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α-ketoglutarate (αKG) is a central metabolic node with a broad influence on cellular physiology. The αKG analogue N-oxalylglycine (NOG) and its membrane-permeable pro-drug derivative dimethyl-oxalylglycine (DMOG) have been extensively used as tools to study prolyl hydroxylases (PHDs) and other αKG-dependent processes. In cell culture media, DMOG is rapidly converted to MOG, which enters cells through monocarboxylate transporter MCT2, leading to intracellular NOG concentrations that are sufficiently high to inhibit glutaminolysis enzymes and cause cytotoxicity. Therefore, the degree of (D)MOG instability together with MCT2 expression levels determine the intracellular targets NOG engages with and, ultimately, its effects on cell viability. Here we designed and characterised a series of MOG analogues with the aims of improving compound stability and exploring the functional requirements for interaction with MCT2, a relatively understudied member of the SLC16 family. We report MOG analogues that maintain ability to enter cells via MCT2, and identify compounds that do not inhibit glutaminolysis or cause cytotoxicity but can still inhibit PHDs. We use these analogues to show that, under our experimental conditions, glutaminolysis-induced activation of mTORC1 can be uncoupled from PHD activity. Therefore, these new compounds can help deconvolute cellular effects that result from the polypharmacological action of NOG.

The plant shikimate pathway directs bulk carbon flow toward biosynthesis of aromatic amino acids (AAAs, i.e. tyrosine, phenylalanine, and tryptophan) and numerous aromatic phytochemicals. The microbial shikimate pathway is feedback inhibited by AAAs at the first enzyme, 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DHS). However, AAAs generally do not inhibit DHS activities from plant extracts and how plants regulate the shikimate pathway remains elusive. Here, we characterized recombinant Arabidopsis thaliana DHSs (AthDHSs) and found that tyrosine and tryptophan inhibit AthDHS2, but not AthDHS1 or AthDHS3. Mixing AthDHS2 with AthDHS1 or 3 attenuated its inhibition. The AAA and phenylpropanoid pathway intermediates chorismate and caffeate, respectively, strongly inhibited all AthDHSs, while the arogenate intermediate counteracted the AthDHS1 or 3 inhibition by chorismate. AAAs inhibited DHS activity in young seedlings, where AthDHS2 is highly expressed, but not in mature leaves, where AthDHS1 is predominantly expressed. Arabidopsis dhs1 and dhs3 knockout mutants were hypersensitive to tyrosine and tryptophan, respectively, while dhs2 was resistant to tyrosine-mediated growth inhibition. dhs1 and dhs3 also had reduced anthocyanin accumulation under high light stress. These findings reveal the highly complex regulation of the entry reaction of the plant shikimate pathway and lay the foundation for efforts to control the production of AAAs and diverse aromatic natural products in plants.

Aging impairs the IGF-I signaling of bone marrow mesenchymal stem cells (bmMSCs), but the mechanism is unclear. Here, we found that the ability to auto-phosphorylate IGF-I receptor (IGF-IR) in response to IGF-I was decreased in the bmMSCs of aged donors. Conversely, data showed that decorin (DCN) expression was prominently increased in aged bmMSCs, and that under IGF-I treatment, DCN knockdown in serum-starved aged bmMSCs potentiated their mitogenic activity and IGF-IR auto-phosphorylation, whereas DCN overexpression in serum-starved adult bmMSCs decreased both activities. Co-immunoprecipitation assays suggested that IGF-I and DCN bound to IGF-IR in a competitive manner. Online MethPrimer predicted 4 CpG islands (CGIs) in the introns of DCN gene. RT-qPCR and bisulfite sequencing showed that dimethyloxalylglycine, an inhibitor of DNA demethylation, increased DCN mRNA expression and CGI-I methylation in adult bmMSCs, whereas 5-aza-2’-deoxycytidine, a DNA methylation inhibitor, decreased DCN mRNA expression and CGI-I methylation in aged bmMSCs, and ultimately enhanced the proliferation of serum-starved aged bmMSCs under IGF-I stimulation. Thus, IGF-IR could be the prime target of aging in down-regulating the IGF-I signaling of bmMSCs, where DCN could be a critical mediator.

Cells respond to reduced oxygen availability predominately by activation of the hypoxia-inducible factor (HIF) pathway. HIF activation upregulates hundreds of genes that help cells survive in the reduced oxygen environment. The aim of this study is to determine whether chemical-induced HIF accumulation mimics all aspects of the hypoxic response of cells. We compared the effects of dimethyloxalylglycine (DMOG) (a HIF stabiliser) on PC12 cells cultured in air oxygen (20.9% O2, AO) with those cultured in either intermittent 20.9% O2 to 2% O2 (IH) or constant 2% O2 (CN). Cell viability, cell cycle, HIF accumulation, reactive oxygen species (ROS) formation, mitochondrial function and differentiation were used to characterise the PC12 cells and evaluate the impact of DMOG. IH and CN culture reduced the increase in cell numbers after 72 and 96 h and MTT activity after 48 h compared to AO culture. Further, DMOG supplementation in AO induced a dose-dependent reduction in the increase in PC12 cell numbers and MTT activity. IH-cultured PC12 cells displayed increased and sustained HIF-1 expression over 96 h. This was accompanied by increased ROS and mitochondrial burden. PC12 cells in CN displayed little changes in HIF-1 expression or ROS levels. DMOG (0.1 mM) supplementation resulted in an IH-like HIF-1 profile. The mitochondrial burden and action potential of DMOG-supplemented PC12 cells did not mirror those seen in other conditions. DMOG significantly increased S phase cell populations after 72 and 96 h. No significant effect on PC12 cell differentiation was noted with IH and CN culture without induction by nerve growth factor (NGF), while DMOG significantly increased PC12 cell differentiation with and without NGF. In conclusion, DMOG and reduced oxygen levels stabilise HIF and affect mitochondrial activity and cell behaviour. However, DMOG does not provide an accurate replication of the reduced oxygen environments.

OBJECTIVE: To study the effect of dimethyloxalylglycine (DMOG) on angiogenesis in Choke Ⅱ zone of rats cross-zone perforator flaps and its mechanism. METHODS: One hundred and twenty-six adult male Sprague Dawley rats were randomly divided into DMOG group, YC-1 group, and control group, with 42 rats in each group. Cross-zone perforator flap model with size of 12 cm×3 cm was made on the back of rats in the three groups. DMOG group was intraperitoneally injected with DMOG (40 mg/kg) at 1 day before operation, 2 hours before operation, and 1, 2, and 3 days after operation; YC-1 group and control group were intraperitoneally injected with YC-1 (10 mg/kg) and the same amount of normal saline at the same time points, respectively. The survival of flap was observed after operation. At 7 days after operation, the survival area of flap in each group was measured and the survival rate of flap was calculated. Flap transmittance test, gelatin-lead oxide angiography, and HE staining were used to observed the angiogenesis in the Choke Ⅱ zone of flaps in each group. Immunohistochemical staining and Western blot were used to detect the expressions of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor 1α (HIF-1α) in Choke Ⅱ zone of flaps in each group. The expressions of VEGF and HIF-1α were also determined by ELISA at 3, 5, and 7 days. RESULTS: At 7 days after operation, there was no obvious necrosis at the distal end of the flap in DMOG group, while necrosis occurred in both the control group and YC-1 group, mainly located at the distal end. The flap survival rate of DMOG group was 90.28%±1.37%, which was significantly higher than that of YC-1 group (84.28%±1.45%) and control group (85.83%±1.60%) (P

Objective Articular cartilage exists in a hypoxic environment, which motivates the use of hypoxia-simulating chemical agents to improve matrix production in cartilage tissue engineering. The aim of this study was to investigate whether dimethyloxalylglycine (DMOG), a HIF-1α stabilizer, would improve matrix production in 3-dimensional (3D) porcine synovial-derived mesenchymal stem cell (SYN-MSC) co-culture with chondrocytes. Design Pellet cultures and scaffold-based engineered cartilage were grown in vitro to determine the impact of chemically simulated hypoxia on 2 types of 3D cell culture. DMOG-treated groups were exposed to DMOG from day 14 to day 21 and grown up to 6 weeks with n = 3 per condition and time point. Results The addition of DMOG resulted in HIF-1α stabilization in the exterior of the engineered constructs, which resulted in increased regional type II collagen deposition, but the stabilization did not translate to overall increased extracellular matrix deposition. There was no increase in HIF-1α stabilization in the pellet cultures. DMOG treatment also negatively affected the mechanical competency of the engineered cartilage. Conclusions Despite previous studies that demonstrated the efficacy of DMOG, here, short-term treatment with DMOG did not have a uniformly positive impact on the chondrogenic capacity of SYN-MSCs in either pellet culture or in scaffold-based engineered cartilage, as evidenced by reduced matrix production. Such 3D constructs generally have a naturally occurring hypoxic center, which allows for the stabilization of HIF-1α in the interior tissue. Thus, short-term addition of DMOG may not further improve this in cartilage tissue engineered constructs.

BACKGROUND. Prolyl hydroxylase inhibitors (PHI) promote stabilization of hypoxia-inducible factor-1 alpha and affect signaling cascades of inflammation and cell death. Their beneficial use in experimental models of ulcerative colitis and lung allograft rejection led us to test the effect of the PHI dimethyl oxalyl glycine (DMOG) in the pathophysiology of graft versus host disease (GVHD). METHODS. Acute GVHD was induced in lethally irradiated BALB/c mice. DMOG was administered intraperitoneally on alternate days for the first 2-weeks posttransplant, and then twice a week till day +50, while controls received vehicle only. Animals were monitored for clinical GVHD and analyzed at day +7 and at day +50. RESULTS. DMOG treatment of allogeneic recipients improved survival by day +50, which was associated with decreased early gut injury and serum tumor necrosis factor-α compared with allogeneic controls. DMOG treatment of allogeneic recipients resulted in increased hypoxia-inducible factor-1 alpha expression and reduced apoptosis in the terminal ileum via Fas-associated protein with death domain protein repression along with decreased T-cell infiltration. Reduced pathology in colon after DMOG treatment associates with intestinal epithelium integrity and reduced damage caused by diminished recruitment of neutrophils. CONCLUSIONS. Taken together, we show protective effects of DMOG on early gut GVHD and improved survival in a model of allogeneic hematopoietic cell transplantation, providing the rationale for further evaluation of PHIs, in the prevention and treatment of acute GVHD.

Mitochondrial dysfunction leads to osteoarthritis (OA) and disc degeneration. Hypoxia inducible factor-1α (HIF-1α) mediated mitophagy has a protective role in several diseases. However, the underlying mechanism of HIF-1α mediated mitophagy in OA remains largely unknown. This current study was performed to determine the effect of HIF-1α mediated mitophagy on OA. Therefore, X-ray and tissue staining including HE staining, safranin O-fast green (S-O) and Alcian Blue were used to assess imageology and histomorphology differences of mouse knee joint. Transcriptional analysis was used to find the possible targets in osteoarthritis. Western blot analysis, RT-qPCR and immunofluorescence staining were used to detect the changes in gene and protein levels in the vitro experiment. The expression of HIF-1α was increased in human and mouse OA cartilage. HIF-1α knockdown by siRNA further impair the hypoxia-induced mitochondrial dysfunction; In contrast, HIF-1α mediated protective role was reinforced by prolylhydroxylase (PHD) inhibitor dimethyloxalylglycine (DMOG). In addition, HIF-1α stabilization could alleviate apoptosis and senescence via mitophagy in chondrocytes under hypoxia condition, which could also ameliorate surgery-induced cartilage degradation in mice OA model. In conclusion, HIF-1α mediated mitophagy could alleviate OA, which may serve as a promising strategy for OA treatment.

Small molecules including sodium butyrate (SB) and dimethyloxalylglycine (DMOG) can promote bone regeneration via inhibitive effects eliciting cellular responses through signaling cascades. The purpose of this study was to determine the synergistic effects of SB and DMOG loaded on calcium sulfate (CaS) on bone regeneration in the challenging vertical augmentation model in the rabbit calvarium. Four plastic cylinders screwed on the calvarium of each of 10 rabbits were randomly grafted with CaS, CaS/SB, CaS/DMOG, or CaS/DMOG/SB. All specimens were assessed by radiographic, histologic, and histomorphometric analyses. In the radiographic analysis, three different layers (new bone, degraded CaS, and pristine CaS layers) could be distinguished within the cylinder in all groups at 2 weeks. Newly formed bone grew up from basal bone, and CaS in contact with newly formed bone was degraded into small particles to form a different layer. At 8 weeks, most of the pristine CaS had been absorbed and hardly seen within the cylinder. In the histomorphometric analysis, all groups showed comparable new bone areas and heights at 2 and 8 weeks. The DMOG group showed a significant increase in new bone area at 8 weeks compared with 2 weeks, but there was no significant difference among the groups at 8 weeks. The DMOG group showed significantly lower values for the residual material area than the control group at 2 weeks. Within the limitations of this study, SB and DMOG seem to exert smaller synergistic effects on bone regeneration compared to CaS alone in vertical bone augmentation.

Phenylacetic acid (PAA) is one type of natural auxin and widely exists in plants. Previous biochemical studies demonstrate that PAA in plants is synthesized from phenylalanine (Phe) via phenylpyruvate (PPA), but the PAA biosynthetic genes and its regulation remain unknown. In this article, we show that the AROGENATE DEHYDRATASE (ADT) family, which catalyzes the conversion of arogenate to Phe, can modulate the levels of PAA in Arabidopsis. We found that overexpression of ADT4 or ADT5 remarkably increased the amounts of PAA. Due to an increase in PAA levels, ADT4ox and ADT5ox plants can partially restore the auxin-deficient phenotypes caused by treatments with an inhibitor of the biosynthesis of indole-3-acetic acid (IAA), a main auxin in plants. In contrast, the levels of PAA were significantly reduced in adt multiple knockout mutants. Moreover, the levels of PPA are substantially increased in ADT4 or ADT5 overexpression plants but reduced in adt multiple knockout mutants, suggesting that PPA is a key intermediate of PAA biosynthesis. These results provide an evidence that members of the ADT family of Arabidopsis can modulate PAA level via the PPA-dependent pathway.

Synaptic N-methyl-d-aspartate receptor subtype 2B(NR2B) is significantly reduced in prefrontal cortex (PFC) in the neurodevelopmental methylazoxymethanol (MAM) model of schizophrenia (SCZ). Recent research has shown that LY395756 can effectively restore NR2B levels and improve cognitive performance in juvenile MAM mice model. However, the underlying mechanisms of these beneficial effects remain unclear.Juvenile MAM mice model of SCZ is used in our study. Synaptic membrane protein levels were examined by western blotting under different treatment conditions. Interaction of cAMP-response element binding protein (CREB) and the promoter of NR2B was detected by the chromatin immunoprecipitation (ChIP) assay. Further examination of signaling pathway that mediates NR2B expression was also investigated by western blotting.In the PFC of the juvenile MAM mice schizophrenia model, CREB was found to directly bind with the promoter of NR2B. LY395756 activated the phosphorylation of AKT. Phosphorylated AKT subsequently induced the phosphorylation of CREB, and the activated CREB promoted the expression of NR2B. Subsequent experiments showed that the dephosphorylation of CREB induced by protein phosphatase 1 (PP1) can inhibit NR2B levels. Taken together, these findings support that the AKT/CREB signaling pathway is essential for the promoting effect of LY395756 on synaptic NR2B in PFC in juvenile MAM mice SCZ model.Our investigation has identified a novel mechanism by which LY395756 increases NR2B expression in juvenile MAM mice SCZ model. The AKT/CREB signaling pathway warrants further research as a potential direction for clinical treatment of SCZ.

Prolyl hydroxylase (PHD) inhibitors are being developed as alternatives to recombinant human erythropoietin (rHuEPO) for the treatment of anemia in patients with chronic kidney disease (CKD). However, the effects of PHD inhibitors and rHuEPO on blood pressure and CKD in animal models susceptible to hypertension and nephropathy have not been studied. The present study compared the effects of dimethyloxaloylglycine (DMOG), a PHD inhibitor, and rHuEPO on the development of hypertension and renal injury in Dahl salt-sensitive rats fed an 8% salt diet for 3 weeks. DMOG and rHuEPO were equally effective at raising hemoglobin levels. Systolic blood pressure rose to a greater extent in rHuEPO-treated rats (267 ± 10 vs. 226 ± 4 mm Hg) than in rats given DMOG (189 ± 8 mm Hg). Urinary protein excretion increased to 568 ± 54 versus 353 ± 25 mg/day in rats treated with rHuEPO and vehicle; however, it only rose to 207 ± 21 mg/day in rats receiving DMOG. DMOG significantly attenuated the degree of glomerulosclerosis and renal interstitial fibrosis as compared with that in vehicle and rHuEPO-treated rats. This was associated with lower renal levels of monocyte chemoattractant protein-1 and interleukin-1β and increased vascular endothelial growth factor expression in cortex and medulla. These results indicate that DMOG and rHuEPO are equally effective in increasing hemoglobin levels in Dahl S rats; however, rHuEPO aggravates hypertension and renal injury, whereas DMOG has marked renoprotective effects. These results suggest that PHD inhibitors may have a therapeutic advantage for the treatment of anemia in CKD. SIGNIFICANCE STATEMENT: Prolyl hydroxylase (PHD) inhibitors are in phase 3 clinical trials as alternatives to recombinant human erythropoietin (rHuEPO) for the treatment of anemia in chronic kidney disease (CKD). The present study reveals that dimethyloxaloylglycine (DMOG), a PHD inhibitor, and rHuEPO are equally effective in increasing hemoglobin levels in Dahl S rats; however, rHuEPO aggravated hypertension and renal injury, whereas DMOG attenuated the development of hypertension and prevented renal injury. PHD inhibitors may provide a safer therapeutic option for the treatment of anemia in CKD.

Placental hypoxia and increased levels of maternal blood anti-angiogenic protein, soluble fms-like tyrosine kinase-1 (sFLT1), are associated with the pathogenesis of pre-eclampsia. We have demonstrated that hypoxia-inducible factor (HIF)-2α mediates the upregulation of the hypoxia-induced FLT1 gene in trophoblasts and their cell lines. Here, we investigated the involvement of HIF-1β, which acts as a dimerization partner for HIF-α, in the upregulation of the FLT1 gene via hypoxia. We confirmed the interactions between HIF-1β and HIF-2α in the nuclei of BeWo, JAR and JEG-3 cells under hypoxia via co-immunoprecipitation. We found that hypoxia-induced upregulation of the FLT1 gene in BeWo cells and secretion of sFLT1 in human primary trophoblasts were significantly reduced by siRNAs targeting HIF-1β. Moreover, the upregulation of the FLT1 gene in BeWo cells induced by dimethyloxaloylglycine (DMOG) was also inhibited by silencing either HIF-2α or HIF-1β mRNA. It was recently shown that DNA demethylation increases both basal and hypoxia-induced expression levels of the FLT1 gene in three trophoblast-derived cell lines. In the demethylated BeWo cells, siRNAs targeting HIF-2α and HIF-1β suppressed the further increase in the expression levels of the FLT1 gene due to hypoxia or treatment with DMOG. However, luciferase reporter assays and bisulfite sequencing revealed that a hypoxia response element (−966 to −962) of the FLT1 gene is not involved in hypoxia or DMOG-induced upregulation of the FLT1 gene. These findings suggest that HIF-1β is essential for the elevated production of sFLT1 in the hypoxic trophoblasts and that the HIF-2α/HIF-1β complex may be a crucial therapeutic target for pre-eclampsia.

Elevation of hypoxia‐inducible factor 1 protein has been shown to be protective in acute kidney injury and HIF1α enhancing drug therapies are currently in clinical trials for the treatment of anemia of chronic kidney disease. Despite its benefits, long‐term HIF1 elevation seems to be associated with additional effects in the kidneys such as tubulointerstitial fibrosis. To better understand the effects of prolonged HIF1 exposure, assessment of baseline and post‐therapy levels of HIF1α and other related biomarkers is essential. In this study, we assessed the effect of HIF1α enhancement using prolyl hydroxylase inhibitor (PHD‐I) DMOG, on a key profibrotic marker of kidney disease. In specific, we examined the change in expression of Collagen 4 subunit A2 in cultured urinary cells of CKD patients pre and post 24‐hour exposure to 1mM DMOG. Our results show that besides HIF1α enhancement, COL4A2 protein is suppressed in presence of DMOG. To determine if this effect is mediated by HIF1, we used HIF1α gene silencing in HEK293 cells and examined the effect of DMOG on protein and gene expression of COL4A2 post 24‐hour exposure. We showed that silencing HIF1α reverses and amplifies the expression of COL4A2 in HEK293 cells. Our data suggest that HIF1 directly regulates the expression of COL4A2 in kidney cells and that HIF1α enhancing therapy has suppressive effects on COL4A2 that may be clinically relevant and must be considered in determining the safety and efficacy of these drugs in the treatment of anemia., In this report we have shown that PHD‐I treatment suppresses COL4A2 expression in CKD renal cells. The effect was shown to be transcriptionally regulated by HIF‐1 and silencing HIF‐1a gene reversed the suppression induced by PHD‐I in HEK293 cells.

The hypoxia-induced transcription factor HIF1 inhibits cell growth in normoxia through poorly understood mechanisms. A constitutive upregulation of hypoxia response is associated with increased malignancy, indicating a loss of antiproliferative effects of HIF1 in cancer cells. To understand these differences, we examined the control of cell cycle in primary human cells with activated hypoxia response in normoxia. Activated HIF1 caused a global slowdown of cell cycle progression through G1, S and G2 phases leading to the loss of mitotic cells. Cell cycle inhibition required a prolonged HIF1 activation and was not associated with upregulation of p53 or the CDK inhibitors p16, p21 or p27. Growth inhibition by HIF1 was independent of its Asn803 hydroxylation or the presence of HIF2. Antiproliferative effects of hypoxia response were alleviated by inhibition of lactate dehydrogenase and, more effectively, by boosting cellular production of NAD(+), which was decreased by HIF1 activation. In comparison to normal cells, various cancer lines showed several fold-higher expressions of NAMPT, which is a rate-limiting enzyme in the main biosynthetic pathway for NAD(+). Inhibition of NAMPT activity in overexpressor cancer cells sensitized them to antigrowth effects of HIF1. Thus, metabolic changes in cancer cells, such as enhanced NAD(+) production, create resistance to growth-inhibitory activity of HIF1 permitting manifestation of its tumor-promoting properties.Abbreviations: DMOG: dimethyloxalylglycine, DM-NOFD: dimethyl N-oxalyl-D-phenylalanine, NMN: β-nicotinamide mononucleotide

The patients with mantle cell lymphoma (MCL) have translocation t(11;14) associated with cyclin D1 overexpression. We observed that iron (an essential cofactor of dioxygenases including prolyl hydroxylases [PHDs]) depletion by deferoxamine blocked MCL cells’ proliferation, increased expression of DNA damage marker γH2AX, induced cell cycle arrest and decreased cyclin D1 level. Treatment of MCL cell lines with dimethyloxalylglycine, which blocks dioxygenases involving PHDs by competing with their substrate 2‐oxoglutarate, leads to their decreased proliferation and the decrease of cyclin D1 level. We then postulated that loss of EGLN2/PHD1 in MCL cells may lead to down‐regulation of cyclin D1 by blocking the degradation of FOXO3A, a cyclin D1 suppressor. However, the CRISPR/Cas9‐based loss‐of‐function of EGLN2/PHD1 did not affect cyclin D1 expression and the loss of FOXO3A did not restore cyclin D1 levels after iron chelation. These data suggest that expression of cyclin D1 in MCL is not controlled by ENGL2/PHD1‐FOXO3A pathway and that chelation‐ and 2‐oxoglutarate competition‐mediated down‐regulation of cyclin D1 in MCL cells is driven by yet unknown mechanism involving iron‐ and 2‐oxoglutarate‐dependent dioxygenases other than PHD1. These data support further exploration of the use of iron chelation and 2‐oxoglutarate‐dependent dioxygenase inhibitors as a novel therapy of MCL.

Hypoxia inducible factor-1α (HIF-1α) is a key transcription factor that maintains oxygen homeostasis. Hypoxic stress is related to the pathogenesis of amyotrophic lateral sclerosis (ALS), and impaired HIF-1α induces motor neuron degeneration in ALS. Dimethyloxalylglycine (DMOG) upregulates the stability of HIF-1α expression and shows neuroprotective effects, but has not been used in ALS as an anti-hypoxic stress treatment. In the present study, we investigated hypoxic stress in ALS model mice bearing G93A-human Cu/Zn superoxide dismutase by in vivo HIF-1α imaging, and treated the ALS mice with DMOG. In vivo HIF-1α imaging analysis showed enhanced hypoxic stress in both the spinal cord and muscles of lower limbs of ALS mice, even at the pre-symptomatic stage. HIF-1α expression decreased as the disease progressed until 126 days of age. DMOG treatment significantly ameliorated the decrease in HIF-1α expression, the degeneration of both spinal motor neurons and myofibers in lower limbs, gliosis and apoptosis in the spinal cord. This was accompanied by prolonged survival. The present study suggests that in vivo bioluminescence resonance energy transfer (BRET) HIF-1α imaging is useful for evaluating hypoxic stress in ALS, and that the enhancement of HIF-1α is a therapeutic target for ALS patients.

Background/aims Hypoxia Inducible Factor-1α (HIF-1α) is involved in cancer progression and is stabilized by the chaperone HSP90 (Heat Shock Protein 90), preventing degradation. Previously identified HSP90 inhibitors bind to the N-terminal pocket of HSP90, which blocks binding to HIF-1α and induces HIF-1α degradation. N-terminal inhibitors have failed in the clinic as single therapy treatments partially because they induce a heat shock response. SM molecules are HSP90 inhibitors that bind to the C-terminus of HSP90 and do not induce a heat shock response. The effects of these C-terminal inhibitors on HIF-1α are unreported. Methods HCT116, MDA-MB-231, PC3, and HEK293T cells were treated with HSP90 inhibitors. qRT-PCR and western blotting was performed to assess mRNA and protein levels of HIF-1α, HSP- and RACK1-related genes. siRNA was used to knockdown RACK1, while MG262 was used to inhibit proteasome activity. Dimethyloxalylglycine (DMOG) was used to inhibit activity of the prolyl hydroxylases (PHDs). Anti-angiogenic activity of HSP90 inhibitors was assessed using a HUVEC tubule formation assay. Results We show that SM compounds decrease HIF-1α target expression at the mRNA and protein level under hypoxia in colorectal, breast and prostate cancer cells, leading to cell death, without inducing a heat shock response. Surprisingly, we found that when the C-terminal of HSP90 is inhibited, HIF-1α degradation occurs through the proteasome and prolyl hydroxylases in an oxygen-dependent manner even in very low levels of oxygen (tumor hypoxia levels). RACK1 was not required for proteasomal degradation of HIF-1α. Conclusion Our results suggest that by targeting the C-terminus of HSP90 we can exploit the prolyl hydroxylase and proteasome pathway to induce HIF-1α degradation in hypoxic tumors.

The hypoxia-inducible factor (HIF) pathway, regulated by prolyl hydroxylase, is central to tissue adaptation to ischemia. The authors tested whether the prolyl hydroxylase inhibitor dimethyloxalylglycine reduces skin flap necrosis.Dorsal skin flaps were raised on hairless rats, with dimethyloxalylglycine delivered intraperitoneally and/or topically for 7 days before and after surgery. After 14 treatment days, percentage of flap necrosis was compared grossly and tissue perfusion compared with an in vivo imaging system. Angiogenesis was compared using immunohistochemical CD31 staining and enzyme-linked immunosorbent assay for tissue vascular endothelial growth factor. Expression levels of HIF-1α and terminal deoxynucleotidyl transferase-mediated dUDP end-labeling were compared using immunohistochemical staining. Complete blood counts and gross necropsy specimens were obtained to assess systemic toxicity.Dimethyloxalylglycine administration significantly improved postoperative flap viability, with combined topical and intraperitoneal dimethyloxalylglycine administration leading to reduced necrosis on postsurgical day 7 at 6 mg/kg/day, 12 mg/kg/day, 24 mg/kg/day, and 48 mg/kg/day versus controls (all p0.05). Compared with controls (unperfused, 39.9 ± 3.8 percent), dimethyloxalylglycine treatment led to a dose-dependent decrease in unperfused tissue at 6 mg/kg/day (11.4 ± 1.7 percent), 12 mg/kg/day (9.4 ± 4.2 percent), 24 mg/kg/day (4.7 ± 2.6 percent), and 48 mg/kg/day (1.4 ± 0.9 percent) (all p0.001). Topical dimethyloxalylglycine application alone administered at 48 mg/kg/day was sufficient to improve flap viability (p = 0.005). Dimethyloxalylglycine-treated flaps exhibited higher CD31 staining (p = 0.004), tissue vascular endothelial growth factor (p = 0.007), HIF-1α staining (p0.001), and reduced terminal deoxynucleotidyl transferase-mediated dUDP end-labeling staining (p = 0.045). There were no differences in hematocrit or macroscopic organ changes on gross necropsy.Topical and systemic targeting of the HIF-1 pathway may be a promising therapeutic approach to improve flap resistance to ischemia.

Oxygen sensing revisited The cellular response to hypoxia (oxygen deficiency) is a contributing factor in many human diseases. Previous studies examining the way in which hypoxia alters gene expression have focused on oxygen-sensing enzymes that regulate the activity of a transcription factor called hypoxia-inducible factor (see the Perspective by Gallipoli and Huntly). Chakraborty et al. and Batie et al. now show that hypoxia can also affect gene expression through direct effects on chromatin regulators. Certain histone demethylases, such as KDM6A and KDM5A, were found to be direct sensors of oxygen. In cell-culture models, hypoxia diminished the activity of these enzymes and caused changes in the expression of genes that govern cell fate. Science , this issue p. 1217 , p. 1222 ; see also p. 1148

The integrity of the intestinal barrier is critical for protecting the host against the pathogen. The role of hypoxia-inducible factor-1α (HIF-1α) in the intestinal barrier disfunction related to sepsis remained unclear. The purpose of the present study is to investigate the role of HIF-1α on oxidative damage, the intestinal mucosal permeability, structural and morphological changes during sepsis. Twenty-four Sprague Dawley (SD) rats were randomly divided into four groups of 6 rats each: the sham group (sham), sepsis group (subjected to cecal ligation and perforation, CLP), sepsis + DMOG group (40 mg/kg of DMOG by intraperitoneal injection for 7 consecutive days before CLP), and sepsis + BAY 87–2243 group (9 mg/kg of BAY 87–2243 orally administered for 3 consecutive days before CLP). Sepsis increased plasma levels of inflammatory mediators, oxidative stress markers and HIF-1α expression; caused pathological damage; increased permeability (P < 0.05); and decreased TJ protein expression in the intestinal mucosa of rats with sepsis (P < 0.05). The addition of DMOG up-regulated HIF-1α, then decreased the plasma levels of inflammatory mediators, oxidative stress markers, alleviated pathological damage to the intestinal mucosa and decreased intestinal permeability (P < 0.05); while BAY 87–2243 treatment had the opposite effects. Our findings showed that HIF-1α protects the intestinal barrier function of septic rats by inhibiting intestinal inflammation and oxidative damage, our results provide a novel insight for developing sepsis treatment.

Background Diastolic dysfunction is emerging as a leading cause of heart failure in aging population. Induction of hypoxia tolerance and reprogrammed cell metabolism have emerged as novel therapeutic strategies for the treatment of cardiovascular diseases. Methods and results In the present study, we showed that deletion of sirtuin 3 (SIRT3) resulted in a diastolic dysfunction together with a significant increase in the expression of prolyl hydroxylases (PHD) 1 and 2. We further investigated the involvement of PHD in the development of diastolic dysfunction by treating the 12–14 months old mice with a PHD inhibitor, dimethyloxalylglycine (DMOG) for 2 weeks. DMOG treatment increased the expression of hypoxia-inducible factor (HIF)-1α in the endothelium of coronary arteries. This was accompanied by a significant improvement of coronary flow reserve and diastolic function. Inhibition of PHD altered endothelial metabolism by increasing glycolysis and reducing oxygen consumption. Most importantly, treatment with DMOG completely reversed the pre-existing diastolic dysfunction in the endothelial-specific SIRT3 deficient mice. Conclusions Our findings demonstrate that inhibition of PHD and reprogrammed cell metabolism can reverse the pre-existed diastolic dysfunction in SIRT3 deficient mice. Our study provides a potential therapeutic strategy of induction of hypoxia tolerance for patients with diastolic dysfunction associated with coronary microvascular dysfunction, especially in the aging population with reduced SIRT3.

Anastomotic leakage (AL) accounts for a major part of in-house mortality in patients undergoing colorectal surgery. Local ischemia and abdominal sepsis are common risk factors contributing to AL and are characterized by upregulation of the hypoxia-inducible factor (HIF) pathway. The HIF pathway is critically regulated by HIF-prolyl hydroxylases (PHDs). Here, we investigated the significance of PHDs and the effects of pharmacologic PHD inhibition (PHI) during anastomotic healing. Ischemic or septic colonic anastomoses were created in mice by ligation of mesenteric vessels or lipopolysaccharide-induced abdominal sepsis, respectively. Genetic PHD-deficiency (Phd1-/-, Phd2+/-, and Phd3-/-) or PHI were applied to manipulate PHD activity. Pharmacologic PHI and genetic PHD2-haplodeficiency (Phd2+/-) significantly improved healing of ischemic or septic colonic anastomoses, as indicated by increased bursting pressure and reduced AL rates. Only Phd2+/- (but not PHI or Phd1-/-) protected from sepsis-related mortality. Mechanistically, PHI and Phd2+/- induced immuno-modulatory (M2) polarization of macrophages, resulting in increased collagen content and attenuated inflammation-driven immune cell recruitment. We conclude that PHI improves healing of colonic anastomoses in ischemic or septic conditions by Phd2+/--mediated M2 polarization of macrophages, conferring a favourable microenvironment for anastomotic healing. Patients with critically perfused colorectal anastomosis or abdominal sepsis could benefit from pharmacologic PHI.

Liver fibrosis is characterized by the accumulation of extracellular matrix proteins, mainly composed of collagen. Hepatic stellate cells (HSCs) mediate liver fibrosis by secreting collagen. Vitamin C (ascorbic acid) is a cofactor of prolyl-hydroxylases that modify newly synthesized collagen on the route for secretion. Unlike most animals, humans cannot synthesize ascorbic acid and its role in liver fibrosis remains unclear. Here, we determined the effect of ascorbic acid and prolyl-hydroxylase inhibition on collagen production and secretion by human HSCs. Primary human HSCs (p-hHSCs) and the human HSCscell line LX-2 were treated with ascorbic acid, transforming growth factor-beta (TGFβ) and/or the pan-hydroxylase inhibitor dimethyloxalylglycine (DMOG). Expression of collagen-I was analyzed by RT-qPCR (COL1A1), Western blotting, and immunofluorescence microscopy. Collagen secretion was determined in the medium by Western blotting for collagen-I and by HPLC for hydroxyproline concentrations. Expression of solute carrier family 23 members 1 and 2 (SLC23A1/SLC23A2), encoding sodium-dependent vitamin C transporters 1 and 2 (SVCT1/SVCT2) was quantified in healthy and cirrhotic human tissue. In the absence of ascorbic acid, collagen-I accumulated intracellularly in p-hHSCs and LX-2 cells, which was potentiated by TGFβ. Ascorbic acid co-treatment strongly promoted collagen-I excretion and enhanced extracellular hydroxyproline concentrations, without affecting collagen-I (COL1A1) mRNA levels. DMOG inhibited collagen-I release even in the presence of ascorbic acid and suppressed COL1A1 and alpha-smooth muscle actin (αSMA/ACTA2) mRNA levels, also under hypoxic conditions. Hepatocytes express both ascorbic acid transporters, while p-hHSCs and LX-2 express the only SVCT2, which is selectively enhanced in cirrhotic livers. Human HSCs rely on ascorbic acid for the efficient secretion of collagen-I, which can be effectively blocked by hydroxylase antagonists, revealing new therapeutic targets to treat liver fibrosis.

We developed a versatile stereoselective route for the synthesis of new 2′-(S)-CCG-IV analogues. The route allows for late stage diversification and thereby provides access to a great variety of conformationally restricted cyclopropyl glutamate analogues. A selection of the 2′-(S)-CCG-IV analogues were evaluated using two-electrode voltage-clamp electrophysiology at recombinant GluN1/GluN2A-D receptors, demonstrating that agonists can be developed with GluN2 subunit-dependent potency and agonist efficacy. We also describe a crystal structure of the GluN2A agonist binding domain in complex with 2′-butyl-(S)-CCG-IV that determines the position of 2′-substituents in (S)-CCG-IV agonists in the glutamate binding site and provides further insight to the structural determinants of their agonist efficacy. The stereoselective synthesis described here enables versatile and straight-forward modifications to diverse analogues of interest for the development of potent subtype-specific NMDA receptor agonists and other applications.

Therapeutic strategies against HBV focus, among others, on the activation of the immune system to enable the infected host to eliminate HBV. Hypoxia-inducible factor 1 alpha (HIF1α) stabilization has been associated with impaired immune responses. HBV pathogenesis triggers chronic hepatitis-related scaring, leading inter alia to modulation of liver oxygenation and transient immune activation, both factors playing a role in HIF1α stabilization.We addressed whether HIF1α interferes with immune-mediated induction of the cytidine deaminase, apolipoprotein B mRNA editing enzyme catalytic subunit 3B (APOBEC3B; A3B), and subsequent covalently closed circular DNA (cccDNA) decay. Liver biopsies of chronic HBV (CHB) patients were analyzed by immunohistochemistry and in situ hybridization. The effect of HIF1α induction/stabilization on differentiated HepaRG or mice ± HBV ± LTβR-agonist (BS1) was assessed in vitro and in vivo. Induction of A3B and subsequent effects were analyzed by RT-qPCR, immunoblotting, chromatin immunoprecipitation, immunocytochemistry, and mass spectrometry. Analyzing CHB highlighted that areas with high HIF1α levels and low A3B expression correlated with high HBcAg, potentially representing a reservoir for HBV survival in immune-active patients. In vitro, HIF1α stabilization strongly impaired A3B expression and anti-HBV effect. Interestingly, HIF1α knockdown was sufficient to rescue the inhibition of A3B up-regulation and -mediated antiviral effects, whereas HIF2α knockdown had no effect. HIF1α stabilization decreased the level of v-rel reticuloendotheliosis viral oncogene homolog B protein, but not its mRNA, which was confirmed in vivo. Noteworthy, this function of HIF1α was independent of its partner, aryl hydrocarbon receptor nuclear translocator.In conclusion, inhibiting HIF1α expression or stabilization represents an anti-HBV strategy in the context of immune-mediated A3B induction. High HIF1α, mediated by hypoxia or inflammation, offers a reservoir for HBV survival in vivo and should be considered as a restricting factor in the development of immune therapies.

Rationale: Antenatal inflammation with placental dysfunction is strongly associated with high bronchopulmonary dysplasia (BPD) risk in preterm infants. Whether antenatal or postnatal HIF (hypoxia-inducible factor) augmentation can preserve lung structure and function and prevent pulmonary hypertension after intrauterine inflammation is controversial.Objectives: To determine whether antenatal or postnatal prolyl-hydroxylase inhibitor (PHi) therapy increases lung HIF expression, preserves lung growth and function, and prevents pulmonary hypertension in a rat model of chorioamnionitis-induced BPD caused by antenatal inflammation.Methods: Endotoxin (ETX) was administered to pregnant rats by intraamniotic injection at Embryonic Day 20, and pups were delivered by cesarean section at Embryonic Day 22. Selective PHi drugs, dimethyloxalylglycine or GSK360A, were administered into the amniotic space at Embryonic Day 20 or after birth by intraperitoneal injection for 2 weeks. Placentas and lung tissue were collected at birth for morphometric and Western blot measurements of HIF-1a, HIF-2a, VEGF (vascular endothelial growth factor), and eNOS (endothelial nitric oxide synthase) protein contents. At Day 14, lung function was assessed, and tissues were harvested to determine alveolarization by radial alveolar counts, pulmonary vessel density, and right ventricle hypertrophy (RVH).Measurements and Main Results: Antenatal PHi therapy preserves lung alveolar and vascular growth and lung function and prevents RVH after intrauterine ETX exposure. Antenatal administration of PHi markedly upregulates lung HIF-1a, HIF-2a, VEGF, and eNOS expression after ETX exposure.Conclusions: HIF augmentation improves lung structure and function, prevents RVH, and improves placental structure following antenatal ETX exposure. We speculate that antenatal or postnatal PHi therapy may provide novel strategies to prevent BPD due to antenatal inflammation.

Aspergillus fumigatus is one of the most common fungal infections involved in the pulmonary diseases. Hypoxia-inducible factor-1α (HIF-1α) is important for antifungal immunity. Diabetes is a risk factor of pulmonary A. fumigatus infection and could affect the expression of HIF-1α. The aim of this investigation was to evaluate the role of HIF-1α in pulmonary A. fumigatus infection in diabetes. In murine model, we found diabetic mice had aggravated pulmonary A. fumigatus infection and declined expression of HIF-1α following pulmonary A. fumigatus infection. And these changes could be corrected by dimethyloxalylglycine (DMOG), the agonist of HIF-1α. In cell experiment, after A. fumigatus stimulation, hyperglycemic state was with a decreased HIF-1α expression and increased NLRP3/IL-1β signal pathway. The percentages of Th1 and Treg cells decreased, while percentages of Th2 and Th17 increased in hyperglycemic group. DMOG suppressed A. fumigatus-stimulated NLRP3 and IL-1β expressions in hyperglycemic group and corrected Th and Treg cells differentiation. These regulatory effects of DMOG could be dampened by activating of NLRP3. These data indicated that hyperglycemia suppressed the regulatory effect of HIF-1α in pulmonary A. fumigatus infection, which can affect Th and Treg cells differentiation by regulating the NLRP3/IL-1β signal pathway.

This study compared effects of five hypoxia-inducible factor (HIF) prolyl hydroxylases (PHD) inhibitors on PC12 cells and primary rat neurons following oxygen-glucose deprivation (OGD). At 100 µM, the PHD inhibitors did not cause cytotoxicity and apoptosis. MTT activity was only significantly reduced by FG4592 or Bayer 85–3934 in PC12 cells. The PHD inhibitors at 100 µM significantly increased the LC3-II/LC3-I expression ratio and downregulated p62 in PC12 cells, so did FG4592 (30 µM) and DMOG (100 µM) in neurons. HIF-1α was stabilised in PC12 cells by all the PHD inhibitors at 100 µM except for DMOG, which stabilised HIF-1α at 1 and 2 mM. In primary neurons, HIF-1α was stabilised by FG4592 (30 µM) and DMOG (100 µM). Pretreatment with the PHD inhibitors 24 hours followed by 24 hour reoxygenation prior to 6 hours OGD (0.3% O2) significantly reduced LDH release and increased MTT activity compared to vehicle (1% DMSO) pretreatment. In conclusion, the PHD inhibitors stabilise HIF-1α in normoxia, induce autophagy, and protect cells from a subsequent OGD insult. The new class of PHD inhibitors (FG4592, FG2216, GSK1278863, Bay85-3934) have the higher potency than DMOG. The interplay between autophagy, HIF stabilisation and neuroprotection in ischaemic stroke merits further investigation.

α-Ketoglutarate (αKG) is a key node in many important metabolic pathways. The αKG analog N-oxalylglycine (NOG) and its cell-permeable prodrug dimethyloxalylglycine (DMOG) are extensively used to inhibit αKG-dependent dioxygenases. However, whether NOG interference with other αKG-dependent processes contributes to its mode of action remains poorly understood. Here we show that, in aqueous solutions, DMOG is rapidly hydrolyzed, yielding methyloxalylglycine (MOG). MOG elicits cytotoxicity in a manner that depends on its transport by monocarboxylate transporter 2 (MCT2) and is associated with decreased glutamine-derived tricarboxylic acid–cycle flux, suppressed mitochondrial respiration and decreased ATP production. MCT2-facilitated entry of MOG into cells leads to sufficiently high concentrations of NOG to inhibit multiple enzymes in glutamine metabolism, including glutamate dehydrogenase. These findings reveal that MCT2 dictates the mode of action of NOG by determining its intracellular concentration and have important implications for the use of (D)MOG in studying αKG-dependent signaling and metabolism. High intracellular concentrations of the α-ketoglutarate analog N-oxalylglycine, owing to MCT2-mediated transport of its newly described prodrug MOG, inhibit multiple enzymes in glutamine metabolism and selectively kill MCT2-expressing cancer cells.

The cystine transporter, system xIn vitro tracer uptake experiments and Western blotting were performed in breast cancer (MDA-MB-231), glioblastoma (U-87), and prostate (PC-3) cancer cell lines. PET imaging and biodistribution studies were conducted in xenograft-bearing immunocompromised Rag2M female mice.All three tracer conformations allowed for the visualization of tumor xenografts at 1 h (for U-87 and PC-3 tumors) or 2 h (in the case of MDA-MB-231 xenografts) post-injection, with the racemate (2S,5R/S-) displaying similar image contrast as compared to the 5- position diastereomers and the 2S,5S-[Our data suggest that pure 2S-[

Background & aims Leucine-rich repeat G-protein-coupled receptor 4 (LGR4) and its ligands R-spondin1-4 (Rspos) have been vastly investigated in embryonic development. The biological functions of Rspos-LGR4 system in liver remains largely unknown. Here, we explored whether it protects hepatocytes against hypoxia/reoxygenation (H/R) induced damage. Methods H/R injury was induced by dimethyloxalylglycine (DMOG) in AML12 cells and the effects of Rspo3 on cell proliferation and apoptosis were assessed. Specific shRNAs were used to interfere LGR4 or β-catenin. Results DMOG caused hepatocytes damage evidenced by increase in HIF-1α, cell death and apoptosis genes p27 and Bax, with concurrent decrease of cell proliferation genes PCNA and CyclinD1. Of all the Rspos, Rspo3 is predominantly expressed in AML12 hepatocytes. Importantly, Rspo3 demonstrated an alteration in a manner similar to proliferation-related genes during H/R injury. Rspo3 pretreatment rendered hepatocytes less vulnerable to DMOG induced H/R injury. Ablation of LGR4 using shRNA attenuated the protective effects of Rspo3. Wnt3a also protected AML12 cells from damages caused by H/R, showing enhanced proliferation activity. Notably, knockdown of β-catenin in hepatocytes completely abolished the effect of Rspo3 pretreatment on the expression levels of PCNA and CyclinD1. Conclusion Rspo3-LGR4 axis protects hepatocytes from H/R injury via activating β-catenin.

Studies on human intestinal injury induced by acute exposure to γ-radiation commonly rely on use of animal models because culture systems do not faithfully mimic human intestinal physiology. Here we used a human Gut-on-a-Chip (Gut Chip) microfluidic device lined by human intestinal epithelial cells and vascular endothelial cells to model radiation injury and assess the efficacy of radiation countermeasure drugs in vitro. Exposure of the Gut Chip to γ-radiation resulted in increased generation of reactive oxygen species, cytotoxicity, apoptosis, and DNA fragmentation, as well as villus blunting, disruption of tight junctions, and compromise of intestinal barrier integrity. In contrast, pre-treatment with a potential prophylactic radiation countermeasure drug, dimethyloxaloylglycine (DMOG), significantly suppressed all of these injury responses. Thus, the human Gut Chip may serve as an in vitro platform for studying radiation-induced cell death and associate gastrointestinal acute syndrome, in addition to screening of novel radio-protective medical countermeasure drugs.

Retinitis pigmentosa (RP) is a group of inherited retinal dystrophies characterized by progressive and irreversible loss of vision due to rod and cone degeneration. Evidence suggests that an inappropriate oxygen level could contribute to its pathogenesis. Rod cell death could increase oxygen concentration, reduce hypoxia-inducible factor 1 (HIF-1α) and contribute to cone cell death. The purposes of this study were: 1) to analyze the temporal profile of HIF-1α, its downstream effectors VEGF, endothelin-1 (ET-1), iNOS, and glucose transporter 1 (GLUT1), and neuroinflammation in retinas of the murine model of rd10 ( retinal degeneration 10) mice with RP; 2) to study oxygen bioavailability in these retinas; and 3) to investigate how stabilizing HIF-1α proteins with dimethyloxaloglycine (DMOG), a prolyl hydroxylase inhibitor, affects retinal degeneration, neuroinflammation, and antioxidant response in rd10 mice. A generalized down-regulation of HIF-1α and its downstream targets was detected in parallel with reactive gliosis, suggesting high oxygen levels during retinal degeneration. At postnatal d 18, DMOG treatment reduced photoreceptor cell death and glial activation. In summary, retinas of rd10 mice seem to be exposed to a hyperoxic environment even at early stages of degeneration. HIF-1α stabilization could have a temporal neuroprotective effect on photoreceptor cell survival, glial activation, and antioxidant response at early stages of RP.-Olivares-González, L., Martínez-Fernández de la Cámara, C., Hervás, D., Millán, J. M., Rodrigo, R. HIF-1α stabilization reduces retinal degeneration in a mouse model of retinitis pigmentosa.

Hypoxia after traumatic injuries to a tooth is one of the causes of subsequent root resorption. Inflammatory cytokines produced under hypoxic conditions are associated with root resorption, but the mechanism has not been fully understood. In this study, the role of hypoxia-inducible factor-1 (HIF-1) signaling in the regulation of CCAAT (cytosine-cytosine-adenosine-adenosine-thymidine)/enhancer-binding protein-β (C/EBPβ) and the receptor activator of nuclear factor kappa-B ligand (RANKL) expressions in immortalized human periodontal ligament (PDL) cells was investigated. PDL cells cultured under a hypoxic condition showed an increase in the expression of C/EBPβ and RANKL messenger RNAs (mRNAs), whereas the expression of osteoprotegerin and HIF-1α mRNAs was unaffected. Hypoxia had no effects on the secretion of interleukin (IL)-1β, IL-6, IL-8, IL-17A, tumor necrosis factor-alpha, macrophage migration inhibitory factor, monocyte chemoattractant protein-1, and macrophage colony-stimulating factor in the culture media. Treatment of the cells with dimethyloxaloylglycine, a competitive HIF prolyl hydroxylase inhibitor, significantly increased the expression of C/EBPβ and RANKL mRNAs. This suggested that the hypoxia-induced elevation of C/EBPβ and RANKL mRNAs was dependent on the HIF-1 activity. PDL cells transfected with a specific small interfering RNA designed to target the C/EBPβ gene showed a significant suppression of the RANKL mRNA. These findings indicated that C/EBPβ may play an important role in tooth root resorption via RANKL activation in hypoxia-exposed PDL cells.

Graded levels of molecular oxygen (O2) exist within developing mammalian embryos and can differentially regulate cellular specification pathways. During differentiation, cells acquire distinct epigenetic landscapes, which determine their function, however the mechanisms which regulate this are poorly understood. The demethylation of 5-methylcytosine (5mC) is achieved via successive oxidation reactions catalysed by the Ten-Eleven-Translocation (Tet) enzymes, yielding the 5-hydroxymethylcytosine (5hmC) intermediate. These require O2 as a co-factor, and hence may link epigenetic processes directly to O2 gradients during development. We demonstrate that the activities of Tet enzymes display distinct patterns of [O2]-dependency, and that Tet1 activity, specifically, is subject to differential regulation within a range of O2 which is physiologically relevant in embryogenesis. Further, differentiating embryonic stem cells displayed a transient burst of 5hmC, which was both dependent upon Tet1 and inhibited by low (1%) [O2]. A GC-rich promoter region within the Tet3 locus was identified as a significant target of this 5mC-hydroxylation. Further, this region was shown to associate with Tet1, and display the histone epigenetic marks, H3K4me3 and H3K27me3, which are characteristic of a bivalent, developmentally ‘poised’ promoter. We conclude that Tet1 activity, determined by [O2] may play a critical role in regulating cellular differentiation and fate in embryogenesis.

The cerebellum undergoes rapid growth during the third trimester and is vulnerable to injury and deficient growth in infants born prematurely. Factors associated with preterm cerebellar hypoplasia include chronic lung disease and postnatal glucocorticoid administration. We modeled chronic hypoxemia and glucocorticoid administration in neonatal mice to study whole cerebellar and cell type-specific effects of dual exposure. Chronic neonatal hypoxia resulted in permanent cerebellar hypoplasia. This was compounded by administration of prednisolone as shown by greater volume loss and Purkinje cell death. In the setting of hypoxia and prednisolone, administration of a small molecule Smoothened-Hedgehog agonist (SAG) preserved cerebellar volume and protected against Purkinje cell death. Such protective effects were observed even when SAG was given as a one-time dose after dual insult. To model complex injury and determine cell type-specific roles for the hypoxia inducible factor (HIF) pathway, we performed conditional knockout of von Hippel Lindau (VHL) to hyperactivate HIF1α in cerebellar granule neuron precursors (CGNP) or Purkinje cells. Surprisingly, HIF activation in either cell type resulted in no cerebellar deficit. However, in mice administered prednisolone, HIF overactivation in CGNPs resulted in significant cerebellar hypoplasia, whereas HIF overactivation in Purkinje cells caused cell death. Together, these findings indicate that HIF primes both cell types for injury via glucocorticoids, and that hypoxia/HIF + postnatal glucocorticoid administration act on distinct cellular pathways to cause cerebellar injury. They further suggest that SAG is neuroprotective in the setting of complex neonatal cerebellar injury. Electronic supplementary material The online version of this article (10.1007/s12311-017-0895-0) contains supplementary material, which is available to authorized users.

Periodontitis is initiated by serious and sustained bacterial infection and ultimately results in chronic immune-mediated inflammation, tissue destruction, and bone loss. The pathogenesis of periodontitis remains unclear. Host immunological responses to periodontal bacteria ultimately determine the severity and mechanisms governing periodontitis progression. This study aimed to clarify the effect of the hypoxia-inducible factor-1α (HIF-1α) activator dimethyloxalylglycine (DMOG) on a mouse periodontitis model and its underlying role in macrophage polarization. qRT-PCR analysis showed that DMOG inhibited the M1-like polarization of both RAW264.7 macrophages and murine bone marrow macrophages (BMMs) and downregulated TNF-α, IL-6, CD86, and MCP-1 expression in vitro. Immunofluorescence staining and flow cytometry also confirmed the less percentage of F4/80 + CD86 + cells after DMOG treatment. The phosphorylation of NF-κB pathway was also inhibited by DMOG with higher level of HIF-1α expression. Furthermore, mice treated with DMOG showed decreased alveolar bone resorption in the experimental periodontitis model, with significant increases in alveolar bone volume/tissue volume (BV/TV) and bone mineral density (BMD). DMOG treatment of mice decreased the ratio of M1/M2 (CD86+/CD206+) macrophages in periodontal tissues, resulting in the downregulation of proinflammatory cytokines such as TNF-α and IL-6 and increased levels of anti-inflammatory factors such as IL-4 and IL-10. DMOG treatment promoted the number of HIF-1α-positive cells in periodontal tissues. This study demonstrated the cell-specific roles of DMOG in macrophage polarization in vitro and provided insight into the mechanism underlying the protective effect of DMOG in a model of periodontitis.

BackgroundNecrotizing enterocolitis (NEC) is a devastating neonatal disease characterized by intestinal necrosis. Hypoxia-inducible factor-1α (HIF-1α) has a critical role in cellular oxygen homeostasis. Here, we hypothesized that prolyl hydroxylase (PHD) inhibition, which stabilizes HIF-1α, protects against NEC by promoting intestinal endothelial cell proliferation and improving intestinal microvascular integrity via vascular endothelial growth factor (VEGF) signaling.MethodsTo assess the role of PHD inhibition in a neonatal mouse NEC model, we administered dimethyloxalylglycine (DMOG) or vehicle to pups before or during the NEC protocol, and determined mortality and incidence of severe intestinal injury. We assessed intestinal VEGF by western blot analysis and quantified endothelial cell and epithelial cell proliferation following immunofluorescence.ResultsDMOG decreased mortality and incidence of severe NEC, increased intestinal VEGF expression, and increased intestinal villus endothelial and epithelial cell proliferation in experimental NEC. Inhibiting VEGFR2 signaling eliminated DMOG's protective effect on intestinal injury severity, survival, and endothelial cell proliferation while sparing DMOG's protective effect on intestinal epithelial cell proliferation.ConclusionDMOG upregulates intestinal VEGF, promotes endothelial cell proliferation, and protects against intestinal injury and mortality in experimental NEC in a VEGFR2 dependent manner. DMOG's protective effect on the neonatal intestinal mucosa may be mediated via VEGFR2 dependent improvement of the intestinal microvasculature.

Exosomes are nano-sized vesicles produced and secreted by cells to mediate intercellular communication. The production and function of exosomes in kidney tissues and cells remain largely unclear. Hypoxia is a common pathophysiological condition in kidneys. This study was designed to characterize exosome production during hypoxia of rat renal proximal tubular cells (RPTCs), investigate the regulation by hypoxia-inducible factor-1 (HIF-1), and determine the effect of the exosomes on ATP-depletion-induced tubular cell injury. Hypoxia did not change the average sizes of exosomes secreted by RPTCs, but it significantly increased exosome production in a time-dependent manner. HIF-1 induction with dimethyloxalylglycine also promoted exosome secretion, whereas pharmacological and genetic suppression of HIF-1 abrogated the increase of exosome secretion under hypoxia. The exosomes from hypoxic RPTCs had inhibitory effects on apoptosis of RPTCs following ATP depletion. The protective effects were lost in the exosomes from HIF-1α knockdown cells. It is concluded that hypoxia stimulates exosome production and secretion in renal tubular cells. The exosomes from hypoxic cells are protective against renal tubular cell injury. HIF-1 mediates exosome production during hypoxia and contributes to the cytoprotective effect of the exosomes.

SDF-1/CXCR4 activation facilitates myocardial repair. Therefore, we aimed to activate the HIF-1α target genes SDF-1 and CXCR4 by dimethyloxalylglycine (DMOG)-induced prolyl-hydroxylase (PH) inhibition to augment CXCR4+ cell recruitment and myocardial repair. SDF-1 and CXCR4 expression was analyzed under normoxia and ischemia ± DMOG utilizing SDF-1-EGFP and CXCR4-EGFP reporter mice. In bone marrow and heart, CXCR4-EGFP was predominantly expressed in CD45+/CD11b+ leukocytes which significantly increased after myocardial ischemia. PH inhibition with 500 μM DMOG induced upregulation of SDF-1 mRNA in human microvascular endothelial cells (HMEC-1) and aortic vascular smooth muscle cells (HAVSMC). CXCR4 was highly elevated in HMEC-1 but almost no detectable in HAVSMC. In vivo, systemic administration of the PH inhibitor DMOG without pretreatment upregulated nuclear HIF-1α and SDF-1 in the ischemic mouse heart associated with increased recruitment of CD45+/CXCR4-EGFP+/CD11b+ cell subsets. Enhanced PH inhibition significantly upregulated reparative M2 like CXCR4-EGFP+ CD11b+/CD206+ cells compared to inflammatory M2-like CXCR4-EGFP+ CD11b+/CD86+ cells associated with reduced apoptotic cell death, increased neovascularization, reduced scar size, and an improved heart function after MI. In summary, our data suggest increased PH inhibition as a promising tool for a customized upregulation of SDF-1 and CXCR4 expression to attract CXCR4+/CD11b+ cells to the ischemic heart associated with increased cardiac repair. Key messages DMOG-induced prolyl-hydroxylase inhibition upregulates SDF-1 and CXCR4 in human endothelial cells.Systemic application of DMOG upregulates nuclear HIF-1α and SDF-1 in vivo.Enhanced prolyl-hydroxylase inhibition increases mainly CXCR4+/CD11b+ cells.DMOG increased reparative M2-like CD11b+/CD206+ cells compared to M1-like cells after MI.Enhanced prolyl-hydroxylase inhibition improved cardiac repair and heart function. Electronic supplementary material The online version of this article (doi:10.1007/s00109-017-1543-3) contains supplementary material, which is available to authorized users.

Prolyl hydroxylase inhibitors induce a proangiogenic response and are therefore proposed to optimize regenerative approaches in periodontics and oral surgery. Here the effect of the prolyl hydroxylase inhibitors dimethyloxalylglycine and deferoxamine, released from collagen barrier membranes, on osteoclastogenesis and osteoblastogenesis was evaluated. Collagen barrier membranes were loaded with dimethyloxalylglycine and deferoxamine. Release studies were performed and supernatants were taken after 1, 3, 6, 24, and 48 h. The effect of these supernatants on osteoblast- and osteoclast-precursor cells was evaluated. Furthermore, dose response studies for dimethyloxalylglycine and deferoxamine were performed. Osteoclastogenesis was evaluated with RAW 264.7 cells based on the number of multinuclear tartrate-resistant acid phosphatase positive cells. Osteoblastogenesis was evaluated with MC3T3-E1 cells based on alkaline phosphatase. Metabolic activity and cell proliferation were assessed based on MTT and BrdU assays. Vascular endothelial growth factor production was evaluated using an immunoassay. We found that supernatants taken in the first hour from collagen barrier membranes loaded with dimethyloxalylglycine or deferoxamine reduced osteoclastogenesis. Osteoblastogenesis was not reduced significantly. Cell proliferation and metabolic activity of RAW 264.7 and MC3T3-E1 cells were inhibited by supernatants of collagen barrier membranes loaded with deferoxamine but not dimethyloxalylglycine. In RAW 264.7 cell culture, vascular endothelial growth factor production was increased only by supernatants of collagen barrier membranes loaded with dimethyloxalylglycine, but not deferoxamine. In MC3T3-E1 cell culture, supernatants of collagen barrier membranes loaded with dimethyloxalylglycine and deferoxamine both increased vascular endothelial growth factor production. Direct measurements showed that the majority of dimethyloxalylglycine and deferoxamine is released in the first hours. Dose-response studies supported the divergent effects of deferoxamine and dimethyloxalylglycine in RAW 264.7 and MC3T3-E1 cultures. Our findings show diverse effects of dimethyloxalylglycine- and deferoxamine-loaded collagen barrier membranes during osteoclastogenesis and osteoblastogenesis. Preclinical studies will reveal if the increase in vascular endothelial growth factor together with the inhibitory effect on osteoclasts can stimulate oral tissue regeneration.

Schizophrenia (SCZ) is a neurodevelopmental psychiatric disorder, in which cognitive function becomes disrupted at early stages of the disease. Although the mechanisms underlying cognitive impairments remain unclear, N-methyl-D-aspartate receptors (NMDAR) hypofunctioning in the prefrontal cortex (PFC) has been implicated. Moreover, cognitive symptoms in SCZ are usually unresponsive to treatment with current antipsychotics and by onset, disruption of the dopamine system, not NMDAR hypofunctioning, dominates the symptoms. Therefore, treating cognitive deficits at an early stage is a realistic approach. In this study, we tested whether an early treatment targeting mGluR2 would be effective in ameliorating cognitive impairments in the methylazoxymethanol acetate (MAM) model of SCZ. We investigated the effects of an mGluR2 agonist/mGluR3 antagonist, LY395756 (LY39), on the NMDAR expression and function in juveniles, as well as cognitive deficits in adult rats after juvenile treatment. We found that gestational MAM exposure induced a significant decrease in total protein levels of the NMDAR subunit, NR2B, and a significant increase of pNR2BTyr1472 in the juvenile rat PFC. Treatment with LY39 in juvenile MAM-exposed rats effectively recovered the disrupted NMDAR expression. Furthermore, a subchronic LY39 treatment in juvenile MAM-exposed rats also alleviated the learning deficits and cognitive flexibility impairments when tested with a cross-maze based set-shifting task in adults. Therefore, our study demonstrates that targeting dysfunctional NMDARs with an mGluR2 agonist during the early stage of SCZ could be an effective strategy in preventing the development and progression in addition to ameliorating cognitive impairments of SCZ.

Cancer cells have an altered redox status, with changes in intracellular signaling pathways. The knowledge of how such processes are regulated in 3D spheroids, being well-established tumor models, is limited. To approach this question we stably transfected HCT116 cells with a pTRAF reporter that enabled time- and cell-resolved activity monitoring of three redox-regulated transcription factors Nrf2, HIF and NF-κB in spheroids enriched for cancer stem cells. At the first day of spheroid formation, these transcription factors were activated and thereafter became repressed. After about a week, both HIF and Nrf2 were reactivated within the spheroid cores. Further amplifying HIF activation in spheroids by treatment with DMOG resulted in a dominant quiescent stem-cell-like phenotype, with high resistance to stress-inducing treatments. Auranofin, triggering oxidative stress and Nrf2 activation, had opposite effects with increased differentiation and proliferation. These novel high-resolution insights into spatiotemporal activation patterns demonstrate a striking coordination of redox regulated transcription factors within spheroids not occurring in conventional cell culture models., Graphical abstract fx1, Highlights • Redox regulated Nrf2, HIF and NF-κB are important in cancer and during development. • Nrf2, HIF and NF-κB are activated in conjunction with markers of cancer stem cells. • Nrf2 and HIF are concurrently induced in the core of growing spheroids. • Induction of HIF promotes a quiescent and resistant stem cell like phenotype.

Fibrosis is a complication of chronic inflammatory disorders such as inflammatory bowel disease (IBD), a condition which has limited therapeutic options and often requires surgical intervention. Pharmacologic inhibition of oxygen-sensing prolyl hydroxylases (PHD), which confer oxygen-sensitivity upon the hypoxia inducible factor (HIF) pathway, has recently been shown to have therapeutic potential in colitis, although the mechanisms involved remain unclear. Here, we investigated the impact of hydroxylase inhibition on inflammation-driven fibrosis in a murine colitis model. Mice exposed to dextran sodium sulfate followed by period of recovery developed intestinal fibrosis characterized by alterations in the pattern of collagen deposition and infiltration of activated fibroblasts. Treatment with the hydroxylase inhibitor dimethyloxalylglycine (DMOG) ameliorated fibrosis. TGF-β1 is a key regulator of fibrosis which acts through the activation of fibroblasts. Hydroxylase inhibition reduced TGF-β1-induced expression of fibrotic markers in cultured fibroblasts suggesting a direct role for hydroxylases in TGF-β1 signalling. This was at least in part due to inhibition of non-canonical activation of extracellular signal-regulated kinase (ERK) signalling. In summary, pharmacologic hydroxylase inhibition ameliorates intestinal fibrosis, through suppression of TGF-β1-dependent ERK activation in fibroblasts. We hypothesize that in addition to previously reported immunosupressive effects, hydroxylase inhibitors independently suppress pro-fibrotic pathways

Sestrin2 (SESN2) is an antioxidant protein that modulates cellular redox homeostasis through regeneration of peroxiredoxins. It has beneficial effects in oxidative or metabolic stress conditions as an upstream regulator of AMP-activated protein kinase (AMPK). Since hypoxia causes oxidative and metabolic stress, this study investigated the effect of SESN2 on signaling pathways altered by hypoxia in colon cancer cells. SESN2 overexpression in HEK293 cells inhibited hypoxia-inducible factor-1α (HIF-1α), which plays a crucial role in tumor growth and development in hypoxia. Moreover, infection with adenovirus-SESN2 (Ad-SESN2) decreased hypoxia or CoCl2-induced HIF-1α accumulation in colorectal cancer cells. Ad-SESN2 also reduced CoCl2-induced hypoxia response element (HRE)-luciferase activity and mRNA level of HIF-1α-driven genes. Furthermore, Ad-SESN2 infected cells showed anti-metastatic effects in serum-induced cell migration and invasion in vitro. Ad-SESN2 facilitated the ubiquitination of HIF-1α protein and increased hydroxyl-HIF-1α (OH-HIF-1α) level. In contrast, treatment with dimethyloxalylglycine (DMOG), an inhibitor of prolyl hydroxylase (PHD), reversed Ad-SESN2-induced OH-HIF-1α and subsequently suppressed HIF-1α level. The inhibitory effects of SESN2 on the serum-induced in vitro cell migration and invasion were also abrogated by DMOG treatment. Furthermore, knockdown of AMPKα reversed Ad-SESN2-mediated increase of OH-HIF-1α and inhibition of HIF-1α. Dominant-negative form of AMPK also restored the Ad-SESN2 mediated decrease in HIF-1α accumulation. Lastly, Ad-SESN2 suppressed tumor growth in a mouse xenograft model. Taken together, these results suggest that SESN2 increases degradation of HIF-1α via AMPK-PHD regulation that contributes to inhibition of in vitro and in vivo tumorigenesis.

Nuclear factor (NF)-κB is a transcription factor that plays an important role in many biological functions. Regulation of NF-κB activity is complicated, and ubiquitination is essential for NF-κB activation. Hypoxia can activate NF-κB. However, the underlying mechanism remains unclear. pVHL is a tumour suppressor and functions as an adaptor of E3-ligase. In this study, we demonstrated that pVHL inhibits NF-κB by mediating K63-ubiquitination of IKKβ, which is dependent on oxygen. We found that pVHL mediates K63-linked ubiquitination of IKKβ, which is an upstream regulator of NF-κB. The pVHL-mediated K63-ubiquitination of IKKβ prevents TAK1 binding, which leads to the inhibition of IKKβ phosphorylation and NF-κB activation. pVHL-mediated K63-ubiquitination of IKKβ is inhibited under hypoxia. DMOG, which is a specific inhibitor of prolyl hydroxylases, also suppresses K63-ubiquitination of IKKβ. Prolyl hydroxylase (PHD) 1 enhances K63-ubiquitination of IKKβ and inhibits IKKβ phosphorylation. These results suggest a novel function for pVHL in mediating K63-linked ubiquitination of IKKβ, which plays a role in the regulation of IKK/NF-κB signalling. The results also provide new insight into the mechanism of NF-κB activation through hypoxia.

Spinal cord injury (SCI) is a devastating neurological disorder that usually leads to a loss of motor and sensory function in patients. The expression of hypoxia inducible factor-1α (HIF-1α) is increased, and exerts a protective role after traumatic SCI. However, the endogenous activity of HIF-1α is insufficient for promoting functional recovery. The present study tested the potential effect of the sustained activation of HIF-1α by the prolylhydroxylase (PHD) inhibitor dimethyloxalylglycine (DMOG) on anti-apoptotic process and the regulation of axonal regeneration after SCI. Here, we found that treatment with DMOG significantly increased the expression of HIF-1α and that the stabilization of HIF-1α induced by DMOG not only decreased the expression of apoptotic proteins to promote neural survival, but also enhanced axonal regeneration by regulating microtubule stabilization