Human ESC-derived vascular cells promote vascular regeneration in a HIF-1α dependent manner

Hypoxia-inducible factor (HIF-1α), a core transcription factor responding to changes in cellular oxygen levels, is closely associated with a wide range of physiological and pathological conditions. However, its differential impacts on vascular cell types and molecular programs modulating human vascular homeostasis and regeneration remain largely elusive. Here, we applied CRISPR/Cas9-mediated gene editing of human embryonic stem cells and directed differentiation to generate HIF-1α-deficient human vascular cells including vascular endothelial cells, vascular smooth muscle cells, and mesenchymal stem cells (MSCs), as a platform for discovering cell type-specific hypoxia-induced response mechanisms. Through comparative molecular profiling across cell types under normoxic and hypoxic conditions, we provide insight into the indispensable role of HIF-1α in the promotion of ischemic vascular regeneration. We found human MSCs to be the vascular cell type most susceptible to HIF-1α deficiency, and that transcriptional inactivation of ANKZF1, an effector of HIF-1α, impaired pro-angiogenic processes. Altogether, our findings deepen the understanding of HIF-1α in human angiogenesis and support further explorations of novel therapeutic strategies of vascular regeneration against ischemic damage.

Spontaneous spheroids from alveolar bone-derived mesenchymal stromal cells maintain pluripotency of stem cells by regulating hypoxia-inducible factors

BACKGROUND: Spontaneous spheroid culture is a novel three-dimensional (3D) culture strategy for the rapid and efficient selection of progenitor cells. The objectives of this study are to investigate the pluripotency and differentiation capability of spontaneous spheroids from alveolar bone-derived mesenchymal stromal cells (AB-MSCs); compare the advantages of spontaneous spheroids to those of mechanical spheroids; and explore the mechanisms of stemness enhancement during spheroid formation from two-dimensional (2D) cultured cells. METHODS: AB-MSCs were isolated from the alveolar bones of C57BL/6 J mice. Spontaneous spheroids formed in low-adherence specific culture plates. The stemness, proliferation, and multi-differentiation capacities of spheroids and monolayer cultures were investigated by reverse transcription quantitative polymerase chain reaction (RT-qPCR), immunofluorescence, alkaline phosphatase (ALP) activity, and oil-red O staining. The pluripotency difference between the spontaneous and mechanical spheroids was analyzed using RT-qPCR. Hypoxia-inducible factor (HIFs) inhibition experiments were performed to explore the mechanisms of stemness maintenance in AB-MSC spheroids. RESULTS: AB-MSCs successfully formed spontaneous spheroids after 24 h. AB-MSC spheroids were positive for MSC markers and pluripotency markers (Oct4, KLF4, Sox2, and cMyc). Spheroids showed higher Ki67 expression and lower Caspase3 expression at 24 h. Under the corresponding conditions, the spheroids were successfully differentiated into osteogenic and adipogenic lineages. AB-MSC spheroids can induce neural-like cells after neurogenic differentiation. Higher expression of osteogenic markers, adipogenic markers, and neurogenic markers (NF-M, NeuN, and GFAP) was found in spheroids than in the monolayer. Spontaneous spheroids exhibited higher stemness than mechanical spheroids did. HIF-1α and HIF-2α were remarkably upregulated in spheroids. After HIF-1/2α-specific inhibition, spheroid formation was significantly reduced. Moreover, the expression of the pluripotency genes was suppressed. CONCLUSIONS: Spontaneous spheroids from AB-MSCs enhance stemness and pluripotency. HIF-1/2α plays an important role in the stemness regulation of spheroids. AB-MSC spheroids exhibit excellent multi-differentiation capability, which may be a potent therapy for craniomaxillofacial tissue regeneration.

Advances in regulation of hypoxia on adipocyte development and lipid metabolism / 生物工程学报

The growth, differentiation and proliferation of adipose cells run through the whole life process. Dysregulation of lipid metabolism in adipose cells affects adipose tissue immunity and systemic energy metabolism. Increasingly available data suggest that lipid metabolism is involved in regulating the occurrence and development of various diseases, such as hyperlipidemia, nonalcoholic fatty liver disease, diabetes and cancer, which pose a major threat to human and animal health. Hypoxia inducible factor (HIF) is a major transcription factor mediating oxygen receptors in tissues and organs. HIF can induce disease by regulating lipid synthesis, fatty acid metabolism and lipid droplet formation. However, due to the difference of hypoxia degree, time and mode of action, there is no conclusive conclusion whether it has harmful or beneficial effects on the development of adipocytes and lipid metabolism. This article summarizes the regulation of hypoxia stress mediated transcription regulators and regulation of adipocyte development and lipid metabolism, aiming to reveal the potential mechanism of hypoxia induced changes in adipocyte metabolism pathways.

Neuroglobin Facilitates Neuronal Oxygenation through Tropic Migration under Hypoxia or Anemia in Rat: How Does the Brain Breathe? / 神经科学通报·英文版

The discovery of neuroglobin (Ngb), a brain- or neuron-specific member of the hemoglobin family, has revolutionized our understanding of brain oxygen metabolism. Currently, how Ngb plays such a role remains far from clear. Here, we report a novel mechanism by which Ngb might facilitate neuronal oxygenation upon hypoxia or anemia. We found that Ngb was present in, co-localized to, and co-migrated with mitochondria in the cell body and neurites of neurons. Hypoxia induced a sudden and prominent migration of Ngb towards the cytoplasmic membrane (CM) or cell surface in living neurons, and this was accompanied by the mitochondria. In vivo, hypotonic and anemic hypoxia induced a reversible Ngb migration toward the CM in cerebral cortical neurons in rat brains but did not alter the expression level of Ngb or its cytoplasm/mitochondria ratio. Knock-down of Ngb by RNA interference significantly diminished respiratory succinate dehydrogenase (SDH) and ATPase activity in neuronal N2a cells. Over-expression of Ngb enhanced SDH activity in N2a cells upon hypoxia. Mutation of Ngb at its oxygen-binding site (His64) significantly increased SDH activity and reduced ATPase activity in N2a cells. Taken together, Ngb was physically and functionally linked to mitochondria. In response to an insufficient oxygen supply, Ngb migrated towards the source of oxygen to facilitate neuronal oxygenation. This novel mechanism of neuronal respiration provides new insights into the understanding and treatment of neurological diseases such as stroke and Alzheimer's disease and diseases that cause hypoxia in the brain such as anemia.

A comprehensive review of natural products with anti-hypoxic activity / 中国天然药物

Natural products exhibit substantial impacts in the field of anti-hypoxic traetment. Hypoxia can cause altitude sickness and other negative effect on the body. Headache, coma, exhaustion, vomiting and, in severe cases, death are some of the clinical signs. Currently, hypoxia is no longer just a concern in plateau regions; it is also one of the issues that can not be ignored by urban residents. This review covered polysaccharides, alkaloids, saponins, flavonoids, peptides and traditional Chinese compound prescriptions as natural products to protect against hypoxia. The active ingredients, effectiveness and mechanisms were discussed. The related anti-hypoxic mechanisms involve increasing the hemoglobin (HB) content, glycogen content and adenosine triphosphate (ATP) content, removing excessive reactive oxygen species (ROS), reducing lipid peroxidation, regulating the levels of related enzymes in cells, protecting the structural and functional integrity of the mitochondria and regulating the expression of apoptosis-related genes. These comprehensive summaries are beneficial to anti-hypoxic research and provide useful information for the development of anti-hypoxic products.

Bloodletting Acupuncture at Jing-Well Points Alleviates Myocardial Injury in Acute Altitude Hypoxic Rats by Activating HIF-1α/BNIP3 Signaling-Mediated Mitochondrial Autophagy and Decreasing Oxidative Stress / 中国结合医学杂志

OBJECTIVE@#To explore the protective effect and possible mechanisms of bloodletting acupuncture at Jing-well points (BAJP) pre-treatment on acute hypobaric hypoxia (AHH)-induced myocardium injury rat.@*METHODS@#Seventy-five rats were randomly divided into 5 groups by a random number table: a control group (n=15), a model group (n=15), a BAJP group (n=15), a BAJP+3-methyladenine (3-MA) group (n=15), and a BANA (bloodletting at nonacupoint; tail bleeding, n=15) group. Except for the control group, the AHH rat model was established in the other groups, and the corresponding treatment methods were adopted. Enzyme-linked immunosorbent assay (ELISA) was used to detect creatine kinase isoenzyme MB (CK-MB) and cardiac troponins I (CTnI) levels in serum and superoxide dismutase (SOD) and malondialdehyde (MDA) levels in myocardial tissue. Hematoxylin-eosin (HE) staining was used to observe myocardial injury, and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) staining was used to observe cell apoptosis. Transmission electron microscopy detection was used to observe mitochondrial damage and autophagosomes in the myocardium. The mitochondrial membrane potential of the myocardium was analyzed with the fluorescent dye JC-1. Mitochondrial respiratory chain complex (complex I, III, and IV) activities and ATPase in the myocardium were detected by mitochondrial respiratory chain complex assay kits. Western blot analysis was used to detect the autophagy index and hypoxia inducible factor-1α (HIF-1α)/Bcl-2 and adenovirus E1B 19k Da-interacting protein 3 (BNIP3) signaling.@*RESULTS@#BAJP reduced myocardial injury and inhibited myocardial cell apoptosis in AHH rats. BAJP pretreatment decreased MDA levels and increased SOD levels in AHH rats (all P<0.01). Moreover, BAJP pretreatment increased the mitochondrial membrane potential (P<0.01), mitochondrial respiratory chain complex (complexes I, III, and IV) activities (P<0.01), and mitochondrial ATPase activity in AHH rats (P<0.05). The results from electron microscopy demonstrated that BAJP pretreatment improved mitochondrial swelling and increased the autophagosome number in the myocardium of AHH rats. In addition, BAJP pretreatment activated the HIF-1α/BNIP3 pathway and autophagy. Finally, the results of using 3-MA to inhibit autophagy in BAJP-treated AHH rats showed that suppression of autophagy attenuated the treatment effects of BAJP in AHH rats, further proving that autophagy constitutes a potential target for BAJP treatment of AHH.@*CONCLUSION@#BAJP is an effective treatment for AHH-induced myocardial injury, and the mechanism might involve increasing HIF-1α/BNIP3 signaling-mediated autophagy and decreasing oxidative stress.

Effect of Hypoxia-Supported Umbilical Cord Mesenchymal Stem Cells on the Expansion of Cord Blood Mononuclear Cells in vitro / 中国实验血液学杂志

OBJECTIVE@#To explore the effect of hypoxia-supported umbilical cord mesenchymal stem cell (UC-MSC) on the expansion of cord blood mononuclear cell （MNC） in vitro.@*METHODS@#The isolated cord blood mononuclear cells were inoculated on the preestablished umbilical cord mesenchymal stem cell layer and cultured under hypoxic conditions (3％ O2) and the experimental groups were normoxia (MNCs were cultured under normoxic conditions), hypoxia (MNCs were cultured under hypoxic conditions), UC-MSC (MNCs were cultured with UC-MSC under normoxic conditions), and UC-MSC+hypoxia (MNCs were cultured with UC-MSC under hypoxic conditions). To further investigate the combinational effect of 3 factors of SCF+FL+TPO (SFT) on expansion of cord blood MNCs in vitro in hypoxia-supported UC-MSC culture system, the experiments were further divided into group A (MNCs were cultured with UC-MSC and SFT under normoxic conditions), group B (MNCs were cultured with UC-MSC under hypoxic conditions), group C (MNCs were cultured with UC-MSC and SFT under hypoxic conditions). The number of nucleated cells (TNC), CD34+ cell, CFU and CD34+CXCR4+, CD34+CD49d+, CD34+CD62L+ cells of each groups were detected at 0, 7, 10 and 14 days, respectively.@*RESULTS@#Compared with group hypoxia and UC-MSC, group UC-MSC+hypoxia effectively promoted the expansion of TNC, CD34+ cell and CFU, and upregulated the expression level of adhesion molecule and CxCR4 of the cord blood CD34+ cell（P<0.05）. After culturing for 14 days, compared with group A and group B, group C effectively promoted the expansion of cord blood MNC at different time points（P<0.05）, and the effect of group A was better than that of group B at 7 and 10 days（P<0.05）.@*CONCLUSION@#Hypoxia-supported UC-MSC efficiently promoted the expansion and expression of adhesion molecule and CXCR4 of cord blood CD34+ cell, and the effect of expansion could be enhanced when SFT 3 factors were added.

iPSCs ameliorate hypoxia-induced autophagy and atrophy in C2C12 myotubes via the AMPK/ULK1 pathway

BACKGROUND: Duchenne muscular dystrophy (DMD) is an X-linked lethal genetic disorder for which there is no effective treatment. Previous studies have shown that stem cell transplantation into mdx mice can promote muscle regeneration and improve muscle function, however, the specific molecular mechanisms remain unclear. DMD suffers varying degrees of hypoxic damage during disease progression. This study aimed to investigate whether induced pluripotent stem cells (iPSCs) have protective effects against hypoxia-induced skeletal muscle injury. RESULTS: In this study, we co-cultured iPSCs with C2C12 myoblasts using a Transwell nested system and placed them in a DG250 anaerobic workstation for oxygen deprivation for 24 h. We found that iPSCs reduced the levels of lactate dehydrogenase and reactive oxygen species and downregulated the mRNA and protein levels of BAX/BCL2 and LC3II/ LC3I in hypoxia-induced C2C12 myoblasts. Meanwhile, iPSCs decreased the mRNA and protein levels of atrogin-1 and MuRF-1 and increased myotube width. Furthermore, iPSCs downregulated the phosphorylation of AMPKA and ULK1 in C2C12 myotubes exposed to hypoxic damage. CONCLUSIONS: Our study showed that iPSCs enhanced the resistance of C2C12 myoblasts to hypoxia and inhibited apoptosis and autophagy in the presence of oxidative stress. Further, iPSCs improved hypoxia-induced autophagy and atrophy of C2C12 myotubes through the AMPK/ULK1 pathway. This study may provide a new theoretical basis for the treatment of muscular dystrophy in stem cells.

Efectos biológicos de la exposición a distintas concentraciones de oxígeno: desde la hipoxia hipobárica al oxígeno hiperbárico / Biological effects due to exposure to different concentrations of oxygen from hypo to hyperoxemia

The systemic effects of oxygen deficiency or excess are not thoroughly described. Knowledge is evolving towards the description of beneficial and detrimental effects of both extremes of partial pressure of oxygen (PaO2). The cellular and tissue mediators derived from the modulation of the oxidative tone and the production of reactive oxygen species (ROS) are widely characterized biochemically, but the pathophysiological characterization is lacking. Preclinical models support the use of hypobaric hypoxia preconditioning, based on its beneficial effects on ventricular function or its reduction in infarct size. A very important use of oxygen today is in commercial diving. However, novel clinical indications for oxygen such as the healing of diabetic foot ulcers and bone injury caused by radiotherapy are increasingly used. On the other hand, the modulation of the hypoxic response associated with exposure to high altitude environments (hypobaric), favors Chile and its highlands as a natural laboratory to determine certain cardiovascular, cerebral and metabolic responses in the resident population. Also, the consequences of the intermittent exposure to high altitudes in workers also deserves attention. This review discusses the physiopathological response to hypo and hyperoxemia, associated with environments with different oxygen concentrations, and brings back the concept of oxygen as a pharmacological mediator in extreme environments such as high altitudes and hyperbaric medicine in divers, decompression sickness, osteonecrosis associated with radiotherapy and sudden sensorineural hearing loss.

Qili Qiangxin, a compound herbal medicine formula, alleviates hypoxia-reoxygenation-induced apoptotic and autophagic cell death via suppression of ROS/AMPK/mTOR pathway in vitro / 中西医结合学报

OBJECTIVE@#Qili Qiangxin (QLQX), a compound herbal medicine formula, is used effectively to treat congestive heart failure in China. However, the molecular mechanisms of the cardioprotective effect are still unclear. This study explores the cardioprotective effect and mechanism of QLQX using the hypoxia-reoxygenation (H/R)-induced myocardial injury model.@*METHODS@#The main chemical constituents of QLQX were analyzed using high-performance liquid chromatography-evaporative light-scattering detection. The model of H/R-induced myocardial injury in H9c2 cells was developed to simulate myocardial ischemia-reperfusion injury. Apoptosis, autophagy, and generation of reactive oxygen species (ROS) were measured to assess the protective effect of QLQX. Proteins related to autophagy, apoptosis and signalling pathways were detected using Western blotting.@*RESULTS@#Apoptosis, autophagy and the excessive production of ROS induced by H/R were significantly reduced after treating the H9c2 cells with QLQX. QLQX treatment at concentrations of 50 and 250 μg/mL caused significant reduction in the levels of LC3II and p62 degradation (P < 0.05), and also suppressed the AMPK/mTOR signalling pathway. Furthermore, the AMPK inhibitor Compound C (at 0.5 μmol/L), and QLQX (250 μg/mL) significantly inhibited H/R-induced autophagy and apoptosis (P < 0.01), while AICAR (an AMPK activator, at 0.5 mmol/L) increased cardiomyocyte apoptosis and autophagy and abolished the anti-apoptotic effect of QLQX. Similar phenomena were also observed on the expressions of apoptotic and autophagic proteins, demonstrating that QLQX reduced the apoptosis and autophagy in the H/R-induced injury model via inhibiting the AMPK/mTOR pathway. Moreover, ROS scavenger, N-Acetyl-L-cysteine (NAC, at 2.5 mmol/L), significantly reduced H/R-triggered cell apoptosis and autophagy (P < 0.01). Meanwhile, NAC treatment down-regulated the ratio of phosphorylation of AMPK/AMPK (P < 0.01), which showed a similar effect to QLQX.@*CONCLUSION@#QLQX plays a cardioprotective role by alleviating apoptotic and autophagic cell death through inhibition of the ROS/AMPK/mTOR signalling pathway.

Danlou Tablet Improves Chronic Intermittent Hypoxia-Induced Dyslipidemia and Arteriosclerosis by HIF-1α-Angptl4 mRNA Signaling Pathway / 中国结合医学杂志

OBJECTIVE@#To detect whether Danlou Tablet (DLT) regulates the hypoxia-induced factor (HIF)-1α-angiopoietin-like 4 (Angptl4) mRNA signaling pathway and explore the role of DLT in treating chronic intermittent hypoxia (CIH)-induced dyslipidemia and arteriosclerosis.@*METHODS@#The mature adipocytes were obtained from 3T3-L1 cell culturation and allocated into 8 groups including control groups (Groups 1 and 5, 0.1 mL of cell culture grade water); DLT groups (Groups 2 and 6, 0.1 mL of 1,000 µg/mL DLT submicron powder solution); dimethyloxalylglycine (DMOG) groups (Groups 3 and 7, DMOG and 0.1 mL of cell culture grade water); DMOG plus DLT groups (Groups 4 and 8, DMOG and 0.1 mL of 1,000 µg/mL DLT submicron powder solution). Groups 1-4 used mature adipocytes and groups 5-8 used HIF-1 α-siRNA lentivirus-transfected mature adipocytes. After 24-h treatment, real-time polymerase chain reaction and Western blot were employed to determine the mRNA and protein expression levels of HIF-1 α and Angptl4. In animal experiments, the CIH model in ApoE-/- mice was established. Sixteen mice were complete randomly divided into 4 groups including sham group, CIH model group [intermittent hypoxia and normal saline (2 mL/time) gavage once a day]; Angptl4 Ab group [intermittent hypoxia and Angptl4 antibody (30 mg/kg) intraperitoneally injected every week]; DLT group [intermittent hypoxia and DLT (250 mg/kg) once a day], 4 mice in each group. After 4-week treatment, enzyme linked immunosorbent assay was used to detect the expression levels of serum total cholesterol (TC) and triglyceride (TG). Hematoxylin-eosin and CD68 staining were used to observe the morphological properties of arterial plaques.@*RESULTS@#Angptl4 expression was dependent on HIF-1 α, with a reduction in mRNA expression and no response in protein level to DMOG or DLT treatment in relation to siHIF-1 α -transfected cells. DLT inhibited HIF-1 α and Angptl4 mRNA expression (P<0.05 or P<0.01) and reduced HIF-1 α and Angptl4 protein expressions with DMOG in mature adipocytes (all P<0.01), as the effect on HIF-1 α protein also existed in the presence of siHIF-1 α (P<0.01). ApoE-/- mice treated with CIH had increased TG and TC levels (all P<0.01) and atherosclerotic plaque. Angptl4 antibody and DLT both reduce TG and TC levels (all P<0.01), as well as reducing atherosclerotic plaque areas, narrowing arterial wall thickness and alleviating atherosclerotic lesion symptoms to some extent.@*CONCLUSION@#DLT had positive effects in improving dyslipidemia and arteriosclerosis by inhibiting Angptl4 protein level through HIF-1 α-Angptl4 mRNA signaling pathway.

Hypoxia promotes differentiation of human induced pluripotent stem cells into embryoid bodies in vitro / 南方医科大学学报

OBJECTIVE@#To investigate effects of physiological hypoxic conditions on suspension and adherence of embryoid bodies (EBs) during differentiation of human induced pluripotent stem cells (hiPSCs) and explore the underlying mechanisms.@*METHODS@#EBs in suspension culture were divided into normoxic (21% O2) and hypoxic (5% O2) groups, and those in adherent culture were divided into normoxic, hypoxic and hypoxia + HIF-1α inhibitor (echinomycin) groups. After characterization of the pluripotency with immunofluorescence assay, the hiPSCs were digested and suspended under normoxic and hypoxic conditions for 5 days, and the formation and morphological changes of the EBs were observed microscopically; the expressions of the markers genes of the 3 germ layers in the EBs were detected. The EBs were then inoculated into petri dishes for further culture in normoxic and hypoxic conditions for another 2 days, after which the adhesion and peripheral expansion rate of the adherent EBs were observed; the changes in the expressions of HIF-1α, β-catenin and VEGFA were detected in response to hypoxic culture and echinomycin treatment.@*RESULTS@#The EBs cultured in normoxic and hypoxic conditions were all capable of differentiation into the 3 germ layers. The EBs cultured in hypoxic conditions showed reduced apoptotic debris around them with earlier appearance of cystic EBs and more uniform sizes as compared with those in normoxic culture. Hypoxic culture induced more adherent EBs than normoxic culture (P < 0.05) with also a greater outgrowth rate of the adherent EBs (P < 0.05). The EBs in hypoxic culture showed significantly up-regulated mRNA expressions of β-catenin and VEGFA (P < 0.05) and protein expressions of HIF-1 α, β-catenin and VEGFA (P < 0.05), and their protein expresisons levels were significantly lowered after treatment with echinomycin (P < 0.05).@*CONCLUSION@#Hypoxia can promote the formation and maturation of suspended EBs and enhance their adherence and post-adherent proliferation without affecting their pluripotency for differentiation into all the 3 germ layers. Our results provide preliminary evidence that activation of HIF-1α/β-catenin/VEGFA signaling pathway can enhance the differentiation potential of hiPSCs.

Overexpression of bHLH domain of HIF-1 failed to inhibit the HIF-1 transcriptional activity in hypoxia

BACKGROUND: Hypoxia inducible factor-1 (HIF-1) is considered as the most activated transcriptional factor in response to low oxygen level or hypoxia. HIF-1 binds the hypoxia response element (HRE) sequence in the promoter of different genes, mainly through the bHLH domain and activates the transcription of genes, especially those involved in angiogenesis and EMT. Considering the critical role of bHLH in binding HIF-1 to the HRE sequence, we hypothesized that bHLH could be a promising candidate to be targeted in hypoxia condition. METHODS: We inserted an inhibitory bHLH (ibHLH) domain in a pIRES2-EGFP vector and transfected HEK293T cells with either the control vector or the designed construct. The ibHLH domain consisted of bHLH domains of both HIF-1a and Arnt, capable of competing with HIF-1 in binding to HRE sequences. The transfected cells were then treated with 200 µM of cobalt chloride (CoCl2) for 48 h to induce hypoxia. Real-time PCR and western blot were performed to evaluate the effect of ibHLH on the genes and proteins involved in angiogenesis and EMT. RESULTS: Hypoxia was successfully induced in the HEK293T cell line as the gene expression of VEGF, vimentin, and ß-catenin were significantly increased after treatment of untransfected HEK293T cells with 200 µM CoCl2. The gene expression of VEGF, vimentin, and ß-catenin and protein level of ß-catenin were significantly decreased in the cells transfected with either control or ibHLH vectors in hypoxia. However, ibHLH failed to be effective on these genes and the protein level of ß-catenin, when compared to the control vector. We also observed that overexpression of ibHLH had more inhibitory effect on gene and protein expression of N-cadherin compared to the control vector. However, it was not statistically significant. CONCLUSION: bHLH has been reported to be an important domain involved in the DNA binding activity of HIF. However, we found that targeting this domain is not sufficient to inhibit the endogenous HIF-1 transcriptional activity. Further studies about the function of critical domains of HIF-1 are necessary for developing a specific HIF-1 inhibitor.

Effect of miR-134 against myocardial hypoxia/reoxygenation injury by directly targeting NOS3 and regulating PI3K/Akt pathway

Abstract Purpose To reveal the function of miR-134 in myocardial ischemia. Methods Real-time PCR and western blotting were performed to measure the expression of miR-134, nitric oxide synthase 3 (NOS3) and apoptotic-associated proteins. Lactic dehydrogenase (LDH) assay, cell counting kit-8 (CCK-8), Hoechst 33342/PI double staining and flow cytometry assay were implemented in H9c2 cells, respectively. MiR-134 mimic/inhibitor was used to regulate miR-134 expression. Bioinformatic analysis and luciferase reporter assay were utilized to identify the interrelation between miR-134 and NOS3. Rescue experiments exhibited the role of NOS3. The involvement of PI3K/AKT was assessed by western blot analysis. Results MiR-134 was high regulated in the myocardial ischemia model, and miR-134 mimic/inhibitor transfection accelerated/impaired the speed of cell apoptosis and attenuated/exerted the cell proliferative prosperity induced by H/R regulating active status of PI3K/AKT signaling. LDH activity was also changed due to the different treatments. Moreover, miR-134 could target NOS3 directly and simultaneously attenuated the expression of NOS3. Co-transfection miR-134 inhibitor and pcDNA3.1-NOS3 highlighted the inhibitory effects of miR-134 on myocardial H/R injury. Conclusion This present work puts insights into the crucial effects of the miR-134/NOS3 axis in myocardial H/R injury, delivering a potential therapeutic technology in future.

The long non-coding RNA H19 induces hypoxia/reoxygenation injury by up-regulating autophagy in the hepatoma carcinoma cells

BACKGROUND: Long non-coding RNA H19 (H19) plays an important role by regulating protein expression in different tissues and organs of the body. However, whether H19 induces hypoxia/reoxygenation (h/R) injury via increase of autophagy in the hepatoma carcinoma cells is unknown. RESULTS: H19 was expressed in the hepatoma carcinoma cells (Hep G2 and HCCLM3 cells) and its expression was most in 8 h/24R. The knockdown of H19 and 3-MA (an autophagy inhibitor) protected against h/R-induced apoptosis, cell damage, the expression of cleaved caspase-3 and cleaved caspase-9, the release of cytochrome c (Cyt c). The knockdown of H19 and 3-MA also decreased the autophagic vesicles (AVs) and the expression of Beclin-1 and the ration of LC3-II/LC3-I, and increased cell viability, the expression of Bcl-2 and P62 and the phosphorylation of PI3K, Akt and mTOR. In addition, chloroquine (CQ, an inhibitor of autophagy flux) markedly decreased formation of autophagy flux (the ration of LC3-II/LC3-I). The results of the knockdown of H19 group were similar to those of the 3-MA (or CQ) group. Rapamycin (a mTOR inhibitor, an autophagy activator) further down-regulated h/R-induced decrease of the phosphorylated PI3K, Akt and mTOR. The knockdown of H19 cancelled the effect of rapamycin. The overexpression of H19 further expanded h/R-induced increase of the ration of LC3-II/LC3-I and decrease of the phosphorylated PI3K, Akt and mTOR. CONCLUSIONS: Our results suggest that the long non-coding RNA H19 induces h/R injury by up-regulation of autophagy via activation of PI3K-Akt-mTOR pathway in the hepatoma carcinoma cells.

Oxidative stress assessment in intestine of newborn rats submitted to hypoxia and reoxygenation with tadalafil

Abstract Purpose: To evaluate the functional and structural response of tadalafil effects in the intestinal mucosa, using an experimental model of hypoxia and reoxygenation injury in rats. Methods: The animals were divided into 4 groups: CTL, H/R, H/R+Td and M+Td. The newborn rats allocated in groups H/R, H/R+Td and M+Td were submitted twice a day, to a gas chamber with CO2 at 100% for 10 minutes and afterward reoxygenation with O2 at 98% for 10 minutes, in the three first days of life. Tadalafil dose was given to newborn of group H/R+Td and to the pregnant rat of group M+Td. Histological analysis was made with hematoxylin-eosin technique and oxidative stress through nitrite and nitrate levels and lipid peroxidation. Results: The histological analysis showed a reduction of mucosa alterations in the groups that received tadalafil. In the oxidative stress evaluation, occurred an increase of NO levels and less lipidic peroxidation in the ileum segments that received tadalafil. Conclusion: Tadalafil provides tissue protection when administered independently to both, pregnant or newborns.

Activación de STAT3 por hipoxia en modelos in vitro de cáncer de cuello uterino y en células endoteliales / STAT3 activation by hypoxia in in vitro models of cervix cancer and endothelial cells

resumen Introducción: El microambiente tumoral influye en el comportamiento de las células cancerosas. Especialmente, el estímulo de agentes estresantes, como la hipoxia, se convierte en un factor crítico para la evolución y el tratamiento del cáncer. La reacción celular frente a diversos estímulos se manifiesta en la activación de vías de señalización como la JAK/STAT, una de las más importantes por sus efectos en la diferenciación y proliferación celular. Objetivo: Evaluar el estado de la vía JAK/STAT mediante la expresión o activación de la proteína STAT3 en células de cáncer de cuello uterino (HeLa) y en células endoteliales (EA.hy926) sometidas a hipoxia. Materiales y métodos: Las líneas celulares se sometieron a condiciones de hipoxia física (1 % de O2) o química (100 μM de deferoxamina, DFO) durante dos, seis y 24 horas. Mediante Western blot se determinó el cambio en la expresión y activación de STAT3, y mediante inmunofluorescencia indirecta su localización subcelular. Resultados:. La hipoxia se evidenció por la activación y translocación al núcleo del HIF-1. Ni la hipoxia física ni la química alteraron la expresión de STAT3, pero sí la activación, según se comprobó por su fosforilación y su translocación al núcleo en los dos modelos bajo estudio. Conclusiones: Se evidenció la importancia de la hipoxia como un estímulo que modifica la activación de la proteína STAT3 en las células HeLa y EA.hy926, lo cual la convierte en un elemento importante en el diseño de estrategias terapéuticas contra el cáncer.

Abstract Introduction: The biological behavior of cancer cells is influenced by the tumor microenvironment in which they develop. In this context, stressor stimuli such as hypoxia are considered critical for tumor development and therapeutic management. Cellular response to various stimuli is evidenced in the activation of intracellular signaling pathways such as JAK/STAT, which is one of the most important for its effects in differentiation and cell proliferation. Objective: To evaluate the condition of the JAK/STAT pathway through the expression/activation of the STAT3 protein in cervix cancer cells (HeLa) and endothelial cells (EA.hy926) subjected to ypoxia. Material and methods: Cell lines were subjected to physical (1% O2) or chemical (deferoxamine, DFO, 100 μM) hypoxia for 2, 6 and 24 hours. Changes in the expression and activation of STAT3, and its subcellular localization by indirect immunofluorescence, were determined by western blot. Results: Hypoxia was evidenced by the activation and translocation to the nucleus of HIF-1. Neither physical nor chemical hypoxia altered STAT3 expression, but it did affect its activation, as seen in its phosphorylation and translocation to the nucleus in the two models under study. Conclusions: The present study highlights the importance of hypoxia as a stimulus that modifies the activation of the STAT3 protein in HeLa and EA.hy926 cells, which makes it an important factor in the design of therapeutic strategies against cancer.

Importance of SERCA2a on early isolated diastolic dysfunction induced by supravalvular aortic stenosis in rats

Cardiac remodeling is defined as changes in shape and function of the heart in response to aggression (pressure overload). The sarcoplasmic reticulum calcium ATPase cardiac isoform 2a (SERCA2a) is a known factor that influences function. A wide spectrum of studies report a decrease in SERCA2a in heart failure, but none evaluate it's the role in early isolated diastolic dysfunction in supravalvular aortic stenosis (AoS). Our hypothesis was that SERCA2a participates in such dysfunction. Thirty-day-old male Wistar rats (60-80 g) were divided into AoS and Sham groups, which were submitted to surgery with or without aorta clipping, respectively. After 6 weeks, the animals were submitted to echocardiogram and functional analysis by isolated papillary muscle (IPM) in basal condition, hypoxia, and SERCA2a blockage with cyclopiazonic acid at calcium concentrations of 0.5, 1.5, and 2.5 mM. Western-blot analyses were used for SERCA2a and phospholamban detection. Data analysis was carried out with Student's t-test and ANOVA. AoS enhanced left atrium and E and A wave ratio, with preserved ejection fraction. Basal condition in IPM showed similar increases in developed tension (DT) and resting tension (RT) in AoS, and hypoxia was similar between groups. After cyclopiazonic acid blockage, final DT was equally decreased and RT was similar between groups, but the speed of relaxation was decreased in the AoS group. Western-blot was uniform in all evaluations. The hypothesis was confirmed, since functional parameters regarding SERCA2a were changed in the AoS group.

Inactivated Pseudomonas aeruginosa inhibits hypoxia-induced pulmonary hypertension by preventing TGF-beta1/Smad signaling

Pseudomonas aeruginosa is one of the common colonizing bacteria of the human body and is an opportunistic pathogen frequently associated with respiratory infections. Inactivated P. aeruginosa (IPA) have a variety of biological effects against inflammation and allergy. Transforming growth factor-β (TGF-β) signaling plays a critical role in the regulation of cell growth, differentiation, and development in a wide range of biological systems. The present study was designed to investigate the effects of IPA on TGF-β/Smad signaling in vivo, using a hypoxia-induced pulmonary hypertension (PH) rat model. Sprague Dawley rats (n=40) were exposed to 10% oxygen for 21 days to induce PH. At the same time, IPA was administered intravenously from day 1 to day 14. Mean pulmonary artery pressure (mPAP) and the right ventricle (RV) to left ventricle plus the interventricular septum (LV+S) mass ratio were used to evaluate the development of PH. Vessel thickness and density were measured using immunohistochemistry. Primary arterial smooth muscle cells (PASMCs) were isolated and the proliferation of PASMCs was assayed by flow cytometry. The production of TGF-β1 in cultured supernatant of PASMCs was assayed by ELISA. The expression levels of α-smooth muscle actin (α-SMA), TGF-β1 and phospho-Smad 2/3 in PASMCs were assayed by western blot. Our data indicated that IPA attenuated PH, RV hypertrophy and pulmonary vascular remodeling in rats, which was probably mediated by restraining the hypoxia-induced overactive TGF-β1/Smad signaling. In conclusion, IPA is a promising protective treatment in PH due to the inhibiting effects on TGF-β1/Smad 2/3 signaling.