Chronic Hypoxia Exposure Research Articles

DDIT4 promotes erythroid differentiation and coordinates with SIPA1 to regulate erythroid proliferation in bone marrow of high altitude erythrocytosis

Erythrocytosis moderately enhances the oxygen-carrying capacity of the blood and is considered a characteristic response of individuals adapting from low-altitude regions to high-altitude regions. Nevertheless, erythrocytosis can also turn excessive and result in maladaptive syndromes, such as high altitude polycythemia (HAPC). The increased differentiation or proliferation of erythroid cells in the bone marrow may be a crucial factor leading to accumulation of peripheral erythroid cells. However, the mechanism of erythroid regulation within the bone marrow of high-altitude erythrocytosis remains insufficiently systematically observed. We utilized single-cell transcription sequencing to characterize bone marrow cells following chronic hypoxic exposure and found that bone marrow erythrocytosis is associated with the accumulation of Baso-E, Poly-E, and Ortho-E cells at the terminal stage of erythroid lineage differentiation. Through analysis of differential gene expression and localization in differentiated cells within the erythroid lineage, we confirmed that DDIT4 expression was localized in advanced differentiated erythroblast including Baso-E, Poly-E and Ortho-E, its expression was significantly enhanced by hypoxia exposure. We demonstrated that overexpression of DDIT4 could promote K562 cell differentiation, and through the IP pull-down interaction protein profile, we found that DDIT4 might participate in regulating the cell cycle by interacting with SIPA1 to promote the proliferation of erythroid cells and may be involved in HAPC.

Ndufs4 inactivation in glutamatergic neurons reveals swallow-breathing discoordination in a mouse model of Leigh Syndrome.

Swallowing, both nutritive and non-nutritive, is highly dysfunctional in children with Leigh Syndrome (LS) and contributes to the need for both gastrostomy and tracheostomy tube placement. Without these interventions aspiration of food, liquid, and mucus occur resulting in repeated bouts of respiratory infection. No study has investigated whether mouse models of LS, a neurometabolic disorder, exhibit dysfunctions in neuromuscular activity of swallow and breathing integration. We used a genetic mouse model of LS in which the NDUFS4 gene is knocked out (KO) specifically in Vglut2 or Gad2 neurons. We found increased variability of the swallow motor pattern, disruption in breathing regeneration post swallow, and water-induced apneas only in Vglut2 KO mice. These physiological changes likely contribute to weight loss and premature death seen in this mouse model. Following chronic hypoxia (CH) exposure, swallow motor pattern, breathing regeneration, weight, and life expectancy were not changed in the Vglut2-Ndufs4-KO CH mice compared to control, indicating a rescue of phenotypes. These findings show that like patients with LS, Ndufs4 mouse models of LS exhibit swallow impairments as well as swallow-breathing dyscoordination alongside the other phenotypic traits described in previous studies. Understanding this aspect of LS will open roads for the development of future more efficacious therapeutic intervention for this illness.

Redefining chronic mountain sickness: insights from high-altitude research and clinical experience

Abstract Chronic Mountain Sickness (CMS), characterized by increased red blood cells above average values traditionally attributed to chronic hypobaric hypoxia exposure, is being redefined in light of recent research and clinical experience. We propose a shift in perspective, viewing CMS not as a singular entity but as Poly-erythrocythemia (PEH), as the Hematocrit/Hemoglobin/Red Blood Cells (Ht/Hb/RBCs) increase constitutes a sign, not a disease reflecting a spectrum of oxygen transport alterations in multiple diseases in the chronic hypoxia environment in high-altitude populations. Drawing on over five decades of experience at the High Altitude Pulmonary and Pathology Institute (HAPPI-IPPA) in Bolivia, we advocate for altitude-specific blood parameter norms and emphasize the importance of correct etiological diagnosis for effective management. This updated understanding not only aids in managing chronically hypoxemic patients at various altitudes but also offers valuable insights into global health challenges, including the recovery from COVID-19.

Recovery of ventilatory and metabolic responses to hypoxia in neonatal rats after chronic hypoxia

Chronic hypoxia (CH) during postnatal development attenuates the hypoxic ventilatory response (HVR) in mammals, but there are conflicting reports on whether this plasticity is permanent or reversible. This study tested the hypothesis that CH-induced respiratory plasticity is reversible in neonatal rats and investigated whether the initial plasticity or recovery differs between sexes. Rat pups were exposed to 3 d of normobaric CH (12 % O2) beginning shortly after birth. Ventilation and metabolic CO2 production were then measured in normoxia and during an acute hypoxic challenge (12 % O2) immediately following CH and after 1, 4–5, and 7 d in room air. CH pups hyperventilated when returned to normoxia immediately following CH, but normoxic ventilation was similar to age-matched control rats within 7 d after return to room air. The early phase of the HVR (minute 1) was only blunted immediately following the CH exposure, while the late phase of the HVR (minute 15) remained blunted after 1 and 4–5 d in room air; recovery appeared complete by 7 d. However, when normalized to CO2 production, the late phase of the hypoxic response recovered within only 1 d. The initial blunting of the HVR and subsequent recovery were similar in female and male rats. Carotid body responses to hypoxia (in vitro) were also normal in CH pups after approximately one week in room air. Collectively, these data indicate that ventilatory and metabolic responses to hypoxia recover rapidly in both female and male neonatal rats once normoxia is restored following CH.

Significance of Nitric Oxide Derived from the Nitric Oxide Synthases System in Cardiovascular Inter-Organ Cross-Talk.

Inter-organ cross-talk contributes to the pathogenesis of various disorders, and drug development based on inter-organ cross-talk is attracting attention. The roles of nitric oxide (NO) derived from the NO synthases system (NOSs) in inter-organ cross-talk remain unclear. We have investigated this issue by using our mice deficient in all three NOSs (triple n/i/eNOSs-/- mice). We reported that 2/3 nephrectomized triple n/i/eNOSs-/- mice die suddenly because of early onset of myocardial infarction, suggesting the protective role of NO derived from NOSs in the cross-talk between the kidney and the heart. We studied the role of NO derived from NOSs expressed in the bone marrow in vascular lesion formation. Constrictive arterial remodeling and neointimal formation following unilateral carotid artery ligation were prominently aggravated in wild-type mice transplanted with triple n/i/eNOSs-/- bone marrow cells as compared with those with wild-type bone marrow cells, suggesting the protective role of NO derived from NOSs in the cross-talk between the bone marrow and the blood vessel. We further investigated the role of NO derived from NOSs expressed in the bone marrow in pulmonary hypertension. The extent of pulmonary hypertension after chronic hypoxic exposure was markedly exacerbated in wild-type mice underwent triple n/i/eNOSs-/- bone marrow transplantation as compared with those underwent wild-type bone marrow transplantation, suggesting the protective role of NO derived from NOSs in the cross-talk between the bone marrow and the lung. These lines of evidence demonstrate that systemic and myelocytic NOSs could be novel therapeutic targets for myocardial infarction, vascular disease, and pulmonary hypertension. Significance Statement We demonstrated in studies with triple n/i/eNOSs-/- mice that partial nephrectomy accelerates the occurrence of myocardial infarction induced by systemic NOSs deficiency, that myelocytic NOSs deficiency aggravates vascular lesion formation after unilateral carotid artery ligation, and that myelocytic NOSs deficiency exacerbates chronic hypoxia-induced pulmonary hypertension. These results suggest that NO derived from NOSs plays a protective role in cardiovascular inter-organ cross-talk, indicating that systemic and myelocytic NOSs could be important therapeutic targets for myocardial infarction, vascular disease, and pulmonary hypertension.

Simple model of pulmonary hypertension secondary to left heart pressure overload induced by partial intravascular occlusion of the ascending aorta.

Pulmonary hypertension is a group of diseases characterized by elevated pulmonary artery pressure and pulmonary vascular resistance with significant morbidity and mortality. The most prevalent type is pulmonary hypertension secondary to left heart disease (PH-LHD). The available experimental models of PH-LHD use partial pulmonary clamping by technically nontrivial open-chest surgery with lengthy recovery. We present a simple model in which the reduction of the cross-sectional area of the ascending aorta is achieved not by external clamping but by partial intravascular obstruction without opening the chest. In anesthetized rats, a blind polyethylene tubing was advanced from the right carotid artery to just above the aortic valve. The procedure is quick and easy to learn. Three weeks after the procedure, left heart pressure overload was confirmed by measuring left ventricular end-diastolic pressure by puncture (1.3 ± 0.2 vs. 0.4 ± 0.3 mmHg in controls, mean ± SD, P < 0.0001). The presence of pulmonary hypertension was documented by measuring pulmonary artery pressure by catheterization (22.3 ± 2.3 vs. 16.9 ± 2.7 mmHg, P = 0.0282) and by detecting right ventricular hypertrophy and increased muscularization of peripheral pulmonary vessels. Contributions of a precapillary vascular segment and vasoconstriction to the increased pulmonary vascular resistance were demonstrated, respectively, by arterial occlusion technique and by normalization of resistance by a vasodilator, sodium nitroprusside, in isolated lungs. These changes were comparable, but not additive, to those induced by an established pulmonary hypertension model, chronic hypoxic exposure. Intravascular partial aortic obstruction offers an easy model of pulmonary hypertension induced by left heart disease that has a vasoconstrictor and precapillary component.NEW & NOTEWORTHY We present a new, simple model of a clinically important type of pulmonary hypertension, that induced by left heart failure. Left ventricular pressure overload is induced in rats by inserting a blinded cannula into the ascending aorta via carotid artery access. This partial intravascular aortic obstruction, which does not require opening of the chest and prolonged recovery, causes pulmonary hypertension, which has a precapillary and vasoconstrictor as well as a vascular remodeling component.

Development of CRID3-Based Anti-inflammatory Agents to Ameliorate Chronic Hypoxia-Induced Memory Impairment in Zebrafish Models.

Chronic hypoxic exposure triggers the onset and progression of cognitive dysfunction; however, the mechanisms underlying chronic hypoxia-induced neuroinflammation and its contribution to cognitive dysfunction remain poorly understood. Although inflammation and hypoxia are interdependent, numerous recent studies have linked the development of various human diseases to hypoxia-induced inflammation. In this study, we focused on the NLRP3 inflammasome with novel analogues of cytokine release inhibitory drug 3 (CRID3), a class of small molecule inhibitors for the NLRP3 inflammasome, to investigate their potential contribution to alleviating chronic hypoxia-induced neuroinflammation using the zebrafish model. The designed CRID3 analogues 6a-q were prepared from 2-methyl furan-3-carboxylate, following a four-step reaction sequence and fully characterized by NMR and mass spectral analysis. The administration of CRID3 analogues 6a-q led to a notable reduction in neuroinflammation and an increase in glial proliferation markers in both sexes. In addition, we investigated the potential effects of CRID3 analogues 6a-q through various behavioral tasks to assess their role in ameliorating post-hypoxic behavioral deficits and cognitive impairment. Notably, the study revealed that post-chronic hypoxia, male zebrafish exhibited significantly higher levels of inflammatory marker expression than females. Furthermore, we observed that the neurogenic response to treatment with CRID3 derivative 6o varied depending on the sex, with females showing a sex-specific differential increase in neurogenesis compared to males. This work emphasizes the significance of considering sex differences into account in developing therapeutic strategies for neurological disorders, as shown by the sex-specific molecular and behavioral changes in zebrafish cognitive impairment and neuroinflammation.

Intermittent hypoxia increases lipid insulin resistance in healthy humans: A randomized crossover trial.

Sympathetic overactivity caused by chronic intermittent hypoxia is a hallmark of obstructive sleep apnea. A high sympathetic tone elicits increases in plasma free fatty acid and insulin. Our objective was to assess the impact of 14 nights of chronic intermittent hypoxia exposure on sympathetic activity, glucose control, lipid profile and subcutaneous fat tissue remodelling in non-obese healthy humans. In this prospective, double-blinded crossover study, 12 healthy subjects were randomized, among them only nine underwent the two phases of exposures of 14 nights chronic intermittent hypoxia versus air. Sympathetic activity was measured by peroneal microneurography (muscle sympathetic nerve activity) before and after each exposure. Fasting glucose, insulin, C-peptide and free fatty acid were assessed at rest and during a multisampling oral glucose tolerance test. We assessed histological remodelling, adrenergic receptors, lipolysis and lipogenesis genes expression and functional changes of the adipose tissue. Two weeks of exposure of chronic intermittent hypoxia versus ambient air significantly increased sympathetic activity (p = 0.04). Muscle sympathetic nerve activity increased from 24.5 [18.9; 26.8] before to 21.7 [13.8; 25.7] after ambient air exposure, and from 20.6 [17.4; 23.9] before to 28.0 [24.4; 31.5] bursts per min after exposure to chronic intermittent hypoxia. After chronic intermittent hypoxia, post-oral glucose tolerance test circulating free fatty acid area under the curve increased (p = 0.05) and free fatty acid sensitivity to insulin decreased (p = 0.028). In adipocyte tissue, intermittent hypoxia increased expression of lipolysis genes (adipocyte triglyceride lipase and hormone-sensitive lipase) and lipogenesis genes (fatty acid synthase; p < 0.05). In this unique experimental setting in healthy humans, chronic intermittent hypoxia induced high sympathetic tone, lipolysis and decreased free fatty acid sensitivity to insulin. This might participate in the trajectory to systemic insulin resistance and diabetes for patients with obstructive sleep apnea.

Exosomes secreted by adipose mesenchymal stem cells overexpressing circPIP5K1C exert

BackgroundErectile dysfunction (ED) seriously affects men's normal life, and obstructive sleep apnoea (OSA) has been diagnosed as a causative factor. Currently, exosomes secreted by adipose mesenchymal stem cells (ADSC) have been used in the non-clinical experimental treatment of ED disease with prominent efficacy due to the advantages of high stability and no immune exclusion. MethodsIn this study, chronic intermittent hypoxia (CIH) exposure was used to induce ED-corresponding phenotypes in Sprague Dawley (SD) rats as well as in cavernous smooth muscle cells (CCSMCs). ED symptoms were treated using exosomes secreted by ADSCs overexpressing circPIP5K1C (EXO-circ) injected into the rat corpus cavernosum. ResultsEXO-circ has the effect of ameliorating ED induced by CIH exposure in rats, the mechanism of which is to promote the expression of the downstream target gene SMURF1 after adsorption of miR-153-3p through the sponge so that SMURF1 and PFKFB3 occur protein-protein binding and ubiquitination degradation of PFKFB3 appears to inhibit the occurrence of spongiotic smooth muscle cells glycolysis, and to restore the function of the smooth muscle. ConclusionsThese findings show that EXO-circ have a promising therapeutic potential in OSA-induced ED.

Living with hypoxia: Residence and site fidelity by golden perch (Macquaria ambigua) in habitats affected by methane seeps and chronic hypoxia

Abstract We conducted an acoustic telemetry study of native golden perch (Macquaria ambigua) to examine movement behaviour in areas affected by methane seeps and hypoxia in the intermittent Condamine River, Murray‐Darling Basin (MDB), Australia. Fish were collected during periods of no flow and hypoxia (dissolved oxygen [DO] &lt;1 mg/L). Despite these conditions, 38 of 43 fish tagged with acoustic transmitters were detected for &gt;3 months post‐tagging in the study reach and 27 fish were being detected after 14 months. During periods of elevated river flow and relatively high DO, 30 fish moved away from their original tagging locations, with three undertaking movements (&gt;7 km) outside the study reach and not returning. Generalised additive mixed models showed a significant increase in the probability of movement as soon as flow commenced and when water temperatures exceeded 19°C. As flows receded, most fish that had moved exhibited accurate homing behaviour to their original tagging location. The patterns of movement and site fidelity exhibited by golden perch correspond with previous studies of the species in intermittent rivers not affected by methane seeps and severe hypoxia, suggesting that the methane seeps and hypoxia did not inhibit fish movement nor render the affected habitats unsuitable for habitation. Golden perch can survive and remain active in water with much lower DO (&lt;1 mg/L) than previously described for large‐bodied native fishes in the MDB. However, fish condition in the study reach was slightly lower than other regions of the MDB, providing preliminary evidence that fish residing in habitats affected by chronic hypoxia and methane seepage may experience sub‐lethal stress. Our results demonstrate the importance of field‐based data on the behavioural and physiological responses of fish to chronic hypoxia and methane exposure to guide appropriate management responses.

Hypoxia-inducible factors activator, roxadustat, increases pulmonary vascular resistance in rats.

Activators of hypoxia inducible factors (HIFs), such as roxadustat, are promising agents for anemia treatment. However, since HIFs are also involved in the regulation of the pulmonary circulation, we hypothesized that roxadustat increases pulmonary vascular resistance and vasoconstrictor reactivity. Using isolated, cell-free solution perfused rat lungs, we found perfusion pressure-flow curves to be shifted to higher pressures by 2 weeks of roxadustat treatment (10 mg/kg every other day), although not as much as by chronic hypoxic exposure. Vasoconstrictor reactivity to angiotensin II and acute hypoxic challenges was not altered by roxadustat. Since roxadustat may inhibit angiotensin-converting enzyme 2 (ACE2), we also tested a purported ACE2 activator, diminazene aceturate (DIZE, 0.1 mM). It produced paradoxical, unexplained pulmonary vasoconstriction. We conclude that the risk of serious pulmonary hypertension is not high when roxadustat is given for 14 days, but monitoring is advisable.

The role of hypoxia and radiation in developing a CTCs-like phenotype in murine osteosarcoma cells.

Introduction: Cancer treatment has evolved significantly, yet concerns about tumor recurrence and metastasis persist. Within the dynamic tumor microenvironment, a subpopulation of mesenchymal tumor cells, known as Circulating Cancer Stem Cells (CCSCs), express markers like CD133, TrkB, and CD47, making them radioresistant and pivotal to metastasis. Hypoxia intensifies their stemness, complicating their identification in the bloodstream. This study investigates the interplay of acute and chronic hypoxia and radiation exposure in selecting and characterizing cells with a CCSC-like phenotype. Methods: LM8 murine osteosarcoma cells were cultured and subjected to normoxic (21% O2) and hypoxic (1% O2) conditions. We employed Sphere Formation and Migration Assays, Western Blot analysis, CD133 Cell Sorting, and CD133+ Fluorescent Activated Cell Sorting (FACS) analysis with a focus on TrkB antibody to assess the effects of acute and chronic hypoxia, along with radiation exposure. Results: Our findings demonstrate that the combination of radiation and acute hypoxia enhances stemness, while chronic hypoxia imparts a cancer stem-like phenotype in murine osteosarcoma cells, marked by increased migration and upregulation of CCSC markers, particularly TrkB and CD47. These insights offer a comprehensive understanding of the interactions between radiation, hypoxia, and cellular responses in the context of cancer treatment. Discussion: This study elucidates the complex interplay among radiation, hypoxia, and cellular responses, offering valuable insights into the intricacies and potential advancements in cancer treatment.

Non-Interferon-Dependent Role of STING Signaling in Pulmonary Hypertension.

Patients with constitutive activation of DNA-sensing pathway through stimulator of IFN (interferon) genes (STING), such as those with STING-associated vasculopathy with onset in infancy, develop pulmonary hypertension (PH). However, the role of STING signaling in general PH patients is heretofore undescribed. Here, we seek to investigate the role of STING in PH development. STING expression in patient lung samples was examined. PH was induced in global STING-deficient mice and global type I IFN receptor 1-deficient mice using bleomycin or chronic hypoxia exposure. PH development was evaluated by right ventricular systolic pressure and Fulton index, with additional histological and flow cytometric analysis. VEGF (vascular endothelial growth factor) expression on murine immune cells was quantified and evaluated with multiplex and flow cytometry. Human myeloid-derived cells were differentiated from peripheral blood mononuclear cells and treated with either STING agonist or STING antagonist for evaluation of VEGF secretion. Global STING deficiency protects mice from PH development, and STING-associated PH seems independent of type I IFN signaling. Furthermore, a role for STING-VEGF signaling pathway in PH development was demonstrated, with altered VEGF secretion in murine pulmonary infiltrated myeloid cells in a STING-dependent manner. In addition, pharmacological manipulation of STING in human myeloid-derived cells supports in vivo findings. Finally, a potential role of STING-VEGF-mediated apoptosis in disease development and progression was illustrated, providing a roadmap toward potential therapeutic applications. Overall, these data provide concrete evidence of STING involvement in PH, establishing biological plausibility for STING-related therapies in PH treatment.

Abstract 19092: Endothelial Caveolin-1 Deficiency Increases Susceptibility to Lethal Heart Failure in Male Mice With Hypoxia-Induced Pulmonary Hypertension

Introduction: Pulmonary arterial hypertension (PAH) is an incurable disease with high mortality. Reduced endothelial cell caveolin-1 (EC Cav1) expression contributes to idiopathic PAH, and in animal models, can be alleviated by inhibiting or deleting endothelial nitric oxide synthase (eNOS). Hypothesis: As females are at greater risk of developing PAH but exhibit a more favorable outcome compared to males, we hypothesized that EC Cav1 deficiency in male mice would contribute to EC Cav1-dependent sex differences following chronic hypoxia exposure. Methods: EC-Cav1 -/- mice ( Cdh5.Cre ERT2 ;mTmG;Cav1 f/f injected with tamoxifen; ECKO) vs. wild-type liter mates injected with oil (ECWT), and Cav1 KO, Cav1-RC, and WT mice (6-8 week old) were exposed to 10% O 2 for up to 8 weeks (hypoxia-induced PH (HPH)). Echocardiography, right ventricular systolic pressure (RVSP) and hypertrophy (RVH), lung histology, Western blot, and survival were used to quantify pulmonary and cardiac function. Data were analyzed by two-way ANOVA and Tukey’s post hoc analysis. Results: After 8-weeks of hypoxia exposure, over 90% of male and 40% of female Cav1 KO became moribund, whereas lethality was less than 10% for WT and Cav1 RC mice (p &lt; 0.01). Cav1 ECKO mice exhibited 30% lethality, whereas all ECWT siblings survived. Both Cav1 KO and ECKO mice exhibited a significant increase in pulmonary artery wall thickness, RVH, and RVSP. Hypoxia also reduced PAT and TAPSE, as well as LV systolic and diastolic function, including IVRT, CO, EF, FS, and global longitudinal strain in Cav1 KO and ECKO as compared to WT, ECWT, and Cav1-RC mice indicating EC Cav1 expression is cardioprotective in the context of HPH. In whole lung lysates, we observed reduced eNOS dimerization and an increase in eNOS pSer1179 indicative of eNOS dysfunction under normoxia which was exaggerated following hypoxia exposure to a greater extent in male Cav1 KO and ECKO as compared to Cav1-RC or WT mice. Conclusions: Chronic hypoxia-induced PH and mortality observed in Cav1 KO and ECKO mice is associated with both RV and LV failure and eNOS dysfunction to a greater extent in male mice, which was largely prevented by EC Cav1 expression.

Brain-aging related protein expression and imaging characteristics of mice exposed to chronic hypoxia at high altitude.

To determine changes in protein expression related to brain aging and imaging features in mice after chronic hypoxia exposure at high altitude. A total of 24 healthy 4-week-old mice were randomly divided into high altitude hypoxia (HH) and plain control (PC) groups (n = 8 per group). HH mice were transported from Xi'an (450 m above sea level) to Maduo (4,300 m above sea level) while PC mice were raised in Xi'an. After 6 months, 7.0T magnetic resonance imaging (MRI) was performed. All mice completed T2-weighted imaging (T2WI), diffusion tensor imaging (DTI), resting-state functional MRI (rs-fMRI), arterial spin labeling (ASL), and magnetic resonance angiography (MRA) examinations. Next, brain slices were prepared and Nissl staining was used to observe morphological changes in neurons. Ultrastructural changes in neurons were observed by transmission electron microscopy. Expression changes of Caspase-3, klotho, P16, P21, and P53 at the gene and protein levels were detected by real-time PCR (RT-PCR) and Western blot. The number of neuronal Nissl bodies in the hippocampus and frontal cortex was significantly decreased in the HH group compared to the PC group. Some hippocampal and frontal cortical neurons were apoptotic, the nuclei were wrinkled, chromatin was aggregated, and most mitochondria were mildly swollen (crista lysis, fracture). Compared with the PC group, the HH group showed elevated expression of caspase-3 mRNA, P16 mRNA, P21 mRNA, and P53 mRNA in the hippocampus and frontal cortex. Expression of Klotho mRNA in the frontal cortex was also significantly decreased. Western blot results showed that caspase-3 protein expression in the hippocampus and frontal cortex of the HH group was increased compared with the PC group. Moreover, there was decreased Klotho protein expression and significantly increased P-P53 protein expression. Compared with the PC group, expression of P16 protein in the frontal cortex of the HH group was increased and the gray matter (GM) volume in the left visceral area, left caudate nucleus, and left piriform cortex was decreased. Furthermore, the amplitude of low frequency fluctuation was decreased in the left posterior nongranular insular lobe, right small cell reticular nucleus, left flocculus, left accessory flocculus, and left primary auditory area, but increased in the GM layer of the left superior colliculus. Regional homogeneity was decreased in the left and right olfactory regions, but increased in the left bed nucleus. After exposure to high altitude, functional connectivity (FC) between the bilateral caudate nucleus and thalamus, corpus callosum, cingulate gyrus, anterior limbic cortex, globus pallidus, and hippocampus was weakened. FC between the right caudate nucleus and hypothalamus and entorhinal cortex was also weakened. The fractional anisotropy value of the left hippocampus was decreased in the HH group. Compared with the PC group, the HH group showed significantly increased inner diameters of the bilateral common carotid artery and left internal carotid artery. The cerebral blood flow values of the bilateral cortex and bilateral hippocampus in the HH group did not change significantly. Taken together, our findings show that chronic hypoxia exposure at high altitude may promote neuronal apoptosis and abnormal expression of related proteins, changing the structure and function of brain. These changes may contribute to brain aging.

Comprehensive metabolomics and transcriptomics analysis reveals protein and amino acid metabolic characteristics in liver tissue under chronic hypoxia.

At high altitudes, oxygen deprivation can cause pathophysiological changes. Liver tissue function is known to impact whole-body energy metabolism; however, how these functions are affected by chronic hypoxia remains unclear. We aimed to elucidate changing characteristics underlying the effect of chronic hypoxia on protein and amino acid metabolism in mouse livers. Mice were maintained in a hypobaric chamber simulating high altitude for 4 weeks. Livers were collected for metabolomic analysis via ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry. For transcriptomics analysis, we conducted RNA sequencing of hepatic tissues followed by Gene Ontology and KEGG pathway enrichment analyses. Chronic hypoxic exposure caused metabolic disorders of amino acids and their derivatives in liver tissue. We identified a number of metabolites with significantly altered profiles (including amino acids, peptides, and analogues), of which serine, phenylalanine, leucine, proline, aspartic acid, L-glutamate, creatine, 5-aminovaleric acid, L-hydroxyarginin, and g-guanidinobutyrate showed great potential as biomarkers of chronic hypoxia. A total of 2124 genes with significantly different expression levels were identified in hypoxic liver tissue, of which 1244 were upregulated and 880 were downregulated. We found pathways for protein digestion and absorption, arginine and proline metabolism, and mineral absorption related to amino acid metabolism were affected by hypoxia. Our findings surrounding the regulation of key metabolites and differentially expressed genes provide new insights into changes in protein and amino acid metabolism in the liver that result from chronic hypoxia.

Quercetin improves pulmonary arterial hypertension in rats by regulating the HMGB1/RAGE/NF-κB pathway

To explore the mechanism through which quercetin improves pulmonary arterial hypertension (PAH). Rat models of hypoxic pulmonary hypertension were established by exposure to hypoxia for 8-10 h each day (6 days a week for 4 weeks), and before each hypoxic exposure, the rats were given intragastric administration of 100 mg/kg quercetin or saline. After the treatments, the right ventricular systolic pressure (RVSP) and pulmonary artery systolic pressure of the rats were recorded. The right ventricular hypertrophy index (RVHI) was measured to evaluate right ventricular hypertrophy. HE staining was used to observe the remodeling of the pulmonary arterioles. The right cardiac function of the rats was evaluated by ultrasound. The protein levels of HMGB1, RAGE, NF-κB, Bax, Bcl-2 and cleaved caspase-3 in the lung tissue of the rats were detected using Western blotting. Compared with the rats maintained in normoxia, the rats with chronic hypoxic exposure showed significantly increased RVHI and RVSP (P<0.01), which were obviously lowered by quercetin treatment (P<0.01). HE staining showed significant pulmonary artery wall thickening with reduced lumen diameter in hypoxia group, and quercetin treatment effectively improved pulmonary vascular remodeling. Ultrasound examination revealed a significantly increased RVFW and a lowered PAT/PET ratio in hypoxia group (P<0.01), and such changes were ameliorated by quercetin treatment (P<0.01). Chronic hypoxia significantly increased the protein expressions of HMGB1 (P<0.01), RAGE, NF-κB and Bcl-2 (P<0.01) and lowered the protein expressions of Bax and cleaved caspase-3 (P<0.01); Quercetin treatment obviously lowered the protein expressions of HMGB1, NF-κB (P<0.05), RAGE (P<0.01) and (P<0.05) and increased the expressions of Bax and cleaved caspase-3 in the rat models (P<0.01). Quercetin improves pulmonary hypertension in rats possibly by promoting apoptosis through the HMGB1/RAGE/NF-κB pathway.

Abstract P3193: Cardiac Glutamine Metabolism As NAD+ Generating Pathway During Chronic Hypoxia

Cardiovascular disease morality relies in the inability of the adult mammalian heart to regenerate damaged tissue due to the inability of the adult cardiomyocytes to proliferate. This lack of proliferation in the adult heart is due to an increased DNA damage and activation of the DNA Damage response upon an increase of mitochondrial ROS generation. We have previously shown that modulation of mitochondrial metabolism towards glucose oxidation at expenses of fatty acid utilization can induce adult cardiomyocyte proliferation and support cardiac regeneration. Moreover, by modulating the oxygen levels and, thus, cardiomyocyte metabolism, by chronic hypoxia exposure, we were also able to induce cardiomyocyte proliferation and cardiac regeneration in adult mice. Remarkably, our metabolomic studies of the hypoxic hearts did not show increase of lactate production, a hallmark of hypoxic metabolism in order to regenerate NAD+ required for glycolysis and ATP production under low oxygen conditions. Interestingly, glutamine metabolism is one of the most compensatory pathways in these conditions. Glutamine can be metabolized to its conversion into glutamate, which can enter in the Krebs’ Cycle, but also feed the malate-aspartate shuttle. This shuttle has the ability to generate cytoplasmic NAD+. Here, we hypothesize that, during chronic hypoxia exposure, the heart uses glutamine as a fuel to regenerate cytoplasmic NAD+ as an alternative to lactate production. By means of carbon labeling and metabolic flux studies in vivo, we have proven that glutamine contribution to the malate-aspartate shuttle is enhanced during hypoxia in the heart. Moreover, we have determined that glutamine supplementation during hypoxia in cell culture increases cytoplasmid NAD+ levels, confirming the contribution of glutamine catabolism to NAD/NADH balance during hypoxia. Overall, we show that glutamine metabolism is an important adaptation to hypoxic metabolism in the heart and describe a new mechanism of metabolic rewiring under hypoxia beyond the classic lactate production upon glycolysis upregulation.

Influence of chronic hypoxia on the hypoxic ventilatory response of juvenile and adult rats

Chronic hypoxia (CH) from birth attenuates the acute hypoxic ventilatory response (HVR) in rats and other mammals, but CH is often reported to augment the HVR in adult mammals. To test the hypothesis that this transition – from blunting to augmenting the HVR – occurs in the third or fourth postnatal week in rats, juvenile and adult rats were exposed to normobaric CH (12% O2) for 7 days and the HVR was assessed by whole-body plethysmography. No transition was observed, however, and the acute HVR was reduced by 61 – 85% across all ages studied. The failure to observe an augmented HVR in adult rats could not be explained by the substrain of Sprague Dawley rats used, the duration of the CH exposure, the order in which test gases were presented, the level of hypoxia used for CH and to assess the HVR, or the effects of CH on the metabolic response to hypoxia and the hypercapnic ventilatory response. A literature survey revealed several distinct patterns of ventilatory acclimatization to hypoxia (VAH) in adult rats, with most studies (77%) revealing a decrease or no change in the acute HVR after CH. In conclusion, the effects of CH on respiratory control are qualitatively similar across age groups, at least within the populations of Sprague Dawley rats used in the present study, and there does not appear to be one “typical” pattern for VAH in adult rats.

Chronic hypoxia disrupts memory T regulatory cell phenotype which contributes to the pathogenesis of pulmonary hypertension

Pulmonary hypertension (PH) caused by chronic hypoxia (CH) is a multifaceted disease most often caused by sleep apnea, prolonged high-altitude exposure, or chronic obstructive pulmonary disease (COPD), with minimal treatment options, leading to right heart failure and death. One major contributor in the pathogenesis of PH that requires more characterization is the accompanying immune response.Our previous studies have demonstrated the importance of inflammation and self-immunity in CH-induced PH. We demonstrated pulmonary-perivascular localization of pro-inflammatory Th17 cells as well as an increase in total Th17 cells in the lung, while finding no overall changes in the number of anti-inflammatory T regulatory (Treg) cells present in the lung. Imbalance between Th17 and Treg cells has been shown to drive multiple inflammatory and metabolic diseases. One area of interest related to this balance has been the recent discovery and attempted characterization of exTregs, or cells that, at one point in time, were Tregs but lost their phenotype transitioning to pro-inflammatory Th17 cells, due to decreases in the expression of the transcription factor FoxP3. Therefore, we hypothesized that the increase in Th17 cells seen in CH is due to a decrease in Tregs and Treg suppressive capacity as well as a switching of Tregs to Th17 cells. Using a mouse model that allows the study of lineage stability and genetic mapping of Tregs, together with single-cell RNA sequencing (scRNA SEQ), we initially established that CH resulted in a gene expression profile consistent with exTregs or Tregs with less suppressive activity, such as downregulation of Il2ra gene and upregulation of Tnfrsf4 (adjusted p-value 0.03). To further establish a loss in Tregs as well as an increase in exTregs following CH exposure, we performed spectral flow cytometry of splenocytes on a Cytek Aurora to look for changes in protein expression and cell prevalence. Finally, we used a similar lineage mapping mouse model to look at Th17 cells. Following these experiments, we found supporting evidence of our scRNA SEQ data, finding a decrease in Tregs in both male and female mice following CH exposure, as well as an increase in exTregs in both groups exposed to CH. We also further supported this data finding Th17 cells tracked through our model to be increased following CH. These results support the importance of immune cell balance and inflammation in the pathogenesis of CH-induced PH. R56 HL153065-01 NIH, AHA 18TPA34170037, AHA967925 &amp; T32 HL07736 NIH 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.