Notch1 signaling modulates hypoxia-induced multidrug resistance in human laryngeal cancer cells.

BACKGROUND: Laryngeal carcinoma is one of the common malignant tumors of the head and neck. Multidrug resistance (MDR) remains a critical problem in the chemotherapy of patients with laryngeal cancer. This study aims to clarify the role and mechanisms of Notch1 signaling in MDR induced by hypoxia in laryngeal cancer cells. METHODS AND RESULTS: Laryngeal carcinoma cells were cultured under normoxia or hypoxia. Notch1 expression was inhibited by small interfering RNA (siRNA). The mRNA expression of Notch1, Hes1, Hey1, MDR1 and survivin was analyzed by real-time PCR. The protein expression of Notch1, the Notch1 intracellular domain (N1ICD), MDR1/P-gp and survivin was analyzed by Western blotting. Current research has shown that hypoxia can upregulate Notch1 expression and Notch1 signaling activity. Furthermore, suppression of Notch1 expression effectively downregulated Notch1 signaling activity and the expression of the MDR and survivin genes in laryngeal cancer cells under hypoxic conditions (P < 0.05). The Cell Counting Kit-8 (CCK-8) assay results confirmed that the sensitivity of hypoxic laryngeal cancer cells to a variety of drugs could be upregulated by suppressing Notch1 expression (P < 0.05). Additionally, flow cytometry (FCM) showed that suppression of Notch1 expression significantly increased drug-induced apoptosis and intracellular rhodamine 123 (Rh123) accumulation in hypoxic laryngeal carcinoma cells (P < 0.05). CONCLUSIONS: Notch1 signalling could be regarded as a pivotal regulator of hypoxia-induced MDR in laryngeal cancer cells through the regulation of survivin-mediated apoptosis resistance and MDR1/P-gp-mediated drug transport.

Effect of Notch1 signaling on cellular proliferation and apoptosis in human laryngeal carcinoma.

BACKGROUND: The occurrence and development of malignancies include excessive proliferation and apoptosis resistance in tumor cells. This study aimed to identify the effects of Notch1 signaling on proliferation and apoptosis of laryngeal cancer cells in a hypoxic microenvironment. METHODS: Notch1 and Ki-67 expression in laryngeal squamous cell carcinoma (LSCC) tissues was detected by immunohistochemistry. The apoptotic index (AI) of LSCC was evaluated by the TUNEL method. Small interfering RNA (siRNA) was used to inhibit Notch1 expression in laryngeal cancer cells. Real-time PCR was used to measure Notch1, Hes1, and Hey1 mRNA expression, and Western blotting was used to measure Notch1 and Notch1 intracellular domain (N1ICD) protein expression. Annexin V-FITC/propidium iodide staining and Cell Counting Kit-8 assays were used to measure cell apoptosis and proliferation, respectively. RESULTS: Notch1 expression was significantly related to the proliferation index (PI) and AI in LSCC tissues. Hypoxia could induce proliferation and inhibit apoptosis in cancer cells. Notch1 expression and Notch1 signaling activity could be upregulated by hypoxia. Suppressing Notch1 signaling activity in hypoxic cells could decrease proliferation and increase apoptosis. CONCLUSIONS: Our study has demonstrated that hypoxia may promote proliferation and inhibit apoptosis of laryngeal cancer cells. Notch1 signaling may play a pivotal role in regulating the proliferation and apoptosis resistance of laryngeal cancer cells under hypoxic conditions.

Lower limit of adequate oxygen delivery for the maintenance of aerobic metabolism during cardiopulmonary bypass in neonates.

BACKGROUND: The objective of cardiopulmonary bypass (CPB) is to maintain an adequate balance between oxygen delivery (Do2) and consumption. The critical Do2 is that at which consumption becomes supply dependent. This study aimed to identify the critical Do2 in neonates, who have higher metabolic rates than adults. METHODS: In a retrospective cohort of neonates, Do2 was calculated from CPB parameters recorded during aortic cross-clamping. High lactate concentration measured after aortic unclamping (lactOFF) was used to identify anaerobic metabolism. Data were analysed using mixed linear and proportional odds regression models. The relationship between Do2 and temperature was analysed in a subgroup of patients with lactOFF <2.5 mM, thought to have had balanced oxygen delivery and consumption. The estimated regression coefficient was further used to adjust hypothetical Do2 thresholds, and Do2 excursions below the threshold were quantified as magnitude-durations. The lowest threshold that provided magnitude-durations and linked with an increase in lactOFF was used as the lowest suitable (critical) Do2 at 37°C. RESULTS: Overall, 22 896 time points were analysed in 180 neonates. In 40 patients with lactOFF <2.5 mM, Do2 varied by 22.87 (0.70) ml min-1 m-2 °C-1. When varying the Do2 threshold between 340 and 380 ml min-1 m-2, excursions below the threshold were linked with incremental lactOFF. A 100 ml m-2 excursion below the 340 ml min-1 m-2Do2 threshold increased the risk of a 1 mM increment in lactOFF by 22% (odds ratio: 1.22; 95% confidence interval: 1.02-1.45). CONCLUSIONS: It was found that 340 ml min-1 m-2 is likely to represent the lowest suitable Do2 required in neonates to maintain aerobic metabolism during normothermic CPB.

Hyperoxygenated Hydrogen-Rich Solution Suppresses Lung Injury Induced by Hemorrhagic Shock in Rats.

BACKGROUND: Hemorrhagic shock could induce acute lung injury (ALI), which is associated with cell hypoxia, lung tissue inflammation, free radical damage, and excessive cell apoptosis. Our previous studies demonstrated that hyperoxygenated solution could alleviate cell hypoxia. Furthermore, hydrogen-rich solution (HS) could relieve lung tissue inflammation, free radical damage and excessive cell apoptosis. Therefore we hypothesize that Hyperoxygenated Hydrogen-rich solution (HOHS) can protect the lung against ALI. MATERIALS AND METHODS: SD rats were randomly divided into five groups (n = 6 at each time point in each group) and were exposed to Hemorrhagic shock induced ALI, and then treated with lactated Ringer's solution (LRS), hyperoxygenated solution, HS, and HOHS, respectively. The protective effects of these solutions were assessed using methods as follows: arterial blood samples were collected for blood gas analysis; Bronchoalveolar lavage fluid was collected for cell count and protein quantification; lung tissue samples were collected to measure wet/dry ratio, as well as levels of T-SOD, MDA, TNF-α, and IL-6; Caspase-3 and TUNEL-positive cells, and pathological changes were observed under light microscope; ALI was scored using the Smith scoring method; ultrastructural changes of lung tissues were further observed with transmission electron microscopy. RESULTS: The results indicated that PaO2, PaCO2, and T-SOD increased in the three treatment groups (P < 0.05), most significantly in the HOHS group (P < 0.01) compared with the LRS group; and conversely that the levels of lactate, MDA, TNF-α and IL-6, cell count, protein content, caspase-3 and TUNEL-positive cells as well as ALI score decreased in the three treatment groups (P < 0.05), most significantly in the HOHS group (P < 0.01) compared with the LRS group. Morphological observation with optical microscope and electron microscopy showed that compared with the LRS group, cell damage in the three treatment groups improved to a varying extent, especially evident in the HOHS group. CONCLUSIONS: These findings demonstrate that HOHS can protect the lung against ALI induced by hemorrhagic shock.

[Effect of HIF-2α mediated FoxM1 expression on proliferation of pulmonary artery smooth muscle cells in hypoxic rats].

Objective: To investigate the effect of hypoxia-inducible factor 2α (HIF-2α) gene on the expression of Forkhead box M1 (FoxM1) protein in the proliferation of hypoxic rat pulmonary artery smooth muscle cells (PASMC). Methods: HIF-2α overexpression lentiviral vector (LV-HIF-2α) and silencing RNA (siRNA) were constructed and transfected into rat PASMC under normoxia and hypoxia, respectively. The PASMC under normoxia were classified into normoxic control group, normoxia + LV-HIF-2α empty group, normoxia + LV-HIF-2α group; the PASMC under hypoxia were classified into hypoxic control group, hypoxia + siRNA-HIF-2α empty group, hypoxia + siRNA-HIF-2α group. The expression of HIF-2α and its downstream proteins FoxM1, cyclin D1 and Aurora A expressions were detected by Western blot. 5-Ethyny-2'-deoxyuridine (EdU) cell proliferation assay was used to evaluate the effect of overexpression and inhibition of HIF-2α expression on the proliferation of rat PASMC. Results: The expression of HIF-2α in normoxia + LV-HIF-2α group was significantly higher than that in normoxic control group and normoxia+LV-HIF-2α empty group (0.17±0.02 vs 0.09±0.01 and 0.07±0.00), while the expression of HIF-2α in PASMC of hypoxia + siRNA-HIF-2α group was significantly lower than that of hypoxic control group and hypoxia + siRNA-HIF-2α empty group (0.28±0.01 vs 0.35±0.02 and 0.30±0.01) (all P<0.05); the expression of FoxM1 protein, cyclinD1 and cell proliferation-related Aurora A protein in normoxia+LV-HIF-2α group were significantly higher than that in normoxic control group and normoxia+LV-HIF-2α empty group (0.40±0.03 vs 0.24±0.01 and 0.30±0.01, 0.22±0.02 vs 0.09±0.01 and 0.08±0.02, 0.29±0.02 vs 0.04±0.01 and 0.07±0.01, respectively) (all P<0.05); the expressions of FoxM1 protein, cyclinD1 and Aurora A protein in hypoxia + siRNA-HIF-2α group were significantly lower than those in hypoxic control group and hypoxia + siRNA-HIF-2α empty group (0.23±0.01 vs 0.36±0.02 and 0.32±0.01, 0.15±0.01 vs 0.31±0.01 and 0.28±0.03, 0.14±0.02 vs 0.33±0.03 and 0.27±0.02, respectively) (all P<0.05); the positive expression rate of EdU in the normoxic control group was significantly lower than that in the normoxia+LV-HIF-2α group [(30.77±2.43)% vs (55.56±3.01)%], while the hypoxic control group was significantly higher than the hypoxic+siRNA-HIF-2α group [(65.28±3.21)% vs (44.64±2.78)%] (both P<0.05). Conclusion: HIF-2α up-regulates the expression of FoxM1 and promotes the proliferation of pulmonary artery smooth muscle cells in hypoxic rats.

[Human umbilical mesenchymal stem cells-derived exosomes modulate the proliferation, apoptosis and migration of human retinal pigment epithelial cells in hypoxia].

Objective: To study the effects of human umbilical mesenchymal stem cells (HUMSCs) exosomes on the proliferation and apoptotic as well as migration of human retinal pigment epithelial cells (HRPE) in hypoxia, and explore its mechanism. Method: Direct adherent culture was adopted to cultivate umbilical cord mesenchymal stem cells and amplified to the fourth generation. Markers on the cell surface were identified by flow cytometry. Culture medium was collect without serum from the 4th generation umbilical cord mesenchymal stem cells. Exosomes were separated and extracted, then the ultrastructure was observed under electron microscope and examined expression of CD63 and CD9 protein by Western blot method with isolated and extracted exosomes. HRPE was cultivated in vitro culture, proliferation was detected at the time point of 0, 1, 2, 3, 4, 5 d with MTT assay under hypoxic condition. Meanwhile, the cell migration was quantified by Wound-Healing Assay under hypoxic condition at 0, 24, 48 and 72 h respectively combined with apoptosis test. The HRPE cells in the growth period were divided into 5 groups: the control group, the hypoxia group and the pretreated exosomes group (100, 200, 300 µg/ml). In all groups, apoptosis was observed by Annexin V/PI dual-dye flow cytometry after 48 h's incubation. Proliferation was observed by MTT assay and the migration was observed with Wound-Healing Assay. Results: Flow cytometry detection of the surface marker of HUMSCs in the 4th generation showed strong positive expression of CD105, CD73, CD90. It was suggested that HUMSCs with isolated culture had MSC specific phenotype with duction of lipids and osteoblasts in vitro. The separated exosomes were observed with spherical membranous structures in different sizes by scanning electron microscopy, and Western blot detected positive expression of CD63 and CD9. In vitro culture of HRPE detected by MTT assay for cell proliferation at the time of hypoxic 0, 1, 2, 3, 4, 5 d, the results showed that, comparing with time point 0 d, other groups had statistically significant OD values. In the first 2 days, the proliferation ability of RPE cells gradually increased as the time of hypoxia prolonged(1.862±0.135, 2.278±0.244). After 3 d, the proliferation ability of RPE cells gradually decreased(1.419±0.124, 1.599±0.156). Wound-Healing Assay results showed that the migration distance gradually increased as[(29.883±4.504), (36.200±1.928) µm] the time of hypoxia increased from 0 to 72 h. The cells were fully covering at the point of 72 h [(1.223±0.194), (0.430±0.299) µm]. Apoptosis test results showed that the number of apoptotic cells was different(3.628%±1.348%, 20.123%±1.183%) with the extension of hypoxia Oxygen before 2 d from 0 to 72 h. At the time of d3, there were more apoptotic cells(42.290%±3.217%). There is a significant difference from pre-2d.RPE cells were divided into 5 groups: the control group, the hypoxia group and the pretreated exosomes group (100, 200, 300 µg/ml).After 48 h hypoxia incubation, MTT assay results showed that, compared with the control group (1.870±0.499), the number of cell proliferation was significantly increased (t=-3.116, P<0.05), while compared with the hypoxia group(2.616±0.307), the proliferation number of exosomes was significantly reduced [(2.041±0.115), (1.931±0.205), (1.929±0.025); t=-4.920, -4.540, -5.286, P<0.01], and there was no significant difference between groups with different doses of the exosomes (F=1.181,P>0.05). Annexin V/PI dual-dye flow cytometry was used to observe the apoptosis results. Compared with the control group 1.180%±0.689%, the number of apoptosis in hypoxia group was significantly increased (19.273%±1.194%, t=-32.141, P<0.01), while compared with the hypoxia group, the number of apoptosis in the exosomes was significantly decreased (12.318%±1.087%, 11.878%±1.348%, 11.090%±1.716%; t=-10.547, -10.057, 9.589, P<0.01). There was no significant difference between the groups with different doses of exosomes (F=1.173, P>0.05). Wound-Healing Assay results showed that, compared with the control group(68.047±2.851) µm, the migration distance of the hypoxia group was significantly increased [(13.470±2.255)µm, t=36.778, P<0.01] while compared with the hypoxia group, the migration distance of the exosomes was reduced (33.110±1.774, 24.650±1.175, 26.440±1.674; t=11.766, 10.770, 11.311, P<0.01), and there was no significant difference between the groups of the exosomes (F=1.179, P>0.05). Conclusion: Human umbilical cord mesenchymal stem cells can effectively inhibit the apoptosis and migration of HRPE cells in hypoxia. It provides a theoretical basis for the research and treatment of RPE related diseases. (Chin J Ophthalmol, 2019, 55: 933-941).

Induction of the mitochondrial NDUFA4L2 protein by HIF-1a regulates heart regeneration by promoting the survival of cardiac stem cell.

The adult mammalian heart doesn't regenerate after cardiomyocyte injury, which was mainly caused by the severe and persistent effects of cardiomyopathy. Recently, some studies reported that the mammalian heart can regenerate under low oxygen environment. However, the mechanism that the mammalian heart can regenerate remains unknown. Here, we used cardiac stem cells (CSCs) to be planted in serum-free medium under hypoxia environment to understand the mechanism of HIF1α/NDUFA4L2 in the regulation of hypoxia-alleviated apoptosis. Our results revealed that hypoxia can alleviated CSCs apoptosis. Hypoxia inhibited the level of cleaved-caspase3 and stimulated the expression of stabilized HIF-1α. DMOG promotes the survival of CSCs and the protein expression of NDUFA4L2. 2-ME repressed the survival of CSCs and the protein expression of NDUFA4L2. CHIP assay showed that HIF-1α regulated the survival of CSCs by augmenting the combination of HIF-1α and NDUFA4L2's HRE. Knockdown of NDUFA4L2 reversed the role of hypoxia in the survival of CSCs. Taken together, hypoxia promotes the viability of CSCs in serum-free medium by HIF-1α/NDUFA4L2 signaling pathway.

[MicroRNA-1249 regulates the apoptosis of myocardial cells in rats with chronic intermittent hypoxia by autophagy].

Objective: To investigate the regulation and possible mechanism of microRNA (miR)-1249 on myocardial apoptosis in chronic intermittent hypoxia rats. Methods: A total of 16male SD rats aged 8 weeks were randomly divided into 2 groups by the random number table: normoxia control group and chronic intermittent hypoxia group (CIH) (n=8 each). The CIH group was exposed to intermittent hypoxia every day from 9: 00 to 17: 00 for 8 consecutive weeks, while the control group received the same frequency of pulse air. Hemodynamic values were measured via a cannula inserted into right common carotid artery. The expressions of miR-1249 and microtubule-associated protein light chain 3 (LC3) mRNA were observed by real-time PCR. The expressions of LC3 and Cleaved Caspase-3 were detected by Western bolt. TUNEL staining was performed to detect myocardial apoptosis. The rat cardiomyocyte cell H9C2 was divided into normoxia group, intermittent hypoxia (IH) group and miR-1249 inhibitor transfected and IH treatment group (inhibitor group). At the end of the experiment, the activation of LC3 protein in each group of cells was determined. Results: Compared with normoxia control group, left ventricle end diastolic pressure (LVEDP) increased [(4.6±0.4) vs (2.2±0.1) mmHg (1 mmHg=0.133 kPa)], left ventricular systolic pressure (LVSP) , maximal rate of pressure decline (-dp/dtmax), and maximal rate of pressure development (+ dp/dtmax) decreased in CIH group [(92.7±4.1) vs (135.3±3.2) mmHg, (4 247±108) vs (7 626±235) mmHg/s, and (3 168±105) vs (6 028±81) mmHg/s] (all P<0.001). The expression of miR-1249 and LC3 mRNA were significantly higher in CIH group than that in normoxia control group (all P<0.001), and a positive correlation was found between the expression of LC3 mRNA and miR-1249. The expression of LC3 and Cleaved Caspase-3 protein in myocardial tissue of CIH rats were significantly higher than that of the normoxia control group (all P<0.001). The proportion of myocardial cell apoptosis in CIH rats was significantly higher than that in the normoxia control group [(23.84±4.94)% vs (2.93±0.73)%] (P<0.001). The activation of LC3 in myocardial cells of inhibitor group was significantly lower than that of IH group, but higher than that in normoxia group. Conclusions: CIH could induce LC3 by raising the expression of miR-1249, and then induce the activation of apoptosis protein Caspase3. It ultimately induces myocardial apoptosis.

[TGF-ß3 improves bone mesenchymal stem cells toward chondrogenic differentiation under hypoxia environment].

Objective: To investigate the impact of TGF-ß3 on the chondrogenesis of bone marrow mesenchymal stem cells (BM-MSCs) under hypoxia environment. Methods: BM-MSCs were obtained from SD rat tibias and femora and cultured with whole bone marrow adherent method. Cell surface antigens were analyzed by flow cytometry and the multiple-directional differentiation capabilities were detected with special differentiation agents to affirm the reality of BM-MSCs. Under normoxia or hypoxia condition, BM-MSCs were induced with TGF-ß3 or not. Then, alcian blue and immunofluorescence staining were performed to evaluate the expression level of aggrecan, collagen Ⅱ. qRT-PCR analysis were performed to analyze the expression of aggrecan, collagen Ⅱ and collagen Ⅹ. qRT-PCR and Western blot analysis was performed to detect the mRNA and protein level of HIF-1α, collagenⅡ and ß-catenin. Results: BM-MSCs were fibroblast-like shape and had ablities of osteogeic, adipogenic and chondrogenic differentiation, with the expression of CD(29, )CD(44) and CD(90) but not CD(45). Alcian blue and immunofluorescence staining showed that BM-MSCs strongly expressed the aggrecan and collagen Ⅱ with the presence of TGF-ß3 under hypoxia condition. qRT-PCR analysis showed the mRNA expression levels of collagen Ⅱ, aggrecan and collagen Ⅹ were up-regulated at 2.46, 2.20 and 1.80 folds, comparing with control group (all P<0.05). Western blot analysis showed that the protein levels of HIF-1α, collagenⅡ in BM-MSCs were up-regulated with the presence of TGF-ß3 under hypoxia condition, but ß-catenin level was down-regulated. Conclusion: TGF-ß3 promotes the chondrogenic differentiation ability of BM-MSCs under hypoxia condition, which may be relative with the inhibition of Wnt/ß-catenin signaling pathway.

[Expression of high-mobility group box-â and N-methyl-D-aspartate receptor in status epilepticus].

Objective: To investigate the expression changes of high mobility group box-1 (HMGB1) and the 2B receptor of N- methyl -D- aspartate receptor (NR2B) in status epilepticus (SE). Methods: (1) Primary hippocampal neurons from SD rats with 16 to 18 days of fetal age were cultured in vitro for 7 days, and exposed to Mg(2+) free media for 3 hours. Those cultured neurons were randomly divided into control group and intermittent hypoxia group. (2) SD rats with similar weight were selected and randomly divided into control group and SE model group. The rat model with SE was established by an intraperitoneal injection of lithium chloride-piloearpine (LI-PILO). Real-time PCR technique was used to detect the expression of HMGB1 and NR2B mRNA. Results: In Sombati's cell model cultured in normal concentration of oxygen, the HMGB1 mRNA expression levels were 0.005 01±0.000 54, 0.026 76±0.003 75, 0.003 52±0.000 33, and the NR2B mRNA expression levels were 0.008 84±0.000 69, 0.012 23±0.000 90, 0.029 11±0.000 71, respectively, at 2, 4 and 6 h; compared with the expressions of HMGB1 and NR2B mRNA at the same time points of Sombatis cell model groups, the differences were also significant (all P<0.05). After the successful establishment of epilepsy model, the HMGB1 mRNA expression levels were 0.000 11±0.000 09, 0.000 18±0.000 01, 0.000 11±0.000 01, and the NR2B mRNA expression levels were 0.196 12±0.009 41, 0.232 11±0.006 27, 0.272 48±0.005 84, respectively, at 6, 8 and 10 h; compared with the expressions of HMGB1 and NR2B mRNA at the same time points of control groups, the differences were all significant (all P<0.05). Conclusion: HMGB1 mRNA expression levels increase at 2, 4 h, decrease at 6 h in the Sombati's cell model in normal oxygen culture, while increase at 6, 8 h, and decrease at 10 h in LI-PILO induced rat model with SE; the NR2B mRNA relative expression increases with time in both the Sombati's cell model in normal oxygen culture and rat model of SE.

Endothelial cells co-cultured with renal carcinoma cells significantly reduce RECK expression under chemical hypoxia.

Renal cell carcinoma (RCC) is characterized by excessive angiogenesis, while chronic kidney disease (CKD) suffers from the opposite problem-failure of reparative angiogenesis. It can be due to their different responses to hypoxic environment. But the specific molecular regulators are still unclear. This study is aimed to explore the influence of human renal cell cancer cells (786-0) and human renal tubular epithelial cells (HK-2) on RECK expression, proliferation, and angiogenesis of adjacent microvascular endothelial cells (HMEC-1) under chemical hypoxia. Cobalt chloride (CoCl2 ) treatment was used to simulate the hypoxia environment in RCC and CKD. Co-culture, cell proliferation assay, and tube formation assay were used to evaluate the influence of 786-0 or HK-2 cells on proliferation and angiogenesis of adjacent HMEC-1 cells. Effects of different environments on RECK expressions in 786-0, HK2, or HMEC-1 cells were determined by Western blot. We found that both 786-0 cells and HK2 cells can upregulate RECK expression of adjacent HMEC-1 cells in normoxic conditions. However, under hypoxia, the HMEC-1 cells co-cultured with 786-0 significantly reduced RECK expression and there was no significant change in HMEC-1 cells co-cultured with HK2 cells. We also found that 786-0 significantly enhanced the proliferation and angiogenesis of adjacent HMEC-1 cells. Our results suggested that some paracrine substances produced by 786-0 cells may reduce RECK expression of adjacent HMEC-1 cells and enhance their proliferation and in vitro angiogenic capacity.

Hypoxic Gene Expression of Donor Bronchi Linked to Airway Complications after Lung Transplantation.

RATIONALE: Central airway stenosis (CAS) after lung transplantation has been attributed in part to chronic airway ischemia; however, little is known about the time course or significance of large airway hypoxia early after transplantation. OBJECTIVES: To evaluate large airway oxygenation and hypoxic gene expression during the first month after lung transplantation and their relation to airway complications. METHODS: Subjects who underwent lung transplantation underwent endobronchial tissue oximetry of native and donor bronchi at 0, 3, and 30 days after transplantation (n = 11) and/or endobronchial biopsies (n = 14) at 30 days for real-time polymerase chain reaction of hypoxia-inducible genes. Patients were monitored for 6 months for the development of transplant-related complications. MEASUREMENTS AND MAIN RESULTS: Compared with native endobronchial tissues, donor tissue oxygen saturations (Sto2) were reduced in the upper lobes (74.1 ± 1.8% vs. 68.8 ± 1.7%; P < 0.05) and lower lobes (75.6 ± 1.6% vs. 71.5 ± 1.8%; P = 0.065) at 30 days post-transplantation. Donor upper lobe and subcarina Sto2 levels were also lower than the main carina (difference of -3.9 ± 1.5 and -4.8 ± 2.1, respectively; P < 0.05) at 30 days. Up-regulation of hypoxia-inducible genes VEGFA, FLT1, VEGFC, HMOX1, and TIE2 was significant in donor airways relative to native airways (all P < 0.05). VEGFA, KDR, and HMOX1 were associated with prolonged respiratory failure, prolonged hospitalization, extensive airway necrosis, and CAS (P < 0.05). CONCLUSIONS: These findings implicate donor bronchial hypoxia as a driving factor for post-transplantation airway complications. Strategies to improve airway oxygenation, such as bronchial artery re-anastomosis and hyperbaric oxygen therapy merit clinical investigation.

[Viability of osteoblasts under cell hypoxia condition].

Objective: To investigate the effects of hypoxia condition and hypoxia-reoxygenation condition on the cell viability, apoptosis rate and gene expression of osteoblasts cultured in vitro. Methods: The cranium osteoblasts from newborn Sprague Dawley rats within 48 hours were cultured and purified through tissue block method.The morphological changes of cells were evaluated by the Alizarin Red S staining and Alkaline phosphatase staining.The third-generation osteoblasts were cultured in normal condition for 36 hours (group A), in hypoxic condition for 24hours (group B), in hypoxic condition for 24hours thereafter reoxygenated for 12 hours (group D), in hypoxic condition for 36 hours (group C). The cell viability of osteoblasts was tested via MTT assay.The apoptosis rate of osteoblasts was tested by FCM (flow cytometry). Quantitative PCR and Western blot methods were used to determine Collagen type Ⅰ, Bone morphogenetic protein 2 (BMP-2), Runt-related transcription factor 2 (RUNX-2), Transforming growth factor-ß1(TGF-ß1) expression levels. Results: The cell viability of osteoblasts decreased, group A(99.1%±8.3%), group B(90.9%±9.4%), group C(79.9%±8.7%), group D(73.0%±8.2%), group D group C >group B >group A(F=26.198, P=0.000); the mRNA expressions of Col Ⅰ, BMP-2, RUNX-2, TGF-ß1 decreased under hypoxia and hypoxia-reoxygenation condition, and group D< group C< group B< group A (F=13.082, P=0.006; F=7.088, P=0.017; F=6.857, P=0.038; F=51.368, P=0.000); the protein expressions of Col Ⅰ, BMP-2, RUNX-2, TGF-ß1 decreased under hypoxia and hypoxia-reoxygenation condition, and group D< group C< group B< group A (F=8.114, P=0.013; F=28.935, P=0.000; F=9.857, P=0.007; F=46.541, P=0.000). Conclusion: Hypoxia condition and hypoxia-reoxygenation condition decreased the cell viability, increase the apoptosis rate and suppress the expressions of associated genes.Hypoxia-reoxygenation condition aggravate the injure of osteoblasts preconditioning under hypoxia condition.

[Effects of chronic intermittent hypoxia on left ventricular myocardial contractibility in a rabbit model of obstructive sleep apnea].

Objective: To explore the effects of chronic intermittent hypoxia (CIH) on left ventricular myocardial contractibility in a rabbit model of obstructive sleep apnea (OSA). Methods: Based on random number table, twenty-four rabbits were randomly divided into three groups: operation, sham, and control groups, each with 8 rabbits. The rabbit model for OSA in operation group was established by repeatedly closing the airway and then reopening it. Upper airway obstructions were conducted on rabbits every day, which were alternately closed for 15 s and then reopened for 75 s in a 90 s-long cycle, for 8 h each day over 3 months. The sham rabbits were subjected to the same surgical procedure but no airway obstructions were applied. The control animals were subjected to no intervention. The blood pressure, left ventricular function parameters were assessed before and after the experiment. And the relative expressions of myosin heavy chain α/ß (α-MHC and ß-MHC) mRNAs in myocardium were observed for all rabbits by real time fluorescent quantitative polymerase chain reaction 3 months later. Results: After 3 months, all rabbits in the operation group manifested sleepiness and the blood pressure rose gradually [(114.25±4.20) vs (93.88±2.10) mmHg, P<0.01]. Compared with the sham operation and the control groups, the left ventricular end-systolic volume [(6.05±1.62) vs (2.83±0.49) and (2.74±0.32) ml, P<0.001] and the left ventricular end-diastolic volume [(1.61±0.78) vs (0.83±0.13) and (0.82±0.10) ml] in operation group were obviously higher, the left ventricular ejection fraction [(63.9±4.2) % vs (74.3±2.5) % and (75.8±3.8) %], left ventricular fractional shortening [(32.2±2.1) % vs (41.8±1.8) % and (42.1±1.8) %] and stroke volume [(1.46±0.13) vs (1.93±0.21) and (1.98±0.24) ml/s] were decreased (all P<0.001). Besides, the maximal rate of the increase of left ventricular pressure [(4 154±360) vs (6 802±492) and (6 759±206) mmHg/s], the maximal rate of the decrease of left ventricular pressure [(4 994±621) vs (6 330±314) and (6 591±225) mmHg/s] in the operation group decreased markedly, left ventricular end diastolic pressure (LVEDP) increased [(6.5±1.6) vs (3.3±0.8) and (3.2±0.9) mmHg] (all P<0.001). The relative expression of α-MHC mRNA in left ventricular myocardial tissue was lower and the relative expression of ß-MHC mRNA was higher in operation group than those in the sham operation and the control groups (P<0.05). There were no significant difference in the relevant indicators of cardiac function, and in the relative expressions of α-MHC mRNA and the ß-MHC mRNA between the control group and the sham operation group (P>0.05). Conclusions: Repeated intermittent hypoxia can induce hypertension and myocardial contractibility damage in OSA model rabbit simulating upper airway obstruction.

[Effects of Prescription Yiqi Huatan Quyu on oxidative stress level and pathological changes in chronic intermittent hypoxia rat liver].

Objective: To investigate the effects of Yiqi Huatan Quyu prescription on oxidative stress and pathological changes in chronic intermittent hypoxia rats liver. Methods: A total of 24 male SD rats aged eight weeks were randomly divided into 3 groups by the random number table: normal control group (NC), chronic intermittent hypoxia group (CIH) and chronic intermittent hypoxia plus Yiqi Huatan Quyu prescription supplement group (Chinese medicine group, CM)(n=8 each). The CIH group and CIHN group were exposed to intermittent hypoxia every day for 8 consecutive weeks. In addition, the CIHN group received Yiqi Huatan Quyu prescription via gavage 30 min before the CIH. Eight weeks later, serums were collected to test alanine aminotransferase (ALT) and aspartate aminotransferase (AST) level. Meanwhile, rat livers were extracted to evaluate malondialdehyde (MDA), total superoxide dismutase (T-SOD), glutathione (GSH) and Nuclear factor κB (NF-κB) and observe the pathological changes of liver. Results: The levels of serum ALT and AST in the CIH group were significantly higher than those of NC group[(112.1±31.7) vs (41.3±6.6) U/L, (295.4±39.8) vs (104.5±12.3) U/L], the contents of the MDA and NF-κB in the CIH group were notably higher than those of NC group[(7.9±1.1) vs (3.6±0.7) nmol/mgprot, (16.2±3.3) vs (5.7±2.2) ng/L], the activities of T-SOD and GSH in the CIH group were significantly lower than those of control group[(181.6±28.6) vs (304.3±42.9) U/mgprot, (5.5±1.3) vs (14.9±2.4) mg/gprot](all P<0.001). The levels of the serum ALT and AST in CM group were significantly lower than those of CIH group[(52.2±8.6) vs (112.1±31.7) U/L, (148.1±20.4) vs (295.4±39.8) U/L], the contents of liver MDA in CM group was notably lower than those of CIH group[(4.5±0.7) vs (7.9±1.1) nmol/mgprot], the activities of liver T-SOD and GSH in CM group were significantly higher than those of CIH group[(226.9±38.9) vs (181.6±28.6) U/mgprot, (10.3±1.7) vs (5.5±1.3) mg/gprot](all P<0.05). Compared with CIH group, declining tendency was observed from contents of NF-κB in CM group, but there was no statistical difference[(15.8±2.1) vs (16.2±3.3) ng/L, P>0.05]. Pathology observation showed the CIH group had abundant inflammatory cells infiltration in the liver portal area and the inflammatory cells infiltration decreased following Chinese medicine treatments. Conclusion: Yiqi Huatan Quyu prescription has some protection to CIH rat liver, which might mediate by the reduction of oxidative stress and inflammatory mediator.

[Role of reactive oxygen species in hypoxia-induced non-small cell lung cancer migration].

Objective: To explore reactive oxygen species (ROS) generation and its role on A549 cell migration under hypoxic condition. Methods: Human non-small cell lung cancer (NSCLC) cell line A549 was incubated in a hypoxic environment (1%O(2), hypoxia group) or in a normoxic environment (21%O(2), normoxia group). The generation of ROS was measured by flow cytometry. The cell motility of A549 cells was detected by Transwell assay. The protein levels of protein kinase B (AKT), p38 mitogen-activated protein kinase (p38) were determined by Western blot analysis. Results: After 16 h hypoxic treatment, the migration of A549 cells in hypoxia group was significantly more than that of normoxia group [(85±10) vs (56±7) per lower magnification, P<0.001]. Besides, the generation of ROS was in a time-depended manner in hypoxia group. The ROS level was increased with the prolonged hypoxia time. It was significantly higher at 24 h than that in normoxia group [(273±4)% vs (102±6)%, P<0.001]. The migrated cells in hypoxia group co-treated with 2 mmol/L NAC for 16 h were less than that with hypoxic treatment alone [(47±13) vs (105±14) per lower magnification, P=0.011]. Meanwhile, the phosphorylation of AKT and p38 increased after 12 h hypoxic treatment in hypoxia group, however, 2 mmol/L NAC co-treatment attenuated this effect. Furthermore, inhibition of phosphorylated AKT with 0.1 µmol/L allosteric AKT inhibitor (MK-2206) in hypoxia group for 16 h reversed the hypoxia-induced A549 cell migration. The migrated cells in hypoxia+ MK-2206 group were significantly less than that in hypoxia group [(155±21) vs (249±32) per lower magnification, P<0.001]. Conclusions: Hypoxia increases the generation of ROS in A549, resulting from oxidative stress under hypoxia. The increased ROS level promotes cell motility through the activation of AKT.

Upregulation of steroidogenic acute regulatory protein by hypoxia stimulates aldosterone synthesis in pulmonary artery endothelial cells to promote pulmonary vascular fibrosis.

BACKGROUND: The molecular mechanism(s) regulating hypoxia-induced vascular fibrosis are unresolved. Hyperaldosteronism correlates positively with vascular remodeling in pulmonary arterial hypertension, suggesting that aldosterone may contribute to the pulmonary vasculopathy of hypoxia. The hypoxia-sensitive transcription factors c-Fos/c-Jun regulate steroidogenic acute regulatory protein (StAR), which facilitates the rate-limiting step of aldosterone steroidogenesis. We hypothesized that c-Fos/c-Jun upregulation by hypoxia activates StAR-dependent aldosterone synthesis in human pulmonary artery endothelial cells (HPAECs) to promote vascular fibrosis in pulmonary arterial hypertension. METHODS AND RESULTS: Patients with pulmonary arterial hypertension, rats with Sugen/hypoxia-pulmonary arterial hypertension, and mice exposed to chronic hypoxia expressed increased StAR in remodeled pulmonary arterioles, providing a basis for investigating hypoxia-StAR signaling in HPAECs. Hypoxia (2.0% FiO2) increased aldosterone levels selectively in HPAECs, which was confirmed by liquid chromatography-mass spectrometry. Increased aldosterone by hypoxia resulted from enhanced c-Fos/c-Jun binding to the proximal activator protein-1 site of the StAR promoter in HPAECs, which increased StAR expression and activity. In HPAECs transfected with StAR-small interfering RNA or treated with the activator protein-1 inhibitor SR-11302 [3-methyl-7-(4-methylphenyl)-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraenoic acid], hypoxia failed to increase aldosterone, confirming that aldosterone biosynthesis required StAR activation by c-Fos/c-Jun. The functional consequences of aldosterone were confirmed by pharmacological inhibition of the mineralocorticoid receptor with spironolactone or eplerenone, which attenuated hypoxia-induced upregulation of the fibrogenic protein connective tissue growth factor and collagen III in vitro and decreased pulmonary vascular fibrosis to improve pulmonary hypertension in vivo. CONCLUSION: Our findings identify autonomous aldosterone synthesis in HPAECs attributable to hypoxia-mediated upregulation of StAR as a novel molecular mechanism that promotes pulmonary vascular remodeling and fibrosis.

Impact of S-adenosylmethionine decarboxylase 1 on pulmonary vascular remodeling.

BACKGROUND: Pulmonary hypertension (PH) is a life-threatening disease characterized by vascular remodeling and increased pulmonary vascular resistance. Chronic alveolar hypoxia in animals is often used to decipher pathways being regulated in PH. Here, we aimed to investigate whether chronic hypoxia-induced PH in mice can be reversed by reoxygenation and whether possible regression can be used to identify pathways activated during the reversal and development of PH by genome-wide screening. METHODS AND RESULTS: Mice exposed to chronic hypoxia (21 days, 10% O2) were reoxygenated for up to 42 days. Full reversal of PH during reoxygenation was evident by normalized right ventricular pressure, right heart hypertrophy, and muscularization of small pulmonary vessels. Microarray analysis from these mice revealed s-adenosylmethionine decarboxylase 1 (AMD-1) as one of the most downregulated genes. In situ hybridization localized AMD-1 in pulmonary vessels. AMD-1 silencing decreased the proliferation of pulmonary arterial smooth muscle cells and diminished phospholipase Cγ1 phosphorylation. Compared with the respective controls, AMD-1 depletion by heterozygous in vivo knockout or pharmacological inhibition attenuated PH during chronic hypoxia. A detailed molecular approach including promoter analysis showed that AMD-1 could be regulated by early growth response 1, transcription factor, as a consequence of epidermal growth factor stimulation. Key findings from the animal model were confirmed in human idiopathic pulmonary arterial hypertension. CONCLUSIONS: Our study indicates that genome-wide screening in mice from a PH model in which full reversal of PH occurs can be useful to identify potential key candidates for the reversal and development of PH. Targeting AMD-1 may represent a promising strategy for PH therapy.

Inhibition of endogenous hydrogen sulfide production in clear-cell renal cell carcinoma cell lines and xenografts restricts their growth, survival and angiogenic potential.

Clear cell renal cell carcinoma (ccRCC) is characterized by Von Hippel-Lindau (VHL)-deficiency, resulting in pseudohypoxic, angiogenic and glycolytic tumours. Hydrogen sulfide (H2S) is an endogenously-produced gasotransmitter that accumulates under hypoxia and has been shown to be pro-angiogenic and cytoprotective in cancer. It was hypothesized that H2S levels are elevated in VHL-deficient ccRCC, contributing to survival, metabolism and angiogenesis. Using the H2S-specific probe MeRhoAz, it was found that H2S levels were higher in VHL-deficient ccRCC cell lines compared to cells with wild-type VHL. Inhibition of H2S-producing enzymes could reduce the proliferation, metabolism and survival of ccRCC cell lines, as determined by live-cell imaging, XTT/ATP assay, and flow cytometry respectively. Using the chorioallantoic membrane angiogenesis model, it was found that systemic inhibition of endogenous H2S production was able to decrease vascularization of VHL-deficient ccRCC xenografts. Endogenous H2S production is an attractive new target in ccRCC due to its involvement in multiple aspects of disease.

Prolyl hydroxylase domain protein 2 plays a critical role in diet-induced obesity and glucose intolerance.

BACKGROUND: Recent studies suggest that the oxygen-sensing pathway consisting of transcription factor hypoxia-inducible factor and prolyl hydroxylase domain proteins (PHDs) plays a critical role in glucose metabolism. However, the role of adipocyte PHD in the development of obesity has not been clarified. We examined whether deletion of PHD2, the main oxygen sensor, in adipocytes affects diet-induced obesity and associated metabolic abnormalities. METHODS AND RESULTS: To delete PHD2 in adipocyte, PHD2-floxed mice were crossed with aP2-Cre transgenic mice (Phd2(f/f)/aP2-Cre). Phd2(f/f)/aP2-Cre mice were resistant to high-fat diet-induced obesity (36.7±1.7 versus 44.3±2.0 g in control; P<0.01) and showed better glucose tolerance and homeostasis model assessment-insulin resistance index than control mice (3.6±1.0 versus 11.1±2.1; P<0.01). The weight of white adipose tissue was lighter (epididymal fat, 758±35 versus 1208±507 mg in control; P<0.01) with a reduction in adipocyte size. Macrophage infiltration into white adipose tissue was also alleviated in Phd2(f/f)/aP2-Cre mice. Target genes of hypoxia-inducible factor, including glycolytic enzymes and adiponectin, were upregulated in adipocytes of Phd2(f/f)/aP2-Cre mice. Lipid content was decreased and uncoupling protein-1 expression was increased in brown adipose tissue of Phd2(f/f)/aP2-Cre mice. Knockdown of PHD2 in 3T3L1 adipocytes induced a decrease in the glucose level and an increase in the lactate level in the supernatant with upregulation of glycolytic enzymes and reduced lipid accumulation. CONCLUSIONS: PHD2 in adipose tissue plays a critical role in the development of diet-induced obesity and glucose intolerance. PHD2 might be a novel target molecule for the treatment of obesity and associated metabolic abnormalities.