HIF-1α Protein Stability Research Articles

Development of Inflammatory Hypoxia and Prevalence of Glycolytic Metabolism in Progressing Herpes Stromal Keratitis Lesions.

Chronic inflammation in tissues often causes the development of hypoxia. Herpes stromal keratitis (HSK) is a corneal chronic inflammatory condition that develops in response to recurrent HSV-1 infection. In this study, we investigated the development of hypoxia, the expression of hypoxia-associated glycolytic genes in HSV-1 infected corneas, and the outcome of blocking hypoxia-inducible factor (HIF) dimerization on the severity of HSK. Our results showed the development of hypoxia, an elevated expression of hypoxia-associated glycolytic genes, and an increased level of lactate in corneas with progressing HSK lesions. The magnitude of hypoxia correlated with the extent of neutrophils infiltrating the infected corneas, and the depletion of neutrophils reduced the development of hypoxia in infected corneas. Additionally, in progressing HSK lesions, nuclear localization of HIF-2α protein was detected in corneal epithelial cells, whereas HIF-1α protein stabilization was observed in infiltrating immune cells. Administration of acriflavine drug to HSV-1-infected mice inhibited nuclear accumulation of HIF-1α and HIF-2α protein in immune cell types and epithelial cells, respectively, in infected corneas. As a result, a decreased influx of CD4 T cells and nongranulocytic myeloid cells, but an increased influx of neutrophils, was noted in developing HSK lesions. Interestingly, acriflavine treatment given during the clinical disease period decreased neovascularization but increased the opacity in HSV-1-infected corneas. Taken together, the results of our study lay the foundation to dissect the role of inflammatory hypoxia and hypoxia-associated genes in the pathogenesis of HSK.

Thyroid axis activity and dopamine function in major depression

Vitamin B1, or thiamine is a critical enzyme cofactor required for metabolic function and energy production. Thiamine deficiency (TD) is common in various diseases, and results in severe neurological complications due to diminished mitochondrial function, oxidative stress, excitotoxicity and inflammation. These pathological sequelae result in apoptotic cell death in both neurons and astrocytes in distinct regions, in particular the thalamus and mammillary bodies. Comparable histological injuries in patients with hypoxia/ischemia (H/I) have also been described, suggesting a congruency between the cellular responses to these stresses. Analogous to H/I, TD stabilizes and activates Hypoxia Inducible Factor-1α (HIF-1α) even without changes in physiological oxygen levels. However, the mechanism of HIF-1α stabilization in TD is currently unknown. Using a pyruvate assay, we have demonstrated that TD induces pyruvate accumulation in mouse primary astrocytes which correlates to an increase in HIF-1α expression. Additionally, we utilized an enzymatic assay for pyruvate dehydrogenase to demonstrate a reduction in catalytic activity during TD due to lack of available thiamine pyrophosphate cofactor, resulting in the observed pyruvate accumulation. Finally, a pyruvate kinase inhibitor which limited pyruvate accumulation was utilized to demonstrate the role of pyruvate accumulation in HIF-1α stabilization during TD. These results reveal that stabilization of HIF-1α protein in TD centralizes on pyruvate accumulation in mouse primary astrocytes due to metabolic disruption of PDH.

Immunometabolic Regulation of Anti-Tumor T-Cell Responses by the Oncometabolite D-2-Hydroxyglutarate

Abstract Since the discovery of gain-of-function mutations in the tricarboxylic acid (TCA) cycle enzyme isocitrate dehydrogenase (IDH) and the resulting accumulation of the metabolite d-2-hydroxyglutarate (d-2HG) in several tumor entities (such as glioma, acute myeloid leukemia (AML), and cholangiocarcinoma) about 10 years ago research has focused on the tumor cell-intrinsic consequences. d-2HG acting as an oncometabolite was shown to promote proliferation, anoikis, tumorigenesis, and differentiation block of hematopoietic cells in an autocrine fashion. Although the prognostic value of the different types of IDH mutations remains controversial the development of inhibitors against mutated IDH is flourishing. On the other hand, serum levels of d-2HG proved to be a more robust adverse prognostic marker in AML and glioma. Surprisingly, until recently only few studies on the paracrine effects of this oncometabolite on the tumor microenvironment with particular focus on the innate or adaptive immunity were available. Now, three recent publications focused on the paracrine effects of tumor-derived d-2HG on T-cells in the context of AML and glioma. It was shown that T-cells are capable of efficiently taking up d-2HG in vitro, which was mirrored by 2HG-enriched T-cells exclusively found in samples from patients with IDH-mutated AML and glioma. Furthermore, all three studies describe an impairment of T-cell activation (although to different extents). The published effects could be at least partly attributed to metabolic alterations evoked by d-2HG influencing amongst others mTOR signaling, Hif-1α protein stability, the balance between aerobic glycolysis and oxidative phosphorylation, and the abundance of ATP (with according changes of AMPK activation). In the context of glioma it was further shown that IDH mutations and high d-2HG levels lead to reduced T-cell migration and consequently lowered T-cell infiltration at the tumor site. Moreover, two of the studies showed an increased frequency of FoxP3+ Tregs. Nevertheless, effects on downstream mechanisms and consequences have been differently addressed in the independent studies, and taken together the findings shed more light on the potentially targetable sites for improving therapeutic approaches. While the work by our group demonstrated profound impairments of Th17 polarization resulting from d-2HG-triggered Hif-1α protein destabilization the comprehensive analyses by Bunse et al. highlighted an inhibitory effect of d-2HG on the intracellular calcium signaling (downstream of the T-cell receptor) and an activation of AMPK signaling with resulting NFAT inhibition leading to the aforementioned activation defects. The summarized results of all three studies emphasized the importance of d-2HG not only as an autocrine but also a paracrine oncometabolite capable of shaping the tumor microenvironment in several facets adding to the common concept of tumor immune escape mechanisms. Those findings could lead to further improvements of the current targeted treatment strategies applied to patients harboring IDH mutations especially in view of the increasing importance of (combined) immune-based therapeutic approaches.

Aqp-1 Gene Knockout Attenuates Hypoxic Pulmonary Hypertension of Mice.

Objective- Hypoxic pulmonary hypertension (HPH) is characterized by proliferative vascular remodeling. Abnormal pulmonary artery smooth muscle cells proliferation and endothelial dysfunction are the primary cellular bases of vascular remodeling. AQP1 (aquaporin-1) is regulated by oxygen level and has been observed to play a role in the proliferation and migration of pulmonary artery smooth muscle cells. The role of AQP1 in HPH pathogenesis has not been directly determined to date. To determine the possible roles of AQP1 in the pathogenesis of HPH and explore its possible mechanisms. Approach and Results- Aqp1 knockout mice were used, and HPH model was established in this study. Primary pulmonary artery smooth muscle cells, primary mouse lung endothelial cells, and lung tissue sections from HPH model were used. Immunohistochemistry, immunofluorescence and Western blot, cell cycle, apoptosis, and migration analysis were performed in this study. AQP1 expression was upregulated by chronic hypoxia exposure, both in pulmonary artery endothelia and medial smooth muscle layer of mice. Aqp1 deficiency attenuated the elevation of right ventricular systolic pressures and mitigated pulmonary vascular structure remodeling. AQP1 deletion reduced abnormal cell proliferation in pulmonary artery and accompanied with accumulation of HIF (hypoxia-inducible factor). In vitro, Aqp1 deletion reduced hypoxia-induced proliferation, apoptosis resistance, and migration ability of primary cultured pulmonary artery smooth muscle cells and repressed HIF-1α protein stability. Furthermore, Aqp1 deficiency protected lung endothelial cells from apoptosis in response to hypoxic injury. Conclusions- Our data showed that Aqp1 deficiency could attenuate hypoxia-induced vascular remodeling in the development of HPH. AQP1 may be a potential target for pulmonary hypertension treatment.

Highlight on Endothelial Activation and Beyond.

Highlight on Endothelial Activation and Beyond.

Implication of Hypoxia-Inducible Factor-1 (HIF-1) As a New Therapeutic Target in JAK2V617F Positive Myeloproliferative Neoplasms (MPN)

Myeloproliferative neoplasms (MPN) are a heterogeneous group of malignancies including polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). The JAK2V617F mutation can be found in 90% of PV and approximately 50% of ET and PMF patients. Hypoxia-inducible factors (HIFs) are master transcriptional regulators of the response to decreases in cellular oxygen levels. Unveiling the function of deregulated HIF-1 signaling in normal and malignant hematopoiesis was the aim of several recent publications, highlighting the importance of HIF-1 for the maintenance of leukemic stem cells (LSCs) in acute and chronic myeloid leukemia (AML/CML). In a JAK2V617F knock-in mouse model and in patients, JAK2V617F was shown to induce the accumulation of reactive oxygen species (ROS) in the hematopoietic stem cell compartment, leading to a stabilization of HIF-1α protein. Further, aberrant STAT5 and PI3K/AKT/mTOR signaling induced HIF-1α expression on the transcriptional and translational level. Ruxolitinib treatment inhibited growth and reduced the expression of HIF-1α and its target gene VEGF in the JAK2V617F human erythroleukemia cell line HEL. In several leukemic cell lines constitutive expression of HIF-1α was reported, even under normoxic conditions. However, it still remains unknown whether HIF-1α plays a role in JAK2V617F positive MPN. In this study, we investigated the role HIF-1α signaling in JAK2V617F positive MPN in vitro.We retrovirally transduced the murine bone marrow cell line 32D with JAK2V617F or JAK2WT. Western blot analysis revealed significant increases in HIF-1α protein levels in JAK2V617F positive cells compared to JAK2WT controls after cultivation in normoxic conditions and this effect was abrogated by treatment with the JAK1/JAK2 inhibitor ruxolitinib. Inhibition of HIF-1, binding to hypoxia response elements (HRE), by low doses of echinomycin (1 nM), significantly impaired proliferation and survival. Using an Annexin-V/7-AAD flow cytometry assay apoptosis was found to be selectively induced in JAK2V617F positive, but not JAK2WT cells after echinomycin treatment. Additionally, BrdU/7-AAD cell cycle analysis revealed that only JAK2V617F positive cells were significantly arrested in G0/1 phase. These findings were consistent with shRNA-mediated knockdown (KD) of HIF-1α in JAK2V617F transduced 32D cells in presence but not the absence of HIF-2 antagonist 2. Inhibition of HIF-2 was necessary due to a compensatory increase of HIF-2α protein levels, shown by Western Blot analysis, counteracting HIF-1α-KD mediated effects. We isolated PBMCs and BMMNCs from JAK2V617F positive patients or healthy controls using Ficoll density gradient centrifugation. Echinomycin significantly abrogated the colony formation ability alone and in combination with ruxolitinib. In vitro treatment with echinomycin significantly decreased cell number and viability of 8 JAK2V617F positive BMMNC samples (4 PV, 3 PMF, 1 preMF; p[1nM]=0.0169, p[5nM]=0.0009) and 7 PBMC samples (6 PV, 1 PMF; p[1nM]=0.0156, p[5nM]=0.0156) in a dose-dependent manner. In contrast, PBMCs from 6 healthy donors were unaffected by the treatment. The same effect was observed in heterozygous and homozygous iPS cell-derived progenitors from JAK2V617F positive PV patients, whereas JAK2WT cells were unaffected by the treatment. Collectively, our data indicate that targeting HIF-1 might represent a novel therapeutic approach in classical Philadelphia-chromosome-negative MPN. DisclosuresBrümmendorf:Pfizer: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Janssen: Consultancy; Merck: Consultancy; Takeda: Consultancy.

β-TrCP upregulates HIF-1 in prostate cancer cells.

The substantial availability of hypoxia-inducible factor 1 (HIF-1) for pathophysiological states, such as malignancies and ischemia, is primarily regulated post-translationally through the ubiquitin proteolytic system. The balance between degradation and stabilization of HIF-1α protein is determined by specific E3 ligases. In our search for new E3 ligases that might affect HIF-1α protein expression, we studied the effects of beta-transducin repeat-containing protein (β-TrCP) on the hypoxic pathway in cancer cells. β-TrCP is overexpressed in many tumors and regulates various cellular processes through mediating the degradation of important targets. Unexpectedly, we found that β-TrCP overexpression increases HIF-1α protein expression level as well as HIF-1 transcriptional activity by stabilizing HIF-1α protein and preventing its ubiquitination and proteasomal degradation in prostate cancer cells. By using a proteomic approach, we succeeded in demonstrating that β-TrCP interferes with the association between HIF-1α and HSP70/CHIP, a HIF-1α established E3 ligase complex. Whereas the E3 ligase activity of β-TrCP is well known, antagonizing another E3 ligase is a new mechanism of action of this important E3. We suggest that destroying or suppressing β-TrCP and thereby interrupting the HIF-1 pathway, could be valuable antitumor therapy.

Yes-associated protein (YAP) binds to HIF-1\u03b1 and sustains HIF-1\u03b1 protein stability to promote hepatocellular carcinoma cell glycolysis under hypoxic stress

BackgroundHypoxia-inducible factor 1α (HIF-1α) is essential in hepatocellular carcinoma (HCC) glycolysis and progression. Yes-associated protein (YAP) is a powerful regulator and is overexpressed in many cancers, including HCC. The regulatory mechanism of YAP and HIF-1α in HCC glycolysis is unknown.MethodsWe detected YAP expression in 54 matched HCC tissues and the adjacent noncancerous tissues. The relationship between YAP mRNA expression and that of HIF-1α was analyzed using The Cancer Genome Atlas HCC tissue data. We cultured HepG2 and Huh7 HCC cells under normoxic (20% O2) and hypoxic (1% O2) conditions, and measured the lactate and glucose levels, migration and invasive capability, and the molecular mechanism of HCC cell glycolysis and progression.ResultsIn this study, we detected YAP expression in 54 matched HCC tissues and the adjacent noncancerous tissues. We observed that hypoxia-induced YAP activation is crucial for accelerating HCC cell glycolysis. Hypoxia inhibited the Hippo signaling pathway and promoted YAP nuclear localization, and decreased phosphorylated YAP expression in HCC cells. YAP knockdown inhibited HCC cell glycolysis under hypoxic. Mechanistically, hypoxic stress in the HCC cells promoted YAP binding to HIF-1α in the nucleus and sustained HIF-1α protein stability to bind to PKM2 gene and directly activates PKM2 transcription to accelerate glycolysis.ConclusionsOur findings describe a new regulatory mechanism of hypoxia-mediated HCC metabolism, and YAP might be a promising therapeutic target in HCC.

Fatty Acids Prevent Hypoxia-Inducible Factor-1α Signaling Through Decreased Succinate in Diabetes

Hypoxia-inducible factor (HIF)-1α is essential following a myocardial infarction (MI), and diabetic patients have poorer prognosis post-MI. Could HIF-1α activation be abnormal in the diabetic heart, and could metabolism be causing this? Diabetic hearts had decreased HIF-1α protein following ischemia, and insulin-resistant cardiomyocytes had decreased HIF-1α-mediated signaling and adaptation to hypoxia. This was due to elevated fatty acid (FA) metabolism preventing HIF-1α protein stabilization. FAs exerted their effect by decreasing succinate concentrations, a HIF-1α activator that inhibits the regulatory HIF hydroxylase enzymes. Invivo and invitro pharmacological HIF hydroxylase inhibition restored HIF-1α accumulation and improved post-ischemic functional recovery in diabetes.

Stabilization of the hypoxia-inducible transcription Factor-1 alpha (HIF-1α) in thiamine deficiency is mediated by pyruvate accumulation

Vitamin B1, or thiamine is a critical enzyme cofactor required for metabolic function and energy production. Thiamine deficiency (TD) is common in various diseases, and results in severe neurological complications due to diminished mitochondrial function, oxidative stress, excitotoxicity and inflammation. These pathological sequelae result in apoptotic cell death in both neurons and astrocytes in distinct regions, in particular the thalamus and mammillary bodies. Comparable histological injuries in patients with hypoxia/ischemia (H/I) have also been described, suggesting a congruency between the cellular responses to these stresses. Analogous to H/I, TD stabilizes and activates Hypoxia Inducible Factor-1α (HIF-1α) even without changes in physiological oxygen levels. However, the mechanism of HIF-1α stabilization in TD is currently unknown. Using a pyruvate assay, we have demonstrated that TD induces pyruvate accumulation in mouse primary astrocytes which correlates to an increase in HIF-1α expression. Additionally, we utilized an enzymatic assay for pyruvate dehydrogenase to demonstrate a reduction in catalytic activity during TD due to lack of available thiamine pyrophosphate cofactor, resulting in the observed pyruvate accumulation. Finally, a pyruvate kinase inhibitor which limited pyruvate accumulation was utilized to demonstrate the role of pyruvate accumulation in HIF-1α stabilization during TD. These results reveal that stabilization of HIF-1α protein in TD centralizes on pyruvate accumulation in mouse primary astrocytes due to metabolic disruption of PDH.

Abstract 1439: Asparagine stimulates growth and endoplasmic reticulum stress response in a fumarate hydratase-deficient renal cell carcinoma cell line

Abstract Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is a syndrome characterized by leiomyomas and an aggressive form of papillary kidney cancer. The genetic driver of HLRCC is a loss-of-function mutation of the Krebs cycle enzyme fumarate hydratase (FH), resulting in increased levels of the Krebs cycle intermediate fumarate, and resulting impaired oxidative phosphorylation. as well as in stabilization of HIF1α protein, which is the final common pathway of many different types of renal cell carcinomas (RCCs). To better understand the energy sources utilized by FH-deficient cells, we examined the nutrient requirements of FH-deficient RCC UOK262 (UOK262D) cell line in comparison to UOK262 cells transfected with an FH transgene (UOK262R). Experiments with selective addition of nonessential amino acids (NEAA) from the media revealed that both UOK262D and UOK262R cell lines required asparagine (A), but not other NEAA, for growth after 96 hours of incubation in the absence of glutamine (G). A (100 uM) and G (2 mM) demonstrated an approximately equal effect on the growth rate of both FH deficient and repleted cells, which was only observed after a minimum of 96 hrs of incubation. The growth effects of A and G were additive in both cell lines. While G increased the oxygen consumption rate (OCR) and decreased glycolysis (ECAR) in both cell lines, addition of A had no additive effect on both parameters, suggesting that G is used for energy production and A is required for biomass production. Similar effects of A and G, alone and in combination, on proliferation were seen in FH deficient and repleted UOK268 cell lines and in the SDH deficient and repleted UOK269 cells. Both A and G activated the mTOR pathway in both UOK262D and UOK262R cell lines as indicated by increased phosphorylation of mTOR, S6K and S6RP at 48h. However, mTOR pathway activation was not observed after A and/or G exposure in UOK268 or UOK269 pairs of cell lines. To determine the genomic response of UOK262 cell lines to A and/or G, we conducted RNASeq analysis in cells incubated for 96 hours to A, G, both or neither. In UOK262D cells, A+G, but not A or G individually, increased RNA levels of many genes associated with endoplasmic reticulum stress response. These gene expression changes were not seen in the FH repleted cell line UOK262R incubated in A+G. Therefore, although the growth effects of A+G in UOK262R and UOK262D cells were similar, and the effects on mTOR signaling pathway effects were similar between the two cell lines, the RNASeq experiment revealed a fundamentally different response to the combination of the 2 amino acids in the FH deficient cells vs. the FH repleted cells. In summary, we have identified asparagine as a key nutrient in HLRCC, establishing it as a potential target for a metabolically rational treatment approach. This research was supported by the Intramural Research Program of the National Cancer Institute, NIH. Citation Format: Rony Panarsky, Youfeng Yang, W. Marston Linehan, Jeffrey A. Moscow. Asparagine stimulates growth and endoplasmic reticulum stress response in a fumarate hydratase-deficient renal cell carcinoma cell line [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 1439.

Abstract 676: Hypoxia Upregulates NADPH Oxidase 4-Mediated Hydrogen Peroxide Release by a HIF-Independent Mechanism in Human Endothelial Cells

NADPH oxidases are important sources of reactive oxygen species in the vascular wall. Recent evidence supports a vasoprotective role of H 2 O 2 produced by the main endothelial isoform Nox4. The impact of hypoxia on NOX4 expression in human endothelial cells and the underlying mechanism remains to be elucidated. In this study, we show that NOX4 mRNA and protein expression was upregulated by hypoxia (1 % O 2 ) in human umbilical vein endothelial cells (HUVEC). Correspondingly, H 2 O 2 production was 2-fold elevated in HUVEC after hypoxia. In contrast to rotenone and oxypurinol, lentiviral downregulation via shNOX4 abolished the elevated hypoxic hydrogen peroxide levels to normoxic values. Hypoxia stabilized the hypoxia-inducible factor (HIF)-1α protein in endothelial cells. Furthermore, VEGF promoter activity and a control promoter containing 3 hypoxia-responsive elements (HRE) were induced by hypoxia. NOX4 promoter deletions up to -119/+239 had an increased basal activity compared to control vector. A full-length and a terminally deleted NOX4 promoter construct missing a putative HRE showed a comparable activity under hypoxic and normoxic conditions, suggesting that NOX4 is not induced on the transcriptional level by hypoxia in endothelial cells. In addition, stabilization of HIF-1α protein under normoxic conditions using DMOG did not change NOX4 mRNA expression in HUVEC. Furthermore, overexpression of HIF-1α did not alter NOX4 promoter activity. Blockade of active transcription by actinomycin D revealed an increased stability of the NOX4 mRNA under hypoxic conditions. In conclusion, this study demonstrates an HIF-independent upregulation of NOX4 as major source of endothelial hydrogen peroxide generation in response to hypoxia. Our data support as a novel mechanism an increased NOX4 mRNA stability under hypoxic conditions in human endothelial cells.

Hepatitis transactivator protein X promotes extracellular matrix modification through HIF/LOX pathway in liver cancer

Hepatocellular carcinoma (HCC), accounting for 90% of primary liver cancer, is a lethal malignancy that is tightly associated with chronic hepatitis B virus (HBV) infection. HBV encodes a viral onco-protein, transactivator protein X (HBx), which interacts with proteins of hepatocytes to promote oncogenesis. Our current study focused on the interaction of HBx with a transcription factor, hypoxia-inducible factor-1α (HIF-1α), which is stabilized by low O2 condition (hypoxia) and is found to be frequently overexpressed in HCC intra-tumorally due to poor blood perfusion. Here, we showed that overexpression of HBx by tetracycline-inducible systems further stabilized HIF-1α under hypoxia in HBV-negative HCC cell lines. Reversely, knockdown of HBx reduced HIF-1α protein stabilization under hypoxia in HBV-positive HCC cell lines. More intriguingly, overexpression of HBx elevated the mRNA and protein expression of a family of HIF-1α target genes, the lysyl oxidase (LOX) family in HCC. The LOX family members function to cross-link collagen in the extracellular matrix (ECM) to promote cancer progression and metastasis. By analyzing the collagens under scanning electron microscope, we found that collagen fibers were significantly smaller in size when incubated with conditioned medium from HBx knockdown HCC cells as compared to control HCC cells in vitro. Transwell invasion assay further revealed that less cells were able to invade through the matrigel which was pre-treated with conditioned medium from HBx knockdown HCC cells as compared to control HCC cells. Orthotopic and subcutaneous HCC models further showed that knockdown of HBx in HCC cells reduced collagen crosslinking and stiffness in vivo and repressed HCC growth and metastasis. Taken together, our in vitro and in vivo studies showed the HBx remodeled the ECM through HIF-1α/LOX pathway to promote HCC metastasis.

Minoxidil Induction of VEGF Is Mediated by Inhibition of HIF-Prolyl Hydroxylase.

The topical application of minoxidil may achieve millimolar concentrations in the skin. We investigated whether millimolar minoxidil could induce vascular endothelial growth factor (VEGF), a possible effector for minoxidil-mediated hair growth, and how it occurred at the molecular level. Cell-based experiments were performed to investigate a molecular mechanism underlying the millimolar minoxidil induction of VEGF. The inhibitory effect of minoxidil on hypoxia-inducible factor (HIF) prolyl hydroxylase-2 (PHD-2) was tested by an in vitro von Hippel–Lindau protein (VHL) binding assay. To examine the angiogenic potential of millimolar minoxidil, a chorioallantoic membrane (CAM) assay was used. In human keratinocytes and dermal papilla cells, millimolar minoxidil increased the secretion of VEGF, which was not attenuated by a specific adenosine receptor antagonist that inhibits the micromolar minoxidil induction of VEGF. Millimolar minoxidil induced hypoxia-inducible factor-1α (HIF-1α), and the induction of VEGF was dependent on HIF-1. Moreover, minoxidil applied to the dorsal area of mice increased HIF-1α and VEGF in the skin. In an in vitro VHL binding assay, minoxidil directly inhibited PHD-2, thus preventing the hydroxylation of cellular HIF-1α and VHL-dependent proteasome degradation and resulting in the stabilization of HIF-1α protein. Minoxidil inhibition of PHD-2 was reversed by ascorbate, a cofactor of PHD-2, and the minoxidil induction of cellular HIF-1α was abrogated by the cofactor. Millimolar minoxidil promoted angiogenesis in the CAM assay, an in vivo angiogenic test, and this was nullified by the specific inhibition of VEGF. Our data demonstrate that PHD may be the molecular target for millimolar minoxidil-mediated VEGF induction via HIF-1.

Mechanical Activation of Hypoxia-Inducible Factor 1α Drives Endothelial Dysfunction at Atheroprone Sites.

Atherosclerosis develops near branches and bends of arteries that are exposed to low shear stress (mechanical drag). These sites are characterized by excessive endothelial cell (EC) proliferation and inflammation that promote lesion initiation. The transcription factor HIF1α (hypoxia-inducible factor 1α) is canonically activated by hypoxia and has a role in plaque neovascularization. We studied the influence of shear stress on HIF1α activation and the contribution of this noncanonical pathway to lesion initiation. Quantitative polymerase chain reaction and en face staining revealed that HIF1α was expressed preferentially at low shear stress regions of porcine and murine arteries. Low shear stress induced HIF1α in cultured EC in the presence of atmospheric oxygen. The mechanism involves the transcription factor nuclear factor-κB that induced HIF1α transcripts and induction of the deubiquitinating enzyme Cezanne that stabilized HIF1α protein. Gene silencing revealed that HIF1α enhanced proliferation and inflammatory activation in EC exposed to low shear stress via induction of glycolysis enzymes. We validated this observation by imposing low shear stress in murine carotid arteries (partial ligation) that upregulated the expression of HIF1α, glycolysis enzymes, and inflammatory genes and enhanced EC proliferation. EC-specific genetic deletion of HIF1α in hypercholesterolemic apolipoprotein E-defecient mice reduced inflammation and endothelial proliferation in partially ligated arteries, indicating that HIF1α drives inflammation and vascular dysfunction at low shear stress regions. Mechanical low shear stress activates HIF1α at atheroprone regions of arteries via nuclear factor-κB and Cezanne. HIF1α promotes atherosclerosis initiation at these sites by inducing excessive EC proliferation and inflammation via the induction of glycolysis enzymes.

Over expression of HIF1α is associated with inactivation of both LimD1 and VHL in renal cell carcinoma: Clinical importance

It is evident that the association of LimD1 and VHL could regulate stabilization of HIF1α protein. In this study, the expression of LimD1, VHL and HIF1α was analyzed in primary renal cell carcinoma (RCC) samples to understand their association with development of the disease. For this purpose, immunohistochemical expression analysis of LimD1, VHL and HIF1α was performed along with proliferation marker PCNA in 32 primary RCC samples in different subtypes at different clinical stages of Indian patients (year: 2014–2016). Significant decrease (P<0.05) in LimD1 and VHL expression was observed in different subtypes of RCC and in early invasive lesions. High nuclear expression of HIF1α was seen in different subtypes as well as in early invasive lesions. The LimD1 and VHL expression strongly correlated with each other, while inversely correlated with HIF1α. On the other hand, increased expression of PCNA was seen in different sub types and in early invasive lesions. The PCNA expression was negatively correlated with LimD1 and VHL expression and positively correlated with HIF1α. Thus, our data indicates that the reduced expression of LimD1 and VHL might have synergistic effect on induction of HIF1α resulting increased cellular proliferation and progression of the disease.

Machine Perfusion at 20°C Prevents Ischemic Injury and Reduces Hypoxia-Inducible Factor-1α Expression During Rat Liver Preservation.

BACKGROUND Ischemic cholangitis is the main cause of liver failure after transplantation and subnormothermic machine perfusion may represent a better strategy than conventional cold storage, minimizing preservation injury. We compared livers preserved by machine perfusion at 20°C (MP20) or by cold storage at 4°C (CS4) with regard to hypoxia-inducible factor (HIF)-1α mRNA expression and protein stabilization in hypoxic conditions. MATERIAL AND METHODS Livers from male Wistar rats were stored on ice at 4°C in UW solution (CS4) or perfused with oxygenated Krebs-Henseleit buffer at 20°C (MP20) for six hours. After preservation, the livers were reperfused for two hours with oxygenated Krebs-Henseleit buffer at 37°C to simulate reimplantation. We collected bile, perfusate, and tissue samples. Transaminases, lactate dehydrogenase, bilirubin, and lactic acid were assayed in the perfusate and bile. ATP/ADP, glycogen, HIF-1α mRNA, and protein expression were measured in the tissue homogenates. RESULTS At the end of preservation, as well as after reperfusion, HIF-1α mRNA expression was significantly higher in the ischemic CS4 livers. Although the hypoxic conditions found in CS4 preservation stabilized HIF-1α protein was significantly higher in the CS4 livers at the end of preservation, no difference was observed after reperfusion, likely because of the oxygen in the reperfusion medium. After reperfusion, the MP20 livers released less transaminases and LDH. The MP20 livers had higher ATP/ADP, glycogen, and biliary bilirubin after both preservation and reperfusion when compared with the CS4 livers. CONCLUSIONS The data demonstrated that MP20 was associated with a lower HIF-1α expression and organ injury with respect to CS4, suggesting that oxygen provided by this preservation setting might approximate the organ request, thus avoiding the ischemic injury usually observed during organ preservation by cold storage.

Selective HIF-1 Regulation under Nonhypoxic Conditions by the p42/p44 MAP Kinase Inhibitor PD184161.

Hypoxia-inducible factor-1 (HIF-1) is a key gene regulator for cellular adaptation to low oxygen. In addition to hypoxia, several nonhypoxic stimuli, including hormones and growth factors, are essential for cell-specific HIF-1 regulation. Our studies have highlighted angiotensin II (AngII), a vasoactive hormone, as a potent HIF-1 activator in vascular smooth muscle cells (VSMC). AngII increases HIF-1 transcriptional activity by modulating specific signaling pathways. In VSMC, p42/p44 mitogen-activated protein kinase (MAPK) pathway activation is essential for HIF-1-mediated transcription during AngII treatment. The present study shows that PD184161, a potent MEK1/2 inhibitor, is an HIF-1 blocker in Ang II-treated VSMC. Unlike PD98059, a widely-used MEK1/2 inhibitor, we found that PD184161 blocked AngII-driven HIF-1α protein induction in a dose-dependent manner. Interestingly, the effect of PD184161 was specific to nonhypoxic activators, since HIF-1α induction by hypoxia (1% O2) was unaffected under similar conditions. VSMC treatment with MG132, a proteasome inhibitor, indicated that PD184161 influenced HIF-1α protein stability. PD184161 also increased HIF-1α binding to von Hippel-Lindau tumor suppressor protein, an E3 ligase component and an indication of HIF-1α hydroxylation. Finally, we show that PD184161 blocked mitochondrial ROS (mtROS) production and cellular ATP levels, at the same time enhancing ascorbate availability in AngII-treated VSMC. Taken together, our study indicates that, independently of p42/p44 MAPK activation, PD184161 blocks mtROS generation by AngII, leading to re-establishment of cellular ascorbate levels, increased VHL binding, and decreased HIF-1α stability. Therefore, this study reveals a previously unsuspected role for PD184161 as an HIF-1 inhibitor in VSMC under nonhypoxic conditions.

Abstract 4520: Hepatitis B virus X protein regulates hypoxia-inducible factor-1alpha (HIF-1 alpha) and lysyl oxidase like 2 (LOXL2) pathway in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC), the most common form of primary liver cancer, is a fatal malignancy and is prevalent in HBV-endemic geographical areas. Hepatitis B virus (HBV) infection is a major etiologic factor of HCC. HBV is a double-stranded DNA virus and encodes for a viral onco-protein called transactivator protein X (HBx) which interacts with the host proteins to enhance proliferative potential of the host cells. Here, using Tet-ON and Tet-OFF HBx inducible systems in HBV-negative HCC cell lines, we showed that induced expression of HBx stabilizes hypoxia-inducible factor-1α (HIF-1α) protein under hypoxia. Reversely, knockdown of HBx in HBV-positive HCC cell lines reduced HIF-1α protein stabilization under hypoxia. More intriguingly, we found that induced HBx expression up-regulated the mRNA and protein expression a known HIF-1α transcriptional target, lysyl oxidase like 2 (LOXL2) in HCC. Our previous study showed that LOXL2 cross-linked collagen in the extracellular matrix to promote HCC metastasis. Scanning electron microscopy demonstrated that knockdown of HBx in HCC cell lines markedly reduced formation of collagen fibers in vitro. Transwell invasion assay showed that knockdown of HBx reduced HCC invasive ability. Picrosirius red staining further showed that knockdown of HBx reduced collagen cross-linking in vivo and repressed HCC growth and metastasis. Taken together, our study unprecedentedly show the HBx remodels the ECM through HIF-1α/LOXL2 pathway to promote HCC metastasis. Citation Format: Cerise Yuen-Ki Chan, Aki Pui-Wah Tse, Elley Yong-Tuen Chiu, Karen Man-Fong Sze, Hui-Yu Koh, Irene Oi-Lin Ng, Carmen Chak-Lui Wong. Hepatitis B virus X protein regulates hypoxia-inducible factor-1alpha (HIF-1 alpha) and lysyl oxidase like 2 (LOXL2) pathway in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4520. doi:10.1158/1538-7445.AM2017-4520

Regulation of SIRT3 signal related metabolic reprogramming in gastric cancer by Helicobacter pylori oncoprotein CagA.

Injection of the Helicobacter pylori cytotoxin-associated gene A (CagA) is closely associated with the development of chronic gastritis and gastric cancer. Individuals infected with H. pylori possessing the CagA protein produce more reactive oxygen species (ROS) and show an increased risk of developing gastric cancer. Sirtuins (SIRTs) are nicotinamide adenine dinucleotide (NAD+)-dependent deacetylases and mitochondrial SIRT3 is known to be a tumor suppressor via its ability to suppress ROS and hypoxia inducible factor 1α (HIF-1α). However, it is unclear whether increased ROS production by H. pylori is regulated by SIRT3 followed by HIF-1α regulation and whether intracellular CagA acts as a regulator thereof. In this study, we investigated correlations among SIRT3, ROS, and HIF-1α in H. pylori-infected gastric epithelial cells. We observed that SIRT3-deficient AGS cells induce HIF-1α protein stabilization and augmented transcriptional activity under hypoxic conditions. In CagA+ H. pylori infected cells, CagA protein localized to mitochondria where it subsequently suppressed SIRT3 proteins. CagA+ H. pylori infection also increased HIF-1α activity through the ROS production induced by the downregulated SIRT3 activity, which is similar to the hypoxic condition in gastric epithelial cells. In contrast, overexpression of SIRT3 inhibited the HIF-1α protein stabilization and attenuated the increase in HIF-1α transcriptional activity under hypoxic conditions. Moreover, CagA+ H. pylori attenuated HIF-1α stability and decreased transcriptional activity in SIRT3-overexpressing gastric epithelial cells. Taken together, these findings provide valuable insights into the potential role of SIRT3 in CagA+ H. pylori-mediated gastric carcinogenesis and a possible target for cancer prevention via inhibition of HIF-1α.