Dimethyloxalylglycine Research Articles

HIF-1α Ameliorates Diabetic Neuropathic Pain via Parkin-Mediated Mitophagy in a Mouse Model.

Mitochondrial dysfunction, which can be regulated by mitophagy, plays a central role in diabetic neuropathic pain (DNP). Mitophagy that was involved in nerve damage-induced neuropathic pain has been reported. Hyperglycemia and cellular hypoxic were the two main characters of diabetes. Hypoxia-inducible factor 1α subunit (HIF-1α) plays a vital role in mitochondrial homeostasis under hypoxia. However, it remains unclear whether mitophagy was changed and could be regulated by HIF-1α in DNP. In this study, the results showed that mitophagy was activated and HIF-1α was upregulated in the spinal cord of diabetic mice. HIF-1α agonist dimethyloxalylglycine (DMOG) could further elevate HIF-1α and Parkin protein, enhance mitophagy, decrease mitochondrial dysfunction, and hyperalgesia. Furthermore, Park2 (encoding Parkin) knockout aggravated hyperalgesia and mitochondrial dysfunction in diabetic mice. Furthermore, mitophagy could not be activated and induced by HIF-1α agonist DMOG in Park2−/− diabetic mice. In this study, we first demonstrated that HIF-1α could upregulate mitophagy in the spinal cord of mice with DNP through modulating the Parkin signaling pathway, promoting new insights into the mechanisms and research of treatment strategies for patients with DNP.

Hypoxia aggravates ferroptosis in RPE cells by promoting the Fenton reaction

Oxidative stress and hypoxia in the retinal pigment epithelium (RPE) have long been considered major risk factors in the pathophysiology of age-related macular degeneration (AMD), but systematic investigation of the interplay between these two risk factors was lacking. For this purpose, we treated a human RPE cell line (ARPE-19) with sodium iodate (SI), an oxidative stress agent, together with dimethyloxalylglycine (DMOG) which leads to stabilization of hypoxia-inducible factors (HIFs), key regulators of cellular adaptation to hypoxic conditions. We found that HIF stabilization aggravated oxidative stress-induced cell death by SI and iron-dependent ferroptosis was identified as the main cell death mechanism. Ferroptotic cell death depends on the Fenton reaction where H2O2 and iron react to generate hydroxyl radicals which trigger lipid peroxidation. Our findings clearly provide evidence for superoxide dismutase (SOD) driven H2O2 production fostering the Fenton reaction as indicated by triggered SOD activity upon DMOG + SI treatment as well as by reduced cell death levels upon SOD2 knockdown. In addition, iron transporters involved in non-transferrin-bound Fe2+ import as well as intracellular iron levels were also upregulated. Consequently, chelation of Fe2+ by 2’2-Bipyridyl completely rescued cells. Taken together, we show for the first time that HIF stabilization under oxidative stress conditions aggravates ferroptotic cell death in RPE cells. Thus, our study provides a novel link between hypoxia, oxidative stress and iron metabolism in AMD pathophysiology. Since iron accumulation and altered iron metabolism are characteristic features of AMD retinas and RPE cells, our cell culture model is suitable for high-throughput screening of new treatment approaches against AMD.

Hydroxylation of the NOTCH1 intracellular domain regulates Notch signaling dynamics

Notch signaling plays a pivotal role in the development and, when dysregulated, it contributes to tumorigenesis. The amplitude and duration of the Notch response depend on the posttranslational modifications (PTMs) of the activated NOTCH receptor – the NOTCH intracellular domain (NICD). In normoxic conditions, the hydroxylase FIH (factor inhibiting HIF) catalyzes the hydroxylation of two asparagine residues of the NICD. Here, we investigate how Notch-dependent gene transcription is regulated by hypoxia in progenitor T cells. We show that the majority of Notch target genes are downregulated upon hypoxia. Using a hydroxyl-specific NOTCH1 antibody we demonstrate that FIH-mediated NICD1 hydroxylation is reduced upon hypoxia or treatment with the hydroxylase inhibitor dimethyloxalylglycine (DMOG). We find that a hydroxylation-resistant NICD1 mutant is functionally impaired and more ubiquitinated. Interestingly, we also observe that the NICD1-deubiquitinating enzyme USP10 is downregulated upon hypoxia. Moreover, the interaction between the hydroxylation-defective NICD1 mutant and USP10 is significantly reduced compared to the NICD1 wild-type counterpart. Together, our data suggest that FIH hydroxylates NICD1 in normoxic conditions, leading to the recruitment of USP10 and subsequent NICD1 deubiquitination and stabilization. In hypoxia, this regulatory loop is disrupted, causing a dampened Notch response.

Hypoxia triggers the outbreak of infectious spleen and kidney necrosis virus disease through viral hypoxia response elements

ABSTRACT Hypoxia frequently occurs in aquatic environments, especially in aquaculture areas. However, research on the relationship between hypoxic aquatic environments with viral diseases outbreak is limited, and its underlying mechanisms remain elusive. Herein, we demonstrated that hypoxia directly triggers the outbreak of infectious spleen and kidney necrosis virus (ISKNV) disease. Hypoxia or activated hypoxia-inducible factor (HIF) pathway could remarkably increase the levels of viral genomic DNA, titers, and gene expression, indicating that ISKNV can response to hypoxia and HIF pathway. To reveal the mechanism of ISKNV respond to HIF pathway, we identified the viral hypoxia response elements (HREs) in ISKNV genome. Fifteen viral HREs were identified, and four related viral genes responded to the HIF pathway, in which the hre-orf077r promoter remarkably responded to the HIF pathway. The level of orf077r mRNA dramatically increased after the infected cells were treated with dimethyloxalylglycine (DMOG) or the infected cells/fish subjected to hypoxic conditions, and overexpressed orf077r could remarkably increase the ISKNV replication. These finding shows that hypoxic aquatic environments induce the expression of viral genes through the viral HREs to promote ISKNV replication, indicating that viral HREs might be important biomarkers for the evaluation of the sensitivity of aquatic animal viral response to hypoxia stress. Furthermore, the frequencies of viral HREs in 43 species aquatic viral genomes from 16 families were predicted and the results indicate that some aquatic animal viruses, such as Picornavirdea, Dicistronviridae, and Herpesviridae, may have a high risk to outbreak when the aquatic environment encounters hypoxic stress.

Effect of Eicosapentaenoic Acid on sFlt-1 and HIF-1α Expression Under Induced Hypoxia Conditions in Trophoblast Tumor Cell Line (JEG-3)

Background: Previous studies have shown the altered levels of long-chain polyunsaturated fatty acids (LCPUFAs) in pathological hypoxic conditions. Elevated soluble fms-like tyrosine kinase-1 (sFlt-1) expression in hypoxia plays an important role in the pathogenesis of placental as preeclampsia. Objectives: The eicosapentaenoic acid (EPA; 20:5, n-3) as LCPUFAs (omega-3) might attenuate sFlt-1 and hypoxia-inducible factor-1α (HIF-1α) expressions and secretions. Methods: JEG-3 cells were incubated with dimethyloxalylglycine (DMOG) and EPA. The SFlt-1 gene expression was determined using a real-time polymerase chain reaction. The protein secretion of sFlt-1 and HIF-1α were analyzed using Western blot. Results: The incubation of JEG-3 cells with DMOG significantly elevated messenger ribonucleic acid (mRNA) expression and protein secretion of sFlt-1 (P < 0.05); nevertheless, EPA decreased mRNA expression and protein secretion of sFlt-1 (P < 0.05). Moreover, EPA inhibited the effect of DMOG on sFlt-1 (P = 0.0361) gene expression and protein secretion and HIF-1α (P = 0.0241) protein secretion. Conclusions: The sFlt-1 expression decreased by n-3 fatty acids in trophoblast tumor cell line under induced hypoxia conditions. It seems that changes in sFlt-1 expression are mediated by the transcription factor HIF-1α.

Dimethyloxalylglycine (DMOG), a Hypoxia Mimetic Agent, Does Not Replicate a Rat Pheochromocytoma (PC12) Cell Biological Response to Reduced Oxygen Culture.

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.

Lactate Transporter SLC16A3 (MCT4) as an Onco-Immunological Biomarker Associating Tumor Microenvironment and Immune Responses in Lung Cancer

PurposeLactate, a marker of tumor metabolic reprogramming, maintains the acidic microenvironment and also affects the metabolism and function of immune cells. SLC16A3 is responsible for the extracellular transport of lactate, which is a key component of glycolysis. However, the role of SLC16A3 in immune infiltration and immunosuppression of lung cancer is largely unknown. Our study explored the therapeutic and prognostic value of SLC16A3 in predicting immune infiltration and immune checkpoint efficacy of lung cancer.MethodsSLC16A3 expression was evaluated with TCGA database. Kaplan–Meier analysis was performed for survival rates. GO and KEEG enrichment was conducted to determine predictive signaling pathways. We utilized TIMER and CIBERSORT to analyze the correlation between SLC16A3 and immunocyte infiltration as well as immune checkpoint. Interleukin and HIF-1a expression was measured with ELISA kit and flow cytometry separately.ResultsIn comparison with normal tissues, SLC16A3 expression was significantly upregulated in both lung adenocarcinoma (LUAD) and squamous carcinoma (LUSC), which was closely related to poor prognosis. GO analysis indicated that SLC16A3 involved in different signal pathways in LUAD and LUSC and linked to HIF-1 signaling in LUAD. High SLC16A3 was correlated with immunosuppressive cells (Treg, Th2 and iDC), immune checkpoint (PD1, PD-L1, PVR, Tim-3, ITGAM) and immunosuppressive factors (foxp3, TGF-β) in LUAD not LUSC. Furthermore, SLC16A3 was identified to tightly interact with IL-8 which may induce microenvironment immune tolerance. Based on the clinical prediction, we performed experiments with LUAD A549 cells and showed reduced IL-8 and HIF-1a when treated with SLC16A3 knockdown. HIF-1a stimulation by dimethyloxalylglycine (DMOG) could restore IL-8 secretion in SLC16A3 downregulated cells.ConclusionTaken together, our results suggest that SLC16A3 contributes to a worse prognosis in lung cancer and may play an important role in immune microenvironment and evasion through HIF-1a-IL8 axis, which could be a novel therapeutic target for immunotherapy in lung cancer.

Effect of dimethyloxalylglycine on angiogenesis in Choke Ⅱ zone of cross-zone perforator flap and its mechanism

To study the effect of dimethyloxalylglycine (DMOG) on angiogenesis in Choke Ⅱ zone of rats cross-zone perforator flaps and its mechanism. 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. 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<0.05). DMOG group had more angiogenesis in Choke Ⅱ zone and the vascular structure was clear and complete. In YC-1 group and control group, the vessels in Choke Ⅱ zone was less and the vascular structure was disordered. The number of vessels was (25.56±1.29)/field in the DMOG group, which was significantly higher than that in the YC-1 group [(7.38±0.54)/field] and the control group [(14.48±0.91)/field] ( P<0.05). At 3, 5, and 7 days after operation, HIF-1α and VEGF expressions in ChokeⅡzone of DMOG group were significantly higher than those in YC-1 group and control group ( P<0.05). DMOG can promote angiogenesis in Choke Ⅱ zone, accelerate the early angiogenesis of the flap, improve the microcirculation and blood supply in the potential zone of the flap, reduce the injury of flap ischemia and hypoxia, and increase the survival rate of the flap.

Bone Microenvironment‐Mimetic Scaffolds with Hierarchical Microstructure for Enhanced Vascularization and Bone Regeneration

AbstractMicrochannel networks within engineered 3D scaffold can allow nutrient exchange and rapid blood vessels formation. However, fabrication of a bone microenvironment‐mimicking scaffold with hierarchical micro/nanofibrous and microchannel structures is still a challenge. Herein, inspired by structural and functional cues of bone remodeling, a microchannel networks‐enriched nanofibrous scaffold by using 3D printing and thermally induced phase separation techniques, which can facilitate cells migration and nutrients transportation, is developed. The customizable vascular‐like structure of polycaprolactone within the nanofibrous gelatin‐silica scaffold is fabricated using 3D‐printed sacrificial templates, while dimethyloxalylglycine (DMOG)‐loaded mesoporous silica nanoparticles (MSNs) located on the scaffold surface and bone forming peptide‐1 (BFP)‐loaded MSNs embedded in the scaffold are implemented for sequential release of DMOG and BFP. The cell experiments show that dual‐drug delivery scaffold (DBM/GP) promotes angiogenesis by stimulating migration, tube formation, and angiogenesis‐related genes/protein expression of endothelial cells, and osteogenesis by promoting osteo‐related genes expression and mineral deposition of osteoblasts. Additionally, DBM/GP scaffold facilitates the angiogenic activity of osteoblasts by activating phosphatidylinositol 3‐kinase/protein kinase B/hypoxia inducible factor‐1α pathway. Furthermore, enhanced vascularization and bone regeneration of DBM/GP scaffold are demonstrated via subcutaneous and skull defect models. Overall, this study reveals that the bone microenvironment‐mimetic dual‐drug delivery scaffold provides a promising strategy for bone defects treatment.

Effects of PHD and HSP90 on erythropoietin production in yak (Bos grunniens) renal interstitial fibroblast-like cells under hypoxia.

Erythropoietin (EPO), a central protein of erythropoiesis, plays an important role during hypoxia adaptation and is regulated by hypoxia-inducible factor (HIF). However, there is no report on EPO-producing cells and their regulatory mechanisms in yak (Bos grunniens). To understand EPO production and regulation of yak, kidneys from different age of yak were collected and expression of EPO, hypoxia-inducible factor 1 alpha (HIF-1α), and hypoxia-inducible factor 2 alpha (HIF-2α) were detected. Then renal tubule epithelial cells (RTECs) and peritubular interstitial fibroblast-like (RIFs) cells were isolated and cultured to determine their EPO production abilities. Subsequently, the cells were treated with dimethyloxalylglycine (DMOG) and Geldanamycin (GA), which are inhibitors of prolyl-4-hydroxylase domain (PHD) and heat shock protein 90 (HSP90) respectively, and siRNAs of HIF-1α and HIF-2α to explore their effect on EPO production and regulation. The results showed that expressions of EPO, HIF-1α, and HIF-2α were different in the different age groups of yak. High DMOG concentration caused a corresponding increase in the levels of HIF-1α and HIF-2α in RIFs and RTECs, however, EPO levels increased in RIFs only and was not detected at any concentration in RTECs; suggesting that EPO was produced in RIFs. Upon treating RIFs with siRNAs of HIF-1α and HIF-2α, we found that EPO was regulated by PHD through HIF-2α. In addition, increasing GA concentration caused a decrease in expression of HSP90, HIF-1α, HIF-2α, and EPO in RIFs. In conclusion, these findings support our proposition that PHD regulates EPO via HIF-2α in yak RIFs, while HSP90 impelled EPO expression.

Structure-Activity Relationships and Transcriptomic Analysis of Hypoxia-Inducible Factor Prolyl Hydroxylase Inhibitors.

To evaluate the differences in action of commercially available 2-oxoglutarate mimetics and “branched-tail” oxyquinoline inhibitors of hypoxia-inducible factor prolyl hydroxylase (HIF PHD), the inhibitors’ IC50 values in the activation of HIF1 ODD-luciferase reporter were selected for comparative transcriptomics. Structure–activity relationship and computer modeling for the oxyquinoline series of inhibitors led to the identification of novel inhibitors, which were an order of magnitude more active in the reporter assay than roxadustat and vadadustat. Unexpectedly, 2-methyl-substitution in the oxyquinoline core of the best HIF PHD inhibitor was found to be active in the reporter assay and almost equally effective in the pretreatment paradigm of the oxygen-glucose deprivation in vitro model. Comparative transcriptomic analysis of the signaling pathways induced by HIF PHD inhibitors showed high potency of the two novel oxyquinoline inhibitors (#4896-3249 and #5704-0720) at 2 μM concentrations matching the effect of 30 μM roxadustat and 500 μM dimethyl oxalyl glycine in inducing HIF1 and HIF2-linked pathways. The two oxyquinoline inhibitors exerted the same activation of HIF-triggered glycolytic pathways but opposite effects on signaling pathways linked to alternative substrates of HIF PHD 1 and 3, such as p53, NF-κB, and ATF4. This finding can be interpreted as the specificity of the 2-methyl-substitute variant for HIF PHD2.

Synergetic protective effect of remote ischemic preconditioning and prolyl 4‑hydroxylase inhibition in ischemic cardiac injury.

It has been reported that hypoxia-inducible factor 1α (HIF-1α) serves a key role in the protective effect of remote ischemic preconditioning (RIP) in ischemia/reperfusion (I/R)-induced cardiac injury. Moreover, inhibition of prolyl 4-hydroxylase (PHD), an enzyme responsible for HIF-1α degradation, prevents I/R-induced cardiac injury. However, whether their protective effects are synergetic remains to be elucidated. The present study aimed to investigate the protective effect of RIP, PHD inhibition using dimethyloxalylglycine (DMOG) and their combination on I/R-induced cardiac injury. Rabbits were randomly divided into seven groups: i) Sham; ii) I/R; iii) lung RIP + I/R; iv) thigh RIP + I/R; v) DMOG + I/R; vi) DMOG + lung RIP + I/R; and vii) DMOG + thigh RIP + I/R. I/R models were established via 30 min left coronary artery occlusion and 3 h reperfusion. For lung/thigh RIP, rabbits received left pulmonary artery (or left limb) ischemia for 25 min and followed by release for 5 min. Some rabbits were administered 20 mg/kg DMOG. The results demonstrated that both lung/thigh RIP and DMOG significantly decreased myocardial infarct size, creatine kinase activity and myocardial apoptosis in I/R rabbits. Furthermore, the combination of RIP and PHD inhibition exerted synergetic protective effects on these aforementioned changes. The mechanistic study indicated that both treatments increased mRNA and protein expression levels of HIF-1α and its downstream regulators, including vascular endothelial growth factor (VEGF), AKT and endothelial nitric oxide synthase (eNOS). In conclusion, the present study demonstrated that RIP and PHD inhibition exerted synergetic protective effects on cardiac injury via activation of HIF-1α and the downstream VEGF/AKT-eNOS signaling pathway.

EGLN1 induces tumorigenesis and radioresistance in nasopharyngeal carcinoma by promoting ubiquitination of p53 in a hydroxylase-dependent manner.

Egl-9 Family Hypoxia Inducible Factor 1 (EGLN1) is a proline hydroxylase mediating degradation of hypoxia-inducible factor α (HIFα) through the ubiquitination system. Studies have indicated an essential role for EGLN1 in angiogenesis and tumorigenesis. However, there is no consensus on the regulation of EGLN1 and its mechanism of action on nasopharyngeal carcinoma (NPC). This study explored the association of the expression of EGLN1 with characteristics of NPC tumors and its underlying mechanism. We found that the expression of EGLN1 showed a positive correlation with tumor T classification and clinical staging of patients with NPC. EGLN1 could promote cell proliferation, invasion and migration, and even enhance the cancer stem cells (CSCs) prosperity and radioresistance of NPC cells. Mechanistically, EGLN1 facilitated degradation of tumor protein p53 through the ubiquitination system. This effect could be weakened in the presence of dimethyloxalylglycine (DMOG), suggesting that EGLN1 down-regulated p53 based on its hydroxylase activity. In conclusion, overexpression of EGLN1 promoted oncogenesis and induced a CSC-like phenotype in NPC cells, then enhancing the ability for radioresistance by interacting with p53 in a hydroxylase-dependent manner. Thus, EGLN1 might serve as a potential therapeutic target for NPC.

PHD2 attenuates high-glucose-induced blood retinal barrier breakdown in human retinal microvascular endothelial cells by regulating the Hif-1α/VEGF pathway.

Diabetic macular edema (DME) is one of the most frequent causes of severe vision loss. The pathogenesis of DME is still not fully understood; however, it is hypothesized to result from breakdown of the blood-retinal barrier (BRB) due to retinal inflammation by vascular endothelial growth factor (VEGF) secretion under hyperglycemic conditions. In this investigation, we discovered that Prolyl-4-hydroxylase 2 (PHD2), an upstream regulator of hypoxia-inducible factor 1 (HIF-1) modulates VEGF expression and thus preserves BRB function in the mouse retina. Primary human retinal microvascular endothelial cells (hRMECs) were cultured in human endothelial serum-free growth medium and exposed to hyperglycemia. Changes in cell viability were investigated by an MTT assay. BRB function in each group was revealed by a paracellular permeability assay and trans-endothelial electrical resistance (TEER). Morphological changes in the BRB were investigated by immunofluorescence staining of occludin and zonula occludens-1 (ZO-1). The mRNA and protein levels of the tight junction proteins, PHD2, HIF-1α, and VEGF were measured by reverse transcription-quantitative PCR (RT-qPCR), western blot analysis and ELISA. Under hyperglycemic conditions, the viability of hRMECs was decreased, and PHD2 expression was downregulated, accompanied by increased paracellular permeability and decreased trans-endothelial electrical resistance. Additionally, HIF-1α and VEGF expression levels were increased, whereas the expression levels of tight junction proteins, including occludin and ZO-1, were decreased and BRB function was compromised. The PHD2 activator R59949 (diacylglycerol kinase inhibitor II), altered these pathological changes, and the PHD2 inhibitor dimethyloxalylglycine (DMOG) resulted in the opposite effects. These results demonstrated that PHD2 inhibited HIF-1 activity by inhibiting HIF-1α expression in hRMECs under hyperglycemic conditions, which led to the downregulation of the expression of the angiogenic factor VEGF, and thus helped to maintain the functions of hRMECs. Therefore, it is reasonable to propose that PHD2 could be a potential novel target for the treatment of DME or other diseases with a similar pathogenesis.

Isoform-specific Roles of Prolyl Hydroxylases in the Regulation of Pancreatic β-Cell Function.

Pancreatic β-cells can secrete insulin via 2 pathways characterized as KATP channel -dependent and -independent. The KATP channel–independent pathway is characterized by a rise in several potential metabolic signaling molecules, including the NADPH/NADP+ ratio and α-ketoglutarate (αKG). Prolyl hydroxylases (PHDs), which belong to the αKG-dependent dioxygenase superfamily, are known to regulate the stability of hypoxia-inducible factor α. In the current study, we assess the role of PHDs in vivo using the pharmacological inhibitor dimethyloxalylglycine (DMOG) and generated β-cell-specific knockout (KO) mice for all 3 isoforms of PHD (β-PHD1 KO, β-PHD2 KO, and β-PHD3 KO mice). DMOG inhibited in vivo insulin secretion in response to glucose challenge and inhibited the first phase of insulin secretion but enhanced the second phase of insulin secretion in isolated islets. None of the β-PHD KO mice showed any significant in vivo defects associated with glucose tolerance and insulin resistance except for β-PHD2 KO mice which had significantly increased plasma insulin during a glucose challenge. Islets from both β-PHD1 KO and β-PHD3 KO had elevated β-cell apoptosis and reduced β-cell mass. Isolated islets from β-PHD1 KO and β-PHD3 KO had impaired glucose-stimulated insulin secretion and glucose-stimulated increases in the ATP/ADP and NADPH/NADP+ ratio. All 3 PHD isoforms are expressed in β-cells, with PHD3 showing the most distinct expression pattern. The lack of each PHD protein did not significantly impair in vivo glucose homeostasis. However, β-PHD1 KO and β-PHD3 KO mice had defective β-cell mass and islet insulin secretion, suggesting that these mice may be predisposed to developing diabetes.

Low-intensity pulsed ultrasound prevents prolonged hypoxia-induced cardiac fibrosis through HIF-1α/DNMT3a pathway via a TRAAK-dependent manner.

Hypoxia-induced cardiac fibrosis is an important pathological process in cardiovascular disorders. This study aimed to determine whether low-intensity pulsed ultrasound (LIPUS), a novel and safe apparatus, could alleviate hypoxia-induced cardiac fibrosis, and to elucidate the underlying mechanisms. Hypoxia (1% O2 ) and transverse aortic constriction (TAC) were performed on neonatal rat cardiac fibroblasts and mice to induce cardiac fibrosis, respectively. LIPUS irradiation was applied for 20minutes every 6hours for a total of 2 times in vitro, and every 2days from 1week before surgery to 4weeks after surgery in vivo. We found that LIPUS dose-dependently attenuated hypoxia-induced cardiac fibroblast phenotypic conversion in vitro, and ameliorated TAC-induced cardiac fibrosis in vivo. Hypoxia significantly upregulated the nuclear protein expression of hypoxia-inducible factor-1α (HIF-1α) and DNA methyltransferase 3a (DNMT3a). LIPUS pre-treatment reversed the elevated expression of HIF-1α, and DNMT3a. Further experiments revealed that HIF-1α stabilizer dimethyloxalylglycine (DMOG) hindered the anti-fibrotic effect of LIPUS, and hampered LIPUS-mediated downregulation of DNMT3a. DNMT3a small interfering RNA (siRNA) prevented hypoxia-induced cardiac fibrosis. Results also showed that the mechanosensitive protein-TWIK-related arachidonic acid-activated K+ channel (TRAAK) messenger RNA (mRNA) expression was downregulated in hypoxia-induced cardiac fibroblasts, and TAC-induced hearts. TRAAK siRNA impeded LIPUS-mediated anti-fibrotic effect and downregulation of HIF-1α and DNMT3a. Above results indicated that LIPUS could prevent prolonged hypoxia-induced cardiac fibrosis through TRAAK-mediated HIF-1α/DNMT3a signalling pathway.

Chemically induced hypoxia by dimethyloxalylglycine (DMOG)-loaded nanoporous silica nanoparticles supports endothelial tube formation by sustained VEGF release from adipose tissue-derived stem cells.

Inadequate vascularization leading to insufficient oxygen and nutrient supply in deeper layers of bioartificial tissues remains a limitation in current tissue engineering approaches to which pre-vascularization offers a promising solution. Hypoxia triggering pre-vascularization by enhanced vascular endothelial growth factor (VEGF) expression can be induced chemically by dimethyloxalylglycine (DMOG). Nanoporous silica nanoparticles (NPSNPs, or mesoporous silica nanoparticles, MSNs) enable sustained delivery of molecules and potentially release DMOG allowing a durable capillarization of a construct. Here we evaluated the effects of soluble DMOG and DMOG-loaded NPSNPs on VEGF secretion of adipose tissue-derived stem cells (ASC) and on tube formation by human umbilical vein endothelial cells (HUVEC)-ASC co-cultures. Repeated doses of 100 µM and 500 µM soluble DMOG on ASC resulted in 3- to 7-fold increased VEGF levels on day 9 (P < 0.0001). Same doses of DMOG-NPSNPs enhanced VEGF secretion 7.7-fold (P < 0.0001) which could be maintained until day 12 with 500 µM DMOG-NPSNPs. In fibrin-based tube formation assays, 100 µM DMOG-NPSNPs had inhibitory effects whereas 50 µM significantly increased tube length, area and number of junctions transiently for 4 days. Thus, DMOG-NPSNPs supported endothelial tube formation by upregulated VEGF secretion from ASC and thus display a promising tool for pre-vascularization of tissue-engineered constructs. Further studies will evaluate their effect in hydrogels under perfusion.

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-1α Attenuates Ischemic Brain Damage by Modulating Inflammatory Response and Glial Activity.

Hypoxia-inducible factor 1 can sufficiently control the progress of neurological symptoms after ischemic stroke owing to their actions associated with its downstream genes. In this study, we evaluated the role of HIF-1α in attenuating brain damage after endothelin-1 injection. Focal cerebral ischemia in mice were induced by endothelin-1 microinjection. Hypoxia-inducible factor 1 activator, dimethyloxalylglycine (DMOG), and HIF-1α inhibitor, acriflavine (ACF), were used to evaluate the hypoxia-inducible factor 1 activity during cerebral ischemia. The expression levels of HIF-1α, glial fibrillary acidic protein (GFAP), interleukin-10 (IL-10), inducible nitric oxide synthase (iNOS), phosphorylated I-kappa-B-alpha/total I-kappa-B-alpha (p-IκBα/IκBα) and nuclear factor kappa B (NF-kB) were assessed. Besides, mRNA levels of IL-10, tumor necrosis factor- alpha (TNF-α), and NF-kB were also analyzed. Results showed a noticeable increase in hypoxia-inducible factor 1 and IL-10 levels in the DMOG group with a decline in iNOS, TNF-α, and NF-kB levels, implying the anti-inflammatory role of hypoxia-inducible factor 1 activator following stroke. These findings were further corroborated by GFAP immunostaining that showed astrocytic activation to be inhibited 12 days post-ischemia, as well as histological and TEM analyses that demonstrated hypoxia-inducible factor 1 induction to alleviate neuronal soma damage and cell death. Based on our study, HIF-1α could be a potential therapeutic target for ischemic stroke.

The effect of hypoxia-mimicking responses on improving the regeneration of artificial vascular grafts

Cellular transition to hypoxia following tissue injury, has been shown to improve angiogenesis and regeneration in multiple tissues. To take advantage of this, many hypoxia-mimicking scaffolds have been prepared, yet the oxygen access state of implanted artificial small-diameter vascular grafts (SDVGs) has not been investigated. Therefore, the oxygen access state of electrospun PCL grafts implanted into rat abdominal arteries was assessed. The regions proximal to the lumen and abluminal surfaces of the graft walls were normoxic and only the interior of the graft walls was hypoxic. In light of this differential oxygen access state of the implanted grafts and the critical role of vascular regeneration on SDVG implantation success, we investigated whether modification of SDVGs with HIF-1α stabilizer dimethyloxalylglycine (DMOG) could achieve hypoxia-mimicking responses resulting in improving vascular regeneration throughout the entirety of the graft wall. Therefore, DMOG-loaded PCL grafts were fabricated by electrospinning, to support the sustained release of DMOG over two weeks. In vitro experiments indicated that DMOG-loaded PCL mats had significant biological advantages, including: promotion of human umbilical vein endothelial cells (HUVECs) proliferation, migration and production of pro-angiogenic factors; and the stimulation of M2 macrophage polarization, which in-turn promoted macrophage regulation of HUVECs migration and smooth muscle cells (SMCs) contractile phenotype. These beneficial effects were downstream of HIF-1α stabilization in HUVECs and macrophages in normoxic conditions. Our results indicated that DMOG-loaded PCL grafts improved endothelialization, contractile SMCs regeneration, vascularization and modulated the inflammatory reaction of grafts in abdominal artery replacement models, thus promoting vascular regeneration.