Hypoxia Mimetic Cobalt Chloride Research Articles

Macrophage-Myofibroblast Transition Contributes to Myofibroblast Formation in Proliferative Vitreoretinal Disorders.

Inflammation and fibrosis are key features of proliferative vitreoretinal disorders. We aimed to define the macrophage phenotype and investigate the role of macrophage-myofibroblast transition (MMT) in the contribution to myofibroblast populations present in epiretinal membranes. Vitreous samples from proliferative diabetic retinopathy (PDR), proliferative vitreoretinopathy (PVR) and nondiabetic control patients, epiretinal fibrovascular membranes from PDR patients and fibrocellular membranes from PVR patients, human retinal Müller glial cells and human retinal microvascular endothelial cells (HRMECs) were studied by ELISA, immunohistochemistry and flow cytometry analysis. Myofibroblasts expressing α-SMA, fibroblast activation protein-α (FAP-α) and fibroblast-specific protein-1 (FSP-1) were present in all membranes. The majority of CD68+ monocytes/macrophages co-expressed the M2 macrophage marker CD206. In epiretinal membranes, cells undergoing MMT were identified by co-expression of the macrophage marker CD68 and myofibroblast markers α-SMA and FSP-1. Further analysis revealed that CD206+ M2 macrophages co-expressed α-SMA, FSP-1, FAP-α and ß-catenin. Soluble (s) CD206 and sFAP-α levels were significantly higher in vitreous samples from PDR and PVR patients than in nondiabetic control patients. The proinflammatory cytokine TNF-α and the hypoxia mimetic agent cobalt chloride induced upregulation of sFAP-α in culture media of Müller cells but not of HRMECs. The NF-ĸß inhibitor BAY11-7085 significantly attenuated TNF-α-induced upregulation of sFAP-α in Müller cells. Our findings suggest that the process of MMT might contribute to myofibroblast formation in epiretinal membranes, and this transition involved macrophages with a predominant M2 phenotype. In addition, sFAP-α as a vitreous biomarker may be derived from M2 macrophages transitioned to myofibroblasts and from Müller cells.

AhR and HIF-1α Signaling Pathways in Benign Meningioma under Hypoxia.

The role of hypoxia in benign meningiomas is less clear than that in the malignant meningiomas. Hypoxia-induced transcription factor 1 subunit alpha (HIF-1α) and its downstream signaling pathways play a central role in the mechanism of hypoxia. HIF-1α forms a complex with the aryl hydrocarbon receptor nuclear translocator (ARNT) protein and can compete for ARNT with aryl hydrocarbon receptor (AhR). In this work, the status of HIF-1α- and AhR-dependent signaling pathways was investigated in World Health Organization (WHO) grade 1 meningioma and patient-derived tumor primary cell culture under hypoxic conditions. mRNA levels of HIF-1α, AhR, and of their target genes as well as of ARNT and nuclear receptor coactivator NCOA2 were determined in tumor tissues from patients in whom the tumor was promptly removed either with or without prior endovascular embolization. Using the patient-derived nonembolized tumor primary cell culture, the effects of a hypoxia mimetic cobalt chloride (CoCl2) and an activator of the AhR signaling pathway benzo(α)pyrene (B[a]P) on mRNA levels of HIF-1α, AhR, and their target genes were investigated. Our findings show active functioning of AhR signaling in meningioma tissue of patients with tumor embolization and crosstalk between HIF-1α and AhR signaling in meningeal cells under hypoxia.

Tissue Inhibitor of Metalloproteinase-3 Ameliorates Diabetes-Induced Retinal Inflammation.

Purpose: Endogenous tissue inhibitor of matrix metalloproteinase-3 (TIMP-3) has powerful regulatory effects on inflammation and angiogenesis. In this study, we investigated the role of TIMP-3 in regulating inflammation in the diabetic retina.Methods: Vitreous samples from patients with proliferative diabetic retinopathy (PDR) and non-diabetic patients were subjected to Western blot analysis. Streptozotocin-treated rats were used as a preclinical diabetic retinopathy (DR) model. Blood-retinal barrier (BRB) breakdown was assessed with fluorescein isothiocyanate (FITC)-conjugated dextran. Rat retinas, human retinal microvascular endothelial cells (HRMECs) and human retinal Müller glial cells were studied by Western blot analysis and ELISA. Adherence of human monocytes to HRMECs was assessed and in vitro angiogenesis assays were performed.Results: Tissue inhibitor of matrix metalloproteinase-3 in vitreous samples was largely glycosylated. Intravitreal injection of TIMP-3 attenuated diabetes-induced BRB breakdown. This effect was associated with downregulation of diabetes-induced upregulation of the p65 subunit of NF-κB, intercellular adhesion molecule-1 (ICAM-1), and vascular endothelial growth factor (VEGF), whereas phospho-ERK1/2 levels were not altered. In Müller cell cultures, TIMP-3 significantly attenuated VEGF upregulation induced by high-glucose (HG), the hypoxia mimetic agent cobalt chloride (CoCl2) and TNF-α and attenuated MCP-1 upregulation induced by CoCl2 and TNF-α, but not by HG. TIMP-3 attenuated HG-induced upregulation of phospho-ERK1/2, caspase-3 and the mature form of ADAM17, but not the levels of the p65 subunit of NF-κB and the proform of ADAM17 in Müller cells. TIMP-3 significantly downregulated TNF-α-induced upregulation of ICAM-1 and VCAM-1 in HRMECs. Accordingly, TIMP-3 significantly decreased spontaneous and TNF-α- and VEGF-induced adherence of monocytes to HRMECs. Finally, TIMP-3 significantly attenuated VEGF-induced migration, chemotaxis and proliferation of HRMECs.Conclusion: In vitro and in vivo data point to anti-inflammatory and anti-angiogenic effects of TIMP-3 and support further studies for its applications in the treatment of DR.

A Functional Variant Rs492554 Associated With Congenital Heart Defects Modulates SESN2 Expression Through POU2F1.

Hypoxia exposure is responsible for the high incidence of congenital heart defects (CHDs) in high-altitude areas, which is nearly 20 times higher than that in low-altitude areas. However, the genetic factors involved are rarely reported. Sestrin2 (SESN2), a hypoxia stress-inducible gene, protects cardiomyocyte viability under stress; thus, SESN2 polymorphism may be a potential risk factor for CHD. We performed an association study of the SESN2 polymorphisms with CHD risk in two independent groups of the Han Chinese population from two different altitude areas. The allele-specific effects of lead single-nucleotide polymorphisms (SNPs) were assessed by expression quantitative trait locus, electrophoretic mobility shift, and luciferase reporter assays. The molecular mechanism of Sesn2 action against hypoxia-induced cell injury was investigated in embryonic rat-heart-derived H9c2 cells treated with or without hypoxia-mimetic cobalt chloride. SNP rs492554 was significantly associated with reduced CHD risk in the high-altitude population, but not in the low-altitude population. The protective T allele of rs492554 was correlated with higher SESN2 expression and showed a preferential binding affinity to POU2F1. We then identified SNP rs12406992 in strong linkage disequilibrium with rs492554 and mapped it within the binding motif of POU2F1. The T-C haplotype of rs492554-rs12406992 could increase luciferase expression, whereas POU2F1 knockdown effectively suppressed it. Mechanistically, increased Sesn2 protects against oxidative stress and cell apoptosis and maintains cell viability and proliferation. In summary, CHD-associated SNP rs492554 acts as an allele-specific distal enhancer to modulate SESN2 expression via interaction with POU2F1, which might provide new mechanistic insights into CHD pathogenesis.

Apocynin ameliorates NADPH oxidase 4 (NOX4) induced oxidative damage in the hypoxic human retinal Müller cells and diabetic rat retina.

NADPH oxidase (NOX) is a main producers of reactive oxygen species (ROS) that may contribute to the early pathogenesis of diabetic retinopathy (DR). ROS has harmful effects on endogenous neuro-survival factors brain-derived neurotrophic factor (BDNF) and sirtuin 1 (SIRT1) are necessary for the growth and survival of the retina. The role of NOX isoforms NOX4 in triggering ROS in DR is not clear. Here we determine the protective effects of a plant-derived NOX inhibitor apocynin (APO) on NOX4-induced ROS production which may contribute to the depletion of survival factors BDNF/SIRT1 or cell death in the diabetic retinas. Human retinal Müller glial cells (MGCs) were treated with hypoxia mimetic agent cobalt chloride (CoCl2) in the absence or presence of APO. Molecular analysis demonstrates that NOX4 is upregulated in CoCl2-treated MGCs and in the diabetic retinas. Increased NOX4 was accompanied by the downregulation of BDNF/SIRT1 expression or in the activation of apoptotic marker caspase-3. Whereas, APO treatment downregulates NOX4 and subsequently upregulates BDNF/SIRT1 or alleviate caspase-3 expression. Accordingly, in the diabetic retina we found a positive correlation in NOX4 vs ROS (p = 0.025; R2 = 0.488) and caspase-3 vs ROS (p = 0.04; R2 = 0.428); whereas a negative correlation in BDNF vs ROS (p = 0.009; R2 = 0.596) and SIRT1 vs ROS (p = 0.0003; R2 = 0.817) respectively. Taken together, NOX4-derived ROS could be a main contributor in downregulating BDNF/SIRT1 expression or in the activation of caspase-3. Whereas, APO treatment may minimize the deleterious effects occurring due to hyperglycemia and/or diabetic mimic hypoxic condition in early pathogenesis of DR.

Abstract 4098: Topotecan-mediated inhibition of HIF1α/ARV7 heterodimers in 22RV1 prostate cancer cells prevents ARV7 nuclear entry, reversing enzalutamide resistance

Abstract Background: Although enzalutamide (ENZ) is an effective treatment for prostate cancer (PC), development of ENZ resistance is inevitable. One mechanism for resistance is upregulation of ARV7, a splice variant of the full-length androgen receptor (FLAR) that lacks the androgen binding and hinge region domains. Nuclear translocation of FLAR requires the hinge region for coupling to tubulin or importin. ARV7 gains nuclear entry independent of the canonical pathway. FLAR has been reported to bind to Hypoxia Inducible Factor-1α (HIF1α) under normoxic conditions in the presence of dihydrotestosterone (DHT). We hypothesized that ARV7 may also heterodimerize with HIF1α in the cytoplasm, enabling nuclear localization and transcriptional activity of both transcription factors. We reported that topotecan (TPT) blocks HIF1α mRNA translation by targeting topoisomerase I (TOPO1) resident on HIF1α mRNA. We determined if ARV7 was bound to HIF1α in ENZ resistant cells and if inhibition of HIF1α translation by topotecan (TPT) could block ARV7 nuclear localization, thereby resensitizing ARV7 expressing cells to ENZ. We also evaluated if TOPO1 resided on FLAR/ARV7 mRNA, providing an additional target for TPT. Methods: 22Rv1 ENZ resistant PC cells that express FLAR and ARV7 protein were treated with the hypoxia mimetic cobalt chloride to stabilize HIF1α. Cytoplasmic and nuclear extracts from 22Rv1 cells were used for co-immunoprecipitation (Co-IP) using anit-HIF1α antibody. The effect of DHT, ENZ, and TPT on HIF1α and AR/ARV7 expression, and interactions between Topo-1 and HIF1α and AR mRNA were determined by western blot and RNA-IP. The effects of ENZ and TPT on 22Rv1 cell viability were evaluated using the XTT viability assay. Results: Topo-I RNA IP demonstrated its linkage to HIF-1α, FLAR and ARV7 mRNA. Co-IP of 22Rv1 whole cell lysates and nuclear extracts with HIF1α-specific antibodies yielded both full length AR and ARV7 protein bands on western blots under normoxic conditions in the presence of DHT and under hypoxic conditions. TPT and ENZ treatment of 22Rv1 cells under hypoxic conditions resulted in significantly reduced HIF1α, FLAR and ARV7 nuclear localization. TPT had an IC50 of 20 nM in normoxia and 91 nM in hypoxia. ENZ had an IC50 of 24.4 uM in normoxia and 388.7 uM in hypoxia. Dose-response curves for TPT / ENZ combinations at concentrations achieved clinically demonstrated synergistic activity, with combination index values of 0.54 under normoxic and 0.19 under hypoxic conditions. Conclusions: Our data indicate that Topo I resides on HIF-1α, FLAR and ARV7 mRNA, providing a potential target for TPT-mediated inhibition of their translation. Clinically achievable TPT concentrations caused HIF1α knockdown in hypoxic 22Rv1 cells in association with diminished nuclear localization of ARV7. 22Rv1 cells showed a synergistic dose-response to the combination of TPT and enzalutamide. These findings support exploration of TPT in combination with ENZ for treatment of PC. Citation Format: John P. Fruehauf, Diana C. Gomez, Leslie Spitalny, Jai-Hyun Kim. Topotecan-mediated inhibition of HIF1α/ARV7 heterodimers in 22RV1 prostate cancer cells prevents ARV7 nuclear entry, reversing enzalutamide resistance [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4098.

Apple Flavonols Mitigate Adipocyte Inflammation and Promote Angiogenic Factors in LPS- and Cobalt Chloride-Stimulated Adipocytes, in Part by a Peroxisome Proliferator-Activated Receptor-γ-Dependent Mechanism

Adipose tissue (AT) expansion induces local hypoxia, a key contributor to the chronic low-grade inflammation that drives obesity-associated disease. Apple flavonols phloretin (PT) and phlorizin (PZ) are suggested anti-inflammatory molecules but their effectiveness in obese AT is inadequately understood. Using in vitro models designed to reproduce the obese AT microenvironment, 3T3-L1 adipocytes were cultured for 24 h with PT or PZ (100 μM) concurrent with the inflammatory stimulus lipopolysaccharide (LPS; 10 ng/mL) and/or the hypoxia mimetic cobalt chloride (CoCl2; 100 μM). Within each condition, PT was more potent than PZ and its effects were partially mediated by peroxisome proliferator-activated receptor (PPAR)-γ (p < 0.05), as tested using the PPAR-γ antagonist bisphenol A diglycidyl ether (BADGE). In LPS-, CoCl2-, or LPS + CoCl2-stimulated adipocytes, PT reduced mRNA expression and/or secreted protein levels of inflammatory and macrophage chemotactic adipokines, and increased that of anti-inflammatory and angiogenic adipokines, which was consistent with reduced mRNA expression of M1 polarization markers and increased M2 markers in RAW 264.7 macrophages cultured in media collected from LPS + CoCl2-simulated adipocytes (p < 0.05). Further, within LPS + CoCl2-stimulated adipocytes, PT reduced reactive oxygen species accumulation, nuclear factor-κB activation, and apoptotic protein expression (p < 0.05). Overall, apple flavonols attenuate critical aspects of the obese AT phenotype.

Hypoxia-induced apoptosis of astrocytes is mediated by reduction of Dicer and activation of caspase-1.

Hypoxia is a condition in which the whole body or a region of the body is deprived of oxygen supply. The brain is very sensitive to the lack of oxygen and cerebral hypoxia can rapidly cause severe brain damage. Astrocytes are essential for the survival and function of neurons. Therefore, protecting astrocytes against cell death is one of the main therapeutic strategies for treating hypoxia. Hence, the mechanism of hypoxia-induced astrocytic cell death should be fully elucidated. In this study, astrocytes were exposed to hypoxic conditions using a hypoxia work station or the hypoxia mimetic agent cobalt chloride (CoCl2 ). Both the hypoxic gas mixture (1% O2 ) and chemical hypoxia-induced apoptotic cell death in T98G glioblastoma cells and mouse primary astrocytes. Reactive oxygen species were generated in response to the hypoxia-mediated activation of caspase-1. Active caspase-1 induced the classical caspase-dependent apoptosis of astrocytes. In addition, the microRNA processing enzyme Dicer was cleaved by caspase-3 during hypoxia. Knockdown of Dicer using antisense oligonucleotides induced apoptosis of T98G cells. Taken together, these results suggest that astrocytic cell death during hypoxia is mediated by the reactive oxygen species/caspase-1/classical caspase-dependent apoptotic pathway. In addition, the decrease in Dicer levels by active caspase-3 amplifies this apoptotic pathway via a positive feedback loop. These findings may provide a new target for therapeutic interventions in cerebral hypoxia.

Curry to treat Alzheimer’s disease?

AbstractPurposeAlzheimer’s disease (AD) is the major cause of dementia in the world with increasing evidence of the retina being affected. Since no cure exists, the development of new treatments is an urgent unmet need. Curcumin, a natural polyphenol found in curry, has been advocated as a potential neuroprotectant in AD. However, its poor solubility in water and low bioavailability, have limited its clinical translation. In this study we describe a novel curcumin nanoparticle (CN) formulation and evaluate its neuroprotective efficacy in vitro and in vivo.MethodsCNs were formulated using a thin film rehydration technique. R28 cells were used to assess CN neuroprotective activity. For in vivo studies 10‐month‐old 3xTg‐AD mice were treated intranasally 5 days/week with 3 μl of either CNs (n = 7) or vehicle (n = 6). After three months of treatment, animals had DARC (Detection of Apoptosing Retinal Cells) imaging and their brains (amyloid‐beta) were immunostained. All results are given with the standard error and the statistical test used is a student T‐test.ResultsCN formulation incorporating over 4 mg/ml of curcumin was stable over 90 days. CNs protected R28 cells against a) glutamate excitotoxicity with an IC50 of 22.36 ± 0.50 mm versus 4.54 ± 0.32 mm for control group (p &lt; 0.0001) and b) hypoxia mimetic cobalt chloride with an IC50 of 427.9±37.62 μm versus 259.4 ± 15.34 μm for control group (p &lt; 0.05). No adverse effects were seen in vivo either systemically or in the eye. 3xTg‐AD mice receiving CNs were found to have a significant reduction in the DARC count (p &lt; 0.05) compared to vehicle controls (17.36 ± 3.34 versus 49.08 ± 13.28). Their brains had a reduction in Aβ deposition score compared to vehicle controls (3.61 ± 0.17 versus 4.47 ± 0.23, p &lt; 0.01).ConclusionsThese promising results suggest that systemic CN has a potential therapeutic effect in AD, and that the effects of AD therapy can be assessed through the eye.

Hypoxia induction in cultured pancreatic islets enhances endothelial cell morphology and survival while maintaining beta-cell function.

Pancreatic islets are heavily vascularized in vivo yet lose this vasculature after only a few days in culture. Determining how to maintain islet vascularity in culture could lead to better outcomes in transplanting this tissue for the treatment of type 1 diabetes as well as provide insight into the complex communication between beta-cells and endothelial cells (ECs). We previously showed that islet ECs die in part due to limited diffusion of serum albumin into the tissue. We now aim to determine the impact of hypoxia on islet vascularization. We induced hypoxia in cultured mouse islets using the hypoxia mimetic cobalt chloride (100 μM CoCl2). We measured the impact on islet metabolism (two-photon NAD(P)H and Rh123 imaging) and function (insulin secretion and survival). We also measured the impact on hypoxia related transcripts (HIF-1α, VEGF-A, PDK-1, LDHA, COX4) and confirmed increased VEGF-A expression and secretion. Finally, we measured the vascularization of islets in static and flowing culture using PECAM-1 immunofluorescence. CoCl2 did not induce significant changes in beta cell metabolism (NAD(P)H and Rh123), insulin secretion, and survival. Consistent with hypoxia induction, CoCl2 stimulated HIF-1α, PDK-1, and LDHA transcripts and also stimulated VEGF expression and secretion. We observed a modest switch to the less oxidative isoform of COX4 (isoform 1 to 2) and this switch was noted in the glucose-stimulated cytoplasmic NAD(P)H responses. EC morphology and survival were greater in CoCl2 treated islets compared to exogenous VEGF-A in both static (dish) and microfluidic flow culture. Hypoxia induction using CoCl2 had a positive effect on islet EC morphology and survival with limited impact on beta-cell metabolism, function, and survival. The EC response appears to be due to endogenous production and secretion of angiogenic factors (e.g. VEGF-A), and mechanistically independent from survival induced by serum albumin.

Interleukin-11 Overexpression and M2 Macrophage Density are Associated with Angiogenic Activity in Proliferative Diabetic Retinopathy

ABSTRACT Purpose To investigate the expression of IL-11 and its receptor IL-11Rα and to quantify density of CD163+ M2 macrophages in proliferative diabetic retinopathy (PDR). Methods Vitreous samples from 29 PDR and 19 nondiabetic patients, epiretinal fibrovascular membranes from 15 patients with PDR and Müller cells were studied by enzyme-linked immunosorbent assay, immunohistochemistry and Western blot analysis. Results We showed a significant increase in expression of IL-11, soluble(s) IL-11Rα, sCD163 and VEGF in vitreous samples from PDR patients compared to nondiabetic controls. Significant positive correlations were found between levels of VEGF and levels of IL-11 and sCD163. Significant positive correlations were found between microvessel density and number of blood vessels and stromal cells expressing IL-11, IL-11Rα and CD163 in PDR epiretinal membranes. The hypoxia mimetic agent cobalt chloride induced upregulation of IL-11 and IL-11Ra in cultured Müller cells. Conclusions IL-11/IL-11Rα signaling and CD163+ M2 macrophages might be involved in PDR angiogenesis.

Impairment of chemical hypoxia-induced sphingosine kinase-1 expression and activation in rheumatoid arthritis synovial fibroblasts: A signature of exhaustion?

Sphingosine kinase 1 (SphK1) and 2 (SphK2) have been shown contribute to synovial inflammation in animal models of arthritis. However, low levels of intracellular sphingosine-1 phosphate (S1P) were reported in fibroblast-like synoviocytes (FLS) from patients in the end stage of rheumatoid arthritis (RA) compared to normal FLS. Moreover, the S1P receptor-mediated chemokine synthesis was altered in RAFLS in response to chemical hypoxia. Since the mechanisms responsible for low levels of intracellular S1P in RAFLS are not fully identified, we evaluated the contribution of SphKs to the S1P-induced synthesis of chemokines under conditions of chemical hypoxia. Our results show that a chemical hypoxia mimetic cobalt chloride (CoCl2) increased SphK1 expression and activation in normal FLS but not in RAFLS. Using selective inhibitors of SphKs and gene silencing approaches, we provide evidence that both SphK1 and SphK2 are involved in hypoxia-induced chemokine production in normal FLS. In contrast, only SphK2 mediates hypoxia-induced chemokine production in RAFLS. Moreover, CoCl2 increased S1P2 and S1P3 receptor mRNA levels in normal FLS but not in RAFLS. The data suggest that altered expression and/or activation of SphK1 combined with reduced induction of S1P receptor expression by CoCl2 impaired the CoCl2-mediated autocrine S1P receptor signaling loop and chemokine production in RAFLS.

Investigation of Endogenous Retrovirus Sequences in the Neighborhood of Genes Up-regulated in a Neuroblastoma Model after Treatment with Hypoxia-Mimetic Cobalt Chloride.

Human endogenous retroviruses (ERVs) have been found to be associated with different diseases, e.g., multiple sclerosis (MS). Most human ERVs integrated in our genome are not competent to replicate and these sequences are presumably silent. However, transcription of human ERVs can be reactivated, e.g., by hypoxia. Interestingly, MS has been linked to hypoxia since decades. As some patterns of demyelination are similar to white matter ischemia, hypoxic damage is discussed. Therefore, we are interested in the association between hypoxia and ERVs. As a model, we used human SH-SY5Y neuroblastoma cells after treatment with the hypoxia-mimetic cobalt chloride and analyzed differences in the gene expression profiles in comparison to untreated cells. The vicinity of up-regulated genes was scanned for endogenous retrovirus-derived sequences. Five genes were found to be strongly up-regulated in SH-SY5Y cells after treatment with cobalt chloride: clusterin, glutathione peroxidase 3, insulin-like growth factor 2, solute carrier family 7 member 11, and neural precursor cell expressed developmentally down-regulated protein 9. In the vicinity of these genes we identified large (>1,000 bp) open reading frames (ORFs). Most of these ORFs showed only low similarities to proteins from retro-transcribing viruses. However, we found very high similarity between retrovirus envelope sequences and a sequence in the vicinity of neural precursor cell expressed developmentally down-regulated protein 9. This sequence encodes the human endogenous retrovirus group FRD member 1, the encoded protein product is called syncytin 2. Transfection of syncytin 2 into the well-characterized Ewing sarcoma cell line A673 was not able to modulate the low immunostimulatory activity of this cell line. Future research is needed to determine whether the identified genes and the human endogenous retrovirus group FRD member 1 might play a role in the etiology of MS.

Protective effect of zinc preconditioning against renal ischemia reperfusion injury is dose dependent.

ObjectivesIschemia-reperfusion injury (IRI) is a major cause of acute kidney injury and chronic kidney disease. Two promising preconditioning methods for the kidney, intermittent arterial clamping (IC) and treatment with the hypoxia mimetic cobalt chloride, have never been directly compared. Furthermore, the protective efficacy of the chemically related transition metal Zn2+ against renal IRI is unclear. Although Co2+ ions have been shown to protect the kidney via hypoxia inducible factor (HIF), the effect of Zn2+ ions on the induction of HIF1α, HIF2α and HIF3α has not been investigated previously.Materials and methodsThe efficacy of different preconditioning techniques was assessed using a Sprague-Dawley rat model of renal IRI. Induction of HIF proteins following Zn2+ treatment of the human kidney cell lines HK-2 (immortalized normal tubular cells) and ACHN (renal cancer) was measured using Western Blot.ResultsFollowing 40 minutes of renal ischemia in rats, cobalt preconditioning offered greater protection against renal IRI than IC as evidenced by lower peak serum creatinine and urea concentrations. ZnCl2 (10 mg/kg) significantly lowered the creatinine and urea concentrations compared to saline-treated control rats following a clinically relevant 60 minutes of ischemia. Zn2+ induced expression of HIF1α and HIF2α but not HIF3α in HK-2 and ACHN cells.ConclusionZnCl2 preconditioning protects against renal IRI in a dose-dependent manner. Further studies are warranted to determine the possible mechanisms involved, and to assess the benefit of ZnCl2 preconditioning for clinical applications.

Abstract A37: Blockade of ARV7:HIF1α; heterodimers after toptoecan reverses enzalutamide resistance in 22Rv1 cells

Abstract Background: Although enzalutamide (ENZ) is initially effective for the treatment of castration-resistant prostate cancer (CRPC), development of ENZ resistance is inevitable. One mechanism for resistance is the emergence of ARV7, an alternative splicing variant of the full length androgen receptor (FL AR) which lacks the androgen binding and hinge region domains. Nuclear translocation of FL AR utilizes the hinge region for coupling to tubulin or importin. ARV7 protein appears to gain nuclear entry independent of the canonical pathway. FL AR has been reported to bind to Hypoxia Inducible Factor-1α (HIF1α). We hypothesized that ARV7 may also heterodimerize with HIF1α in the cytoplasm, facilitating its nuclear localization and transcriptional activity. We previously found that TPT blocks HIF1α translation by targeting topoisomerase I resident on HIF1α mRNA. We therefor determined if ARV7 was bound to HIF1α in ENZ resistant cells and if inhibition of HIF1α translation by topotecan (TPT) could block ARV7 nuclear localization, thereby resensitizing ARV7 expressing cells to ENZ. Methods: 22Rv1 ENZ resistant prostate cancer cells express comparable levels of full length AR and truncated ARV7 protein. 22Rv1 cells were treated with the hypoxia mimetic cobalt chloride to stabilize HIF1α. Cytoplasmic and nuclear extracts from 22Rv1 cells were used for co-immunoprecipitation (Co-IP) using anit-HIF1α antibody. The effect of TPT on HIF1α and AR/ARV7 expression, and interactions between Topo-1 and HIF1α and AR mRNA were determined by western blot and RNA-IP. The effects of ENZ and TPT on 22Rv1 cell viability were evaluated using the Luminescent cell viability assay. Results: Co-IP of 22Rv1 whole cell lysates and nuclear extracts with HIF1α-specific antibodies yielded both full length AR and ARV7 protein bands on western blots. Compared with control cells, TPT treatment of 22Rv1 cells under hypoxic conditions resulted in significantly reduced HIF1α accompanied by decreased ARV7 nuclear localization. Topo-I immunoprecipitation confirmed its linkage to HIF-1α mRNA. Surprisingly, we found that Topo-I RNA-IP pulled down AR mRNA as well. Dose-response curves for the TPT / ENZ combination demonstrated synergistic activity, with combination index values of 0.6. Under hypoxic conditions, TPT had an IC50 of 120 nM. pretreatment of 22Rv1 cells with a minimally toxic dose of 15 nM TPT reduced the ENZ IC50 from 93 nM to 60 nM. Conclusions: Our data indicate that Topo I residing on HIF-1α mRNA provided a druggable target for TPT-mediated inhibition of HIF1α mRNA translation. Clinically achievable TPT concentrations caused HIF1α knockdown in hypoxic 22Rv1 cells in association with diminished nuclear localization of ARV7. 22Rv1 cells showed a synergistic dose-response to the combination of TPT and enzalutamide. These findings support further exploration of TPT modulation of ENZ resistance in the clinic. Citation Format: John P. Fruehauf, Nathan Farrokh, Sarmen Sarkissian, Jai-Hyun Kim. Blockade of ARV7:HIF1α; heterodimers after toptoecan reverses enzalutamide resistance in 22Rv1 cells. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr A37.

Hypoxia mimetic induces lipid accumulation through mitochondrial dysfunction and stimulates autophagy in murine preadipocyte cell line

BackgroundHypoxia occurs within adipose tissue of obese human and mice. However, its role in adipose tissue regulation is still controversial. MethodsWe used murine preadipocyte 3T3-L1 cells and hypoxia was induced by using hypoxia mimetic agents, as CoCl2. To study adipocyte differentiation, we evaluated the adipocyte markers (PPARγ, C/EBPα and aP2), and a preadipocyte marker (pref-1) by qPCR, western blotting and immunofluorescence. Lipid accumulation was evaluated by Oil red-O assay and perilipin levels by western blotting and immunofluorescence. The effect of CoCl2 in microRNA, miR-27a and miR-27b, levels was evaluated by qPCR. We also assessed the mitochondrial membrane potential and reactive oxygen species (ROS), superoxide and ATP production. The effect of hypoxia mimetic in autophagy was determined by LC3B and p62 level evaluation by western blotting. ResultsOur results show that the hypoxia mimetic cobalt chloride increases lipid accumulation with no expression of PPARγ2. Furthermore, using qPCR we observed that the hypoxia mimetic increases microRNAs miR-27a and miR-27b, which are known to block PPARγ2 expression. In contrast, cobalt chloride induces mitochondrial dysfunction, and increases ROS production and autophagy. Moreover, an antioxidant agent, glutathione, prevents lipid accumulation induced by hypoxia mimetic indicating that ROS are responsible for hypoxia-induced lipid accumulation. ConclusionsAll these results taken together suggest that hypoxia mimetic blocks differentiation and induces autophagy. Hypoxia mimetic also induces lipid accumulation through mitochondrial dysfunction and ROS accumulation. General significanceThis study highlights the importance of adipocyte response to hypoxia, which might impair adipocyte metabolism and compromise adipose tissue function.

Hypoxia Drives AML Proliferation in the Bone Marrow Microenvironment Via Macrophage Inhibitory Factor

Abstract Introduction Hypoxia is an important component of the bone marrow microenvironment and the hematopoietic stem cell niche. Studies have shown that hypoxia contributes to the development and maintenance of acute myeloid leukemia (AML) cells within the bone marrow microenvironment. Hypoxia is principally maintained by members of the hypoxia-inducible factor (HIF), in particular HIF1a and its target genes, including MIF. We have previously shown that AML cells express constitutively high macrophage migration inhibitory factor (MIF) which drives IL-8 expression by the BM-MSC which in turn supports AML cell survival and proliferation. The aim of the present study is to determine if there is a connection between the role of hypoxia in regulating AML survival and MIF survival signals. Furthermore we investigate the role BM-MSC in regulating the hypoxic response. Methods Primary AML and BM-MSC were isolated from AML patients following informed consent and under approval from the UK National Research Ethics Service (LRCEref07/H0310/146). AML cell lines and primary AML blasts were cultured under normoxic (20% oxygen) or hypoxic conditions (1% oxygen) for 4 - 24 hours, mRNA expression of MIF, HIF1a, VEGF and IL-8 were determined by RT-PCR. MIF and IL-8 protein was determined using target specific ELISA. HIF1a protein expression was determined by western blotting. Hypoxia-mimetic agents, cobalt chloride (CoCl2) and desferrioxamine (DFO) were used. Cell proliferation was determines using CellTiter Glo and trypan blue exclusion. CFU-assays were performed using complete methylcellulose media. Results To determine If MIF is regulated by HIF1a in AML cells, we mimicked hypoxic conditions using CoCl2 and DFO in AML cells. Both CoCl2 and DFO upregulate MIF transcription and protein expression in OCI-AML3 cell lines and in primary AML blasts. Moreover, hypoxia increases both MIF mRNA expression and MIF chemokine expression compared to normoxic conditions. Lentiviral mediated knockdown of MIF in AML cells show significantly reduced cell proliferation and colony formation in methylcellulose media. Recombinant MIF induced interleukin-8 in AML blasts and the MIF inhibitor blocked MIF induced IL-8 release. Lentiviral mediated KD of HIF1a decreased MIF expression in human AML cells and a significant reduced their proliferative capacity. Finally we found that hypoxia increased MIF in AML blasts which was further increased when in co-culture with BM-MSC. Conclusions The results reported here suggest that hypoxia significantly affects the expression of the survival cytokine MIF in AML blasts. Furthermore, we show that BM-MSC regulate HIF1a induced MIF expression in AML under hypoxic conditions. We propose this hypoxia regulated HIF1a/MIF axis is essential to blast survival in the bone marrow niche. Disclosures Rushworth: Infinity Pharmaceuticals: Research Funding.

Silencing HIF-1α aggravates growth inhibition and necrosis of proximal renal tubular epithelial cell under hypoxia

The kidney is particularly susceptible to ischemia/hypoxia insult while dysfunction of proximal tubular epithelial cells (PTEC) is a primary pathologic hallmark in acute kidney injury. Hypoxia-inducible factor-1 (HIF-1) is a key regulator responsible for cellular hypoxic responses. Therefore, we investigated the effects of HIF-1 suppression, using small interference RNA (siRNA), upon the cell fate of PTEC under hypoxia, and explored the underlying possible molecular mechanism. Hypoxia was induced with hypoxia mimetic cobalt chloride. Our data showed that, in HIF-1α siRNA group, the HK-2 cells growth inhibition and necrosis became worse than those in hypoxia group. However, for apoptosis, no significant difference was observed between them. Consistent with the downregulation of HIF-1α in HIF-1α siRNA group, both mRNA and protein expression of glucose transporter-1 (Glut-1) and vascular endothelial growth factor (VEGF) also reduced more significantly than those in hypoxia group. In conclusion, silencing HIF-1α gene could aggravate growth inhibition and necrosis of PTEC under hypoxia. We provide evidence, from the opposite direction, that HIF-1 activation under hypoxia may facilitate adaptation and survival of proximal renal tubular cells, and the beneficial effects may be related to its downstream genes, such as Glut-1 and VEGF.

Abstract 4228: Imaging and targeting of hypoxic microenvironments in prostate cancer

Abstract Cancer cells display an adaptive response to hypoxia through the activation of several genes mediated by the binding of hypoxia inducible factors (HIFs) to hypoxia response elements (HRE) in the promoter region of target gene that results in their increased transcription [1]. HIFs promote key steps in tumorigenesis, including angiogenesis, metabolism, proliferation, metastasis, and differentiation [1]. Bacterial or yeast cytosine deaminase (yCD) converts the nontoxic prodrug 5-fluorocytosine (5-FC) to the anti-cancer drug 5-fluorouracil (5-FU) that is widely used in cancer treatment [2]. Using a lentivirus approach, we established controlled expression of yCD by HRE in prostate cancer cells (PC-3). These cells also report on HIF-1α expression with regulated luciferase (Luc) expression, allowing detection of hypoxia, and the generation of 5-FU from 5-FC by yCD in the presence of hypoxia. Transduction efficiency and reporter activity in response to hypoxia was evaluated by performing luciferase assays, and bioluminescence imaging (BLI) of cells in vitro or in vivo using a Xenogen IVIS Spectrum system. Cell viability in vitro in response to hypoxia in the presence of 5-FC was assessed by MTS assay. In vivo studies were performed by inoculating 2×10⁁6 PC-3-HRE-Luc cells and PC-3-HRE-yCD+Luc cells on either flank of 5-week-old male severely combined immune deficient (SCID) mice. BLI was performed once tumors reached ∼200mm3 followed by 5-FC injection through the tail vein (200mg/kg) and intraperitoneally (250mg/kg). BLI was performed 3 days after the first 5-FC injection and continued through the treatment protocol. At the end of the treatment protocol, tumors were excised, and a part of the tumor was processed for immunohistochemistry. Bioluminescence was detected in both PC3-HRE-Luc and PC-3-HRE-yCD+Luc cells only in response to the hypoxia mimetic cobalt chloride or hypoxia (1% O2) confirming the regulation of luciferase by hypoxia and activation of CD. Expression of yCD and its ability to convert the prodrug 5-FC to 5-FU, with increased cell kill was evident under hypoxia. In vivo, engineered PC-3-HRE-yCD+Luc cells reported hypoxia, and showed significant reduction of hypoxic regions and tumor volume. Morphologically, PC-3-HRE-yCD+Luc tumors exhibited extensive necrosis. We are currently evaluating the effects of eliminating hypoxic cancer cells on distant metastasis as well as on aggressive subpopulations such as cancer stem cells in the primary tumor.

Upregulation of Thrombin/Matrix Metalloproteinase-1/Protease-Activated Receptor-1 Chain in Proliferative Diabetic Retinopathy

ABSTRACTPurpose: Selective proteolytic activation of protease-activated receptor-1 (PAR1) by thrombin and matrix metalloproteinase-1 (MMP-1) plays a central role in enhancing angiogenesis. We investigated the expression levels of thrombin, MMP-1, and PAR1 and correlated these levels with vascular endothelial growth factor (VEGF) in proliferative diabetic retinopathy (PDR). In addition, we examined the expression of PAR1 and thrombin in the retinas of diabetic rats and PAR1 in human retinal microvascular endothelial cells (HRMEC) following exposure to high-glucose, the proinflammatory cytokines interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and the hypoxia mimetic agent cobalt chloride (CoCl2).Methods: Vitreous samples from 32 PDR and 23 nondiabetic patients, epiretinal membranes from 10 patients with PDR, retinas of rats, and HRMEC were studied by enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, and Western blot analysis. An assay for in vitro cell migration angiogenesis was performed in HRMEC.Results: In epiretinal membranes, PAR1 was expressed in vascular endothelial cells, CD45-expressing leukocytes, and myofibroblasts. ELISA and Western blot assays revealed significant increases in the expression levels of thrombin, MMP-1, and VEGF in vitreous samples from PDR patients compared to nondiabetic controls. Significant positive correlations were found between the levels of VEGF and the levels of thrombin (r = 0.41; p = 0.006) and MMP-1 (r = 0.66; p < 0.0001). Significant increases of cleaved PAR1 (approximately 50 kDa) and the proteolytically active thrombin (approximately 50 kDa) were detected in rat retinas after induction of diabetes. The proinflammatory cytokines IL-1β and TNF-α, but not high-glucose and CoCl2, induced upregulation of cleaved PAR1 (approximately 30 kDa) in HRMEC. In addition, thrombin and MMP-1 induced VEGF in HRMEC and vorapaxar, a PAR1 inhibitor, inhibited thrombin-induced migration in HRMEC.Conclusions: Interactions among thrombin, MMP-1, PAR1, and VEGF might facilitate angiogenesis in PDR.