Hypoxia For 6h Research Articles

Abstract TP314: The α5 Integrin Inhibitor ATN161 Mitigates SARS-CoV-2- Induced Brain Endothelial Barrier Disruption and Neuroinflammation α5 Integrin Inhibitor ATN 161

Introduction: Although SARS-CoV-2 infection can cause a wide range of mild to severe symptoms, the pathophysiology of acute and long-term neurological manifestations remains elusive. The arginine-glycine-aspartic acid motif of the viral spike protein may use to bind integrins receptors in the CNS, which play an important role in cerebrovascular integrity. Here we investigate the role of integrin α5β1 in mediating brain endothelial damage and inflammation during SARS-CoV-2 infection. Method: Mouse brain microvascular endothelial cells (bEnd.3) were treated with SARS-CoV-2 (Isolate USA-WA1/2020) or delta variant spike protein for 24h then later exposed to hypoxia for 6h (to model the effects of in vivo pulmonary infection). Cells were pretreated with ATN-16, 1h before SARS-CoV-2 and hypoxia challenge. Further, BALB/c mice were inoculated intranasally with 2x10 4 -PFU of the MA-10 strain of SARS-CoV-2 and treated with 1mg/kg of ATN-161, an α5β1 integrin inhibitor, retro-orbitally. The brains were collected 3 days post-infection for neuropathological evaluation. Results: SARS-CoV-2 and delta variant spike protein inoculations induced integrin α5 and decreased claudin-5 expression in bEnd.3 cells in a dose-dependent manner. SARS-CoV-2 spike protein challenge at 0.5 μg for 24h followed by hypoxia for 6h resulted in increased α5 and decreased claudin-5 expression in either hypoxia or SARS-CoV-2+hypoxia combination. ATN-161 (10μM) pretreatment inhibited SARS-CoV-2+hypoxia-induced α5 upregulation and restored claudin-5 loss in bEnd.3 cells. The in vivo studies showed a significant increase in the pro-inflammatory response as measured by cytokine IL-6 expression and a decrease in barrier integrity by tight junction claudin-5 expression in the brains of MA10-infected mice compared to mock controls. ATN-161 treatment decreased IL-6 expression and increased claudin 5 expression in infected mice. Conclusion: Therefore, we propose that targeting integrin α5β1 through inhibitors such as ATN-161 offers a promising therapeutic strategy for attenuating SARS-CoV-2 and its immunological impact on brain vasculature. This approach may be pivotal in reducing both acute and chronic neurological morbidities associated with COVID-19.

CircRNA02318 Exerts Therapeutic Effects on Myocardial Ischemia-Reperfusion Injury Rats by Regulating the Nox1/Akt Through Inhibiting Drebrin

Our research aims to explore the therapeutic effect of circRNA02318 on MIRI rats and the functional mechanism. The MIRI model was constructed in rats. CircRNA02318 overexpressing adenovirus was injected in situ during MIRI perfusion. H9C2 cells were treated with hypoxia for 6 h and reoxygenation for 3 h. Overexpression of circRNA02318 downregulated Drebrin, Nox1, cleaved caspase-3 and Bax in H/R H9C2 cells and MIRI rat heart tissues, promoted the expression of p-Akt/Akt and Bcl-2, and inhibited the apoptosis of H9C2 cells. The volume of myocardial infarction and the release of LDH and TnI in MIRI rats were suppressed by circRNA02318. The Nox1, cleaved caspase-3 and Bax levels were promoted, the level of p-Akt/Akt and Bcl-2 was repressed, and the apoptosis was facilitated by the Drebrin overexpression. Furthermore, the effect of Drebrin overexpression on H9C2 cells was abolished by circRNA02318. Collectively, circRNA02318 exerted therapeutic effects on MIRI rats by inhibiting Drebrin.

Abstract 10357: Pyruvate Dehydrogenase Kinase 4 is a Novel Regulator of Endothelial Cell Mitochondrial Respiration in Diabetes-Impaired Angiogenesis: Implications for Preventing Diabetic Vascular Complications

Introduction: Diabetic patients have a greater risk of experiencing chronic wounds and amputation than non-diabetic patients. Ischaemia-driven angiogenesis is essential for tissue repair but is impaired in diabetes due to aberrant endothelial cell (EC) responses to ischaemia. Pyruvate dehydrogenase kinase 4 (PDK4) normally suppresses mitochondrial respiration in ischaemia by phosphorylating the pyruvate dehydrogenase complex (pPDC) to preserve EC function. However, the role of the PDK4/PDC axis in angiogenesis and the effects of diabetes are unexplored. Methods and Results: In human coronary artery endothelial cells (HCAECs), siRNA-mediated PDK4 knockdown reduced tubulogenesis (49%, P <0.01) and migration (51%, P <0.01), revealing an essential role for PDK4 in angiogenesis. HCAECs were exposed to 5 or 25mM glucose for 72h and hypoxia for 6h. High glucose attenuated the induction of PDK4 and pPDC in response to hypoxia. This was associated with an aberrant increase in mitochondrial respiration (20%, P <0.01), measured by the Seahorse Bioanalyser system, and impaired tubulogenesis (52%, P <0.001) and migration (79%, P <0.0001). Lentiviral overexpression of PDK4 rescued this impairment in high glucose and hypoxia. Similarly, treatment with reconstituted high-density lipoprotein (rHDL), a known proangiogenic agent, restored the induction of PDK4 and pPDC in hypoxia and high glucose, and rescued tubulogenesis and migration. Mechanistically, rHDL increased binding of FOXO1 to the PDK4 promoter (78%, P <0.05), assessed by chromatin immunoprecipitation, compared to PBS controls. In a murine model of diabetic wound healing, diabetes impaired the early induction of PDK4 and pPDC in response to wound ischaemia, and reduced wound neovessels. Topical rHDL enhanced wound PDK4 and pPDC levels over 72h post-wounding (68-165%, P <0.05), and increased wound neovascularisation (62%, P <0.05) and wound closure (45%, P <0.001), compared to PBS controls. Conclusion: PDK4 is essential for hypoxia-driven angiogenesis. This response is impaired in diabetes and can be corrected by PDK4 overexpression and rHDL. We have identified a novel regulatory pathway for diabetes-impaired angiogenesis that can be targeted to prevent diabetic vascular complications.

Hypoxia Inducible Factor is partially involved in mitochondrial and metabolic responses to hypoxia in rats DEMAREST Maud 1, MARCOUILLER François 1, SOLIZ Jorge 1, JOSEPH Vincent 11 Institut universitaire de cardiologie et de pneumologie, Université Laval, Québec

High altitude environments present many constraints for mammals, the most well‐known being the lack of oxygen or hypoxia. Nevertheless, several ecological reports show that mice (Mus musculus) unlike rats (Rattus norvegicus) are present above 4000m1 although they are not native to this environment. The associated general hypothesis is that mice are pre‐adapted to high altitude environments. There are also differences in mitochondrial2, ventilatory and metabolic3 responses to hypoxia between these two species. One of the mechanisms involved in many of these responses is the hypoxia‐inducible factor HIF, which helps maintain the cell's oxygen homeostasis. What precisely are the different roles of HIF in mitochondrial, ventilatory, and metabolic responses to hypoxia in rats and mice, two species more or less adapted to high‐altitude environments? Does the pharmacological stabilization of HIF in normoxia mimic the responses observed in hypoxia? Conversely, does blocking HIF in hypoxia suppress these same responses? We measured the ventilatory and metabolic responses (whole body plethysmography) of rats (SD) and mice (FVB) under the following conditions: (i) 6h normoxia (21% O2); (ii) 6h hypoxia (10% O2); (iii) 6h normoxia with injection of deferoxamine, a HIF stabilizer (200mg/kg) and (iv) 6h hypoxia with injection of 2‐methoxyestradiol, a HIF blocker (5mg/kg). In addition, we evaluated mitochondrial respiration in homogenates of liver and cerebral cortex, two organs sensitive to hypoxia. Our preliminary results show that hypoxia increases ventilation and reduces metabolism and complex I efficiency in the cerebral cortex. Furthermore, pharmacological stabilization of HIF has no effect on the ventilatory response, but seems to mimic some mitochondrial and metabolic responses to hypoxia, whereas blocking HIF has no effect on these same responses. If in mice blocking HIF in hypoxia reduces the amplitude of the observed responses, our data will demonstrate fundamental differences in the mechanisms of responses to hypoxia between these two species that would strongly support our general hypothesis.

Connexin43 Overexpression Exacerbates Myocardial Ischemic Reperfusion Injury in Diabetes via Modulating Cardiac Autophagy

BackgroundPatients with diabetes are more vulnerable to myocardial ischemia/reperfusion injury (MIRI). Abnormal increases of cardiac Connexin43 (Cx43) were shown to be associated with a variety of pathological conditions including myocardial ischemia and diabetic cardiomyopathy, concomitant with various degrees of changes in autophagy. However, the role of Cx43 in MIRI in diabetic conditions and in particular its potential interplay with cardiac autophagy in this pathology is unknown. Thus, the present study was aimed to explore the role and mechanism of Cx43 in diabetic MIRI.Methods and ResultsStreptozotocin (STZ)‐induced diabetic and age‐matched control rats were subjected to MIRI by occluding the left coronary artery for 30 minutes followed by 2 hours of reperfusion at 5 weeks of diabetes induction. In vitro, rat origin H9C2 cardiomyocytes (H9C2) were exposed to 35mmol/L high glucose(HG)for 48 hours(h) in the absence or presence of Cx43 gene knockdown with siRNA, followed by hypoxia for 6h and reoxygenation for 12h. Myocardial infarct size was assessed by TTC staining. Cell viability was detected by the CCK‐8 assay, apoptosis by TUNEL assay. Cardiac Cx43 protein, apoptosis‐related proteins and autophagy‐related LC3, p62 and mTOR proteins were assessed by Western blotting. Post‐ischemic myocardial infarct size and cardiac CX43 protein expression were significantly increased in diabetic rats as compared with that in the control (p<0.05), concomitant with increases in the ratio of LC3 II/I and in the protein expression of P62 and mTOR (all p<0.05). In vitro, exposure of H9C2 cells to HG significantly increased cell injury evidenced by increased LDH release and reduced cell viability, and significantly enhanced cell apoptosis and reactive oxygen species(ROS) production that was accompanied by increased cardiomyocyte CX43 overexpression and enhanced autophagy but paradoxically also enhanced mTOR activation, similar to that seen in vivo. All these changes were further exacerbated following H/R under HG. Cx43 Knockdown in H9C2 results in decreased ROS production and decreased apoptosis after H/R under HG leading to increased cell viability (all p<0.05), while Cx43 knockdown attenuated autophagy. Application of the selective mTOR inhibitor rapamycin abolished the abovementioned beneficial effects of Cx43 Knockdown.ConclusionOverexpressed CX43 in the diabetic heart exacerbates MIRI in diabetes through enhanced cardiac autophagy under hyperglycemic condition.

Abstract TMP19: Atn-161 Prevents α5 Integrin-mediated Tight Junction Disruption Caused By Sars-cov-2 And Its Delta Variant In Brain Endothelial Cells

Patients with significant cerebrovascular comorbidities (e.g. brain ischemia, vascular dementia) are more affected and are more likely to have worsened post-acute neurologic sequelae after SARS-CoV-2 infection. This may be due to viral invasion and propagation in brain endothelial cells (BECs) and disruption of the blood-brain barrier (BBB). Viral spike protein used to bind and infect host cells encodes an arginine-glycine-aspartic acid (RGD) motif that it may use to bind integrins cell receptors that play an important role in cerebrovascular integrity. Therefore, integrins may represent an acute and post-acute SARS-CoV-2 therapeutic target. However, the interplay between vascular dysregulation, integrin function, and COVID-19 is unclear. As we have previously demonstrated that activation of the integrin α5 plays a key role in BBB breakdown, stroke injury, OGD/R, SARS-CoV-2 infection, and its inhibition with the clinically validated peptide ATN-161 is therapeutic in these conditions, we hypothesize that SARS-CoV-2 alters BEC α5 integrin (and associated tight junction protein) expression as a means of infecting and altering cerebrovascular integrity, and this can be prevented by ATN-161. Methods: Mouse BECs (bEnd3) were inoculated with heat-inactivated SARS-CoV-2 (Isolate USA-WA1/2020) or delta variant of SARS-CoV-2 spike protein for 24 h then later exposed to hypoxia for 6h to model the effects of in vivo pulmonary infection. Cells were pretreated with ATN-161 (1, 5, and 10μM) 1h before SARS-CoV-2 challenge and during hypoxia. α5 and claudin-5 proteins were analyzed by immunoblotting. Results: Both SARS-CoV-2 inoculations induced integrin α5 and decreased claudin-5 expression (delta > SARS-CoV-2) in a dose-dependent fashion, although higher doses of SARS-CoV-2 (2.5 and 5 μg) had no effect on these proteins. SARS-CoV-2 spike protein challenge at 0.5 μg followed by hypoxia resulted in increased α5 and decreased claudin-5 expression in either hypoxia or SARS-CoV-2+hypoxia combination. ATN-161 (10μM) pretreatment inhibited SARS-CoV-2+hypoxia-induced α5 upregulation and restored claudin-5 loss. In addition to its demonstrated anti-viral effects, ATN-161 may be an important therapy for SARS-CoV-2-mediated cerebrovascular injury.

Preliminary discussion on the potential mechanism of follistatin-like protein 1 in the process of proliferative diabetic retinopathy

Objective To observe the changes of follistatin-like protein 1 (FSTL1) in serum of patients with proliferative diabetic retinopathy (PDR). Methods Twenty PDR patients confirmed by clinical examination and 20 normal people were included in the study. Human retinal vascular endothelial cells (HRCEC) were divided into HRCEC blank control group, 3 h hypoxia group, 6 h hypoxia group. Human umbilical vein endothelial cell (HUVEC) were divided into HUVEC blank control group, 3h hypoxia group, 6h hypoxia group. Real-time quantitative PCR (RT-PCR) and ELISA were used to determine the expression of FSTL1, TGF-β, VEGF, connective tissue growth factor (CTGF) mRNA and protein in peripheral blood and cells of all groups from all subjects. Results The expressions of FSTL1, TGF-β1, CTGF, VEGF mRNA in blood samples of patients with PDR were 1.79±0.58, 0.97±0.21, 1.85±0.69 and 1.38±0.44. The expressions of FSTL1, TGF-β1 protein were 1.19±0.50, 0.71±0.24 ng/ml and 734.03±116.45, 649.36±44.23 ng/L. Compared with normal people, the differences were statistically significant (tmRNA=0.90, 0.21, 2.85, 1.77; P=0.00, 0.00, 0.04, 0.02. tprotein=1.88, 7.68; P=0.00, 0.02). The cell viability of HRCEC cells in the 3 h hypoxia group and the 6 h hypoxia group were 0.66±0.05 and 0.64±0.04, respectively. Compared with the blank control group, the difference was statistically significant (F=13.02, P=0.00). The cell viability of HUVEC cells in the 3 h hypoxia group and the 6 h hypoxia group were 0.63±0.06 and 0.68±0.06, respectively. Compared with the blank control group, the difference was statistically significant (F=26.52, P=0.00). Comparison of FSTL1, TGF-β1, CTGF, and VEGF mRNA expression in HRCEC blank control group and 3 h hypoxia group, the differences were statistically significant (F=14.75, 44.93, 85.54, 6.23; P=0.01, 0.00, 0.00, 0.03). Compared with the HRCEC blank control and 3 h hypoxia group, the expressions of FSTL1 and TGF-β1 protein were statistically significant (P<0.05). There was a statistically significant difference in TGF-β1 protein expression in the hypoxic 6 h group (P=0.03) and no significant difference in FSTL1 protein expression (P=0.68). Comparison of FSTL1, TGF-β1, CTGF, and VEGF mRNA expression in HUVEC blank control group and 3h hypoxia group, the differences were statistically significant (F=19.08, 25.12, 22.89, 13.07; P=0.00, 0.00, 0.00, 0.01). Immunofluorescence staining results showed that FSTL1, TGF-β1, CTGF, and VEGF proteins were positively expressed in cells in the 3h hypoxia and 6h hypoxia groups. Conclusion The expression of FSTL1 gene and protein in serum of PDR patients was significantly higher than that of normal people. Key words: Follistatin-related proteins; Human umbilical vein endothelial cells; Diabetic retinopathy

Xenogeneic and Allogeneic Mesenchymal Stem Cells Effectively Protect the Lung Against Ischemia-reperfusion Injury Through Downregulating the Inflammatory, Oxidative Stress, and Autophagic Signaling Pathways in Rat

This study tested the hypothesis that both allogenic adipose-derived mesenchymal stem cells (ADMSCs) and human inducible pluripotent stem cell-derived MSCs (iPS-MSCs) offered a comparable effect for protecting the lung against ischemia-reperfusion (IR) injury in rodent through downregulating the inflammatory, oxidative stress, and autophagic signaling pathways. Adult male Sprague–Dawley rats (n = 32) were categorized into group 1 (sham-operated control), group 2 (IRI), group 3 [IRI + ADMSCs (1.0 × 106 cells)/tail-vein administration at 0.5/18/36 h after IR], and group 4 [IRI + iPS-MSCs (1.0 × 106 cells)/tail-vein administration at 0.5/18/36 h after IR], and lungs were harvested at 72 h after IR procedure. In vitro study demonstrated that protein expressions of three signaling pathways in inflammation (TLR4/MyD88/TAK1/IKK/I-κB/NF-κB/Cox-2/TNF-α/IL-1ß), mitochondrial damage/cell apoptosis (cytochrome C/cyclophilin D/DRP1/ASK1/APAF-1/mitochondrial-Bax/caspase3/8/9), and autophagy/cell death (ULK1/beclin-1/Atg5,7,12, ratio of LCB3-II/LC3B-I, p-AKT/m-TOR) were significantly higher in lung epithelial cells + 6h hypoxia as compared with the control, and those were significantly reversed by iPS-MSC treatment (all P < 0.001). Flow cytometric analysis revealed that percentages of the inflammatory cells in bronchioalveolar lavage fluid and circulation, and immune cells in circulation/spleen as well as circulatory early and late apoptotic cells were highest in group 2, lowest in group 1, and significantly higher in group 3 than in group 4 (all P < 0.0001). Microscopy showed the lung injury score and numbers of inflammatory cells and Western blot analysis showed the signaling pathways of inflammation, mitochondrial damage/cell apoptosis, autophagy, and oxidative stress exhibited an identical pattern of flow cytometric results among the four groups (all P < 0.0001). Both xenogeneic and allogenic MSCs protected the lung against IRI via suppressing the inflammatory, oxidative stress, and autophagic signaling.

Inhibition of ATM/Chk2 Pathway Ameliorates Hypoxia-induced Myocardium Injury

Background: Acute myocardial infarction (AMI) is a kind of acute myocardial ischemic disease based on atherosclerosis that has high global morbidity and mortality. Recent work suggests the possible presence of DNA damage response (DDR) in AMI. However, the evidence is insufficient, and it remains elusive whether DDR can be used as a therapeutic target of AMI. Methods: The inductions of DDR were confirmed in rat myocardial ischemia model and cell model. Then, the specific inhibitor or siRNA target Chk1, ATM and Chk2 were used in H9C2 and NRVMs to assess their protective effect under hypoxia. Results: The induction of phosphorylation of H2A.X was observed in a rat model, suggesting that DDR occurs in myocardial tissues. DDR was also induced in vitro in H9C2 and NRVMs hypoxia models by the upregulation of p-H2A.X, p-Chk1, and p-Chk2. Then, we used UCN-01, a specific Chk1 inhibitor, to block Chk1 phosphorylation and find it has no effect on p-H2A.X expression, MTT, LDH, and cell apoptosis after 6h hypoxia. However, KU-55933, an ATM inhibitor, reversed the hypoxia-induced phosphorylation of ATM, H2A.X, and Chk2, and it also reduced apoptosis of H9C2 and NRVMs. Conclusion: Our results suggested that DDR occurs during myocardial ischemia in vivo and oxygen depletion injury in vitro. Inhibition of the ATM/Chk2 pathway might play a cytoprotective role in guarding against myocardial cell injury.

Polydatin protects cardiomyocytes against myocardial infarction injury by activating Sirt3

Myocardial infarction (MI), which is characterized by chamber dilation and left ventricular (LV) dysfunction, represents a major cause of morbidity and mortality worldwide. Polydatin (PD), a monocrystalline and polyphenolic drug isolated from a traditional Chinese herb (Polygonum cuspidatum), alleviates mitochondrial dysfunction. We investigated the effects and underlying mechanisms of PD in post-MI cardiac dysfunction. We constructed an MI model by left anterior descending (LAD) coronary artery ligation using wild-type (WT) and Sirt3 knockout (Sirt3−/−) mice. Cardiac function, cardiomyocytes autophagy levels, apoptosis and mitochondria biogenesis in mice that underwent cardiac MI injury were compared between groups. PD significantly improved cardiac function, increased autophagy levels and decreased cardiomyocytes apoptosis after MI. Furthermore, PD improved mitochondrial biogenesis, which is evidenced by increased ATP content, citrate synthase (CS) activity and complexes I/II/III/IV/V activities in the cardiomyocytes subjected to MI injury. Interestingly, Sirt3 knockout abolished the protective effects of PD administration. PD inhibited apoptosis in cultured neonatal mouse ventricular myocytes subjected to hypoxia for 6h to simulate MI injury. PD increased GFP-LC3 puncta, and reduced the accumulation of protein aggresomes and p62 in cardiomyocytes after hypoxia. Interestingly, the knock-down of Sirt3 nullified the PD-induced beneficial effects. Thus, the protective effects of PD are associated with the up-regulation of autophagy and improvement of mitochondrial biogenesis through Sirt3 activity.

Selenoprotein M gene expression, peroxidases activity and hydrogen peroxide concentration are differentially regulated in gill and hepatopancreas of the white shrimp Litopenaeus vannamei during hypoxia and reoxygenation

In many organisms, episodes of low O2 concentration (hypoxia) and the subsequent rise of O2 concentration (reoxygenation) result in the accumulation of reactive oxygen species and oxidative stress. Selenoprotein M (SelM), is a selenocysteine containing protein with redox activity involved in the antioxidant response. It was previously shown that in the white shrimp Litopenaeus vannamei, the silencing of SelM by RNAi decreased peroxidase activity in gill. In this work, we report the structure of the SelM gene (LvSelM) and its relative expression in hepatopancreas and gill after 24h of hypoxia followed by 1h of reoxygenation. The gene is composed by four exons interrupted by tree introns. In gills and hepatopancreas, SelM expression increased after 24h of hypoxia followed by 1h of reoxygenation, while peroxidases activity diminished in hepatopancreas but increased in gills. Hydrogen peroxide (H2O2) concentration was higher in hepatopancreas in response to hypoxia for 6h and did not change after 24 of hypoxia followed by reoxygenation; conversely, no change was detected in gill. SelM appears to be a key enzyme in gill oxidative stress regulation, since the higher expression is associated with an increase in peroxidases activity while maintaining H2O2 concentration. In contrast, in hepatopancreas there is a higher expression after hypoxia and reoxygenation for 24h, but peroxidases activity was lower and the change in H2O2 occurred after 6h of hypoxia and this level was maintained during reoxygenation.

Rapid ingress of gaseous 1-MCP and acute suppression of ripening following short-term application to midclimacteric tomato under hypobaria

Our previous studies demonstrated that tomato fruit (breaker or pink) exposed at the midclimacteric stage to hypobaric hypoxia for 6h exhibited transient increased sensitivity to subsaturating levels of 1-methylcyclopene (1-MCP). In the present study, we examined the effect of gaseous 1-MCP (500nLL−1, 20.8μmolm−3) applied to mid-climacteric (>60% peak ethylene production) tomato fruit under hypobaric hypoxia (10kPa, 2.1kPa O2,) for 1h. Application of 500nLL−1 1-MCP under atmospheric conditions had little effect on softening and timing and magnitude of peak ethylene production, and moderate effects on respiration and lycopene and PG accumulation. By contrast, midclimacteric fruit exposed to 500nLL−1 gaseous 1-MCP under hypobaric hypoxia for 1h showed acute disturbance of ripening. Firmness and hue angle declines were delayed for ten days and peak ethylene production for eleven days compared with trends for the other treatments. Maximum ethylene production did not exceed 50% of maxima for the other treatments and a definitive respiratory climacteric was not observed. Accumulation of internal gaseous 1-MCP was enhanced under hypobaric hypoxia. Internal 1-MCP in fruit exposed to 20μLL−1 1-MCP (831μmolm−3) under hypobaric hypoxia for 2 or 10min averaged 7.5±0.5 and 8.7±1.4μLL−1, respectively, compared with 0.8±0.3 and 3.9±0.7μLL−1 in fruit exposed under atmospheric conditions. After 1h exposure, internal 1-MCP averaged 10.8±2.2μLL−1 under hypobaric hypoxia compared with 5.3±1.4μLL−1 under atmospheric conditions. The results indicate that high efficacy of 1-MCP applied under hypobaric hypoxia is due to rapid ingress and accumulation of internal gaseous 1-MCP.

Exposure to supernatants of macrophages that phagocytized dead mesenchymal stem cells improves hypoxic cardiomyocytes survival

ObjectiveTo observe the impact of supernatants from macrophages that phagocytized dead MSCs (pMΦ) on the survival of hypoxic cardiomyocytes. MethodsMSCs were isolated from bone marrow of mice and dead MSCs were harvested after 6h hypoxia. Macrophages were obtained from thioglycolate-elicited peritoneal cavity. Macrophages and dead MSCs were co-cultured for 2days in the presence or absence of LPS (1μg/ml). Cardiomyocytes obtained from neonatal mice were exposed to various medium including supernatants from pMΦ. MTT cell proliferation assay and mitochondria membrane potential were used to evaluate the viability of cardiomyocytes. Cytokines and chemokines (TNF-α, IFN-γ, IL-6, IL-12, PGE2, VEGF-α, Ang-1, KGF, IGF-1, PDGF-BB, and EPO) in culture medium of macrophages, MSCs and pMΦ were detected by ELISA and Real-Time-PCR. Results: phagocytic activity of macrophages to dMSC was significantly enhanced by LPS. PGE2, VEGF-α, Ang-1, KGF, IGF-1, PDGF-BB, and EPO levels were significantly increased in supernatants of pMΦ. Exposure to supernatants of pMΦ significantly improved viability and survival time of hypoxic cardiomyocytes. ConclusionExposure to supernatants of pMΦ significantly improved viability and survival time of hypoxic cardiomyocytes, which might be linked to increased cytokines and chemokines secretion by pMΦ.

Exhaled nitric oxide is associated with acute mountain sickness susceptibility during exposure to normobaric hypoxia

Nitric oxide is a gaseous signaling molecule that participates in a large variety of physiological functions and may have a role in the pathology of altitude illnesses, such as acute mountain sickness (AMS). The effect of normobaric hypoxia on the fraction of exhaled NO ( [Formula: see text] ) is a controversial area of high altitude physiology, with the effect varying widely across studies. We exposed 19 male subjects to normobaric hypoxia for 6h and measured [Formula: see text] and AMS (via Lake Louise Score) each hour. For data analysis, subjects were divided into AMS-positive and AMS-negative groups based on their Lake Louise Scores during exposure. Eighteen subjects completed the study, and the incidence of AMS was 50%. Mean [Formula: see text] was unchanged at hour 1 but was significantly elevated above baseline for the remainder of the normobaric hypoxia exposure (p<0.001). Subjects who developed AMS had a significantly lower mean [Formula: see text] at baseline compared to resistant subjects (p=0.013). Further investigations are warranted to confirm our results and to understand the physiological basis of this association.

Research of HIF-1 Mediated SDF-1/CXCR4 Axis on the Directional Migration and Neural Differentiation of Mesenchymal Stem Cells

AbstractAbstract 4699 Introduction:Mesenchymal stem cells (MSCs) are under study as therapeutic delivery agents that assist in the repair of damaged tissues. At present, the mechanisms of targeted therapy of MSCs are known to relate with the hypoxia-inducible factor-1 (HIF-1) and its regulated biological axis stromal cell-derived factor-1/chemokine receptor 4 (SDF-1/CXCR4). SDF-1 and CXCR4 have a wide distribution in various cells and tissues, which plays an important role in the development of immune system, circulatory system and central nervous system. We thus inferred that SDF-1/CXCR4 may participate in the differentiation as well as the migration of stem cells. Our research aims to explore the effect of HIF-1α and its mediated SDF-1/CXCR4 axis on the directional migration and neural differentiation of MSCs, which may lead to a break in the efficiency and target distribution of MSCs therapy. Method:(1) Influence of hypoxia, CXCR4 antagonist (AMD3100) and SDF-1α on the proliferation of rat Mesenchymal Stem Cells (rMSCs): rMSCs were isolated from bone marrow of rats, and expanded in vitro. The growth feature of rMSCs exposed to hypoxia (PO2=1%) or normoxia was identified by growth curve, while the effect of AMD3100 (5ug/ml) and SDF-1α (10ng/ml and 100ng/ml) on the proliferation ability of rMSCs was detected by cell counting kit-8. (2) Effect of hypoxia on the expression of HIF-1α, CXCR4 and SDF-1α: Firstly, we used RT-PCR, western blotting and flow cytometry to detect the expression of HIF-1α and CXCR4 mRNA and protein levels in rMSCs which treated with hypoxia for 0h, 6h, 12h, 24h, 48h and 72h. Secondly, HIF-1α, SDF-1α mRNA and protein level in the hippocampus of rats which suffered hypoxia- ischemia for 1d, 3d, 5d, 7d, 14d and 21d were also detected by the same assays. (3) Research of HIF-1α and SDF-1/CXCR4 axis on the migration of rMSCs: We first detected the change of CXCR4 mRNA and protein levels in rMSCs treated with AMD3100 (5ug/ml) and SDF-1α (10ng/ml) by RT-PCR, western blotting and flow cytometry, and then studied SDF-1/CXCR4 axis on the migration of rMSCs using Transwell assay. (4) Effect of HIF-1α and SDF-1/CXCR4 axis on the differentiation of rMSCs: protein level of NSE and GFAP as well as positive rate of neural-induced rMSCs which have been pretreated with AMD3100 (5ug/ml) were detected by western blotting and immunocytochemistry. Results:Persistent hypoxia promoted the proliferation of rMSCs, while AMD3100 and SDF-1α at the concentration mentioned above had no effect. Compared to normal control, the protein expression of HIF-1α in rMSCs increased in hypoxic condition while the mRNA of HIF-1α did not change. Furthermore, the mRNA and protein level of CXCR4 both increased in rMSCs exposed to hypoxia for 6h and 12h, and the results confirmed by flow cytometry. We found HIF-1α mRNA was stably expressed in hippocampus, and increased significantly in hypoxia-ischemia brain damaged (HIBD) rats in a time dependent manner, which reached the peak on 7d. As expected, SDF-1α mRNA in hippocampus of HIBD rats was higher than that of normal control group, which reached the peak on 7d (P<0.01) and stably expressed till 21d, while the protein level is mainly in concordance. Moreover, CXCR4 mRNA was extremely up-regulated in rMSCs treated with SDF-1α (10ng/ml), however, in 5 ug/ml AMD3100 treated rMSCs, which decreased markedly (P<0.01), and the results were confirmed by western blotting and flow cytometry assays (P<0.05). Transwell assay manifested that SDF-1α had obvious chemotaxis to rMSCs. Protein level and positive cell number of NSE and GFAP were extremely down-regulated in rMSCs which pretreated with 5ug/ml AMD3100. Conclusion:Increased expression of HIF-1α led to the up-regulation of SDF-1/CXCR4 axis, and rMSCs displayed chemotaxis migration ascribed to the receptor-ligand interactions of SDF-1α and CXCR4, suggesting that HIF-1 and its mediated SDF-1/CXCR4 axis are of great significant on the directional migration of rMSCs. We also showed that CXCR4 antagonisation reduced the neural differentiation capabilities of rMSCs, thus suggested that SDF-1/CXCR4 axis may deeply involve in the neural differentiation of rMSCs. Disclosures:No relevant conflicts of interest to declare.

E0049 E2F1 stabilises p53 to suppress VEGF expression and neovascularization in the ischaemic myocardium

ObjectiveWe have previously shown that genetic deletion of the transcription factor E2F1 increases the expression of vascular endothelial growth factor (VEGF) and enhances blood flow recovery in the ischaemic limb...

Abstract 3785: An Involvement of a Novel G-protein Activator on Hypoxia-Induced Apoptosis of Cardiomyocytes and its Interaction with Connexin 43

Recently, we identified a novel receptor-independent G-protein activator, Activator of G-protein signaling 8 (AGS8), from a cDNA library of rat hearts subjected to repetitive transient ischemia. AGS8 mRNA was induced in the ischemic myocardium and it regulated Gβγ signaling. In the face of myocardial ischemia and/or ischemia/reperfusion, AGS8 may be involved in signaling events associated with adaptation of the cardiomyocyte to ischemic events. As an initial step to address this issue, we examined the influence of AGS8 on survival of cultured cardiomyocyte following hypoxia. Cultured rat neonatal cardiomyocytes (NCM) were exposed to hypoxia for 6h, 24 h or hypoxia (6h)/reoxygenation (18h) following transfection of AGS8 siRNA. Apoptosis was determined by DNA end-labeling or detection of changes of mitochondrial membrane potential. Interestingly, silencing of AGS8 inhibited apoptosis of cardiomyocytes induced by hypoxia alone (57.8±3.4%, p&lt;0.05 vs. control-siRNA, at 24 h, mean±SEM) as well as hypoxia/reoxygenation (36.4±4.0%, p&lt;0.05 vs. control-siRNA, mean±SEM). Transfection of pcDNA::AGS8 by virus envelope increased apoptosis of NCM (151%±9.6%, p&lt;0.05 vs. vector group, mean±SEM) following hypoxia/reoxygenation, but not NCM cultured in normoxia. Affinity purified AGS8 antibody recognized immunoreactive signals enriched at the cell-cell interface of ventricular cardiomyocytes and NCM, regions also enriched in the gap junction protein connexin 43 (CX43). Indeed, AGS8 and CX43 co-immunoprecipitated from left ventricle lysates. The level of CX43 protein was decreased by overexpression of AGS8 in NCM, whereas suppression of AGS8 expression increased the level of CX43. Subsequent studies indicated that AGS8 stimulated phosphorylation of CX43 in a Gβγ dependent manner following transient expression in COS7 cells. As the level and/or phosphorylation of CX43 may influence apoptosis of cardiomyocytes, these studies suggest a potential mechanism by which AGS8 induction in the face of ischemia may provide a component of the signaling events involved in adaptation and suggest that targeted disruption of the AGS8 cascade may protect the myocardium from ischemic injury.

Effects of hypoxia, hyperoxia on the regulation of expression and activity of matrix metalloproteinase-2 in hepatic stellate cells.

To study the effects of hypoxia, hyperoxia on the regulation of expression and activity of matrix metalloproteinase-2 (MMP-2) in hepatic stellate cells (HSC). The expressions of MMP-2, tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) and membrane type matrix metalloproteinase-1 (MT1-MMP) in cultured rat HSC were detected by immunocytochemistry (ICC) and in situ hybridization (ISH). The contents of MMP-2 and TIMP-2 in culture supernatant were detected with ELISA and the activity of MMP-2 in supernatant was revealed by zymography. In the situation of hypoxia for 12h, the expression of MMP-2 protein was enhanced (hypoxia group positive indexes: 5.7 +/- 2.0, n=10; control: 3.2 +/- 1.0, n = 7; P<0.05), while TIMP-2 protein was decreased in HSC (hypoxia group positive indexes: 2.5 +/- 0.7, n = 10; control: 3.6 +/- 1.0, n = 7; P < 0.05), and the activity (total A) of MMP-2 in supernatant declined obviously (hypoxia group: 7.334 +/- 1.922, n = 9; control: 17.277 +/- 7.424, n = 11; P < 0.01). Compared the varied duration of hypoxia, the changes of expressions including mRNA and protein level as well as activity of MMP-2 were most notable in 6h group. The highest value(A(hypoxia)-A(control)) of the protein and the most intense signal of mRNA were in the period of hypoxia for 6h, along with the lowest activity of MMP-2. In the situation of hyperoxia for 12h, the contents (A(450)) of MMP-2 and TIMP-2 in supernatant were both higher than those in the control, especially the TIMP-2 (hyperoxia group: 0.0499 +/- 0.0144, n = 16; control: 0.0219 +/- 0.0098, n = 14; P < 0.01), and so was the activity of MMP-2 (hyperoxia group: 5.252 +/- 0.771, n = 14; control: 4.304 +/- 1.083, n = 12; P < 0.05), and the expression of MT1-MMP was increased. HSC is sensitive to the oxygen, hypoxia enhances the expression of MMP-2 and the effect is more marked at the early stage; hyperoxia mainly raises the activity of MMP-2.

Effect of hypoxia on monoamine synthesis in brains of developing rats. II. Different length of exposure.

Tyrosine and tryptophan hydroxylase activity was studied &lt;i&gt;in vivo&lt;/i&gt; in the postnatal rat brain by measuring the accumulation of dihydroxyphenylalanine (DOPA) and 5-hydroxytryptophan (5-HTP) respectively after inhibition of Z-aromatic amino acid decarboxylase with NSD 1015. 1-, 4-, 14- and 28-day-old rats were exposed to a 12% oxygen environment for a period of 30 min, 2 and 6 h. After 30 min in the 12% oxygen environment the tryptophan hydroxylase activity decreased significantly in all age groups studied, but increased significantly after 6h hypoxia compared to controls in 1- and 4-day-old rats. Tyrosine hydroxylase activity in 1-day-old rats was significantly decreased only after 30 min hypoxia as it returned to normal after 2 and 6 h. In the 4-day-old rats a decrease was noted after 30 min and 6 h. In the 14- and 28-day-old rats a significant reduction of tyrosine hydroxylase activity was found after all the time intervals studied. It is concluded that the oxygen dependent, first, rate-limiting step in the synthesis of the neurotransmitters dopamine, noradrenaline and 5-hydroxytryptamine seems to be less vulnerable in the brains of early postnatal rats. Decreases and increases of the levels of tryptophan correlated well to the found changes in 5-HTP levels. This correlation did not seem to be present in the case of tyrosine and DOPA accumulation. It is thus suggested that the observed changes in tryptophan hydroxylation may in part depend on the changes in tryptophan availability. This did not seem to be the case concerning tyrosine hydroxylation and tyrosine availability.