Days Of Hypoxia Research Articles

Pathobiological characterization of pulmonary hypertension associated with heart failure with preserved ejection fraction

Abstract Introduction Heart failure with preserved ejection fraction (HFpEF) is an age-related cardiometabolic disease associated with vascular inflammation in cardiac tissue, pulmonary congestion and right ventricular dysfunction. Patients with HFpEF often develop pulmonary hypertension (PH) and right ventricular (RV) hypertrophy. Pulmonary vascular remodeling contributes to combined post- and precapillary PH (CpcPH), which is associated with worse outcome in humans. A recently developed meta-inflammatory murine HFpEF model approximates the characteristics of human HFpEF. Herein, HFpEF is induced by metabolic (high-fat diet (HFD)) and hypertensive stress (inhibition of constitutive NO synthase by Nω-nitro-1-arginomethyl ester (L-NAME)) over a 12-week period of time. With a focus on PH, we have developed a 'three-hit' model based on this model with a supplementary 21- or 42-day hypoxia phase (HFpEF-CpcPH). Methods Adult C57BL/6N mice were fed HFD for 15 or 18 weeks and supplied with L-NAME (0,5g/L or 1g/L) via drinking water, including a final period of 21 or 42 days, respectively, of hypoxia (normobaric, 10 % oxygen) or normoxia. Transthoracic echocardiography was performed using a small animal ultrasound device (Vevo 3100, Visual Sonics) and measurement of right ventricular systolic pressure (RVSP) using a Millar® pressure catheter (Mil-SPR-1000) inserted into the right ventricle. Right ventricular hypertrophy was measured as the ratio of the wet weight of the right ventricle to the left ventricle including the septum (RV/LV+S). Results Administration of HFD and L-NAME had no significant effect on RVSP in either HFpEF-normoxia group (15 weeks (n=8) or 18 weeks (n=5)) compared to the control group (n=8, n=5). 21 days of hypoxia increased RV systolic pressure (RVSP) to 37.05±1.6 (PH, n=8) and 40.33±0.87 mmHg (HFpEF-CpcPH, n=8) respectively, while 42 days of hypoxia caused no further increase (35.1±3.5 mmHg (PH, n=5) and 35.24±3.47 mmgHg (HFpEF-CpcPH, n=4)), respectively. RV hypertrophy (RV/LV+S) was significantly increased after 21 days of hypoxia in HFpEF-CpcPH compared to HFpEF (n=8) (34.42±1.75 vs. 24.93±1.1). Again, 42 days of hypoxia did not lead to a further increase in RV hypertrophy (35.2±3.9 vs. 25.80±4.39) in HFpEF-CpcPH, even though there was a significant difference compared to the HFpEF group. As expected, the heart weight/body weight ratio was significantly increased in HFpEF-CpcPH compared to HFpEF at both time points. However, mice under the prolonged hypoxia phase showed a significantly greater decrease in body weight, which complicates the interpretation of the results. Conclusion In summary, we could show that in the meta-inflammatory HFpEF model no significant PH can be observed within the investigated periods of time. However, a 21 day hypoxia phase was sufficient to induce a HFpEF-CpcPH phenotype. This model provides an important basis to develop new therapeutic approaches.

Ultrasound Neuromodulation of an Anti-Inflammatory Pathway at the Spleen Improves Experimental Pulmonary Hypertension.

Inflammation is pathogenically implicated in pulmonary arterial hypertension; however, it has not been adequately targeted therapeutically. We investigated whether neuromodulation of an anti-inflammatory neuroimmune pathway involving the splenic nerve using noninvasive, focused ultrasound stimulation of the spleen (sFUS) can improve experimental pulmonary hypertension. Pulmonary hypertension was induced in rats either by Sugen 5416 (20 mg/kg SQ) injection, followed by 21 (or 35) days of hypoxia (sugen/hypoxia model), or by monocrotaline (60 mg/kg IP) injection (monocrotaline model). Animals were randomized to receive either 12-minute-long sessions of sFUS daily or sham stimulation for 14 days. Catheterizations, echocardiography, indices of autonomic function, lung and heart histology and immunohistochemistry, spleen flow cytometry, and lung single-cell RNA sequencing were performed after treatment to assess the effects of sFUS. Splenic denervation right before induction of pulmonary hypertension results in a more severe disease phenotype. In both sugen/hypoxia and monocrotaline models, sFUS treatment reduces right ventricular systolic pressure by 25% to 30% compared with sham treatment, without affecting systemic pressure, and improves right ventricular function and autonomic indices. sFUS reduces wall thickness, apoptosis, and proliferation in small pulmonary arterioles, suppresses CD3+ and CD68+ cell infiltration in lungs and right ventricular fibrosis and hypertrophy and lowers BNP (brain natriuretic peptide). Beneficial effects persist for weeks after sFUS discontinuation and are more robust with early and longer treatment. Splenic denervation abolishes sFUS therapeutic benefits. sFUS partially normalizes CD68+ and CD8+ T-cell counts in the spleen and downregulates several inflammatory genes and pathways in nonclassical and classical monocytes and macrophages in the lung. Differentially expressed genes in those cell types are significantly enriched for human pulmonary arterial hypertension-associated genes. sFUS causes dose-dependent, sustained improvement of hemodynamic, autonomic, laboratory, and pathological manifestations in 2 models of experimental pulmonary hypertension. Mechanistically, sFUS normalizes immune cell populations in the spleen and downregulates inflammatory genes and pathways in the lung, many of which are relevant in human disease.

Effects of environmental hypoxia on the goldfish skeletal muscle: Focus on oxidative status and mitochondrial dynamics

The skeletal muscle is a highly plastic tissue. Its ability to respond to external stimuli and challenges allows it to face the functional needs of the organism. In the goldfish Carassius auratus, a model of hypoxia resistance, exposure to reduced oxygen is accompanied by an improvement of the swimming performance, relying on a sustained contractile behavior of the skeletal muscle. At the moment, limited information is available on the mechanisms underlying these responses.We here evaluated the effects of short- (4 days) and long- (20 days) term exposure to moderate water hypoxia on the goldfish white skeletal muscle, focusing on oxidative status and mitochondrial dynamics. No differences in lipid peroxidation, measured as 2-thiobarbituric acid-reacting substances (TBARS), and oxidatively modified proteins (OMP) were detected in animals exposed to hypoxia with respect to their normoxic counterparts. Exposure to short-term hypoxia was characterized by an enhanced SOD activity and expression, paralleled by increased levels of Nrf2, a regulator of the antioxidant cell response, and HSP70, a chaperone also acting as a redox sensor. The expression of markers of mitochondrial biogenesis (TFAM) and abundance (VDAC) and of the mtDNA/nDNA ratio was similar under normoxia and under both short- and long-term hypoxia, thus excluding a rearrangement of the mitochondrial apparatus. Only an increase of PGC1α (a transcription factor involved in mitochondrial dynamics) was detected after 20 days of hypoxia. Our results revealed novel aspects of the molecular mechanisms that in the goldfish skeletal muscle may sustain the response to hypoxia, thus contributing to adequate tissue function to organism requirements.

A ring N(CH3)2-based derivative of resveratrol inhibits pulmonary vascular remodeling in hypoxia pulmonary hypertension

Pulmonary artery smooth muscle cells (PASMCs) phenotypic switching and pulmonary artery endothelial cells (PAECs) endothelial-mesenchymal transition (EndMT) are important in promoting pulmonary hypertension (PH)-pulmonary vascular remodeling (PVR). Resveratrol can efficiently inhibit the proliferation of PASMCs, but its application is limited due to its low bioavailability and solubility. In this study, we modified resveratrol to assess the role of A ring N(CH3)2-based derivatives of resveratrol (Res4) in PVR-PASMCs phenotypic switching and PVR-PAECs EndMT. Chemical methods were used for the preparation of Res4; NMRS and HPLC were used to authenticate Res4. Mice developed PVR after 4 weeks of hypoxia (10% O2). Res4 (50 mg/kg/d) attenuated right ventricular systolic pressure, right ventricular hypertrophy, and PVR. PASMCs developed phenotypic switching and PAECs developed EndMT after 2 days of hypoxia (3% O2). Res4 (10 μM) could inhibit PASMCs and PAECs viability. Res4 could decrease proliferating cell nuclear antigen (PCNA) and osteopontin (OPN) expression, and increase α-smooth muscle actin (α-SMA) and vimentin expression in PASMCs. It could also decrease PCNA, α-SMA, vimentin expression and increase platelet endothelial cell adhesion molecule (CD31) expression in PAECs. Notably, Res4 inhibited the phosphorylation levels of mitogen-activated protein kinase kinase (MEK), extracellular signal-regulated protein kinase (ERK), Jun-N-terminal kinase (JNK), and p38 kinase in hypoxia-treated PASMCs and PAECs, indicating MAPK pathway may be involved in Res4-induced inhibition of PASMCs phenotypic switching and PAECs EndMT. Our data demonstrated that Res4 exerts antiproliferative effects by regulating PASMCs phenotypic switching and PAECs EndMT. Res4 may be potentially used as a drug against PH-PVR.

Fourteen Days Of Hypoxia And Apnea Training Does Not Modify Pulmonary Gas Exchange Efficiency

Fourteen Days Of Hypoxia And Apnea Training Does Not Modify Pulmonary Gas Exchange Efficiency

SAFETY AND FEASIBILITY OF HYPOXIA EXPOSURE IN PATIENTS WITH PRIOR MYOCARDIAL INFARCTION

Objective: In mice, normobaric hypoxia exposure corresponding to 8,000 m altitude after myocardial infarction reactivated cardiomyocyte proliferation and improved left ventricular ejection fraction (LVEF). Translating these findings to patients might be limited by hypoxia-induced pulmonary hypertension and myocardial ischemia. Design and method: We conducted a feasibility and safety study at the: envihab facility in 4 patients who had experienced myocardial infarction with fully revascularized LAD-stenosis as culprit lesion 37-104 months prior study entry. Their peak VO2 ranged from 32 - 43 ml/kg/min. The study comprised a two-days baseline, 19 days normobaric hypoxia, and two-days recovery. We gradually lowered atmospheric oxygen to 0.118 FIO2, which is equivalent to 4500 m, and maintained that level for 4.5 days. We obtained transthoracic echocardiography, magnetic resonance imaging, and determined Troponin I, NTproBNP at baseline, during 0.118 FIO2, and recovery. Results: Except for symptoms of acute mountain sickness, hypoxia was well tolerated as severe adverse reactions did not occur and pulmonary hypertension rapidly abated during recovery. During the last 10 days of hypoxia, peripheral oxygen saturation was continuously <90%. LVEF was 50.7±11.0 % (mean±SD) baseline, 57.6±11.2% at 0.118 FIO2, and 57.3±11.2% during recovery. Furthermore, echocardiographic LV global longitudinal strain was -15.28±5.4% at baseline and -17.28±7.56 % in recovery. Magnetic resonance imaging confirmed these findings. NTproBNP diminished (baseline: 203±209 pg/ml, 0.118 FIO2: 122±101/pg/ml, recovery: 85±66 pg/ml) and Troponin I concentrations remained in the reference range throughout the study. In the patient with the largest LVEF increase following hypoxia, an additional 12 weeks follow-up measurement showed stable improvements in LVEF (baseline: 36%, recovery: 48%, 12 weeks follow-up: 47%), left ventricular strain, NTproBNP, and exertional dyspnea. Conclusions: Our study provides evidence regarding safety and potential of hypoxia exposure after myocardial infarction and lays the foundation for future studies assessing hypoxia-influences on cardiac regeneration. However, our findings have to be interpreted with caution given the small sample size and the selected patient population.

The combined effects of 14 days of hypoxia and apnea training on hemoglobin mass and reticulocyte counts

INTRODUCTION: Indigenous diving populations like the Bajau have extraordinary breath-hold abilities, which includes tolerating arterial partial pressures that are low in oxygen and high in carbon dioxide. While these populations have genetic adaptations that improve their hypoxemic and hypercapnic tolerances, other populations can improve these tolerances through training adaptations that allow for increases in oxygen carrying and pH buffering capacities. For example, two weeks of residence at altitude stimulates increases in hemoglobin mass (Hbmass) via erythropoiesis, while completing apnea training daily for 14 days elevates reticulocyte count. However, it is unclear whether relatively brief (<4 h) daily exposures to moderate hypoxia and maximal apneas, such as may occur with diving, meaningfully stimulates erythropoiesis in people without breath hold diving experience or ancestry. PURPOSE: Test the hypothesis that 14 d of moderate hypoxia for 3 h/d and 10 maximal apneas/d augments circulating Hbmass and reticulocyte count. METHODS: Eighteen healthy adults (27±6 y; 11 women) completed 14 days of training within a 16-day period. Training days consisted of 10 maximal apneas and 3 h of exposure to normobaric hypoxia (FiO2=0.14). Average apnea duration was recorded and presented as mean ± SD. On Days 1, 7, and 14 subjects completed a carbon monoxide rebreathe procedure pre and post training to estimate Hbmass (n=11) and provided a venous sample of blood for reticulocyte count (n=14) pre and post training. Repeated measures linear mixed models were used to assess change in Hbmass and reticulocytes from Day 1. These data are presented as mean difference (diff.) from Day 1 and 95% confidence intervals. RESULTS: Average apnea duration increased from Day 1 (69±29 s) to Day 7 (83±30 s, p<0.01) and to Day 14 (90±29 s, p<0.01), but there was no change from Day 7 to Day 14 (p=0.26). There was no interaction (p=0.79) or main effects of Day (p=0.54) or pre/post training (p=0.94) on Hbmass. Specifically, pre training Hbmass did not differ from Day 1 on Day 7 (mean diff. 23 (–131, 178) g, p=0.91) or Day 14 (mean diff. 5 (–154, 164) g, p=0.99) and post training Hbmass did not differ Day 1 on Day 7 (mean diff. 29 (–152, 210) g, p=0.90) or Day 14 (mean diff. 5 (–154, 164) g, p=0.99). An interaction effect (p=0.02) for reticulocyte count, showed a mean increase from pre to post training on Day 14 (mean diff. 9 (3, 15) #·μL-1, p<0.01). However, there was no change in reticulocyte count between Days (p=0.53). Pre training Day 1 reticulocyte count did not differ from Day 7 (mean diff. -3 (–9, 4) #·μL-1, p=0.48) or Day 14 (mean diff. -2 (–9, 6) #·μL-1, p=0.79). CONCLUSION: Fourteen days of brief daily exposures to moderate hypoxia and maximal apnea training did not augment circulating Hbmass or reticulocytes. Supported by Office of Naval Research award N00014-20-1-2593. This is the full abstract presented at the American Physiology Summit 2023 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.

Insight into the role of myokines and myogenic regulatory factors under hypobaric hypoxia induced skeletal muscle loss

ABSTRACT Background The present study aimed to analyse the role of myokines and the regeneration capacity of skeletal muscle during chronic hypobaric hypoxia (CHH). Methods Male Sprague-Dawley rats were exposed to hypobaric hypoxia (HH) for 1d, 3d and 7d. Results Exposure to HH enhanced the levels of decorin, irisin, IL-6 and IL-15 till 3 days of hypoxia and on 7 day of exposure, no significant changes were observed in relation to control. A significant upregulation in myostatin, activated protein kinase, SMAD3, SMAD4, FOXO-1, MURF-1 expression was observed with prolonged HH exposure as compared to normoxic control. Further, myogenesis-related markers, PAX-7, Cyclin D1 and myogenin were downregulated during CHH exposure in comparison to control. Energy metabolism regulators such as Sirtuin 1, proliferator-activated receptor gamma coactivator-1α and GLUT-4, were also increased on 1-d HH exposure that showed a declining trend on CHH exposure. Conclusions These results indicated the impairment in the levels of myokines and myogenesis during prolonged hypoxia. CHH exposure enhanced the levels of myostatin and reduced the regeneration or repair capacity of the skeletal muscles. Myokine levels could be a predictive biomarker for evaluating skeletal muscle performance and loss at high altitudes.

Cardioprotective Antioxidant and Anti-Inflammatory Mechanisms Induced by Intermittent Hypobaric Hypoxia.

More than 80 million people live and work (in a chronic or intermittent form) above 2500 masl, and 35 million live in the Andean Mountains. Furthermore, in Chile, it is estimated that 100,000 people work in high-altitude shifts, where stays in the lowlands are interspersed with working visits in the highlands. Acute exposure to high altitude has been shown to induce oxidative stress in healthy human lowlanders due to increased free radical formation and decreased antioxidant capacity. However, intermittent hypoxia (IH) induces preconditioning in animal models, generating cardioprotection. Here, we aim to describe the responses of a cardiac function to four cycles of intermittent hypobaric hypoxia (IHH) in a rat model. The twelve adult Wistar rats were randomly divided into two equal groups, a four-cycle of IHH and a normobaric hypoxic control. Intermittent hypoxia was induced in a hypobaric chamber in four continuous cycles (1 cycle = 4 days of hypoxia + 4 days of normoxia), reaching a barometric pressure equivalent to 4600 m of altitude (428 Torr). At the end of the fourth cycle, cardiac structural and functional variables were also determined by echocardiography; furthermore, cardiac oxidative stress biomarkers (4-Hydroxynonenal, HNE; nitrotyrosine, NT), antioxidant enzymes, and NLRP3 inflammasome panel expression are also determined. Our results show a higher ejection and a shortening fraction of the left ventricle function by the end of the fourth cycle. Furthermore, cardiac tissue presented a decreased expression of antioxidant proteins. However, a decrease in IL-1β, TNF-αn, and oxidative stress markers is observed in IHH compared to normobaric hypoxic controls. Non-significant differences were found in protein levels of NLRP3 and caspase-1. IHH exposure determines structural and functional heart changes. These findings suggest that initial states of IHH are beneficial for cardiovascular function and protection.

The survival and responses of blue mussel Mytilus edulis to 16-day sustained hypoxia stress

The blue mussel Mytilus edulis, which is a worldwide commercial species distributed mainly from the intertidal zone to tens of meters deep, has been previously studied regarding its acute defense responses to air exposure and intermittent hypoxia. However, the effects of sustained hypoxia, such as caused by coastal eutrophication, remain to be explored. In the present study, the critical threshold of dissolved oxygen (DO) for experimental mussels exposed to 16 days of hypoxia was DO 0.7–0.8 mg L−1, below which survival dropped drastically from nearly 80% to <38%. When hypoxia was combined with DO fluctuations or with poor water quality, the threshold rose to an average of DO 1.0 mg L−1, which resulted in less than 80% survival. To find possible clues of physiological stress to account for mortalities, the metabolic rate and enzyme activities of Na+/K+ ATPase, superoxide dismutase, acid phosphatase, and alkaline phosphatase were further recorded.

Pharmacodynamic of cilostazol for anti-altitude hypoxia.

The plateau environment is characterized by low oxygen partial pressure, leading to the reduction of oxygen carrying capacity in alveoli and the reduction of available oxygen in tissues, and thus causing tissue damage. Cilostazol is a phosphodiesterase III inhibitor that has been reported to increase the oxygen release of hemoglobin (Hb) in tissues. This study aims to explore the anti-hypoxic activity of cilostazol and its anti-hypoxic effect. A total of 40 male BALB/C mice were randomly divided into a low-dose cilostazol (6.5 mg/kg) group, a medium-dose (13 mg/kg) group, a high-dose (26 mg/kg) group, and a control group. The atmospheric airtight hypoxia experiment was used to investigate the anti-hypoxic activity of cilostazol and to screen the optimal dosage. Twenty-four male Wistar rats were randomly divided into a normoxia control group, a hypoxia model group, an acetazolamide (22.33 mg/kg) group, and a cilostazol (9 mg/kg) group. After 3 days of hypoxia in the 4 010 m high altitude, blood from the abdominal aorta was collected to determine blood gas indicators, the levels of IL-6 and TNF-α in plasma were determined by enzyme-linked immunosorbent assay, and the levels of malondialdehyde (MDA), superoxide dismutase (SOD), and glutataione (GSH) were measured. The degree of pathological damage for rat tissues was observed with HE staining. Compared with the control group, the survival time of mice in the low, medium, and high dose group of cilostazol was significantly prolonged, and the survival time of mice in the medium dose group was the longest, with an extension rate at 29.34%, so the medium dose was the best dose. Compared with the hypoxia model group, the P50 (oxygen partial pressure at Hb oxygen saturation of 50%) value of rats in the cilostazol group was significantly increased by 1.03%; Hb and Hct were significantly reduced by 8.46% and 8.43%, and the levels of IL-6 and TNF-α in plasma were reduced by 50.65% and 30.77%. The MDA contents in heart, brain, lung, liver, and kidney tissues were reduced by 37.12%, 29.55%, 25.00%, 39.34%, and 21.47%, respectively. The SOD activities were increased by 94.93%, 9.14%, 9.42%, 13.29%, and 20.80%, respectively. The GSH contents were increased by 95.24%, 28.62%, 28.57%, 20.80%, and 44.00%, respectively. The results of HE staining showed that compared with the hypoxia model group, cilostazol significantly improved the damage of heart, lung, and kidney tissues in rats after hypoxia. Cilostazol can significantly improve the oxidative stress and inflammatory reaction caused by rapid altitude hypoxia, and it has a significant protective effect on tissue damage caused by hypoxia, suggesting that it has obvious anti-hypoxic activity.

Abstract 14009: Longitudinal Analysis Supports Reproducibility Of Treatment With Sildenafil In The Semaxanib/Hypoxia Rat Model Of Pulmonary Arterial Hypertension

Rats develop translatable pulmonary arterial hypertension (PAH) after exposure to the VEGF antagonist semaxanib (SU5416) concurrent with hypoxia. Sildenafil is used clinically for PAH and as a positive control in this preclinical model. A longitudinal analysis was constructed by pooling data from discrete rat PAH studies conducted over an 8-year period to evaluate the stability and reproducibility of the SU5416/Hypoxia rodent model as well as the response to sildenafil. Sprague-Dawley rats were administered SU5416 on Day 1 and subsequently maintained in a low oxygen environment for 28 days. Sildenafil was given as a positive control in either prevention or treatment mode. Pulmonary hemodynamics were obtained on the final day as appropriate by design; hearts were also assessed for right ventricular hypertrophy. Prevention studies (≥17) were terminated following 28 days of hypoxia while most treatment studies (≥17) had a protocol-defined period of normoxia following hypoxia. In prevention studies, the mean of both systolic pulmonary arterial pressure (sPAP) and the right ventricular hypertrophy Fulton’s Index (FI) remained consistent across studies over the 7-year period: sPAP: PAH vehicle control (PAH-VC) 70 ± 19 mmHg (n = 190), sildenafil 51 ± 15 mmHg (n = 165), p&lt;.05; FI: PAH-VC 0.5573 ± 0.1048 (n = 210), sildenafil 0.4984 ± 0.1007 (n = 172), p&lt;.05. Sildenafil elicited mean reductions in sPAP of 28% and FI of 11% over the 7-year period. Mean survival rate was comparable for PAH-VC (96%) and sildenafil groups (99%).In treatment studies, the mean of both sPAP and FI remained consistent across studies over the 8-year period: sPAP: PAH-VC 89 ± 24 mmHg (n = 148) vs. sildenafil 67 ± 22 mmHg (n = 168), p&lt;.05 vs PAH-VC; FI: PAH-VC 0.5938 ± 0.0944 (n = 168) vs. sildenafil 0.5336 ± 0.1077 (n = 173), p&lt;.05 vs PAH-VC. Sildenafil elicited mean reductions in sPAP of 25% and FI of 10% over the 8-year period. Mean survival rate was comparable for PAH-VC (91%) and sildenafil groups (95%).The effects of sildenafil on reduction of PAH were stable and of similar magnitude in both prevention and treatment studies over an 8-year period. Thus, this longitudinal analysis indicates a reproducible and consistent effect of both the SU5416/Hypoxia PAH model and the use of sildenafil as a positive control.

Sildenafil Citrate Downregulates PDE5A mRNA Expression in Women with Recurrent Pregnancy Loss without Altering Angiogenic Factors-A Preliminary Study.

In our previous study, we showed that sildenafil citrate (SC), a selective PDE5A blocker, modulated NK cell activity in patients with recurrent pregnancy loss, which correlated with positive pregnancy outcomes. It was found that NK cells had a pivotal role in decidualization, angiogenesis, spiral artery remodeling, and the regulation of trophoblast invasion. Thus, in the current study, we determined the effects of SC on angiogenic factor expression and production, as well as idNK cell activity in the presence of nitric synthase blocker L-NMMA. Methods: NK cells (CD56+) were isolated from the peripheral blood of 15 patients and 15 fertile women on MACS columns and cultured in transformation media containing IL-15, TGF-β, and AZA—a methylation agent—for 7 days in hypoxia (94% N2, 1% O2, 5% CO2). Cultures were set up in four variants: (1) with SC, (2) without SC, (3) with NO, a synthase blocker, and (4) with SC and NO synthase blocker. NK cell activity was determined after 7 days of culturing as CD107a expression after an additional 4h of stimulation with K562 erythroleukemia cells. The expression of the PDE5A, VEGF-A, PIGF, IL-8, and RENBP genes was determined with quantitative real-time PCR (qRT-PCR) using TaqMan probes and ELISA was used to measure the concentrations of VEGF-A, PLGF, IL-8, Ang-I, Ang-II, IFN–γ proteins in culture supernatants after SC supplementation. Results: SC downregulated PDE5A expression and had no effect on other studied angiogenic factors. VEGF-A expression was increased in RPL patients compared with fertile women. Similarly, VEGF production was enhanced in RPL patients’ supernatants and SC increased the concentration of PIGF in culture supernatants. SC did not affect the expression or concentration of other studied factors, nor idNK cell activity, regardless of NO synthase blockade.

Arginine:Glycine Amidinotransferase Is Essential for Creatine Supply in Mice During Chronic Hypoxia.

Objective: Chronic hypoxia induces pulmonary and cardiovascular pathologies, including pulmonary hypertension (PH). L-arginine:glycine amidinotransferase (AGAT) is essential for homoarginine (hArg) and guanidinoacetate synthesis, the latter being converted to creatine by guanidinoacetate methyltransferase. Low hArg concentrations are associated with cardiovascular morbidity and predict mortality in patients with PH. We therefore aimed to investigate the survival and cardiac outcome of AGAT knockout (Agat−/−) mice under hypoxia and a possible rescue of the phenotype.Methods:Agat−/− mice and wild-type (WT) littermates were subjected to normoxia or normobaric hypoxia (10% oxygen) for 4 weeks. A subgroup of Agat−/− mice was supplemented with 1% creatine from weaning. Survival, hematocrit, blood lactate and glucose, heart weight-to-tibia length (HW/TL) ratio, hArg plasma concentration, and Agat and Gamt expression in lung, liver, and kidneys were evaluated.Results: After 6 h of hypoxia, blood lactate was lower in Agat−/−-mice as compared to normoxia (p < 0.001). Agat−/− mice died within 2 days of hypoxia, whereas Agat−/− mice supplemented with creatine and WT mice survived until the end of the study. In WT mice, hematocrit (74 ± 4 vs. 55 ± 2%, mean ± SD, p < 0.001) and HW/TL (9.9 ± 1.3 vs. 7.3 ± 0.7 mg/mm, p < 0.01) were higher in hypoxia, while hArg plasma concentration (0.25 ± 0.06 vs. 0.38 ± 0.12 μmol/L, p < 0.01) was lower. Agat and Gamt expressions were differentially downregulated by hypoxia in lung, liver, and kidneys.Conclusion:Agat and Gamt are downregulated in hypoxia. Agat−/− mice are nonviable in hypoxia. Creatine rescues the lethal phenotype, but it does not reduce right ventricular hypertrophy of Agat−/− mice in hypoxia.

Possible Adaptation of the Adrenal Gland Hsp72 Expression to Hypoxic Stress.

Akin, Senay, Metin Bastug, Ridvan Colak, Hakan Ficicilar, Betul Simten Saglam, Nazan S. Kosar, and Haydar Demirel. Possible adaptation of the adrenal gland Hsp72 expression to hypoxic stress. High Alt Med Biol. 22:293-299, 2021. Background: Adrenal glands play a central role in the general response to stress and controlling wholebody homeostasis. One of the most severe environmental stresses encountered by high-altitude climbers is hypoxia. Since the 72 kDa heat shock protein (Hsp72) has a critical role in cellular homeostasis, regulation of Hsp72 in adrenal glands seems to be crucial for maintaining cellular integrity of the gland and sustaining an adequate whole-body stress response in a hypoxic environment. Therefore, this study investigated if 15 days of hypoxia results in the induction of Hsp72 in adrenal glands. In addition, we examined whether heat treatment had any effect on adrenal Hsp72 expression to hypoxia, as cellular and systemic physiological cross-adaptation was suggested between heat stress and hypoxic stress. Materials and Methods: Male 4-month-old Wistar rats were randomly assigned to one of the four experimental groups (n = 8 each group): (1) control (C), (2) heat treatment (15H), (3) heat treatment and 15 days of normobaric hypoxia (15HHp), and (4) 15 days of normobaric hypoxia (15Hp). Three one-hour heat treatment sessions at 41°C were applied on the first two days before hypoxic exposure and on the day 7. Hypoxic exposure was consisting of normobaric hypoxia containing 9.7% O2. Results: Fifteen days of hypoxia did not increase the adrenal Hsp72 levels (p = 0.99). Furthermore, when hypoxia was added to the heat treatment, heat-related increases in adrenal Hsp72 levels disappeared. Adrenal weight to body weight ratio was not different among groups (p = 0.11). Plasma corticosterone levels were significantly lower in all experimental groups compared with control (p < 0.05), and addition of hypoxia resulted in further significant reduction of the plasma corticosterone levels (C > 15H>15HHp >15Hp; p < 0.05). Conclusions: These data demonstrate the adaptation of the adrenal gland to 15-day chronic normobaric hypoxic stress as well as possible cross-adaptation between heat and hypoxic stress in the adrenal gland.

Comparison of effects in sustained and diel-cycling hypoxia on hypoxia tolerance, histology, physiology and expression of clock genes in high latitude fish Phoxinus lagowskii

Phoxinus lagowskii is a popular fish in Chinese cuisine. Though it is found mainly in China's high-latitude regions, where diel-cycling hypoxia (DCH) is known to have unique impacts on aquatic organisms, there is little known about its response to hypoxia. Currently, nothing is known about the changes in blood parameters, gill and liver morphology, glucose and lipid metabolism, or expression of genes involved in clock and glucose metabolism in response to sustained hypoxia (SH) and diel-cycling hypoxia (DCH). To elucidate the influence of sustained and diel-cycling hypoxia on fish hypoxia tolerance, resting oxygen consumption (MO2) analysis was performed after ten days of hypoxia. This analysis revealed that hypoxia tolerance profoundly improved after ten days of either sustained or diel-cycling hypoxia acclimation, with DCH groups showing greater improvements than SH groups. Additionally, an increase in RBCs was found in P. lagowskii, suggesting an increase in the O2-carrying capacity of the blood to tolerate hypoxia. Hemoglobin (Hb) concentrations in P. lagowskii were increased at four days of diel-cycling hypoxia, confirming that physiological and metabolic adaptation to hypoxia is based on the duration of O2 exposure. Increased Hb and hematocrit (Hct) were found in DCH-exposed fish, both of which have been directly linked to high-latitude hypoxia tolerance. In the gills, lamella surface area increased in SH-exposed fish more than DCH-exposed fish, and these increases were accompanied by a decrease in the volume of interlamellar cell mass (ILCM). Histology changes in the liver showed a higher frequency of cytoplasmic vacuolization in DCH-exposed fish. PK increases in SH-exposed fish suggest that fish can use more energy sources in persistent hypoxia. Meanwhile, DCH-exposed fish use TG as an energy source. In SH-exposed fish, self-regulation of Cry1a was observed, whereas Cry1b gene was up-regulated significantly. In DCH-exposed fish, three of eight clock genes studied had increased expression, including Per1a, Clocka, and Cry1b, suggesting that SH and DCH result in different hypoxic responses. This study presents a novel approach to the study of fish responses to hypoxia in high latitude and shows that sustained hypoxia and diel-cycling hypoxia induce large differences in fish physiology.

Fifteen days of moderate normobaric hypoxia does not affect mitochondrial function, and related genes and proteins, in healthy men.

To investigate within the one study potential molecular and cellular changes associated with mitochondrial biogenesis following 15 days of exposure to moderate hypoxia. Eight males underwent a muscle biopsy before and after 15 days of hypoxia exposure (FiO2 = 0.140-0.154; ~ 2500-3200 m) in a hypoxic hotel. Mitochondrial respiration, citrate synthase (CS) activity, and the content of genes and proteins associated with mitochondrial biogenesis were investigated. Our main findings were the absence of significant changes in the mean values of CS activity, mitochondrial respiration in permeabilised fibers, or the content of genes and proteins associated with mitochondrial biogenesis, after 15 days of moderate normobaric hypoxia. Our data provide evidence that 15 days of moderate normobaric hypoxia have negligible influence on skeletal muscle mitochondrial content and function, or genes and proteins content associated with mitochondrial biogenesis, in young recreationally active males. However, the increase in mitochondrial protease LON content after hypoxia exposure suggests the possibility of adaptations to optimise respiratory chain function under conditions of reduced O2 availability.

Transcriptome Analysis of Hypoxic Lymphatic Endothelial Cells Indicates Their Potential to Contribute to Extracellular Matrix Rearrangement.

Lymphedema (LE) affects millions of people worldwide. It is a chronic progressive disease with massive development of fibrosclerosis when untreated. There is no pharmacological treatment of lymphedema. The disease is associated with swelling of the interstitium of the affected organ, mostly arm or leg, impressive development of adipose tissue, fibrosis and sclerosis with accumulation of huge amounts of collagen, and Papillomatosis cutis. Malnutrition and reduced oxygenation of the affected tissues is a hallmark of lymphedema. Here, we investigated if the hypoxia of lymphatic endothelial cells (LECs) might contribute to fibrosis. We applied RNASeq and qPCR to study the concordant changes of the exome of three human foreskin-derived LEC isolates after 4 days of hypoxia (1% O2) vs. normoxia (21% O2). Of the approximately 16,000 genes expressed in LECs, 162 (1%) were up- or down-regulated by hypoxia. Of these, 21 genes have important functions in the production or modification of the extracellular matrix (ECM). In addition to the down-regulation of elastin, we found up-regulation of druggable enzymes and regulators such as the long non-coding RNA H19, inter-alpha-trypsin inhibitor heavy chain family member 5 (ITIH5), lysyl-oxidase (LOX), prolyl 4-hydroxylase subunit alpha 1 (P4HA1), procollagen-lysine 2-oxoglutarate 5-dioxygenase 2 (PLOD2), and others that are discussed in the paper. Initial lymphatics do not produce a continuous basement membrane; however, our study shows that hypoxic LECs have an unexpectedly high ability to alter the ECM.

Differences in the microcirculation disturbance in the right and left ventricles of neonatal rats with hypoxic pulmonary hypertension

Microcirculation disturbance is a crucial pathological basis of heart damage; however, microcirculation alterations induced by hypoxic pulmonary hypertension (HPH) remain unknown, and the left ventricle (LV) in HPH is conventionally ignored. Herein, we investigated the changes in the cardiac structure, function and microcirculation after HPH and further compared the differences between the right ventricle (RV) and LV. Using a neonatal rat model of HPH, we found RV myocardial hypertrophy, dysfunction and poor myocardial perfusion in HPH rats. Additionally, RV microcirculation disturbance manifested as the abnormal expression of endothelin-1/eNOS and increased expression of intercellular cell adhesion molecule-1 (ICAM-1) or E-selectin 3 days after hypoxia, followed by vascular inflammation, coronary arterial remodeling and microvascular sparseness. Impairment in LV vasodilation was detected in rats after 3 days of hypoxia; however, no obvious microvascular rarefaction or inflammatory reaction was observed in the LV. In conclusion, our results suggest that HPH mainly triggers RV microcirculation disturbances, causing low myocardial perfusion damage and cardiac dysfunction. Despite the differences in the RV and LV, their impaired microvascular function, mediated by endothelial cells, occurs almost simultaneously after HPH, earlier than cardiac functional or structural abnormalities.

Mice but not rats modulate liver mitochondrial machinery in chronic hypoxia

Mice (FVB) permanently living at high‐altitude have increased respiratory frequencies and sustained or increased metabolic rates. On the other hand, rats permanently living at high‐altitude (SD) have impaired respiratory control, high hematocrit and hemoglobin, signs of pulmonary hypertension, and reduced metabolism; all these considered to be detrimental. This is coincident with the fact that common rats are not found in natural high‐altitude environments. At sea level, we reported that mice, but nor rats, exposed to 21 days of hypoxia (12% O2) increase their metabolic rate (O2 consumption and CO2 production), similarly to other rodent species that are considered well adapted to high‐altitude. As mitochondria is the final user of O2 in metabolism, we were interested in the role of mitochondria behind this divergence in physiological adjustments. We used saponin‐permeabilized samples of liver (most energy‐consuming organ in rodents), from male adult FVB mice and SD rats after exposure to hypoxia (12% O2, for 0, 1, 7 or 21 days), to measure the mitochondrial O2 consumption rate (OCR) following the SUIT‐01 protocol in the high‐resolution respirometer O2K (OROBOROS instruments). OCR was measured with substrates activating the NADH, succinate , and fatty acid pathways of the electron transport chain (ETC). We also measured the maximum capacity of ETC by uncoupling the electron transport from the oxidative phosphorylation with CCCP. The level of activation of complexes I and II was reported as the OCR for the N or S pathway correspondingly, divided by the maximum capacity (flux control ratio).Our preliminary results show that, compared to rats, mice have significantly higher ETC maximum capacities in normoxia and hypoxia. Moreover, after 21 days of hypoxia, mice showed an increase of 43% in maximum capacity in comparison to normoxic controls, while no increase in maximum capacity was evidenced in rats. In mice, we observed that complex I is transiently hyperactivated after 1 day of hypoxia and then, its activity reduces down below control levels by day 21 of hypoxia. Contrastingly, complex II activity is reduced after one day of hypoxia, getting back to control levels by day 21 of hypoxia. Furthermore, in rats, an increased activity of complex I occurred after 21 days of hypoxia. Regardless of normoxic or hypoxic exposure, rats showed higher activation levels of complex II than mice.These results show that mice, but not rats, have the ability to modulate their liver metabolic mitochondrial machinery under chronic hypoxia. The higher and adjustable maximum capacity in mice, suggest a more powerful and plastic liver mitochondrial machinery than in rats. This might explain in part their ability to keep sustained metabolic rates under hypoxic conditions.Support or Funding InformationSupported by NSERC, the Health Respiratory Network of Québec and CIHR.