nitric oxide (NO) on the expression of hypoxia inducible factor 1 alpha (HIF 1α) mRNA in hypoxic pulmonary hypertension (HPH) rats, 30 healthy male Wistar rats were randomly divided into normoxic control group, chronic hypoxic group and hypoxia plus L argine (L Arg) group. The animal model of HPH was developed. The mean pulmonary arterial pressure (mPAP) was measured by inserting a microcatheter into the pulmonary artery. The HIF 1α mRNA expression levels were detected by in situ hybridization (ISH) and semiquantitative RT PCR. It was found that after 14 days hypoxia, the mPAP in normoxic control group (17.6±2 7 mmHg,1 mmHg=0 133 kPa) was significantly lower than that in chronic hypoxic group(35.8±6.1 mmHg, t =0.2918, P <0.05) and mPAP in chronic hypoxic group was higher than that in hypoxia plus L argine group(24.4±3.8 mmHg, t =0.2563, P <0.05). ISH showed that the expression of HIF 1α mRNA in the intraacinar pulmonary arteriolae (IAPA) in normoxic control group (0.1076±0.0205) was markedly weaker than that in chronic hypoxic group (0.3317±0.0683, t =3.125, P <0.05) and that in chronic hypoxic group was stronger than that in hypoxia plus L argine group (0.1928±0.0381, t =2.844, P <0.05). RT PCR showed that the content of HIF 1α mRNA in chronic hypoxic group (2.5395±0.6449) was 2.16 times and 1.75 times higher than that in normoxic control group (1.1781±0.3628) and hypoxia plus L argine group (1.4511±0.3981), respectively. It is concluded that NO can reduce the mPAP by the inhibition of the expression of HIF 1α mRNA, which may be one of the mechanisms through which NO affects the pathogenesis of HPH.

Puerarin decreases hypoxia inducible factor-1 alpha in the hippocampus of vascular dementia rats

In this study, a rat vascular dementia model was established by permanent bilateral common carotid arterial occlusion. Rats were intraperitoneally injected with puerarin 3 days before modeling, for 45 successive days. Results demonstrated that in treated animals hippocampal structures were clear, nerve cells arranged neatly, and cytoplasm was rich in Nissl bodies. The number of cells positive for hypoxia inducible factor-1 alpha, erythropoietin and endothelial nitric oxide synthase was reduced; and the learning and memory abilities of rats were significantly improved. Our experimental findings indicate that puerarin can significantly improve learning and memory in a vascular dementia model, and that the underlying mechanism may be associated with the regulation of the expression of hypoxia inducible factor-1 alpha.

缺氧诱导因子-1α和BCL-2/腺病毒E1B19 kDa相关蛋白3在中耳胆脂瘤表达及意义

目的探讨缺氧诱导因子-1α(hypoxia inducible factor-1,HIF-1α)和BCL-2/腺病毒E1B19KDa相关蛋白3(Bcl2/adenovirus E1B 19 kD interacting protein 3,BNIP3)在中耳胆脂瘤中的表达及胆脂瘤上皮的凋亡情况。方法采用免疫组织化学方法检测30例中耳胆脂瘤标本与18例外耳道皮肤标本中HIF-1α和BNIP3蛋白的表达情况,使用末端脱氧核苷酸转移酶介导的dUTP缺口末端标记(terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling,Tunel)检测20例中耳胆脂瘤标本和18例外耳道皮肤标本的凋亡情况。使用Pearson相关分析检验HIF-1α和BNIP3蛋白之间的相关性。结果 HIF-1α在胆脂瘤组和对照组的平均光密度分别为0.16±0.07和0.08±0.03,两组比较差异有统计学意义(t=4.279,P<0.01);BNIP3在胆脂瘤组和对照组的平均光密度分别为0.16±0.08和0.11±0.06,两组比较差异有统计学意义(t=2.463,P=0.0185);经pearson相关分析,在胆脂瘤上皮中,HIF-1α和BNIP3之间呈正相关(r=0.418,P=0.003);Tunel染色中,凋亡指数在胆脂瘤组和对照组分别为(52.8±12.5)%和(9.99±2.97)%,两组比较差异有统计学意义(t=14.166,P<0.01)。结论 HIF-1α和BNIP3在中耳胆脂瘤中的异常表达可能与胆脂瘤的高凋亡特性有关。

Correlations of hypoxia-inducible factor-1α/hypoxia-inducible factor -2α expression with angiogenesis factors expression and prognosis in non-small cell lung cancer

Background Hypoxia-inducible factor （HIF） may play an important role in the process of tumorigenesis as well as tumor progression. The aim of this study was to compare the expression between HIF-1α and HIF-2α in tumor angiogenesis and the overall impact on patient prognosis in human non-small cell lung cancer （NSCLC）. Methods In the current work we compared the immunohistochemical expression of HIF-1α and HIF-21 in surgical specimens of 140 patients with NSCLC in a tissue microarray study. Relationships between HIF-α expression and clinicopathological or angiogenic factors, including prognosis, were analyzed. Results High HIF-1α and HIF-2α expression was noted in 49/140 （35.0%） and in 64/140 （45.7%） of the cases, respectively. There was no direct correlation between HIF-la and HIF-2α expression. Patients with advanced stage tumors had frequent high expression of HIF-2a （P=0.007）, and we also found a significant correlation between HIF-2α and T or N stage （P=0.030 and 0.043, respectively）. HIF-1α showed a marginal association with T stage （P=0.084）, which showed a higher expression in early stage tumors. A significant correlation （p=0.045） was noticed between HIF-1α and vascular endothelial growth factor （VEGF） expression while the expression levels of thymidine phosphorylase （TP）, cyclooxygenase （COX）-2 and microvessel density （MVD） were significantly higher in high HIF-2a tumors （P=0.020, 0.004 and 0.046, respectively）. In addition, univariate analysis of overall survival demonstrated that HIF-2a expression, but not HIF-la, was related to poor outcome （P=-0.001） and it retained significant in multivariate analysis （P=0.036）. Conclusions Taken together, we conclude that HIF-1α and HIF-2α may differentially regulate the major angiogenic factors in different stages of the tumor process in NSCLC. HIF-2α may play a dominant role in tumor angiogenesis and appears to be of obvious value as a significant prognostic factor in NSCLC.

Tongxinluo Inhibits Cyclooxygenase-2, Inducible Nitric Oxide Synthase, Hypoxia-inducible Factor-2α/Vascular Endothelial Growth Factor to Antagonize Injury in Hypoxia-stimulated Cardiac Microvascular Endothelial Cells

Background： Endothelial dysflinction is considered as the initiating process and pathological basis of cardiovascnlar disease. Cyclooxygenase-2 （COX-2） and prostacyclin synthase （PGIS）, inducible nitric oxide synthase （iNOS） and endothelial NOS （eNOS） are key enzymes with opposing actions in inflammation and oxidative stress, which are believed to be the major driver of endothelial dysfunction. And in hypoxia （Hx）, Hx-inducible factor （HIF）-1α and HIF-2α are predominantly induced to activate vascular endothelial growth factor （VEGF）, restllting in abnormal proliferation. Whether and how Tongxinluo （TXL） modulates COX-2, PGIS, iNOS, eNOS, HIF-1α, HIF-2α, and VEGF in Hx-stimulated human cardiac microvascular endothelial cells （HCM ECs） have not been clarified. Methods： HCMEC were treated with CoCl2 to mimic Hx and the mRNA expressions of COX-2, PGIS, iNOS, eNOS, HIF-1α, HIF-2α. and VEGF were first confirmed, and then their mRNA expression and protein content as well as the cell pathological alterations were evaluated for TXL treatment with different concentrations, In addition, the effector molecular of inflammation prostaglandin E2 （PGE2） and the oxidative marker nitrotyrosine （NT） was adopted to reflect HCMEC in.jury. Results： Hx could induce time-dependent increase of COX-2, iNOS, HIF-2α, and VEGF in HCMEC. Based on the Hx-induced increase, TXL could mainly decrease COX-2, iNOS, HIF-2α, and VEGF in a concentration-dependent manner, with limited effect on the increase of PGIS and eNOS. Their protein contents verified the mRNA expression changes, which was consistent with the cell morphological alterations. Furthermore, high dose TXL could inhibit the Hx-induced increase of PG E, and NT contents, attenuating the inflammatory and oxidative injury. Conclusions： TXL could inhibit inflammation-related COX-2, oxidative stress-related iNOS, and H IF-2α/VEGF to antagonize Hx-induced HCMEC injury.

Effect of environmental factors on chemoresistance of HepG2 cells by regulating hypoxia-inducible factor-1α

Background Accumu1αting evidence demonstrates that the microenvironment of the host has an important effect on the chemoresistance of tumors. We also found that the formation of intrinsic multidrug resistance is re1αted to environmental factors that are common with tumor growth of hepatocellu1αr carcinoma. The aim of this study was to explore the molecu1αr mechanisms by which multidrug resistance of hepatocellu1αr carcinoma is induced by the microenvironment. In particu1αr, the regu1αtion of nuclear transcription factor （hypoxia-inducible factor-1α, HIF-1α） activation in the process of multidrug resistance formation was investigated. Methods HepG2 cells were exposed to different microenvironmental conditions respectively, such as hypoxia, stimu1αtion of glucose deprivation and transfection of p1αsmid PcDNA3/HBx. In the HepG2 cells, the expression of the re1αted MDR proteins, HIF-1α protein expression and localization, activity of extracellu1αr signal-regu1αted kinase /mitogen-activated protein kinase （ERK/MAPK） were detected. Specific inhibitor U0126 was used to block ERK/MAPK signal pathway, the alteration of HIF-1α and the re1αted MDR proteins were investigated. Multivariate analysis of variance （MANOVA） repeated measures and one-way analysis of variance （ANOVA） followed by Tukey test or t-test were used to determine differences over time and effects of the treatments. Results The above three microenvironment factors increase the expression of the re1αted MDR proteins （including P-gp, LRP, and MRP1） and induce MDR of HepG2 cells. HIF-1α was induced at the protein and mRNA levels and the nuclear translocation was also increased. The activity of ERK/MAPK was also increased in HepG2 cells. But when ERK/MAPK pathway was inhibited, the mRNA and protein decreased. Inhibition of ERK/MAPK significantly reduced HIF-1α, whereas HIF-1α mRNA levels were not affected. expression of MDR1, MRP1, and LRP was to some extent activated HIF-1α protein and the nuclear translocation of Conclusions The microenvironmental factors could induce MDR of HepG2 cells by the activity of HIF-1α. The activity of HIF-1α is regu1αted by the ERK/MAPK pathway at the phosphory1αtion level. As an important nuclear transcription factor, HIF-1α controls the transcription of MDR-re1αted genes and the synthesis of their corresponding proteins by ERK/MAPK signal pathway in HepG2 cells.

HIF-2α regulates CD44 to promote cancer stem cell activation in triple-negative breast cancer via PI3K/AKT/mTOR signaling

BACKGROUND Breast cancer is a common malignant tumor that seriously threatens women’s health.Breast cancer stem cell(CSC)-like cell population may be the main factor for breast cancer metastasis.Therefore,targeted therapy for CSCs has great potential significance.Hypoxia-inducible factor is a transcription factor widely expressed in tumors.Studies have shown that down-regulation of the hypoxia signaling pathway inhibits tumor stem cell self-renewal and increases the sensitivity of stem cells to radiotherapy and chemotherapy mediated by hypoxiainducible factor-2α(HIF-2α).However,the specific mechanism remains unclear and further research is necessary.AIM To investigate the effect of HIF-2αdown-regulation on stem cell markers,microsphere formation,and apoptosis in breast cancer cell line MDA-MB-231 under hypoxia and its possible mechanism.METHODS Immunohistochemistry was used to detect the expression of HIF-2αand CD44 in triple-negative breast cancer(TNBC)and non-TNBC tissues.Double-labeling immunofluorescence was applied to detect the co-expression of HIF-2αand CD44 in MDA-MB-231 cells and MCF-7 cells.HIF-2αwas silenced by RNA interference,and the expression of CD44 and transfection efficiency were detected by real-time fluorescent quantitative PCR.Further,flow cytometry,TdT-mediated X-dUTP nick end labeling,and mammosphere formation assays were used to evaluate the effect of HIF-2αon CSCs and apoptosis.The possible mechanisms were analyzed by Western blot.RESULTS The results of immunohistochemistry showed that HIF-2αwas highly expressed in both TNBC and non-TNBC,while the expression of CD44 in different molecular types of breast cancer cells was different.In in vitro experiments,it was found that HIF-2αand CD44 were expressed almost in the same cell.Compared with hypoxia+negative-sequence control,HIF-2αsmall interfering ribonucleic acid transfection can lower the expression of HIF-2αand CD44 mRNA(P<0.05),increase the percentage of apoptotic cells(P<0.05),and resulted in a reduction of CD44+/CD24−population(P<0.05)and mammosphere formation(P<0.05)in hypoxic MDA-MB-231 cells.Western blot analysis revealed that phosphorylated protein-serine-threonine kinase(p-AKT)and phosphorylated mammalian target of rapamycin(p-mTOR)levels in MDA-MB-231 decreased significantly after HIF-2αsilencing(P<0.05).CONCLUSION Down-regulation of HIF-2αexpression can inhibit the stemness of human breast cancer MDA-MB-231 cells and promote apoptosis,and its mechanism may be related to the CD44/phosphoinosmde-3-kinase/AKT/mTOR signaling pathway.

Mitochondrial malic enzyme 2 promotes breast cancer metastasis via stabilizing HIF-1α under hypoxia

Objective: α-ketoglutarate(α-KG) is the substrate to hydroxylate collagen and hypoxia-inducible factor-1α(HIF-1α), which are important for cancer metastasis. Previous studies have shown that the upregulation of collagen prolyl 4-hydroxylase in breast cancer cells stabilizes the expression of HIF-1α by depleting α-KG levels. We hypothesized that mitochondrial malic enzyme 2(ME2) might also affect HIF-1α expression via modulating α-KG levels in breast cancer cells.Methods: We evaluated ME2 protein expression in 100 breast cancer patients using immunohistochemistry and correlated with clinicopathological indicators. The effect of ME2 knockout on cancer metastasis was evaluated using an orthotopic breast cancer model. The effect of ME2 knockout or knockdown on the levels of α-KG and HIF-1α proteins in breast cancer cell lines was determined both in vitro and in vivo.Results: ME2 was found to be upregulated in the human breast cancerous tissues compared with the matched precancerous tissues(P<0.001). The elevated expression of ME2 was associated with a poor prognosis(P=0.019).ME2 upregulation was also related to lymph node metastasis(P=0.016), pathological staging(P=0.033), and vascular cancer embolus(P=0.014). Also, ME2 knockout significantly inhibited lung metastasis in vivo. In the tumors formed by ME2 knockout cells, the levels of α-KG were significantly increased and collagen hydroxylation level did not change significantly but HIF-1α protein expression was significantly decreased, compared to the control samples. In cell culture, cells with ME2 knockout or knockdown demonstrated significantly higher α-KG levels but significantly lower HIF-1α protein expression than control cells under hypoxia. Exogenous malate and α-KG exerted similar effect on HIF-1α in breast cancer cells to ME2 knockout or knockdown. Additionally,treatment with malate significantly decreased 4 T1 breast cancer lung metastasis. ME2 expression was associated with HIF-1α levels in human breast cancer samples(P=0.008).Conclusions: Our results provide evidence that upregulation of ME2 is associated with a poor prognosis of breast cancer patients and propose a mechanistic understanding of a link between ME2 and breast cancer metastasis.

Constitutive renal Rel/nuclear factor-κB expression in Lewis polycystic kidney disease rats

AIM： To determine the temporal expression and pattern of Rel/nuclear factor （NF）-κB proteins in renal tissue in polycystic kidney disease （PKD）. METHODS： The renal expression of Rel/NF-κB proteins was determined by immunohistochemistry, immunofuorescence and immunoblot analysis in Lewis polycystic kidney rats （LPK, a genetic ortholog of human nephronopthsis-9） from postnatal weeks 3 to 20. At each timepoint, renal disease progression and the mRNA expression of NF-κB-dependent genes （TNFa and CCL2） were determined. NF-κB was also histologically assessed in human PKD tissue.RESULTS： Progressive kidney enlargement in LPK rats was accompanied by increased renal cell proliferation and interstitial monocyte accumulation （peaking at weeks 3 and 10 respectively）, and progressive interstitial fibrosis （with a smooth muscle actin and Sirius Red deposition significantly increased compared to Lewis kidneys from weeks 3 to 6 onwards）. Rel/NF-κB proteins （phosphorylated-p105, p65, p50, c-Rel and RelB） were expressed in cystic epithelial cells （CECs） of LPK kidneys as early as postnatal week 3 and sustained until late-stage disease at week 20. From weeks 10 to 20, nuclear p65, p50, RelB and cytoplasmic IκBa protein levels, and TNFa and CCL2 expression, were upregulated in LPK compared to Lewis kidneys. NF-κB proteins were consistently expressed in CECs of human PKD. The DNA damage marker γ-H2AX was also identifed in the CECs of LPK and human polycystic kidneys. CONCLUSION： Several NF-κB proteins are consistently expressed in CECs in human and experimental PKD. These data suggest that the upregulation of both the canonical and non-canonical pathways of NF-κB signaling may be a constitutive and early pathological feature of cystic renal diseases.

The impact of ^(60)Co γ-ray on cycles to Hep-2 human laryngeal cancer cell in the condition of hypoxia

Objective： The aim of the study was to investigate the impact of 60Co y-ray on apoptosis, cell cycles and the expression of protein hypoxia-inducible factor-1α （HIF-1α） to Hep-2 cell line in the conditions of normoxia and hypoxia. Methods： Hep-2 cell were divided into 2 groups： group A （normoxia） and group B （hypoxia）. All of the ceils were exposed to y-ray with dosage being 0, 1, 3, 5, 10, 20, and 40 Gy. Flow cytometry was used to measure the protein level of HIF-1α and to detect apoptosis and cell cycles. The protein level of HIF-1α was also determined by immunohistochemistry and Western blotting. Results： The protein level of HIF-1α in group B was significantly higher than that in group A. In group A, low doses （1-5 Gy） of y-ray had caused G0/G1 cell cycle arrest and high doses （10-40 Gy） had caused G2/M cell cycle arrest. In group B, without exposure of y-ray （0 Gy） had caused G0/G1 cell cycle arrest, all of the different dosage of y-ray could cause G2/M cell cycle arrest. The curve of apoptosis rate in group A was a parabola, the apoptotic rate was related to the dosage of y-ray in a dosage dependent manner. The peak was at the point of 5 Gy. The apoptosis rate in group A was significantly higher than that in group B. Conclusion： Different doses of y-ray could cause different cell cycles arrest then make different impact on apoptosis to Hep-2 ceil. The lower apoptosis rate in condition of hypoxia maybe has a relationship with G2/M cell cycle arrest. Up-regulated HIF-1α protein may be one of the reasons for G2/M cell cycle arrest.

Expression of cyclooxyenase-2 protain and its relationship with HIF-1α in HCC

Objective： To investigate the clinical significance of COX-2 （Cyclooxygenase-2） expression in HCC （Primary hepatocellular carcinoma） and clarify whether COX-2 is correlated with hypoxia-inducible factor-1α （HIF-1α） in the development of HCC. Methods： Tumor tissues were obtained from 53 patients with HCC. COX-2 and HIF-1α were determined by immunohistochemistry. All 53 patients were regularly followed up and the data were collected prospectively. Results： Immunostaining showed the expression of COX-2 （ n = 33, 62.3 % ） and HIF-1α （ n = 36, 67.9% ） in most tumor cells. The level of COX-2 was correlated with HIF-1α levels（ r = 0.4413, P 〈0.01 ）. There were significant correlation between clinicopathological features and higher tumor cytosolic COX-2 level was in the presence of multiple tumors （ P = 0.01）, venous invasion （ P = 0.03）, advanced tumor stage （ P = 0.01）, and well-different tumor grade （P =0.03）. High-tumor cytosolic COX-2 level was correlated with patient＇s worse prognosis （P = 0.0085）. Conclusion： Elevated tumor COX-2 level is correlated with elevated HIF-1α levels and invasiveness in HCC, suggesting COX-2 plays an important role in the progression of HCC, and may be an important therapeutic target in HCC.

A Correlative Study between CT Perfusion Parameters and Angiogenesis in Rabbit VX2 Liver Tumors

Objective: The purpose of this study was to evaluate the correlation between CT perfusion parameters and the hypoxia-inducible factor-1 alpha (HIF-1α), vascular en-dothelial growth factor (VEGF), matrix metalloproteinase-2 (MMP-2) and microvessel density (MVD) marked by CD34 molecular of rabbit VX2 liver tumors and to investigate the value of CT perfusion imaging in evaluating tumor angiogenesis. Material and methods: Twenty-four cases of rabbit VX2 liver tumor were performed by CT perfusion scanning. Hepatic artery perfusion (HAP), portal vein perfusion (PVP), total hepatic blood flow (THBF) and hepatic perfusion index (HPI) were measured by perfusion software. HIF-1α, VEGF and MMP-2 expression and MVD were detected in the 24 rabbit VX2 liver tumor tissue samples using immunohistochemical method. The correlation between the HIF-1α, VEGF, MMP-2 expression and MVD and CT perfusion parameters were analyzed. Results: Correlation analysis revealed that the expression of HIF-1α, MMP-2, MVD were positively related to the HAP, THBF, HPI (p < 0.01), but no relations with PVP (p > 0.05);and correlation analysis revealed that the expression of VEGF was positively related to the HAP, HPI (p 0.05). There was a positive relationship between the expression of HIF-1α, VEGF, MMP-2 and MVD (p < 0.01). Conclusions: CT perfusion imaging can reflect the blood perfusion of the rabbit VX2 liver tumors and evaluate the information of angiogenesis about tumors.

Hippocampus hypoxia-inducible factor-1 alpha and heme oxygenase-1 expression in the delayed encephalopathy after acute carbon monoxide poisoning rat model

Objective To research the expression of hypoxia-inducible factor-1 alpha(HIF-1α)and heme oxygenase-1(HO-1)in hippocampus of rats with delayed encephalopathy after acute carbon monoxide poisoning(DEACMP)and its functions.Methods One hundred and fiftysix rats were selected and randomly divided into

Hypoxia-inducible factor-lαand semaphorin4D genes involved with tumor-associated macrophage-induced metastatic behavior and clinical significance in colon cancer

Background Hypoxia promotes tumor angiogenesis and hypoxia-inducible factor-1 alpha （HIF-lg） plays a pivotal role in this process. Recently identified pro-angiogenic factor, semaphorin4D （Sema4D） also promotes angiogenesis and enhances invasive proliferation in some tumors. Furthermore, tumor-associated macrophages （TAMs） can increase the expression of HIF-la and Sema4D in cancer cells and thus influence tumor growth and progression. The purpose of this study was to evaluate the effect of TAMs on the expression of Sema4D and HIF-la and the impact of biologic behavior in colon cancer cells. Methods Immunohistochemistry was used to analyze HIF-la and Sema4D expression in 86 curatively resected colon cancer samples and 52 normal colon tissues samples. The relationship between their expression and clinicopathological factors was analyzed. Furthermore, macrophage-tumor cell interactions, such as metastasis, angiogenesis, were also studied using in vitro co-culture systems. Statistical analysis was performed using SPSS 17.0 software （SPSS Inc., USA）. Differences between two groups were analyzed with Student＇s t test. Results HIF-la （58%） and Sema4D （60%） were expressed at a significantly higher level in tumors than in normal tissues （P 〈0.01, for both）. Furthermore, HIF-la and Sema4D expression was significantly correlated with lymphatic metastasis, specific histological types and TNM stages （P 〈0.05）, but not with age and tumor size （P 〉0.05）. Sema4D expression was correlated with that of HIF-la （r=0.567, P 〈0.01）. TAMs markedly induced HIF-la and Sema4D expression in colon cancer calls and subsequently increased their migration and invasion. Conclusions HIF-la and Sema4D expression are closely related to lymphatic metastasis, specific histological types and TNM stages in colon cancer. Furthermore, TAMs promote migration and invasion of colon cancer cells and endothelial tube formation, possibly through up-regulation of HIF-la and Sema4D.

Forensic Significance of Messenger RNA and Protein Expression of Genes Downstream of Hypoxia Inducible Factor 2 in Myocardial Tissue for Death Discrimination

Background: As a heterodimeric transcription factor, hypoxia-inducible factor 2 alpha subunit (HIF2A), is an important member of the HIF family. It plays a significant role in the hypoxia adaptation process by regulating the different types of downstream transcription factors and auxiliary regulatory factors. HIF2A-related factors are believed to participate in the progression of myocardial injury or myocardial ischemia, support the protection of ischemic myocardium, and provide guiding significance for the diagnosis and discrimination of sudden cardiac death in forensic pathology. Aim and Objectives: This study aimed to explore the discriminability and applicability of HIF2A-related factors in myocardial infarction cases compared with other causes of death, provide further insights for the forensic diagnosis of heart failure (HF) cases with myocardial infarction, and support the clinical treatment of patients with HF after myocardial infarction. Materials and Methods: The relative expression levels of HIF2A, amphiregulin (AREG), potassium large conductance calcium-activated channel subfamily M β1 (KCNMB1), peroxisome proliferator-activated receptor α (PPARA), vascular endothelial growth factor (VEGF), and VEGFR2 messenger RNAs (mRNAs) in myocardial tissue samples were performed using quantitative reverse transcriptase-polymerase chain reaction. A partial least squares-discriminant analysis model was constructed to select the indicators with better identification effects for myocardial infarction cases. The protein levels of HIF2A, AREG, KCNMB1, and PPARA were further detected by immunohistochemistry. The forensic autopsy cases (27 cases in total, postmortem interval <72 h) included seven cases of acute myocardial infarction and ten cases of myocardial ischemia. There were ten cases in the control group, including four cases of traffic injury, one case of injury by fall from height, and five cases of blunt force injury. Results: Characteristic results were observed in the myocardial ischemia/infarction samples. Compared with the control group, the relative mRNA expression levels of AREG, KCNMB1, and PPARA were significantly increased during the progression of myocardial ischemia, but this was not observed for HIF2A, VEGF, or VEGFR2 mRNA. Immunohistochemistry assays further verified the expression levels of the related factors at the protein level, and H and E staining showed signs of angiogenesis and inflammation in the ischemia/infarction group. Conclusions: By controlling the expression of downstream target genes (AREG, KCNMB1, and PPARA) during myocardial cell hypoxia adaptation, HIF2A has a potential significance in the diagnosis of myocardial infarction in forensic medicine. We believe that HIF2A, AREG, KCNMB1, and PPARA can be used as molecular pathological biomarkers for the discrimination of causes of death in myocardial infarction cases.