Levels Of Hypoxia-inducible Factor-1alpha Research Articles

NIMG-36. VISUALIZATION OF TUMOR HETEROGENEITY AND PREDICTION OF ISOCITRATE DEHYDROGENASE MUTATION STATUS FOR HUMAN GLIOMAS BY USING MULTIPARAMETRIC PHYSIOLOGIC AND METABOLIC MRI

Abstract Preoperative prediction of isocitrate dehydrogenase mutation status is clinically meaningful, but remains challenging. This study aimed to predict the isocitrate dehydrogenase (IDH) status of gliomas by using the machine learning voxel-wise clustering method of multiparametric physiologic and metabolic magnetic resonance imaging (MRI) and to show the association of the created cluster labels with the glucose metabolism status of the tumors. Sixty-nine patients with diffuse glioma were scanned by pH-sensitive MRI, diffusion-weighted imaging, fluid-attenuated inversion recovery, and contrast-enhanced T1-weighted imaging at 3 T. An unsupervised two-level clustering approach, including the generation of a self-organizing map followed by the K-means clustering, was used for voxel-wise feature extraction from the acquired images. The logarithmic ratio of the labels in each class within tumor regions was applied to a support vector machine to differentiate IDH mutation status. Bootstrapping and leave-one-out cross-validation were used to calculate the area under the curve (AUC) of receiver operating characteristic curves, accuracy, sensitivity, and specificity for evaluating performance. Targeted biopsies were performed for 14 patients to explore the relationship between clustered labels and the expression of key glycolytic proteins determined using immunohistochemistry. The highest prediction performance to differentiate IDH status was found for 10-class clustering, with a mean AUC, accuracy, sensitivity, and specificity of 0.94, 0.91, 0.90, and 0.91, respectively. The tissues with labels 7 + 8 + 9 + 10 showed high expression levels of hypoxia-inducible factor 1-alpha, glucose transporter 3, and hexokinase 2, which are typical of IDH wild-type glioma, whereas those with labels 1 showed low expression of these proteins. Our machine learning model successfully predicted the IDH mutation status of gliomas, and the resulting clusters properly reflected the metabolic status of the tumors.

The prognostic value of hypoxia biomarkers in older patients with chronic heart failure

Abstract Chronic kidney disease (CKD) worsens the prognosis of chronic heart failure (CHF). Hypoxia is the leading link in pathogenesis, especially in older patients with comorbidity. The aim of this study was to investigate the prognostic value of biomarkers of myocardial, renal dysfunction and hypoxia in older patients with CHF. Materials and methods 80 older hypertensive patients with CHF (48 females, mean age 70.7±8.7 years) were examined. CHF was defined according to ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure, 2016. CKD was diagnosed and classified according to the KDIGO guidelines (2012). Serum levels of hypoxia-inducible factor 1-alpha (HIF-1α), endogenous erythropoietin (eEPO), N-terminal propeptide of type B natriuretic hormone (NT-proBNP), cystatin C were assessed. The follow-up period was 12 months; the primary endpoint was total mortality. Results CKD was diagnosed in 49 (61.3%) older patients with CHF. The HIF-1α level was significantly higher in the group of deceased patients than in the survivors (0.08 (IQR 0.06; 0.11) and 0.05 (IQR 0.04; 0.07) ng/ml, p=0.02), as well as the level of NT-proBNP (1126.3 (IQR 551.8; 2750.0) and 164.4 (IQR 135.5; 1100.9) pg/ml, p<0.0001), eEPO (16.92 (ICR 5.43; 64.57) and 5.36 (IQR 1.65; 8.85) mIU/ml, p<0.0001), cystatin C (1.49 (ICR 0.86; 2.13) and 0.99 (IQR 0.82; 1.32) Mg/l, p=0.0005). Cox regression analysis adjusted for sex, age and comorbidity (χ2=36.8, p<0.0001) showed that endogenous erythropoietin, independently of other factors and biomarkers, determined the prognosis of annual mortality in patients with chronic heart failure (HR 3.27 (95% CI 1.08–9.91, p=0.03); χ2=30.7, p=0.0002). When constructing classification trees, in older patients with CHF in the presence of eEPO less than 16.19 mIU/ml NT-proBNP more than 232.5 pg/ml is an unfavorable factor (in patients with NT-proBNP <232.5 pg/ml the risk decreased to 0) (for model: sensitivity – 57.1%; specificity – 92.3% (AUC=0.87); p=0.0015). Conclusions The level of eEPO in older patients with CHF has an independent and closer relationship with the annual mortality of patients than the NT-proBNP – currently accepted biomarker of the severity and prognosis of CHF. Funding Acknowledgement Type of funding sources: None.

COX2 Enhances Neovascularization of Inflammatory Tenocytes Through the HIF-1α/VEGFA/PDGFB Pathway.

Tendon injuries are among the most challenging in orthopedics. During the early tendon repair, new blood vessel formation is necessary. However, excessive angiogenesis also exacerbates scar formation, leading to pain and dysfunction. A significantly worse outcome was associated with higher expression levels of hypoxia-inducible factor-1 alpha (HIF-1α), and its transcriptional targets vascular endothelial growth factor A (VEGFA) and platelet-derived growth factor B (PDGFB), but the underlying molecular mechanisms remain unclear. In this study, lipopolysaccharide (LPS) was used to induce an inflammatory response in tenocytes. LPS increased the tenocytes’ inflammatory factor COX2 expression and activated the HIF-1α/VEGFA/PDGFB pathway. Moreover, the conditioned medium from the tenocytes boosted rat aortic vascular endothelial cell (RAOEC) angiogenesis. Furthermore, Trichostatin A (TSA), an inhibitor of histone deacetylase, was used to treat inflammatory tenocytes. The expression levels of HIF-1α and its transcriptional targets VEGFA and PDGFB decreased, resulting in RAOEC angiogenesis inhibition. Finally, the dual-luciferase reporter gene assay and chromatin immunoprecipitation (ChIP) assay proved that the HIF-1α/PDGFB pathway played a more critical role in tenocyte angiogenesis than the HIF-1α/VEGFA pathway. TSA could alleviate angiogenesis mainly through epigenetic regulation of the HIF-1α/PDGFB pathway. Taken together, TSA might be a promising anti-angiogenesis drug for abnormal angiogenesis, which is induced by tendon injuries.

Serum Hypoxia-Inducible Factor 1alpha Levels Correlate with Outcomes After Intracerebral Hemorrhage.

BackgroundSerum hypoxia-inducible factor 1alpha (HIF-1α) is a key regulator in hypoxic and ischemic brain injury. We determined the relationship between serum HIF-1α levels and long-term prognosis plus severity of intracerebral hemorrhage (ICH).MethodsA total of 97 ICH cases and 97 healthy controls were enrolled. Glasgow Coma Scale (GCS) score and hematoma volume were used to assess hemorrhagic severity. Glasgow Outcome Scale (GOS) score of 1–3 at post-stroke 90 days was defined as a poor outcome.ResultsSerum HIF-1α levels of ICH patients were significantly higher than those of healthy controls (median, 218.8 vs 105.4 pg/mL; P<0.001) and were substantially correlated with GCS score (r=−0.485, P<0.001), hematoma volume (r=0.357, P<0.001) and GOS score (r=−0.436, P<0.001). Serum HIF-1α levels >239.4 pg/mL discriminated patients at risk of 90-day poor outcome with sensitivity of 65.9% and specificity of 79.3% (area under the receiver operating characteristic curve, 0.725; 95% confidence interval, 0.625–0.811; P<0.001). Moreover, serum HIF-1α levels >239.4 pg/mL were independently associated with a poor 90-day outcome (odds ratio, 5.133; 95% confidence interval, 1.117–23.593; P=0.036).ConclusionSerum HIF-1α, in close correlation with hemorrhagic severity and poor 90-day outcome, may serve as a potential prognostic biomarker for ICH.

Modulation of the Dental Pulp Stem Cell Secretory Profile by Hypoxia Induction Using Cobalt Chloride.

The action of stem cells is mediated by their paracrine secretions which comprise the secretory profile. Various approaches can be used to modify the secretory profile of stem cells. Creating a hypoxic environment is one method. The present study aims to demonstrate the influence of CoCl2 in generating hypoxic conditions in a dental pulp stem cell (DPSCs) culture, and the effect of this environment on their secretory profile. DPSCs that were isolated from human permanent teeth were characterized and treated with different concentrations of CoCl2 to assess their viability by an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and proliferation by a cell counting kit (CCK)-8 assay. The gene expression level of hypoxia-inducible factor 1-alpha (HIF-1α) was analyzed by quantitative real time polymerase chain reaction (qRT-PCR) to demonstrate a hypoxic environment. Comparative evaluation of the growth factors and cytokines were done by cytometric bead array. Gene expression levels of transcription factors OCT4 and SOX2 were analyzed by qRT-PCR to understand the effect of CoCl2 on stemness in DPSCs. DPSCs were positive for MSC-specific markers. Doses of CoCl2, up to 20 µM, did not negatively affect cell viability; in low doses (5 µM), it promoted cell survival. Treatment with 10 µM of CoCl2 significantly augmented the genetic expression of HIF-1α. Cells treated with 10 µM of CoCl2 showed changes in the levels of growth factors and cytokines produced. It was very evident that CoCl2 also increased the expression of OCT4 and SOX2, which is the modulation of stemness of DPSCs. A CoCl2 treatment-induced hypoxic environment modulates the secretory profile of DPSCs.

Sirtuin-1 inhibits endothelin-2 expression in human granulosa-lutein cells via hypoxia inducible factor 1 alpha and epigenetic modifications†.

Endothelin-2 (EDN2) expression in granulosa cells was previously shown to be highly dependent on the hypoxic mediator, hypoxia inducible factor 1 alpha (HIF1A). Here, we investigated whether sirtuin-1 (SIRT1), by deacetylating HIF1A and class III histones, modulates EDN2 in human granulosa-lutein cells (hGLCs). We found that HIF1A was markedly suppressed in the presence of resveratrol or a specific SIRT1 activator, SRT2104. In turn, hypoxia reduced SIRT1 levels, implying a mutually inhibitory interaction between hypoxia (HIF1A) and SIRT1. Consistent with reduced HIF1A transcriptional activity, SIRT1 activators, resveratrol, SRT2104, and metformin, each acting via different mechanisms, significantly inhibited EDN2. In support, knockdown of SIRT1 with siRNA markedly elevated EDN2, whereas adding SRT2104 to SIRT1-silenced cells abolished the stimulatory effect of siSIRT1 on EDN2 levels further demonstrating that EDN2 is negatively correlated with SIRT1. Next, we investigated whether SIRT1 can also mediate the repression of the EDN2 promoter via histone modification. Chromatin immunoprecipitation (ChIP) analysis revealed that SIRT1 is indeed bound to the EDN2 promoter and that elevated SIRT1 induced a 40% decrease in the acetylation of histone H3, suggesting that SIRT1 inhibits EDN2 promoter activity by inducing a repressive histone configuration. Importantly, SIRT1 activation, using SRT2104 or resveratrol, decreased the viable numbers of hGLC, and silencing SIRT1 enhanced hGLC viability. This effect may be mediated by reducing HIF1A and EDN2 levels, shown to promote cell survival. Taken together, these findings propose novel, physiologically relevant roles for SIRT1 in downregulating EDN2 and survival of hGLCs.

EGFR in enterocytes & endothelium and HIF1α in enterocytes are dispensable for massive small bowel resection induced angiogenesis.

BackgroundShort bowel syndrome (SBS) results from significant loss of small intestinal length. In response to this loss, adaptation occurs, with Epidermal Growth Factor Receptor (EGFR) being a key driver. Besides enhanced enterocyte proliferation, we have revealed that adaptation is associated with angiogenesis. Further, we have found that small bowel resection (SBR) is associated with diminished oxygen delivery and elevated levels of hypoxia-inducible factor 1-alpha (HIF1α).MethodsWe ablated EGFR in the epithelium and endothelium as well as HIF1α in the epithelium, ostensibly the most hypoxic element. Using these mice, we determined the effects of these genetic manipulations on intestinal blood flow after SBR using photoacoustic microscopy (PAM), intestinal adaptation and angiogenic responses. Then, given that endothelial cells require a stromal support cell for efficient vascularization, we ablated EGFR expression in intestinal subepithelial myofibroblasts (ISEMFs) to determine its effects on angiogenesis in a microfluidic model of human small intestine.ResultsDespite immediate increased demand in oxygen extraction fraction measured by PAM in all mouse lines, were no differences in enterocyte and endothelial cell EGFR knockouts or enterocyte HIF1α knockouts by POD3. Submucosal capillary density was also unchanged by POD7 in all mouse lines. Additionally, EGFR silencing in ISEMFs did not impact vascular network development in a microfluidic device of human small intestine.ConclusionsOverall, despite the importance of EGFR in facilitating intestinal adaptation after SBR, it had no impact on angiogenesis in three cell types–enterocytes, endothelial cells, and ISEMFs. Epithelial ablation of HIF1α also had no impact on angiogenesis in the setting of SBS.

Plantamajoside inhibits hypoxia-induced migration and invasion of human cervical cancer cells through the NF-κB and PI3K/akt pathways

Plantamajoside (PMS) is a major compound of Plantago asiatica and possesses anti-tumor property in several types of cancers. However, the effect of PMS on cervical cancer has not been investigated. This study aimed to investigate the effect of PMS on the migration and invasion of cervical cancer cell lines under hypoxic condition. Our results demonstrated that PMS significantly inhibited hypoxia-caused increases in cell migration and invasion of cervical cancer cells. The hypoxia-induced epithelial-mesenchymal transition (EMT) process was prevented by PMS with increased E-cadherin expression, and decreased expression levels of N-cadherin and vimentin in cervical cancer cells. Besides, the expression levels of transcription factors slug and snail were suppressed by PMS in hypoxia-induced cervical cancer cells. The increased mRNA and protein levels of hypoxia-inducible factor 1alpha (HIF-1α) in hypoxia-induced cervical cancer cells were prevented by PMS. Furthermore, PMS blocked the hypoxia-induced activation of NF-κB and PI3K/Akt pathway in cervical cancer cells. Taken together, these findings suggest that PMS exerted an anti-tumor activity in cervical cancer through preventing the hypoxia-induced EMT. Thus, PMS might serve as a therapeutic agent for the treatment of cervical cancer.

Altered VEGF, Bcl-2 and IDH1 expression in patients with adenomyosis.

Adenomyosis is a benign uterine disease resulting from the myometrial invasion of the endometrial gland and stroma. In the current study, angiogenesis, apoptosis and energy metabolism were investigated in adenomyosis. A retrospective study was performed using paraffin archival tissues. Three groups were included in the study: Group I and Group II; ectopic and eutopic endometrial tissues of patients with adenomyosis, respectively, and Control Group; endometrial tissue of individuals without adenomyosis. Vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), intercellular adhesion molecule 1 (ICAM-1) and hypoxia-inducible factor 1 alpha (HIF-1A) levels were evaluated as angiogenic markers. Bcl-2, caspase-9 and caspase-3 levels were investigated as apoptotic indicators, and isocitrate dehydrogenase 1 (IDH1), succinate dehydrogenase complex subunit C (SDHC) and fumarate hydratase (FH) levels were also examined as energy metabolism markers. Gene expression levels of all parameters were determined by RT-PCR. VEGF expression levels were found to be increased in Group I according to the control group and Group II. Bcl-2 expression levels were found to be increased in the Group I compared to the Group II. It was determined that expression levels of IDH1 were decreased in the Group I and Group II compared to the Control Group. There was no significant difference in the other examined parameters. Although we did not find a significant difference in HIF-1A levels between the groups, we found a positive correlation between VEGF and HIF-1A in the Group I. These results point out that VEGF, HIF-1A, Bcl-2 and IDH1 may be associated with the etiology of adenomyosis.

Loss of HIF1A From Pancreatic Cancer Cells Increases Expression of PPP1R1B and Degradation of p53 to Promote Invasion and Metastasis

Pancreatic ductal adenocarcinomas (PDACs) are hypovascular, resulting in the up-regulation of hypoxia inducible factor 1 alpha (HIF1A), which promotes the survival of cells under low-oxygen conditions. We studied the roles of HIF1A in the development of pancreatic tumors in mice. We performed studies with KrasLSL-G12D/+;Trp53LSL-R172H/+;Pdx1-Cre (KPC) mice, KPC mice with labeled pancreatic epithelial cells (EKPC), and EKPC mice with pancreas-specific depletion of HIF1A. Pancreatic and other tissues were collected and analyzed by histology and immunohistochemistry. Cancer cells were cultured from PDACs from mice and analyzed in cell migration and invasion assays and by immunoblots, real-time polymerase chain reaction, and liquid chromatography-mass spectrometry. We performed studies with the human pancreatic cancer cell lines PATU-8988T, BxPC-3, PANC-1, and MiaPACA-2, which have no or low metastatic activity, and PATU-8988S, AsPC-1, SUIT-2 and Capan-1, which have high metastatic activity. Expression of genes was knocked down in primary cancer cells and pancreatic cancer cell lines by using small hairpin RNAs; cells were injected intravenously into immune-competent and NOD/SCID mice, and lung metastases were quantified. We compared levels of messenger RNAs in pancreatic tumors and normal pancreas in The Cancer Genome Atlas. EKPC mice with pancreas-specific deletion of HIF1A developed more advanced pancreatic neoplasias and PDACs with more invasion and metastasis, and had significantly shorter survival times, than EKPC mice. Pancreatic cancer cells from these tumors had higher invasive and metastatic activity in culture than cells from tumors of EKPC mice. HIF1A-knockout pancreatic cancer cells had increased expression of protein phosphatase 1 regulatory inhibitor subunit 1B (PPP1R1B). There was an inverse correlation between levels of HIF1A and PPP1R1B in human PDAC tumors; higher expression of PPP1R1B correlated with shorter survival times of patients. Metastatic human pancreatic cancer cell lines had increased levels of PPP1R1B and lower levels of HIF1A compared with nonmetastatic cancer cell lines; knockdown of PPP1R1B significantly reduced the ability of pancreatic cancer cells to form lung metastases in mice. PPP1R1B promoted degradation of p53 by stabilizing phosphorylation of MDM2 at Ser166. HIF1A can act a tumor suppressor by preventing the expression of PPP1R1B and subsequent degradation of the p53 protein in pancreatic cancer cells. Loss of HIF1A from pancreatic cancer cells increases their invasive and metastatic activity.

Crosstalk between EP2 and PPARα Modulates Hypoxic Signaling and Myopia Development in Guinea Pigs

PurposeCyclic adenosine monophosphate (cAMP) and peroxisome proliferator-activated receptor alpha (PPARα) levels mediate extracellular matrix (ECM) changes by altering the levels of hypoxia-inducible factor 1-alpha (HIF-1α) in various tissues. We aimed to determine, in the sclera of guinea pigs, whether a prostanoid receptor (EP2)-linked cAMP modulation affects PPARα and HIF-1α signaling during myopia.MethodsThree-week-old guinea pigs (n = 20 in each group), were monocularly injected with either an EP2 agonist (butaprost 1 µmol/L/10 µmol/L), an antagonist (AH6809 10 µmol/L/30 µmol/L) or a vehicle solution for two weeks during normal ocular growth. Separate sets of animals received these injections and underwent form deprivation (FD) simultaneously. Refraction and axial length (AL) were measured at two weeks, followed by scleral tissue isolation for quantitative PCR (qPCR) analysis (n = 10) and cAMP detection (n = 10) using a radioimmunoassay.ResultsButaprost induced myopia development during normal ocular growth, with proportional increases in AL and cAMP levels. FD did not augment the magnitude of myopia or cAMP elevations in these agonist-injected eyes. AH6809 suppressed cAMP increases and myopia progression during FD, but had no effect in a normal visual environment. Of the diverse set of 27 genes related to cAMP, PPARα and HIF-1α signaling and ECM remodeling, butaprost differentially regulated 15 of them during myopia development. AH6809 injections during FD negated such differential gene expressions.ConclusionsEP2 agonism increased cAMP and HIF-1α signaling subsequent to declines in PPARα and RXR mRNA levels, which in turn decreased scleral fibrosis and promoted myopia. EP2 antagonism instead inhibited each of these responses. Our data suggest that EP2 suppression may sustain scleral ECM structure and inhibit myopia development.

Hypoxic training improves blood pressure, nitric oxide and hypoxia-inducible factor-1 alpha in hypertensive patients.

To examine the effects of intermittent hypoxic breathing at rest (IHR) or during exercise (IHT) on blood pressure and nitric oxide metabolites (NOx) and hypoxia-inducible factor-1 alpha levels (HIF-1α) over a 6-week period. 47 hypertensive patients were randomly allocated to three groups: hypertensive control (CON: n = 17; IHR: n = 15 and IHT: n = 15. The CON received no intervention; whereas, IH groups received eight events of hypoxia (FIO2 0.14), and normoxia (FIO2 0.21), 24-min hypoxia and 24-min normoxia, for 6weeks. The baseline data were collected 2days before the intervention; while, the post-test data were collected at days2 and 28 after the 6-week intervention. We observed a significant decrease of the SBP in both IH groups: IHR (- 12.0 ± 8.0mmHg, p = 0.004 and - 9.9 ± 8.8mmHg, p = 0.028, mean ± 95% CI) and IHT (- 13.0 ± 7.8mmHg, p = 0.002 and - 10.0 ± 8.4mmHg, p = 0.016) at days2 and 28 post-intervention, respectively. Compared to CON, IHR and IHT had increased of NOx (IHR; 8.5 ± 7.6μmol/L, p = 0.031 and IHT; 20.0 ± 9.1μmol/L, p < 0.001) and HIF-1α (IHR; 170.0 ± 100.0pg/mL, p = 0.002 and IHT; 340.5 ± 160.0pg/mL, p < 0.001). At 2days post-intervention, NOx and HIF-1α were negatively correlated with SBP in IHT. IH programs may act as an alternative therapeutic strategy for hypertension patients probably through elevation of NOx and HIF-1α production.

Abstract 170: Deferoxamine Treatment Decreases Levels Of Reactive Oxygen Species And Cellular Apoptosis In Irradiated Skin

Purpose: Radiation therapy is a cornerstone of oncologic treatment. Unfortunately, radiation can lead to pain and ulceration in the acute phase, as well as stiffness and fibrosis in the chronic phase. Deferoxamine (DFO) is an FDA approved iron chelator that has been shown to increase skin vascularization by stabilizing hypoxia-inducible factor 1-alpha levels and improve radiation-induced fibrosis. However, it is unclear whether there are other mechanisms underlying the therapeutic effects that DFO exhibits on the skin. Previous work has shown that DFO delivered transdermally improves healing in diabetic ulcer wounds by decreasing oxidative stress and cellular apoptosis. This study evaluates the effects of transdermal DFO on reactive oxygen species and cell death in the context of irradiation (IR) to skin. Methods: CD-1 nude immunodeficient mice (n=8) were randomized to one of two conditions: 1. Control patch (IR only) (n=4) and 2. DFO patch (n=4). The study period consisted of a 14-day pre-IR period, followed by a 12-day course of fractionated IR which delivered a total of 30 Gray to all mice. The control group received patches without DFO and the DFO group received patches containing DFO throughout the experiment with patches changed daily. Mouse scalp skin was harvested one day after the completion of IR. Tissue samples were either fixed in 4% PFA, snap frozen in OCT, or directly stored at -80°C. Fixed samples were embedded in paraffin, sectioned, and stained for iron with Prussian blue. Snap frozen samples were cryosectioned at 10um, mounted, stained with dihydroethidium (DHE), and imaged with confocal microscopy. DHE detects reactive oxygen species. Stored samples were mechanically homogenized and enzyme-linked immunosorbent assays (ELISAs) were performed to measure levels of human Bax and Cleaved Caspase-3, markers of apoptosis. Results: Treatment with DFO was associated with decreased levels of iron in the skin, confirming DFO’s role as an iron chelator. DHE fluorescence was more intense in the control patch group than in the DFO patch group (***p<0.0001), indicating that ROS generation decreased with DFO treatment. Similarly, apoptosis was highest in the control group and lowest in the DFO group as shown by higher human Bax protein (*p=0.028) and Cleaved Caspase-3 (**p=0.0038) levels. Conclusions: DFO delivered transdermally leads to a dose-dependent decrease in reactive oxygen species and apoptotic markers. These results provide further insight into the mechanisms underlying its restorative effects on irradiated skin. Future work will investigate the relationship of reactive oxygen species and fibroblast subpopulations in the skin.

The Hypoxic Response Expression as a Survival Biomarkers in Treatment-Naive Advanced Breast Cancer.

Objective:Hypoxia-associated biomarkers profiling may provide information for prognosis, staging, and subsequent therapy. We aim to evaluate whether the quantitative gene and protein expression of hypoxic response tumor markers — carbonic anhydrase IX (CAIX) and hypoxia- inducible factor 1 alpha (HIF1A) — may have a role in predicting survival in advanced breast cancer of Indonesian population. Methods:Tumor tissues and peripheral blood samples were collected from treatment - naïve locally advanced (LABC) or metastatic breast cancer patients (MBC) at Wahidin Sudirohusodo General Hospital (Makassar, South Sulawesi) and its referral network hospitals from July 2017 to March 2019. The level of mRNA (of blood and tumor tissue samples) and soluble protein (of blood samples) of CAIX and HIF1A were measured by RT-qPCR and ELISA methods, respectively, besides the standard histopathological grading and molecular subtype assessment. The CAIX and HIF1A expression, patients’ age, tumor characteristics, surgery status, and neoadjuvant chemotherapy drug classes were further involved in survival analyses for overall survival (OS) and progression-free survival (PFS). Results:Forty (30 LABC, 10 MBC) eligible patients examined were 21 hormone-receptors positives (15 Luminal A, 6 Luminal B) and 19 hormone-receptors negatives (10 HER2-enriched, 9 triple-negative). The CAIX blood mRNA and CAIX soluble protein levels in hormone-receptors negative patients were higher than in hormone-receptor-positive patients (p < 0.05). In univariate analysis, both CAIX and HIF1A levels predict OS (except HIF1A protein) with CAIX tissue mRNA has the highest hazard ratio (HR 8.04, 95%CI:2.45-26.39), but not PFS. Cox proportional hazard model confirmed that CAIX tissue mRNA is the independent predictor of OS (HR 6.10, 95%CI: 1.16-32.13) along with surgical status and tumor advancement type (LABC or MBC). Conclusions:CAIX mRNA expression of tumor tissue in treatment-naïve advanced breast cancer has a predictive value for OS.

Protodioscin protects PC12 cells against oxygen and glucose deprivation-induced injury through miR-124/AKT/Nrf2 pathway.

The purpose of the current study was to demonstrate the neuroprotective effect of protodioscin (Prot) in an in vitro model of ischemia/reperfusion (I/R) and investigate the underlying molecular mechanism. After PC12 cells were exposed to oxygen and glucose deprivation (OGD) reperfusion, PI staining by flow cytometry was used to quantify the rate of apoptosis. The levels of hypoxia-inducible factor 1-alpha (HIF-1α), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) were determined using commercially available kits. Intracellular reactive oxygen species (ROS) level was detected using the 20,70-dichlorodihy-drofluorescein diacetate (DCFH-DA) fluorescence assay. The expression levels of heat-shock proteins (HSP), PI3K, AKT, Nrf2, and miR-124 were tested by western blot or quantitative PCR. Prot significantly attenuated oxygen-glucose deprivation/reperfusion (OGD/R)-induced apoptotic death. Prot also reduced the oxidative stress as revealed by increasing the activities of SOD and GSH-Px, decreasing the levels of ROS and MDA. Moreover, mechanism investigations suggested that Prot prevented the decrease of HSP70, HSP32 (hemeoxygenase-1, HO-1), and PI3K protein expression, phosphorylation of AKT, and the accumulation of nuclear Nrf2. The level of miR-124 was decreased in PC12 cells, which was also effectively reversed by Prot treatment. Prot protected PC12 cells against OGD/R-induced injury through inhibiting oxidative stress and apoptosis, which could be associated with increasing HSP proteins expression via activating PI3K/AKT/Nrf2 pathway and miR-124 modulation.

Dental hypofunction alters subgingival microorganisms: a pilot study.

The aim of this study was to evaluate dental plaque compositions, vascular endothelial growth factor (VEGF) and hypoxia-inducible factor (HIF) 1-alpha levels in gingival crevicular fluid (GCF) at hypofunctional and normofunctional teeth in healthy individuals and chronic periodontitis patients. Sixty systemically healthy individuals were enrolled. Study groups were: group 1 hypofunctional healthy group (group 1, N.=15); group 2 hypofunctional periodontitis group (group 2, N.=15); group 3 normofunctional healthy group (group 3, N.=15); and group 4 normofunctional periodontitis group (group 4, N.=15). Clinical periodontal measurements (plaque index, gingival index and clinical attachment level) were recorded. Dental plaque and GCF samples were taken. VEGF and HIF 1-alpha levels in GCF were determined. Subgingival plaque samples were evaluated for 11 different bacterial species as, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, Prevotella intermedia, Peptostreptococcus micros, Fusobacterium nucleatum, Campylobacter rectus, Eubacterium nodatum, Eikenella corrodens and Capnocytophaga species. Tannerella forsythia, Peptostreptococcus micros, Eubacterium nodatum levels decreased in hypofunctional healthy and periodontitis groups (P<0.05). Porphyromonas gingivalis levels increased in hypofunctional healthy group and decreased in hypofunctional periodontitis group (P<0.05). There was also a decrease in Eikenella corrodens levels in hypofunctional periodontitis group (P<0.05). There were no difference regarding the Aggregatibacter actinomycetemcomitans, Capnocytophaga spp., Prevotella intermedia and Fusobacterium nucleatum levels among the groups (P>0.05). VEGF and HIF-1α levels in both GCF and serum samples were also similar (P>0.05). Within the limits of this study, the authors found that the levels of four significant bacterial strains were decreased in both hypofunctional healthy and hypofunctional periodontitis groups compared to normofunctional equivalents. Though not evaluated in this study, this situation could be due to periodontal ligament atrophy and related physiological alterations.

Single injection of sustained-release prostacyclin analog ONO-1301-MS ameliorates hypoxic toxicity in the murine model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by several pathologies including oxidative stress, apoptosis, neuroinflammation, and glutamate toxicity. Although multiple reports suggest that ischemia and hypoxia in the spinal cord plays a pivotal role in the pathogenesis of ALS, the precise role of hypoxia in disease progression remains unknown. In this study, we detected higher expression levels of Hypoxia-inducible factor 1-alpha (HIF-1α), a key regulator of cellular responses to hypoxia, in the spinal cord of ALS patients and in the transgenic mice overexpressing the familial ALS-associated G93A SOD1 mutation (mSOD1G93A mice) compared to controls. Single subcutaneous administration of sustained-release prostacyclin analog ONO-1301-MS to mSOD1G93A mice abrogated the expression of HIF-1α in their spinal cords, as well as erythropoietin (EPO) and vascular endothelial growth factor (VEGF), both of which are downstream to HIF-1α. Furthermore, ONO-1301-MS increased the level of mature brain-derived neurotrophic factor (BDNF) and ATP production in the spinal cords of mSOD1G93A mice. At late disease stages, the motor function and the survival of motor neurons of ONO-1301-MS-treated mSOD1G93A mice was significantly improved compared to vehicle-treated mSOD1G93A mice. Our data suggest that vasodilator therapy modulating local blood flow in the spinal cord has beneficial effects against ALS disease progression.

HIF1A and VEGF regulate each other by competing endogenous RNA mechanism and involve in the pathogenesis of peritoneal fibrosis

BackgroundPeritoneal fibrosis is a major intractable complication of long-term peritoneal dialysis, and would eventually lead to peritoneal ultrafiltration failure and the termination of peritoneal dialysis. Hypoxia-inducible factor 1-alpha (HIF1A) has been reported to regulate vascular endothelial growth factor (VEGF) and involves in peritoneal fibrosis, but the exact molecular regulation mechanism remains unknown. MethodsHIF1A and VEGF protein levels were measured in 42 peritoneal patients using enzyme linked immunosorbent assay. Bioinformatics, reverse transcription-polymerase chain reaction, correlation analysis, RNA interference, gene over-expression and luciferase assays were performed to clarify the competing endogenous RNA (ceRNA) regulation between HIF1A and VEGF. ResultsBoth HIF1A and VEGF levels were elevated in the peritoneal effluent of peritoneal dialysis patients with ultrafiltration problems, and were positively correlated with each other at protein level and mRNA level. Bioinformatics analysis identified 8 common targeted miRNAs for HIF1A and VEGF, including miR-17-5p, 20a, 20b, 93, 106a, 106b, 199a-5p and 203. MiR-17-5p was proved to be present in patients’ peritoneal effluent and selected for further studies. HIF1A mRNA and VEGF mRNA could regulate each other, and miR-17-5p was required in the regulations. Down/up regulation of HIF1A mRNA and VEGF mRNA resulted in up/down regulation of miR-17-5p. Furthermore, down/up regulation of miR-17-5p was associated with up/down regulation of HIF1A mRNA and VEGF mRNA. Luciferase assay indicated that HIF1A and VEGF regulated each other through 3′UTR. ConclusionHIF1A and VEGF could regulate each other in peritoneal mesothelial cell in the mediation of miR-17-5p and 3′UTR, indicating HIF1A and VEGF might regulate each other through competing endogenous RNA mechanism in the development of peritoneal fibrosis.

Tamoxifen mechanically deactivates hepatic stellate cells via the G protein-coupled estrogen receptor

Tamoxifen has been used for many years to target estrogen receptor signalling in breast cancer cells. Tamoxifen is also an agonist of the G protein-coupled estrogen receptor (GPER), a GPCR ubiquitously expressed in tissues that mediates the acute response to estrogens. Here we report that tamoxifen promotes mechanical quiescence in hepatic stellate cells (HSCs), stromal fibroblast-like cells whose activation triggers and perpetuates liver fibrosis in hepatocellular carcinomas. This mechanical deactivation is mediated by the GPER/RhoA/myosin axis and induces YAP deactivation. We report that tamoxifen decreases the levels of hypoxia-inducible factor-1 alpha (HIF-1α) and the synthesis of extracellular matrix proteins through a mechanical mechanism that involves actomyosin-dependent contractility and mechanosensing of tissue stiffness. Our results implicate GPER-mediated estrogen signalling in the mechanosensory-driven activation of HSCs and put forward estrogenic signalling as an option for mechanical reprogramming of myofibroblast-like cells in the tumour microenvironment. Tamoxifen, with half a century of safe clinical use, might lead this strategy of drug repositioning.

Combined Metabolo-Volumetric Parameters of 18F-FDG-PET and MRI Can Predict Tumor Cellularity, Ki67 Level and Expression of HIF 1alpha in Head and Neck Squamous Cell Carcinoma: A Pilot Study

BACKGROUND: Our purpose was to evaluate associations of combined 18F-FDG-PET and MRI parameters with histopathological features in head and neck squamous cell carcinoma (HNSCC). METHODS: Overall, 22 patients with HNSCC were acquired (10 with G1/2 tumors and 12 with G3 tumors).18F-FDG-PET/CT and MRI was performed and maximum standardized uptake value (SUVmax), total lesion glycolysis (TLG) and metabolic tumor volume (MTV) were estimated. Neck MRI was obtained on a 3 T scanner. Diffusion weighted imaging was performed with estimation of apparent diffusion coefficient (ADC). Perfusion parameters Ktrans,Ve, and Kep were derived from dynamic contrast-enhanced (DCE) imaging. Different combined PET/MRI parameters were calculated as ratios: PET parameters divided by ADC or DCE MRI parameters. The following histopathological features were estimated: Ki 67, EGFR, VEGF, p53, hypoxia-inducible factor (HIF)-1α, and cell count. Spearman's correlation coefficient (p) was used for correlation analysis. P < .05 was taken to indicate statistical significance. RESULTS: In overall sample, cellularity correlated with SUVmax/ADCmin (P = .558, P = .007), TLG/ADCmin (P = .546, P = .009), and MTV/ADCmin (P = .468, P = .028). MTV/Kep correlated with expression of HIF-1α (P = .450, P = 0,047). In G1/2 tumors, SUVmax/ADCmin correlated with HIF-1α (P = −.648, P = .043); MTV/Kep (P = −.669, P = .034) and TLG/Kep (P = −.644, P = .044) with Ki67. In G3 tumors, cellularity correlated with SUVmax/ADCmin (P = .832, P = .001), SUVmax/ADCmean (P = .741, P = .006), and TLG/ADCmin (P = .678, P = .015). MTV/ADCmin and TLG/ADCmin tended to correlate with HIF-1α. CONCLUSION: Combined parameters of 18F-FDG-PET and MRI can reflect Ki 67, tumor cellularity and expression of HIF-1α in HNSCC. Associations between parameters of 18F-FDG-PET and MRI and histopathology depend on tumor grading.