Hypoxia-inducible Protein Research Articles

Carbonic Anhydrase IX-Targeted Near-Infrared Dye for Fluorescence Imaging of Hypoxic Tumors.

Use of tumor-targeted fluorescence dyes to help surgeons identify otherwise undetected tumor nodules, decrease the incidence of cancer-positive margins, and facilitate localization of malignant lymph nodes has demonstrated considerable promise for improving cancer debulking surgery. Unfortunately, the repertoire of available tumor-targeted fluorescent dyes does not permit identification of all cancer types, raising the need to develop additional tumor-specific fluorescent dyes to ensure localization of all malignant lesions during cancer surgeries. By comparing the mRNA levels of the hypoxia-induced plasma membrane protein carbonic anhydrase IX (CA IX) in 13 major human cancers with the same mRNA levels in corresponding normal tissues, we document that CA IX constitutes a nearly universal marker for the design of tumor-targeted fluorescent dyes. Motivated by this expression profile, we synthesize two new CA IX-targeted near-infrared (NIR) fluorescent imaging agents and characterize their physical and biological properties both in vitro and in vivo. We report that conjugation of either acetazolamide or 6-aminosaccharin (i.e., two CA-IX-specific ligands) to the NIR fluorescent dye, S0456, via an extended phenolic spacer creates a brightly fluorescent dye that binds CA IX with high affinity and allows rapid visualization of hypoxic regions of solid tumors at depths >1 cm beneath a tissue surface. Taken together, these data suggest that a CA IX-targeted NIR dye can constitute a useful addition to a cocktail of tumor-targeted NIR dyes designed to image all human cancers.

Oxygen-Carrying Micro/Nanobubbles: Composition, Synthesis Techniques and Potential Prospects in Photo-Triggered Theranostics.

Microbubbles and nanobubbles (MNBs) can be prepared using various shells, such as phospholipids, polymers, proteins, and surfactants. MNBs contain gas cores due to which they are echogenic and can be used as contrast agents for ultrasonic and photoacoustic imaging. These bubbles can be engineered in various sizes as vehicles for gas and drug delivery applications with novel properties and flexible structures. Hypoxic areas in tumors develop owing to an imbalance of oxygen supply and demand. In tumors, hypoxic regions have shown more resistance to chemotherapy, radiotherapy, and photodynamic therapies. The efficacy of photodynamic therapy depends on the effective accumulation of photosensitizer drug in tumors and the availability of oxygen in the tumor to generate reactive oxygen species. MNBs have been shown to reverse hypoxic conditions, degradation of hypoxia inducible factor 1α protein, and increase tissue oxygen levels. This review summarizes the synthesis methods and shell compositions of micro/nanobubbles and methods deployed for oxygen delivery. Methods of functionalization of MNBs, their ability to deliver oxygen and drugs, incorporation of photosensitizers and potential application of photo-triggered theranostics, have also been discussed.

Abstract A083: Cell adhesion to laminin, by integrin α6β1, mediates drug resistance to PI3K in castration-resistant prostate cancer through AR-dependent induction of BNIP3

Abstract Pten loss leading to constitutive PI3K/Akt/mTor activation is a well-characterized PCa survival pathway frequently activated in castration-resistant prostate cancer (CRPC). Unfortunately, targeted mono-therapies to PI3K/Akt/mTor with or without anti-androgen therapy failed to achieve significant response in clinical trials with CRPC patients. The mechanisms that drive PI3K resistance in CRPC are unknown. The androgen receptor (AR) is also a major driver of prostate cancer (PCa) survival, even in CRPC. However, surprisingly very little is known about how AR promotes PCa survival. We previously identified an AR-dependent survival pathway in androgen-dependent tumor cells whereby AR induction of integrin α6β1 and adhesion to laminin in the tumor microenvironment activates NF-κB to induce Bcl-xL. This pathway acts in parallel to the PI3K/Akt pathway in Pten-null tumor cells such that combined inhibition of both PI3K and integrin α6β1 is required to effectively kill tumor cells and suppress tumor growth. While this drug combination was effective in androgen-responsive tumors in vivo, it poorly suppressed CRPC. Thus, we hypothesized there are additional survival mechanisms operating in CRPC. To identify CRPC survival pathways, we mined the gene expression data comparing androgen-sensitive to CRPC tumors in a series of human PDX LuCaP models. We found the hypoxia-inducible protein, BNIP3, to be preferentially elevated in CRPC tumors, and this correlated with increased integrin α6β1 expression and enhanced AR signaling. Furthermore, elevated BNIP3 predicts for poor outcome in PCa patients. We discovered that androgen induces BNIP3 expression specifically in CRPC C4-2 cell lines, but not the androgen-dependent parental LNCaP cells. BNIP3 expression is dependent on AR induction of integrin α6β1, adhesion to laminin, and nuclear translocation of HIF-1α. The CRPC C4-2 cells are highly resistant to PI3K inhibition by PX866 compared to LNCaP when adherent to laminin, and siRNA-mediated loss of integrin α6 or BNIP3 sensitizes these cells to PI3K inhibition. Blocking Bnip3 expression in C4-2 cells injected orthotopically into mice reduced tumor growth and induced apoptosis. BNIP3 can promote survival by activating autophagy and/or mitophagy. Even though autophagic flux was specifically enhanced in laminin-adherent CRPC C4-2 cells, but not in LNCaP cells, this was not dependent on BNIP3. WT BNIP3 can restore PI3K drug resistance in shBNIP3-expressing cells, but the BNIP3 ΔLIR mutant, which is unable to target mitochondria to autophagosomes, cannot restore drug resistance. We have identified an AR-dependent survival pathway in CRPC involving BNIP3-induced mitophagy that is dependent on and mediated by integrin α6β1 adhesion to laminin that promotes resistance to PI3K inhibition. Laminin is abundant in prostate tissue, lymph nodes, and bone—the primary sites for PCa growth. Thus, laminin within the tumor microenvironment of both primary and metastatic PCa drives drug resistance in CRPC. Citation Format: Eric Nollet, Sourik Ganguly, Veronique Schulz, Eva Corey, Cindy K. Miranti. Cell adhesion to laminin, by integrin α6β1, mediates drug resistance to PI3K in castration-resistant prostate cancer through AR-dependent induction of BNIP3 [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A083.

Relationships between expression of hypoxia-inducible factor-1α protein and clinicopathological characte-ristics and prognosis of patients with gastric cancer

Objective To investigate the expression level of hypoxia-inducible factor-1α (HIF-1α) in gastric cancer and its relationship with clinicopathological characteristics and prognosis of patients with gastric cancer. Methods From March 3, 2011 to September 28, 2012, 49 patients with gastric adenocarcinoma tissue chips were selected from pathology department of our hospital. They were matched with paracancerous tissues. The expression levels of HIF-1α were measured by immunohistochemistry method in gastric cancer tissues and paracancerous tissues chips. Kaplan-Meier was used to evaluate the progression-free survival (PFS) and overall survival (OS), and the Cox proportional hazards model was used to analyze whether HIF-1α was a prognostic factor. Results The high expression rate of HIF-1α protein in gastric cancer was significantly higher than that in paired para-carcinoma group (42.9% vs. 4.1%, χ2=20.509, P<0.001). The expression of HIF-1α protein was related to TNM stage (χ2=4.601, P=0.032), vascular invasion (χ2=6.766, P=0.009) and degree of differentiation (χ2=7.969, P=0.005). Compared with patients with low expression of HIF-1α, the median PFS (16.2 months vs. 27.3 months) and median OS (34.8 months vs. 43.8 months) were shorter in the patients with high expression of HIF-1α, and the differences were statistically significant (median PFS: χ2=4.661, P=0.002; median OS: χ2=6.903, P=0.009). The results of single factor analysis showed that overexpre-ssion of HIF-1α was correlated with PFS and OS (PFS: HR=4.461, 95%CI: 1.969-10.802, P<0.001; OS: HR=3.109, 95%CI: 1.274-7.588, P=0.013). The results of Cox multivariate analysis showed that overexpression of HIF-1α was the independent risk factor that affected the survival and prognosis of patients with gastric cancer (PFS: HR=4.747, 95%CI: 2.175-10.230, P<0.001; OS: HR=3.171, 95%CI: 1.358-7.404, P=0.008). Conclusion The high expression rate of HIF-1α protein in gastric cancer tissues is significantly higher than that in paracancerous tissues. The expression of HIF-1α is associated with TNM stage, vascular invasion, degree of differentiation in patients with gastric cancer. The high expression of HIF-1α is associa-ted with the shorter median OS and PFS. The high expression of HIF-1α is an independent risk factor for the survival and prognosis of patients with gastric cancer, which is expected to be an independent marker of poor prognosis. Key words: Stomach neoplasms; Hypoxia-inducible factor 1; Pathology, clinical; Prognosis

Upregulation of Angiogenic Factors via Protein Kinase C and Hypoxia-induced Factor-1α Pathways under High-glucose Conditions in the Placenta.

Abnormal glucose metabolism during pregnancy is an established risk factor for preeclampsia (PE). Disruption of the balance between placental angiogenic factors is linked to PE pathophysiology. We examined whether hypoxia-induced factor-1α (HIF-1α) and protein kinase Cβ (PKCβ) are involved in the regulation of placental angiogenic factors under high-glucose conditions in vitro. The human choriocarcinoma cell lines BeWo and JEG-3, and the human trophoblast cell line HTR-8/SVneo were cultured with 10 and 25 mmol/L glucose [control glucose group (CG) and high-glucose group (HG), respectively]. We examined the changes in HIF-1α, soluble fms-like tyrosine kinase-1 (sFlt-1), placental growth factor (PlGF), and vascular endothelial growth factor (VEGF) expression in the CG and HG by real-time PCR and ELISA. PKC activation was also measured by ELISA. The expressions of HIF-1α, sFlt-1, PlGF, and VEGF were significantly higher in the HG than in the CG. PKC activity was significantly increased in the HG. High glucose affected the expression of angiogenic factors in choriocarcinoma cells via the PKCβ and HIF-1α pathways, suggesting their involvement in PE pathogenesis.

Abstract 668: Electron paramagnetic resonance (EPR) pO2 image-guided tumor biopsies to analyze hypoxia-induced proteins

Abstract Stereotactic MCa4 and FSa tumor tissue biopsies registered with electron paramagnetic resonance (EPR) O2 imaging were used to identify sampling sites suitable to measure protein levels of three hypoxic cell biomarkers, hypoxia-induced factor 1-alpha (HIF-1α), carbonic anhydrase IX (CA9), and vascular endothelial growth factor (VEGF). EPR imaging provided quantitative localization of pO2 levels in tumors grown in the legs of C3H mice. Since hypoxic cells (pO2 &amp;lt;10 torr) are up to three times more resistant than normoxic cells to radiation lethality, identification of hypoxic populations to predict radiotherapeutic outcome is important. The oxygen broadening of narrow EPR spectral lines, or, equivalently, the increase in relaxation rates of electron magnetization, displays pO2 with 1-2 torr resolution in image voxels less than 1 mm3. The pO2 reporter molecule OX063 was used to image hypoxic areas in tumors. The molecule is selectively retained in tumors with a half-life of ~30 minutes. We used inversion recovery electron spin echo (IRESE) to measure the T1 rate of the trityl inside the tumor-bearing leg of mice. Voxel maps with O2 levels less than 10 torr (HF10) were used to guide biopsy cannulas to obtain tumor cell samples. Each of these biopsy cores were further subdivided into samples from which protein amounts could be determined via ELISA. Results from MCa4 and FSa tumor cells indicate a strong correlation between EPR pO2-identified hypoxic areas (&amp;lt;10 torr) and HIF-1α, CA9, and VEGF protein. The activation of various genes, including stem-cell activation, angiogenesis, vasodilation, glucose metabolism, reduced apoptotic activity, and cell cycle changes, contributes to poor radiotherapeutic outcome. EPR pO2 imaging may be used to precisely identify hypoxic areas harboring cells that protect tumor tissue from the toxicity of conventional radiation leading to radiotherapeutic failure. Grant Support: This work was supported by grants from the NIH, including R01 CA98575 and P41 EB002034. Citation Format: Richard Miller, Boris Epel, Martyna Elas, Martyna Krzykawska-Serda, Matthew Maggio, Eugene Barth, Mihai Giurcanu, Howard Halpern. Electron paramagnetic resonance (EPR) pO2 image-guided tumor biopsies to analyze hypoxia-induced proteins [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 668.

Luseogliflozin Inhibits HIF-1α Expression in Renal Proximal Tubular Epithelial Cells

Recent clinical trials have demonstrated the renoprotective effects of SGLT2 inhibitors in diabetic nephropathy (DN). However, the mechanisms of SGLT2 inhibitors on prevention of DN have not been fully elucidated. Hypoxia-induced tubulointerstitial fibrosis is considered to be a common pathway for various progressive kidney diseases including DN. Hypoxia inducible factor (HIF)-1α plays an important role in these pathological processes. In this study, we assessed the effects of luseogliflozin, an SGLT2 inhibitor, on HIF-1α expression in both cultured human renal proximal tubular epithelial cells (HRPTECs) and kidney of diabetic mice. Hypoxia (1%O2 for 24h) markedly increased HIF-1α protein in HRPTEC, whereas luseogliflozin inhibited expression of hypoxia-induced HIF-1α protein. In addition, luseogliflozin inhibited mRNA expression for HIF-1α-targeted genes, PAI-1, VEGF and GLUT-1 under hypoxia. The inhibitors of mitochondrial respiratory complex I, rotenone or complex III, antimycin A equally suppressed hypoxia-induced HIF-1α expression. We next treated db/db mice with 15 mg/kg/day luseogliflozin for 8 weeks. Luseogliflozin administration lowered plasma glucose levels and decreased glomerular mesangial matrix expansion as well as glomerular and interstitial fibronectin accumulation in db/db mice. Electron microscopic study revealed that luseogliflozin attenuated the thickening of glomerular basement membrane in db/db mice. Intriguingly, luseogliflozin decreased the length of mitochondria in the renal proximal tubules of the S1 segment. Furthermore, luseogliflozin attenuated diabetes-induced HIF-1α accumulation in the kidney of db/db mice. These results suggest that luseogliflozin inhibits hypoxia-induced HIF-1α accumulation at least partly by suppressing mitochondrial respiration. SGLT2 inhibitors may protect diabetic kidney from hypoxia-induced renal fibrosis by attenuating the expression of HIF-1α and profibrotic molecules. Disclosure R. Bessho: None. Y. Takiyama: None. T. Ota: None.

Functional Plasticity of Gamma Delta T Cells and Breast Tumor Targets in Hypoxia.

Interactions between immune and tumor cells in the tumor microenvironment (TME) often impact patient outcome, yet remain poorly understood. In addition, the effects of biophysical features such as hypoxia [low oxygen (O2)] on cells within the TME may lead to tumor evasion. Gamma delta T cells (γδTcs) naturally kill transformed cells and are therefore under development as immunotherapy for various cancers. Clinical trials have proven the safety of γδTc immunotherapy and increased circulating γδTc levels correlate with improved patient outcome. Yet, the function of γδTc tumor infiltrating lymphocytes in human breast cancer remains controversial. Breast tumors can be highly hypoxic, thus therapy must be effective under low O2 conditions. We have found increased infiltration of γδTc in areas of hypoxia in a small cohort of breast tumors; considering their inherent plasticity, it is important to understand how hypoxia influences γδTc function. In vitro, the cell density of expanded primary healthy donor blood-derived human γδTc decreased in response to hypoxia (2% O2) compared to normoxia (20% O2). However, the secretion of macrophage inflammatory protein 1α (MIP1α)/MIP1β, regulated on activation, normal T cell expressed and secreted (RANTES), and CD40L by γδTc were increased after 40 h in hypoxia compared to normoxia concomitant with the stabilization of hypoxia inducible factor 1-alpha protein. Mechanistically, we determined that natural killer group 2, member D (NKG2D) on γδTc and the NKG2D ligand MHC class I polypeptide-related sequence A (MICA)/B on MCF-7 and T47D breast cancer cell lines are important for γδTc cytotoxicity, but that MIP1α, RANTES, and CD40L do not play a direct role in cytotoxicity. Hypoxia appeared to enhance the cytotoxicity of γδTc such that exposure for 48 h increased cytotoxicity of γδTc against breast cancer cells that were maintained in normoxia; conversely, breast cancer lines incubated in hypoxia for 48 h prior to the assay were largely resistant to γδTc cytotoxicity. MICA/B surface expression on both MCF-7 and T47D remained unchanged upon exposure to hypoxia; however, ELISAs revealed increased MICA shedding by MCF-7 under hypoxia, potentially explaining resistance to γδTc cytotoxicity. Despite enhanced γδTc cytotoxicity upon pre-incubation in hypoxia, these cells were unable to overcome hypoxia-induced resistance of MCF-7. Thus, such resistance mechanisms employed by breast cancer targets must be overcome to develop more effective γδTc immunotherapies.

High salt intake shifts the mechanisms of flow-induced dilation in the middle cerebral arteries of Sprague-Dawley rats.

The goal of the present study was to examine the effect of 1 wk of high salt (HS) intake and the role of oxidative stress in changing the mechanisms of flow-induced dilation (FID) in isolated pressurized middle cerebral arteries of male Sprague-Dawley rats ( n = 15-16 rats/group). Reduced FID in the HS group was restored by intake of the superoxide scavenger tempol (HS + tempol in vivo group). The nitric oxide (NO) synthase inhibitor Nω-nitro-l-arginine methyl ester, cyclooxygenase inhibitor indomethacin, and selective inhibitor of microsomal cytochrome P-450 epoxidase activity N-(methylsulfonyl)-2-(2-propynyloxy)-benzenehexanamide significantly reduced FID in the low salt diet-fed group, whereas FID in the HS group was mediated by NO only. Cyclooxygenase-2 mRNA (but not protein) expression was decreased in the HS and HS + tempol in vivo groups. Hypoxia-inducible factor-1α and VEGF protein levels were increased in the HS group but decreased in the HS + tempol in vivo group. Assessment by direct fluorescence of middle cerebral arteries under flow revealed significantly reduced vascular NO levels and increased superoxide/reactive oxygen species levels in the HS group. These results suggest that HS intake impairs FID and changes FID mechanisms to entirely NO dependent, in contrast to the low-salt diet-fed group, where FID is NO, prostanoid, and epoxyeicosatrienoic acid dependent. These changes were accompanied by increased lipid peroxidation products in the plasma of HS diet-fed rats, increased vascular superoxide/reactive oxygen species levels, and decreased NO levels, together with increased expression of hypoxia-inducible factor-1α and VEGF. NEW & NOTEWORTHY High-salt (HS) diet changes the mechanisms of flow-induced dilation in rat middle cerebral arteries from a combination of nitric oxide-, prostanoid-, and epoxyeicosatrienoic acid-dependent mechanisms to, albeit reduced, a solely nitric oxide-dependent dilation. In vivo reactive oxygen species scavenging restores flow-induced dilation in HS diet-fed rats and ameliorates HS-induced increases in the transcription factor hypoxia-inducible factor-1α and expression of its downstream target genes.

Hypoxia-stressed cardiomyocytes promote early cardiac differentiation of cardiac stem cells through HIF-1α/Jagged1/Notch1 signaling

Hypoxia is beneficial for the differentiation of stem cells transplanted for myocardial injury, but mechanisms underlying this benefit remain unsolved. Here, we report the impact of hypoxia-induced Jagged1 expression in cardiomyocytes (CMs) for driving the differentiation of cardiac stem cells (CSCs). Forced hypoxia-inducible factor 1α (HIF-1α) expression and physical hypoxia (5% O2) treatment could induce Jagged1 expression in neonatal rat CMs. Pharmacological inhibition of HIF-1α by YC-1 attenuated hypoxia-promoted Jagged1 expression in CMs. An ERK inhibitor (PD98059), but not inhibitors of JNK (SP600125), Notch (DAPT), NF-κB (PTDC), JAK (AG490), or STAT3 (Stattic) suppressed hypoxia-induced Jagged1 protein expression in CMs. c-Kit+ CSCs isolated from neonatal rat hearts using a magnetic-activated cell sorting method expressed GATA4, SM22α or vWF, but not Nkx2.5 and cTnI. Moreover, 87.3% of freshly isolated CSCs displayed Notch1 receptor expression. Direct co-culture of CMs with BrdU-labeled CSCs enhanced CSCs differentiation, as evidenced by an increased number of BrdU+/Nkx2.5+ cells, while intermittent hypoxia for 21 days promoted co-culture-triggered differentiation of CSCs into CM-like cells. Notably, YC-1 and DAPT attenuated hypoxia-induced differentiation. Our results suggest that hypoxia induces Jagged1 expression in CMs primarily through ERK signaling, and facilitates early cardiac lineage differentiation of CSCs in CM/CSC co-cultures via HIF-1α/Jagged1/Notch signaling.

NDRG1 and FOXO1 regulate endothelial cell proliferation in infantile haemangioma.

The etiopathogenesis of infantile haemangioma has not been well understood, and it is accepted that angiogenic mediator dysregulation is the main contributor to the abnormal haemangioma capillary formation. The role of NDRG1, a hypoxia-inducible protein; FOXOs, which are tumor suppressor proteins; and the mTOR complex 2 pathway in infantile haemangioma have not been studied yet. The purpose of this study was to investigate NDRG1 and FOXO1 expression in the infantile haemangioma and the correlation of these proteins with proliferation and involution. Primary endothelial cells were obtained, with parental agreement, from 12 infantile haemangioma patients during surgery; 6 patients had proliferating infantile haemangiomas and 6 had involuting IHs. We compared the infantile haemangioma tissues and primary endothelial cells with human vein endothelial cells using microarrays, real-time PCR, Western blotting and immunohistochemical staining. Our data indicated that FOXO1 expression was downregulated in proliferating infantile haemangioma tissue. We found that the expression of NDRG1, a molecule upstream of the FOXO1 pathway, increased during haemangioma proliferation. NDRG1 knockdown decreased haemangioma endothelial cell proliferation and downregulated c-MYC oncoprotein levels. Our findings suggest that NDRG1 positively regulates haemangioma proliferation. FOXO1 dysregulation plays an important role in infantile haemangiomas pathogenesis.

127 NDRG1 regulates proliferation of endothelial cells of infantile hemangioma

The etiopathogenesis of infantile hemangioma has not been well understood and is accepted that dysregulation of angiogenic mediators mainly contributes to abnormal formation of hemangioma capillaries. The role of NDRG1, a hypoxia-inducible protein, and FOXOs, a tumor suppressor, and mTOR complex 2 pathway in infantile hemangioma have not been studied yet. The purpose of this study is to investigate NDRG1 and FOXO1 expression in the infantile hemangioma and their correlation with proliferation and involution. Primary endothelial cells were obtained during surgery from 12 infantile hemangioma patients, 6 proliferating infantile hemangiomas and 6 involuting IHs with parental agreement. We compared the infantile hemangioma tissues and primary endothelial cells with human vein endothelial cells using microarray, real-time PCR, western blot, and immunohistochemical stain. Our data indicated that FOXO1 expression was downregulated in proliferating infantile hemangioma tissue. We found that increased NDRG1, a upstream molecule of FOXO1 pathway, expression observed during proliferation of hemangioma. NDRG1 knockdown decreased proliferation of hemangioma endothelial cells and down-regulated c-MYC oncoprotein. Our findings suggest that NDRG1 positively regulate the proliferation of hemangioma. Dysregulation by FOXO1 plays an important role in the pathogenesis of infantile hemangioma.

A lesson from the oxidative metabolism of hibernator heart: Possible strategy for cardioprotection.

In the present study we hypothesized that myocardial adaptive phenotype in mammalian hibernation involves rearrangement of mitochondria bioenergetic pathways providing protective pattern in states of reduced metabolism and low temperature. European ground squirrels (Spermophilus citellus) were exposed to low temperature (4 ± 1 °C) and then divided into two groups: (1) animals that fell into torpor (hibernating group) and (2) animals that stayed active and euthermic for 1, 3, 7, 12, or 21 days (cold-exposed group). Protein levels of selected components of the electron transport chain and ATP synthase in the heart increased after prolonged cold acclimation (mainly from day 7-21 of cold exposure) and during hibernation. Peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) was also upregulated under both cold exposure and hibernating conditions. The phosphorylation state (Thr172) of 5'-AMP-activated protein kinase α increased early in cold exposure (at day 1 and 3) along with increased protein levels of phosphofructokinase and pyruvate dehydrogenase, whereas hypoxia inducible factor 1α protein levels showed no changes in response to cold exposure or hibernation. Hibernation also resulted in protein upregulation of three antioxidant defense enzymes (manganese and copper/zinc superoxide dismutases and glutathione peroxidase) and thioredoxin in the heart. Cold-exposed and hibernation-related phenotypes of the heart are characterized by improved molecular basis for mitochondrial energy-producing and antioxidant capacities that are achieved in a controlled manner. The recapitulation of such adaptive mechanisms found in hibernators could have broad application for myocardial protection from ishemia/reperfusion to improve hypothermic survival and cold preservation of hearts from non-hibernating species, including humans.

MiR-375 down-regulation of the rearranged L-myc fusion and hypoxia-induced gene domain protein 1A genes and effects on Sertoli cell proliferation.

ObjectiveThis study aimed to screen and identify the target genes of miR-375 in pig Sertoli (ST) cells and to elucidate the effect of miR-375 on the proliferation of ST cells.MethodsIn this study, bioinformatics software was used to predict and verify miR-375 target genes. Quantitative polymerase chain reaction (PCR) was used to detect the relationship between miR-375 and its target genes in ST cells. Enzyme-linked immunosorbent assay (ELISA) of rearranged L-myc fusion (RLF) and hypoxia-induced gene domain protein 1A (HIGD1A) was performed on porcine ST cells, which were transfected with a miR-375 mimics and inhibitor to verify the results. Dual luciferase reporter gene assays were performed to assess the interactions among miR-375, RLF, and HIGD1A. The effect of miR-375 on the proliferation of ST cells was analyzed by CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS).ResultsFive possible target genes of miR-375, including RLF, HIGD1A, colorectal cancer associated 2, POU class 3 homeobox 1, and WW domain binding protein 1 like, were found. The results of quantitative PCR suggested that mRNA expression of RLF and HIGD1A had a negative correlation with miR-375, indicating that RLF and HIGD1A are likely the target genes of miR-375. The ELISA results revealed that RLF and HIGD1A were negatively correlated with the miR-375 protein level. The luminescence results for the miR-375 group co-transfected with wild-type RLF and HIGD1A vector were significantly lower than those of the miR-375 group co-transfected with the blank vector or mutant RLF and HIGD1A vectors. The present findings suggest that RLF and HIGD1A are target genes of miR-375 and that miR-375 inhibits ST cell proliferation according to MTS analysis.ConclusionIt was speculated that miR-375 affects cell proliferation through its target genes, which play an important role in the development of testicular tissue.

Hypoxia-inducible lipid droplet-associated protein inhibits adipose triglyceride lipase

Elaborate control mechanisms of intracellular triacylglycerol (TAG) breakdown are critically involved in the maintenance of energy homeostasis. Hypoxia-inducible lipid droplet-associated protein (HILPDA)/hypoxia-inducible gene-2 (Hig-2) has been shown to affect intracellular TAG levels, yet, the underlying molecular mechanisms are unclear. Here, we show that HILPDA inhibits adipose triglyceride lipase (ATGL), the enzyme catalyzing the first step of intracellular TAG hydrolysis. HILPDA shares structural similarity with G0/G1 switch gene 2 (G0S2), an established inhibitor of ATGL. HILPDA inhibits ATGL activity in a dose-dependent manner with an IC50 value of ∼2 μM. ATGL inhibition depends on the direct physical interaction of both proteins and involves the N-terminal hydrophobic region of HILPDA and the N-terminal patatin domain-containing segment of ATGL. Finally, confocal microscopy combined with Förster resonance energy transfer-fluorescence lifetime imaging microscopy analysis indicated that HILPDA and ATGL colocalize and physically interact intracellularly. These findings provide a rational biochemical explanation for the tissue-specific increased TAG accumulation in HILPDA-overexpressing transgenic mouse models.

P022 Severe disease activity in UC is associated with mucosal hypoxia measured endoscopically

Tissue hypoxia has been shown to be a key microenvironmental regulator of mucosal barrier function and inflammation in pre-clinical models of colitis. However, the role of mucosal hypoxia in the pathogenesis of UC has yet to be extensively studied in IBD patients. Visible light spectroscopy can be utilised to measure the haemoglobin (Hb) oxygen saturations (%) colonic mucosa during endoscopy. Carbonic anhydrase 9 (CA-9) is a hypoxia-inducible protein and endogenous marker of hypoxia. The aim of this study was to compare mucosal oxygen saturations in normal and inflamed colonic mucosa in UC patients and assess relationship with disease activity. Patients undergoing colonoscopy were prospectively recruited in a single academic centre, with >3800 IBD patients. Endoscopies were performed using CO2 insufflation. Measurements of colonic mucosal Hb saturations were performed segmentally and from zones of transition from inflamed to normal mucosa, using an endoscopic probe (applied to intact mucosa) and tissue oximeter (T-Stat System, Spectros).Endoscopic severity was graded using the endoscopic Mayo score. Mucosal biopsies were obtained to grade histologic activity and for protein analysis. 38 patients were included in this study; n = 7 controls, n = 31 UC; male=25 (65.7%). The median age of UC patients was 39 years (IQR 34.6- 55.9). n = 5 had Mayo 0, n = 9 Mayo 1, n = 11 Mayo 2 and n = 6 Mayo 3 activity. At the time of the procedure, five patients were on biologic therapy, five immunomodulators and six on steroids. The median rectal saturation in the control group was 76.2% (IQR 73–80),compared with 77% (IQR 73.8–80.6) in the UC cohort (Mayo 0–3 inclusive) (p = 1.00). There was no significant difference across any colonic segment between these two cohorts. The median rectal mucosal O2 saturation in Mayo 0 was 77.6% (77.4–81.2),Mayo 1: 80.6% (77–83.8), Mayo 2: 75.4% (73.5–79.2) and Mayo 3:73.3% (68.7–74.8) (p = 0.02). Mucosal Hb Saturations (%) by disease severity. CA9 protein expression was significantly higher in biopsies with Mayo 2–3 activity compared with Mayo 0-1 disease. CA-9 protein expression in rectal biopsies. Mucosal O2 saturation is significantly decreased in severely active UC and oxygen signalling pathways are activated suggesting that mucosal hypoxia is an important determinant of disease severity in UC. These observations suggest the potential for oxygen signalling pathways as novel therapeutic targets.

Hypoxia induces selective modifications to the acetylome in the brain of zebrafish (Danio rerio).

Reversible protein acetylation is an important regulatory mechanism for modulating protein function. The cellular protein acetylome is in large part dictated by the cellular redox balance, and in particular [NAD+]. While the relationship between hypoxia, redox balance, energy charge and resulting mitochondrial dysfunction has been examined in the context of hypoxia-linked pathologies, little is known about the direct effects of decreases in environmental oxygen on reversible lysine acetylation, and the resulting modifications to mitochondrial metabolism. To address this knowledge gap, we exposed zebrafish (Danio rerio) to 16 h of hypoxia (2.21 kPa) and quantified acetylation levels of 1220 proteins using whole-cell proteomics in samples of brain taken from normoxic and hypoxic zebrafish. In addition, we examined the effects of hypoxia on cytoplasmic and mitochondrial redox status, whole-cell energetics, the activity of the mitochondrial NAD+-dependent deacetylase SIRT3, and electron transport chain complex activities to determine if there is an association between hypoxia-induced metabolic disturbances, protein acetylation, and mitochondrial function. Our results (1) reveal several key changes in the acetylation status of proteins in the brain, primarily within the mitochondria; (2) show significant fluctuations in cytoplasmic and mitochondrial redox status within the brain during hypoxia exposure; and (3) provide evidence that lysine acetylation may be related to large changes in electron transport and ATP-synthase complex activities and adenylate status in zebrafish exposed to hypoxic stress. Together, these data provide new insights into the role of protein modifications in mitochondrial metabolism during hypoxia.

Long Non-Coding RNA PVT1/miR-150/ HIG2 Axis Regulates the Proliferation, Invasion and the Balance of Iron Metabolism of Hepatocellular Carcinoma

Background/Aims: To investigate the biological roles and underlying molecular mechanisms of long non-coding RNA (lncRNA) PVT1 in Hepatocellular carcinoma (HCC). Methods: qRT-PCR was performed to measure the expression of miRNA and mRNA. Western blot was performed to measure the protein expression. CCK-8 assay was performed to determine cell proliferation. Flow cytometry was performed to detect cell apoptosis. Wounding-healing assay and Transwell assay was performed to detect cell migration and invasion. Dual luciferase reporter assay was performed to verify the target relationship. Quantichrom iron assay was performed to check uptake level of cellular iron. Results: PVT1 expression was up-regulated in HCC tissues and cell lines. Function studies revealed that PVT1 knockdown significantly suppressed cell proliferation, migration and invasion, and induced cell apoptosis in vitro. Furthermore, PVT1 could directly bind to microRNA (miR)-150 and down-regulate miR-150 expression. Hypoxia-inducible protein 2 (HIG2) was found to be one target gene of miR-150, and PVT1 knockdown could inhibit the expression of HIG2 through up-regulating miR-150 expression. In addition, the expression of miR-150 was down-regulated, while the expression of HIG2 was up-regulated in HCC tissues and cell lines. Moreover, inhibition of miR-150 could partly reverse the biological effects of PVT1 knockdown on proliferation, motility, apoptosis and iron metabolism in vitro, which might be associated with dysregulation of HIG2. In vivo results showed that PVT1 knockdown suppressed tumorigenesis and iron metabolism disorder by regulating the expression of miR-150 and HIG2. Conclusion: Taken together, the present study demonstrates that PVT1/miR-150/HIG2 axis may lead to a better understanding of HCC pathogenesis and provide potential therapeutic targets for HCC.

Expression of programmed death-ligand 1 and hypoxia-inducible factor-1α proteins in endometrial carcinoma.

Programmed death-ligand 1 (PD-L1) and hypoxia-inducible factor-1α (HIF-1α) proteins mediate major alterations of tumor microenvironment including generation of immunosuppressive microenvironment and tumor hypoxia, respectively. These alterations play a crucial role in carcinogenesis and tumor progression. The present study was designed to investigate the correlation between the expression of PD-L1 and HIF-1α proteins and the clinicopathologic variables in endometrial carcinoma. Tumor tissue sections from 95 endometrial carcinomas were evaluated for PD-L1 and HIF-1α immunohistochemical protein expression. The statistical analyses were performed using Chi-square and Fisher's exact tests when appropriate. Two-sided P < 0.05 was considered statistically significant. PD-L1 and HIF-1α expression were detected in 48.4% and 68.4% of endometrial carcinomas, respectively. PD-L1 expression was significantly associated with lymph node metastasis (P = 0.027). HIF-1α expression was significantly associated with tumor grade, depth of myometrial invasion, and lymph node metastasis (P = 0.014, 0.012, and 0.046, respectively). A significant positive correlation was detected between PD-L1 and HIF-1α immunoexpression (P = 0.015). PD-L1 and HIF-1α proteins are promising potential prognostic biomarkers in endometrial carcinomas since their overexpression is associated with clinicopathologic variables of advanced disease. A potential role of HIF-1α in upregulation of PD-L1 expression is suggested based on the finding of positive correlation between PD-L1 and HIF-1α expression in endometrial carcinoma. These findings point to a potential role of biomarkers inhibitors in controlling endometrial cancer progression.

Temporal Response of Angiogenesis and Hypertrophy to Resistance Training in Young Men.

In this double-blind, randomized control trial, 36 men (22 ± 1 yr) were randomized to placebo or protein supplementation, and participated in 12 wk of resistance training. Skeletal muscle biopsies were collected before and after 2, 4, 8, and 12 wk of training. Immunohistochemistry assessed fiber type-specific size and capillarization. Western blot and reverse transcription polymerase chain reaction assessed proteins involved in the molecular regulation of angiogenesis. Resistance training effectively increased Type I (15% ± 4%; P < 0.01) and Type II fiber cross-sectional area (28% ± 5%; P < 0.0001), an effect that tended to be further enhanced with protein supplementation in Type II fibers (P = 0.078). Capillary-to-fiber ratio significantly increased in Type I (P = 0.001) and II (P = 0.015) fibers after 12 wk of resistance exercise training and was evident after only 2 wk. Capillary-to-fiber perimeter exchange index increased in the Type I fibers only (P = 0.054) after 12 wk of training. Training resulted in a reduction in vascular endothelial growth factor mRNA. A (P = 0.008), while vascular endothelial growth factor receptor 2 (P = 0.016), hypoxia-inducible factor 1α (P = 0.016), and endothelial nitric oxide synthase (P = 0.01) increased in both groups. Hypoxia-inducible factor 1α protein content was higher in the protein group (main group effect, P = 0.02), and endothelial nitric oxide synthase content demonstrated a divergent relationship (time-group interaction, P = 0.049). This study presents novel evidence that microvascular adaptations and the molecular pathways involved are elevated after 2 wk of a 12-wk resistance training program. Increases in muscle fiber cross-sectional area are effectively matched by the changes in the microvasculature, providing further support for resistance training programs to optimize metabolic health.