The protective effects of HIF-1α activation on sepsis induced intestinal mucosal barrier injury in rats model of sepsis.

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.

Hypoxia-inducible factor-1ß is essential for upregulation of the hypoxia-induced FLT1 gene in placental trophoblasts.

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-1ß, which acts as a dimerization partner for HIF-α, in the upregulation of the FLT1 gene via hypoxia. We confirmed the interactions between HIF-1ß 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-1ß. Moreover, the upregulation of the FLT1 gene in BeWo cells induced by dimethyloxaloylglycine (DMOG) was also inhibited by silencing either HIF-2α or HIF-1ß 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-1ß 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-1ß is essential for the elevated production of sFLT1 in the hypoxic trophoblasts and that the HIF-2α/HIF-1ß complex may be a crucial therapeutic target for pre-eclampsia.

Suppression of collagen IV alpha-2 subunit by prolyl hydroxylase domain inhibition via hypoxia-inducible factor-1 in chronic kidney disease.

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.

NAD+ metabolism controls growth inhibition by HIF1 in normoxia and determines differential sensitivity of normal and cancer 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: ß-nicotinamide mononucleotide.

HIF-1α activator DMOG inhibits alveolar bone resorption in murine periodontitis by regulating macrophage polarization.

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.

Hypoxia-Inducible Factor 1 Alpha-Mediated RelB/APOBEC3B Down-regulation Allows Hepatitis B Virus Persistence.

BACKGROUND AND AIMS: 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. APPROACH AND RESULTS: 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 ± LTßR-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. CONCLUSIONS: 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.

Stereoselective synthesis of novel 2'-(S)-CCG-IV analogues as potent NMDA receptor agonists.

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.

Dimethyloxalylglycine (DMOG) and the Caspase Inhibitor "Ac-LETD-CHO" Protect Neuronal ND7/23 Cells of Gluocotoxicity.

It well known that long-lasting hyperglycaemia disrupts neuronal function and leads to neuropathy and other neurodegenerative diseases. The α-ketoglutarate analogue (DMOG) and the caspase-inhibitor "Ac-LETD-CHO are potential neuroprotective molecules. Whether their protections may also extend glucotoxicity-induced neuropathy is not known. Herein, we evaluated the possible cell-protective effects of DMOG and Ac-LETD-CHO against hyperglycaemia-induced reactive oxygen species and apoptosis in ND7/23 neuronal cells. The impact of glucotoxicity on the expression of HIF-1α and a panel of micro-RNAs of significance in hyperglycaemia and apoptosis was also investigated.ND7/23 cells cultured under hyperglycaemic conditions showed decreased cell viability and elevated levels of ROS production in a dose- and time-dependent manner. However, presence DMOG (500 µM) and/or Ac-LETD-CHO (50 µM) counteracted this effect and increase cell viability concomitant with reduction in ROS production, DNA damage and apoptosis. AcLETD-CHO suppressed hyperglycaemia-induced caspase 3 activation in ND7/23 cells. Both DMOG and Ac-LETD-CHO increased HIF-1α expression paralleled with the suppression of miR-126-5p, miR-128-3p and miR-181 expression and upregulation of miR-26b, 106a-5p, 106b-5p, 135a-5p, 135b-5p, 138-5p, 199a-5p, 200a-3p and 200c-3p expression.We demonstrate a mechanistic link for the DMOG and Ac-LETD-CHO protection against hyperglycaemia-induced neuronal dysfunction, DNA damage and apoptosis and thereby propose that pharmacological agents mimicking these effects may represent a promising novel therapy for the hyperglycaemia-induced neuropathy.

Collagen release by human hepatic stellate cells requires vitamin C and is efficiently blocked by hydroxylase inhibition.

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 (TGFß) 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 TGFß. 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.

Delivery of dimethyloxalylglycine in calcined bone calcium scaffold to improve osteogenic differentiation and bone repair.

As hypoxia plays a vital role in the angiogenic-osteogenic coupling, using proline hydroxylase inhibitors to manipulate hypoxia-inducible factors has become a strategy to improve the osteogenic properties of biomaterials. Dimethyloxallyl glycine (DMOG) is a 2-ketoglutarate analog, a small molecular compound that competes for 2-ketoglutaric acid to inhibit proline hydroxylase. In order to improve the osteogenic ability of calcined bone calcium (CBC), a new hypoxia-mimicking scaffold (DMOG/Collagen/CBC) was prepared by immersing it in the DMOG-Collagen solution, followed by freeze-drying. All coated CBC scaffolds retained the inherent natural porous architecture and showed excellent biocompatibility. A slow release of DMOG by the DMOG-loaded CBC scaffolds for up to one week was observed inin vitroexperiments. Moreover, the DMOG/Collagen/CBC composite scaffold was found to significantly stimulate bone marrow stromal cells to express osteogenic and angiogenic genesin vitro. In addition, the osteogenic properties of three kinds of scaffolds, raw CBC, Collagen/CBC, and DMOG/Collagen/CBC, were evaluated by histology using the rabbit femoral condyle defect model. Histomorphometric analyses showed that the newly formed bone (BV/TV) in the DMOG/Collagen/CBC group was significantly higher than that of the Collagen/CBC group. However, immunostaining of CD31 and Runx2 expression between these two groups showed no significant difference at this time point. Our results indicate that DMOG-coated CBC can promote osteogenic differentiation and bone healing, and show potential for clinical application in bone tissue engineering.

Decorin inhibits the insulin-like growth factor I signaling in bone marrow mesenchymal stem cells of aged humans.

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.

Systemic hypoxia mimicry enhances axonal regeneration and functional recovery following peripheral nerve injury.

Despite the ability of peripheral nerves to regenerate after injury, failure occurs due to an inability of supporting cells to maintain growth, resulting in long-term consequences such as sensorimotor dysfunction and neuropathic pain. Here, we investigate the potential of engaging the cellular adaptive response to hypoxia, via inhibiting its negative regulators, to enhance the regenerative process. Under normoxic conditions, prolyl hydroxylase domain (PHD) proteins 1, 2, and 3 hydroxylate the key metabolic regulator hypoxia inducible factor 1α (HIF1α), marking it for subsequent proteasomal degradation. We inhibited PHD protein function systemically via either individual genetic deletion or pharmacological pan-PHD inhibition using dimethyloxalylglycine (DMOG). We show enhanced axonal regeneration after sciatic nerve crush injury in PHD1-/- mice, PHD3-/- mice, and in DMOG-treated mice, and in PHD1-/- and DMOG-treated mice a reduction in hypersensitivity to cooling after permanent sciatic ligation. Electromyographically, PHD1-/- and PHD3-/- mice showed an increased CMAP amplitude one-month post-injury, probably due to protection against denervation induced muscle atrophy, while DMOG-treated and PHD2+/- mice showed reduced latencies, indicating improved motor axon function. DMOG treatment did not affect the growth of dorsal root ganglion neurites in vitro, suggesting a lack of direct effects of DMOG on axonal regrowth. Enhanced regeneration in vivo was concurrent with an increase in macrophage density, and a shift in macrophage polarization state ratios (from M1-like toward M2-like) in DMOG-treated animals. These results indicate PHD proteins as a novel therapeutic target to improve regenerative and functional outcomes after peripheral nerve injury without manipulating molecular O2.

The Prolyl Hydroxylase Inhibitor Dimethyl Oxalyl Glycine Decreases Early Gastrointestinal GVHD in Experimental Allogeneic Hematopoietic Cell Transplantation.

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.

SerpinB1 expression in Th17 cells depends on hypoxia-inducible factor 1-alpha.

SerpinB1, previously known as MNEI (monocyte/neutrophil elastase inhibitor), has been well established to maintain the survival of neutrophils. Our recent studies showed that SerpinB1 is also the signature gene of IL-17-producing γδT cells and Th17 cells, and its expression is maintained by IL-23 signaling. Deficiency of SerpinB1 largely ameliorates the experimental autoimmune encephalomyelitis (EAE) with enhanced granule protease-mediated mitochondrial damage leading to suicidal cell death of pathogenic CD4 T cells. However, the mechanism that induces SerpinB1 expression in Th17 cells still remains elusive. Here, we showed that SerpinB1 was induced in Th17 cells, and plays a pivotal role to maintain the pathogenic signature of IL-23-primed Th17 cells in vitro. Its expression in Th17 cells was independent of Th17-lineage specific transcript factor retinoic acid-related orphan receptor γ t (RORγt), but was controlled by glycolysis and the mammalian target of rapamycin (mTOR) signaling. Finally, by using two specific pharmacological inhibitors, our study further deciphered that hypoxia-inducible factor 1α (HIF-1α) specifically controlled the SerpinB1 expression in Th17 cells. On the other side, when HIF-1α stabilizer Dimethyloxalylglycine (DMOG) was applied, SerpinB1 expression was significantly increased in Th17 cells. Taken together, this study is the first to report that SerpinB1 expression in Th17 cells is mediated by glycolysis/mTOR/HIF-1α pathway.

Hyperglycemia suppresses the regulatory effect of hypoxia-inducible factor-1α in pulmonary Aspergillus fumigatus infection.

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-1ß 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-1ß 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-1ß signal pathway.

Perinatal Hypoxia-Inducible Factor Stabilization Preserves Lung Alveolar and Vascular Growth in Experimental Bronchopulmonary Dysplasia.

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.

Stabilization of HIF-1α alleviates osteoarthritis via enhancing mitophagy.

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.

Hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitors induce autophagy and have a protective effect in an in-vitro ischaemia model.

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.

Quantitative MS-Based Proteomics: Comparing the MCF-7 Cellular Response to Hypoxia and a 2-Oxoglutarate Analogue.

The hypoxia-inducible factors (HIFs) are key transcription factors in determining cellular responses involving alterations in protein levels in response to limited oxygen availability in animal cells. 2-Oxoglutarate-dependent oxygenases play key roles in regulating levels of HIF and its transcriptional activity. We describe MS-based proteomics studies in which we compared the results of subjecting human breast cancer MCF-7 cells to hypoxia or treating them with a cell-penetrating derivative (dimethyl N-oxalylglycine; DMOG) of the stable 2OG analogue N-oxalylglycine. The proteomic results are consistent with reported transcriptomic analyses and support the proposed key roles of 2OG-dependent HIF prolyl- and asparaginyl-hydroxylases in the hypoxic response. Differences between the data sets for hypoxia and DMOG might reflect context-dependent effects or HIF-independent effects of DMOG.

Bone augmentation using small molecules with biodegradable calcium sulfate particles in a vertical onlay graft model in the rabbit calvarium.

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.