Hours Of Hypoxia Research Articles

Hypoxia induces reversible gill remodeling in largemouth bass (Micropterus salmoides) through integrins-mediated cell adhesion

Gill remodeling is an important strategy for fish to cope with hypoxia, and many of the teleost possess this ability, but the underlying mechanism is not well understood. To investigate the mechanism of hypoxia-induced gill remodeling, largemouth bass (Micropterus salmoides) exposed to hypoxia (dissolved oxygen level: 2.0 ± 0.2 mg L−1) for 7 days, followed by 7 days of reoxygenation. Hypoxia tests were also performed on primary gill cells from largemouth bass. We found that hypoxia-induced gill remodeling increased the respiratory surface area of the gills. This change in gill morphology was reversible and recovered after reoxygenation. A reduction in the number of mucous cells and rearrangement of mitochondria-rich cells (MRCs) were observed during gill remodeling. After 7 days of reoxygenation, the number of mucous cells and the position of the MRCs were restored. Hypoxia resulted in a 2.92-fold increase in the number of primary gill cells that underwent migration over a 12-h period. The mRNA levels of nine integrin subunits (α1, α2, α5, α7, α8, α10, αL, β1 and β2) were significantly up-regulated after 12 h of hypoxia in vivo, and the changes in the expression of these subunits were consistent with the HIF-1α trend. Immunohistochemistry showed that integrin β1 protein levels were significantly increased and were abundantly expressed in the interlamellar cell mass after exposure to hypoxia. Taken together, the results of the present study demonstrated that changes in mucosal cells and MRCs play an important role in hypoxia-induced gill remodeling in largemouth bass and that these changes are regulated by integrins.

Identification of cardioprotective substances in Panax ginseng/P. notoginseng based on mitochondrial morphological characteristics and UPLC-Triple-TOF-MS

Panax ginseng is reputed to be capable of replenishing healthy Qi and bolstering physical strength, and P. notoginseng can resolve blood stasis and alleviate pain. P. ginseng and P. notoginseng are frequently employed to treat ischemic heart diseases caused by blockages in the heart vessels. Mitochondrial dysfunction often coexists with abnormal mitochondrial morphology, and mitochondrial plasticity and dynamics play key roles in cardiovascular diseases. In this study, primary neonatal rat cardiomyocytes were exposed to 4 hours of hypoxia(H) followed by 2 hours of reoxygenation(R). MitoTracker Deep Red and Hoechst 33342 were used to label mitochondria and nuclei, respectively. Fluorescence images were then acquired using ImageXpress Micro Confocal. Automated image processing and parameter extraction/calculation were carried out using ImagePro Plus. Subsequently, representative parameters were selected as indicators to assess alterations in mitochondrial morphology and function. The active compounds of P. ginseng and P. notoginseng were screened out and identified based on the UPLC-Triple-TOF-MS results and mitochondrial morphometric parameters. The findings demonstrated that RS-2, RS-4, SQ-1, and SQ-4 significantly increased the values of three key morphometric parameters, including mitochondrial length, branching, and area, which might contribute to rescuing morphological features of myocardial cells damaged by H/R injury. Among the active components of the two medicinal herbs, 20(R)-ginsenoside Rg_3, ginsenoside Re, and gypenoside ⅩⅦ exhibited the strongest protective effects on mitochondria in cardiomyocytes. Specifically, 20(R)-ginsenoside Rg_3 might upregulate expression of optic atrophy 1(OPA1) and mitofusin 2(MFN2), and ginsenoside Re and gypenoside ⅩⅦ might selectively upregulate OPA1 expression. Collectively, they promoted mitochondrial membrane fusion and mitigated mitochondrial damage, thereby exerting protective effects on cardiomyocytes. This study provides experimental support for the discovery of novel therapeutic agents for myocardial ischemia-reperfusion injury from P. ginseng and P. notoginseng and offers a novel approach for large-scale screening of bioactive compounds with cardioprotective effects from traditional Chinese medicines.

Inhibition of microtubule function prevents hypoxia-induced glycolysis

In normoxic conditions where oxygen supply exceeds demand, mammalian cells produce 38 molecules of adenosine triphosphate (ATP) per molecule of glucose through cellular respiration which is primarily oxygen-dependent. In hypoxia where oxygen demand exceeds supply, the cell enters a bioenergetic crisis due to the decrease in oxygen-dependent ATP synthesis. The cell relies on overcoming this deficiency in ATP through upregulation of glycolytic gene expression via the hypoxia inducible factor 1-a (HIF-1a). However, it is highly unlikely, if not impossible, that the bioenergetic requirements of the hypoxic cell depend solely on the increased expression of glycolytic enzymes distributed randomly in the cytoplasm. We have recently shown that in response to hypoxia, glycolytic enzymes coalesce to form a glycolytic metabolon to effciently increase ATP production in response to this metabolic challenge (Kierans SJ, et al., 2023). While the function of this metabolon has been characterised, the mechanisms underpinning formation, scaffolding, and intracellular distribution remains poorly understood. Here we investigate the possible role of the cytoskeleton as a scaffold or transport system. Microtubules are a primary method of intracellular transport and may alter the energy distribution strategies of cells in bioenergetic crisis. It is hypothesized that the microtubular network transports glycolytic enzymes and/or facilitates increased glycolytic ATP production by promoting metabolon formation to induce glycolysis in hypoxia. Through western blots, it was successfully shown that upon exposure to 1% oxygen, caco2 intestinal epithelial cells induce a robust hypoxic response by stabilising HIF-1a. To investigate the role of the microtubules, two microtubule inhibitors were used: nocodazole, a microtubule assembly and disassembly inhibitor and dynarrestin, a dynein-1 motor transport protein inhibitor. Non-toxic concentrations of nocodazole and dynarrestin were determined using cell viability assays (trypan blue and resazurin assays). Upon exposure to 24 hours of hypoxia, caco2 cells elicit a statistically significant increase in intracellular lactate (185.9% ± 7.5% SEM, n = 3), which was then supressed when treated with nocodazole or dynarrestin (20.6% ± 10.2% SEM and 74.9% ± 8.87 SEM respectively, n = 3). These results thus implicate a role for the transport and structural capabilities of the microtubules in hypoxia-induced glycolysis. Kierans, S. J., Fagundes, R. R., Malkov, M. I., Sparkes, R., Dillon, E. T., Smolenski, A.,... & Taylor, C. T. (2023). Hypoxia induces a glycolytic complex in intestinal epithelial cells independent of HIF-1-driven glycolytic gene expression. Proceedings of the National Academy of Sciences, 120(35), e2208117120. University College Dublin, School of Medicine. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 5418: Lamellipodial protrusions induced by hypoxia depend upon kindlin-2 in prostate cancer cells

Abstract Hypoxia is a physiologically relevant feature of the prostate microenvironment that promotes migration and invasion resulting in extracapsular extension, the first step toward metastatic progression. Prostate cancer invasion depends on integrins as mechanosensing membrane receptors during creation of membrane lamellipodial protrusions and focal adhesions (FAs). The objective of this study was to determine the molecular events that promote membrane protrusions under hypoxia and whether this was dependent upon kindlin-2, an essential integrin adapter that marks activated β1 integrin heterodimers. DU145 cells depleted of one copy of FERMT2+/- (50% kindlin-2 expression) by CRISPR/Cas9 or DU145 FERMT2wt (normal kindlin-2 expression) were grown under acute exposure to hypoxia (1% O2) and compared to cells grown under normal tissue culture conditions. Immunofluorescence microscopy experiments were performed to analyze the spatial temporal expression of kindlin-2 complexes. Kindlin-2 complexes were confirmed by immunoprecipitation using an anti-kindlin-2 3A3-antibody from Sigma-Aldrich. Colocalization was determined by obtaining 2D immunofluorescence microscopy images analyzed using ImageJ 2.1.0/1.53c and Nikon NIS-Elements 5.30.04. Under hypoxic conditions, analysis over four time points (4h, 8h, 12h & 16h) increased the number and area of FAs (marked by paxillin (PXN)) containing kindlin-2 in a time-dependent manner by 2-fold and 1.5-fold, respectively, but not in DU145 FERMT2+/- cells. Additionally, hypoxia increased membrane area, perimeter, and the plasma membrane intensity of kindlin-2 exclusive of FAs in DU145 FERMT2wt cells which was maximal after 8 hours of hypoxia. Interestingly, limiting the kindlin-2 expression in FERMT2+/- cells resulted in a loss of hypoxia-induced lamellipodial protrusions (marked by lamellipodin (RAPH1)) containing kindlin-2 while hypoxia-induced kindlin-2 FA changes were preserved. The current working hypothesis is that lamellipodial protrusions are dependent upon kindlin-2 expression whereas the established FAs are stable under conditions of reduced kindlin-2 expression in hypoxia. This data suggests that an early stage of migration, lamellipodial extensions are sensitive to kindlin-2 availability. Further studies are required to determine the dynamic interplay between protrusive events and focal adhesions in relation to kindlin-2 for prostate cancer cells to migrate and invade. Citation Format: Colin Nelson, Daniel Hernandez-Cortes, Kendra D. Marr, Jaime MC Gard, Allan I. Paxson, William L. Harryman, Natalya K. Seppanen, John M. Ryniawec, Anne E. Cress. Lamellipodial protrusions induced by hypoxia depend upon kindlin-2 in prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5418.

Knockdown of NEAT1 prevents post-stroke lipid droplet agglomeration in microglia by regulating autophagy

BackgroundLipid droplets (LD), lipid-storing organelles containing neutral lipids like glycerolipids and cholesterol, are increasingly accepted as hallmarks of inflammation. The nuclear paraspeckle assembly transcript 1 (NEAT1), a long non-coding RNA with over 200 nucleotides, exerts an indispensable impact on regulating both LD agglomeration and autophagy in multiple neurological disorders. However, knowledge as to how NEAT1 modulates the formation of LD and associated signaling pathways is limited.MethodsIn this study, primary microglia were isolated from newborn mice and exposed to oxygen-glucose-deprivation/reoxygenation (OGD/R). To further explore NEAT1-dependent mechanisms, an antisense oligonucleotide (ASO) was adopted to silence NEAT1 under in vitro conditions. Studying NEAT1-dependent interactions with regard to autophagy and LD agglomeration under hypoxic conditions, the inhibitor and activator of autophagy 3-methyladenine (3-MA) and rapamycin (RAPA) were used, respectively. In a preclinical stroke model, mice received intraventricular injections of ASO NEAT1 or control vectors in order to yield NEAT1 knockdown. Analysis of readout parameters included qRT-PCR, immunofluorescence, western blot assays, and behavioral tests.ResultsMicroglia exposed to OGD/R displayed a temporal pattern of NEAT1 expression, peaking at four hours of hypoxia followed by six hours of reoxygenation. After effectively silencing NEAT1, LD formation and autophagy-related proteins were significantly repressed in hypoxic microglia. Stimulating autophagy in ASO NEAT1 microglia under OGD/R conditions by means of RAPA reversed the downregulation of LD agglomeration and perilipin 2 (PLIN2) expression. On the contrary, application of 3-MA promoted repression of both LD agglomeration and expression of the LD-associated protein PLIN2. Under in vivo conditions, NEAT1 was significantly increased in mice at 24 h post-stroke. Knockdown of NEAT1 significantly alleviated LD agglomeration and inhibited autophagy, resulting in improved cerebral perfusion, reduced brain injury and increased neurological recovery.ConclusionNEAT1 is a key player of LD agglomeration and autophagy stimulation, and NEAT1 knockdown provides a promising therapeutic value against stroke.Graphical abstract

Effect of hypoxia on Cystic Fibrosis Transmembrane conductance Regulator channel corrected by Elexacaftor/Tezacaftor/Ivacaftor

The accumulation of mucus resulting from the obstruction of bronchi of cystic fibrosis (CF) patients, induces a reduction of the oxygen (O2) pressure and produces a hypoxic environment for the epithelial cells of the lungs. Our study aims to better characterize the impact of hypoxia on CFTR function in the pathophysiological context of cystic fibrosis. We used Human airway epithelial cells from two CF donors and human bronchial epithelial cell lines non-CF and CF, grown and expended in normoxia (21% O2) and then switched to hypoxia (1% O2) for 2 to 24 hours. Cells were treated by dimethyl sulfoxide or Elexacaftor/ Tezacaftor/Ivacaftor for 24 hours. We show that the peak of Hypoxia Inducible Factor 1α is reached in a range of 4 to 6 hours post-hypoxia induction. We also demonstrate that the global amount of ETI corrected F508del-CFTR is significantly decreased after 24 hours of hypoxia. A decreased ETI corrected F508del-CFTR activity was recorded by both patch-clamp and Ussing chamber recordings. Our results show that hypoxia, despite the effectiveness of Elexacaftor/ Tezacaftor/Ivacaftor correction, impacts the downstream effects of the F508del mutation, which suggests that oxygen availability in the lungs is a factor to take into account for the administration of Trikafta to patients.

Normobaric hypoxia shows enhanced FOXO1 signaling in obese mouse gastrocnemius muscle linked to metabolism and muscle structure and neuromuscular innervation.

Skeletal muscle relies on mitochondria for sustainable ATP production, which may be impacted by reduced oxygen availability (hypoxia). Compared with long-term hypoxia, the mechanistic in vivo response to acute hypoxia remains elusive. Therefore, we aimed to provide an integrated description of the Musculus gastrocnemius response to acute hypoxia. Fasted male C57BL/6JOlaHsd mice, fed a 40en% fat diet for six weeks, were exposed to 12% O2 normobaric hypoxia or normoxia (20.9% O2) for six hours (n = 12 per group). Whole-body energy metabolism and the transcriptome response of the M. gastrocnemius were analyzed and confirmed by acylcarnitine determination and Q-PCR. At the whole-body level, six hours of hypoxia reduced energy expenditure, increased blood glucose and tended to decreased the respiratory exchange ratio (RER). Whole-genome transcriptome analysis revealed upregulation of forkhead box-O (FOXO) signalling, including an increased expression of tribbles pseudokinase 3 (Trib3). Trib3 positively correlated with blood glucose levels. Upregulated carnitine palmitoyltransferase 1A negatively correlated with the RER, but the significantly increased in tissue C14-1, C16-0 and C18-1 acylcarnitines supported that β-oxidation was not regulated. The hypoxia-induced FOXO activation could also be connected to altered gene expression related to fiber-type switching, extracellular matrix remodeling, muscle differentiation and neuromuscular junction denervation. Our results suggest that a six-hour exposure of obese mice to 12% O2 normobaric hypoxia impacts M. gastrocnemius via FOXO1, initiating alterations that may contribute to muscle remodeling of which denervation is novel and warrants further investigation. The findings support an early role of hypoxia in tissue alterations in hypoxia-associated conditions such as aging and obesity.

Differential miRNA expression of hypoxic MCF7 and PANC-1 cells.

Hypoxia plays a critical role in the tumor microenvironment by affecting cellular proliferation, metabolism, apoptosis, DNA repair, and chemoresistance. Since hypoxia provokes a distinct shift of microRNA, it is important to illustrate the relative contribution of each hypoxamiR to cancer progression. The present study aims to shed light on the hypoxamiRs that are involved in pancreatic and breast cancer progression to highlight novel targets for the development of new therapies. For 20 cycles, MCF7 breast cancer cells and PANC-1 pancreatic cancer cells were subjected to chronic cyclic hypoxia, which consisted of 72 hours of hypoxia followed by 24 hours of reoxygenation. After 10 and 20 cycles of hypoxia, miRNA expression alterations were profiled using RT-PCR array and further analyzed using a visual analytics platform. The MTT cell proliferation assay was used to determine hypoxic cells' chemoresistance to doxorubicin. Under chronic cyclic hypoxia, hypoxic PANC-1 cells have a comparable doubling time with their normoxic counterparts, whereas hypoxic MCF7 cells show a massive increase in doubling time when compared to their normoxic counterparts. Both hypoxic cell lines developed EMT-like phenotypes as well as doxorubicin resistance. According to the findings of miRNet, 6 and 10 miRNAs were shown to play an important role in enriching six hallmarks of pancreatic cancer in the 10th and 20th cycles of hypoxia, respectively, while 7 and 11 miRNAs were shown to play an important role in enriching the four hallmarks of breast cancer in the 10th and 20th cycles of hypoxia, respectively. miR-221, miR-21, miR-155, and miR-34 were found to be involved in the potentiation of hypoxic PANC-1 hallmarks at both the 10th and 20th cycles, while miR-93, miR-20a, miR-15, and miR-17 were found to be involved in the potentiation of hypoxic MCF7 hallmarks at both the 10th and 20th cycles. This variation in miRNA expression was also connected to the emergence of an EMT-like phenotype, alterations in proliferation rates, and doxorubicin resistance. The chemosensitivity results revealed that chronic cyclic hypoxia is critical in the formation of chemoresistant phenotypes in pancreatic and breast cancer cells. miR-181a and let-7e expression disparities in PANC1, as well as miR-93, miR-34, and miR-27 expression disparities in MCF7, may be associated with the formation of chemoresistant MCF7 and PANC-1 cells following 20 cycles of chronic cyclic hypoxia. Indeed, further research is needed since the particular mechanisms that govern these processes are unknown.

Persistent Proclivity to a Proinflammatory State in a Human Enteroid Model of Necrotizing Enterocolitis.

Background: Necrotizing enterocolitis (NEC) is a devastating disease of premature neonates with substantial morbidity and mortality. Necrotizing enterocolitis is associated with prematurity, a hyperinflammatory response, and dysregulation of intestinal barrier function. We hypothesize that patients with NEC will have an increased hyperinflammatory intestinal response compared with those without NEC. Patients and Methods: Enteroids were generated from intestinal tissue from neonates undergoing resection. They were treated with 100 mcg/mL lipopolysaccharide (LPS), subjected to 24 hours of hypoxia inducing experimental NEC, then compared with untreated controls. Expression of tumor necrosis factor (TNF-α) and interleukin 8 (IL-8) were evaluated via reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA) to measure inflammatory response. Analysis of variance (ANOVA) determined statistical significance (p < 0.05). Results: Treated NEC-derived enteroids expressed significantly higher levels of IL-8 (RT-qPCR, p = 0.003; ELISA, p = 0.0002) compared with untreated NEC-derived enteroids with an increase in inflammatory marker concentration in those with a greater degree of prematurity (ELISA, p = 0.0015). A higher level of IL-8 was seen in NEC-derived enteroids compared with control after treatment (RT-qPCR, p = 0.024). Tumor necrosis factor-α levels were elevated in treated NEC-derived enteroids compared with untreated NEC-derived enteroids (RT-qPCR, p = 0.006; ELISA, p = 0.002) and compared with treated non-NEC-derived enteroids (RT-qPCR, p = 0.025; ELISA, p < 0.0001). Conclusions: Enteroids generated from neonates with NEC have an elevated hyperinflammatory response in response to NEC-inducing stimuli compared with controls. Enteroids generated from neonates with NEC with a greater degree of prematurity have a larger increase in inflammatory markers. This tendency toward a hyperinflammatory state may be correlated with an infant's proclivity to develop NEC and further demonstrates the hyperinflammatory state of prematurity.

Fish Carboxyl Esterase Activity as a Possible Hypoxia Biomarker

Physiology and biochemistry adaptations studied in fish are important to comprehend how they endure the challenges of living in aquatic environments. Oxygen variations are a frequent natural and cyclic phenomenon that occurs in the Pantanal and Amazon watersheds and it is the limiting factor to survival of the aerobic species. The growing of pollution in these water basins threaten many species as anthropic hypoxia intensifies the production of reactive oxygen species (ROS) and lipid peroxidation inside the cells, leading organisms to a condition known as oxidative stress. Our research group, studying the effects of the oxygen variations on the carboxyl esterase (CarbE) enzyme of pacu, a typical fish from Pantanal, observed that CarbE activity diminished by half in pacus exposed to 42 hours of hypoxia. Based on this finding, we decided to investigate this phenomenon in other fish species, including hypoxia-tolerant fishes. CarbE activities were determined using p-nitrophenyl acetate (p-NPA) as a substrate. All five species of fish studied had both serum and liver CarbE activity significantly decreased. Pacus had its serum and liver activities decreased by approximately 50% under severe hypoxia.

PS-B08-4: CHRONIC INTERMITTENT HYPOXIA INDUCES RENAL FIBROSIS BY MR ACTIVATION

Purpose: Obstructive sleep apnea is mainly characterized by chronic intermittent hypoxia (CIH), which is associated with chronic kidney disease. Macrophage is known to play an important role in renal fibrosis. However, little is known of the potential role of macrophage-to-myofibroblast transition (MMT) in hypoxic conditions. The aim of this study was to understand how infiltrating macrophages may contribute to renal fibrosis in hypoxic conditions. Methods: In this study, 30 adult male sprague dawley (SD) rats were randomly divided into normoxic control(NC), chronic intermittent hypoxia(CIH), and eplerenone(EPL) groups-each containing ten rats. The CIH and EPL groups were exposed to hypoxia in a commercial hypoxic chamber for 5 weeks. Rats in the EPL group were administered EPL orally via diet for 5 weeks. All rats were then sacrificed and serum, 24-hour urine and tissue specimens were collected for renal function, histopathology, western blot, immunohistochemistry. The RAW264.7 macrophages were subjected to 24 hours of hypoxia and 12 hours of reoxygenation. Cells were collected for immunofluorescence staining. Results: In addition to blood pressure, renal aldosterone level was also increased in CIH rats. Massons trichrome and Sirius red staining showed significantly higher collagen deposition and fibrosis in CIH rats. A markedly increased co-expression of α-SMA and CD68 in vitro and in vivo after CIH exposure. MR nuclear translocation was seen in hypoxic macrophages. Western blot showed that the expression of SGK-1 and p-SGK-1 was upregulated. These effects were reversed by MR blocker (EPL). Conclusions: CIH-induced macrophage infiltration, an important source of myofibroblasts, might participate in the development of renal interstitial fibrosis. These effects were reversed by EPL. Therefore, we speculate MR activation may be related to CIH-induced renal fibrosis.

Hypoxia Primes Human ISCs for Interleukin-Dependent Rescue of Stem Cell Activity

Hypoxia in the intestinal epithelium can be caused by acute ischemic events or chronic inflammation in which immune cell infiltration produces inflammatory hypoxia starving the mucosa of oxygen. The epithelium has the capacity to regenerate after some ischemic and inflammatory conditions suggesting that intestinal stem cells (ISCs) are highly tolerant to acute and chronic hypoxia; however, the impact of hypoxia on human ISC (hISC) function has not been reported. Here we present a new microphysiological system (MPS) to investigate how hypoxia affects hISCs from healthy donors and test the hypothesis that prolonged hypoxia modulates how hISCs respond to inflammation-associated interleukins (ILs). hISCs were exposed to <1.0% oxygen in the MPS for 6, 24, 48, and 72 hours. Viability, hypoxia-inducible factor 1a (HIF1a) response, transcriptomics, cell cycle dynamics, and response to cytokines were evaluated in hISCs under hypoxia. HIF stabilizers and inhibitors were screened to evaluate HIF-dependent responses. The MPS enables precise, real-time control and monitoring of oxygen levels at the cell surface. Under hypoxia, hISCs maintain viability until 72 hours and exhibit peak HIF1a at 24 hours. hISC activity was reduced at 24 hours but recovered at 48 hours. Hypoxia induced increases in the proportion of hISCs in G1 and expression changes in 16 IL receptors. Prolyl hydroxylase inhibition failed to reproduce hypoxia-dependent IL-receptor expression patterns. hISC activity increased when treated IL1β, IL2, IL4, IL6, IL10, IL13, and IL25 and rescued hISC activity caused by 24 hours of hypoxia. Hypoxia pushes hISCs into a dormant but reversible proliferative state and primes hISCs to respond to a subset of ILs that preserves hISC activity. These findings have important implications for understanding intestinal epithelial regeneration mechanisms caused by inflammatory hypoxia.

Does severe hypoxia during irradiation of Aedes aegypti pupae improve sterile male performance?

BackgroundThe yellow fever mosquito, Aedes aegypti, vectors several pathogens responsible for human diseases. As a result, this mosquito species is a priority for control by mosquito control districts in Florida. With insecticide resistance development becoming a concern, alternative control strategies are needed for Ae. aegypti. Sterile insect technique (SIT) is an increasingly popular option that is being explored as a practical area-wide control method. However, questions about sterile male performance persist. The objectives of this study were to determine the extent to which hypoxia exposure prior to and during irradiation effects the longevity, activity and mating competitiveness of sterile male Ae. aegypti.MethodsMale longevity was monitored and analyzed using Cox regression. Mosquito activity was recorded by an infrared beam sensor rig that detected movement. Competing models were created to analyze movement data. Fecundity and fertility were measured in females mated with individual males by treatment and analyzed using one-way ANOVAs. Mating competition studies were performed to compare both hypoxia and normoxia treated sterile males to fertile males. Competitiveness of groups was compared using Fried’s competitiveness index.ResultsFirst, we found that subjecting Ae. aegypti pupae to 1 h of severe hypoxia (< 1 kPa O2) did not directly increase mortality. One hour of hypoxia was found to prevent decreases in longevity of irradiated males compared to males irradiated in normoxic conditions. Exposure to hypoxia prior to irradiation did not significantly improve activity of sterile males except at the highest doses of radiation. Hypoxia did significantly increase the required dose of radiation to achieve > 95% male sterility compared to males irradiated under normoxic conditions. Males sterilized after an hour in hypoxic conditions were significantly more competitive against fertile males compared to males irradiated under normoxic conditions despite requiring a higher dose of radiation to achieve sterility.ConclusionsHypoxia was found to greatly improve key performance metrics in sterile male Ae. aegypti without any significant drawbacks. Little work other than increasing the target dose for sterility needs to be conducted to incorporate hypoxia into SIT programs. These results suggest that SIT programs should consider including hypoxia in their sterile male production workflow.Graphical

Cannabidiol Attenuates Chronic and Acute Hyperalgesia in Sickle Mice By Reducing Inflammation and Oxidative Stress

Pain is a major comorbidity of sickle cell disease (SCD) leading to hospitalization, opioid use and reduced survival. It can present as both chronic pain and/or unpredictable episodes of intense acute pain. Hydroxyurea (HU) is the mainstream FDA approved treatment for SCD, but its effect on reducing pain remains to be understood. A larger number of people with SCD use cannabis products for pain relief compared to those with other conditions. Cannabidiol (CBD) is one of the major constituents of Cannabis sativa, second to tetrahydrocannabinol, and has gained attention for its ability to treat pain without intoxicating side effects. CBD has been shown to decrease inflammation, oxidative stress, and inhibit mast cell activation, all of which occur in SCD. Thus, we hypothesize that CBD will ameliorate both chronic and acute hyperalgesia in humanized homozygous BERK sickle mice (called sickle) by inhibiting mast cell activation and decreasing inflammation and oxidative stress. To simulate the clinical condition, we treated female sickle mice intraperitoneally with [i] vehicle (1:1:18 DMSO:Tween-20:saline), [ii] HU (50 mg/kg/d HU), [iii] CBD (20 mg/kg/d) or [iv] HU + CBD and determined their effect on chronic and acute hyperalgesia (pain). Sickle mice treated with synthetic CBD alone or cotreatment with HU+CBD daily showed significantly reduced chronic mechanical (p<0.001) and cold (p<0.001) hyperalgesia compared to baseline after 7 days of treatment, which was maintained for up to 14 days. HU decreased mechanical (p<0.01) but not cold hyperalgesia after 14 days compared to baseline. Thus, CBD independently attenuates chronic hyperalgesia, whereas HU may have a modest effect on chronic pain. Following the initial 14 days of treatment above, we incited acute hyperalgesia by treating sickle mice with hypoxia/reoxygenation (H/R; 3 hours of hypoxia at 8% O2 and 92% N2, followed by 1 hour of reoxygenation at normoxia) on days 15 (H/R-1) and 18 (H/R-2). We observed that CBD, HU, and HU+CBD treatments were able to prevent an increase in H/R-incited hyperalgesia (acute response). CBD alone and HU+CBD significantly reduced mechanical (p<0.001) and cold hyperalgesia (p<0.001) compared to baseline following incitement of both H/R-1 and H/R-2, and up to 25 days, the last period of observation. HU alone did not show a significant decrease in mechanical or cold hyperalgesia compared to baseline following incitement of either H/R event, except for cold hyperalgesia 72h post-H/R-2 (p<0.05). CBD and HU+CBD treatments also reduced non-evoked cold avoidance compared to baseline in sickle mice, which spent significantly more time (p<0.01) in a cold chamber (4°C) 7d after H/R-2 (day 25), but HU alone did not. Thus, HU has a modest or no effect on different features of sickle pain, while CBD is able to ameliorate both chronic and acute pain. We next investigated the mechanism of CBD's analgesic effect in sickle mice. Following 9 days of daily treatment, we observed that CBD decreased total and degranulating mast cells (p<0.05 and p<0.01, respectively) in the skin compared to vehicle, and a reduction in tryptase (p<0.05), a marker of mast cell activation, in the skin secretagogue. CBD treatment decreased substance P (p=0.05) and malondialdehyde, (p<0.05), markers of neuroinflammation and oxidative stress respectively, in the skin secretagogue, and serum amyloid P (p<0.05), a marker of inflammation, in plasma. CBD significantly decreased inflammatory cytokines IL-3, which promotes mast cell proliferation, TNFα, a hallmark feature of cytokine storm, and MIP-1α and RANTES, which contribute to leukocyte recruitment. Thus, CBD attenuates inflammation, neuroinflammation and oxidative stress in sickle mice and may ameliorate leukocyte activities, a hallmark feature of SCD. We demonstrate that CBD is effective in attenuating chronic hyperalgesia and preventing acute pain by decreasing inflammation and oxidative stress. The effect of HU on reducing pain following H/R and chronic pain are modest and specific to certain characteristic features of sickle pain. Importantly, HU does not appear to interfere with the analgesic effect of CBD in sickle mice, which shows promise for CBD treatment in persons with SCD on HU. The availability of a >99% CBD-extract (Epidiolex) approved by the FDA to treat some forms of epilepsy may help facilitate clinical trials for assessing the effects of CBD on pain in persons with SCD.

Evaluation of targeted oxidative stress induced by oxygen-ozone in vitro after ischemic induction

ABSTRACT Encephalic vascular accident, or stroke, is the most common pathology of the central nervous system in humans, the second leading cause of death and physical and cognitive disabilities, in developing countries. It presents as an ischemic (more common) or hemorrhagic form. Ozone therapy has been shown to be effective in neuromodulation, neuroprotection, and nerve regeneration. The present study aimed to evaluate the effect of targeted mild ozone after inducing cerebral ischemia in vitro. Neuroblastoma lineage cells (SH-SY5Y) and canine amniotic membrane stem cells were subjected to 24 hours of hypoxia in an incubator culture chamber. The cells were evaluated by MTT assay, colorimetric assay spectrophotometry, fluorescence microscopy, and flow cytometry. Treatment with low concentrations of ozone (2–10 µg/mL), indicated a possible neuroregenerative effect at low concentrations, correlated with lower levels of apoptosis and oxidative stress compared to cells not subjected to hypoxia. High concentrations of ozone (18–30 µg/mL) promoted an increase in rate of apoptosis and cell death. We developed a novel protocol that mimics ozone therapy for ischemic stroke, using ozonized culture medium after hypoxia induction. Although more studies are needed, we conclude that ozone has a dose-dependent hormetic effect and can reverse the effect of ischemia in vitro at low concentrations.

Environmental Hypoxia is a Driver of Sonic Hedgehog-dependent Troglomorphic Evolution in Astyanax Cavefish

Astyanax mexicanus cavefish (CF) have evolved numerous troglomorphic traits since their divergence from surface fish (SF) ancestors about 20,000 years ago. A large number of these traits are under the control of Sonic Hedgehog (Shh), a signaling morphogen that is overexpressed along the midline in CF relative to SF embryos. The Shh-dependent traits include eye degeneration and increases in the olfactory lobes, hypothalamus, jaws, and oral taste buds. Eye degeneration and tastebud enhancement are antagonistic tradeoffs governed by the pleiotropic shh gene. CF have also evolved multiple changes in circulatory system development, highlighted by larger hematopoietic domains, more erythrocytes, and reversals in normal heart asymmetry, which are also controlled by Shh signaling. Although Shh signaling is an important factor in CF troglomorphism, the shh gene is not associated with any of the many QTL identified by genetic analysis, suggesting that upstream processes, rather than mutation of the shh locus itself, may be important in Shh regulation. Due to the absence of primary productivity, cave pools harboring Astyanax CF are deficient in dissolved oxygen. Accordingly, the Hypoxia Inducing Factor (HIF) transcription factors and their downstream targets are constitutively active during CF development. To determine if oxygen deficiency is an upstream regulator of Shh signaling, we subjected early SF embryos to 8 or 16 hours of hypoxia in the laboratory and assayed the effects on shh expression, erythrocyte formation, heart asymmetry, and eye development. The results indicated that hypoxia can cause shh overexpression, increase erythropoiesis, reverse heart asymmetry, and induce eye degeneration in SF embryos. We conclude that environmental hypoxia may be responsible for the evolution of troglomorphic traits in Astyanax CF through hyperactivation of the Shh signaling pathway.

P1483: EPELEUTON, A NOVEL SYNTHETIC SECOND GENERATION W-3 FATTY ACID, PROTECTS HUMANIZED SICKLE CELL MICE AGAINST HYPOXIA/REOXYGENATION ORGAN DAMAGE

Background: Sickle cell disease (SCD) is a hereditary red cell disorder with high mortality and morbidity. Although crizanlizumab, a P-selectin inhibitor, and voxelotor, an oral anti-sickling agent, have been recently approved for clinical management of SCD, therapeutic options to limit acute events and to reduce disease progression are still required. An attempt to target inflammatory vasculopathy and to modulate inflammatory response has been made based on evidence in other diseases such as in cardiovascular disease looking to administration of ω-3 fatty acids. In humanized SCD mice, we previously showed that polyunsaturated fatty acid supplementation protects against acute sickle cell-related lung and liver damages during hypoxia/reoxygenation (H/R) induced vaso-occlusive crisis (VOCs) (Kalish B et al. Haematologica 2016). This agrees with the observation that the resolution process actively controlled by specialized pro-resolving lipid mediators is defective in SCD (Matte A et al, Blood 2019). Epeleuton (15-HEPE, 15 hydroxy eicosapentaenoic acid) is a novel synthetic second generation w-3 fatty acid which (i) has a favorable clinical safety profile, (ii) modulates systemic inflammation and triglyceride synthesis in patients with cardiometabolic disease (Climax J et al J Am Heart As 2020) and (ii) beneficially affects red cell indices in long-term studies in rats. Aims: To evaluate the impact of epeleuton on acute organ damage in a humanized mouse model of SCD. Methods: 3-4 month old male and female mice healthy control (Hbatm1(HBA)Tow Hbbtm3(HBG1,HBB)Tow) and Tim Townes sickle mice (SCD, Hba tm1(HBA)Tow Hbb tm2(HBG1,HBB*)Tow) were used. Animals were treated for 6 weeks with epeleuton 1,000 mg/kg once daily or vehicle by gavage. Mice underwent 10 hours of hypoxia (8% oxygen), followed by 3 hours of reoxygenation (21% oxygen) to mimic acute VOCs (Matte A et al, Blood 2019). Hematologic parameters and molecular analysis of target organs for SCD such as lung were carried out. Results: In SCD mice exposed to H/R stress, we found that epeleuton (i) reduces H/R induced hemolysis; (ii) normalizes tyrosine-phosphorylation profile of red cell membrane proteins; and (iii) decreases neutrophil count. In lungs of SCD mice exposed to H/R stress, we observed that epeleuton prevents the H/R induced activation of inflammatory and redox related transcription factors NF-kB, p65 and Nrf2 local inflammatory response compared with vehicle-treated animals. Epeleuton treated SCD mice exposed to H/R stress showed down-regulation of VCAM-1 and E-selectin, markers of inflammatory vasculopathy as well as reduction of lung protein oxidation and expression of anti-oxidant systems such as heme-oxygenase-1 (HO-1) or peroxiredoxin-2. Similar changes were also observed in livers of epeleuton-treated SCD mice exposed to H/R stress compared with vehicle-treated SCD animals. Of note, epeleuton prevented the H/R induced up-regulation of both VCAM-1 and ICAM-1, suggesting a direct impact of treatment on vascular activation and inflammation. Summary/Conclusion: In humanized SCD mice exposed to H/R stress to mimic acute VOCs, epeleuton acts as a multimodal agent targeting red cells, hemolysis, inflammatory response, and vascular dysfunction. Our data provide the rationale for epeleuton as a potential novel therapeutic option for clinical management of patients with SCD.

Mechanism of ALDH2 improves the neuronal damage caused by hypoxia/reoxygenation.

To investigate the protective effect and mechanism of ALDH2 on PC12 cells and brain nerve tissue injury under hypoxia. The hypoxia model of PC12 cells with low ALDH2 expression was established and screened. The eukaryotic expression vector of wild type pEGFP-N1-ALDH2 and blank plasmid pEGFP-N1 were constructed and transfected into PC12 hypoxia cells respectively. After reoxygenation culture, the morphology, quantity, ALDH2 expression level and apoptosis rate of the two groups were observed, and the role of ALDH2 in cell hypoxia injury was analyzed. Eighty SD rats were randomly divided into model group (ischemia-reperfusion injury group), Alda-1 group (intraperitoneal injection of alda-1 12 hours before and after modeling), DMSO group (intraperitoneal injection of dimethyl sulfoxide) and sham operation group, with 20 rats in each group. The neurobehavioral score, apoptosis rate of nerve cells, the content and activity of ALDH2 in active cerebral cortex and hippocampal CA1 area were compared. The number of PC12 cells in hypoxia group was lower than that in control group. The expression level of ALDH2 protein in PC12 cells after 4 hours of hypoxia was lower than that in normal culture group. The number of PC12 cells transfected with wild-type recombinant plasmid was significantly more than that of blank plasmid group. Compared with the hypoxia group, the pre apoptotic and post apoptotic cells in wild type transfection group decreased after hypoxia treatment. Compared with sham operation group, nerve injury and apoptosis were increased in group M and DMSO, while ALDH2 activity and expression did not change significantly. Compared with M group and DMSO group, the nerve injury and apoptosis in Alda-1 group were improved, ALDH2 activity was increased, and ALDH2 expression was not significantly changed in Alda-1 group. Increasing the expression of ALDH2 or enhancing the activity of ALDH2 can improve the injury of neurons induced by hypoxia/reoxygenation.

Knockdown of TRIM8 Protects HK-2 Cells Against Hypoxia/Reoxygenation-Induced Injury by Inhibiting Oxidative Stress-Mediated Apoptosis and Pyroptosis via PI3K/Akt Signal Pathway.

BackgroundAcute kidney injury (AKI) emerges as an acute and critical disease. Tripartite motif 8 (TRIM8), one number of the TRIM protein family, is proved to participate in ischemia/reperfusion (I/R) injury. However, whether TRIM8 is involved in renal I/R injury and the associated mechanisms are currently unclear.PurposeThis study aimed to investigate the precise role of TRIM8 and relevant mechanisms in renal I/R injury.Materials and MethodsIn this study, human renal proximal tubular epithelial cells (HK-2 cells) underwent 12 hours of hypoxia and 2 h, 3 h or 4 h of reoxygenation to establish an in vitro hypoxia/reoxygenation (H/R) model. The siRNAs specific to TRIM8 (si-TRIM8) were transfected into HK-2 cells to knockdown TRIM8. The cell H/R model included various groups including Control, H/R, H/R+DMSO, H/R+NAC, si-NC+H/R, si-TRIM8+H/R and si-TRIM8+LY294002+H/R. The cell viability and levels of reactive oxygen species (ROS), hydrogen peroxide (H2O2), mRNA, apoptotic proteins, pyroptosis-related proteins and PI3K/AKT pathway-associated proteins were assessed.ResultsIn vitro, realtime-quantitative PCR and western-blot analysis showed that the mRNA and protein expression of TRIM8 were obviously upregulated after H/R treatment in HK-2 cells. Compared with the H/R model group, knockdown of TRIM8 significantly increased cell viability and reduced the levels of ROS, H2O2, apoptotic proteins (Cleaved caspasebase-3 and BAX) and pyroptosis-related proteins (NLRP3, ASC, Caspase-1, Caspase-11, IL-1β and GSDMD-N). Western-blot analysis also authenticated that PI3K/AKT pathway was activated after TRIM8 inhibition. The application of 5 mM N-acetyl-cysteine, one highly efficient ROS inhibitor, significantly suppressed the expression of apoptotic proteins and pyroptosis-related proteins. Moreover, the combined treatment of TRIM8 knockdown and LY294002 reversed the effects of inhibiting oxidative stress.ConclusionKnockdown of TRIM8 can alleviate H/R-induced oxidative stress by triggering the PI3K/AKT pathway, thus attenuating pyropyosis and apoptosis in vitro.

Abstract 11972: Ivabradine Attenuates Hypoxia/Reoxygenation-Induced Excessive Autophagy in H9c2 Cardiomyocyte by Modulation of the PI3K/Akt/mTOR Pathway

Introduction: Despite recent advances in resuscitation techniques, the mortality associated with survival from cardiac arrest (CA) still remains low. Cardiovascular ischemia/reperfusion injury (IRI) is one of the primary pathophysiology involved. Hypothesis: We assessed the hypothesis that ivabradine could attenuate hypoxia/reoxygenation injury of H9c2 cardiomyocytes by inhibiting excessive autophagy through PI3K/Akt/mTOR Pathway. Methods: Cultured H9c2 were randomly divided into 3 groups: CON (normoxia), H/R (hypoxia reoxygenation) and IVA. The IVA was divided into 4 subgroups, in which H9c2 were treated with or without ivabradine(20μM or 100μM) or PI3-kinase inhibitor LY294002(10μM) for 12 hours and then subjected to 12 hours of hypoxia and 24 hours of reoxygenation. Hypoxia was achieved by a hypoxia chamber filled with 5%CO 2 and 95% N 2 at 37°C. Cell viability were measured with CCK-8 assay kits. Cell autophagy was assessed by transmission electron microscopy (TEM). The expressions of autophagy marker protein (LC3, Beclin-1), PI3K, Akt and mTOR were determined by Western-blot assay. Results: A decrease of cell viability and an increase formulation of autophagosomes /autophagy lysozymes occurred after H/R. Significant improvement was noted in cells treated with ivabradine compared to H/R(Figure 1). Ivabradine promoted pmTOR/mTOR expression and lower expressions of LC3II/LC3I and Beclin 1. LY294002 antagonized the effects of ivabradine on antophagy (Figure 2). Conclusions: Ivabradine could protect H9c2 against H/R injury via inhibiting excessive autophagy through PI3K/Akt/mTOR pathway.